natural-products-in-plant-pest-management

NATURAL PRODUCTS IN PLANT PESTMANAGEMENT

NATURAL PRODUCTSIN PLANT PEST MANAGEMENTEdited byNawal K. DubeyCentre for Advanced Studies in BotanyBanaras Hindu University, Varanasi, India

CABI is a trading name of CAB InternationalCABI Head OfficeCABI North American OfficeNosworthy Way875 Massachusetts AvenueWallingford7th FloorOxfordshire OX10 8DE Cambridge, MA 02139UKUSATel: +44 (0)1491 832111 Tel: +1 617 395 4056Fax: +44 (0)1491 833508 Fax: +1 617 354 6875E-mail: cabi@cabi.orgE-mail: cabi-nao@cabi.orgWebsite: www.cabi.org© CAB International 2011. All rights reserved. No part of this publication maybe reproduced in any form or by any means, electronically, mechanically, byphotocopying, recording or otherwise, without the prior permission of thecopyright owners.A catalogue record for this book is available from the British Library,London, UK.Library of Congress Cataloging-in-Publication DataNatural products in plant pest management / edited by N.K. Dubey.p. cm.Includes bibliographical references and index.ISBN 978-1-84593-671-6 (alk. paper)1. Natural pesticides. 2. Agricultural pests–Control. 3. Plant products.I. Dubey, N. K. II. Title.SB951.145.N37N394 2011632′.96–dc22ISBN-13: 978 1 84593 671 6Commissioning editor: Sarah MellorProduction editor: Fiona ChippendaleTypeset by AMA Dataset, Preston, UK.Printed and bound in the UK by CPI Antony Rowe, Chippenham.2010020068

ContentsContributorsPrefaceviiix1. Global Scenario on the Application of Natural Productsin Integrated Pest Management Programmes 1N.K. Dubey, Ravindra Shukla, Ashok Kumar,Priyanka Singh and Bhanu Prakash2. Plant Products in the Control of Mycotoxinsand Mycotoxigenic Fungi on Food Commodities 21Sonia Marín, Vicente Sanchis and Antonio J. Ramos3. Natural Products from Plants: Commercial Prospectsin Terms of Antimicrobial, Herbicidal and Bio-stimulatoryActivities in an Integrated Pest Management System 42J.C. Pretorius and E. van der Watt4. Antimicrobials of Plant Origin to Prevent the Biodeteriorationof Grains 91K.A. Raveesha5. Some Natural Proteinaceous and Polyketide Compoundsin Plant Protection and their Potential in GreenConsumerization 109L.A. Shcherbakova6. Natural Products as Allelochemicals in Pest Management 134Roman Pavelav

viContents7. Potency of Plant Products in Control of Virus Diseases of Plants 149H.N. Verma and V.K. Baranwal8. Phytochemicals as Natural Fumigants and Contact InsecticidesAgainst Stored-product Insects 175Moshe Kostyukovsky and Eli Shaaya9. Prospects of Large-scale Use of Natural Products as Alternativesto Synthetic Pesticides in Developing Countries 191D.B. Olufolaji10. Current Status of Natural Products in Pest Managementwith Special Reference to Brassica carinata as a Biofumigant 205María Porras11. Fungal Endophytes: an Alternative Source of BioactiveCompounds for Plant Protection 218R.N. Kharwar and Gary Strobel12. Suppressive Effects of Compost Tea on Phytopathogens 242Mila Santos, Fernando Diánez and Francisco Carretero13. Biotechnology: a Tool for Natural Product Synthesis 263Sanath HettiarachiIndex 280

ContributorsV.K. Baranwal, Advanced Centre of Plant Virology, I.A.R.I, New Delhi, India.E-mail: vbaranwal2001@yahoo.comFrancisco Carretero, Plant Production Department. University of Almeria,La Cañada de San Urbano. 04120 Almería, SpainN.K. Dubey, Department of Botany, Banaras Hindu University, Varanasi-221005,India. E-mail: nkdubey2@rediffmail.comFernando Diánez, Plant Production Department. University of Almeria,La Cañada de San Urbano. 04120 Almería, SpainSanath Hettiarachi, Department of Botany, University of Ruhuna, Matara,Sri Lanka. Email: sanath@bot.ruh.ac.lkR.N. Kharwar, Mycopathology and Microbial Technology Lab, Departmentof Botany, Banaras Hindu University, Varanasi-221005, India.E-mail: rnkharwar@yahoo.comMoshe Kostyukovsky, Agricultural Research Organization, the Volcani Center,Israel, Bet Dagan, P.O. Box 6, 50250, Israel. E-mail: inspect@volcani.agri.gov.ilAshok Kumar, Department of Botany, Banaras Hindu University, Varanasi-221005, IndiaSonia Marín, Food Technology Department, Lleida University, XaRTA-UTPV,Lleida, Spain. E-mail: smarin@tecal.udl.catD.B. Olufolaji, Department of Crop, Soil and Pest Management, TheFederal University of Technology, P.M.B. 704, Akure. Nigeria.E-mail: tundeolufolaji@yahoo.co.ukRoman Pavela, Crop Research Institute, Drnovska 507, Prague 6 – Ruzyne, CzechRepublic. E-mail: pavela@vurv.czMaría Porras, Department of Crop Protection, IFAPA Centro LasTorres – Tomejil, Aptdo. 41200-Alcalá del Río, Sevilla, Spain.E-mail: mariaa.porras@juntadeandalucia.esBhanu Prakash, Department of Botany, Banaras Hindu University,Varanasi-221005, Indiavii

viiiContributorsJ.C. Pretorius, Department of Soil, Crop and Climate Sciences, Universityof the Free State, P.O. Box 339, Bloemfontein 9300, South Africa.E-mail: pretorjc.sci@ufs.ac.zaAntonio J. Ramos, Food Technology Department, Lleida University, XaRTA-UTPV, Lleida, SpainK.A. Raveesha, Herbal Drug Technology Laboratory, Department of Studies inBotany, University of Mysore, Manasagangotri, Mysore-570 006, India.E-mail: karaveesha@gmail.comVicente Sanchis, Food Technology Department, Lleida University, XaRTA-UTPV,Lleida, SpainMila Santos, Plant Production Department. University of Almeria, La Cañada deSan Urbano, 04120 Almería, Spain. E-mail: msantos@ual.esEli Shaaya, Agricultural Research Organization, the Volcani Center, Israel,Bet Dagan, P.O. Box 6, 50250, IsraelL.A. Shcherbakova, Russian Research Institute of Phytopathology, VNIIF, B.,Vyazyomy, Moscow reg., 143050, Russia. E-mail: larisa@vniif.rosmail.comRavindra Shukla, Department of Botany, Banaras Hindu University, Varanasi-221005, IndiaPriyanka Singh, Department of Botany, Banaras Hindu University, Varanasi-221005, IndiaGary Strobel, Department of Plant Sciences, Montana State University, Bozeman,MT 59717, USAE. van der Watt, Department of Soil, Crop and Climate Sciences, University of theFree State, P.O. Box 339, Bloemfontein 9300, South AfricaH.N. Verma, Jaipur National University, Jaipur, India.E-mail: vermalko@yahoo.co.uk

PrefaceThe ever increasing global population needs substantial resources for foodproduction. However, food production as well as its protection is imperative.The situation gets particularly critical in developing countries where the netfood production rate is slowing down relative to the population rise. Theworld food situation is aggravated by the fact that, in spite of all the availablemeans of plant protection, a major fraction of the yearly output of food commoditiesgets destroyed by various pests including bacteria, fungi, viruses,insects, rodents and nematodes. The production of mycotoxins by fungi hasadded new dimensions to the gravity of the problem. Losses at times aresevere enough to lead to famine in large areas of the world that are denselypopulated and dependent on agriculture.The use of synthetic pesticides has undoubtedly contributed to a greenrevolution in different countries through increased crop protection. However,recent years witnessed considerable pressure on consumers and farmersto reduce or even eliminate the deployment of synthetic pesticides inagriculture owing to environmental risks emerging from their indiscriminateuse. Thus, there has been renewed interest in botanical pesticides as the alternativeand eco-chemical option in pest management. It is also imperative forsustainable agriculture to reduce the incidence of pests and crop diseases toa degree that does not seriously damage the farmer’s products and also todevelop cost-effective strategies with minimal ecological side effects.The use of locally available plants in the control of pests is an age-oldtechnology in many parts of the world. Some plants, namely Derris, Nicotianaand Ryania, were used to combat agricultural pests during the prehistoricera. Used widely until the 1940s, such botanical pesticides have been partiallyreplaced by synthetic pesticides that are easier to procure and longerlasting.Higher plants, in this respect, harbour numerous compounds that mayoffer resistance to pathogens. There has been a renewed interest in botanicalix

xPrefaceantimicrobials owing to various distinct advantages. Botanicals, being thenatural derivatives, are biodegradable, and do not leave toxic residues orbyproducts to contaminate the environment. The main thrust of recentresearch in the area has been to evolve alternative control strategies thateventually reduce dependency on synthetic fungicides. Recently, in differentparts of the world, emphasis has been placed on exploitation of higher plantproducts as the novel chemotherapeutants for plants because of their nontoxicity,systemicity and biodegradability. To date, different plant productshave been formulated as botanical pesticides for large-scale applications forthe eco-friendly management of plant pests and as an alternative to syntheticpesticides in crop management. These products are cost-effective and havelow toxicity to humans and livestock. Therefore, such products from higherplants may be exploited as the eco-chemical and biorational approach inintegrated plant protection programmes. The interests and trends in themodern society for ‘green consumerism’ involving fewer synthetic ingredientsin the agriculture industry may favour the approval of plant-basedproducts as the safe alternative strategy in managing plant pests. Naturalplant chemicals will certainly play a pivotal role in the near future for pestcontrol in industrialized and developing countries as well. Because of theirbiodegradable nature, systemicity following application, ability to alter thebehaviour of target pests and their favourable safety profile, the botanicalpesticides are considered the vital tools in achieving the evergreen revolutionand in the ecological strategy of controlling agricultural pests.To date, natural plant products for pest management form the frontierareas of research on a global scale. The present book deals with the currentstate and future prospects of botanical pesticides in the eco-friendly managementof plant pests. Looking at the growing interest of professionals and thegeneral public alike, it seemed imperative to produce a book that encompassesas much information as possible, with an emphasis on the exploitation ofplant products in the possible management of agricultural pests. The bookmay be useful to many, including plant pathologists, microbiologists, entomologists,plant scientists and natural product chemists. It is a compilationof invited chapters from eminent scientists and professors from educationalinstitutions and research organizations of different countries. The bookaddresses different topics under the domain of natural compounds inagricultural pest management and is an attempt to include notable anddiversified scientific works in the field carried out by leading scientists inthe world.The book harbours a total of 13 chapters contributed by eminent scientistsworking in the field so as to offer relevant and practical information oneach topic. Different issues, including the global scenario on the applicationof botanical pesticides, plant products in the control of mycotoxins, the commercialapplication of botanical pesticides and their prospects in green consumerism,natural products as allelochemicals, their efficacy against viraldiseases and storage pests, and bioactive products from fungal endophytes,have been covered in the book. I am very grateful to the contributors for theirgenerous and timely responses in spite of their busy academic schedules.

PrefacexiI wish to extend my appreciation to all the contributors for their cooperationand encouragement. Without their enthusiasm and the timely submission oftheir excellent chapters, this work would not have been possible.The book focuses on newer developments in agricultural pest managementusing natural products and provides up-to-date information fromworld experts in different fields. It is expected that the book will be a sourceof inspiration to many for future developments in the field. It is also hopedthat the book will become useful for those engaged in such an extraordinaryand attractive area. The book would serve as the key reference for recentdevelopments in frontier research on natural products in the management ofagricultural pests and also for the scientists working in this area.I convey the strength of my feelings to my wife Dr Nirmala Kishore,daughter Dr Vatsala Kishore and son Navneet Kishore who have alwaysbeen the excellent intellectual companions without whose constant lovingsupport, patience, and unmatched help and sacrifices it would have beendifficult to bring out the book on time. I also bow my head to my parents(Sri G.N. Dubey and Smt Shanti Devi) for their blessings. My sincere thanksare also due to my current research students, especially Ravindra Shukla, forhelp and cooperation.Thanks are due to CABI Publishers for publishing the book with utmostinterest; I am thankful to the staff members of CABI for sparing no pains toensure a high standard of publication. Special thanks goes to Sarah Mellor,the Commissioning Editor, who initially motivated me to bring out this bookduring the ICPP 2008 held in Turin, Italy.N.K. Dubey

1 Global Scenario on theApplication of Natural Productsin Integrated Pest ManagementProgrammesN.K. DUBEY, RAVINDRA SHUKLA, ASHOK KUMAR,PRIYANKA SINGH AND BHANU PRAKASHDepartment of Botany, Banaras Hindu University, Varanasi, IndiaAbstractIn recent years there has been considerable pressure in agriculture to reduce chemicalpesticides and to look for their better alternatives. The plant kingdom is recognizedas the most efficient producer of different biologically active compounds, which providethem with resistance against different pests. Some higher plant products havebeen currently formulated as botanical pesticides and are used on a large scale as ecofriendlyand biodegradable measures in managing agricultural pests. Botanicals usedin agricultural pest management are safer to the user and the environment. The interestin the possible use of natural compounds to control agricultural pests has notablyincreased in response to consumer pressure to reduce or eliminate chemically synthesizedadditives in foods. There is a wide scope of use of plant-based pesticides in theintegrated management of different agricultural pests. A consolidated and continuoussearch of natural products may yield safer alternative control measures comparableto azadirachtin and pyrethryoids, which are being used in different part of theworld as ideal natural fungicides. The products from higher plants are safe and economicaland would be in high demand in the global pesticide market because of theirdiverse mode of application.1.1 IntroductionAgriculture plays an important role in the survival of humans and animals.It is the driving force for broad-based economic growth, particularly in developingcountries. Tropical and subtropical regions have a greater potential forfood production and can grow multiple crops annually. Agricultural cropssuffer a colossal loss due to the ravages of insects and diseases thus causinga serious threat to our agricultural production. In some years, losses are muchgreater, producing catastrophic results for those who depend on the crop forfood. Major disease outbreaks among food crops have led to famines and© CAB International 2011. Natural Products in Plant Pest Management(ed. N.K. Dubey) 1

2 N.K. Dubey et al.mass migrations throughout history. Loss of crops from plant diseases mayresult in hunger and starvation, especially in less developed countries whereaccess to disease-control methods is limited and annual losses of 30–50 % arecommon for major crops. Owing to the congenial climatic conditions andparticular environment, the agriculture in tropical and subtropical countriessuffers severe losses due to pests (Varma and Dubey, 2001; Roy, 2003).Even during storage, foods are severely destroyed by fungi, insects andother pests. The deterioration in the stored food commodities is mainly causedby three agents, fungi, insects and rodents, under different conditions of storage.Insect pests cause heavy losses to stored grains, especially in humid andwarm areas of the world. The production of mycotoxins by several fungi hasadded a new dimension to the gravity of the problem. Fungi are significantdestroyers of foodstuffs during storage, rendering them unfit for human consumptionby retarding their nutritive value and sometimes by the productionof mycotoxins. According to FAO estimates, 25% of the world food crops areaffected by mycotoxins each year (Dubey et al., 2008). Generally, tropical conditionssuch as high temperature and moisture, unseasonal rains during harvest,and flash floods lead to mycotoxins. Poor harvesting practices andimproper storage during transport and marketing can also contribute to theproliferation of mycotoxins. Among the mycot oxins, aflatoxins raise the mostconcern posing a great threat to human and livestock health as well as internationaltrade. Aflatoxins are the most dangerous and about 4.5 billion people indeveloping countries are exposed to aflatoxicoses (Williams et al., 2004;Srivastava et al., 2008). Aflatoxins are potent toxic, carcinogenic, mutagenic,immunosuppressive agents, produced as secondary metabolites by the fungiAspergillus flavus and A. parasiticus on a variety of food products.The entire effort of growing a crop will be lost in the absence of cropprotection, resulting in financial loss to the grower. Therefore crop protectionagainst various pests is a must in agriculture. There is a need to reduce if noteliminate these losses by protecting the crops from different pests throughappropriate techniques. Currently, the role of crop protection in agricultureis of great importance and is a more challenging process than before. Hence,there is an urgent need to pay proper attention to control quantitative lossesdue to pest infestations of crops and their produce as well as qualitativelosses due to mycotoxin contamination.1.2 The Need to Search for Eco-friendly Control MeasuresThe most conventional and common method of pest and disease control isthrough the use of pesticides. Pesticides are the substances or mixture of substancesused to prevent, destroy, repel, attract, sterilize or mitigate the pests.Generally pesticides are used in three sectors, viz. agriculture, public healthand consumer use. These pesticides are largely synthetic compounds that killor deter the destructive activity of the target organism.Many farmers and crop growers use insecticides to kill infestinginsects. The consumption of pesticide in some of the developed countries is

Global Scenario and Natural Products 3almost 3000 g/hectare. Unfortunately, there are reports that these compoundspossess inherent toxicities that endanger the health of the farm operators,consumers and the environment (Cutler and Cutler, 1999). Pesticides aregenerally persistent in nature. Upon entering the food chain they destroy themicrobial diversity and cause ecological imbalance. Their indiscriminate usehas resulted in the development of a resistance problem among insects toinsecticides, pesticide residue hazards, upsetting the balance of nature and aresurgence of treated populations. Insect resistance to phosphine is a matterof serious concern (Rajendran, 2001; Benhalima et al., 2004; Thie and Mills,2005; Dubey et al., 2008). The repeated use of certain chemical fungicides inpacking houses has led to the appearance of fungicide-resistant populationsof storage pathogens (Brent and Hollomon, 1998). The ability of some ofthese pests to develop resistance curbs the effectiveness of many commercialchemicals. Resistance has accelerated in many insect species and it wasreported by the World Resources Institute that more than 500 insect and mitespecies are immune to one or more insecticides (WRI, 1994). Similarly about150 plant pathogens such as fungi and bacteria are now shielded againstfungicides (Shetty and Sabitha, 2009). Moreover, the control of such pests hasbecome increasingly difficult because of reduced effectiveness of pesticidescaused by the emergence of pesticidal resistance in arthropod pests.Reliance on synthetic chemicals to control pests has also given rise to thedestruction of beneficial non-target organisms (parasitoids and predators),thereby affecting the food chain and impacting on biological diversity. Therehave also been cases of pests becoming tolerant to insecticides, resulting inthe use of double and triple application rates (Stoll, 2000).Furthermore, the use of synthetic chemicals has also been restrictedbecause of their carcinogenicity, teratogenicity, high and acute residualtoxicity, ability to create hormonal imbalance, spermatotoxicity, long degradationperiod, environmental pollution and their adverse effects on foodand side effects on humans (Omura, et al., 1995; Unnikrishnan and Nath,2002; Xavier, et al., 2004; Konstantinou, et al., 2006; Feng and Zheng, 2007).Pesticides can be remarkably persistent in biological systems. They havebeen reported to be accumulated in ecosystems. Osprey eggs in the QueenCharlotte Islands, polar bear fat in the high Arctic, and the blubber of whalesin all the oceans of the world are contaminated with pesticide residues, eventhough all these creatures live far from point sources of pesticide application(CAPE, 2009). Water and wind, as well as the bodies of animals that serveas prey for others (including humans) higher on the food chain, are theuniversal vectors for pesticide dispersal. Highest on the food chain, humanbreast milk contamination is of great concern because of high levels ofbio-accumulated pesticides. The World Health Organization (WHO) estimatesthat 200,000 people are killed worldwide, every year, as a direct resultof pesticide poisoning (CAPE, 2009). Pest control strategies, therefore, needproper regulation in the interest of human health and environment. In recentyears there has been considerable pressure on consumers to reduce or eliminatechemical fungicides in foods. There is increasing public concern over thelevel of pesticide residues in food. This concern has encouraged researchers

4 N.K. Dubey et al.to look for alternative solutions to synthetic pesticides (Sharma andMeshram, 2006).Effort is therefore needed to find alternatives or formulations forcurrently used pesticides. There is, therefore, still a need for new methods ofreducing or eliminating food-borne pathogens, possibly in combination withexisting methods (Leistner, 1978). At the same time, Western society appearsto be experiencing a trend of ‘green consumerism’ (Tuley de Silva, 1996; Smidand Gorris, 1999), desiring fewer synthetic food additives and products witha smaller impact on the environment.Considering all these drawbacks, many components of the integratedpest management (IPM) concept were developed in the late 19th and early20th centuries, and biopesticides and bioagents became important toolsin pest management strategies that are practical, economical, eco-friendlyand protective for both public health and the environment. Sustainableagriculture aims to reduce the incidence of pests and diseases to such adegree that they do not seriously damage the farmer’s crops — but withoutupsetting the balance of nature. One of the aims of sustainable agricultureis to rediscover and develop methods whose cost and ecological side-effectsare minimal.1.3 Historical Use of Botanical Products in Pest ManagementPlant diseases are known from times preceding the earliest writings.The Bible and other early writings mention diseases, such as rusts, mildews,blights and blast. Disease control measures are as old as modern civilizationsand were first recorded long before the Renaissance in both the Western andthe Oriental worlds. The use of locally available plants in the control of pestsis an ancient technology in many parts of the world. Rotenone was originallyemployed in South America to paralyse fish, causing them to come towardsthe surface and be easily captured (Fig. 1.1a; Ware, 2002). It has been used infruit gardens to combat such pests as Japanese beetles, mites and stinkbugs.Preparations of roots from the genera Derris elliptica, Lonchocarpus nicou andTephrosia sp. containing rotenone were commercial insecticides in the 1930s.Rotenone is a flavonoid derivative that strongly inhibits mitochondrialfunction by inhibiting NADH-dependent coenzyme Q oxidoreductase(Singer and Ramsay, 1994). Rotenone persists 3–5 days on the foliage afterapplication and is easily biodegradable. Nicotine obtained from severalmembers of the genus Nicotiana has been used commercially as an insecticide(Fig. 1.1b). However, presently it is not accepted by most organic certificationprogrammes because of its residue and the potential harm it can cause tohumans and the agroecosystem (MMVR, 2003). Ryanodine, an alkaloid fromthe tropical shrub Ryania speciosa, has been used as a commercial insecticideagainst the European corn borer (Fig 1.1c). Ryania controls many insects,particularly those with chewing mouthparts (Coats, 1994; Regnault-Rogerand Philogene, 2008).

Global Scenario and Natural Products 5(a) (b) (c)CHH 3C CH 2HOHOOHOOHOOHHONOHNNCH 3OCH 3HOHOOHOOOHOHFig. 1.1. (a) Rotenone (b) Nicotine (c) Ryanodine.Used widely until the 1940s, such botanical pesticides were partly displacedby synthetic pesticides that at the time seemed easier to handle andlonger lasting. With the knowledge of the adverse effects of synthetic pesticides,worldwide attention is currently being given to shifting to nonsyntheticsafer pesticides. There is renewed interest in the application ofbotanical pesticides for crop protection, and scientists are now experimentingand working to protect crops from pest infestations using indigenousplant materials (Roy et al., 2005).1.4 Plant Products in Current Worldwide Use as PesticidesThe plant kingdom is recognized as the most efficient producer of chemicalcompounds, synthesizing many products that are used in defence againstdifferent pests (Prakash and Rao, 1986; 1997; Charleston et al., 2004; Ismanand Akhtar, 2007). Higher plants contain a wide spectrum of secondarymetabolites such as phenolics, flavonoids, quinones, tannins, essential oils,alkaloids, saponins and sterols. Tens of thousands of secondary products ofplants have been identified and there are estimates that hundreds of thousandsof such compounds exist. These secondary compounds represent alarge reservoir of chemical structures with biological activity. Therefore,higher plants can be exploited for the discovery of new bioactive productsthat could serve as lead compounds in pesticide development because oftheir novel modes-of-action (Philogene et al., 2005). The rainforest plantsare particularly thought to have developed a complete array of defenceprovidingchemicals. This resource is largely untapped for use as pesticides(Tripathi et al., 2004).Extracts prepared from different plants have been reported from time totime to have a variety of properties including insecticidal activity, repellenceto pests, antifeedant effects, insect growth regulation, toxicity to nematodes,mites and other agricultural pests, and also antifungal, antiviral andantibacterial properties against pathogens (Prakash and Rao, 1986, 1997).

6 N.K. Dubey et al.Natural pest controls using botanicals are safer to the user and theenvironment because they break down into harmless compounds withinhours or days in the presence of sunlight. Botanical pesticides are biodegradable(Devlin and Zettel, 1999) and their use in crop protection is a practicalsustainable alternative. Pesticidal plants have been in nature for millionsof years without any ill or adverse effects on the ecosystem. Botanical pesticidesare also very close chemically to those plants from which they arederived, so they are easily decomposed by a variety of microbes common inmost soils. Their use maintains the biological diversity of predators (Grangeand Ahmed, 1988), and reduces environmental contamination and humanhealth hazards. Botanical pesticides tend to have broad-spectrum activityand are sometimes stimulatory to the host metabolism (Mishra and Dubey,1994). Botanical insecticides can often be easily produced by farmers andsmall-scale industries. Recently, attention has been paid towards the exploitationof higher plant products as novel chemotherapeutics in plant protection.Such plant products have also been formulated for their large-scaleapplication in crop protection, and are regarded as pro-poor and cost-effective(Dubey et al., 2009).NeemNeem (Azadirachta indica) is regarded as the ‘Wonder Tree’, ‘BotanicalMarvel’, ‘Gift of Nature’ and ‘Village Pharmacy’ in India. From prehistorictimes, neem has been used primarily against household and storage pests,and to some extent against pests related to field crops in the Indian subcontinent.Neem oil and seeds are known to have inherent germicidal propertiesand have been in use for Ayurvedic (herbal) medicines in India for a longtime. Burning neem leaves in the evening is a common practice in rural Indiato repel mosquitoes. Neem is widely grown in other Asian countries andtropical and subtropical areas of Africa, America and Australia. It grows wellin poor, shallow, degraded and saline soil. Neem can be considered as themost important among all biopesticides for controlling pests. Neem pesticidesdo not leave any residue on the crop and therefore are preferred overchemical pesticides. In the past decade, neem has become a source of naturalpesticide due to its non-toxicity, environmental safety and so on, therebyreplacing synthetic pesticides. Neem derivatives have been applied againstseveral species of storage pests and crop pests as leaves, oil, cake, extractsand as formulations in neem oil (Gahukar, 2000; Dhaliwal et al., 2004). Neempesticides are thus a potential alternative to chemical-based pesticides andtheir use can avoid the dumping of thousands of tonnes of agrochemicals onEarth every year.Neem-based pesticides are sold under trade names such as Margosan-O,Azatin Rose Defense, Shield-All, Triact and Bio-neem. They have been shownto control gypsy moths, leaf miners, sweet potato whiteflies, western flowerthrips, loopers, caterpillars and mealybugs as well as some of the plantdiseases, including certain mildews and rusts.

Global Scenario and Natural Products 7Neem products also function as insect growth regulators (IGRs). Thetreated insects are usually prevented from moulting to develop into the nextlife stage and they die. The treatment may also deter egg laying. Generally,chewing insects are affected more than sucking insects. Insects that undergocomplete metamorphosis are also generally affected more than those that donot undergo metamorphosis.Neem seeds are a rich storehouse of over 100 tetranortriterpenoids anddiverse non-isoprenoids (Devkumar and Sukhdev, 1993). The neem tree containsmore than 100 different limonoids in its different tissues (Isman et al., 1996).Many of these are biologically active against insects as antifeedants. Themost touted biologically active constituent of neem has been highlyoxygenated azadirachtin and some of its natural analogues and derivatives.Azadirachtin (molecular formula: C 35H 44O 16, chemical structure shown inFig. 1.2), a highly oxidized triterpenoid, is the most widely publicized bioactivemolecule in neem. It is systemic in nature, absorbed into the plant andcarried throughout the tissues, being ingested by insects when they feed onthe plant. This may make it effective against certain foliage-feeders thatcannot be reached with spray applications. Azadirachtin is more effectivewhen formulated in a neem oil medium together with the other naturalproducts of neem. Hence, it is preferable to use neem oil enriched withazadirachtin as a stable feed stock for making pesticide formulations.Neem as a harmless and safe pesticide fits into integrated pest managementand organic farming. In toxicological studies carried out in the USAand Germany, different neem products were neither mutagenic nor carcinogenic,and they did not produce any skin irritations or organic alterations inmice and rats, even at high concentrations. Azadirachtin is considerednon-toxic to mammals, having a low mammalian toxicity with an LD 50of>5000 mg/kg for rat (oral acute) (Raizada et al., 2001), fish (Wan et al., 1996)and pollinators (Naumann and Isman, 1996). Hence it is classified by theHCH 3 CH 3 CCOCH 3CH 3CH 3OOO CCC O OOHCH 3H H 3 C OHOOHOOHHOO C OOFig. 1.2. Azadirachtin.

8 N.K. Dubey et al.PyrethrumSabadillaEnvironmemtal Protection Agency (EPA) in class IV. Regarding its environmentalimpact, neem is sensitive to light and the half-life of azadirachtin isone day (Kleeberg, 2006).Pyrethrum is one of the oldest and safest insecticides, and is extracted fromthe dried flower buds of Chrysanthemum sp. The ground, dried flowers wereused in the early 19th century to control body lice during the NapoleonicWars. Even today, powders of the dried flowers of these plants are sold asinsecticides. Pyrethrum is a mixture of four compounds: pyrethrins I and IIand cinerins I and II (chemical structures shown in Fig. 1.3; Ware, 2002).Chrysanthemum plants, Chrysanthemum cinerariaefolium, are grown primarilyin Kenya, Uganda, Tanzania and Ecuador. Pyrethrins affect the insect oncontact, creating disturbances in the nervous system which eventually resultin convulsions and death. Low doses, however, often cause temporary paralysisfrom which the insect may recover. For this reason, pyrethrums aremixed with a synergist such as piperonyl butoxide (PBO) derived from sassafrasor n-octyl bicycloheptane dicarboximide to increase insect mortalityand to extend their shelf life (Ware, 2002).Pyrethrum products represent 80% of the total market of botanical insecticidesand are favoured by organic growers because of their low mammaliantoxicity and environmental non-persistence (Isman, 1994). Pyrethrum is nontoxicto most mammals, making it among the safest insecticides in use.Pyrethroids versus pyrethrinPyrethroids are synthetic materials designed to imitate natural pyrethrum.They have been developed based on pyrethrins, but are much more toxic andlong lasting (Singh and Srivasava, 1999). They are marketed under varioustrade names, for example Ambush or Decis. Pyrethroids can be useful insecticides,but some pyrethroids are extremely toxic to natural enemies. Pyrethroidsare also toxic to honey bees and fish. Sunlight does not break themdown and they stick to leaf surfaces for weeks, killing any insect that touchesthe leaves. This makes them less specific in action and more harmful to theenvironment than pyrethrin. In addition they irritate the human skin.Sabadilla, also known as cevadilla, is derived from the seeds of the sabadillalily (Schoenocaulon officinale), a tropical lily that grows in Central and SouthAmerica (Soloway, 1976). The active ingredient is an alkaloid known asveratrine which is commonly sold under the trade names ‘Red Devil’ or‘Natural Guard’ (for the structure, see Fig. 1.4). This compound was firstused in the 16th century, and grew in popularity during the Second WorldWar, when other botanicals such as pyrethrum and rotenone were in shortsupply. The dust is made from the seeds and the active components are

Global Scenario and Natural Products 9H 3 CHCH 3CH 2H 3 COPyrethrin IH 3 CH 3 CHH 3 CHH 3 CO HCH 3OCH 3HOOHCH 3OCH 3Cinerin IH 3 COHH 3 CH 3 COH 3 CHH 3 CHH 3 CCH 3OCH 2O H(Z)Pyrethrin IICH 3OCH 3HOCH 3Cinerin IIO HCH 3OFig. 1.3. Pyrethrum.NH OHHHOOHOHOOOOHOH OOHHFig. 1.4. Veratrine.

10 N.K. Dubey et al.Carvonelacking in the other plant parts (roots, bulbs, stems and leaves). It is interestingthat the toxic constituents actually become more powerful after storage.Sabadilla is considered among the least toxic of botanical insecticides,with an oral LD 50of 4000–5000 mg/kg (in mice) (Dayan et al., 2009). It slowsdown the shutting of Na + channels and disturbs membrane depolarization,causing paralysis before death (Bloomquist, 1996). No residue is left after theapplication of sabadilla because it breaks down rapidly in sunlight.Carvone is a monoterpene of the essential oil of Carum carvi (see Fig. 1.5 forthe structure). It is a non-toxic botanical pesticide under the trade nameTALENT. It inhibits the sprouting of potato tubers during storage and protectsthem from bacterial rotting without exhibiting mammalian toxicity.Thus, it enhances the shelf life of stored fruits and vegetables and inhibitsmicrobial deterioration without altering the taste and odour of the fruits aftertreatment (Varma and Dubey, 1999). The LD 50value of carvone (in mice) isreported to be 1640 mg/kg (Isman, 2006).Allyl isothiocyanateAllyl isothiocyanate (Fig. 1.6) is an organosulfur compound that serves theplant as a defence against herbivores. Because it is harmful to the plant itself,it is stored in the harmless form of the glucosinolate, separate from themyrosinase enzyme present in plants. When an animal chews the plant, theallyl isothiocyanate is released due to action of myrosinase enzyme, thusrepelling the animal.Members of the plant family Brassicaceae are chemically linked by thealmost universal presence of glucosinolates, a class of sulfur-containingglycosides, also called mustard oil glycosides or thioglucosides. Thesecompounds are considered the first line of defence of crucifers against insectsand other organisms (Renwick, 1996).OH 3 CCCH 2CH 3Fig. 1.5. Carvone.NCSFig. 1.6. Allyl isothiocyanate.

Global Scenario and Natural Products 111.5 Other Plant ProductsOther environmentally safe botanical pesticides are in use from plants,namely from Annona squamosa (seeds), Pongamia pinnata (seeds) and Vitexnegundo (leaf) (Hiremath et al., 1997). Some plant species are known tobe highly resistant to nematodes. The best documented of these includemarigolds (Tagetes spp.), rattlebox (Crotalaria spectabilis), chrysanthemums(Chrysanthemum spp.), and castor bean (Ricinus communis) (Duke, 1990).These plants may be recommended as intercropping plants to control thenematode population in soil through their exudates and leachates. In additionto protecting crops from infestation, many rainforest plants can be usedas insect repellents. Bright orange berries of Bixa orellana are effective indeterring biting insects, in addition to being used as a body paint and dye(Butler, 2009). In some of the members of Asteraceae, the photodynamiccompound alpha-terthienyl has been shown to account for the strong nematicidalactivity of the roots (Fig. 1.7a). However, no plant-derived productsare sold commercially for the control of nematodes. Strychnine formulationsare used in commercial rodenticides (Fig. 1.7b). Quassia amara (SurinamWood), belonging to the family Simaroubaceae, is a tree species naturallydistributed in Suriname and several tropical countries. Traditionally, the barkand leaves are used in herbal remedies and as medicine because the majorsecondary metabolites of this tree, quassin and neo-quassin, exhibit pharmacologicalproperties such as antimalarial, antifungal, anti-ulcerative, antiedimogenicand anticancer activity. The male reproductive system, particularlyspermatogenesis, sperm maturation and androgen biosynthesis, is highlysensitive to the metabolites of Q. amara, which would be useful for insect pestcontrol but may also affect male reproduction in non-target organisms. Therefore,their pharmacological effects on mammals should be determined beforerecommendation to avoid any handling problems with such chemicals. However,quassin has been recently assessed in trials in Australia to control pestsof Brassicaceae (Thacker, 2002).Some plants have been reported to contain insect growth regulatorychemicals (IGRs), which disrupt insect maturation and emergence asadults. Juvabione (Fig. 1.8a), found in the wood of balsam fir, was discoveredby accident when paper towels made from this source were used to lineinsect-rearing containers resulting in a suppression of insect development(a)(b)NHSSSNOHHHOHFig. 1.7. (a) α-terthienyl (b) Strychnine.

12 N.K. Dubey et al.(a)OCO(b)CHCH 33 OOCH 3Fig. 1.8. (a) Juvabione (b) Precocenes.O(Varma and Dubey, 1999). Analogues of insect juvenile hormones such asjuvocimenes in Ocimum basilicum have also been reported (Balandrin et al.,1985). Precocenes isolated from essential oils of Matricaria recutita interferewith the normal function of insect glands that produce juvenile hormonesresulting in the suppression of insect growth while moulting (Fig. 1.8b).Many plant chemicals deter insects from feeding, thereby showing anantifeedant effect. Azadirachtin and limonoids such as limonin and nomilinfrom different plant species in Meliaceae and Rutaceae (e.g. from Citrusfruits) have long been used successfully for insect control, especially in India.Azadirachtin protects newly grown leaves of crop plants from feeding damage,thereby showing systemic antifeedant properties (Varma and Dubey,1999).1.6 Essential OilsSince the middle ages, essential oils have been widely used for bactericidal,virucidal, fungicidal, antiparasite, insecticidal, medicinal and cosmetic applications,especially nowadays in the pharmaceutical, sanitary, cosmetic, andagricultural and food industries. In nature, essential oils play an importantrole in the protection of the plants as antibacterials, antivirals, antifungals,insecticides and also against herbivores by reducing their appetite for suchplants. They also may attract some insects to favour the dispersion of pollensand seeds, or repel undesirable others. Some essential oils have been recognizedas an important natural source of pesticides. Aromatic plants producemany compounds that are insect repellents or act to alter insect feedingbehaviour, growth and development, ecdysis (moulting), and behaviour duringmating and oviposition. Recently researchers have demonstrated suchcompounds showing larvicidal and antifeedant activity (Adebayo et al., 1999;Larocque et al., 1999; Gbolade, 2001), capacity to delay development, adultemergence and fertility (Marimuthu et al., 1997), deterrent effects on oviposition(Naumann and Isman 1995; Oyedele et al., 2000), and arrestant and repellentaction (Landolt et al., 1999). Plants with strong smells, such as Frenchmarigold and coriander, act as repellents and can protect the crops nearby.Most insect repellents are volatile terpenoids such as terpenen-4-ol. Otherterpenoids can act as attractants. In some cases, the same terpenoid can repelcertain undesirable insects while attracting more beneficial insects. For instance,geraniol will repel houseflies while attracting honey bees (Duke, 1990).

Global Scenario and Natural Products 13Repellents and attractants modify the behavioural response of insects.This is the basis for the principle of behavioural insect control, whereby agiven species is either attracted to a bait or pheromone, or repelled from ahost plant by a repulsive agent (Fagoonee, 1981). Some of the plants containchemicals which alter the behaviour and life cycle of insect pests withoutkilling them. Such chemicals are termed as semio-chemicals by the Organisationfor Economic Cooperation and Development (Jones, 1998). The mostattractive aspect of using essential oils and/or their constituents as crop protectantsis their favourable mammalian toxicity because many essential oilsand their constituents are commonly used as culinary herbs and spices. Suchproducts are generally exempted from toxicity data requirements by theEnvironmental Protection Agency, USA. Some American companies haverecently taken advantage of this situation and have been able to bring essential-oil-basedpesticides to market. Mycotech Corporation producesCinn amite TM , an aphidicide/miticide/fungicide for glasshouse and horticulturalcrops, and Valero TM , a fungicide for use in grapes, berry crops, citrusand nuts. Both products are based on cinnamon oil, with cinnamaldehyde asthe active ingredient. EcoSMART Technologies are aiming to become a worldleader in essential-oil-based pesticides (Shaaya and Kostjukovsky, 1998).Several essential oil constituents are already in use as an alternative to conventionalinsecticides. For example, d-limonene is an active ingredient ofcommercially available flea shampoos, pulegone and citronellal are used asmosquito repellents, and 1,8-cineole is the structural base of the herbicidecinmethylin (Duke et al., 2000). Many commercial products such as BuzzAway (containing oils of citronella, cedarwood, eucalyptus and lemongrass),Green Ban (containing oils of citronella, cajuput, lavender, safrole fromsassafrass, peppermint and bergaptene from bergamot oil) and Sin-So-Soft ®(containing various oils) are in use as insect repellents (Chou et al., 1997).A Push–Pull or stimulo–deterrent diversionary strategy has been developedin South Africa for minimizing damage due to maize stem borer insects(Cook et al., 2006). This strategy involves the selection of plant speciesemployed as trap crops to attract stem borer insects away from maize crops,or some plant species are used as intercrops to repel insects. The trap andrepellent plants contain some semio-chemicals which attract or repel theinsect. Pennisetum purpureum and Sorghum vulgare attract the stem borerinsect, while Milinis minutiflra, Desmodium uncinatum and D. intorium are therepellent plants. The Push–Pull strategy is also employed in the control ofHeliothis sp. in cotton fields (Pyke et al., 1987). The Push–Pull strategy exploitingthe chemical ecology of plants would prove an interesting, indigenousand readily available concept in the management of insect population in fieldcrops. Plant flowers such as marigolds and certain types of vegetables canhelp to control pests in or around the main crop, which is sometimes called‘companion planting’ (Kuepper and Dodson, 2001).Some of the essential oils and their components show chemosterilantactivity making the insect pests sterile. The compound β-asarone extractedfrom rhizomes of Acorus calamus, possesses antigonadial activity causing thecomplete inhibition of ovarian development of different insects (Varma and

14 N.K. Dubey et al.Dubey, 1999) (Fig. 9.1). The products showing chemosterilant activity arehighly required in integrated pest management programmes to limit thechances of physiological (resistant) race development by insects.Some of the essential oils have been found useful against those species ofpests that are resistant towards synthetic pesticides. These essential oils are acomplex mixture of components including minor constituents, in contrast tosynthetic pesticides based on single products, and they act synergisticallywithin the plant as a defence strategy. Hence, it is likely that they are moredurable towards pests evolving resistance (Feng and Isman, 1995). Due totheir largely environmentally friendly nature, they can be efficiently used forpest management in urban areas, homes and other sensitive areas such asschools, restaurants and hospitals (Isman, 2006).Octopamine (a biogenic amine found in insects) has a broad spectrum ofbiological roles in insects, acting as a neurotransmitter, neurohormone andcirculating neurohormone–neuromodulator (Evans, 1980; Hollingworth et al.,1984). Octopamine exerts its effects through interacting with at least twoclasses of receptors which, on the basis of pharmacological criteria, have beendesignated octopamine-1 and octopamine-2 (Evans, 1980). Interrupting thefunction of octopamine results in a total breakdown of the nervous system ininsects. Therefore, the octopaminergic system of insects represents a biorationaltarget for insect control. The lack of octopamine receptors in vertebratesprobably accounts for the profound selectivity of certain essential oilsas insecticides. A number of essential oil compounds have been demonstratedto act on the octopaminergic system of insects (Enan et al., 1998).Mode-of-action studies on monoterpenoids also indicate the inhibition ofacetylcholinesterase enzyme activity as the major site of action in insects(Rajendran and Sriranjini, 2008).Encapsulation is the suitable technology for the formulation of essentialoil-basedpesticides. The method reduces the loss of the active agents andoffers the possibility of a controlled release of oil vapours (Moretti et al., 1998).Essential oils can also be incorporated with polymers into sheets. Attractantadhesive films with essential oils have been prepared to control insects inagriculture and horticulture (Klerk’s Plastic Industries B.V., 1990).Many of the commercial products that include essential oils are on the‘Generally Recognised as Safe’ (GRAS) list fully approved by the Food andDrug Administration (FDA) and Environmental Protection Agency (EPA) inUSA for food and beverage consumption (Burt, 2004).OCH 3H 3 COOCH 3CH 3Fig. 1.9. β-Asarone.

Global Scenario and Natural Products 151.7 Higher Plant Products as Inhibitors of Aflatoxin SecretionsThe post-harvest colonization of various moulds on food commoditiesreduces their shelf life and market value, as well as rendering them unfit forhuman consumption because of the secretion of different types of mycotoxins,including aflatoxin, that cause undesirable effects on human health.Hence, both qualitative as well as quantitative losses of food commoditieshave been reported due to fungal infestations. For the complete protectionof stored food commodities from fungal biodeterioration, a fungitoxicantshould be inhibitory to fungal growth as well as aflatoxin secretion. Reducingaflatoxin residue levels in food or feed can confer international tradeadvantages in developing countries and there may also be long-term benefitsfor the local population through health improvement (Dichter, 1987). Somenatural products from plants such as allicin from garlic and onion extracts,clove oil, and black and white pepper have been reported to control fungaltoxins (Ankri and Mirelman, 1999). Leaves of Garcinia indica (Selvi et al.,2003), Morinda lucida and Azadirachta indica (Bankole, 1997) have been foundeffective in controlling aflatoxin production in food commodities. Some ofthe natural products, such as cinnamon and clove oil (Sinha et al., 1993), phenols(Singh, 1983), some spices (Hasan and Mahmoud, 1993) and many essentialoils (Razzaghi-Abyaneh et al., 2008) have been reported as effective inhibitorsof fungal growth and aflatoxin production. The extracts of several wild andmedicinal plants have also been tested against aflatoxin-producing fungi(Bilgrami et al., 1980). Essential oils from Cymbopogon citratus, Monodora myristica,Ocimum gratissimum, Thymus vulgaris and Zingiber officinale have beenreported for their inhibitory effect on food spoilage and mycotoxin-producingfungi. Recently, the essential oils of Cinnamomum camphora (Singh et al., 2008a),Thymus vulgaris (Kumar et al., 2008) and Pelargonium graveolens (Singh et al.,2008b) have been reported to suppress aflatoxin B 1secretion by different toxigenicstrains of A. flavus. However, there is little in the literature on the abilityto monitor aflatoxin secretions by toxigenic fungal strains on food commodities.1.8 ConclusionSustainable agriculture aims to reduce the incidence of pests and diseases tosuch a degree that they do not seriously damage the farmer’s crop withoutupsetting the balance of nature. One of the aims of sustainable agriculture isto rediscover and develop strategies of which the cost and ecological sideeffectsare minimal. The secondary compounds of plants are a vast repositoryof compounds with a wide range of biological activities. Unlike compoundssynthesized in the laboratory, secondary compounds from plants are virtuallyguaranteed to have biological activity and that activity is likely to functionin protecting the producing plant from a pathogen, herbivore, orcompetitor. Among the variety of nature’s ecosystem services, natural pestcontrol is an important aspect. Hence, it is pertinent to explore the pesticidalactivity of plant products.

16 N.K. Dubey et al.Natural pest controls using botanicals are safer to the user and theenvironment because they break down into harmless compounds withinhours or days in the presence of sunlight. They are also very close chemicallyto those plants from which they are derived, so they are easily decomposedby a variety of microbes common in most soils. Because of greater consumerawareness and negative concerns towards synthetic chemicals, crop protectionusing botanical pesticides is becoming more popular. There is a widescope for the use of plant-based pesticides in the integrated management ofdifferent agricultural pests.ReferencesAdebayo, T.A., Gbolade, A.A. and Olaifa, J.I.(1999) Comparative study of toxicity ofessential oils to larvae of three mosquitospecies. Nigerian Journal of Natural Productsand Medicine 3, 74–76.Ankri, S. and Mirelman, D. (1999) Antimicrobialproperties of allicin from garlic.Microbes and Infection 1, 125–129.Balandrin, M.F., Klocke, J.A., Wurtele, E.S.and Bollinger, W.H. (1985) Natural plantchemicals: sources of industrial and medicinalmaterials. Science 228, 1154–1160.Bankole, S.A. (1997) Effect of essential oilsfrom two Nigerian medicinal plants(Azadirachta indica and Morinda lucida) ongrowth and aflatoxin B 1production inmaize grain by a toxigenic Aspergillusflavus. Letters in Applied Microbiology24, 190–192.Benhalima, H., Chaudhry, M.Q., Mills, K.A.and Price, N.R. (2004) Phosphine resistancein stored-product insects collectedfrom various grain storage facilities inMorocco. Journal of Stored Products Research40, 241–249.Bilgrami, K.S., Prasad, T., Misra, R.S. andSinha, K.K. (1980) Survey and Study ofMycotoxin Producing Fungi Associated withthe Grains in Standing Maize Crops. 98,Final technical Report. ICAR project,Bhagalpur University, India.Bloomquist, J.R. (1996) Pesticides: Chemistriesand characteristics. Radcliffe NationalIPM textbook. .Brent, K.J. and Hollomon, D.W. (1998) Fungicideresistance: the assessment of risk. FRAC,Global Crop Protection Federation,Brussels, Monograph No. 2, pp. 1–48.Burt, S. (2004) Essential oils: their antibacterialproperties and potential applications infoods – a review. International Journal ofFood Microbiology 94, 223–253.Butler, R.A. (2009) Saving the rainforest withMedicinal Plants. .CAPE (2009) Position Statement on SyntheticPesticides. .Charleston, D.S., Dicke, M., Vet, L.E.M. andKfir, R. (2004) Integration of biologicalcontrol and botanical pesticides: evaluationin a tritrophic context. In: Proceedingsof the Fourth International Workshop on theManagement of Diamondback moth andother Crucifer Pests. Melbourne, Australia.26–29 November 2001, pp. 207–216.Chou, J.T., Rossignol, P.A. and Ayres, J.W.(1997) Evaluation of commercial insectrepellents on human skin against Aedesaegypti (Diptera: Culicidae). Journal ofMedical Entomology 34, 624–630.Coats, J.R. (1994) Risks from natural versussynthetic insecticides. Annual Review ofEntomology 39, 489–515.Cook, S.M., Khan, Z.R. and Pickett, J.A. (2006)The Use of Push-Pull Strategies in IntegratedPest Management. Annual Reviewof Entomology 52, 375–400.Cutler, H.G. and Cutler, S.J. (1999) BiologicalActive Natural Products: Agrochemicals.CRS Press, Boca Raton, USA.Dayan, F.E., Cantrell, C.L. and Duke, S.O. (2009)Natural products in crop protection.

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2 Plant Products in the Control ofMycotoxins and MycotoxigenicFungi on Food CommoditiesSONIA MARÍN, VICENTE SANCHIS AND ANTONIO J. RAMOSFood Technology Department, Lleida University, Lleida, SpainAbstractMycotoxins are naturally occurring secondary metabolites of several toxigenic fungithat contaminate the whole food chain, from agricultural products, through to humanconsumption. Restrictions imposed by the food industries and regulatory agencies onthe use of some synthetic food additives have led to a renewed interest in searchingfor alternatives, such as natural antimicrobial compounds, particularly those derivedfrom plants. This chapter summarizes recent work on the antifungal activity of plantproducts and their potential for use as food additives. During the past two decadesmany publications have dealt with the inhibition of mycotoxigenic species by naturalplant products. Most of them showed the high efficacy of such products as antifungals.Their final application to food products is, however, still in its infancy. Thereasons for this are: (i) different origin, varieties and extraction methods of plantproducts result in essential oils and oleoresins that are widely varied in composition,preventing a direct extrapolation of results, unless experiments are carried out usingpure components of these essential oils and oleoresins; (ii) plant products should beapplied in such a way and at a concentration that does not affect sensorial quality offood products. Most in vitro studies used high concentrations of plant extracts anddirect contact as the screening technique, so the application of these extracts to foodshas not always been successful; and (iii) safety issues should be addressed prior to thewidespread application of such extracts.2.1 Mycotoxins in FoodsMycotoxins are naturally occurring secondary metabolites of several toxigenicfungi that contaminate the whole food chain, from agriculturalproducts such as peanuts and other nuts, fruits and dried fruits, and ultimatelyare consumed by man. These toxins can be produced in the field duringthe growth of the fungus on the crop or later, as a result of substandardhandling or storage. Animal-derived foods such as milk, cheese and meatcan be other sources of mycotoxins, if animals have been given contaminated© CAB International 2011. Natural Products in Plant Pest Management(ed. N.K. Dubey) 21

22 S. Marín et al.feed. The occurrence of mycotoxins may differ from year to year. The FAOestimates that mycotoxins contaminate 25% of agricultural crops worldwide.They also have a significant impact on economics, by causing losses in farmanimals or giving rise to difficulties in their management, or by renderingcommodities unacceptable for national or international trade because theydo not conform to existing regulations (EU, Commission Regulation1881/2006 and 1126/2007, 105/2010 and 165/2010).The generic term mycotoxicosis covers a variety of toxicities that targetexposed organs of animals and humans. In this regard, several mycotoxinsare potent animal carcinogens and have been classified by the InternationalAgency for Research in Cancer as potential human carcinogens. The toxiceffects of mycotoxins on human health are acute with a rapid onset and anobvious toxic or chronic response as characterized by low-dose exposure tomycotoxins over a long-time period. So, the real impact of the mycotoxinsin human health depends on the type and the amount of the mycotoxiningested.2.2 Natural Preservatives for Control of Mycotoxigenic Fungiand Mycotoxins in FoodsSynthetic preservatives are nowadays the more effective and widespread wayof chemical control of harmful microorganisms and diseases caused by them.Both field fungicides or antifungal food additives are efficient ways to controlfungal growth in foods and raw materials. Antifungal agents are chemicalsthat prevent or delay mould growth. However, the presence of chemical residuesin foods and labelling of preservatives in food packages are major concernsnowadays; furthermore, increasing fungal resistance has also become aproblem. The interest in the search for natural preservatives and their applicationin food products appear to be stimulated by modern consumer trendsand concerns about the safety of current food preservatives.Restrictions imposed by the food industries and regulatory agencies onthe use of some synthetic food additives have led to renewed interest insearching for alternatives, such as natural antimicrobial compounds, particularlythose derived from plants.2.3 Plant Products as Alternatives for Control of MycotoxigenicFungi and MycotoxinsPlants are continuously exposed to a wide range of pathogens and, eventhough they do not possess an immune system, they have evolved a varietyof defence mechanisms against these pathogens. After the perception of theprimary wound, a signal is transducted locally and systemically throughthe activation of different pathways which are integrated in the regulationof the stress response (Doares et al., 1995). Several plant pathogenesisrelatedproteins such as β-glucanases, chitinases, chitin-binding proteins,

Control of Mycotoxins and Mycotoxigenic Fungi 23polygalacturonase-inhibiting proteins and α-amylases have been shown topossess antifungal activity against toxigenic fungi. When attacked by insectsand pathogens, plants produce, together with defence proteins, a wide varietyof volatile and non-volatile secondary metabolites, such as phytoalexins,alkaloids, terpenes, aldehydes, etc.Much research has been published on the antimicrobial activity ofplant products, including flours, plant extracts, oleoresins and essentialoils. A very interesting option is the use of essential oils as antimicrobialadditives, because they are rich sources of biologically active compounds.Essential oils are mainly obtained by steam distillation from various plantsources.The antimicrobial activity of plant products has been extensively studiedand demonstrated against a number of microorganisms, mostly in vitro ratherthan in tests with foods, and usually using a direct-contact antimicrobialassay. In the direct-contact method, active compounds are brought into contactwith the selected microorganisms, and their inhibition is monitored bymeans of direct inspection or by measuring a physical property that is directlyrelated to microorganism growth, such as optical density, impedance or conductance.The solution of active compound may be added to a test tube or toagar medium, and then microorganisms are inoculated. The disc diffusiontest consists of spiking a sterile disc with the active compound; spiked discsare then added after inoculation of the medium, after which the inhibitionzones are measured, giving an indication of the antimicrobial strength. Forvolatile compounds such as essential oils, the antimicrobial effectiveness inthe vapour phase is of particular interest. This variety of testing techniquesmakes it difficult to compare results obtained by different researchers, mainlybecause of the difficulty in knowing the concentrations of active compoundsapplied.In vitro assays against mycotoxigenic fungiMany plant extracts have been tested in vitro for antifungal activity against awide range of fungi associated with deterioration of food commodities andherbal drugs.Studies carried out by Karthikeyan et al. (2007) revealed that aqueousleaf extract of zimmu (an interspecific hybrid of Allium cepa L. and Alliumsativum L.) exhibited strong antifungal activity against Aspergillus flavus,Fusarium moniliforme and Alternaria alternata and caused in vitro fungalgrowth inhibition of 73.3%, 71.1% and 74.4%, respectively. These mouldscause mouldy sorghum grains. Essential oil and methanol extract of Saturejahortensis had strong activity against A. flavus isolated from lemon fruit(Dikbas et al., 2008).Extracts of Cynara cardunculus were effective in the control of A. flavus,A. niger, A. ochraceus, Fusarium tricinctum, Penicillium funiculosum, P. ochrochloron,Trichoderma viride and Alt. alternata (Kukic et al., 2008). Similar resultswere obtained with aqueous extract of Adenocalymma sativum in order to

24 S. Marín et al.control A. flavus, A. niger, A. terreus, F. oxysporum, F. roseum, P. italicum, Cladosporiumcladosporioides and Alt. alternata. The minimum inhibitory concentrationof extracts against A. flavus and A. niger were superior to those of twocommonly used synthetic fungicides (Shukla et al., 2008).The essential oil of Amomum subulatum exhibited a fungitoxic spectrumagainst a wide range of moulds such as A. niger, A. flavus, A. terreus,A. fumigatus, Alt. alternata, Cladosporium herbarum, Curvularia lunata, F. oxysporum,Helminthosporium oryzae and Trichoderma viride. Their mycelial growthwas significantly inhibited at 750 μg/ml (Singh et al., 2008a).An in vitro initial screening of a range of 37 essential oils (includingcinnamon leaf, Cynnamomum zeylanicum; clove, Syzygium aromaticum; lemongrass,Cymbopogon citrates; oregano, Origanum vulgare; and palma rose,Cymbopogon martinii) on inhibition of mycelial growth of toxigenic strains ofF. verticillioides, F. proliferatum and F. graminearum under different temperatures(20–30°C) and water activities (a w) (0.95–0.995) was made. The basicmedium was a 3% maize meal extract agar. The agar medium was modifiedwith glycerol in order to get the assayed water activity and the essential oilswere incorporated at different concentrations (0, 500, 1000 μg/ml). Althoughwater activity was determinant for the growth of the isolates, in general, thepreservative effects of the oils were not linked to a w. However, a trend to ahigher inhibition by the oils when a wwas low was observed. Temperaturehad a minor importance in the inhibitory effect of the essential oils (Vellutiet al., 2004a).Essential oils of both cinnamon and clove were shown to effectivelyinhibit growth of both A. flavus and P. islandicum by direct contact, and in thevapour phase. In the atmosphere generated by these two essential oils eugenolwas the major compound, while other essential oils with low levels ofeugenol proved to be ineffective, suggesting that eugenol was the antimicrobialagent (López et al., 2005).Guynot et al. (2003) proved that the volatile fractions of cinnamon leaf,clove, bay (Laurus nobilis), lemongrass and thyme (Thymus vulgaris) showedpotential antifungal activity against A. flavus and A. niger.In vitro assays for the inhibition of mycotoxin productionSpecies in Aspergillus section Flavi, mainly A. flavus and A. parasiticus areamong the most commonly occurring spoilage fungi. Some strains of thesespecies have the ability to produce aflatoxins. These fungi invade agriculturalcommodities such as corn, peanuts and cottonseed, and herbal drugs,the resulting contamination with aflatoxin often making these pro ducts unfitfor consumption. Moreover, these mycotoxins are considered carcinogenicsubstances and their presence is a health concern, so their control is one ofthe aims in order to obtain safe products. Little attention has been paid, however,to the efficacy of essential oils in inhibiting aflatoxin production.Recently, work has been carried out to study the effect of these products asanti-aflatoxigenic agents.

Control of Mycotoxins and Mycotoxigenic Fungi 25Bluma et al. (2008) carried out a study with several plant extracts. A totalof 96 extracts from 41 Argentinian plant species were screened against fourstrains of Aspergillus section Flavi. Studies on the percentage of germination,germ-tube elongation rate, growth rate, and aflatoxin B 1(AFB 1) accumulationwere carried out. Clove, mountain thyme (Hedeoma multiflora) andpoleo (penny royal; Lippia turbinate var. integrifolia) essential oils showed themost antifungal effect in all growth parameters analysed as well as AFB 1accumulation.Essential oils from Pelargonium graveolens (Singh et al., 2008b) andArtabotrys odoratissimus (Srivastava et al., 2009) exhibited fungitoxicityagainst toxigenic strains of A. flavus at 0.75 g/l and 750 μl/l, respectively. Theoils of P. graveolens and Aelagonium odoratissimus showed excellent antiaflatoxigenicefficacy as they completely inhibited AFB 1production even at0.50 g/l and 750 μl/l, respectively.In addition, an in vitro study with aqueous extracts of neem (Azadirachtaindica) leaves on A. flavus and A. parasiticus has shown that the extracts fail toinhibit the vegetative growth of these moulds, while aflatoxin biosynthesiswas essentially blocked in vitro (A. flavus 100% and A. parasiticus more than95%, using extract concentrations at 10% v/v). AFB 1synthesis was alsoinhibited at low extract concentrations of Allium sativum in a semi-syntheticmedium (Shukla et al., 2008).The essential oil of Cinnamomum camphora was effective against othertoxigenic A. flavus isolates detected in medicinal plants. Srivastava et al.(2008) evaluated other essential oils. In this work, the growth of a toxigenicstrain of A. flavus decreased progressively with increasing concentrationsof essential oils from leaves of C. camphora and the rhizome of Alpiniagalanga incorporated into SMKY medium. Both oils showed complete inhibitionof growth of the toxigenic strain of A. flavus at 1000 mg/l. The oilssignificantly arrested AFB 1production by A. flavus. The oil of C. camphoracompletely blocked AFB 1production at 750 mg/l, whereas that of Alpinagalanga showed complete inhibition at 500 mg/l only. The combination ofC. camphora and Alpina galanga oils showed more efficacy than the individualoils, showing complete inhibition of AFB 1production even at 250 mg/l.The major components of C. camphora oil, as determined using GC–MS,were fenchone (34.82%), camphene (23.77%), α-thujene (17.45%), l-limolene(7.54%) and cis-p-menthane (5.81%). In case of Alpina galanga oil, bicyclo[4.2.0]oct-1-ene,7-exo-ethenyl (58.46%), trans-caryophyllene (7.05%),α-pinene (14.94%) with camphene (2.15%), germacrene (1.78%) and citronellylacetate (1.41%) were recorded as major components. In this study, theoils showed anti-aflatoxigenic properties at concentrations lower than theirfungitoxic concentration. Thus, the inhibition of fungal mycelia by theseoils may be through a mode other than the aflatoxin inhibition. The differencein antifungal and aflatoxin inhibition efficacy of essential oils may beattributed to the oil composition. The components of the oils may be actingby different modes of action for antifungal activity and aflatoxin inhibition.The interesting finding of this study is the better efficacy of the oil combinationof C. camphora and Alpina galanga in controlling the mycelial growth as

26 S. Marín et al.well as aflatoxin production at a concentration lower than with the individualoils (Srivastava et al., 2008).The results obtained by Sandosskumar et al. (2007) in in vitro experimentsconfirmed the antifungal activity of the zimmu extract against toxigenicstrains of A. flavus. In addition, when the aflatoxigenic strains were grown inmedium containing zimmu extract the production of AFB 1was completelyinhibited, even at a concentration of 0.5%. In addition, when AFB 1was incubatedwith this extract a complete degradation of the toxin was observed5 days after incubation. It is possible that the reduction in AFB 1content maybe due to detoxification or catabolism of AFB 1by root exudates of zimmu.Molyneux et al. (2007) suggested the hypothesis that aflatoxin biosynthesisis stimulated by oxidative stress on the fungus and the compounds capableof relieving oxidative stress should therefore suppress or eliminate aflatoxinbiosynthesis.Srivastava et al. (2008) concluded that, in general, the inhibitory action ofnatural products on fungal cells involves cytoplasm granulation, cytoplasmicmembrane rupture and inactivation and/or inhibition of synthesis of intracellularenzymes. These actions can occur in an isolated or in a concomitantmanner and culminate with mycelium germination inhibition. Phenolic compoundsin the essential oils have been mostly reported to be responsible fortheir biological properties; however, some non-phenolic constituents of oilsare more effective. The aldehyde group is also believed to be responsible forantimicrobial activity. Among the alcohols, longer chain (C6–C10) moleculesin the oils have been reported to be more effective. Such compounds presentin the oils may be held responsible for such biological activities.In addition, the therapeutic use of essential oils and their combinationscomprising more than one fungitoxic ingredient may also provide a solutionfor the rapid development of fungal resistance which is currently noticed incases of different prevalent antifungal therapeutics. Results obtained by otherresearchers (Sidhu et al., 2009) confirmed the synergistic effect of plantextracts. So, the combination of botanicals can be used for control of fungalgrowth and aflatoxin production.Another mycotoxigenic mould is P. expansum, which is mainly responsiblefor decay in apples and pears kept in cold storage rooms. In addition, it isregarded as the major producer of the mycotoxin patulin. When applesinvaded by P. expansum are used in making apple products (e.g. fruit juices),these products will probably be contaminated with patulin. Its presence is ahealth concern.Neem leaf extracts inhibited patulin production at concentrations higherthan 12.5 mg/ml, reaching a 96% inhibition at 50 mg/ml of neem extract.Patulin concentrations were reduced by neem extracts in cultures whose growthwas not inhibited. So, the inhibition of patulin production does not appear tobe simply a function of mycelial weight reduction (Mossini et al., 2004).The aldehydes hexanal, trans-2-hexenal, citral, trans-cinnamaldehydeand p-anisaldehide, the phenols carvacrol and eugenol, and the ketones2-nonanone and (–)-carvone were screened for their ability to controlP. expansum conidia germination and mycelial growth (Neri et al., 2006).

Control of Mycotoxins and Mycotoxigenic Fungi 27The in vitro spore germination and mycelial growth assay showed a consistentfungicidal activity by trans-2-hexenal, whereas (–)-carvone, p-anisaldehyde,eugenol and 2-nonanone exhibited a progressively lower inhibition. Thealdehyde trans-2-hexenal was the best inhibitor of conidial germination witha minimum inhibitory concentration (MIC) of 24.6 μl/l, while carvacrol wasthe best inhibitor of mycelial growth with a MIC of 24.6 μl/l. Other in vitroexperiments carried out with this mould by Venturini et al. (2002) determinedthat thymol and citral were the essential oil components that showed thegreatest inhibitory effects.2.4 Research in Antimicrobial Plant Products Applied to FoodsCerealsBiologicals, because of their natural origin, are biodegradable and they donot leave toxic residues or by-products to contaminate the environment.Spices and herbs have been used for thousands of centuries by many culturesto enhance the flavour and aroma of foods. However, an increasing numberof researchers have demonstrated that commonly used herbs and spices,such as garlic, clove, cinnamon, thyme, oregano, allspice, bay leaves, mustardand rosemary, possess antimicrobial properties. The activity was mainly dueto the presence of essential oils (Table 2.1). Essential oils and their constituentshave been used extensively as flavour ingredients in a wide variety of foods,beverages and confectionery products. Many such products are classified as‘Generally Recognized As Safe’.Cereals are among food commodities that are mostly prone to bear mycotoxins.Fusarium mycotoxins (including fumonisins, zearalenone and trichothecenes,among others) and ochratoxin A are mainly found in cereals. Maize, in particular,is a suitable substrate for aflatoxin accumulation. Therefore, plantextracts, essential oils, or their purified components have been assayed fortheir efficacy controlling fungi when applied directly to cereals. Togetherwith the effect of these compounds on mycotoxigenic fungi developmentand on mycotoxin production, the phytotoxicity of the oils to the seeds hasbeen evaluated, mainly calculated as the percentage of germination of theseeds, their viability or seedling growth. Maize has been the most investigatedcereal, followed by wheat, sorghum, rice and others.MaizeLemongrass as powder or as essential oil has been one of the most frequentlyassayed for its efficacy against mycotoxigenic fungi on maize. Thus, Adegokeand Odesola (1996) found that the application of the essential oil of lemongrassto maize could avoid the development of inoculated A. flavus and P. chrysogenum.Authors suggested that the presence of phytochemical componentssuch as tannins, alkaloids and glycosides, as well as the terpenes found in theessential oil, could play an important role in this effect.

28 S. Marín et al.Table 2.1. Most common essential oils investigated in relation to mycotoxigenic mouldsprevention in foodstuffs.Essential oil Plant species Major compounds ReferencesThymeOreganoCinnamonMustardCloveLemongrassBasilNeemThymusvulgarisOriganumvulgareCinnamomumzeylanicumBrassica hirta,Brassica juncea,Brassica nigra,Brassica rapaSyzygiumaromaticumCymbopongoncitratusOcimumbasilicumAzadirachtaindicaThymol, carvacrol,linaloolCarvacrol,thymolEugenol,cinnamaldehyde,caryophylleneAllylisothiocyanateEugenol,cariophylleneGeranial,neralThymolHexadecanoic acid,oleic acidMontes-Belmont and Carvajal,1998; Soliman and Badeaa,2002Marín et al., 2003; Velluti et al.,2003; López et al., 2004;Marín et al., 2004;Souza et al., 2007Montes-Belmont and Carvajal,1998; Soliman and Badeaa, 2002;Marín et al., 2003; Velluti et al.,2003; Marín et al., 2004Nielsen and Rios, 2000;Mari et al., 2002;Dhingra et al., 2009Montes-Belmont and Carvajal,1998; Nielsen and Rios, 2000;Awuah and Ellis, 2002; Marínet al., 2003; Velluti et al., 2003;Marín et al., 2004; Matan et al.,2006; Bluma and Etcheverry,2008; Reddy et al., 2009Adegoke and Odesola, 1996;Dubey et al., 2000; Marín et al.,2003; Velluti et al., 2003;Fandohan et al., 2004; Vellutiet al., 2004b; Marín et al., 2004;Somda et al., 2007;Souza et al., 2007Montes-Belmont and Carvajal,1998; Soliman and Badeaa,2002; Fandohan et al., 2004;Atanda et al., 2007Montes-Belmont and Carvajal,1998; Owolade et al., 2000;Fandohan et al., 2004;Somda et al., 2007;Reddy et al., 2009Lemongrass essential oil has also been assayed to control the growth andfumonisin B 1(FB 1) production by F. proliferatum (Velluti et al., 2003; Souza et al.,2007). For example, Marín et al. (2003) found that the essential oil (500 or 1000μg/g) inhibited growth of this fungus on artificially inoculated irradiatedgrains at 0.995 a wand 20 or 30°C, but results where not satisfactory at 0.95 a w.Inhibition of FB 1production was only effective when it was applied to grains

Control of Mycotoxins and Mycotoxigenic Fungi 29at 0.995 a wand 30°C. Results were similar when assayed on naturally contaminatedmaize inoculated with F. verticillioides and F. proliferatum, showingthat antimycotoxigenic ability of lemongrass only took place at the higherwater activities (Marín et al., 2003). From this study it was suggested that competingmycobiota play an important role in FB 1accumulation and that theefficacy of essential oils in cereals may be much lower than that observed inthe in vitro experiments using synthetic media. The main components foundin the essential oil used in these studies were geranial and neral, and in muchlower quantities limonene, geranyl acetate, geraniol and methyl heptenone.Fandohan et al. (2004) also assayed lemongrass essential oil againstF. verticillioides, finding a total inhibition of F. verticillioides growth at a concentrationof 8 μl/g over 21 days. At 4.8 μl/g fumonisin production was notaffected under open storage conditions, but a marked reduction was observedin closed conditions. Unfortunately, at this dose the oil adversely affectedkernel germination.When assayed on irradiated maize against a mycotoxigenic strain ofF. graminearum, lemongrass essential oil (500 or 1000 μg/g) had an inhibitoryeffect on growth rate of grains at 0.995 a wand at 20 or 30°C, but at 0.95 a wonly the higher dose was effective. Deoxynivalenol (DON) production wasinhibited at 0.995 a w/30°C at both doses, but no significant effect wasobserved for zearalenone (ZEA) control (Velluti et al., 2004b) (DON and ZEAboth being toxins produced by F. graminearum, amongst other Fusarium species).Similarly, several F. graminearum strains assayed on non-sterilizedmaize with lemongrass essential oil showed a limited efficacy for DON control(total prevention at 0.995 a w/30°C, but no effect at 20°C) and a limitedeffect for ZEA control (prevention at 0.95 a w/30°C) (Marín et al., 2004).Besides lemongrass, a huge number of essential oils have been tested forfungal control or mycotoxin prevention on maize. Thus it has been confirmedthat essential oils from seeds of Azadirachta indica (neem tree; 500 and 1000μg/g) and leaves of Morinda lucida (500 μg/g), two Nigerian medicinal plants,completely inhibited A. flavus aflatoxin synthesis in inoculated maize grains.Similarly, essential oils of cinnamon, peppermint (Mentha piperita), basil (Ocimumbasilicum), oregano, Teloxys ambrosioides (the flavoring herb epazote),clove and thyme caused a total inhibition of A. flavus development on maizekernels (Montes-Belmont and Carvajal, 1998). Only around a 50% reductionwas observed with the oils of Eucalyptus globulus (eucalyptus) and Pipernigrum (black pepper). Some of their constituents, when used at 2% concentration,such as thymol or o-methoxycinnamaldehyde significantly reducedA. flavus maize grain contamination. No effect was observed with the essentialoils of Allium cepa (onion) and Allium sativum (garlic). However, otherauthors have found that Allium extracts at 2.5% have a significant effect on thereduction of the incidence of F. proliferatum in maize grains (Souza et al., 2007).Essential oils of Pimpinella anisum (anise), Pëumus boldus (boldus), mountainthyme, clove and poleo were assayed against Aspergillus section Flavi(A. flavus and A. parasiticus) in sterile maize grain under different water activities(0.982–0.90 a wrange) (Bluma and Etcheverry, 2008). Five essential oilswere shown to influence lag phase, growth rate and AFB 1accumulation, in a

30 S. Marín et al.way that depends on their concentration, substrate water activity and time ofincubation. Only the highest concentration assayed (3000 μg/g) showed theability to maintain antifungal activity during a 35-day incubation period.Ethanolic, methanolic and aqueous extracts of leaves, roots, scape andflowers of Agave asperrima (maguey cenizo) and Agave striate (espadin) havebeen tested for their capacity to inhibit growth and aflatoxin production byA. flavus and A. parasiticus on maize. Leaves and roots showed no inhibitoryeffect, and methanolic extracts from flowers were the most effective. It wasfound that 50% of the minimal inhibitory concentration of extracts (approximately20 mg/ml) produced aflatoxin reductions higher than 99% in maizein storage conditions (Sánchez et al., 2005).Natural maize phenolic acids such as trans-cinnamic acid (CA) andferulic acid (FA), alone or in combination, have been tested to control A. flavusand A. parasiticus growth and aflatoxin production on maize (Nesci et al.,2007). A combination of 25 mM CA + 30 mM FA was very effective in thecontrol of fungal growth and completely inhibited AFB 1production at alla wassayed (0.99–0.93 a wrange). However, some treatments, as a CA–FAmixture at 10 + 10 mM, could lead to stimulation of an A. parasiticus population,and stimulation of AFB 1could be possible in some treatments.Addition of whole or ground dry basil leaves at 50–100 mg/g to maizehas shown to be effective in the reduction of aflatoxin contamination in maizestored for 32 days, with reductions in the range of 75–94% (Atanda et al.,2007). The authors suggested that aflatoxins could be controlled by co-storingwhole dry sweet basil leaves with aflatoxin-susceptible foods in a verysimple manner.Aqueous extracts of leaves of some indigenous plants from Nigeria, suchas Ocimum gratissimum, Acalypha ciliata, Vernonia amyygdalina, Mangiferaindica and Azadirachta indica, had a significant inhibitory effect on F. verticillioidesdevelopment on maize, with Acalypha ciliata being the most effective(Owolade et al., 2000).The effect of cinnamon, clove, oregano and palma rose oils (500–1000 μg/g)on growth and FB 1accumulation by F. verticillioides and F. proliferatum inmaize grain, at 0.995 and 0.950 a wand at 20 and 30°C, have also been evaluated,resulting in different efficacies depending on the treatment conditions(Marín et al., 2003; Velluti et al., 2003). These essential oils have also been usedto study their effects on ZEA and DON production by F. graminearum in nonsterilizedmaize grain in the same initial conditions. In this case the efficacyof essentials oils was found to be poor, clove essential oil being that with abroader applicability (Marín et al., 2004).Ocimum vulgare (30 μg/g) and Aloysia triphylla (lemon verbena; 45 μg/g)essential oils were evaluated on F. verticillioides FB 1production on maizegrain. The oregano essential oil decreased the production level of FB 1, probablybecause of its content of monoterpenes (such as thymol, menthol andcinnamaldehyde) that act as antioxidants and inhibitors of toxicogenesis andsclerotial development. On the other hand, Aloysia triphylla increased theproduction of FB 1, probably because of the presence of myrcenone, alphathujone and isomers of myrcenone in the oil, compounds that showed

Control of Mycotoxins and Mycotoxigenic Fungi 31oxidant properties that increase lipid peroxidation and, consequently, fumonisinproduction (López et al., 2004).Essential oils of O. basilicum and O. gratissimum have been demonstratedto reduce the incidence of F. verticillioides in maize, and totally inhibited fungalgrowth at concentrations of 6.4 and 4.8 μl/g, respectively, although at the4.8 μl/g dose they did not affect fumonisin production. On the other hand,oil of neem seeds has been shown to accelerate the growth of F. verticillioideson maize grains (Fandohan et al., 2004).WheatTo control A. flavus development on wheat, the essential oils of C. citratus,O. gratissimum, Zingiber cassumunar and Caesulia axillaris have been assayed.The oils of Caesulia citratus and Caesulia axillaris showed fungistatic activity,indicating their in vivo applicability as herbal fumigants (Dubey et al., 2000).In the same way, vapours of the essential oil of Caesulia axillaris (1300μg/g, v/v) have been shown to control A. flavus, A. niger, A. fumigatus, A.sulphureus, Rhizopus spp., Mucor spp., Curvularia spp., Penicillium oxalicumand Absidia spp. during 12 months of storage. The essential oil of Menthaarvensis (600 μg/g, v/v) showed similar results. These oils were also effectivein controlling the insect pests Sitophilus oryzae (Varma and Dubey, 2001). Similarly,the practical applicability of essential oil from leaves of wormseed(Chenopodium ambrosioides) as a fumigant for protecting stored wheat hasbeen assayed. A concentration of 100 μg/ml applied as a fumigant was ableto control A. flavus, A. niger, A. parasiticus, A. terreus, A. candidus and P.citrinum during 12 months of storage (Kumar et al., 2007).The essential oils of thyme and cinnamon (≤500 μg/g), Calendula officinalis(marigold) (≤2000 μg/g), Mentha viridis (spearmint), basil and Achilleafragantissima (quyssum) (≤3000 μg/g) completely inhibited A. flavus, A. parasiticus,A. ochraceus and F. moniliforme on wheat grains. Caraway oil (Carumcarvi) was inhibitory at 2000 μg/g against A. flavus and A. parasiticus and at3000 μg/g against A. ochraceus and F. moniliforme. Anise oil completely inhibitedthe four fungi at ≤500 μg/g. Worse results were obtained with essentialoils of chamomile (Matricaria chamomilla) and hazanbul (Achillea millefalium)(Soliman and Badeaa, 2002).Resveratrol, an extract of grape skin, has been demonstrated to be effectivein controlling the ochratoxigenic fungi P. verrucosum and A. westerdijkiaeon naturally contaminated wheat (Aldred et al., 2008). Total populations offungi were significantly reduced by the presence of this compound, often byabout 1–3 log colony forming units (CFU), but CFUs of the mycotoxigenicinoculated fungi were reduced only about 1–2 logs. In experiments developedat different water activities (0.995–0.80 a wrange) and temperatures (15–25°C)during a 28-day period of storage, grain treated with resveratrol (200 μg/g)had significantly less ochratoxin A (OTA) than the untreated controls. OTAcontamination was reduced by >60% in most of the treatment conditions.Extracts of Argyreia speciosa and Oenothera biennis have also beenassayed. Hexadecanyl p-hydroxycinnamate and scopoletin isolated from

32 S. Marín et al.roots of A. speciosa have demonstrated effective antifungal activity againstthe mycotoxigenic species A. alternata on wheat grains (more than 80%inhibi tion at 500 μg/g, and 100% at 1000 μg/g). Gallic acid from O. biennisshowed activity against F. semitectum at 1000 μg/g (96.9% inhibition)(Shukla et al., 1999).SorghumEssential oils of sweet basil, cassia (Cinnamomum cassia), coriander (Coriandrumsativum) and bay leaf (Laurus nobilis) were tested for their efficacy to control anaflatoxigenic strain of A. parasiticus on non-sterilized sorghum grains stored at28°C for 7 days (Atanda et al., 2007). Sweet basil seemed to be the best option,as 5% dosage gave an optimal sorghum protective effect. The use of this oil isadequate in terms of the possibility of practical application, and is within theacceptable sensory levels of 1–5% for food consumption. Good results on fungalgrowth control were also obtained with the essential oils of cassia, and withthe combination of cassia + sweet basil essential oils. It was also suggested thataflatoxin production could be controlled in sorghum grains by co-storingwhole dry sweet basil leaves with the grains; 100 mg/g of basil leaves havebeen shown to reduce the total aflatoxin contamination by 90.6%.The effect of essential oils of lemongrass, eucalyptus and crude oil ofneem has been tested against F. moniliforme, as well as against Colletotrichumgraminicola and Phoma sorgina, on sorghum grains (Somda et al., 2007). Of thethree plant extracts evaluated, lemongrass essential oil was the most potent.Concentrations of 6 and 8% caused the greatest reduction of seed infection atlevels comparable to synthetic fungicides such as Dithane M-45. More than50% of F. moniliforme growth was reduced by lemongrass essential oil.An emulsifiable concentrate (EC) of zimmu has been tested underfield conditions to check its efficacy controlling A. flavus, F. moniliforme andA. alternata on sorghum grains (Karthikeyan et al., 2007). A leaf extract of zimmu,formulated to 50 EC, contaning 50% (v/v) zimmu extract, using an organicsolvent (cyclohexanone), an emulsifier (Tween-80) and a stabilizer (epichlorohydrin),was prepared. Foliar application of zimmu formulation 50 EC at3 ml/l (v/v) concentration 60, 75 and 90 days after sowing significantlyreduced the incidence of grain mould (about 70% reduction) and increasedthe grain weight and grain hardness. The plant extract compared favourablywith foliar applications of the fungicide mancozeb (2.5 g/l). A significantreduction in the AFB 1content in sorghum was observed when plants weresprayed with a 50 EC formulation at 0.3% concentration.RicePlant extracts of different parts of Allium cepa, Allium sativum, Azadirachtaindica, Eucalyptus terticolis, Ocimum sanctum, Annona squamosa, Pongamia glaberrima,Curcuma longa and Syzgium aromaticum have been assayed for theiractivities against aflatoxigenic A. flavus growth and AFB 1production on rice(Reddy et al., 2009). Among the plant extracts tested, Syzgium aromaticum(5 g/kg) showed complete inhibition of A. flavus growth and AFB 1production.

Control of Mycotoxins and Mycotoxigenic Fungi 33Curcuma longa, Azadirachta sativum and O. sanctum also effectively inhibitedthe A. flavus growth (65–78%) and AFB 1production (72.2–85.7%) at 5 g/kg.FruitsPenicillium expansum (blue mould) is the causative agent of the mycotoxinpatulin in apples and pears. Some researchers in this field have focused onthe use of certain compounds of the essential oils of plants (Table 2.1), insteadof applying the essential oil directly. For example, marked fungicidal actionwas obtained with carvacrol against Mucor piriformis at 125 μg/ml, whilep- anisaldehyde stopped mycelial growth of P. expansum at 1000 μg/ml( Caccioni and Guizzardi, 1994). Allyl-isothiocyanate (AITC) can be employedsuccessfully in modified atmosphere packaging or as a gaseous treatmentbefore storage. Blue mould was controlled by exposing inoculated Conferenceand Kaiser pears for 24 h to an AITC-enriched atmosphere. At 5 mg/l,AITC showed a high fungicidal activity on pears inoculated with P. expansumat 10 4 conidia/ml, without causing phytotoxic effects. Disease incidenceappeared to be correlated with inoculum concentration and AITC treatmentconcentration. Increasing the P. expansum inoculum concentration at a constantAITC concentration resulted in increasing disease incidence. In fruitsinoculated with the higher P. expansum concentration (10 6 conidia/ml) andtreated with 5 mg/l AITC, the infected wounds were reduced by only 20%with respect to the control. At such a high inoculum concentration the efficacyof the most active molecules is usually significantly reduced. The goodresults obtained with AITC treatments delayed up to 24–48 h after inoculationsuggest that this compound has potential for postharvest disease control.The efficacy of AITC in controlling P. expansum in pears could allow it tobe used to control blue mould infection during packing-house fruit handling.The ability of AITC to control thiabendazole-resistant strains of P. expansumis particularly useful in this context because the proportion of thiabendazoleresistantstrains is usually high. The results of analysis on the skin and pulpof pears treated with AITC confirmed the extremely low concentration ofAITC residue in fruit, which is unlikely to have any implications for humanhealth. The use of AITC, produced from purified sinigrin or from Brassicajuncea defatted meal, against P. expansum appears very promising as aneconomically viable alternative with a moderately low impact on theenvironment (Mari et al., 2002). The potential use of volatile fungicides tocontrol post harvest diseases requires a detailed examination of theirbiological activity and dispersion in fruit tissues, and the development of aformulation that inhibits growth of pathogens without producing phytotoxiceffects on fruits.Neri et al. (2006) showed that volatile eugenol at concentrations of74 and 984 μl/l was necessary to inhibit mycelial growth and conidial germination,respectively, in P. expansum. Apples harvested at commercialmaturity were subjected to water treatment, lecithin at 50 mg/ml, eugenolethoxylate(2 mg/ml) and pure eugenol (2 mg/ml) combined with

34 S. Marín et al.50 mg/ml lecithin. All treatments were tested at 18 and 50°C. Fruits weredipped in the treatment solutions. After 2 min of treatment, fruits werestored at 2°C at normal atmosphere. Significant disease incidence reductionswere observed in fruits treated with eugenol mixed with lecithin at50°C; this combination reduced the incidence of P. expansum by 60–90%. Thelecithin–eugenol formulation did not induce immediate or delayed phytotoxicityat room temperature. Investigation of its vapour phase propertiesand its applicability in the storage room will surely facilitate the applicationof eugenol as a control agent for long periods and may avoid the problemsof phytotoxicity induced by some liquid formulations (Amiri et al.,2008).Essential oils of Caesulia axillaris and Mentha arvensis were applied at1500 and 1000 μl/l to the storage atmosphere of P. italicum inoculated oranges.The Caesulia-oil-treated oranges showed an increased storage life of 3 days,and the Mentha oil-treated oranges showed an increase of 7 days. No visualsymptoms of possible injury caused by the oils were observed on the peel ofthe fruits (Varma and Dubey, 2001).Aspergillus section Nigri (formerly A. niger) is an ubiquitous fungalcontaminant of foodstuffs, such as fruits, vegetables, nuts and spices. Somespecies in this section have been recently shown to produce ochratoxin A.Pepperfruit (Dennetia tripetala) extracts have been tested in tomato pureeagainst common spoilage fungi, including A. niger. Extracts, as a single hurdle,failed to inhibit fungal growth when compared to counts before treatment.Moreover, the use of extracts alone would entail the use of concentrationsthat may affect the sensorial properties of the tomato; however, combiningwith heat treatment (80°C for 1 min) or NaCl addition (10 mg/g) resulted ineffective treatments (Ejechi et al., 1999). Cinnamon essential oil has beentested against a range of fungi isolated from tomato, including A. niger.Although in vitro experiments showed promising results in terms of colonydevelopment and fungal sporulation inhibition, when cinnamon oil wastested as a volatile in the atmosphere of stored tomatoes and peppers nomajor effects were observed (Tzortzakis, 2009). Tomatoes and strawberriesexposed to an enriched oil vapour showed improved fruit-quality-relatedattributes, confirming the benefits observed after exposure to cinnamon oilvapour at different concentrations. These findings may have considerablecommercial significance.Extracts of Zingiber officinale and Xylopia aetiopica were added (1–3%) toA. niger, A. flavus or Rhizopus stolonifer inoculated orange and apple juices.Although growth was reduced, the extracts either alone or in combinationdid not impose enough stress to stop the growth of the fungi (Akpomeyadeand Ejechi, 1999).The advantage of essential oils is their bioactivity in the vapour phase andthe limitation of aqueous sanitation for several commodities (e.g. strawberriesand grapes) make the essential oils useful as possible fumigants for storedcommodity protection. One limitation of the essential oils is the strongflavour they impart, thus restricting their applicability only to products witha compatible flavour.

Control of Mycotoxins and Mycotoxigenic Fungi 35NutsSalicylic acid, thymol, vanillyl acetate, vanillin and cinnamic acid completelyinhibited the germination of fungi contaminating walnut kernels. All fivecompounds showed somewhat similar activity in inhibiting the growth ofthe potentially mycotoxigenic A. niger, A. flavus and P. expansum at the concentrationstested (up to 25 mM), with thymol showing the highest activity(i.e. complete inhibition of growth at 5 mM) (Kim et al., 2006).Powders from the leaves of O. gratissimum and cloves of Syzgium aromaticumwere used as protectants at 3% in combination with various packagingmethods to store 3.5 kg groundnut kernel samples (9.3% moisture)artificially inoculated with A. parasiticus. Selected treatments were repeatedwith naturally infected kernels. A high level of protection was obtained withSyzygium powder at 3% concentration using 12% moisture kernels (Awuahand Ellis, 2002).Calori-Domingues and Fonseca (1995) found that treatment of unshelledpeanuts with grapefruit seed extract was not efficient in controlling aflatoxinproduction during storage. Peanuts treated with grapefruit seed extract at5,000 and 10,000 mg/kg had mean aflatoxin contamination in the range2,757–56,334 μg/kg and 688–5,092 μg/kg, respectively, while the control had3,362–108,333 μg/kg. Of all of the chemicals tested only propionic acid waseffective in controlling aflatoxin production. Treatments were consideredefficient when the aflatoxin content (B 1+ G 1) remained less than 30 μg/kg.Shelled groundnut samples with moisture contents between 7.5 and10.5% and inoculated with conidia of A. glaucus and A. parasiticus were storedfor 15–90 days at 25°C, and fumigated with synthetic food grade essential oilof mustard (Brassica rapa) (100 μl/l space). Deterioration of the samples wasassessed by estimating the percentage of kernels colonized by fungi, thenumber of CFUs/kernel, and the accumulation of ergosterol and free fattyacids. The values of these variables increased with the moisture content andstorage period, independent of the fumigation treatment; however, the rate ofincrease was significantly lower in fumigated samples (Dhingra et al., 2009).2.5 Use of Plant Products in Active PackagingActive packaging (AP) is an innovative food packaging concept that combinesadvances in food technology, food safety, and packaging and materialsciences in an effort to better meet consumer demands for fresh-like, safeproducts. One specific application is to incorporate essential oil(s) into thepackaging to prevent the growth of microorganisms.The use of preservative food-packaging films offers several advantagescompared to the direct addition of preservatives to food products, since thepreservative agents are applied to the packaging material in such a way thatonly low levels of the preservatives come into contact with the food. Eitherof two approaches can be used to give packaging materials antimicrobialactivities. In preservative-releasing or migrating approaches, preservatives

36 S. Marín et al.are introduced into their bulk mass, or applied to their surfaces, which subsequentlymigrate into the food or the headspace surrounding the food. Innon-migrating approaches, compounds are applied to the packaging surfacesthat inhibit target microorganisms when they come into contact with them.The most widely used materials in food and drink packaging are variouskinds of papers and boards, which are usually wax-coated to improve theirwater-resistance and increase the shelf-life of the packaged products. Addingan active compound to the wax formulation before coating creates an APmaterial. The shelf-life of the packaging manufactured using cinnamaldehyde-fortified-cinnamonessential oil was evaluated against A. flavus, and itwas found to retain its total activity over the whole 71-day test period. Finally,the efficacy of the coatings was tested in trials with two varieties of strawberries.Complete protection was obtained during 7 days storage at 4°C, duringwhich no visible fungal contamination developed and there were no apparentvisible or organoleptic changes in the strawberries (Rodriguez et al., 2007).López et al. (2007) demonstrated the potential utility of polypropylene andpolyethylene/ethylene vinyl alcohol copolymer films with incorporated oreganoor cinnamaldehyde-fortified-cinnamon essential oils at concentrations of4% (w/w) as antifungal packaging materials, also against A. flavus. Theymaintain their antimicrobial properties for more than 2 months, and their usein contact with foodstuffs has been demonstrated not to be harmful to consumers’health. The main drawback could be the organoleptical alteration ofthe packaged food due to the chemicals released by the active package.AP may be an alternative to modified atmosphere packaging (MAP) orcould complement it. For example, the volatile gas phase of combinations ofcinnamon oil and clove oil showed good potential to inhibit growth of spoilagefungi normally found in intermediate moisture foods when combinedwith a modified atmosphere comprising a high concentration of CO 2(40%)and low concentration of O 2(

Control of Mycotoxins and Mycotoxigenic Fungi 37gas phase contained at least 3.5 μg/ml, AITC was fungicidal to all testedfungi. Results of sensory evaluation showed that hot-dog bread was moresensitive to AITC than rye bread. The minimal recognizable concentration ofAITC was 2.4 μg/ml gas phase for rye bread and between 1.8 and 3.5 μg/mlgas phase for hot-dog bread. These findings showed that the required shelflifeof rye bread could be achieved by AP with AITC. AP of hot-dog bread,may nevertheless require the additional effect of other preserving factors toavoid off-flavour formation (Nielsen and Rios, 2000).Comparisons between the effectiveness of the volatile gas phases and theliquid phases of essential oils have shown that oil in the liquid phase is moreeffective in preventing spoilage than when added via the gas phase. Highervolumes are required if the essential oils contact only the contaminatingmicroorganisms in the gas phase. However, advantages of using a volatilegas phase of essential oil for food products are that it may have a lesserinfluence of the final taste and aroma of the product and its release may beregulated more easily.2.6 ConclusionsDuring the past decade many publications have dealt with the inhibition ofmycotoxigenic species by natural plant products. Most of them showed ahigh efficacy of such products as antifungals. Their final application to foodproducts is, however, still in its infancy. Several reasons are involved:●●●Different origin, varieties and extraction methods of plant productsresult in essential oils and oleoresins that are widely varied in their composition.This heterogeneity prevents a direct extrapolation of results,unless experiments are carried out using pure components of theseessential oils and oleoresins.Plant products should be applied in such a way and at concentrationsthat do not affect grain viability or the sensorial quality of food products.Most in vitro studies used high concentrations of plant extracts and useddirect contact as the screening technique, so the application of theseextracts to foods was not always successful.Finally, safety issues should be fully addressed prior to the widespreadapplication of such plant products.ReferencesAdegoke, G.O. and Odesola, B.A. (1996) Storageof maize and cowpea and inhibitionof microbial agents of biodeteriorationusing the powder and essential oil oflemon grass (Cymbopogon citratus). InternationalBiodeterioration & Biodegradation37, 81–84.Akpomeyade, D.E. and Ejechi, B.O. (1999)The hurdle effect of mild heat and twotropical spice extracts on the growth ofthree fungi in fruit juices. Food ResearchInternational 31, 339–341.Aldred, D., Cairns-Fuller, V. and Magan, N.(2008). Environmental factors affect

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3 Natural Products from Plants:Commercial Prospectsin Terms of Antimicrobial,Herbicidal and Bio-stimulatoryActivities in an Integrated PestManagement SystemJ.C. PRETORIUS AND E. VAN DER WATTDepartment of Soil, Crop and Climate Sciences, University of the FreeState, Bloemfontein, South AfricaAbstractThe use of natural products developed from wild plants is gaining interest andmomentum throughout the world in both developed and developing countries. Indeveloping countries the use of natural plant extracts is simply the result of the inabilityof subsistence farmers to afford commercial synthetic pesticides. However, indeveloped countries this is largely due to consumer resistance towards syntheticchemicals, including antimicrobial, herbicidal and bio-stimulatory agents, believed tobe potentially hazardous to the environment and human health. In this chapter anoverview of the latter three pesticide groups is supplied in terms of its current ‘naturalproduct’ status from an integrated pest management perspective as it is applied inthe agricultural industry. First, some background is provided in order to cover thehistory of natural product development in these three pesticide categories. Second, ashort synopsis of screening programmes that identified wild plants containing naturalcompounds which have the potential to be considered in natural product developingprogrammes is supplied. Lastly, the outcome of these development programmesthat realized commercialized natural products is covered.3.1 IntroductionPlant diseases cause large yield losses throughout the world and all importantfood crops are attacked with disastrous consequences for food security.In many cases, plant diseases may be successfully controlled with syntheticfungicides, but this is costly to African peasantry and often has disadvantagesand side effects on the ecosystem (De Neergaard, 2001). It is, however,© CAB International 2011. Natural Products in Plant Pest Management42 (ed. N.K. Dubey)

Natural Products from Plants 43an established fact that the use of synthetic chemical pesticides providesmany benefits to crop producers. These benefits include higher crop yields,improved crop quality and increased food production for an ever increasingworld population. Despite this, synthetic pesticides may pose some hazardsto the environment, especially when improperly used by farmers in developingcountries who lack the technical skill of handling them, and who fail toadapt to this technology easily. This may result in undesirable residues left infood, water and the environment, toxicity to humans and animals, contaminationof soils and groundwater and may lead to the development of croppest populations that are resistant to treatment with agrochemicals.Moreover, in Africa and the Near East obsolete pesticides have become asource of great environmental concern. Some stocks are more than 30 yearsold and are kept in poor conditions because of inadequate storage facilitiesand lack of staff trained in storage management. The Food Agricultural Organization(FAO) estimated that developing countries are holding stocks ofmore than 100,000 tonnes of obsolete pesticides, of which 20,000 tonnes are inAfrica. Many of these chemicals are so toxic that a few grams could poisonthousands of people or contaminate a large area. Most of these pesticideswere left over from pesticide donations provided by foreign aid programmes.In the absence of environmentally sound disposal facilities, stocks are constantlyincreasing (Alemayehu, 1996). Obsolete pesticide stocks are, therefore,potential time bombs. Leakage, seepage and various accidents relatedto pesticides are quite common and widespread. Storage conditions rarelymeet internationally accepted standards. Many pesticide containers deteriorateand leak their contents into the soil, contaminating groundwater and theenvironment. Most stores are in the centres of urban areas or close to publicdwellings. According to the World Health Organization (WHO) there are 25million cases of acute occupational pesticide poisoning in developingcountries each year (Alemayehu, 1996).As a result of the problems outlined above, farmers in developing countriesand researchers alike are seeking less hazardous and cheaper alternatives toconventional synthetic pesticides. One such alternative is the use of naturalproducts from plants to control plant diseases in crops as part of an organicapproach to Integrated Pest Management (IPM) programmes. Justification forpursuing this alternative can be found in the following statement published adecade ago by the Environmental Protection Agency (EPA) regarding theadvantages of natural products from plants in the control of plant diseases:Natural products from plants have a narrow target range and highly-specificmode of action; show limited field persistence; have a shorter shelf life andpresent no residual threats. They are often used as part of Integrated PestManagement (IPM) programmes; are generally safer to humans and theenvironment than conventional synthetic chemical pesticides and can easily beadopted by farmers in developing countries who traditionally use plant extractsfor the treatment of human diseases. (Deer, 1999)A further reason for exploring the use of plant extracts or natural productsas biological pesticides more extensively can be found in the plant

44 J.C. Pretorius and E. van der Wattitself. Plants have evolved highly specific chemical compounds thatprovide defence mechanisms against attack by disease-causing organisms,including fungal attack, microbial invasion and viral infection (Cowan,1999). These bioactive substances occur in plants as secondary metabolites,and have provided a rich source of biologically active compoundsthat may be used as novel crop-protecting agents (Cox, 1990). In naturesome wild plants have the potential to survive both harsh biotic and abioticenvironmental conditions. This has initiated the postulate that suchplants might be utilized as sources for the development of natural productsto be applied in agriculture by man as natural herbicides, bactericides,fungicides or products with bio-stimulatory properties in crude orsemi-purified form.It is estimated that there are more than 250,000 higher plant species onearth (Cowan, 1999) offering a vast, virtually untapped, reservoir of bioactivechemical compounds with many potential uses, including their applicationas pharmaceuticals and agrochemicals. It is generally assumed thatnatural compounds from plants pose less risk to animals and humans andare more environmentally friendly than their synthetic counterparts( Johnson, 2001). As in pharmacology, biochemicals isolated from higherplants may contribute to the development of natural products for the agriculturalindustry in three different ways (Cox, 1990): (i) by acting as naturalpesticides in an unmodified state (crude extracts); (ii) by providing thechemical ‘building blocks’ necessary to synthesize more complex compounds;and (iii) by introducing new modes of pesticidal action that mayallow the complete synthesis of novel analogues in order to counter theproblem of resistance to currently used synthetic products by bacterial andfungal pathogens.However, it is quite common that a natural compound isolated from aplant may be of great biological interest but may not be sufficiently robust foruse (Steglich et al., 1990). Subsequently, a need for the modification of naturalproducts into synthetic analogues that will give the desired effect may stillexist. One can, for example, isolate a natural compound with promising antimicrobialactivity and, by introducing a stable chemical structure with higheractivity synthetically, develop a commercial product (Steglich et al., 1990).The alternative that is now more vigorously being pursued in organic farmingsystems is the application of the plant material itself in a natural form.However, to date and despite statements such as these made almost twodecades ago, the use of natural plant extracts as pesticides to control pathogensin crops, for example, are not widespread while synthetic chemical pesticidesstill remain the major tool in pest management systems. Nevertheless,some natural products have been commercialized recently and the use of acombination of synthetic chemicals and natural products in IPM programmeswill probably become more popular in future.Another related area of organic farming systems is the potential to applynatural plant extracts as either plant growth regulators or natural herbicides.A plant growth regulator is an organic compound, either natural or synthetic,that modifies or controls one or more specific physiological processes within

Natural Products from Plants 45a plant (Salisbury and Ross, 1992). If the compound is produced within theplant it is called a plant hormone e.g. auxins, gibberellins, cytokines, abscissicacid and ethylene. Two decades ago, Roberts and Hooley (1988) stated thatthe potential exists to apply a plant extract as a foliar spray in order to stimulategrowth in crop plants and hence increase yields. According to the authors,a principal objective of the agricultural and horticultural industries is tomanipulate plant growth and development in such a way that the quantityor quality of a crop is increased. After the late-1980s an elevated interestdeveloped in terms of identifying natural plant compounds that possess thepotential to manipulate plant growth and development over a short period,e.g. a growing season.From a crop production perspective, the term ‘integrated pest management’,in the broad sense of the word, means to apply more than one methodor product in order to control ‘pests’ that farmers have to deal with on aregular basis. The term ‘pests’, in the broad sense of the word, includes,among other things, insect and weed pests as well as viral, bacterial andfungal diseases. However, the term ‘integrated pest management’ has a differentmeaning when applied in either conventional cropping systems ororganic agriculture. Pest control in conventional cropping systems is mostlydriven by the large-scale use of many different synthetic chemicals and itcannot be denied that great success has been achieved in this way. Due toconsumer resistance and pressure generated by the ‘green revolution’towards the use of synthetic chemicals, a shift towards organic agriculturewas inevitable. Restrictions in terms of synthetic chemical application inorganic agriculture have, in turn, forced a shift towards the use of natural ororganic products.In conventional cropping systems integrated pest management is ratherstraightforward and entails the use of any registered product on the marketthat a farmer might choose, and this applies for any of the ‘pests’ mentionedearlier. The methods of application of these products are in many cases similar,e.g. foliar application, but might include integrated seed, soil and foliartreatments. IPM in organic agriculture is much more complex and controlledand may include sowing multiple crops, extended rotation cycles, mulching,specific soil cultivation methods (Dayan et al., 2009) and the use of organicallycertified natural products.Be that as it may, the reduction in the number of synthetic products as aresult of more stringent pesticide registration procedures (Dayan et al., 2009),such as the Food Quality Protection Act of 1996 in the United States, hasopened the door for the vigorous pursuit of natural products from plantsover the past two decades. In the meantime, many natural compounds fromwild plants have been isolated, purified, identified and patented but, only afew products are commercially available.In this chapter attention will be given to the integrated pest managementconcept, the rationale for considering natural compounds from plants andtheir potential to be applied as agrochemicals in an IPM system in the agriculturalindustry as well as selected areas where some progress have beenmade over the past three decades. In terms of the latter these will include

46 J.C. Pretorius and E. van der Wattnatural compounds with application potential as antimicrobial, herbicidaland bio-stimulatory agents.3.2 Rationale for Considering Natural Compounds from WildPlants to be Developed as Commercial ProductsWild plants are a valuable source for the development of new naturalproducts with the potential to be used for disease management in organiccrop production systems (Duke et al., 1995). Widespread public concern forlong-term health and environmental effects of synthetic pesticides, especiallyin developed countries, has prompted a renewed effort to search for naturalcompounds of both plant and microbial origin that can be applied as alternativesto existing synthetic pesticides (Ushiki et al., 1996). However, only asmall proportion has been investigated for possible use in plant diseasecontrol in agriculture.World agriculture still encounters huge crop losses annually due to plantdiseases and the situation is more serious for most subsistence farmers indeveloping countries that depend on non-conventional disease managementpractices, often with doubtful results. In contrast, crop producers in developedcountries rely heavily on synthetic fungicides and bactericides to controlplant diseases. Despite their efficacy against plant diseases, syntheticchemicals are considered to cause environmental pollution and are potentiallyharmful to human health. According to the National Academy ofSciences (Wilson, 1997), the carcinogenic risk of fungicide residues in food ismore than insecticides and herbicides put together.The long-term effect of pesticides in contaminating the environment is ofparticular concern, together with the fact that frequent application of fungicideshas resulted in fungal mutation and, subsequently, new resistant strains(Khun, 1989). Moreover, consumer resistance towards the use of syntheticchemicals has escalated, especially in developed countries, supplying a rationalefor the application of natural product alternatives in the agriculturalindustry (Duke et al., 1995). Currently, in many developed countries, the tendencyto shift to organic farming systems has evolved under consumer pressurein an attempt to reduce the risk of pesticide application. As a result,research on the possible utilization of biological resources and its applicationpotential in agriculture has become very relevant. A promising approach inthis regard is the use of natural plant products as an alternative to syntheticchemicals due to their apparently less negative impact on the environment(Ganesan and Krishnaraju, 1995).Conceptually and practically the use of natural plant products inagriculture is not new, but dates back to the time of Democratus (470 bc),where sprinkling of amurca, an olive residue, was recommended to controllate blight disease (David, 1992). In short, plant extracts have been recognizedto solve agricultural problems ever since man took to farming ( Pillmoor,1993). For example, the control of insect pests through the use of naturalplant pro ducts, such as Pyrethrum extracted from the Pyrethrum plant,

Natural Products from Plants 47Chrysanthemum cinerariefolium, and neem seed from Azadirachta indica( Richard, 2000), has a long history in plant protection.Despite the fact that reports on the practical application of natural plantextracts towards the control of plant pathogens in modern agriculture arefound less frequently in the literature (Menzies and Bélanger, 1996), aconsiderable number of reports on natural chemicals that are biologicallyactive against various plant diseases is currently available. These naturalcompounds are usually secondary metabolites and are synthesized in plantsas a result of biotic and abiotic interactions (Waterman and Mole, 1989;Helmut et al., 1994). By means of bioassay guided screening, a number ofthese natural plant compounds, with antimicrobial activity, have beenisolated and progress has also been made towards the identification andstructural elucidation of these bioactive compounds (Grayer and Harborne,1994). Although extractable secondary metabolites have long been consideredas an important source of pharmaceuticals, the evaluation of their applicationpotential in crop production systems has been largely neglected. A widerange of activities with both positive and negative effects, including the controlof microorganism, plant growth regulation (Adam and Marquardt, 1986),the induction of plant resistance to various diseases (Daayf et al., 1995;Schmitt et al., 1996) and promotion of beneficial microorganisms in the soilrhizosphere (Williams, 1992) have been reported. Despite these efforts, theisolation of plant secondary metabolites has led to very few commercialsuccesses in the agricultural industry and more specifically in crop managementpractices.An accelerated search for alternative options to synthetic fungicides,based on natural products from plants, therefore, seems to be an importantconsideration in light of the current restrictions in pesticide use in both developedand developing countries. This especially applies to the search forenvironmentally friendly bioactive components with broad-spectrum antimicrobialactivity (Benner, 1993). This probably needs serious considerationin developing countries where yield losses are high as a result of low-inputproduction systems due to the unaffordability of synthetic fungicides to localfarmers.According to the Natural Antifungal Crop Protectants Research Agency(Hall, 2002) spoilage and plant pathogenic fungi are responsible for some20% loss of the potential global plant production for food and non-food use.The very large amount of chemical crop protectants used to control theselosses can be detrimental to both the environment and human health.Therefore research has been initiated to develop and implement non- syntheticcrop protectants using natural antifungal agents (green chemicals) orantifungal metabolites from plants. These natural crop protectants will bedesigned for use on food or non-food crops vulnerable to fungal deterioration(Hall, 2002). From an agronomic perspective, a secondary aim of researchon natural plant products is to cultivate bioactive plants, as alternativeagricultural crops, to serve as sources for the bioactive compounds.Despite the inherent potential of compounds from plants to be applied asnatural products in agriculture, cognizance has to be taken of problems

48 J.C. Pretorius and E. van der Wattassociated with its use in plant disease control systems that have beenencountered in the past, and that have probably contributed to it not beingregarded as a viable strategy in the agrochemical industry. First, claims havebeen made that the efficacy of plant extract compounds are not comparableto that of synthetic fungicides and lack consistency (Benner, 1993). Second,natural products have, in some cases, been reported to be phytotoxic to cropplants (Benner, 1993). Third, some natural compounds are unstable and canbe broken down by UV-light or oxidation before the desired biological effectshave been produced (Seddon and Schmidt, 1999). However, these argumentscannot be generally accepted as less than 10% of natural plants have beenscreened for their application potential and in most cases only towards onetarget pathogen (Hamburger and Hostettman, 1991). In light of the vast numberof plant species known in the world today, the subsequent chemicaldiversity should allow for the identification of desired biologically activecompounds with sufficient stability.In contrast to the arguments against the use of natural plant compounds,other arguments towards its potential beneficial attributes should be considered.These include the possible reduction in the risk of fungicidal resistance,and the fact that they are potentially less toxic to humans and animals( Ganesan and Krishnaraju, 1995). Most importantly, it is envisaged that crudeplant extracts might be more affordable to subsistence farmers as they arereadily available and are probably cheaper to produce. Hence, attempts todevelop plant-derived natural products and the consideration of its applicationpotential in disease management systems in both developed anddeveloping countries does not seem to be out of line.In developing countries in particular, the consideration of applying naturalplant products in their crude form should be high on the agenda. Theapproach has long been used in most traditional farming systems in manydeveloping countries. Most African farmers possess substantial indigenousknowledge of insect and pathogen control. Although this knowledge is probablynot scientifically based, some examples of natural insecticides appliedin Ethiopian subsistence farming systems are worth mentioning. Extracts ofchinaberry (Melia azedarach), pepper tree (Schiunus molle) and endod (Phytolaccadodecandra) are used to control insects in both organized agricultureand home gardens (Gebre-Amlak and Azerefegne, 1998). Crude extracts ofall three plant species were reported by the authors to be effective againstBusseola fusca (maize stalk borer) larvae. With regard to disease management,a crude extract of Dolichos kilimandscharicus L. (Bosha) has been used as aslurry to treat sorghum seed in the control of covered (Sporisorium sorghi;Ehrenberg) and loose (Sphacelotheca cruenta, Kuhn) kernel smuts in Ethiopia(Tegegne and Pretorius, 2007). Experimentally, treatment of sorghum seedwith D. kilimandscharicus, in a powder form, provided excellent control ofboth pathogens and was as effective as the standard chemical, Thiram ® .However, this has been practised on a small and isolated scale. It seems necessaryto obtain a more scientific base through additional research in order toconsider an expansion of these practices as well as to consider the economicpotential of this approach.

Natural Products from Plants 493.3 Secondary Metabolites: Characteristics, Functions andPossible Applications in AgricultureExcept for primary metabolites such as carbohydrates, proteins, lipids andnucleic acids, plants also contain a large variety of secondary metabolites.Secondary metabolites are classified by different authors in different waysbut for the purpose of this précis, a simple classification into three groups issupplied: (i) isoprenoid; (ii) aromatic; and (iii) alkaloid components (Stumpfand Conn, 1981; Dey and Harborne, 1989; Salisbury and Ross, 1992).Isoprenoid componentsThis group of compounds is relatively diverse and three terms have beenallocated to it, namely isoprenoids, terpenoids and terpenes. The term‘ isoprenoid’ is relatively descriptive in the sense that the common factor thatrelates these compounds is the 5C units, called isoprene units (Fig. 3.1).However, the term ‘terpene’ is probably used more often.Under this group, known hormones such as gibberillic acid (GA), abscisicacid (ABA) and brassinosteroids as well as other components such as sterols,carotenoids, rubber and the phytol ‘tail’ of chlorophyll are classified. Allof these consist of repeating isoprene units. Isoprene units join in differentways to form components with chain, ring or combined chain–ring structures(Fig. 3.2).Terpenes are found abundantly in nature and are, as far as chemicalstructure is concerned, a diverse group of secondary metabolites. On thebasis of the number of isoprene units, terpenes are divided into seven classes.Hemiterpenes consist of one isoprene unit (C 5); they are not found in thisform (freely) in nature, but can be found in the form of alcohols or acids(examples shown in Fig. 3.3).Fig. 3.1. An isoprene unit.Repeating isoprene-units (chain) Ring Ring and chain combinedFig. 3.2. Different ways in which isoprene units can combine.

50 J.C. Pretorius and E. van der WattMonoterpenes, consisting of two isoprene units (C 10), form the maincomponent of essential oils and are therefore economically important due totheir aroma (perfume), e.g. menthol, camphor, geraniol and pinene (Fig. 3.4).Their application potential in agriculture lies in the fact that specific monoterpenesare growth inhibitors of higher plants (e.g. menthol and pinene) andtherefore have the potential to be developed as natural herbicides.Sesquiterpenes consist of three isoprene units (C 15) (Fig. 3.5). One of thebest known sesquiterpenes is absissic acid (ABA), which has a hormonalfunction. Plants showing natural resistance towards drought stress synthesizemore ABA than drought-sensitive crops. Theoretically, the externalapplication of ABA to crops has the potential to manipulate drought resistancein crops. Other sesquiterpenes have medicinal value e.g. as diureticsand as antimicrobials (e.g. kadinene from juniper berry).Diterpenes consist of four isoprene units (C 20) and are the rarest types ofterpenes in plants. Of the most important in this group is the hormone gibberellicacid (GA; Fig. 3.6). Although natural hormones are used in the horticulturaland agricultural industries on a small scale as growth stimulants, itis expensive and not economically justifiable on a large scale.OHCOOHIso-amyl alcoholAngelic acidFig. 3.3. Hemiterpenes found in the form of alcohols or acids.OHOMenthol Camphor Geraniol PineneFig. 3.4. Examples of monoterpenes.OHH 3 COH CHCH 33HOHH COOHCH 3HABAKadineneFig. 3.5. Examples of sesquiterpenes.

Natural Products from Plants 51Triterpenes consist of six isoprene units (C 30) and are divided into fourgroups, namely common triterpenes, steroids, saponins and cardiac glycosides.Common triterpenes are commonly found in plants as a wax layer onleaves and on some fruits e.g. limonene (Fig. 3.7).Most steroids are hydrolysed on carbon 3 and are in fact all sterols. Theyplay important roles either as hormones in plants (e.g. brassinosteroids) orvitamin precursors (e.g. 1-α-25-dihydroxy-vitamin D 3glycoside; Fig. 3.8).Brassinosteroids (e.g. brassinolide; Fig. 3.9) have growth-stimulating andyield-increasing properties and are regarded as the secondary metaboliteswith great application potential in both the agricultural and horticulturalindustries (see below).OOOHHOCOOHFig. 3.6. Gibberellic acid.OOOOOFig. 3.7. Limonene.OH CH 3HOHOHHHOOHOHOA brassinosteroid (brassinolide)HOOH1-α-25-dihydroxy-vitamin D 3 glycosideFig. 3.8. Examples of triterpenes.

52 J.C. Pretorius and E. van der WattOther known sterols are cholesterol that acts as a precursor of othersteroids in plants, and ergosterol that is rare in plants but abundant in fungi(Fig. 3.10). Usually, if the ergosterol concentration increases in plants, it is anindication of fungal infection.Saponins are so-called triterpene-glycosides coupled to a sugar group.The molecule consists of two parts, namely the glycone (sugar) and aglycone(triterpene) moieties (Fig. 3.11). Saponins have soap properties, foam in waterand have a bitter taste. They are also known for their medicinal properties inAsiatic communities.OHCH 3OHHOHOOH OFig. 3.9. A typical brassinosteroid (brassinolide).HOHOCholesterolErgosterolFig. 3.10. Two known sterols, cholesterol and ergosterol.AglyconeHOHHOSugarOOHHCH 2 OHOHOFig. 3.11. A typical saponin.

Natural Products from Plants 53Tetraterpenes consist of eight isoprene units (C 40). The best known in thisgroup are the carotenoids, e.g. β-carotene (Fig. 3.12). It is known as a colourpigment supplying colour to flowers, but also for its role in protectingchlorophyll against over exposure to light.Aromatic componentsAll compounds in the aromatic group of secondary metabolites contain atleast one aromatic ring (benzene ring; Fig. 3.13) in their structure, of whichone or more hydroxyl groups are substituted. Thousands of aromatic compoundshave already been identified in plants. The group can be divided intotwo sections, namely non-phenolic and phenolic aromatic compounds.Non-phenolic aromatic compoundsAlthough most aromatic compounds contain one or more OH groups in thebenzene ring, the OH groups are absent in non-phenolic aromatic compounds.For the sake of convenience, this group is further divided into twogroups, namely non-phenolic amino acids and hormones, as well as tetrapyrroles.In the former group, the two amino acids phenyl alanine and tryptophan,also precursors of many phenolic compounds, and the hormone auxin(indole acetic acid or IAA) are best known (Fig. 3.14). As is the case for GA,auxin is also used as a growth stimulant in the horticultural industry, but ona small scale.In the tetrapyrrole group of non-phenolic aromatic compounds, chlorophyll(Fig. 3.15) is best known and consists of four pyrrole rings forming aporphyrine ‘head’ attached to a phytol ‘tail’. Another example is phytochromethat is a light-sensitive component, allowing plants to distinguishbetween different day lengths.H 3 CH CH 3 H CH 3 H H H H HH 3 C CH 3CH 3CH 3CH 3H 3 CHHHHHCH 3HHFig. 3.12. β-carotene.OHFig. 3.13. A typical aromatic ring (benzene ring).

54 J.C. Pretorius and E. van der WattCOO –COOHNH 3+NH 3+COO –NHNHPhenyl alanine Tryptophan Auxin (IAA)(indole acetic acid)Fig. 3.14. Non-phenolic aromatic compounds.H 2 C=CHHCH 3(H-C=O for chlorophyll b)H 3 CNNCH 2 CH 3HH 3 CHONNHHCO =MgCCH 2 CH 2C–OCH 3CH 3CHHOH H HO - CH 2 -C=C-CH 2 -CH 2 -CH 2 -C-CH 2 -CH 2 -CH 2 -C-CH 2 -CH 2 -CH 2 -C-HCH 3CH 3CH 3CH 3Phytol ‘tail’(C 20 H 39 )Chlorophyll a and bRO OHN NH HHN NS-Protein– OOCCOO –Phytochrome (P f -form)Fig. 3.15. Non-phenolic chlorophyll and phytochrome.

Natural Products from Plants 55Phenolic aromatic compoundsPhenols are the most abundant aromatic and naturally occurring componentsof plants. The phenols are classified in different ways, depending onauthors. We will only briefly discuss the simple phenols, phenyl propanoids,flavonoids, tannins and kinones.Simple phenols are all monomeric (consisting of only one aromaticring; Fig. 3.16). However, the most general simple phenols with growthinhibitingalle lopathic properties that have the potential to be developed asnatural herbicides are vanillin, vanillic acid (a benzoic acid) and hydrokinone(Fig. 3.17). p-Hydroxy-benzoic acid and vanillic acid are the most commongrowth- inhibiting benzoic acids involved with allelopathy and arefound in maize, wheat, sorghum and barley.Phenol is probably the precursor of all other phenolic compounds foundin plants. Hydrokinone, resorsinol and catechol are found in low concentrationsin plants and their functions are not fully understood. They are mostlysecreted by insects as a defence mechanism against other insects and animals.Salicylic acid, on the other hand, possesses anaesthetic properties andis the active ingredient of Aspirin ® . In plants a number of functions havebeen identified for salicylic acid, namely induction of flowering as well as theinduction of ‘pathogenesis related’ (PR) proteins (peroxidase, chitinase andβ-1,3-glucanase) that increases a plant’s resistance to fungal infections. Whensecreted by plants, it also has an allelopathic growth inhibiting effect on otherplants in the environment.Phenyl propanoids are synthesized from the aromatic amino acid phenylalanine and contained in their structure is a 3-carbon side chain coupled to aphenol, e.g. coumaric acid and caffeic acid (Fig. 3.18).OHOHHOOHOHOHOHCOOHOHPhenol Hydrokinone Resorsinol Catechol Salicylic acidFig. 3.16. Simple phenols.OHO–CH 3HOHydrokinoneRR = -COH=vanillinR = -COOH=vanillic acidFig. 3.17. Simple phenols with great potential to be developed as natural products.

56 J.C. Pretorius and E. van der Wattp-Coumaric acid is the direct precursor of other coumarins such asumbelliferone and scopoletin but coumarin itself is synthesized fromcinnamic acid (Fig. 3.19).The potential of phenyl propanoids to be developed into natural productsfor the agricultural industry is probably underestimated at present. Forexample, coumarin, isolated from Mellilotus alba (White Clover; a legume), isa strong inhibitor of seed germination and has the potential to be developedas a pre-emergence herbicide. However, depending on the concentration, itis also known to stimulate IAA activity and therefore growth. Its potential asbio-stimulant has not been researched to date. Coumarin can also combinewith monosaccharide sugars such as glucose to form coumarin glycosides,e.g. umbelliferone glycoside (Fig. 3.20).These glycosides inhibit the activity of the enzyme 6-phosphogluconatedehydrogenase, the regulatory enzyme of the oxidative pentose phosphate(OPP)-pathway, and therefore possess excellent potential to be developed asnatural herbicides. Umbelliferone also has an inhibiting effect on the growthOHRFig. 3.18. Two well-known phenyl propanoids.COOHp-Coumaric acid (R=H)Caffeic acid (R=OH)OOHCH 3 OHOOOHOOOOOUmbelliferoneScopoletinCoumarinCinnamic acidFig. 3.19. Phenyl propanoids synthesized from the precursor, p-coumaric acid.glycosylOOOFig. 3.20. Umbelliferone glycoside.

Natural Products from Plants 57of certain seedlings and shows potential as an herbicide. Scopoletin has astimulating effect on seed germination but an inhibiting effect on seedlinggrowth, indicating a potential as pre-emergence herbicide.Flavonoids are the largest group of natural phenolic compounds foundin plants. More than 5000 different flavonoids have been described. All flavonoidshave one thing in common, namely a 15-carbon ‘skeleton’ structuredin three phenyl rings (A, B and C; Fig. 3.21). Other examples are shownin Fig. 3.22.A large variety of biological activities have been associated with flavonoids,including antiviral, anti-inflammatory, antioxidant, antidiabetic,anticancer, insect-repelling and free-radical-scavenging activities. Whatmakes this group extraordinary from an industrial perspective are the excessivequantities found in both edible and non-edible plants as well as theirpotential to be developed into natural products. It is estimated that about 2%of all carbon photosynthesized by plants is converted to flavonoids and thisamounts to approximately 1 × 10 9 tons per annum. With this enormousamount of flavonoids available, and in light of the numerous bioactivitiesidentified to date, the time is now ripe to consolidate our knowledge of theBACFig. 3.21. Typical fl avonoid structure.HOOHOHOHOHOHOOHOOOHOHOHOHOHOHOHEpicatechin Gallocatechin (+)-CatechinOHOHOHOHOHOHHOOHHOOHOOHOHyperosidegalactoseOOHOQuercitrinrhamnoseFig. 3.22. Flavonoid examples.

58 J.C. Pretorius and E. van der Wattgroup and to evaluate the application potential of these compounds in theagricultural industry (Pretorius, 2003 and references therein).Tannins are generally found in the xylem and bark of vascular plants.Two types of tannins have been distinguished namely hydrolysable and condensedtannins. When hydrolysable tannins are broken down (hydrolysed),the well known gallic and digallic acids are formed (Fig. 3.23). Tannins combinewith proteins to form insoluble co-polymers and this technique is usedto tan animal skins. However, tannins are toxic to humans and animals andnot suitable for natural product development (Borris, 1996).Kinones are divided in three groups, benzokinones, naphtakinones andantrakinones (Fig. 3.24). Most kinones are coloured but do not contribute tocolour in plants as they are found in the inside tissue layers. The best knownexample of benzokinone is ubikinone (Fig. 3.25), also known as Co-enzymeQ, which acts as an electron carrier during the light reaction ofphotosynthesis. Juglone, the well known naphtakinone, has a strongHOHOHOC OHOCOOHHOHOCOOHHOGallic acidHODigallic acidFig. 3.23. Two hydrolysable tannins.OOOOBenzokinoneFig. 3.24. Three groups of kinones.OJuglone(a naphtakinone)OAntrakinoneOH 3 C-OH 3 C-OOUbikinoneHFig. 3.25. Ubikinone, the best known example of a benzokinone.

Natural Products from Plants 59inhibiting effect on seed germination and seedling growth. Antrakinone isknown as an insect repellent.AlkaloidsMore than 3000 alkaloids have been isolated from plants. The best knownalkaloids are morphine (isolated from the poppy flower), nicotine (tobacco)and caffeine (coffee; Fig. 3.26).Alkaloids are best known for their medicinal properties but also for theirtoxicity. Their functions in plants are not well known but are possibly linkedto the natural resistance of plants against insects and pathogens. Alkaloidsare also known to inhibit seed germination and seedling growth of certainplants. Because of their toxicity, chances are slim that alkaloids will beconsidered in natural product development programmes.3.4 Antimicrobial Properties of Plant ExtractsPlants are under constant attack from various microorganisms and fungi.Their survival is testamentary to their ability to defend themselves chemicallyagainst these attacks by producing a myriad of secondary metaboliteswith antimicrobial properties (Cowan, 1999; Richard, 2001). Therefore, inaddition to other functions, one of the most important functions of secondarymetabolites in plants is antimicrobial activity against bacteria, fungi andviruses as well as acting as deterrents towards insects and predators( Lazarides, 1998; Minorsky, 2001). Failure of microorganisms to colonize wildplants has often been attributed to the presence of these inhibitory compoundswithin challenged tissues. However, monoculture crops have losttheir ability to defend themselves against biotic stressors to a large extent.Callow (1983) classified antimicrobial compounds (secondary metabolites)isolated from plants into two categories: (i) constitutive compounds,which are present in healthy plants; and (ii) induced compounds synthesizedfrom remote precursors following infection. The term ‘constitutive’ includescompounds that are released from inactive precursors following tissueHOONCH 3NNCH 3H 3 CONO CH 3NN NHOCH 3Fig. 3.26. Examples of alkaloids.Morphine Nicotine Caffeine

60 J.C. Pretorius and E. van der Wattdamage, for example the release of toxic hydrogen cyanide from cyanogenicglycosides. Induced compounds include the types of secondary metabolitesthat are low molecular weight antimicrobial compounds and are synthesizedby and accumulate in plants that have been exposed to microorganisms(e.g. phytoalexins).Since plants have evolved highly elaborate chemical defences againstattack, these have provided a rich source of biologically active compoundsthat may be used as novel crop-protecting agents. It follows, therefore, thatsecondary metabolites with antimicrobial activity purified and isolated fromplant extracts, possess the potential to be developed into natural products.As a result, many organizations and institutions in different countries of theworld are currently concentrating on natural product research. For example,the aim of the Agrochemical Discovery and Development Program of theNational Centre for Natural Products Research in the USA is to identify leadcompounds for the development of environmentally benign and toxicologicallysafe pest management agents (Borris, 1996). This programme is done incollaboration with scientists in the Natural Products Utilization ResearchUnit of the United States Development Agency (USDA) agricultural researchservice. Emphasis is on the discovery and development of compounds thatare useful in the control of diseases affecting small niche crops. The researchcentre is devoted to improving agricultural productivity through the discovery,development and commercialization of agrochemicals derived fromnatural plant compounds (Johnson, 2001).Moreover, an increasing number of scientists have become involved inintensive plant screening programmes for bioactivity and have contributedto the identification of plant species with the potential to be included inprogrammes for the development of natural products for the agriculturalindustry. It is therefore not surprising that a significant amount of evidenceover the past three decades has demonstrated that plant extracts are activeagainst plant pathogenic fungi (Lawson et al., 1998), soil-borne fungi (Awuah,1994; Bianchi et al., 1997), bacteria (Leksomboon et al., 2001) and nematodes(Oka et al., 2000). In recent years research interest has turned towards isolating,purifying and identifying these active compounds that have applicationpotential in plant disease control.According to Grayer and Harborne (1994), secondary metabolites withantimicrobial properties include terpenoids (e.g. iridoids, sesquiterpenoidsand saponins), nitrogen- and/or sulphur-containing compounds (e.g. alkaloids,amines and amides), aliphatics (especially long-chain alkanes and fattyacids) and aromatics (e.g. phenolics, flavonoids, bi-benzyls, xanthones andbenzo-quinones). Most of these compounds have been studied for their antifungaland antibacterial activities and potential usefulness against plantpathogens in vitro (Verpoorte, 1998; Baldwin, 1999; Paul et al., 2000) and somealso in vivo. Many aromatic compounds, including simple and alkylatedphenols, phenolic acids, phenylpropanoids, coumarins, flavonoids, isoflavonoids,quinones and xanthones, have been reported to show notableantifungal activities (Grayer and Harborne, 1994). For example, Bae et al.(1997) isolated flavonol diglycoside from leaves of Phytolacca americana L.

HOCH 2Compound 3Natural Products from Plants 61which exhibited significant antifungal activity against Botrytis cinerea, Botryosphaeriadothidea and Colletotrichum gloeosporioides (Glomerella cingulata).Flavonoid classes most often associated with antifungal activity are flavanones,flavonols, certain biflavones, chalcones and dihydrochalcones. Forexample, three glycosides isolated from Terminalia alata (T. elliptica) rootsshowed antifungal activity against the plant pathogen Aspergillus niger and thehuman pathogen Candida albicans at extremely low concentrations of between25 and 32 ppm (Srivastava et al., 2001). The three new glycosides identifiedwere: compound 1, 3,3’-di-O-methylellagic acid 4-O-β-d-glucopyranosyl-(1,4)-β-d-glucopyranosyl-(1,2)-α-l-arabinopyranoside; compound 2, 5,7,2’-tri-Omethyl-flavanone4’-O-α-l-rhamnopyranosyl-(1,4)-β-d-glucopyranoside andcompound 3, 2-α,3-β,19-β,23-tetrahydroxyolean-12-en-28-oic acid 3-O-β-d-galacto-pyranosyl-(1,3)-β-d-glucopyrano-side-28-O-β-d-glucopyrano-side(Fig.3.27). Compound 1 was a glycoside of an ellagic acid, whereas compounds 2and 3 were a flavanone glycoside and a triterpene saponin, respectively.Saponins are one of several groups of compounds that originate from triterpenoidsand show a wide range of biological activities. For instance, fungicidaltriterpenoid saponins, which also have molluscidal activity, have beenisolated from roots of Dolichos kilimandscharicus (Marston et al., 1988). Thesaponins from Dolichos were identified as the 3-O-β-d-glucopyranosides ofhederagenin, bayogenin and medicagenic acid (Marston et al., 1988). Saponinsfrom Mimusops elengi and M. littoralis seeds were also reported to be activeagainst Phytophthora palmivora and Colletotrichum capsici (Johri et al., 1994).The anti-infective potency of extracts from plants emphasizes the vastpotential for natural compounds to be developed into commercial products(Duke, 1990). Although many of these plant compounds have been appliedHOOOOCH 3ORH 3 COOOCH 3ORH 3 CO OOH 3 COOCompound 1 Compound 2HOHOCOOR′ROFig. 3.27. Three new glycosides with antifungal properties isolated and identifi ed fromTerminalia alata (redrawn after Srivastava et al., 2001). See text for names of compounds.

62 J.C. Pretorius and E. van der Wattin the pharmaceutical industry (Naseby et al., 2001) and folk medicine( Duncan et al., 1999), their potential for plant disease management has notyet been fully realized. Crop production still depends heavily on syntheticbasedproducts (Philip et al., 1995) despite consumer resistance towards theirpossible residual effects. For this reason the switch to organic farming,including the use of natural products in disease management systems, hasbecome a priority (Benner, 1993; Michael, 1999). The next section provides anoverview of selected plants containing compounds with antimicrobial properties,identified during the past two decades, as well as the current status ofnatural products that have recently been commercialized.Plants with antifungal propertiesTwo decades ago Naidu (1988) assayed young and mature leaf extracts ofCodiaeam variegatum for antifungal activity. All extracts inhibited Alternariaalternata and Fusarium oxysporum in vitro, with the young leaves being moreactive against A. alternata and the old leaves more active against F. oxysporum.The active secondary metabolites from the leaves extracts were identified asphenolic compounds by chromatographic analysis. Phytochemical screeningof the leaves also revealed other metabolites that may be responsible forantifungal activity.In another study in the same year Bandara et al. (1988) reported thatsteam distillates of the leaves of Croton aromaticus and C. lacciferus and rootextracts of C. officinalis inhibited mycelial growth of Cladosporium cladosporioidesin vitro. Root extracts of C. lacciferus were moderately active while thoseof C. aromaticus were inactive. Of the six compounds isolated from rootextracts showing antifungal activity, only 2,6-dimethoxybenzoquinoneobtained from the chloroform extract of C. lacciferus was significantly active.Comparatively small quantities of this compound were required to inhibitgrowth of the pathogens Botryodiplodia theobromae and Colletotrichumgloeosporioides (Glomerella cingulata).Also in the same year Gonzalez et al. (1988) extracted Alnus acuminatanodules with either 5% NaOH or water while constituents were separated bysilica gel column chromatography. The following compounds were isolated:xylose, ribose, an aromatic carboxylic acid, a fatty acid, a phenolic biarylheptanoidand a flavonoid glycoside. The flavonoid glycoside was found toinhibit the growth of Fusarium oxysporum and Pythium species.In the 1990s a number of contributions by natural product researcherswere published. Only a few are mentioned here. Ajoene, a secondary metabolitederived from garlic (Allium sativum), was shown to inhibit spore germinationof some fungi including Alternaria solani and other Alternaria spp.,Collectotrichum spp., Fusarium oxysporum and other Fusarium spp. that causeserious diseases in some important crop plants in India (Singh et al., 1990). Ina study conducted by Hoffmann et al. (1992) a methanol extract of Castela emoryiwas active as both a preventative and curative agent against grape downymildew caused by Plasmopara viticola. An active secondary metabolite was

Natural Products from Plants 63identified as a glycoside, 15-glucopyranosyl-glaucarubolone. Saponins fromMimusops elengi and M. littoralis seeds and crude extract of Ammi majus were86–100% effective against Phytophthora palmivora in vitro and the saponinswere 100% effective against Colletotrichum capsici (Johri et al., 1994). Encouragingresults were obtained in 2 years of field trials using these products for thecontrol of pathogens on Piper betle. No phytotoxicity was observed. Baeet al. (1997) isolated an antifungal secondary metabolite, flavonol diglycoside,from the leaves of Phytolacca Americana L. and identified the compound askaempferol-3-O-β-d-apiofwanosyl-(1,2)-β-d-lucopyranoside by spectral analyses.The compound exhibited significant antifungal activity against Botrytiscinerea, Botryosphaeria dothidea and Colletotrichum gloeosporioides (Glomerellacingulata).Earlier reports on the potential of compounds from plants to be consideredin natural product research most probably played a role in the elevationof large-scale screening programmes that followed during the past decade.For example, Pretorius et al. (2002a) performed a wide search for SouthAfrican plant species with fungitoxic properties against plant pathogens ofeconomic importance in agriculture. For this study, 39 plant species, representing20 families from the subclasses Rosidae, Asteridae, Commelinidae andLiliidae, were collected from the Blyde River Canyon Nature Reserve,Mpumalanga, South Africa. Crude extracts were prepared and bio-assayed,at equal concentrations, for their antifungal potential by determining theinhibitory effects on the mycelial growth of seven economically importantplant pathogenic fungi. Statistically, significant differences between plantsand plant parts were observed as well as the resistance of different fungi totreatment with different plant extracts. The most significant broad spectrummycelial growth inhibition was obtained with extracts from two species ofthe subclass Liliidae, namely Aristea ecklonii and Agapanthus inapertus. Thecrude extract of A. ecklonii performed best of all extracts as it totally inhibitedthe mycelial growth of all seven of the plant pathogenic test organisms andoutperformed the inhibition by a broad-spectrum synthetic fungicide( carbendazim/difenoconazole). Crude extracts of A. inapertus showed completeinhibition of four, and strong inhibition of the remaining three, plantpathogenic fungi.In the same year Pretorius et al. (2002b) also performed an in vivo studyon the control of black spot (Ascochyta blight) in pea leaves, caused byMycosphaerella pinodes, by a crude bulb extract of Eucomis autumnalis. Thefourth internode leaves were removed from 4-week-old pea (cv. Mohanderfer)plants, placed on moist filter paper in Petri dishes and inoculated with anM. pinodes spore suspension before and after treatment with the extract. Thecrude extract prevented M. pinodes spore infection of the leaves when theleaves were inoculated with spores both before and after treatment withthe extract, confirming complete inhibition of spore germination. The crudeE. autumnalis extract showed no phytotoxic effect on the leaves even at thehighest concentration applied.Equally promising was the results of a comprehensive study conductedby Chen et al. (2002) to determine the inhibitory effect of 58 plant extracts on

64 J.C. Pretorius and E. van der Wattspore germination and the effective control of grape downy mildew( Plasmopara viticola). Among the plant extracts, those of Chloris virgata, Dalbergiahupeana, Pinus massoniana, Paeonia suffruticosa and Robinia pseudoacaciainhibited spore germination of the pathogen significantly. An in vivo leaf disctest showed that the infected leaf discs, treated with these five plant extracts,exhibited no disease symptoms. Their effects were the same or better thanthat of the traditional fungicide, liquid Bordeaux.Pandey et al. (2002), similarly, compared the antifungal potential of leafextracts from 49 angiosperms, collected in Uttar Pradesh, India, with commercialfungicides, by screening them against Helminthosoporium sativum(Cochliobolus sativus). The leaf extract of Mangifera indica completely inhibitedthe mycelial growth of the test fungus while four plant species, A. sativum,Azadirachta indica, Lawsonia inermis and Matricaria chamomila (Chamomillarecutita) showed more than 90% inhibition. On assaying different parts ofMangifera indica, the leaf and seed extracts were found to possess the highestactivity and, together with a leaf extract of Matricaria chamomile, performedbetter than the commercial fungicides.An approach to screen for plants with antimicrobial activity against plantpathogens that often results in success is to exploit indigenous knowledge onmedicinal plants and to screen these known plants for likely candidates. Therationale behind this approach is to screen traditional medicinal plantsknown in a specific area for their antimicrobial properties instead of randomlychoosing potential candidates from the long list of currently knownflowering plants, conifers, ferns or bryophytes. Rajiv et al. (2002) conducted astudy to screen for the most effective extracts out of 15 medicinal plantsagainst Helminthosporium nodulosum (Cochliobolus nodulosus) causing blight infinger millet. These included Impatiens balsamina, Solanum nigrum, Tageteserecta, A. sativum, A. indica, Datura metel, Emblica officinalis (Phyllanthusemblica), Eucalyptus citriodora, Euphorbia pulcherrima, Lantana camara, Menthaarvensis, Mimosa pudica, Nerium indicum (N. oleander), Ocimum sanctum(O. tenuiflorum) and Ricinus communis. Extracts were sprayed on the pottedfinger millet plants at 15, 30, 45, 60 and 75 days after sowing. Crude extractsof S. nigrum and I. balsamina showed the highest mycelial growth inhibition,followed by T. erecta. Overall, the crude extract of S. nigrum recorded the bestresult in vitro but was found inferior to the I. balsamina extract in in vivo tests(Rajiv et al., 2002).Besides large-scale screening programmes, data from quite a number ofsmaller projects that included the screening of one or more plants were publishedduring the past decade. In most cases these smaller projects includedthe isolation and identification of active compounds involved, even if onlythe chemical group level. Although large-scale screening programmes areimportant to at least identify plant orders, families, genera or species withpromising potential, the more concerted approach is probably preferable.Only a few approaches are mentioned here.In their study of antimicrobial properties of plant extracts, Orlikowski(2001a) used grapefruit extract (GE; Biosept 33 SL) to control Phytophthoraspp. Amendment of peat with GE at a concentration of 165 μg cm –3 resulted

Natural Products from Plants 65in a drastic decrease in colony-forming units of the pathogen Phytophthoracryptogea and suppression of its development in potted gerbera (Gerbera jamesonii)and cypress (Chamaecyparis lawsoniana). About 40 μg GE ml –1 inhibitedapproximately 50% of the mycelial growth, whereas the pathogen did notdevelop at all in the presence of GE at a concentration of 1000 μg ml –1 . Theantifungal property of GE against P. cryptogea was attributed to the presenceof the active ingredient 7-geranoxycoumarin in GE (Orlikowski et al.,2001b).Kishore et al. (2002) reported on the antimicrobial activity of aqueous leafextracts from Lawsonia inermis and Datura metel against Mycosphaerella berkeleyicausing late leaf spot in groundnuts (Arachis hypogaea). Field experimentswere conducted at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, India during 1999 and 2000 using a susceptiblegroundnut cultivar (TMV2). The D. metel extract continuouslyreduced disease progress up to 115 days after sowing while the severity oflate leaf spot at harvest was significantly less than that of the controls. TheL. inermis extract was slightly less effective, containing disease progress up to95 days after sowing, but disease severity was also considerably less thanthat of the untreated and the positive chlorothalonil treated controls. Podyields in plots sprayed with L. inermis and D. metel extracts were 20 and 48%higher, respectively, than in the control plots.In the same year the aqueous extracts from bird cherry tree (Padus avium[Prunus padus]), aspen (Populus tremula), and celandine (Chelidonium majus)effectively suppressed the germination of Puccinia triticina (Puccinia recondite)uredospores (Karavaev et al., 2002). Fungitoxic activity of the extracts wasattributed to the high phenolic compound content and high peroxidaseactivity in the leaves of these plants.Curir et al. (2003) investigated the phenol compositions of two cultivarsof carnation (Dianthus caryophyllus) namely ‘Gloriana’ and ‘Roland’, whichwere partially and highly resistant, respectively, to Fusarium oxysporum subsp.dianthi. The aim was to determine if endogenous phenols could have an antifungaleffect against the pathogen. Analyses were performed on healthy andF. oxysporum-inoculated tissues in vitro as well as on plants in vivo. Two benzoicacid derivatives, protocatechuic acid (3,4-dihydroxybenzoic acid) andvanillic acid (4-hydroxy-3-methoxybenzoic acid), were found within healthyand inoculated tissues of both cultivars, together with the flavonol glycosidepeltatoside (3-[6-O-(α-l-arabinopyranosyl)-β-d-glucopyranosyl] quercetin).These molecules proved to be only slightly inhibitory towards the pathogen.2,6-Dimethoxybenzoic acid was detected in small amounts only in the inoculatedcultivar ‘Gloriana’, while the highly resistant cultivar ‘Roland’ showedthe presence of the flavone datiscetin (3,5,7,2’-tetrahydroxyflavone). Thelatter compound exhibited an appreciable fungitoxic activity againstF. oxysporum subsp. dianthi.Natural plant extracts have in recent years also received attention as asignificant and safe resource for the control of soil-borne pathogens due tothe phasing out of methyl bromide, which has played a major role as a soilfumigant worldwide (Eshel et al., 2000). Methyl bromide, the major fumigant

66 J.C. Pretorius and E. van der Wattused, is scheduled to be phased out, because it was defined by the MontrealProtocol of 1991 as a chemical that contributes to the depletion of the ozonelayer (Bowers and Locke, 2000). Presently, pre-plant soil fumigation and fungicideapplications are used to control wilt diseases. Subsequently, extractsfrom a number of plants have been identified as possible alternatives tomethyl bromide against soil-borne pathogens.Due to environmental and safety concerns associated with pesticides, aswell as the need for a replacement of methyl bromide, Bowers and Locke(2000) investigated the effect of several formulated plant extracts and essentialoils on soil populations of F. oxysporum. Fusarium wilts are some of themost widespread and destructive diseases of many major ornamental andhorticultural crops (Bowers and Locke, 2000). Treatment of the soil with 10%aqueous emulsions of the formulated extracts of a chilli pepper extract andessential oil of mustard mixture, a cassia tree extract and clove oil reducedpopulations of Fusarium by 99.9, 96.1 and 97.5%, respectively, 3 days after soiltreatment. The same formulations also suppressed disease development inthe greenhouse and resulted in an 80–100% plant stand after 6 weeks. Theobserved reductions in the pathogen population in soil and increase in plantstand in the greenhouse indicated that these natural plant products may playimportant roles in future biologically based management strategies for thecontrol of Fusarium wilt diseases.Similar results were observed with neem tree (Azadirachta indica) extractsagainst soil-borne fungi (Ume et al., 2001). It was shown that extracts fromthe leaves and seed kernels possess antifungal activity against Sclerotium rolfsii(Corticium rolfsii). Both leaf and seed extracts showed some effect againstdifferent growth stages of the fungus, but the effects were fungistatic ratherthan fungitoxic. The non-polar extracts of the seed kernels were reported tobe more effective than those rich in polar terpenoids such as azadirachtinand an aqueous leaf extract was also more effective than the kernel-derivedmaterial. Neem is perhaps the most useful traditional medicinal plant inIndia and each part of the tree has some medicinal property that has made itcommercially exploitable (Kausik et al., 2002).Amadioha (2002) evaluated the antifungal activities of different extractsof neem, both in vitro and in vivo. The oil extract from seeds as well as waterand ethanol leaf extracts of the plant were effective in reducing the radialmycelial growth of Cochliobolus miyabeanus in culture and in controlling thespread of brown spot disease in rice. However, the oil extract was found tobe the most effective, followed by the ethanol leaf extract, in inhibiting thegrowth of the pathogen in vitro and in controlling the development of thedisease in vivo. The oil and ethanol extracts compared favourably with carbendazim(Bavistin) at 0.1% active ingredient and had the potential to controlthe brown spot disease of rice in vivo. Additionally, Bohra and Purohit(2002) studied the effects of the aqueous extracts of 17 plant species collectedfrom Rajasthan, India on a toxigenic strain of Aspergillus flavus. The neemextract recorded the highest mycelial growth inhibition of the fungus.The antimicrobial potential of plant extracts has also been reportedagainst the highly resistant fungi that cause soil-borne damping-off disease

Natural Products from Plants 67in plants. The efficacy of crude, boiled water and acetone extracts of 17 plantspecies against Rhizoctonia solani, causing wet root rot disease in chickpeas,was evaluated under laboratory conditions (Kane et al., 2002). The crude,boiled water and acetone extracts of A. sativum, the crude and boiled waterextract of Eucalyptus sp., as well as the boiled water and acetone extracts ofZingiber officinale contributed to 100% inhibition of the mycelial growth of thepathogen. In the same year Prabha et al. (2002) reported on the antifungalproperties of extracts from Foeniculum vulgare, Coriandrum sativum, Trigonellafoenum-graecum, Anethum graveolens and Cuminum cyminum against threefungi, including Fusarium oxysporum. All extracts showed a relatively greaterinhibitory effect on F. oxysporum, with stem extracts from A. graveolens exhibitingcomplete growth suppression, while the mycelial growth of the othertwo fungi was also inhibited significantly.Om et al. (2001) also reported the effect of essential oils extracted fromCallistemon lanceolatus (Callistemon citrinus), Citrus medica, Eclipta alba, Hyptissuaveolens and Ocimum canum (O. americanum) against Rhizoctonia solani, thecause of damping-off disease of tomato and chilli (Capsicum annuum). Theessential oils of Citrus medica, E. alba and O. canum completely inhibitedthe growth of the fungus within 24 h. The essential oils of C. lanceolatus andO. canum controlled the damping-off disease of tomato by 57 and 71%and that of chilli by 40 and 83%, respectively. The same effect was alsoobserved by applying crude, boiled water and acetone extracts fromA. sativum, and a boiled water extract of Eucalyptus sp., as well as boiledwater and acetone extracts of Zingiber officinale (Kane et al., 2002).In some cases plant extracts not only inhibit fungal mycelial growth, butalso spore germination. Chen et al. (2002) reported that Chloris virgata, Dalbergiahupeana, Pinus massoniana, Paeonia suffruticosa and Robinia pseudoacaciaextracts inhibited spore germination of grape downy mildew (Plasmoparaviticola) and the effect was comparable to that of a traditional fungicide, liquidBordeaux. Other work also demonstrated that leaf extracts of Lawsoniainermis and Datura metel possess antifungal activity against Mycosphaerellaberkeleyi, causing late leaf spot in groundnuts (Kishore et al., 2002). Particularly,the D. metel extract continuously reduced disease progress up to 115days after sowing while the severity of late leaf spot at harvest wassignificantly less than that of the controls.Plant seeds also contain compounds with antimicrobial properties. Seedextracts of 50 plant species, belonging to different families, were evaluatedfor their ability to inhibit the growth of Trichoderma viride in vitro (Bharathimathaet al., 2002). Of the various seed extracts, that of Harpullia cupanioides(Roxb.), belonging to the family Sapindaceae, displayed very high antifungalactivity. The seed extract of H. cupanioides strongly inhibited the growth ofRhizoctonia solani, Curvularia lunata (Cochliobolus lunatus), Colletotrichum musaeand Alternaria alternata and retained its antifungal activity even after heatingat 100°C for 10 min or autoclaving at 121°C for 20 min. Rodriguez and Montilla(2002) reported on the in vitro and in vivo antimicrobial effect of a Citrusparadisi seed extract (Citrex) on F. oxysporum lycopersici causing tomato wilt.Five treatments were evaluated: (i) immersion of plant roots in a solution of

68 J.C. Pretorius and E. van der WattCitrex before transplant; (ii) weekly application to the foliage; (iii) weeklyapplication to the soil; (iv) weekly application to the foliage and to the soil;and (v) immersion of plant roots at transplant plus weekly application to thesoil. The control was infested soil without application of the product. Treatments(i) and (iii) reduced wilting by 85%, indicating that it is possible tocontrol soil-borne pathogens with the C. paradisi seed extract.Extremely promising, from a natural product development perspective,is that many plant extracts compare favourably with commercially synthesizedfungicides. For example, leaf extracts of Azadirachta indica, Atropa belladonna,Calotropis procera, Ocimum basilicum, Eucalyptus amygdalina, Ailantursexcelsa (Ailanthus excelsa) and Lantana camara, at different concentrations,were compared to the fungicides Bavistin (carbendazim), Dithane M-45(mancozeb), captan, thiram and Topsin M (thiophanate-methyl) at standardconcentrations against F. oxysporum inducing fenugreek wilt under greenhouseconditions. Seeds of fenugreek were separately soaked overnight ineach leaf extract as well as the fungicide solutions (Gupta and Bansal, 2003).All the leaf extracts, except that of Ailanturs excelsa, significantly inhibited themycelial growth of Fusarium compared to the untreated control. Maximumgermination of fenugreek seeds was observed with Bavistin (93.33%), followedby Atropa belladonna (90.66%), Azadirachta indica (87.99%), L. camara(87.99%), C. procera (85.99%), O. basilicum (85.33%) and E. amygdalina (82.40%).Dithane M-45 (80.00%), Captan (86.66%), thiram (86.66%) and Topsin M(86.66%) were on par with the leaf extracts.An approach adopted by Solunke et al. (2001), following the increasingavailability of data in the literature on plants with antifungal activity, was toevaluate the potential of plant extracts to be applied in IPM programmes.The authors conducted a study to manage sclerotium rot (Sclerotium rolfsii[Athelia rolfsii]) of potato using a commercial fungicide, carbendazim, separatelyand together with plant extracts. Sensitivity of fungal isolates (SRP-1,SRP-2, SRP-3 and SRP-4) against carbendazim was determined beforehand.Subsequently, aqueous extracts of Azadirachta indica, Allium cepa, Glossocardiabosvallea and Vinca rosea (Catharanthus roseus) were mixed with carbendazimin solution and used to treat potato slices. Based on the minimum inhibitoryconcentration of carbendazim against the four isolates, SRP-4 appeared to betolerant while SRP-1 was sensitive. The percentage control efficacy (PCE) ofcarbendazim alone and in mixture with plant extracts were tested against thetolerant isolate, SRP-4. Using carbendazim in combination with plant extractsincreased its PCE. Application of carbendazim along with A. indica and G.bosvallea recorded a PCE of 100%. Carbendazim with V. rosea and with A. ceparecorded PCEs of 90.77 and 80.53%, respectively. The results showed that it ispossible to reduce the selection pressure of carbendazim when combinedwith plant extracts.Finally, an aspect that needs special reference is that the antimicrobialefficacy of plant extracts compare favourably with that of commercial fungicidesin controlling plant diseases. In fact, research into antimicrobial activitiesof plant extracts has offered some potential solution to disease control inthe absence of effective chemical control. For example, few commercial

Natural Products from Plants 69fungicides have been effective in inhibiting teliospore germination of Tilletiaindica, the causal agent of Karnal bunt of wheat. This disease is becomingmore widespread, in part, because of the lack of effective chemical control.Various extracts of native plants from Sonora, Mexico were evaluated todetermine their antifungal activity against T. indica. Dichloromethane andmethanol extracts were incubated with the fungus to measure the inhibitionof mycelial growth. Dichloromethane extracts from Chenopodium ambrosiodesand Encelia farinosa reduced radial mycelial growth significantly, but totalinhibition occurred at a relatively high concentration of 500 mg ml –1 of thedichloromethane extract from Larrea tridentata. Teliospores subjected to treatmentwith the latter plant extract showed no viability when transferred tofresh culture media, confirming the extract’s potential to be applied ascontrolling agent for T. indica (Rivera et al., 2001).Plants with antibacterial propertiesAlready in the 1990s, the application of natural plant extracts under bothgreenhouse (Blaeser and Steiner, 1999; Nikolov et al., 1999; Scholz et al., 1999)and postharvest (El-Ghaouth et al., 1995; Bhaskara et al., 1998) conditionsagainst various pathogens confirmed the potential of natural compoundsfrom plants to be developed into commercial products. Although the numberof natural products applied in biological control systems is currently arelatively small percentage of the total world market, it is predicted toincrease substantially. This implicates the possible decrease of pesticideapplication in agriculture in the future. In turn, this approach could create asignificant market opportunity for alternative products to be applied on alarger scale in organic crop production systems.A vast number of reports on large-scale screening programmes to evaluatewild plants for antibacterial activity are available in the literature; werefer to only a few. The antibacterial and antifungal activities of 38 plantsbelonging to 17 families were tested by Ghosh et al. (2000). The solventextracts of different morphological parts of these plants were tested against14 bacterial and 18 fungal strains. Out of these, Alpinia mutica, Cephalandraindica, Croton bonplandianum, Curcuma amada, Holarrhena antidysenterica, Moringaoleifera and Zingiber spectabile were found to contain antimicrobial properties(Ghosh et al., 2000). Khan and Omoloso (2002) studied the antibacterialand antifungal activities of methanol extracts of Harpullia petiolaris leaves,stems, root barks and heartwoods (collected from Papua New Guinea). Antibacterialactivities were found to be highest in fractions of root bark, petroland dichloromethane fractions of stem bark, the petrol fraction of heartwoodand the butanol fraction of leaves. Antifungal activity was only observed inthe petrol fractions of the root bark and stem heartwood.Ethanol and water extracts of leaves, flowers, shoots, bark and fruits of30 herbal and woody plant species were tested for in vitro growth inhibitionof Erwinia amylovora by Krupinski and Sobiczewski (2001) using an agar diffusionmethod. Active extracts were found in 23 species, while in 13 of these

70 J.C. Pretorius and E. van der Wattthe active substances were found for the first time. The highest growthinhibition of this bacterium was recorded for extracts of Aloe arborescens,Juglans regia, Rhus typhina (R. hirta), Salvia officinalis and Satureja hortensis. Inalmost all cases ethanol appeared to be a better solvent of active plantsubstances against E. amylovora than water.A similar approach was followed by Morais et al. (2002) in screeningcrude extracts of 45 known medicinal plants against bacterial pathogens oftomato. The antibacterial activity of crude extracts was tested against Xanthomonascampestris pv. vesicatoria, Ralstonia solanacearum and Clavibacter michiganensesubsp. michiganense. Some assays were also performed to verify thecapability of these plant extracts to show antibiosis. Five of the 45 extractsshowed significant activity against the test bacteria, confirming the potentialof using either these extracts or active substances contained in them as naturalproducts under field conditions. In the same year, Devanath et al. (2002)compared the antibacterial effect of extracts from three medicinal plants(Psidium guajava, Aloe vera [A. barbadensis] and Datura stramonium) to twostandard fungicides (Streptocycline and Blitox or copper oxychloride) againstRhizoctonia solanacearum in vitro. The aqueous extract of Aloe vera was mosteffective in suppressing the growth of R. solanacearum followed by the extractof Psidium guajava.Zeller et al. (2002) reported on the effect of an extract from Hedera helixagainst fire blight on pome fruits, caused by Erwinia amylovora. The highestgrowth inhibition of this bacterium was recorded for extracts of Aloe arborescens,Juglans regia, Rhus typhina (R. hirta), Salvia officinalis and Satureja hortensis.In almost all cases ethanol appeared to be a better solvent of active plantsubstances against E. amylovora than water. Also using the bacterium E. amylovoraas a test organism, Jin and Sato (2003) searched for secondary metabolitesin aqueous extracts from succulent young shoots of pear (Pyrus).Aqueous extracts of the tissue of succulent young shoots exhibited strongantibacterial activity against E. amylovora. The active compound was isolatedfrom the extract by steam distillation in vacuo, purified through charcoalpowder column chromatography and identified as benzoquinone (2,5- -cyclohexadiene-1,4-dione) by NMR spectra, mass spectra and HPLC analysis.In the same year, Pretorius et al. (2002a) demonstrated the broad-spectrumantibacterial activity of crude extracts from Acacia erioloba, Senna italica andBuddleja saligna against the plant pathogens Agrobacterium tumefaciens,Clavibacter michiganense pv. michiganense, Erwinia carotovora pv. carotovora,Pseudomonas solanacearum and Xanthomonas campestris pv. phaseoli.Additionally, plant extracts with antibacterial properties can provide analternative to certain antibiotics. Zeller et al. (2002) reported on such alternativesto the antibiotic streptomycin for the control of fire blight on pomefruits, caused by E. amylovora, which is of great economic importance forGerman and European fruit producers. An antagonistic preparation,BIOPRO, showed a control efficacy of up to 60% and the plant extract fromHedera helix revealed a high efficacy in the field in combination with a lowconcentrated copper compound and a metal salt. The control of fire blight inthis manner was comparable to that by the antibiotic streptomycin under

Natural Products from Plants 71artificial and natural infection conditions. The latter is important in light ofthe fact that the use of copper-containing antimicrobials is phasing out inEurope due to recent legislation, while the use of antibiotics, such as streptomycin,is highly opposed.Current status of antimicrobial products from plant extractsExtracts from various plant species appear to have promising potential fortheir application as natural products in the agricultural industry against bothplant pathogenic bacteria and fungi and to be integrated in plant diseasemanagement systems. However, to date, the number of commercially developednatural plant products that emerged from screening programmes isalarmingly low. The natural plant product Milsana ® , extracted from the giantknotweed (Reynoutria sacchalinensis), is probably best known (Daayf et al.,1995). The product has been reported to control powdery mildew, caused bySphaerotheca fuliginea, in the long English cucumber under greenhouse conditions,and also showed broad spectrum activity against powdery mildew oftomato, apple and begonia as well as downy mildew of grapevine and rustof bean (Daayf et al., 1995). It was concluded by Petsikos et al. (2002) thatMilsana ® can substantially contribute to the management of cucumberpowdery mildew in organic or integrated farming systems.A volatile natural product, Carvone, derived from dill and carawayseed, has been developed to inhibit the growth of storage pathogens and tosuppress sprouting of potatoes in the warehouse (Moezelaar et al., 1999). Carvoneis currently marketed as Talent ® in the Netherlands. Additionally,Fungastop and Armorex II, two natural products developed in the USA(Soil Technologies Corp.), are commercially available for the control ofvarious plant diseases in agriculture.Dayan et al. (2009) supplied an excellent summary of new natural antimicrobialproducts that were recently commercialized for use in either conventionalor organic agricultural or both industries. To name a few, these includeBla-S (against rice blast disease in eastern Asia), Kasugamin (against riceblast and other crop diseases in Japan), Mildiomycin (against powderymildews mainly in Japan), Delvolan (against fungal diseases of ornamentals)and Validacin (used for Rhizoctonia spp. control on a variety of crops).All of these products are fermentation secondary products from Actinomycetes,mostly Streptomyces spp., commercialized for use as agriculturalfungicides in Japan, and to a lesser extent in other parts of the world.Several plant essential oils are marketed as fungicides for organic farmers(Dayan et al., 2009). These include E-Rase from jojoba (Simmondsia californica)oil, Sporan from rosemary (Rosemarinus officianalis) oil, Promaxfrom thyme (T. vulgaris) oil, Trilogy from neem (A. indica) oil and GC-3being a mixture of cottonseed (Gossypium hirsutum) oil and garlic (A. sativum)extract. According to Dayan et al. (2009), few scientific papers deal with theseproducts or the actual active components, and their modes of action againstindividual plant pathogens are largely unknown.

72 J.C. Pretorius and E. van der WattA fairly new approach to microbial disease control is via systemicacquired resistance (SAR) or, in other words, the activation of natural defencemechanisms within the plant via elicitors (Dayan et al., 2009). Harpin proteinthat induces SAR in plants is sold as Messenger or ProAct. Brassinosteroidsthat induce the synthesis of PR proteins that resist fungal infection incrops, are sold as ComCat ® (Roth et al., 2000). The latter product seems to beused more often as a bio-stimulatory agent; an aspect that will be dealt withlater in this chapter.These past successes and the public’s current concern over the impact ofsynthetic pesticides on the environment ensures a continued, if not anincreased, interest in searching nature for environmentally friendlier pestmanagement tools (Dayan et al., 2009).3.5 Natural Bio-herbicidesThe herbicide component of all pesticides sold increased from less than 20%in the 1950s to almost 50% of the market in the 1980s (Jutsum, 1988) and thishas since risen to over 60%. Today, herbicides account for more than half ofthe volume of all agricultural pesticides applied in the developed world andtheir use has also contributed to the concern of consumers towards the potentialhealth and environmental impact they may have (Dayan et al., 2009).However, almost three decades ago, McWhorter and Chandler (1982) estimatedthat weeds contribute to a significant 12% loss in worldwide cropproduction, emphasizing the need to control weeds. These two perspectives,namely the need to control weeds and the negative attitude of consumers,have in part fuelled the current elevated interest in the discovery of naturalproducts to be applied as an environmentally friendly method of weedcontrol.However, the complexity of the environment in terms of weed controland especially the possible side effects of herbicides or biological con trolagents on non-targeted plants, calls for a comprehensive study oncontrol strategies, whether biological, chemical, cultural or a combination ofthese in an integrated weed management system. The biological approach toweed control can be separated into three distinct strategies: conservation,inundative and classical depending on the setting (e.g. range, row crops,urban), the extent of the infestation and the biology of the system (McFadyen,1998; Goeden and Andrés, 1999). According to the authors, conservationbiocontrol refers to situations where a natural biocontrol agent is alreadypresent and is able to control the weed but requires assistance in the form ofcultural practices or integrated management decisions that enable the agentto thrive. Although conservation strategies are less common in the biologicalapproach to weed control than in the biocontrol of insects, examples do exist(Goeden and Andrés, 1999).Inundative biocontrol involves the release of large numbers of a biologicalcontrol agent at a time when weed populations are expected to escapecontrol or exceed a critical economic or competitive threshold. When the

Natural Products from Plants 73agent is already present at a level that does not provide adequate, continuouscontrol of the target, this strategy is known as augmentation biocontrol(Goeden and Andrés, 1999). In general, inundative biocontrol relies on thereleased organisms themselves to control the target without any expectationof continued control by future generations of the agent (Elzen and King,1999). In an inundative strategy the agent may need to be released or appliedseveral times during a single crop cycle in the event of re-growth orre- emergence of the target weed. Thus, inundative strategies typically applyto relatively high-input systems.Classical biocontrol, sometimes referred to as inoculative biocontrol,involves the practice of identifying co-evolved natural enemies from thenative range of a target weed species and releasing them into the invadedrange to reduce the presence of the weed to acceptable levels (McFadyen,1998).In classical biocontrol, the agents are expected to reproduce and proliferateon the target weed and disseminate throughout its invaded range, reachingan ecological equilibrium with the target weed and providing continuous,perpetual control. Successful control depends almost wholly on damagecaused by the descendants of the released individuals rather than by thereleased individuals themselves (Elzen and King, 1999). Classical biocontrolis generally practiced in low-input systems. Biologically based weed controlcan also take the form of weed-resistant properties in crop plants, akin tohost-plant resistance versus insect and pathogen pests.Allelopathy, the production by a plant of secondary metabolites thatinhibit growth of nearby plants, is a phenomenon that has been studied forits potential utility in weed control for many years (Rector, 2008). This impliesthe use of natural compounds from plants, referred to as allelochemicals, innatural product development programmes. The term allelopathy is derivedfrom two Greek words ‘allelos’ and ‘patos’ meaning ‘to suffer on each other’(Delabays and Mermillod, 2002). According to the authors, allelopathy isdefined as a chemical process whereby certain plants release natural compoundsinto the environment that can either stimulate or inhibit the growthand development of surrounding plants. In nature the latter is more likely, asa form of competition for growing space, where allelochemicals are releasedfrom the roots of one plant and absorbed by the roots of surrounding plantspreventing them from growing in the same area. There are several ways inwhich an allelopathic plant can release its protective chemicals. These include:(i) volatilization, where a chemical is released in the form of a gas throughthe leaves and, on absorption, sensitive surrounding plants are stunted ordie; (ii) leaching of allelochemicals from decomposing abscised leaves preventingsurrounding plants from establishing in the same area; and (iii) exudationof allelochemicals into the soil through the roots, preventingsurrounding plants from prospering (Delabays and Mermillod, 2002).Despite its long history, little progress has been made in incorporatingallelopathy into mainstream weed management programmes due to a failureto provide adequate weed control while maintaining other agronomic qualitiesof the crop (Belz, 2007). There is a strong possibility that formulation hasplayed a key role in the failure of bio-herbicides in the market and their

74 J.C. Pretorius and E. van der Wattefficacy in the field. This is understandable when the bottom line criterion forany natural product developed from plant extracts, including naturalherbicides, is considered. This criterion is that the product must be effective,safe and have consistent results and must have an adequate shelf life of atleast 1 year.Despite the apparent failure to develop bio-herbicides from plants orother biological sources at present (see current status later in this chapter),the need to do so is increasing due to weeds becoming more resistant towardsthe commercially available synthetic herbicides (Dudai et al., 1999; Duke etal., 2000; Tworkoski, 2002). For example, in New Zealand the problems posedby invasive weeds are among the most severe in the world with an estimatedcost of NZ$100 million each year (Juliena et al., 2007). Of all the weeds currentlyidentified in New Zealand, only a third has been targeted by biologicalcontrol. The latter emphasizes the need to integrate either biological controlagents or chemical bio-herbicides or both into a broader managementprogramme for weeds.Plants with herbicidal propertiesThe greatest potential for developing natural herbicides probably lies in theplant kingdom, a largely untapped reservoir of natural compounds withallelopathic herbicidal properties. Although little success in terms of naturalproduct development has been realized, extensive research has been done inthe past to screen for the growth inhibitory activity of extracts from manyplant species (Wu et al., 2002). Only a few recent examples are supplied toemphasize the principle.Nie et al. (2002) reported that extracts of Wedelia chinensis reduced seedgermination, inhibited seedling growth, resulted in yellowing leaves andreduced resistance to disease in weeds such as Cyperus difformis, Paspalumthunbergii, Alternanthera sessilis and Cynodon dactylon at relative low concentrationof 0.4 g FW (fresh weight) ml –1 water. This study confirmed the potentialof W. chinensis extracts sprayed before crop emergence to control thegermination of weed seeds. Another study by Randhawa et al. (2002) showedthat a sorghum extract reduced seed germination and seedling growth of theweed Trianthema portulacastrum substantially at high concentrations (75–100%) but promoted shoot length of the weed at low concentrations (25%).Similar contrasting results were reported for the effects of extracts fromeight lucerne cultivars on seed germination as well as on root and hypocotyldevelopment of lettuce seedlings (Tran and Tsuzuki, 2002). Extractsfrom some lucerne cultivars had a stimulatory effect in terms of seed germinationas well as root and hypocotyl growth, whereas others showed thedirect opposite effect, confirming that crop plants can also be affected byplant extracts aimed at controlling weed growth. Singh et al. (2003) confirmedthis phenomenon by showing that aqueous leaf leachates of Eucalyptuscitriodora inhibited the germination and seedling growth of all testcrops (Vigna radiata, V. mungo and Arachis hypogaea) investigated. Further,

Natural Products from Plants 75in their study of bioactivity of plant extracts, Deena et al. (2003) demonstratedthe inhibitory effect of leaf, stem and root leachates from Andrographispaniculata on germination and seedling growth in rice. From this it becameclear that the bioactivities of plant extracts are unpredictable and may givedifferent and often contrasting results with regard to inhibition or promotionof growth and development in other plants. The reaction of crops totreatment with plant extracts may depend on the interaction between differenttypes of plant species or even on the concentration of the extracts(Channal et al., 2002a).Research into the allelopathic activity of plant extracts has resulted in theidentification of active ingredients responsible for both inhibition or stimulationof either seed germination or seedling growth. In this respect, Chunget al. (2002) reported on the inhibitory effect of ferulic, p-hydroxybenzoic,p-coumaric and m-coumaric acids isolated from three rice cultivars on thegrowth of barnyard grass. This suggested that these compounds may be, atleast, a key factor in rice allelopathy on barnyard grass, and the informationpresented may contribute to the development of natural herbicides.Similar active allelochemicals were isolated by Sasikumar et al. (2002). Intheir study on the allelopathic effects of Parthenium hysterophorus leachateson cowpea, pigeonpea, greengram, blackgram and horsegram, the authorsreported significant seed germination inhibition for all test crops. Gas chromatographicanalysis showed the presence of phenolic acids, namely caffeic,p-coumaric, ferulic, p-hydroxybenzoic and vanillic acids in the leachatesfrom different plant parts (leaf, stem, flower and root) of P. hysterophorus. Amixture of allelopathic compounds in bioassays significantly inhibited thegermination and vigour index of all test crops. However, leachates fromflowers had no inhibitory effect on the germination of blackgram andgreengram seeds.Kato and Kawabata (2002) isolated a growth-inhibiting compound fromthe acetone extract of 30-day-old lemon balm (Melissa offinalis) shoots bymeans of silica gel column chromatography. This uncharacterized compoundinhibited the growth of cress seeds at concentrations higher than 0.3 µg ml –1 .Iqba et al. (2002) showed that living buckwheat reduced weed biomass comparedto plots without buckwheat. A laboratory study revealed that root exudatesfrom buckwheat (collected from Aomori, Japan) suppressed root andshoot growth of the weeds Trifolium repens, Brassica juncea, Amaranthus palmeri,Echinochloa crus-galli and Digitaria ciliaris but also that of lettuce, andreduced weed dry weight. Fagomine, 4-piperidone and 2-piperidinemethanolwere isolated from a chloroform extract and identified as the activeingredients.Several allelochemicals have also been characterized from Helianthusannuus that inhibit seed germination and seedling growth of Amaranthusalbus, Amaranthus viridis, Agropyron repens (Elymus repens), Ambrosia artemisiifolia,Avena fatua, Celosia cristata (C. argentea var. cristata), Chenopodium album,Chloris barbara (Chloris barbata), Cynodon dactylon, Digitaria sanguinalis, Dactylocteniumaegyptium, D. ciliaris, E. crus-galli, Flaveria australasica, P. hysterophorus,Portulaca oleracea, Sida spinosa, Trianthema portulacastrum and Veronica

76 J.C. Pretorius and E. van der Wattpersica (Macias et al., 2002). The inhibitory effects of this crop may be utilizedfor weed management to attain reduced herbicide usage in sustainableagricultural systems (Azania et al., 2003).Previous studies by Salamci et al. (2007) confirmed that the essentialoils of T. aucheranum and T. chiliophylllum, characterized by the relativelyhigh content of 1,8-cineole, camphor, borneol and α-terpineol, exhibitedpotent inhibitory effects on seed germination and seedling growth ofA. retroflexus. All commercialized essential oils act as non-selective, contactherbicides that can provide good but transient weed control. The use ofessential oils for weed control in organic agriculture seems promising, butthese natural herbicides all act very rapidly and their efficacy is limited bythe fact that they probably volatize relatively quickly (Dayan et al., 2009).Other oils such as pine and clover oils have also been used in organicfarming systems, but with limited success because the relatively high rateof use required for control makes it expensive compared to only onetreatment of glyphosate with the same or even better control of weeds(Dayan et al., 2009).Current status of natural herbicidesAlthough several natural products from various sources are probably currentlyunder development and targeted against specific weeds across theglobe, very little literature is available on the use and environmental impactof natural products in organic agriculture and little success can be reportedat present. According to Babua et al. (2003), reasons why so many promisingbio-herbicides have failed to reach the commercial phase probably include:(i) the economics of patenting and registration; (ii) inadequate or noncommercialmarket sizes; and (iii) technological constraints, especially relatingto formulation chemistry. Discovery and development of a syntheticchemical herbicide can easily cost US$30 million (Heiny and Templeton,1993) or more. It is generally expected that the development of a naturalproduct herbicide should cost less. Indeed, two notable successes includeCOLLEGO and BIOMAL, developed from exotic pathogens, where thedevelopment costs were US$1.5 million and US$2.6 million respectively. Thecosts involved with the development of these two examples of natural productscan probably not be accepted as a rule and there are no guarantees thatprospective entrepreneurs will experience the same. However, from an ecologicalperspective natural products are much more favoured (Heiny andTempleton, 1993) as a result of added advantages over classical syntheticweed control agents, including their narrow host ranges that reduce theirimpact on non-targeted plants in the environment (Rector, 2008), and thisalone serves as a driving force for natural product developers to continuewith this entrepreneurial business enterprise. Additionally, the quicklyexpanding organic agriculture industry does not allow synthetic pesticides,including herbicides (Anonymous, 2009), and this provides more momentumfor the natural product industry.

Natural Products from Plants 77In an excellent recent review by Dayan et al. (2009), a number of naturalcompounds from which bio-herbicides have been commercialized forweed control in organic agriculture have been listed. A few examplesinclude corn gluten meal (e.g. WeedBan, Corn Weed Blocker and BioscapeBioweed), a mixture of essential oils and other organic compounds(e.g. Burnout, Bioorganic, Weed Zap and GreenMatch) as well aspelargonic acid mixed with related short-chain fatty acids and paraffinicpetroleum oil (e.g. Scythe). However, despite the examples of naturalproducts mentioned above, a tripeptide obtained from the fermentationculture of the actinomycete Streptomyces hygroscopis and registered asBialaphos is regarded by Dayan et al. (2009) to be the only true commercializednatural product herbicide to date. It is a pro-herbicide that ismetabolized into the active ingredient l-phosphinothricin (Fig. 3.28) in thetreated plant.Bialaphos and phosphinothricin inhibit the enzyme glutamine synthetasewhich is necessary for the production of glutamine and for ammoniadetoxification. The elevated ammonia levels in tissues of treated plants stopsphotosynthesis and results in plant death. Both Bialaphos and phosphinothricinare broad-spectrum post-emergence herbicides that can be used fortotal vegetation control in many agricultural settings or in non-cultivatedareas and to desiccate crops before harvest (Dayan et al., 2009).The list of commercialized bio-herbicides has expanded substantiallyduring the past two decades. However, their mere existence must be consideredin view of the following very valid comments by Dayan et al. (2009):(i) as opposed to traditional synthetic herbicides, none of the natural herbicidalcompounds allowed for use in organic agriculture are very active andthey must, therefore, be applied in relatively large quantities that may leadto undesirable effects on the environment and the soil fauna and microbes,which is in direct opposition with the philosophical positions and purposeof those who practice organic agriculture; (ii) the use of organic weed managementtools may be enhanced in the context of an integrated pest managementprogramme that includes sowing multiple crops, extendedrotation cycles, mulching, and soil cultivation and cover; (iii) existing naturalherbicides show very little crop selectivity and still require laboriousapplication methods to ensure they do not come in contact with the desiredcrop; and (iv) organic weed management methods may be possible insmall-scale farming and high-value crops but do not seem feasible in theproduction of the agronomic crops such as grains grown in large-scalefarming enterprises.OHOPONH 2OHFig. 3.28. L-phosphinothricin (redrawn from Dayan et al., 2009).

78 J.C. Pretorius and E. van der Watt3.6 Natural Compounds from Plants with Bio-stimulatoryPotentialAllelochemicals found in plants are probably all secondary metabolites thatare distinctive from primary metabolites in that they are generally nonessentialfor the basic metabolic processes such as respiration and photosynthesis(Richard, 2001). They are numerous and widespread, especially inhigher plants (Pillmoor, 1993), and often present in small quantities (1–5%) ascompared to primary metabolites (carbohydrates, proteins and lipids).Approximately 88,000–100,000 secondary metabolites have been identifiedin all plant forms, showing both structural and activity diversity (Verpoorte,1998). Ecologically, these chemicals play essential roles in attracting pollinators,as adaptations to environmental stresses and serve as chemical defencesagainst insects and higher predators as well as microorganisms (Rechcigland Rechcigl, 2000). Although the purpose of the production of secondarymetabolites in plants has long been argued among researchers, it is now universallyaccepted that they are produced as a result of abiotic (Beart et al.,1985) and biotic stresses (Bourgaud, et al., 2001), probably as part of a plantdefence arsenal. Besides the role secondary metabolites play in plant metabolism,the growth promotion or inhibitory properties of certain naturalcompounds from plants have been extensively researched (Wu et al., 2002).Current status of plant products with bio-stimulatory potentialAlready, at the end of the millennium, two successful natural products developedin Moldavia (formerly part of the Soviet Union) are Moldstim andPavstim, extracted from hot peppers (Capsicum annum L.) and leaves ofDigitalis purpurea L., respectively (Waller, 1999). Both products have beenused on a large scale as plant-growth regulators and for disease control.These developments are excellent examples of how natural plant resourcescan be exploited and applied in agriculture.However, from an agricultural perspective, plant extracts containinggrowth-promoting substances have always been of interest to the researchcommunity in terms of the role they could play in addressing future foodsecurity issues. The ideal breakthrough would be to identify a plant or plantsthat contain bio-stimulatory substances promoting growth and resistance topathogens, as well as yields in agricultural and horticultural crops. At thispoint it seems appropriate to consider recent discoveries of bio-stimulatorycompounds from plants that have the potential to adhere to the ‘ideal breakthrough’criterion in terms of their application potential as natural productsin the agricultural industry.Extracts from numerous plant species, with bio-stimulatory properties,were identified and evaluated for their commercial potential. Channal et al.(2002b) reported on seed germination as well as seedling growth enhancementof sunflower and soybean by leaf extracts from three tree species

Natural Products from Plants 79( Tectona grandis, Tamarindus indica and Samanea saman). Terefa (2002) reportedsimilar effects for P. hysterophorus extracts on tef (Eragrostis tef) while Neelamet al. (2002) demonstrated similar effects for Leucaena leucocephala extracts onwheat (Triticum aestivum). However, none of these studies revealed that treatmentwith the different plant extracts had any effect on the final yields of thecrops under investigation.In this regard, a report by Ferreira and Lourens (2002) demonstrating theeffect of a liquid seaweed extract (now trading as a natural product under thename Kelpak) on improving the yield of canola must be regarded as significant.Kelpak applied singly or in combination with the herbicide Clopyralid® at various growth stages of canola (Brassica napus) was assessed in afield experiment conducted in South Africa during 1998–1999. Foliar applicationof 2 l Kelpak ha –1 , applied at the four-leaf growth stage, significantlyincreased the yield of the crop. The active compounds in Kelpak are auxinsand cytokinin.In the same year, a study directed towards identifying bio- stimulatoryproperties in plant extracts was performed by Cruz et al. (2002a). The authorstreated the roots of bean, maize and tomato with an aqueous leachate of Callicarpaacuminata and followed the in vitro effects on radicle growth, proteinexpression, catalase activity, free radical production and membrane lipidperoxidation in the roots. The aqueous extract of C. acuminata inhibited theradicle growth of tomato but had no effect on root growth of maize or beans.However, expression of various proteins in the roots of all treated plants wasobserved. In treated bean roots the expression of an 11.3 kDa protein by theleachate, showing a 99% similarity with subunits of an α-amylase inhibitorfound in other beans, was induced. In treated tomato an induced 27.5 kDaprotein showed 95% similarity to glutathione-S-transferases of other Solanaceaespecies. Spectrophotometric analysis and native gels revealed thatcatalase activity was increased twofold in tomato roots and slightly in beanroots, while no significant changes were observed in treated maize roots.Luminol chemiluminescence levels, a measure of free radicals, increasedfourfold in treated tomato roots and twofold in treated bean roots. Oxidativemembrane damage in treated roots, measured by lipid peroxidation ratesrevealed an almost threefold increase in peroxidation in tomato while noeffect was observed in maize or beans (Cruz et al., 2002a).The significance of this study lies in the fact that various metabolicevents can be manipulated in plants by treatment with certain plant extracts.What has to be established by researchers is whether these altered metabolicevents contribute towards positive or negative physiological changes withinthe treated plants. The rationale for this type of research lies in the search fornatural compounds to be applied in sustainable yield-improving, as well asweed-, pest- and disease-controlling, management systems (Singh et al.,2001). According to the authors, natural compounds isolated from someplants show strong bio-herbicidal activity at high concentrations but at lowconcentrations these extracts can promote crop seed germination and seedlinggrowth, hence showing a potential to be applied as bio-stimulatoryagents or growth-promoting substances in agriculture. There is general

80 J.C. Pretorius and E. van der Wattconsensus amongst scientists that research in this regard should concentrateon both the inhibitory and stimulatory effect of plant extracts on seed germination,seedling growth and the physiology of other test plants in order toverify the action at hand (Khan et al., 2001; Ameena and George, 2002; Cruzet al., 2002b; Duary, 2002; Obaid and Qasem, 2002).Probably the most effective compounds to enhance crop yield, crop efficiencyand seed vigour have been identified as brassinosteroids (BRs;Mandava, 1979; 1988), first extracted from rape (Brassica napus L.) pollen(Adam and Marquard, 1986). In a recent mini-report, Zullo and Adam (2002)confirmed the prospective agricultural uses of BRs. The assumption of theirapplication potential was made from data collected over the past threedecades and only a few examples are presented here. Yield increases thatwere in most instances significant were reported, as cited by Zullo and Adam(2002), in beans and lettuce (Meudt et al., 1983), rice (Lim, 1987), maize (Limand Han, 1988), wheat (Takematsu et al., 1988), chickpea (Ramos, 1995) andtomato (Mori et al., 1986). Many other examples of BR use for increasing cropyield can be found in the literature (Kamuro and Takatsuto, 1999; Khripachet al., 1999; Khripach et al., 2000).Besides their yield-improving effects, BRs have been shown to increaseplant growth in crops (Rao et al., 2002) and especially root growth (Müssiget al., 2003), to increase resistance in crops towards low-temperature injury(Kamuro and Takatsuto, 1999) and to increase resistance of potato to infectionsby Phytophthora infestans and Fusarium sulfureum (Kazakova et al., 1991).Although many BRs, such as 24-epibrassinolide, are commercially availableand employed in some countries, more accurate studies on dosage, methodand time of application, its suitability for the plant or cultivar, and associationwith other phytohormones are needed, because many of the results wereobtained by experiments performed in greenhouses or small fields (Zulloand Adam, 2002).A report on a prototype bio-stimulatory natural product developed froma BR-containing extract of Lychnis viscaria came from Roth et al. (2000). In2003, after 12 years of intensive research under laboratory, greenhouse andfield conditions at the University of the Free State, South Africa, a productwas listed in Germany as a plant-strengthening agent under the trade nameComCat and commercialized by a German company, Agraforum AG.Foliar applications of ComCat have been demonstrated to enhance consistentlyroot development in seedlings and final yields in a number of vegetable,fruit and row crops, as well as to induce resistance in crops towardsabiotic and biotic stress conditions, and the mechanisms of action were elucidatedon both a metabolic and genetic level (unpublished results, Pretorius,J.C. and van der Watt, E., University of the Free State, South Africa). Recently,significant yield increases in tomato, preharvest treated with ComCat,were reported by Workneh et al. (2009). The authors also claimed more than70% shelf life extension and higher marketability in tomato fruit harvestedfrom plants treated with ComCat during the vegetative growth phase comparedto the untreated control under ambient storage conditions. PreharvestComCat treated tomatoes contained lower total soluble sugar levels at

Natural Products from Plants 81OHOHOHCH 3OOHHOHOOOO24-epi-secasterone 24-epicastasteroneOHHOHOOHOBrassinolideOHFig. 3.29. Brassinosteroid active compounds contained in the plant-strengtheningagent ComCat®.harvest and showed better keeping quality in terms of physiological weightloss and juice content compared to untreated controls. In light of the diversepositive effects of ComCat on agricultural and horticultural crops,the product largely adheres to the ‘ideal candidate’ criterion stated earlier.Three BRs have been identified as the main active components of ComCatand these include 24-epi-secasterone, 24-epicastasterone and brassinolide(Fig. 3.29).The future of commercialized bio-stimulants seems positive in light ofthe elevated costs of fertilizer. In this regard, research in terms of the use ofbio-stimulants in combination with fertilizer levels lower than the recommendedstandard for different crops seems to be important in an attempt tolower the input costs that have become a gloomy issue for farmers recently.3.7 ConclusionsDiscovery programmes by the agrochemical industry are mostly driven bylarge-scale synthetic programmes followed by screening to identify potentialnew bio-pesticides, including antimicrobials and herbicides. Most companieshave a more modest effort to evaluate natural products from outsidesources and, to a lesser extent, from in-house isolation efforts. Although theliterature is replete with reports of the isolation and characterization of phytotoxinsfrom many sources, and many of these compounds have been patentedfor potential use, the use of natural or natural-product-derivedherbicides in conventional agriculture is limited (Dayan et al., 2009).Since the Second World War, traditional agricultural practices haveincluded the use of synthetic chemicals for the management of plant pathogens,pests and weeds. This has, without any doubt, increased crop productionbut with some deterioration of the environment and human health(Cutler, 1999). Research indicates that even if one never uses pesticides, onecan still be exposed to them by being a consumer of commodities that othershave treated with pesticides, e.g. through food.

82 J.C. Pretorius and E. van der WattIn addition to the target pathogen, pesticides may kill various beneficialorganisms and their toxic forms can persist in the soil. The increasing incidenceof resistance among pathogens towards synthetic chemicals is also acause for serious concern. The above is not only of major concern to thedeveloped countries, where consumer preferences are for organically producedfoods, but also in the developing world, such as Africa, where syntheticpesticides are too expensive for subsistence farming. Because of theseproblems there is a need to find alternatives to synthetic pesticides.Among the various alternatives, natural plant products that are biodegradableand eco-friendly are receiving the attention of scientists worldwide.Such products derived from higher plants and microbes are relatively bioefficacious,economical and environmentally safe and can be ideal candidatesfor use as agrochemicals (Macias et al., 2002). Additionally, the manufacturersof natural bio-stimulants applied in agriculture claim increased production,profit increases, cutting of operating costs and reduced fertilizer costs withno detrimental effect to the environment (Chen et al., 2002). A number ofplants showing the potential to act as donor plants for these natural productshave been outlined in this chapter.ReferencesAdam, G. and Marquardt, V. (1986) Brassinosteroids.Phytochemistry 25, 1787–1799.Alemayehu, W. (1996) Unused pesticides indeveloping countries: 100 000 tonnesthreaten health and environment. News& Highlights. Food and AgricultureOrganization of the United Nations Page.Amadioha, A.C. (2002) Fungitoxic effects ofextracts of Azadirachta indica againstCochliobolus miyabeanus causing brownspot disease of rice. Archives of Phytopathologyand Plant Protection 35, 37–42.Ameena, M. and George, S. (2002) Allelopathicinfluence of purple nut sedge (Cyperus rotundusL.) on germination and growth ofvegetables. Allelopathy Journal 10, 147–152.Anonymous. (2009) Organic farming..Arras, G., Piga, A. and D’Hallewin, G. (1993)The use of Thymus capitatus essential oilunder vacuum conditions to control Penicilliumdigitatum development on citrusfruit. Acta Horticulturae 344, 147–153.Awuah, R.T. (1994) In vivo use of extracts fromOcimum gratissimum and Cymbopogon citratesagainst Phytophthora palmivoracausing blackpod disease of cocoa.Annals of Applied Biology 124, 173–178.Azania, A., Azania, C., Alves, P., Palaniraj, R.,Kadian, H.S., Sati, S.C., Rawat, L.S.,Dahiya, D.S. and Narwal, S.S. (2003)Allelopathic plants: Sunflower (Helianthusannuus L.). Allelopathy Journal 11, 1–20.Babua, R.M., Sajeenaa, A., Seetharamana, K.,Vidhyasekarana, P., Rangasamyb, P.,Prakashc, M.S., Rajab, A.S and Bijib, K.R.(2008) Molecular biology approaches tocontrol of intractable weeds: New strategiesand complements to existing biologicalpractices Plant Science 175, 437–448.Bae, E.Y., Shin, E., Lee, D.H., Koh, Y.J., Kim,J.H., Bae, E.Y., Shin, E.J., Lee, D.H.,Koh, Y.J. and Kim, J.H. (1997) Antifungalkaempferol-3-O-β-D-apiofuranosyl-(1,2)-β-D-glucopyranoside from leavesof Phytolacca americana L. Korean Journalof Plant Pathology 13, 371–376.Baldwin, I.T. (1999) The Jasmonate cascadeand the complexity of induced defenseagainst herbivore attack. In: Michael, W.(ed.) Function of Plant Secondary Metabolitesand their Exploitation in Biotechnology.CRC Press, Columbus, USA, pp. 155–179.

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4 Antimicrobials of Plant Originto Prevent the Biodeteriorationof GrainsK.A. RAVEESHADepartment of Studies in Botany, University of Mysore,Manasagangotri, IndiaAbstractA significant portion of stored food becomes unfit for human consumption due to thebiodeterioration of grains. The incessant and indiscriminate use of synthetic chemicalsin crop protection has been one of the major factors in polluting soil and waterbodies. Thus, there is a need to search for effective, efficient and eco-friendly alternativemethods for preventing the biodeterioration of grains during storage. Antimicrobialsof plant origin are an important alternative, which could be better exploited toprevent grain biodeterioration. Fungi are significant destroyers of food so the biodeteriorationof grains can be prevented by inhibiting fungal growth. Of late, manyplant extracts have been screened for antifungal activity. Decalepis hamiltonii Wight &Arn. (Asclepiadaceae) and Psoralea corylifolia L. (Leguminosae) have revealed highlysignificant antifungal activity. The antifungal active compounds from these plantshave been isolated by antifungal activity guided assays and characterized usingNMR, IR and mass spectral studies. The biomolecules responsible for the activitywere identified as 2H-Furo [2,3-H]-1-benzopyran-2-one in P. corylifolia and 2-hydroxy-4-methoxybenzaldehyde in D. hamiltonii. In vitro and in vivo evaluations of thesebiomolecules have shown promising inhibitory activity of important biodeterioration-causingfungi. Comparative evaluation of the bioactive compounds with those ofthe routinely used chemical fungicides is highly encouraging. Results suggest thepotential of these biomolecules for commercial exploitation to develop eco-friendlyherbal remedies for preventing the biodeterioration of grains during storage.4.1 IntroductionStorage of food is a necessity to ensure the availability of food throughout theyear. Stored foods are prone to postharvest loss in quality and quantity dueto infestation by different groups of organisms. Biodeterioration may bedefined as the quality and quantity loss of stored food caused by organisms.The organisms broadly responsible for such deterioration are microbes,insects and rodents. Among microbes, fungi are significant destroyers of© CAB International 2011. Natural Products in Plant Pest Management(ed. N.K. Dubey) 91

92 K.A. Raveeshafood, particularly species of Aspergillus and Penicillium. Grain produced isnot a grain until it is consumed without quality loss (Neergaard, 1977). Inspite of the developments in the methods of food preservation, nearly 30% ofthe food produced is lost during storage, due to poor handling and storagepractices.The incessant and indiscriminate use of chemical pesticides in agriculturehas resulted in drug resistance, residual toxicity, and has caused soil andwater pollution. This has necessitated the search for natural eco-friendlyalternatives, which are biodegradable and non-toxic to non-target species.Plants are a repository of novel biomolecules with various biological activities.Among the 250,000–500,000 species of plants available on earth, thenumber of plants screened phytochemically is a small percentage, amongthese the number of plants screened for various biological activities is infinitesimallysmall. In recent years there have been serious efforts to screenplants for various biological activities and the scientific endeavour is focusedmainly on utilizing plants for human health and least towards plants forplant health. Considering the fact that large amounts of synthetic pesticidesknown to cause environmental pollution are used in present-day agriculture,there is an urgent need to search for natural eco-friendly alternatives. Antimicrobialsof plant origin appear to be one of the safest and eco-friendlyalternatives. Hence, there is a need to search for antimicrobials of plant originto manage field crop loss in general and to prevent biodeterioration of grainsduring storage in particular.Clinical microbiologists have two reasons to be interested in antimicrobialsof plant origin. First, it is very likely that these phytochemicals orbiomolecules will find their way into the arsenal of prescribed antimicrobialdrugs. Second, the public is becoming increasingly aware of the problemswith and side effects from the over prescription and traditional use ofantibiotics (Cowan, 1999).Agricultural microbiologists have many more reasons to be interested inantimicrobials of plant origin: (i) it is very likely that these phytochemicalsmay find their way into the array of antimicrobial drugs prescribed; (ii) it isknown that the effective life span of any antimicrobial is limited, hence newerantimicrobials are necessary; (iii) it may help to overcome the rising incidenceof drug resistance amongst pathogenic microbes and the mechanismof action could be different; (iv) the phytochemicals present in plant extractsmay alleviate the side effects that are often associated with synthetic antimicrobials;(v) phytomedicines usually have multiple effects on the plantbody and their actions are often growth promoting, more systemic andbeyond the symptomatic treatments of disease; and (vi) it may help in developingcost effective remedies that are affordable to the population (Cowan,1999; Doughari, 2006).Finding healing powers in plants for human health needs is an ancientidea and practice. Searching herbal remedies for crop protection in generaland prevention of biodeterioration in particular is recently gaining importance.Agricultural microbiologists around the world are evaluating a numberof plant extracts for antimicrobial activity against important plant

Antimicrobials to Prevent Biodeterioration of Grains 93pathogens in vitro. Some attempts are also being made to identify the potentialof plant extracts to prevent biodeterioration of grains during storage.The body of literature available in the area of antimicrobials of plant originis voluminous and concerns different areas of study. In the present context,the literature reviewed concerns mainly the antifungal agents of plantorigin with specific reference to important biodeterioration-causing fungi.The body of literature available may be broadly categorized under the followingheadings:●●●In vitro evaluation of plant extracts for antifungal activity against importantbiodeterioration-causing fungi.In vitro evaluation of the active compounds for antifungal activity againstimportant biodeterioration-causing fungi.Isolation and identification of the antifungal active compounds fromplants.4.2 In Vitro Evaluation of Plant Extracts for Antifungal Activityagainst Important Biodeterioration-causing FungiScreening plant extracts for antimicrobial activity is the first step towards theisolation of the active component. Water and/or different organic solvents,singly or in combination, are generally used in the preparation extracts.Aqueous extractsAntifungal activity assays of leaf extract obtained from Ocimum sanctum,Adhatoda vasica, Emblica officinalis, Saussurea lappa, Glycyrrhiza glabra, Zingiberofficinale, Piper longum, Piper nigrum, Onosma bracteatum, Tinospora cordifolia,Fagonia cretica and Terminalia chebula were tested against Aspergillus flavus at10% and 50% concentration. P. longum and Z. officinale showed significantantifungal activity (Farooq and Pathak, 1998). Crude extracts of 40 Iranianand Canadian plants were tested for antifungal activity against several speciesof Aspergillus. Of these, 26 plants (65%) showed activity and the spectrumof activity was wide in case of Diplotaenia damavandica, Heracleumpessicum, Sanguisorba minor and Zataria multiflora (Sardari et al.,1998).Sinha and Saxena (1999) conducted an antifungal activity assay of leafextract of Allium cepa and Allium sativum (garlic) against Aspergillus niger andreported that the garlic was more effective in inhibiting the germination ofspores and mycelial growth of the fungus. Mahmoud (1999) evaluated theantifungal activity of five different concentrations of aqueous extract of Lupinusalbus, Ammivis naga and Xanthium pungens against A. flavus and productionof aflatoxins and reported the inhibition of mycelial growth of A. flavusat 2, 4, 6, 8 and 10 mg/ml.Aqueous extracts of 50 plants belonging to 27 families were screened forantifungal activity against A. flavus and A. niger and it was found that only

94 K.A. Raveeshafour plants, namely Trachysper neumammi, Allium sativum, Syzygium aromaticumand Plectranthus rugosus were effective against both species of Aspergillus(Singh and Singh, 2000). Aqueous extract of Terminalia australis hasbeen reported to be effective in inhibiting Aspergillus strains by Carpanoet al. (2003).Aqueous extracts of Aloe barbadensis, Datura stramonium, Zingiber officinale,Murraya koenigii and Azadirachta indica were evaluated against species ofAspergillus by employing the poisoned food technique and seed inoculationmethod. All the plant extracts showed significant inhibitory activity.D. stramonium and A. indica showed higher activity (Sharma et al., 2003). Theextracts of Toona ciliata (stem bark) and Amoora rohituka (stem bark) exhibitedsignificant in vitro antifungal activities against A. flavus at 20 and 30%concentration (Chowdhury et al., 2003).Solvent extractsWelsh onion ethanol extract was tested against A. flavus and A. parasiticusfor mycelial growth inhibitory activity and aflatoxins production (Fanand Chen, 1999). Sharma et al. (2002) evaluated an alcoholic extract of Semecarpusanacardium L. against Aspergillus fumigatus at different concentrations(20–400 μg/ml) and observed complete inhibition at 40 μg/ml.Petroleum ether and methanolic extract of Eupatorium ayapana weretested for antifungal activity at 250, 500, 750 and 1000 μg/ml against A. nigerand A. flavus. Petroleum ether extract showed higher antifungal activity thanthe methanolic extract (Gupta et al., 2002). An ethyl acetate soluble fraction ofacidified aqueous mother liquor and buffer soluble fraction of neutral motherliquor of stem bark of Alangium salvifolium were tested for antifungal activityagainst species of Aspergillus by Katyayani et al., (2002) and reportedsignificant activity at 10 mg/ml concentration.The methanolic extract of stem bark of Ailanthus excelsa partitioned withchloroform recorded significant antifungal activity against A. niger, A. fumigatus,Penicillium flequentence, and Penicillium notatum at 300, 90, 70, and 140μg/ml concentrations, respectively (Joshi et al., 2003). Moderate antifungalactivity of ethanolic extracts of the trunk bark of Zanthoxylum fagara, Z. elephantiasisand Z. martinicense was observed against A. niger, and A. flavus at500 and 1000 μg/disc (Hurtado et al., 2003). Ethanolic extract of seeds of Piperguineense and other solvent fractions obtained by column chromatographytested against A. flavus, revealed highly significant antifungal effect (Nganeet al., 2003). Methanol and methyl chloride extracts of 20 Indonesian plantswere tested for antifungal activity against A. fumigatus at 10 mg/ml by thepoisoned food technique. Extracts of six plants, viz. Terminalia catappa, Swieteniamahagoni, Phyllanthus acuminatus, Ipomoea spp., Tylophora asthmatica andHyptis brevipes recorded significant antifungal activity (Goun et al., 2003).Extracts of aerial parts of Achillea clavennae, Achillea holosericea, Achillealingulata and Achillea millefolium (hexane:ether:methanol = 1:1:1) were testedfor antifungal activity by disc diffusion assay against A. niger. All four species

Antimicrobials to Prevent Biodeterioration of Grains 95exhibited antifungal activity against tested strains (Stojanovicet al., 2005).Organic solvent leaf extracts of two Moroccan Cistus L. species Cistusvillosus L. and Cistus monspeliensis L. (Cistaceae) used in traditional medicinewere tested for their antifungal properties against A. fumigatus. The extractsdiffered in their antifungal activities. C. villosus extracts exhibited higheractivity than C. monspeliensis (Bouamama et al., 2006).Antifungal activity of Trapa natans L. fruit rind, extracted in different solventswith increasing polarity was observed and 1,4-dioxan, chloroform,acetone, dimethylformamide, ethanol, and water did not reveal any activityagainst A. niger (Parekh and Chanda, 2007).4.3 In Vitro Evaluation of the Active Components for AntifungalActivity against Important Biodeterioration-causing FungiPlants possess unlimited ability to produce secondary metabolites and morethan 12,000 of them have been isolated, which is probably less than 10% ofthe total. Useful antimicrobial phytochemicals are phenolics and poly phenols,quinones, flavones, flavonoids and flavanols, tannins, coumarins, terpenoidsand essential oils, alkaloids, and lectins and polypeptides. Cowan(1999) has critically reviewed the antimicrobials of plant origin and has listedthe plants with antimicrobial activity.Many secondary metabolites have been identified and many more areyet to be discovered. There is growing evidence that most of these compoundsare involved in the interaction of plants with other species, primarilyin the defence of the plant from plant pests. Secondary compounds representa large reservoir of chemical structures with biological activity (Duke, 1990).This resource is largely untapped for use as pesticides.Considering the advantages of organic pesticides over synthetic pesticides,nowadays, attempts are made by many workers to screen plants forantimicrobial activity, isolate and characterize the bioactive compounds fromdifferent parts. Such attempts to isolate antimicrobials of plant origin andtest their efficacy against different storage fungi to prevent biodeteriorationof grains are, however, very few.The high terpene hydrocarbon content in the oils of chamomile, lavender,eucalyptus, and geranium are responsible for their antifungal activity againstA. niger and Aspergillus ochraceous (Lis-Balchin et al., 1998). Antimicrobialcompounds, such as 3-0-methylveracevine, 3-0-[3-(2,2,2,-trifluoroethoxy)]-5-(methoxybenzoyl) veracevine 3-0-(3,5-diiodobenzoyl) veracevine, 3-0-(3-thienoyl) veracevine, isolated from the seeds of Schoenocaulon officinaleshowed highly significant antifungal activity against A. flavus and A. niger(Oros and Ujvary, 1999).Mathekga et al. (2000) isolated an acylated form of a phloroglucinol fromHelichrysum caespititium. The structural elucidation revealed that the compoundwas 2-methyl-4-2’-4’(6’-trihydroxy-3’-(2-methylpropanoylphenyl)-2-0-enylacetate). This compound completely inhibited the mycelial growth

96 K.A. Raveeshaof A. niger and A. flavus at 1.0 μg/ml. Bioactive compounds12β-hydroxysandarocopimar-15-one and 2-propionoxyo-β-chesorcylic acidisolated from methanolic extract of leaves of Trichilia heudelotti showed highlysignificant antifungal activity against A. niger at 100 μg/ml (Aladesanmi andOdediran, 2000).Investigations on the antifungal active methanol fraction of the root ofEpinetrum villosum (Exell) Troupin (Menispermaceae) led to the isolation ofthe bisbenzylisoquinoline alkaloid cocsoline, which displayed significantantifungal activity with a minimum inhibitory concentration (MIC) forA. flavus and A. niger of 31.25 g/ml (Otshudi et al., 2005).Phytochemical analysis of the leaves of Vernonia amygdalina yielded twoknown sesquiterpene lactones, vernolide and vernodalol, that exhibited significantantifungal activity. Vernolides exhibited high activity and the 50%lethal concentration (LC 50) values ranged from 0.2 to 0.4 mg/ml for P. notatum,A. flavus, A. niger and Mucor hiemalis. Vernodalol showed moderateinhibitory activity against A. flavus, A. niger and P. notatum with LC 50valuesof 0.3, 0.4 and 0.5 mg/ml, respectively (Erasto et al., 2006).4.4 Isolation and Identification of the Antifungal ActiveComponent from PlantsThe isolation and identification of antimicrobials of plant origin involve thefollowing stepwise approach:● Selection of the plant, followed by the selection of the plant part.● Selection of appropriate solvent for extraction.● Separation and purification of the active component.● Characterization and structural elucidation of the active component.● In vitro evaluation of the active component for antimicrobial activityagainst test fungi.● In vivo evaluation of the active component for antifungal activity.Employing bioactivity-directed fractionation and isolation (BDFI) for aspecific antimicrobial activity against biodeterioration-causing fungi isadvantageous. Two plants have been extensively studied using this method,namely Psoralea corylifolia and Daecalepis hamiltonii. The active componentresponsible for antifungal activity has been isolated, characterized and theactivity has been demonstrated in vitro. Further investigations have beendone to prove its efficacy to prevent biodeterioration in vivo.Potential of 2H-furo[2,3-H]-1-benzopyran-2-one isolated from Psoraleacorylifolia L. to prevent the biodeterioration of maizePsoralea corylifolia L. (Leguminosae) is an annual herb (Fig. 4.1) reported tohave medicinal properties. In traditional medicine, the seeds (Fig. 4.2) areused in the treatment of psoriasis, leucoderma and inflammatory disease of

Antimicrobials to Prevent Biodeterioration of Grains 97Fig. 4.1. Psoralea corylifolia L. (plant).Fig. 4.2. Psoralea corylifolia L. (seeds).

98 K.A. Raveeshathe skin. An antifungal activity guided assay of different solvent extractsrevealed that petroleum ether and methanol extracts were highly active.Hence a combination of petroleum ether and methanol was employed for theisolation of the antimicrobial active component from the seeds of P. corylifolia(Kiran and Raveesha, 2004).Isolation and characterization of the antifungal active componentPowdered seeds of P. corylifolia were refluxed in a petroleum ether and methanolmixture [9:1(v/v)] in a Soxhlet apparatus for 8 h at 60°C. An excess ofsolvent was removed by distillation under reduced pressure. The concentratedextract was cooled for 48 h at 5°C to obtain the pure compound astransparent, colourless, rectangular prism-shaped crystals (Fig. 4.3). Theaverage yield of the compound was 5 mg per 25 g of seed. The melting pointwas 138°C. The purity of the compound was confirmed using thin-layerchromatography (TLC) and the R fvalue was 0.47.The bioactive compound was subjected to infra red (IR), 1 H-NMR,13 C-NMR (Al-Fatimi et al., 2006) and gas chromatography–mass spectral(GC–MS) analyses (Yanez et al., 2005) for structural elucidation.The IR spectrum showed an absorption band in the region of 1652.9 cm –1for C–O-stretching. Further absorption bands at 1550.7 cm –1 and 1454 cm –1were due to the presence of a coumarin ring oxygen and a furan ring oxygen,respectively.In 1 H-NMR spectra, the signal due to C 3–H and C 4–H of coumarinappeared at δ 6.41 as a doublet and at δ 7.81 as a singlet. The aromatic protonsFig. 4.3. Stereo micrograph of the active compound [2H-furo[2,3-H]-1-benzopyran-2-one] isolated from seeds of P. corylifolia, showing transparent, colourless, rectangular,prism-shaped crystals.

Antimicrobials to Prevent Biodeterioration of Grains 99(C 7+ C 8) are mingled together and appeared at δ 7.7 as a multiplet. Thesignal due to the C 3’ and C 2protons appeared at 6.84 and 7.49 as a doublet,respectively.13 C-NMR data of the bioactive compound showed peaks at δ 146.85(C 2),143.8 (C 4), 119.77(C 3), 115.5(C 7), 114.76(C 8), 2 106.3(C 3’), and 99.86 (C 2’), whichare inconsistent with structure.GC–MS analysis showed a molecular ion peak at M/z 186.17 consistentwith the molecular formula C 11H 6O 3. The peak at M/z 158 was due to theformation of the coumarin cation. The recorded chromatogram of the plotmatched with the chromatogram of an already known compound, 2H-furo[2,3-H]-1-benzopyran-2-one. Figure 4.4 presents the molecular structure of thebioactive compound.In vitro evaluation of the antifungal active componentTen species of Aspergillus, viz. A. flavus, A. niger, A. terreus, A. tamarii, A. flavusoryzae, A. fumigatus, A. candidus, A. ochraceous, A. flavipes and A. flavus columnaris,and two species of Penicillium, viz., P. chrysogenum and P. notatum, isolatedfrom maize seeds and known to cause biodeterioration of grains served as testfungi for the antifungal activity assay using the poisoned food technique.The poisoned food technique is as follows. Malt extract salt agar (MESA)medium amended with different concentrations of the bioactive compoundwere prepared and poured into sterile Petri plates and allowed to cool andsolidify. Mycelium discs (5 mm diameter) of 7-day-old cultures of species ofAspergillus and Penicillium were placed at the centre of the plates and incubatedat 25 ± 1°C for 7 days. The MESA medium without the bioactive compoundserved as a control. The colony diameter was measured. Similarly the fungicidesCaptan (C 9H 8Cl 3NO 2S) and Thiram (C 6H 12N 2S 4) were also tested againstall the test fungi at the recommended dose of 2000 ppm concentration for comparativeevaluation. The percentage inhibition of mycelial growth if any wasdetermined by the formula PI = C – T/C × 100; where C is the diameter of controlcolony and T is the diameter of treated colony (Pinto et al., 1998). MICs foreach of the test fungi were determined on the basis of the concentration neededto inhibit totally the test fungi (Fig. 4.5).The total inhibition of A. flavus was observed at 100 ppm. A. niger andA. fumigatus were totally inhibited at 500 and 600 ppm, respectively. A. flavusoryzae and A. flavus columnaris were totally inhibited at 700 ppm. A. ochraceousand A. flavipes were totally inhibited at 900 ppm. Total inhibition ofOOOFig. 4.4. Molecular structure of the bioactive compound, 2H-furo [2,3-H]-1-benzopyran-2-one, isolated from seeds of P. corylifolia.

100 K.A. RaveeshaConcentration of the bioactivecompound (ppm)10009008007006005004003002001000Aspergillus flavusA. nigerA. tamariiA. fumigatusA. ochraceousA. flavipesFungiA. terreusA. flavus oryzaeA. candidusA. flavus columnarisPenicillium notatumP. chrysogenumFig. 4.5. Minimal inhibitory concentration (MIC) of the bioactive compound [2H-furo[2,3-H]-1-benzopyran-2-one] isolated from seeds of P. corylifolia L. against species of Aspergillusand Penicillium.A. tamarii, A. terreus and A. candidus was not observed even at 1000 ppm.Penicillium notatum and P. chrysogenum were totally inhibited at 800 ppm and900 ppm concentration, respectively.The concentration of the bioactive compound needed for total inhibitionof Aspergillus and Penicillium species was much lower than the recommendeddose of the test fungicides.In vivo evaluation of the active component to prevent the biodeterioration ofmaizeMaize grains naturally infected with diverse species of Aspergillus and Penicilliumwere treated with 250, 500, 1000 and 1500 ppm concentration of thebioactive compound. Untreated seeds served as a control. The treated anduntreated seeds were stored at room temperature (30 ± 2°C) for 4 months andthe moisture content of the seed sample was maintained at 13.5% ( Janar dhanaet al., 1999). Samples were drawn at regular intervals of 30, 60, 90 and 120days from each treatment and subjected to seed mycoflora analysis employingthe standard method (ISTA, 1999), determination of protein content(Lowry et al., 1951) and carbohydrate content (Dubios et al., 1956) to assessthe level of biodeterioration.Results revealed a significant reduction in the seed mycoflora in all thetreatments with a total elimination of seed-borne fungi including species ofAspergillus and Penicillium in the grains treated with 1000 ppm and 1500 ppmconcentration of the bioactive compound in all the storage periods tested. No

Antimicrobials to Prevent Biodeterioration of Grains 101change in protein and carbohydrate content was observed in the grainstreated with 1000 and 1500 ppm even after 4 months of storage, suggestingthat the active compound, 2H-furo[2,3-H]-1- benzopyran-2-one isolated fromseeds of P. corylifolia L. could be exploited to prevent the biodeterioration ofgrains.Potential of 2-hydroxy-4-methoxybenzaldehyde isolated from Decalepishamiltonii Wight & Arn. to prevent biodeterioration of paddyDecalepis hamiltonii Wight & Arn., a member of the family Asclepiadaceae, isan important medicinal plant widely used in traditional medicine. Therhizome (Fig. 4.6) of this plant is largely used in South India for pickling.An antifungal activity assay of different solvent extracts of the freshrhizome of this plant revealed highly significant activity in the petroleumether extract against seed-borne fungal pathogens (Mohana et al., 2006). Furtherexperimentation conducted to isolate and characterize the antifungalactive principle from the petroleum phenolic fraction using chloroform as aFig. 4.6. Rhizome of Decalepis hamiltonii Wight & Arn.

102 K.A. Raveeshasolvent for TLC revealed the presence of seven bands. An antifungal activityassay of each of these bands revealed that band five with an R fvalue of 0.77showed significant antifungal activity, whereas the other bands did not showany antifungal activity. Light blue fluorescence at 365 nm was observed.The active principle was isolated and subjected to 1 H-NMR, 13 C-NMRand MS analysis to confirm the identity of the compound. The 1 H-NMRanalysis of the compound showed NMR peaks at δ 3.85 (s,-0CH3), 6.52 (dd,J = 2 HZ; 3-H), 6.55(d, J = 7 Hz, 5-H), 7.40 (d, J = 7 Hz; 6-H), 9.70 (s, CHO), 11.6(s,-OH) functional groups. 13 C-NMR analysis of the compounds showedeight carbon signals 135.6 (1-CH), 108.7 (3-CH), 167.2 (C of carbonyl), 101.05(5-CH), 164.8(2-C), 115.5(C), 194.7 (6-CH), and 56.09 (CH3) and its identitywas confirmed by MS analysis [m/z (% abundance): 57(48), 95(46), 108(24),121(20), 151(100), 152(70)]. The strong molecular ion peak (m/z, 152) andstronger M-1 ion peak (m/z, 151) observed were characteristic of an aromaticaldehyde. The melting point of the active compound is 46°C. Resultsrevealed that the active compound responsible for the activity was2-hydroxy-4-methoxybenzaldehyde (Fig. 4.7), reported in the literature byNagaraju et al. (2001).The active component isolated was subjected to an in vitro antifungalactivity assay by the poisoned food technique and in vivo experiments toevaluate the potential of this active compound to prevent the biodeteriorationof paddy during storage.An in vitro antifungal activity assay against important seed-borne fungiassociated with paddy known to cause biodeterioration during storagerevealed a highly significant inhibitory activity against the test fungi. Completeinhibition of the test fungi was observed at 650 μg/ml of the compound(Table 4.1). Further in vivo experimentation done to assess theefficacy of the active principle to prevent biodeterioration of paddy up to90 days of storage revealed a high potency of the compound to preventbiodeterioration of grains. The percentage incidence of seed-borne fungalspecies decreased significantly in the samples treated with 1 g/kg of theactive compound. Comparative efficacy studies revealed that theconcentration of the active compound needed to prevent growth of fungiresponsible for biodeterioration of paddy was much less than that of thiram(2 g/kg) (Table 4.2).C H OO HO C H 3Fig. 4.7. Molecular structure of the bioactive compound, 2-hydroxy-4-methoxybenzaldehydeisolated from the rhizome of Decalepis hamiltonii Wight & Arn.

Antimicrobials to Prevent Biodeterioration of Grains 103Table 4.1. The antifungal activity of 2-hydroxy-4-methoxybenzaldehyde isolated from D. hamiltonii and that of thiram againstphytopathogenic fungi isolated from paddy.Concentration ofactive compound(µg/ml)AlternariaalternataMycelium growth inhibition (%) of seed-borne pathogenic fungi of paddyDrechsleratetrameraFusariumoxysporumFusariumproliferatumPyriculariaoryzaeTrichoconispadwickii40 3.25 ± 0.3 3.34 ± 0.2 11.47 ± 0.3 12.72 ± 0.4 0.00 ± 0.0 2.10 ± 0.560 8.09 ± 0.5 6.6 ± 0.6 24.33 ± 0.3 23.35 ± 0.1 5.66 ± 0.4 3.61 ± 0.580 11.33 ± 0.4 19.72 ± 0.4 39.31 ± 0.4 42.30 ± 0.4 10.34 ± 0.2 8.84 ± 0.610 16.71 ± 0.5 24.20 ± 0.6 53.05 ± 0.3 51.63 ± 0.2 14.36 ± 0.4 11.88 ± 0.4100 21.82 ± 0.7 34.60 ± 0.5 56.90 ± 0.5 57.58 ± 0.4 17.77 ± 0.4 12.64 ± 0.5150 34.34 ± 0.7 45.89 ± 0.6 68.53 ± 0.3 68.06 ± 0.5 22.64 ± 0.2 24.67 ± 0.6200 43.07 ± 0.6 67.10 ± 0.3 71.26 ± 1.2 79.06 ± 0.3 26.60 ± 0.3 26.55 ± 0.5250 59.94 ± 0.7 86.69 ± 0.4 83.14 ± 0.4 86.21 ± 0.3 42.76 ± 0.7 31.41 ± 0.7300 64.78 ± 0.7 91.35 ± 0.5 90.07 ± 0.4 92.78 ± 0.6 56.57 ± 0.6 37.18 ± 0.5350 76.14 ± 0.5 100.0 ± 0.0 98.33 ± 0.7 100.0 ± 0.0 63.54 ± 2.3 41.75 ± 1.0400 91.49 ± 1.1 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 67.94 ± 0.4 49.56 ± 0.5450 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 78.19 ± 0.5 65.00 ± 0.6500 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 85.88 ± 0.7 76.19 ± 0.6550 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 89.79 ± 0.6 90.08 ± 0.8600 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 97.57 ± 0.3 100.0 ± 0.0650 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0Thiram 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0 100.0 ± 0.0Data given are mean of four replicates ± standard error.Analysis of variance (ANOVA) d.f. = 15 at P < 0.0001.

104 K.A. RaveeshaTable 4.2. Effect of 2-hydroxy-4-methoxy-benzaldehyde isolated from D. hamiltonii and thiram on seed-borne fungi of paddy storedup to 90 days.Incidence of seed-borne fungal species in paddy seeds (%)Untreated 0.5 g/kg active compound 1 g/kg active compound Thiram (2 g/kg)Storageperiods (days) 0 30 60 90 0 30 60 90 0 30 60 90 0 30 60 90Alternariaspp.Aspergillusspp.Curvulariaspp.Drechsleraspp.Fusariumspp.Penicilliumspp.Pyriculariaspp.TrichoconispadwickiiTrichotheciumspp.22 ± 0.5 26 ± 0.5 28 ± 0.3 27 ± 0.8 4 ± 0.5 12 ± 0.6 14 ± 0.6 19 ± 0.6 0.0 0.0 7 ± 0.4 11 ± 0.7 0.0 0.0 8 ± 0.6 11 ± 0.742 ± 0.8 51 ± 1.2 64 ± 0.5 72 ± 0.4 9 ± 0.3 14 ± 0.3 26 ± 0.4 35 ± 0.5 0.0 5 ± 0.2 12 ± 0.6 17 ± 1.1 0.0 5 ± 0.6 23 ± 0.5 32 ± 0.528 ± 0.6 27 ± 0.5 31 ± 0.5 39 ± 0.8 0.0 8 ± 0.8 13 ± 0.3 18 ± 0.8 0.0 0.0 4 ± 0.3 6 ± 0.4 0.0 0.0 0.0 3 ± 0.341 ± 0.8 40 ± 0.8 44 ± 0.4 48 ± 0.7 0.0 5 ± 0.5 9 ± 0.5 14 ± 0.8 0.0 0.0 0.0 4 ± 0.3 0.0 0.0 0.0 1 ± 0.236 ± 0.3 42 ± 0.7 49 ± 0.9 54 ± 0.8 0.0 6 ± 0.3 10 ± 0.5 15 ± 0.8 0.0 0.0 6 ± 0.3 13 ± 0.6 0.0 0.0 3 ± 0.4 8 ± 0.521 ± 0.4 34 ± 0.8 42 ± 0.5 58 ± 0.9 4 ± 0.3 11 ± 0.5 19 ± 0.4 28 ± 0.5 0.0 0.0 8 ± 0.3 18 ± 0.5 0.0 8 ± 0.6 15 ± 0.6 22 ± 0.522 ± 0.5 26 ± 0.8 27 ± 0.8 25 ± 0.4 0.0 6 ± 0.5 8 ± 0.8 12 ± 0.5 0.0 0.0 2 ± 0.9 4 ± 0.8 0.0 0.0 0.0 2 ± 0.323 ± 0.7 25 ± 0.5 21 ± 0.8 19 ± 0.5 0.0 0.0 4 ± 0.5 8 ± 0.7 0.0 0.0 0.0 2 ± 0.3 0.0 0.0 0.0 1 ± 0.613 ± 0.4 16 ± 0.8 21 ± 0.5 26 ± 0.5 0.0 0.0 5 ± 0.3 10 ± 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0Per cent incidence is based on four replicates with 100 seeds each, F = 321.64; P < 0.001.

Antimicrobials to Prevent Biodeterioration of Grains 105The evaluation of the bioactive compound to understand the impact on thenutritional quality of the paddy grains stored up to 90 days revealed that1 g/kg treatment is effective in preventing biodeterioration of paddy grainswithout affecting the nutritional quality in terms of dry matter loss, lipid, carbohydrateand protein content (Fig. 4.8a–d), suggesting that 2-hydroxy-4-methoxybenzaldehydeis an important antimicrobial of plant origin that could be exploitedto prevent biodeterioration of grains during storage (Mohana et al., 2009).Even though a few antimicrobial active components have been isolatedand characterized by earlier workers, evaluations of these to assess their potencyto prevent biodeterioration of grains are lacking. The active compounds isolatedfrom the seeds of P. corylifolia L. and from the rhizome of D. hamiltoniiWight & Arn. have been successively evaluated to assess their potency to(a)Carbohydrate content (g/g)0.700.680.660.640.620.600.580.560.540.52Changes in total carbohydrates0 30 60 90Storage period (days)(b)Protein content (mg/g)4.34.24.14.03.93.83.70Changes in proteins30 60 90Storage period (days)Control0.5 gm/kgControl0.5 gm/kg1 gm/kgThiram1.0 gm/kgThiram(c)Lipid contents (mg/g)4035302520151050Changes in lipids0 30 60 90Storage period (days)(d)Dry matter loss (%)65432100Dry matter loss30 60 90Storage period (days)Control0.5 g/kgControl0.5 g/kg1 g/kgThiram1 g/kgThiramFig. 4.8. (a–d) Comparative effi cacy of the bioactive compound 2-hydroxy-4-methoxybenzaldehydeisolated from D. hamiltonii (0.5 g/kg and 1 g/kg) and thiram (2 g/kg) on fungiinducing nutritional losses in paddy grains stored up to 90 days.

106 K.A. Raveeshaprevent the biodeterioration of grains during storage. D. hamiltonii is an importantedible plant and thus has an additional advantage as a herbal, eco-friendlyremedy for the prevention of grain biodeterioration. Further investigations onthe standardization of methods for high yield of the active compounds, toxicologicalaspects, development of formulations, treatment procedures and a packageof practices to be adapted may pave the way for commercial exploitation.4.5 ConclusionPlants have been over exploited for human health needs while underexploited for plant health needs. One of the possible reasons for this may bethe high-input cost that could be made good in human health care, while itmay be difficult in plant health care. Considering the cost of environmentalpollution and other related damages caused by the use of chemical pesticidesin crop protection, the development of eco-friendly natural pesticides is anecessity. Screening plants for antimicrobial activity against phytopathogensin general and against biodeterioration-causing fungi in particular is the firststep towards this goal. Subsequent to demonstrating the science behind theantimicrobial activity, research needs to be focused on developing an appropriate,cost-effective package of practices employing biotechnologicalapproaches. Some of the possible approaches to decreasing the cost of productionare: (i) identify the gene responsible for the production of the activecomponent and considerably enhance the yield of the active componentthrough bioengineering; (ii) isolate and identify the endophytes, if any, associatedwith the production of the active compound and develop fermentativemethods for the large-scale production of the active component; (iii) developa package of practice, preferably utilizing the edible plant part pieces orpowder directly as an amendment in an appropriate quantity during grainstorage; and (iv) the chemical synthesis of the active compound.ReferencesAladesanmi, A.J. and Odediran, S.A. (2000)Antimicrobial activity of Trichilia heudelottileaves. Fitoterapia 71, 179–182.Al-Fatimi, M.A.A., Julich, W.D., Jansen, R.and Lindequist, U. (2006) Bioactive componentsof the traditionally used MushroomsPodaxis pistillaris. Evidence. BasedComplementary and Alternative Medicine 3,87–92.Bouamama, H., Noel, T., Villard, J., Benharref,A. and Jana, M. (2006) Antimicrobialactivities of the leaf extracts of twoMoroccan Cistus L. species. Journal of Ethnopharmacology104, 104–107.Carpano, S.M., Spegazzini, E.D., Rossi, J.S.,Castro, M.T. and Debenedetti, S.L. (2003)Antifungal activity of Terminalia australis.Fitoterapia 74, 294–297.Chowdhury, R., Hasan, C.M. and Rashid, M.A.(2003) Antimicrobial activity of Toonaciliata and Amoora rohituka. Fitoterapia74, 155–158.Cowan, M.M. (1999) Plant products as antimicrobialagents. Clinical MicrobiologyReviews 12, 564–582.Doughari, J.H. (2006) Antimicrobial activityof Tamarindus indica Linn. Tropical Journalof Pharmaceutical Research 5, 592–603.

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5 Some Natural Proteinaceousand Polyketide Compoundsin Plant Protection andtheir Potential in GreenConsumerizationL.A. SHCHERBAKOVARussian Research Institute of Phytopathology, Moscow, RussiaAbstractPlant protection from diseases with the use of natural compounds, which areindigenous and biodegradable in the environment, fits the purpose of green consumerization.Natural compounds controlling plant pathogens belong to different chemicalclasses and are produced by a wide range of organisms. The structural andfunctional diversity of these compounds provides a great potential in crop protectiontechnologies in correspondence with green consumerization objectives. Disease preventionby natural compounds results from either direct or plant-mediated influenceson targeted pathogens. Some natural substances affecting causative agents directlydo not produce a biocidal effect but specifically attack pathways related to theirpathogenicity. Biogenic compounds influencing plants induce resistance or enhancetolerance to diseases. They elicit natural defence responses in plants and, unlikechemical pesticides, do not promote the occurrence of resistant forms in targetedpathogens. Two bacterial proteins, CspD and MF3, that have elicitor properties havebeen well documented to provide resistance against various phytopathogens andplay a promising role in green consumerization. Fungal polyketides, statins, inhibitinga pathogenicity-related pathway in melanin-producing fungi are considered asprospective candidates for developing biopesticides and reducing the impact of plantdiseases without killing pathogenic microorganisms.5.1 IntroductionPlant diseases continue to cause considerable damage to global agricultureresulting in yield losses and deterioration of agricultural products, includingtheir contamination with hazardous substances, e.g. mycotoxins, whichinduce toxicological problems in people and animals. Despite amazing progressachieved in crop protection due to breeding for resistance to diseases© CAB International 2011. Natural Products in Plant Pest Management(ed. N.K. Dubey) 109

110 L.A. Shcherbakovaand availability of effective agrochemicals, a number of problems are facedin managing the diseases successfully. The resistant cultivars provide onlypartial plant protection from a disease because of the occurrence of newpathogenic races that are able to overcome the resistance. Sometimes, breedingfor resistance is too complicated because of the diversity and high variabilityof plant pathogens and the variety of defence responses at differentstages of pathogenesis and plant development.Chemical protection gives a powerful tool for effective control of manydisease-causing agents, but its implementation has a range of undesirableside impacts. Thus, the use of chemical pesticides is associated with environmentalrisks, such as pollution of soil, water and crop plants with xenobioticpesticide residues, killing beneficial microorganisms or insects,thereby interfering with the natural management of pests and pathogens,a reduction in biological diversity, and so on. Sometimes, chemical pesticidesare not always effective and many treatments with several fungicidesare needed to control some fungal pathogens. As a result of continualpesticide application on plants and stored agricultural products, toxic residuesof chemical pesticides may appear and accumulate in food. Lastly,perhaps the most disturbing consequence of chemical protection is thedevelopment of pesticide resistance in pathogens that makes a protectiveeffect changeable and insufficiently effective. The health and ecologicalproblems related to chemical pesticides are now receiving a great deal ofattention. Recognition of these problems has created the phenomenon ofgreen consumerization.The biocontrol of phytopathogens as an additional or alternative plantprotection method looks more natural and environmentally compatible thanchemical treatments. The use of non-pathogenic or beneficial microorganismsand/or natural compounds, which are native and biodegradable in theenvironment, is the main objective of green consumerization. A few hundredbiocontrol products based on numerous beneficial strains predominantlyof Trichoderma, Bacullis, Pseudomonas, Agrobacterium, Streptomyces andGliocladium species are produced, commercialized and used as biopesticidesand/or biofertilizers.Natural compounds effectively controlling plant pathogens and pestsare represented by a diversity of substances belonging to different chemicalclasses and produced by a wide range of living organisms (Varma and Dubey,1999). Currently, the list of these natural compounds is growing longer fromyear to year. Many natural products have already been used or recommendedas biopesticides (e.g. various secondary plant metabolites such pyrethrins,glucosinolate-sinigrin-producing allyl isothiocyanate, carvone, azadirachtin,rotenone, ryania, nicotine, some essential oils and other volatile plant substances).The plant-mediated mode of action of some natural compounds,which does not establish contact with pathogens, decreases the risk of resistantpathogenic form selection. Because the search for natural substances fittingphytosanitary regulations and environmentally compatible croppingmethods is a continuous active process, novel compounds are being discoveredin plants, animals and microorganisms and are marketed in place of

Proteinaceous and Polyketide Compounds in Plant Protection 111chemical pesticides in order to be adopted by so-called ‘cottage industry’ orgovernmentally regulated farming.The disease-preventing effect of natural compounds that are used or canbe used for crop protection is a result of a direct or plant-mediated influenceon targeted pathogens. Affecting phytopathogens directly, the compoundsinterrupt pivotal metabolic pathways and/or disrupt vitally important structuresthat have as a consequence the death of the pathogens (biocidal effect).Some natural substances or their analogues do not produce lethal effects toplant pathogens but specifically influence pathways related to pathogenicityor/and toxigenesis, e.g. mycotoxin production (Kumar et al., 2008; Shuklaet al., 2008). Such non-biocidal suppressors can impair the pathogenicity forplants as well as the toxic risk for people and animals. In the case of aplant-mediated mode of action, natural compounds elicit and activatedefence responses in plants that result in induced resistance to diseases (e.g.Shcherbakova et al., 2007; 2008; Odintsova et al., 2009). Lastly, some of biogeniccompounds influencing plants do not induce resistance but enhancetolerance to pathogens (sometimes by improving the physiological state ofplants). The structural and functional diversity of natural compounds as wellas their abundance provide a great potential for bringing plant protectiontechnologies in line with green consumerization.The main goal of this chapter is to analyse properties of a few chemicallyand functionally different natural compounds and demonstrate their prospectiveas promising candidates for new plant protection products with significantpotential in relation to green consumerization. The chapter will focuson the characterization of two proteinaceous compounds of bacterial origin,the elicitor properties of which provide disease resistance, and also on fungalstatins as natural compounds, which inhibit a pathogenicity-related pathwayin melanin-producing fungi and can be used for developing biopesticidesthat reduce the harmful impact of crop diseases without killingpathogenic organisms. In addition, microbial and plant proteinaceous compoundsthat have been previously well documented to be promising forgreen consumerization will be briefly surveyed.5.2 Proteinaceous Compounds of Microbial and Plant Origin inthe Biocontrol of Plant PathogensProteins are abundant and ubiquitous natural compounds strongly associatedwith all living organisms, hence it is possible to believe their involvementin plant protection will not conflict with green consumerizationprinciples. Because proteinaceous compounds are important constituents ofa plant immune system and take part in the natural plant defence againstpathogens, scientists have emphasized their potential for plant protection fora long time. Basically, two general approaches to the practical realization ofthe protective potential of these compounds are the formulation of producers(biocontrol or non-pathogenic microorganisms) or the engineering of transgenicplants. The compounds synthesized by formulated biocontrol agents

112 L.A. Shcherbakovainteract with pathogenic microflora or induce resistance in plants. Constitutiveor inducible expression of protein-encoding genes transferred into plantsconfer them resistance to many economically important diseases.Several groups of microbial and plant proteinaceous substances thathave well-established potentialities in plant protection will be very brieflyreviewed below. The antipathogenic properties of two proteins, which wereisolated from biocontrol bacteria and relatively recently studied as putativeelicitors of disease resistance in plants, will be described in more detail.The biocontrol potential of proteinaceous compounds will be consideredalmost exclusively by using examples of fungal and bacterial pathogens.Their antipest properties will be not covered within the scope of this chapter.Nevertheless, it should be emphasized that many proteins and peptides discussedin this section (microbial peptides, defensins, proteinase inhibitorsand lectins) are well known as biological insecticides or nematicides of greatpotentiality. Biopesticides based on microbial peptides or transgenic plantscarrying genes of defensins, lectins and proteinase inhibitors are good toolsfor the fight against pests of plants.Antimicrobial peptides produced by microorganisms and plantsThe generation of antimicrobial peptides is one of the widespread naturaldefence mechanisms of innate immunity of living organisms, and these compoundsare interesting targets for green consumerization. Plants, includingagricultural crops, produce numerous defensive peptides. Microorganisms,including those used in plant protection, synthesize and excrete antimicrobialpeptides that are often responsible for the biocontrol effect. In severalauthors’ opinions, peptides have advantages over more elementary organicantimicrobial compounds when used for plant protection goals. Because thepeptide molecules contain from 10 to 50 (sometimes up to 85) amino acidresidues, they can more specifically interact with their protein targets incausative agents (Park et al., 2009). Biopesticidal peptides can work againstpathogens by inhibiting nucleic-acid and protein biosyntheses and enzymeactivity or by interacting with the plasmalemma and destroying its integrity(Huang, 2000).Antimicrobial peptides from biocontrol microorganismsPeptides possessing antimicrobial effects have been reported to be producedby bacteria, fungi, plants, invertebrates and vertebrates (Garcia-Olmedo et al., 1998; Zasloff, 2002; Degenkolb et al., 2003; Bulet et al., 2004;Nybroe and Sorensen, 2004; Carvalho and Gomes, 2009). According to therecent review focused on antimicrobial peptides related to plant-diseasecontrol (Montesinos, 2007), about 900 antimicrobial peptides are producedby living organisms via ribosomal or non-ribosomal synthesis. Biocontrolmicroorganisms have been reported to use both types of the peptide synthesis(Finking and Marahiel, 2004; Montesinos, 2007) and generate a widerange of these compounds. Antimicrobial peptides of microorganisms

Proteinaceous and Polyketide Compounds in Plant Protection 113(AMPM) have been classified into several groups of linear and cyclicpeptides (fungal defensins, bacteriocins, peptaibols, cyclopeptides andpseudopeptides) based on their most essential structural characteristics,and have been inventoried according to name, composition and producermicroorganisms (Montesinos, 2007).The efficacy of the natural antimicrobial peptides produced by biocontrolmicroorganisms towards various plant pathogenic bacteria, fungi andoomycetes in vitro and in vivo has been well documented (Table 5.1).Table 5.1. Instances of plant pathogens sensitive to antimicrobial peptides of microorganisms.Targetedplant pathogens Group of effective AMPM ReferenceFungiAlternaria spp. Pseudopeptides Stein, 2005Botrytis cinereaCyclopeptidesFungal defensinsPeptaibolsPseudopeptidesLavermicocca et al., 1997Ongena et al., 2005Vila et al., 2001Moreno et al., 2003Moreno et al., 2005Xiao-Yan et al., 2006Stein, 2005Bipolaris sorokiniana Peptaibols Xiao-Yan et al., 2006Colletotrichum spp. Peptaibols Xiao-Yan et al., 2006Erysiphe sp. Cyclopeptides Selim et al., 2005Fusarium avenaceum Cyclopeptides Selim et al., 2005Fusarium oxysporum Peptaibols Xiao-Yan et al., 2006Ongena et al., 2005Fusarium solani Cyclopeptides Stein, 2005Fusarium spp. Fungal defensins Vila et al., 2001Moreno et al., 2003Moreno et al., 2005Leptosphaeria maculans Cyclopeptides Pedras et al., 2003Magnaporthe griseaFungal defensinsPseudopeptidesMoreno et al., 2003Moreno et al., 2005Stein, 2005Monilina fructicola Cyclopeptides Gueldner et al., 1988Podosphaera fuca Cyclopeptides Romero et al., 2007Podosphaera sp. Cyclopeptides Stein, 2005Rhizoctonia solaniCyclopeptidesPeptaibolsPseudopeptidesAsaka and Shoda, 1996Bassarello et al., 2004Nielsen and Sorensen, 2003Ongena et al., 2005Xiao-Yan et al., 2006Stein, 2005Sclerotinia sclerotium Cyclopeptides Pedras et al., 2003Sclerotium cepivarum Peptaibols Gouland et al., 1995Continued

114 L.A. ShcherbakovaTable 5.1. Continued.Targetedplant pathogens Group of effective AMPM ReferenceSphaerotheca sp. Pseudopeptides Stein, 2005Uncinula nector Pseudopeptides Stein, 2005Ventura inaequalis Cyclopeptides Burr et al., 1996Phytophthora infestans Cyclopeptides De Bruijn et al., 2007Pythium intermedium Cyclopeptides De Souza et al., 2003Pythium ultimum Cyclopeptides Nielsen et al., 2002BacteriaErwinia amylovora Pseudopeptides Brady et al., 1999Jin et al., 2003Erwinia caratovora Cyclopeptides Selim, 2005Clavibacter michiganesis Peptaibols Xiao-Yan et al., 2006Pseudomonas syringae Cyclopeptides Bais et al., 2004Rhodococcus fascians Cyclopeptides Bassarello et al., 2004Antimicrobial peptides from plantsA broad family comprising antimicrobial peptides produced by plants iscalled defensins. One of their functions in plants is the involvement in defencemechanisms against pathogens, pests and abiotic stresses (Terras et al., 1995;Lay and Anderson, 2005). These are basic peptides structurally and functionallyrelated to the defensins of mammalia and insects. Molecular masses ofplant defensins that are characterized at present range from 5 to 7 kDa. Theirprimary structure is formed with 45–55 amino acid residues. Two structuralcharacteristics of the defensins, the presence of a pattern of conserved cysteineresidues and a domain with extremely variable amino acid sequence, aretypical components for mature peptide molecules of plant defensins. Withrare exceptions, defensins found in plants have a similar globular spatialstructure that is stabilized by a structural motif formed using disulfide bondsbetween eight cysteine residues. Possibly, defensins can aggregate in vivointo dimers or oligomers (Terras et al., 1992). A certain correlation is observedbetween the primary structure and antimicrobial activity of some plantdefensins. In general, the incorporation of basic amino acids (viz. arginine)that add positive charge to a peptide molecule resulted in a considerableincrease in the antimicrobial activity (Terras et al., 1992; De Samblanx et al.,1997; Landon et al., 2000).In the overwhelming majority of cases, plant defensins possess inhibitoryactivity against fungi; however, the growth of Gram-negative bacteria isalso arrested after exposure to these peptides (Terras et al., 1993; Segura et al,1998; Wong et al, 2006). An analysis of publications by Carvalho and Gomes(2009) showed many damaging plant pathogens of economically importantcrops can be inhibited in vitro with plant defensins. Various fungal species(Alternaria brassicola, Alternaria solani, Botrytis cinerea, Cladosporium colocasiae,

Proteinaceous and Polyketide Compounds in Plant Protection 115Cladosporium sphaerospermum, Colletotrichum lindemuthianum, Diploidia maydis,Magnaporthe grisea, Mycosphaerella arachidicola, Mycosphaerella fijinesis,Nectria haematococca, Penicillium digitatum, Penicillium expansum, Phaeoisariopsispersonata, Physalospora piricola, Rhizoctonia solani, Septoria tritici, Verticiliumalbo-atrum, V. dahliae, and the toxigenic species Fusarium culmorum, F. decemcellulare,F. graminearum, F. oxysporum, F. verticillioides and Aspergillus niger);two species of oomycetes (Phytophthora infestans and P. parasitica) and alsobacteria, viz. C. michiganensis and Ralstonia solanacearum, are among them.Effective concentrations of plant defensins differ depending on the testedpeptides and the targeted pathogens (values of IC 50, a protein concentrationthat is required for 50% growth inhibition, vary from 1–100 μg/ml). The levelof antimicrobial activity may be regulated with bivalent ions ( Terras et al.,1992; 1993; Osborn et al., 1995; Segura et al., 1998; Wong and Ng, 2005). Alongwith the growth inhibitory effect, some plant defensins cause morphologicalchanges in fungal mycelia (Carvalho and Gomes, 2009).Studies on the mode of action of antimicrobial plant defensins are inprogress. There are confirmed hypotheses that an interaction with the cellmembrane of microorganisms resulting in ion efflux and reactive oxygenspecies (ROS) generation significantly contributes to the mechanisms responsiblefor antifungal properties of plant defensins. Besides antimicrobial activity,plant defensins possess a range of biological functions (Lay and Anderson,2005; Carvalho and Gomes, 2009).The availability of antimicrobial peptides produced by plants or microorganismsthrough ribosomal synthesis for crop protection has been demonstratedby an increased disease resistance of transgenic plants expressingfungal and plant defensin genes (Montesinos, 2007; Carvalho and Gomes,2009). Several agricultural crops, e.g. tobacco, tomato, rice, aubergine, papayaand canola, which are transformed with these genes and produce the correspondingpeptides, have little or no disease development in laboratory,greenhouse or field experiments. A number of peptides produced by microorganismsare insecticidal or nematicidal. The ability of plant defensins toinhibit α-amylase and proteases can contribute to plant defence against pestinsects (Lin et al., 2007).Enzymes, proteinase inhibitors, lectins and PR proteinsLytic enzymesTo obtain nutrients, microorganisms synthesize various lytic enzymes thatcan attack polymeric compounds of different origin. Biocontrol agents canuse these enzymatic activities on plant pathogens. Microbial chitinases, glucanasesand proteases are lytic enzymes of most importance for the biocontrolof phytopathogens. These enzymes hydrolyse chitin, β-glucans andproteins, which can result in direct suppression of pathogen development orgenerate products that function as resistance inducers. For instance, biocontrolisolates of Trichoderma harzianum and Trichoderma atroviride produceendochitinase, β-1,3-glucanase and alkaline proteinase, which degrade plant

116 L.A. Shcherbakovapathogenic fungi in vitro, halt their growth in planta and play a role inmycoparasitism (Elad et al., 1982; Benitez et al., 1998; Lorito, 1998; Cherninand Chet, 2002). Oligosaccharides or chitosan derived from fungal cell wallsexposed to microbial glucanases and chinases elicit a cascade of defenceresponses in plants: generation of ROS, induction of pathogenesis-relatedproteins (PR proteins) (including plant chitinases and glucanases),phytoalexins and lignification (Dyakov and Ozeratskovskaya, 2007).The potential of lytic enzymes for plant-disease management was welldemonstrated by studying chinolytic systems in the biocontrol bacteria Bacilluscereus, Pantoea agglomerans, Pantoea dispersa, and fungi, especially in thewidely used biocontrol fungus Trichoderma. Chitinases produced by Trichodermaare effective on virtually all chitinous pathogens, non-toxic for plantsand possess higher antifungal activity than such enzymes isolated from othersources. The antifungal activity of chitinases from Trichoderma can reach thelevel of some chemical pesticides (Lorito, 1998; Bonaterra et al., 2003; Changet al., 2003; Gohel et al., 2004).Along with use of enzyme-producing biocontrol agents, there are severalother application strategies for chinolytic enzymes. The most conventionalapproach consists of conferring resistance via engineering transgenic plantscontaining heterologous chitinase and glucanase genes. Overexpression ofthese genes in response to pathogen invasion can cause higher levels of theenzymes in the plant cells followed by a faster and effective neutralization ofthe pathogen. Indeed, transgenic broccoli, potato and tobacco plants expressingthe T. harzianum endochitinase gene have been found to show resistanceagainst A. alternata, A. solani, B. cinerea and R. solani. Transgenic tobacco andcabbage, carrying a bean chitinase gene were protected against R. solani.Transgenic cucumber, rice, grapevine, strawberry and wheat transformedwith chitinase genes from rice (Oryza sativa) were resistant to B. cinerea,R. solani, M. grisea, Sphaerotheca humuli and F. graminearum, respectively(Gohel et al., 2006). Expression of exochitinase genes in transgenic apple treesconfers resistance to apple scab (Venturia inaequalis), a pathogen which is controlledby multiple applications of chemical fungicides during the growingseason (Bolar et al., 2000). These results show the broad potential for themicrobial chitinase transgenesis into plants for controlling fungal phytopathogens.The additional strategies are related to fermentation and differentways of improving the enzyme producers (Gohel et al., 2006).Other cases suggesting feasibility of crop protection with enzymes canbe illustrated by the examples of constructing transgenic potato carryingglucose oxidase gene from A. niger or apple, potato and tobacco plantsexpressing the bacteriophage T4 lysozyme gene (Wu et al., 1997). Glucoseoxidase is an enzyme involved in generating plant ROS. Expression of theglucose oxidase gene led to accumulation of peroxide ions in plant tissuesthat increased resistance to fungal diseases, e.g. to late blight (P. infestans),wilt (Verticillium dahliae) and early blight (A. solani). Lysozymes are widespreadenzymes that hydrolyse peptidoglycan of bacterial cell walls. Appleplants with the T4L gene showed significant resistance to the fire blight agentE. amylovora (Ko et al., 2000), while potato and tobacco was resistant to

Proteinaceous and Polyketide Compounds in Plant Protection 117E. carotovora subsp. carotovora (During et al., 1993). However, lysozyme excretioncan have adverse effect on soil microbiota. Thus, the growth of B. subtilishas been observed to be suppressed in rhizosphere transgenic T4 lysozymeproducingpotato plants (Ahrenholtz et al., 2000).LectinsPlant lectins are a heterogeneous collective of proteins that specifically bindcarbohydrates in a reversible way and take part in phytopathogen recognition.Interacting with like components, lectins can attach to the cell surface. Thestructure and functions of these compounds are discussed in detail (e.g.Chrispeels and Raikhel, 1991). Lectins are well known naturally occurringinsecticides of widespread effect. Some free lectins strongly affect microbegrowth in plants and probably contribute to inhibiting pathogenesis. There arechitin-specific lectins synthesized in the phloem and translocated via vessels.These findings suggest that lectins are potential antifungal agents.Proteinase (protease) inhibitors of plant originPlant inhibitors of proteinases are a large group of peptides or small proteinsable to bind proteolytic enzymes of different organisms with competitiveinhibition of their activity. In plants, they are abundant in seeds and storageorgans, where their content can be up to 10% of water-soluble proteins. Thesecompounds are considered as reserve proteins and regulators of protein statusor enzyme activity in plants. The proteinase inhibitors differ in substratespecificity, have various isoforms, and their oligomers can combine ordissociate with an influence on the inhibitor properties.A defensive function of proteinase inhibitors towards insects was initiallyrevealed when insects, after feeding, became inactive as a result of trypsininactivation. Protease inhibitors of plant origin were also shown to be activeagainst plant pathogenic nematodes. Since many phytopathogenic fungi andbacteria secrete extracellular proteolytic enzymes, which play an importantrole in pathogenesis (Valuyeva and Mosolov, 2004), plants use inhibition ofsuch enzymes as a defence strategy towards these microorganisms (Ryan,1990; Habib and Khalid, 2007).There are now ample data on protease inhibitors effective against phytopathogensin vitro and in vivo. For instance, inhibitors from potato inactivateproteinases secreted by F. solani or F. sambucinum into cultural liquid. Inhibitorsfrom buckwheat and pearl millet suppress spore germination and thegrowth of many fungi including Aspergillus flavus, Aspergillus parasiticus,F. moniliforme, F. oxysporum, A. alternata and Trichoderma reesei. Inhibitors ofproteinases are accumulated in response to pathogen invasion and preventdisease development (e.g. in tomato inoculated with P. infestans). In somecases, correlation between disease resistance and the constitutive inhibitorsis found (e.g. between wheat resistance to smut, or lupine and soybean tofusarial wilt). Cells of potato tubers treated with elicitors, such as salicylic orarachidonic acids, are able to excrete potatin and three chymotrypsininhibitors (Habib and Khalid, 2007).

118 L.A. ShcherbakovaAt least 14 genes encoding different protease inhibitors alone or incombination with other heterologous genes have been reported to be transferredinto cultured plants, which showed increased resistance predominantlyto insects (Valuyeva and Mosolov, 2004). Plant protease inhibitorshave also been used to engineer resistance against viruses in transgenicplants. For example, expression of the gene encoding the cysteine proteinaseinhibitor from rice, oryzacystatin, by transgenic tobacco was found to conferresistance against tobacco etch virus and potato Y virus, replication of whichdepends on cysteine proteinase activity (Valuyeva and Mosolov, 2004; Habiband Khalid, 2007).In the green consumerization context, it is important that some of theavailable proteinase inhibitors are inactive for non-pathogenic microorganismsand do not inhibit activity of proteinases of animal origin. Thus, chestnutcystatin, which strongly inhibits the protease activity and the growth ofpathogenic B. cinerea, Colletotrichum graminicola, and Stagonospora nodorum,has no effect on the protease activity and the growth of the saprophyte Trichodermaviride (Pernas et al., 1999), and a proteinase inhibitor extracted frombean seeds specifically suppresses serine proteinase of pathogenic C. lindemuthianumbut does not influence animal trypsin and chymotrypsin activity(Valuyeva and Mosolov, 2004). Non-specific antipathogenic activity of proteaseinhibitors suggests that transgenic crops producing inhibitors of insecticidalor nematicidal proteinases may be incorporated into integratedsystems of plant protection against pests and pathogens. A further advantageof this approach is the possibility that inhibitory activity against proteinasescould be combined with the insecticidal activity of lectins, resulting in asynergistic antipathogenic effect.PR proteinsPlants have numerous defence mechanisms that are activated in response topathogen attacks, abiotic stresses and chemicals that mimic pathogenchallenge. Production of proteins related to pathogenesis (PR proteins) is onesuch inducible mechanism (Van Loon et al., 1994; Van Loon et al., 2006). Theseproteins are not detectable at all or are present only at basal concentrationsin healthy plant tissues. Since the term PR proteins based on the abovecharacteristics was often used for all constitutive plant proteins for whichcontent or increasing activity was induced by microorganisms, the new term‘inducible defence-related proteins’ was recently introduced (Van Loon et al.,2006).PR proteins consist of a large variety of families with members that differin occurrence, expression and biological activities; they are divided into 17classes (Sels et al., 2008). A range of the above-mentioned plant proteins andpeptides such as chitinases, β-1,3-glucanases, peroxidase, defensins and proteinaseinhibitors are PR proteins and vice versa; some typical PR proteinsare antimicrobial and inhibit pathogen growth in vitro. For instance, tobaccoPR-1a is antifungal, and tomato proteins of the PR-1 family inhibit zoosporegermination and reduce pathogenicity of P. infestans. The ability to disrupt

Proteinaceous and Polyketide Compounds in Plant Protection 119fungal membranes has been shown for toumatin-like proteins (permatins) ofthe PR-5 class. Overexpression of PR protein genes in plants renders diseaseresistance. For example, the high level expression of PR-1 in transgenictobacco plants promotes control of Perenospora tabacina and Phytophthoranicotiana (Dyakov and Ozeratskovskaya, 2007).Biochemical functions of PR proteins, their role in defence mechanisms,engineering of transgenic plants with enhanced resistance to plant pathogensand characteristics of individual PR proteins have been comprehensivelysurveyed by many researchers (Van Loon, 1985; Loon and Van Strien,1999; Van Punja, 2001; De Lucca et al., 2005; Edreva, 2005; Van Loon et al.,2006; Sels et al., 2008).Proteinic inducers of plant resistance as a promising strategy for greenconsumerizationThe protective effect of some natural compounds against plant diseasesresults from the induction of plant resistance to pathogens rather than frombiocidal activity towards the causative agents. This strategy is worthy of specialconsideration because it avoids a direct effect on a pathogen and involvesa plant-mediated mode of action. This activates the natural defence responsesof plants, minimizing the probability of the targeted pathogens developingresistance.Active defence mechanisms are initiated in plants upon recognition ofstructural and chemical characteristics particular to a pathogen, collectivelyreferred to as pathogen-associated molecular pattern (PAMPs). The PAMPcomponents represented by compounds of different chemical origin werenamed as general elicitors (or general inducers). These elicitors initiate a conservedset of plant defence responses such as ROS production, deposition ofcallose, protein phosphorylation, and transcriptional activation of earlyresponse genes, resulting in PAMP-triggered immunity or induced resistance.Natural compounds conferring disease resistance on plants in themanner of general elicitors is of interest because of the non-specific characterof the induced resistance – lots of pathogens can be controlled with one activecompound. The success of applying elicitor compounds is directly dependenton understanding their properties and mechanisms of action. As well asall other biogenic or abiogenic, general or specific inducers, proteinaceouselicitors produced by plant pathogens (e.g. glicoproteins, flagellins, elongationfactor Tu, elicitins and transglutaminases from Phytophthora spp., proteinsand peptides from Cladosporium flavum, monolicollin, cold shockproteins, harpins etc.) are the subject of genetic and biochemical research ofsignalling pathways and molecular mechanisms underlying plant resistanceto diseases. Non-protein small compounds (plant signalling molecules andhormones) participating in signalling as well as chemical compounds thathave been found to mimic elicitors are also being studied in this way. Transgenicplants with genes encoding microbial elicitors (e.g. elicitins) are createdfor research purposes using biotechnological methods.

120 L.A. ShcherbakovaRegarding the application, general elicitors to plant protection, chitosan,arachidonic, salicylic and β-aminobutyric acids may be referred to as examplesof active ingredients that have been formulated and introduced intoagricultural practice as commercial biogenic non-protein inducers of plantresistance. For instance, a supplement of tomato growth substratum withchitosan suppressed the root rot caused by F. oxysporum f. sp. radicis-lycopersici(Lafontaine and Benhamou, 1996). Moreover, several chitosan formulationscollectively known as ‘chitozars’, which are effective against root rots,late blight and powdery mildew on cereals, potato and legumes, have beenincluded in biological protection systems of these crops in Russia. Amongprotein elicitors, bacterial harpins are the most important. Harpins (hypersensitivityresponse and pathogenicity) are components of a transport systemused by bacteria to transfer proteins through bacterial and plantmembranes. In the bacterial genome, harpins are products a hrp-gene cluster.One of the first harpins studied was HrpN, the product of hprN geneexpressed in E. amylovora. HrpN has been shown to be an acidic glycine-richthermostable protein with a molecular mass of 44 kDa that induces resistancein Arabidopsis (Wei and Beer, 1996). Harpin has been commercialized andmarketed as a product that enhances resistance of some field, ornamentaland vegetable crops to many diseases, and that stimulates plant growth andflowering.In the coming years, we can expect the agricultural use of other elicitorproteins for increasing plant resistance to diseases both by engineering ofplants containing elicitor protein genes and by exposure of plants to proteinelicitor-based products. Two bacterial proteins with plant-protecting activityare considered below as examples of very promising compounds for developingboth resistant transgenic crops and products for plant treatment.Cold shock protein CspD as an elicitor of disease resistance in plantsCold shock protein D (CspD) isolated from Bacillus thuringiensis culture brothis a thermostable low molecular weight protein (molecular mass 7.2 kDa).The B. thuringiensis gene encoding CspD was cloned and sequenced (CspD, #AY272058 in GenBank). Nucleotide and amino acid sequences of CspD showhigh homology with other bacterial CSPs (Dzhavakhiya et al., 2000, Krominaand Dzhavakhiya, 2004).CSPs are highly conserved proteins produced by various bacteria constitutivelyor in response to cold shock. They have also been studied in plants,e.g. in Arabidopsis, tobacco and wheat. CSPs are involved in cell growth andadaptation to low temperatures. They bind nucleonic acids and are consideredto be putative translation anti-terminators. Bacterial CPSs contain a specificconserved cold shock domain (CSD), which occurs in CSPs from otherorganisms. For instance, a fragment of high homology to bacterial CSD wasfound in a family of plant CSPs referred to as glycine-rich proteins (GRPs).Expression of grp-genes is dependent on the level of plant hormones (indolylacetic, salicylic, abscisic acids), illumination and the plant developmentphase, and is upregulated by cold and wounding. The synthesis of GRPs can

Proteinaceous and Polyketide Compounds in Plant Protection 121also be enhanced upon pathogen attack both in compatible and incompatibleplant–pathogen combinations. Alien CSPs are recognized by plants as componentsof PAMP and elicit resistance to some pathogens. Thus, CSPs fromMicrococcus lysodeikticus non-specifically induced defence responses inplants. It was found that a peptide consisting of 15 amino acid residues(csp15) that represented a consensus sequence of RNA-binding PNP-1 andRNP-2 motifs was responsible for eliciting activity towards some Solanaceaeplants (Felix and Boller, 2003). The peptide csp15 induced an ‘oxidative burst’in tobacco (Nicotiana tabacum, cv. Havanna 425) and potato (Solanumtuberosum), but was ineffective towards rice and cucumber cells.CspD from B. thuringiensis has been reported to induce resistance both inmonocotyledonous and dicotyledonous crops against a wide range of pathogens:from filamentous microorganisms (fungi and oomycetes) to viruses(Dzhavakhiya et al., 2000). For instance, CspD applied by dropping ontowheat leaves or by spraying potato and rice seedlings with water- or bovineserum albumin (BSA)-stabilized CspD solutions produced resistance againstS. nodorum, P. infestans and M. grisea, respectively. Effective crop protectionwas observed in both greenhouse and field experiments. Exposure of tobaccoplants to CspD induced resistance to tobacco mosaic virus (TMV) and potatoX-virus. Peptide csp15 of CspD protein (VKWFNAEKGFGFITP) also showedresistance-inducing activity on plants and in model plant–pathogen systems.Treatments of tobacco leaf halves with 1–10 μmol csp15 in 0.1% BSA a daybefore inoculation with TMV resulted in a drastic reduction of lesion spotnumber on the treated halves as compared to 0.1% BSA-treated (control)halves or whole control leaves of the same plant (Kromina and Dzhavakhiya,2004).Several tests were carried out to identify which host defence responses tothe pathogen challenge are activated by CspD and csp15. Applying csp15 tothe surface of discs cut from potato tubers (cv. Istrinskiy, R1) increased thehypersensitive response of potato cells to the incompatible P. infestans race r4with a decreased number of dead cells and induced accumulation of salicylicacid in the tuber tissues. Both CspD and csp15 have no fungitoxicity. Additionof the peptide to the suspension of cultured tobacco cells activated theH + pump and caused a reversible change in the extracellular pH. The resistanceinduced with csp15 or CspD in plants has a systemic character (Krominaand Dzhavakhiya, 2004).To transfer the CspD gene into tobacco plants, the pBilt7 plasmid, containingthe CspD expression cassette (P35S/CspD/pACaMV), on pBin19 vectorbackground was constructed and transformed into A. tumefaciens. As a result,seven lines of cv. Xanthi (NN) and five lines of cv. Samsung (nn) were produced.Expression of CspD in these lines was confirmed by real-time PCR.The transgenic tobacco plants had the same habitus as control plants andproduced fertile projeny. The transgenic lines showed increased resistance toAlternaria longipes and TMV that coincided with the range of antipathogenicactivity observed for the elicitor protein per se. Importantly, the level of CspDexpression coincided with the resistance level to the both pathogens amongall tested lines as well as inside any one line. These findings lead to the

122 L.A. Shcherbakovaassumption that disease resistance of transgenic tobacco is due to defenceresponses that are elicited with CspD synthesized inside the plant (Krominaand Dzhavakhiya, 2006).The above results can be used in breeding programmes for production ofcrop cultivars with resistance against a wide range of phytopathogens orCspD-based biopesticides providing complex protective effects against fungi,oomycetes and viruses.MF3 protein, bacterial peptidyl-prolyl cis/trans isomerase, conferring plantresistance to pathogensThe thermostable protein MF3 (16.9 kDa) is produced by biocontrol Pseudomonasfluorescens isolate 197. After cloning and sequencing the MF3-encodinggene from P. fluorescens 197, a full primary structure of the protein wasdetermined, and high homology between amino acid sequences of MF3 andpeptidyl-prolyl cis/trans isomerases of the FK506-binding protein (FKBP)type was found ( Dzhavakhiya et al., 2005; Shumilina et al., 2006).Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that acceleratethe slow cis/trans isomerization of the peptide bond between proline residuesand the adjacent amino acid in protein. Initially, de novo synthesizedpolypeptides have the trans conformation, which stabilizes the polypeptidechain, but to form the native protein structure, about 7% of the peptide bondsformed with proline residues need to be isomerized into the cis conformation.PPIases take part in the renaturation of denatured proteins, in proteinsynthesis de novo and in the formation of biologically active conformations ofsome polypeptides. PPIases are highly conservative enzymes produced byprokaryotes, eukaryotes, viruses and phages. They have been classified intothree families, referred to as cyclophilins, FKBPs, which regulate signalsystems of eukaryotic cells, and parvulins.In plants, FKBPs were initially detected in Vicia fava, Triticum aestivumand Arabidopsis thaliana. It was found that A. thaliana contains 23 FKBPs andFKBP-like low- or high-molecular weight proteins. Plant PPIases of the FKBPtype control plant fertility, cell division and differentiation as well as the perceptionof plant hormones. Plant FKBPs are expressed both constitutivelyand in response to various stresses such as wounding, salinity or heat shock.PPIases produced by phytopathogens can contribute significantly to thepathogenic process. For instance, the plant pathogenic fungi M. grisea and B.cinerea produce cyclophilins. Mutations in the cyclophylin genes impair virulenceof these fungi. The M. grisea mutants form low-turgor appressoriaunable to penetrate through leaf cuticle. Deletion of the cyclophylin genedoes not influence M. grisea growth in vitro but affects formation of fungalconidia (Viaud et al., 2002).It is likely that PPIases are involved in plant–pathogen relationships.On the one hand, some pathogens are in need of host plant PPIases for establishingthe infection process. On the other hand, interaction of plant PPIasewith pathogen proteins can result in induction of disease resistance. Thuscysteine proteinase AvrRpt2, that is transported by Pseudomonas syringae to

Proteinaceous and Polyketide Compounds in Plant Protection 123Arabidopsis cells during pathogen invasion must be folded with the plantcyclophylin ROC1 to form an active conformation able to cleave plantproteins. Along with other proteins, the activated bacterial protease cleavesprotein RIN4, which is combined in Arabidopsis in an inactive complex witha signal protein RPS2. Proteolysis of RIN4 releases RPS2 that triggers defenceresponses (Mackey et al., 2003). PPIases of one organism have been reportedto inhibit the growth of another organism by means of competitive bindingwith a receptor, and probably some PPIases can suppress growth of culturedplant pathogens. For instance, cyclophylin C-CyP isolated from Chinesecabbage (Brassica campestris L. ssp. pekinensis) inhibits in vitro growth ofseveral fungi including Rhizoctonia solani, B. cinerea, F. solani and F. oxysporum(Lee et al., 2007).Various experiments have shown that MF3 induced resistance in variousmonocotyledonous and dicotyledonous plants against several fungal andviral pathogens. MF3 was found to protect tobacco against TMV, potato Yvirus and A. longipes, and barley against Bipolaris sorokiniana. At the sametime, this protein did not influence TMV infectivity, was not fungitoxic andhad no phytotoxicity towards tobacco plants and cereals. Moreover, treatmentof barley and wheat seeds, naturally infected by B. sorokiniana andF. culmorum, with MF3 promoted formation of a well-developed root systemin the diseased barley and wheat seedlings, that conferred plant tolerance toroot rots and was consistent with growth-stimulatory functions for PPIasesin plants (Dzhavakhiya et al., 2005; Shumilina et al., 2006).Comparative analysis of MF3 with 45 proteins of different homology levelsrevealed two conserved sequences in the MF3 polypeptide chain. Proteolysiswith trypsin inside one of these conserved sequences producedfragments that did not induce resistance to TMV in tobacco leaves. Thesedata allowed the assumption that the analysed sequence contained a motifresponsible for the resistance-inducing activity of MF3. Such a motif,IIPGLEKALE GKAVGDDLEVAVEPEDAYG, was detected and named MF3-29 because it was found to consist of 29 amino acid residues. This fragmentwas necessary and sufficient for induction of tobacco resistance to TMV. Biologicaltests on isolated tobacco leaves showed that treatments of tobaccoleaves with chemically synthesized oligopeptide MF3-29 at concentrations0.5, 5 and 50 nM were as effective against the virus as the whole protein at thesame concentrations.In order for products based on protein elicitors to effectively controlplant pathogens, they have to access plant receptors recognizing PAMPs. Thelarge size of the molecules or hydrophobic barriers on the plant surface (suchas cuticle) can impede physical contact or chemically mediated recognitionof elicitor proteins and the subsequent induction of defence responses inplants. To solve this problem, special molecular carriers facilitating elicitortransport to plant cells should be developed. Various polycationic molecules,especially chitosan, which are used now for the delivery of large biologicalmolecules (DNA or proteins) to their outer or intracellular receptors, lookpromising as putative carriers of proteinaceous elicitors. Experiments withthe wheat leaf spot agent S. nodorum and turnip mosaic virus (TuMV)

124 L.A. Shcherbakovademonstrated that MF3 integration with chitosan, a non-toxic andbiodegradable natural polymer with elicitor properties, enhanced resistanceinducingactivity of MF3, and enlarged the scope of the antipathogenic actionof this protein. No inhibitory effect on fungal growth in vitro was observed ifMF3, chitosan or the MF3–chitosan complex was added to culture media.Neither the protein nor chitosan applied separately on wheat leaves protectedplants against S. nodorum. However, leaf treatment with the MF3–chitosan complex before plant inoculation with the pathogen significantlyreduced disease severity. Spraying cabbage seedlings (cv. Krautman) withthe MF3– chitosan-complex resulted in at least a week's delay in TuMV accumulation,whereas virus spread was not delayed in seedlings sprayed withMF3 alone or chitosan alone (Shumilina et al., 2005).Rape lines (cv. Westar) with an mf3 gene insertion showed increasedresistance to fungal pathogens Plasmodiophora brassicae and TuMV. Most ofthe transgenic plants carried more than one gene insertion. The bacterial proteinconcentrations in some lines amounted to 37 pg/mg of fresh weight. Anincreased level of rape resistance to TuMV was kept independently of MF3variation in plant tissues. Identically high resistance was observed at eightdifferent protein concentrations in transgenic lines. This may suggest thattiny amounts of MF3 are sufficient for conferring plant resistance to the virus.It was shown that five transgenic lines of the six that were resistant to TuMValso expressed an enhanced endurance to P. brassicae for 50 days after inoculation.The disease symptoms were significantly less severe on roots of transgenicplants as compared to symptoms developing on non-transgenic raperoots or roots of transgenic lines that had lost the mf3 insertion. It is believedthat the best lines obtained may be included in breeding programmes forcreating rape cultivars with resistance against both pathogens (Dzhavakhiyaet al., 2005; Shumilina et al., 2006).Thus, the wide range of elicitor activity of CspD and MF3, as well as thepossibility to use general approaches of biocontrol, gene engineering technologyand plant treatments in CspD- and MF3-based plant protection demonstratethe great potential of proteinaceous elicitors of natural defencemechanisms for the environmentally safe control of phytopathogens. Toxicologystudies and in-house laboratory tests demonstrated that the proteinsare non-toxic to animals and plants. Both of these compounds are patentedas new proteins with plant protecting properties (EP0868431A1 and PCTWO2005/061533 A1). Due to the systemic character of the resistance inducedwith CspD and MF3, the resistance spreads to the whole plant even if onlythe seeds or leaves are treated. After a single treatment with the protein solutions,induced resistance remained effective for not less than 3 weeks. Inmany cases this can cover the period of highest risk infection. The large-scaleintroduction of transgenic cultivars resistant to a certain pathogen is sometimeslimited because different pathogens dominate when the climatic andsoil conditions change. Moreover, there is the problem of unpredictable seasonalalteration of pathogens, which decreases the effect of pre-sowing treatments.Crop engineering or/and developing new products based on generalelicitors such as CpsD or MF3 inducing ‘universal’ resistance would promote

Proteinaceous and Polyketide Compounds in Plant Protection 125overcoming these limitations. Searching for novel proteins and peptideswould promote progress in this field. Finding elicitor activity in MF3 proteinbelonging to PPIases that were never meant to be designated as disease resistanceinducers shows desired properties may be discovered during screeningof natural biologically active compounds which were previously studiedand characterized as non-hazardous for people and the environment. Thispresents additional prospects for developing new biocontrol productscomplying with green consumerization requests.5.3 Statins as New Promising Candidates for BiopesticidesOne of the effective plant disease management strategies is based on thecontrol of metabolic relationships in a plant–pathogen system. This can bereached by using compounds that alter or partially block specific pathwaysof biosynthesis in plants or microorganisms, resulting in derangements inthe trophic relationships of pathogens with host plants, or in the productionof microbial metabolites related to pathogenicity. Such imbalances may notcause the rapid death of pathogens but can lead to a reduction of their vitalcapacity or/and impair their pathogenic properties. Similar metabolic effectsmay be caused by plant inhibitors of fungal and bacterial proteases asdescribed above, as well as by the inhibitors of insect and nematode enzymes.Statins, secondary metabolites of microbial origin belonging to the polyketidegroup, represent another class of natural compounds that have recentlybeen discovered to modulate metabolic plant–pathogen interactions. Themetabolic pathway targeted by statins is sterol biosynthesis.Sterols are well known to be ubiquitous and important components ofouter membrane and intracellular membranes of eukaryotic organisms andto play essential roles in their physiology. These compounds are required forgrowth, development and reproduction of plant pathogens. A number oforganisms including insects, nematodes and oomycetes are not able to synthesizesterols or even their precursors. Sterol-dependent phytopathogensobtain free sterols or intermediates from host plants and may be controlledby compounds inducing alterations in content, availability or composition ofplant sterols.Currently, several chemical fungicides are available that inhibit the synthesisof ergosterol in fungi. They prevent cellular membrane formation, stopfungal growth and may suppress sporogenesis in established infectionagents. However, statins are natural inhibitors of sterol biosynthesis that presumablycan cause similar effects. Statins were found to be produced bydifferent microorganisms, predominantly by filamentous fungi. They inhibitthe enzyme β-hydroxy-β-methylglutaryl-CoA reductase (HMG-CoA reductase)and prevent sterol biosynthesis at the level of conversion of HMG-CoAto mevalonic acid. Statins are widely applied in medicine as drugs againstatherosclerogenic diseases because they effectively decrease the blood cholesterollevel. Thus, the biological compatibility and safety of statins are wellproven.

126 L.A. ShcherbakovaRecently, studies were made to investigate the potential of two statins,lovastatin and compactin, in plant protection against diseases (Dzhavakhiyaand Petelina, 2008; Ukraintsteva, 2008). In these studies, both statins wereobtained by means of microbial synthesis using the ‘superproducer’ strainsAspergillus terreus 45-50 (lovastatin) and Penicillium citrinum 18-12 (compactin);these statins were examined in greenhouse and field experiments withplant treatments and in vitro tests on several pathogenic fungi. As statins areinsoluble in water, the authors used aqueous solutions of the statin sodiumsalts that are referred to below as compactin and lovastatin.The studied statins were found to possess fungicidal activity in vitroagainst a number of plant pathogenic fungi. The addition of lovastatin atconcentrations from 0.001 to 0.1% to agar media inhibited growth of M. grisea,S. nodorum and Coletotrichum atramentarium. Compactin arrested in vitrogrowth of Cladosporium cucumerinum, S. nodorum and M. grisea at the sameconcentration range. In addition, 0.001% compactin solution significantlyreduced germination of S. nodorum spores while the 0.01% solution completelysuppressed spore germination in this fungus. Among tested plantpathogens, the fungus M. grisea, rice blast agent, showed the highest sensitivityto both statins (for lovastatin IC 50was about 0.003%), while S. nodorumwas most resistant to lovastatin (IC 50= 0.02%) and C. cucumerinum tended tocompactin resistance. Besides a growth-inhibitory effect, the presence of thestatins in nutrition media resulted in a discolouration of fungal mycelia in allfungi, especially in M. grisea and C. atramentarium exposed to lovastatin. Thisobservation suggested suppression of fungal melaninogenesis. In all experiments,dicolouration-inducing concentrations of the statins were at leasttenfold lower than growth-inhibiting concentrations.The studied statins were revealed to possess disease-preventing properties.They delay and decrease disease development on treated plants whenapplied simultaneously or prior to inoculation with pathogens. Lovastatinand compactin protected wheat against S. nodorum and tobacco from A. longipes.In addition, lovastatin showed protective activity against M. grisea onrice, whereas compactin was effective against Puccinia graminis on wheat andP. infestans on potato. Both fungicidal and protective effects were stronglydose-rate dependent, but fungitoxic concentrations also made a toxic impacton plants, whereas protection from diseases was provided by far lower concentrationsthat were non-phytotoxic. Thus, almost total or 27–36% S. nodorumgrowth inhibition in vitro was observed at 0.1% and 0.01% lovastatinconcentrations in nutrition media, respectively. Both doses showed phytotoxicityon wheat leaves, but only the higher concentration caused retardanteffect on growing plants. When 0.1% lovastatin solution was used for wheatseed soaking for 1.5–2 h, reduction of seedling length averaged 50%, and0.01% concentration caused no inhibitory influence on plant growth. A considerablereduction of the disease index was found after application of only0.0005% lovastatin solution on isolated wheat leaves. The applied concentrationdid not suppress S. nodorum germination and in vitro growth but preventeddisease development with 94 and 72% protection efficacy at 3 and7 days after inoculation, respectively. This suggests that a mechanism other

Proteinaceous and Polyketide Compounds in Plant Protection 127than fungicidal activity contributes to the protective effect of statins( Dzhavakhiya and Petelina, 2008).The resistance of fungi to extreme environmental conditions and bioticstresses is largely determined by their ability to produce protective highmolecular-weightpigments. One of the common fungal pigments in cell walls ismelanin. Melanin is a coloured polymer produced by many plant pathogenicfungi, including M. grisea, S. nodorum, C. lagenarium and C. atramentarium,through the pentaketide pathway and depends on the availability of acetyl-CoA. Melanin plays a significant role in the infectivity of these fungi. Forinstance, the ability of the rice pathogen M. grisea to penetrate into tissues of thehost plant is directly associated with the presence of melanin in the fungus.Strains of M. grisea and C. lagenarium defective in melanin formation lose pathogenicityand are incapable of forming mycelial overgrowth in host plants. Revertantsrestoring wild-colour type regain pathogenicity (Dzhavakhiya et al., 1990).Interestingly, antimelanogenic compounds, aminoalkylphosphinates, afamily of phospho-analogues of natural amino acids, suppress biosynthesisof melanin and some toxic metabolites in the polyketide pathway of M. grisea,and are also fungicidal. The aminoalkylphosphinates serve as analogues ofalanine, a precursor to pyruvic acid that is required for melanin biosynthesis.Exposure of fungi to the phospho-analogues of amino acids inactivates pyruvatedehydrogenase, thus inhibiting synthesis of acetyl-CoA and melanin(Zhukov et al., 2004) as well as aflatoxins (Khomutov, Khurs, Shcherbakova,Mikityuk, Dzavakhiya and Zhemchuzhina; unpublished data). The discoverythat non-fungicidal lovastatin and compactin concentrations can inducemycelium de-pigmentation and decrease disease severity on plants suggeststhat their protective effect may be associated with impairing pathogenicitydue to an effect on melanin biosynthesis in causative agents. Statins and fungalmelanin are both polyketides. Thus, the metabolism of the two compoundsis interrelated, and it is not improbable that statins can negativelymediate a stage of the polyketide pathway involved in the melanization ofplant pathogenic fungi.Although the mode of protective action of statin is required for understandingand further research, first small-plot field trials suggest they may beof certain interest from a practical point of view. For example, one preplantingtreatment of potato tubers by soaking in 0.1% compactin solution forhalf an hour resulted in a 1-month delay of late blight (P. infestans) emergenceon plants and a slower course of disease. Only extremely high statin concentrationsof 0.5% undesirably influenced plant physiological characteristics. Bythe end of the growing season, an insignificant reduction of potato late blightwas observed on plants arising from the treated tubers, but these plantsproduced fewer diseased tubers. There is reason to suppose that lovastatinand compactin possess anti-phytoviral activity (Ukraintseva, 2008).Further research of the mechanisms responsible for protective activitycould help statins take a fitting place among the biopesticides of tomorrow.Lovastatin and compactin per se might serve as base molecules for biochemicalengineering of active analogues, and the approaches used to studyingtheir plant-protecting properties might be implicated in screening of other

128 L.A. Shcherbakovapathogen-controlling compounds within the statin group and their derivatives.Such screening might lead to the detection of new natural polyketideswith high protective activity that would not have an adverse effect on plants.Since the agricultural application of statin-based formulations does notdemand as high a level of statin purification as that in the pharmacologicalindustry, using a relatively simple isolation procedure (Dzhavakhiya, 2008)and the ‘superproducer’ strains suggests that statin-based plant- protectiontechnology would be a more economical and ecologically safe strategy thanany method using chemical pesticides.5.4 ConclusionThe disease-preventing effect of natural compounds is a result of a direct orplant-mediated influence on targeted pathogens. The compounds can affectphytopathogens directly, interrupting pivotal metabolic pathways andresults in the death of the pathogens (biocidal effect). Some natural substancesor their analogues specifically influence pathways related to pathogenicityand toxigenesis. In the case of a plant-mediated mode of action,natural compounds elicit and activate defence responses in plants that resultin induced resistance to diseases. In addition, some biogenic compoundsinfluencing plants do not induce resistance but enhance tolerance to pathogensby improving the physiological state of plants. The structural and functionaldiversity of natural compounds as well as their abundance provide agreat potential for bringing plant-protection technologies in line with greenconsumerization.ReferencesAhrenholtz, I., Harms, K., De Vries, J. andWackernagel, W. (2000) Increased killingof Bacillus subtilis on the hair rootsof transgenic T4 lysozyme-producingpotatoes. Applied and EnvironmentalMicrobiology 66, 1862–1865.Asaka, O. and Shoda, M. (1996) Biocontrol ofRhizoctonia solani damping-off of tomatowith Bacillus subtilis RB14. Applied andEnvironmental Microbiology 62, 4081–4085.Bais, H.P., Fall, R. and Vivanco, J.M. (2004)Biocontrol of Bacillus subtilis againstinfection of Arabidopsis roots byPseudomonas syringae is facilitated by biofilmformation and surfactin production.Plant Physiology, 143, 1–13.Bassarello, C., Lazzaroni, S., Bifulco, G., LoCantore, P., Iacobellis, N.S., Riccio, R.,Gomez-Paloma, L. and Evidente, A. (2004)Tolaasins A–E, five new lipodepsipeptidesproduced by Pseudomonastolaasii. Journal of Natural Products 67,811–816.Benitez, T., Limon, C., Delgado-Jarana, J. andRey, M. (1998) Glucanolytic and otherenzymes and their genes. In: Harman,G.E., Kubicek, C.P. (eds) Trichoderma andGliocladium: Enzymes, biological controland commercial application, Taylor &Francis, London, 2, 101–128.Bolar, J.P., Norelli, J.L., Wong, K-W., Hayes,C.K., Harman, G.E. and Aldwinckle, H.S.(2000) Expression of endochitinase fromTrichoderma harzianum in transgenic appleincreases resistance to apple scab andreduces vigor. Phytopathology 90, 72–77.

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6 Natural Products asAllelochemicals in PestManagementROMAN PAVELACrop Research Institute, Prague, Czech RepublicAbstractAllelochemicals are one of the most plentiful groups of substances in the vegetablekingdom. The significance and use of these chemicals in nature are as varied as allelochemicalsthemselves. Allelochemicals also include a group of substances called allomones.These substances are created by plants as a defence against phytophagousinsects and comprise repellents, anti-ovipositants and antifeedants. This group ofplant metabolites can be of practical use in many areas of human activity. This chapterdeals with possibilities of using plant extracts containing allomone in the protectionof plants against pests. Substances with antifeedant effects, in particular, appear to behighly promising for the development of new, environmentally safe insecticides. In thischapter, we have therefore focused mostly on this group of substances and criticallyconsidered the perspective of using antifeedant substances in plant protection.6.1 IntroductionPlants have developed alongside insects since the very beginning of theirexistence. Thanks to their common history, very fragile mutual relationshipshave formed between individual plant and insect species, which we aretrying to clarify today using scientific approaches. On the one hand, plantsutilize insects, especially as their pollinators, and on the other, insects useplants as the source of their food. Therefore, for balanced mutual interactionsbetween plants and insects, communication and mutual influence must existbetween them to prevent uncontrolled excessive reproduction of any speciesin order to maintain equilibrium in the ecosystem with maximum possiblebiodiversity. Plants thus created many strategies in the course of theirco-evolution to protect themselves efficiently against insect pests, and theinsects try to circumvent such strategies. Understanding their mutual relationshipshelps us not only to understand the world around us but alsoprovides information that may lead to a practical use.© CAB International 2011. Natural Products in Plant Pest Management134 (ed. N.K. Dubey)

Allelochemicals in Pest Management 135As mentioned above, there are many types of plant–insect interactions;however, the interaction between plants as a source of food and phytophagousinsects as their pests is one of the most interesting and important onesfor practical applications in agriculture. In this case, it is a relationship between‘food’ on one hand and ‘consumer’ on the other; the plants were forced todevelop numerous defensive mechanisms to prevent uncontrolled destructivepest attacks. The most important defensive plant mechanisms include thesynthesis of biologically active compounds, the so-called secondary metabolites.First, such substances may provide direct insecticide effects, causingmortality of phytophagous insects, and/or second, they may exert indirectinsecticide effects, only influencing insect behaviour in some manner. Knowledgeof such relationships leads not only to useful information necessary forcultivating plants with resistance, but also to the direct utilization of extractsin cultural plant protection using the so-called botanical insecticides.Although the first group of compounds, i.e. those with direct insecticidalactivity, has been used by humans as extracts for millennia, both in fightingphytophagous pests as well as against parasites or storage pests, the othergroup of compounds, generally called allelochemicals, has become thesubject of more profound interest only in recent decades (Pavela, 2007a).The term allelochemicals (from Greek allelon: ‘one another’) is used todescribe the chemicals involved in interspecific interactions. It is defined asa chemical significant to organisms of a species different from its source.Allelochemicals are divided into four subgroups, depending on whether theemitter, the receiver, or both benefit in the interaction.●●●●An allomone (from Greek allos – ‘another’; horman – ‘to stimulate’) isdefined as a chemical substance, produced or acquired by an organism,which evokes in the receiver a reaction adaptively favourable to theemitter, e.g. a plant emits allomones to deter herbivores.A kairomone (from Greek kairos – ‘opportunistic’) is defined as achemical substance, produced or acquired by an organism, which evokesin the receiver a reaction adaptively favourable to the receiver but not tothe emitter, e.g. secondary plant compounds help herbivores in findingplants to feed on.A synomone (from Greek syn – ‘with or jointly’) is defined as a chemicalsubstance, produced or acquired by an organism, which evokes in thereceiver a reaction adaptively favourable to both the emitter and thereceiver. This group of allelochemicals includes floral scents and nectarsthat attract insects and other pollinators and substances that play animportant role in symbiotic relationships.An apneumone (from Greek a-pneum – ‘breathless or lifeless’) is definedas a substance, emitted by a non-living material, which evokes a reactionadaptively favourable to the receiving organism, but detrimental toanother organism that may be found in or on the non-living material.For example, parasites or predators are attracted to non-living substancesin which they may find another organism, their host or prey, byapneumones released from the non-living substance.

136 R. PavelaIn many cases not a single semiochemical has an effect on its own butdifferent groups of chemicals in a precisely defined mixture act in an effectivelycombined manner. In general, it can be said that the whole group ofallelochemicals may find its application in plant protection. Nevertheless,metabolites falling in the allomone group represent the most studied groupwith the most potential at present.6.2 Allomones – A Prospective Group of Substances forAlternative Plant ProtectionAs explained above, this group of substances includes plant metabolites thathave some type of negative effect on insect behaviour providing benefit tothe plants. The chemicals can be divided as follows according to their modeof action:1. Repellents2. Anti-ovipositants3. AntifeedantsRepellents are substances that directly deter insects from settling on theplant. They include an entire group of simple aromatic hydrocarbons, whichmay be released into the environment, thus having a direct effect on insectchemoreceptors (Koul, 2005). Repellency often tends to be connected withanti-oviposition, as such substances deter females from settling on nutritiveplants and prevent oviposition at the same time. However, anti-ovipositionneed not always be connected with repellency. It may be connected with antifeedancyvery often – when upon settling on the plant, the female finds thatthe plant cannot provide food of good quality or acceptable for its descendants,and thus she flies off to seek a more suitable plant. The group of substanceswith an anti-oviposition effect includes a whole range of chemicalsfrom simple aromatic terpenes, phenols or alkaloids to molecules falling inthe group of polyphenols or limonoids (Koul, 2005).The last group of substances – antifeedants – deter phytophagous insectsagainst food consumption. This group of substances has been studied on alarge scale in recent times and the use of such substances in plant protectionagainst pests is connected with their significant potential. This group ofchemicals also range from simple aromatic terpenes, phenols or alkaloids tomolecules falling in the group of polyphenols or limonoids. At present, thereare high expectations for this group of substances due to their practical use inplant protection (Isman, 1994; Koul, 2005; Pavela, 2007b).The good prospects for the practical use of antifeedants are based onseveral factors. Most of the repellent substances fall into the group ofaromatic hydrocarbons, which are volatile in the environment and their efficiencytime is therefore reduced depending on period of application anddosage used. Numerous substances belonging to the polyphenols andhigher terpenes are included in the category of antifeedants, which mayhave a much longer persistence time, thereby extending the efficiency time

Allelochemicals in Pest Management 137of products (Pavela and Herda, 2007a,b). Moreover, their antifeedant efficiencyis often connected with further biological activity such as growth inhibitionand cumulative mortality.Allomones with antifeedant activity include a high percentage ofsubstances that also meet the following requirements for development ofnew botanical insecticides:●●●●They are natural plant metabolites, safe for health and the environment;Such substances provide specific effects on individual pest species – theythus show high selectivity;Mixtures of substances with synergistic effects can be obtained, whichprevent the development of pest resistance;Antifeedant substances usually show other biological effects such asgrowth inhibition, mortality and reduced fertility of surviving individuals,which increase the practical efficiency of the products (Isman, 2002;Koul, 2005).All these characteristics increase the prospects for the use of antifeedantsubstances in plant protection. However, products based on allomonesshould be applied as a preventative measure, for example, at the time of pestinvasion or its initial occurrence or at the time of larval incubation. Althougha large number of biologically active substances and extracts from plantswith antifeedant effects have been tested, only a limited number of productshave been subjected to a detailed field application and in the development ofplant protection products (Isman, 1994).6.3 Current State of Antifeedant ResearchThe history of the research of substances that show antifeedant effects datesback to the 1930s. As early as 1932, Metzger and Grant tested about 500 plantextracts against Popilla japonica, although results were not substantiallyencouraging. Later, Pradhan et al. (1962) evaluated extracts of the Indianneem tree, Azadirachta indica, that prevented feeding by the desert locusts.Although terrestrial plants produce a diverse array of secondary metabolites,probably more than 100,000 unique compounds (Isman, 2002), today, about900 compounds have been identified to possess feeding deterrence againstinsects (Koul, 2005).In addition to various compounds isolated from plants or synthesized asinsect antifeedants, several studies demonstrate the antifeedant efficacy inmetabolite mixtures of plant essential oils or total extracts against a variety ofinsect species.In recent years studies have revealed the antifeedant potential of plantessential oils against postharvest pests, aphids, thrips, lepidopterans, termitesand mite pests (Hori, 1999; Hou-HouaMin et al., 2002; Koul, 2005;Isman, 2006). Similarly, during the past few years much emphasis has beenplaced on demonstrating the antifeedant efficacy in total plant extracts(Mancebo et al., 2000a,b; Wang et al., 2000; Pavela, 2004a,b; Zhang et al., 2004;

138 R. PavelaDebrowski and Seredynska, 2007) as they seem to exhibit the activity asmulticomponent systems. However, it is also well known that antifeedantsshow interspecific variability (Isman, 1993). The existence of such interspecificdifferences, as shown for many insect species, is encouraging with aview to searching selectively for specific feeding deterrents.As already indicated, present research provides very important informationon plant substances and their antifeedant efficiency. Nevertheless, itmust be noted that the antifeedant efficiency of a significant number ofknown antifeedant compounds is dependent on the exposure period andtheir concentration. After a long exposure period, some of these compoundslose their antifeedant efficiency due to the development of resistance in theinsects consuming the contaminated food (Koul, 2005). Nevertheless, suchcompounds may have other biological effects, at the same time, such ascumulative mortality or perhaps larval growth inhibition.However, it should be emphasized that most of the antifeedant research isin the preliminary trial stage, although the activity of more than 900 compoundsand several hundreds of plant extracts are known (Koul, 2005). However,it is expected that in the near future some novel efficacious plant-basedcompounds will be formulated as antifeedants, from the huge bio diversityprovided by nature. Attention is currently being paid to promote the indigenousantifeedant plants by extolling their practical application to the farmers.6.4 Mechanism of Antifeedant ActionFood selection among insect herbivores is a highly specialized phenomenon.While olfactory and physical aspects of plants or their organs can be importantin insect host finding and acceptance (Miller and Strickler, 1984), thechoice of food is based primarily upon contact chemoreception of variousallelochemicals (Frazier, 1986; Stadler, 1992). In particular, dietary experiencehas influenced the ability of insects to taste plant chemicals that mayhave served as signals of suitability or unsuitability. Certain dietary constituentsappeared to suppress the development of taste sensitivity to deterrentsin an insect (Renwick, 2001). Avoidance of allelochemicals, whenlooked at from a behavioural point of view, is the outcome of interactionswith chemoreceptors characterized by broad sensitivity to a spectrum ofdeterrents (Mullin et al., 1994).According to Schoonhoven et al. (1992), there are four basic reasons whythe chemosensory perception of feeding deterrents by phytophagous insectswarrants special attention:●●●●Feeding deterrents are apparently more important in host-plant recognitionthan phagostimulants.A huge number of feeding deterrents exist, with variable molecularstructures adding to their diversity.There are fewer deterrent receptors.Different deterrents may elicit different behavioural reactions, indicatingthe presence of a differential sensory coding system.

Allelochemicals in Pest Management 139Studies of the chemosensoric insect system are only at the initial point ofresearch, and knowledge of the mode of action of the substances is superficialand should be studied in detail. Such a lack of knowledge is particularlycaused by the fact that suitable technologies for chemosensoric investigationshave been available only relatively recently, making it possible to performreliable measurements of response to substance at insect chemoreceptors.Although research of chemoreceptors is important for a general understandingof efficiency of individual antifeedant substances, from the practicalpoint of view, experiments based on simple biological tests are thosemostly used in antifeedancy studies.Bioassays against insects have been used for decades as a means ofelucidating the activity of many chemical components or extracts. The majorgoals achieved by employing bioassay techniques are to determine the rolesof naturally occurring chemicals, identify the mechanism of resistance incrop plants and to find various insect control agents. The basic design tostudy deterrents is to present to an insect a substrate with the candidatechemical and to measure the response of the insect. Therefore, substratechoice and presentation are important factors for a successful bioassay. Bothnatural and artificial substrates are used, depending upon the goal of theexperiment. On one hand one may emphasize that artificial substrates offeruniformity, but at the same time studies have shown that thresholds for thesame deterrent may vary as much as 1000 times between natural and artificialsubstrates (Schoonhoven, 1982), perhaps due to differences in porosity oruptake rates by the insect. For sucking insects, the principal artificial substrateused has been a chemically defined liquid presented between naturalor artificial membranes (Koul, 2005).However, whatever the substrate may be, it is important that no texturaldifferences should occur between the control and test substrates. Colour differencesmay also influence insects during testing. Care is needed to ensurethe least hindrance with the presentation to the insect chemoreceptors, whichshould be in the usual way. Natural substrates could be whole plant, leaves,leaf discs, or specialized substrates such as twigs, blocks of wood, board, andpaper towel discs. Artificial substrates usually include agar-based artificialdiets, simple liquid-based artificial diets, styropors, or discs of foamedpolystyrene, or polyurethane, and glass fibre discs (Koul, 2005).Leaf discs are commonly used in preference or consumption bioassayswith chewing insects. These assays are important in estimating the biologicalpotential of the antifeedant effect of plant extracts in screening studies, andthey correspond as much as possible to the conditions of the practical application.However, it must be emphasized that these assays are short term. Forthe purpose of practical use, further biological assays must be performed inextracts or substances showing the best biological activity, which will be of along-term nature and will provide evidence on the practical applicability ofthe substances or extracts in plant protection. Nevertheless, such assays areirreplaceable as screening assays.Individual types of biological assays used for evaluating antifeedantefficiency are discussed by Koul (2005). However, in general, such assays can

140 R. Pavelabe divided into two groups according to the mode of the experiment: a choiceassay or a no-choice assay.The principle is that insects can choose either control or treated discs(choice) or insects may be exposed to the test substance only (no choice). Theno-choice situation often is more representative of our agricultural system,especially for monophagous species, but at the same time it is very sensitive(Fig. 6.1).The general procedure adopted in this test is that measured leaf discs arepunched out from substrates and treated either on one side or both sideswith a known quantity of test material in a carrier solvent. It is preferable touse emulsified solutions in water in order to avoid interference with leaf disctexture due to solvents (Isman, 2002). A method has been described by whichleaf surfaces can be covered with a uniform amount of a test chemical for bioassaywith leaf-feeding insects. Chemicals are dissolved in gelatine solutions,which can be sprayed evenly and which will adhere well to many leaf surfaces.Upon accurate application, the dosage per leaf area can be determined,which is an important practical viewpoint for the application itself.After application, the leaf discs are dried at room temperature and thenfed to candidate insects. Usually the arenas used are Petri dishes of variablesizes in which one treated and one control disc is placed (choice), or both theleaf discs are treated (no choice). In certain experiments five to ten treatedand untreated leaf discs are used and placed alternately in the Petri dishes ina choice situation. The number of larvae introduced into each arena is variabledepending upon the size and stage of the larvae used. There is also considerablevariation in the duration of experiments, both long term and shortFig. 6.1. No-choice test with extract obtained from Leuzea carthamoides against larvaeLeptinotarsa decemlineata, 48 h after application of 1% extract.

Allelochemicals in Pest Management 141term. The consumption in each experiment is measured using various digitizingleaf area meters.The bioassay is conducted for a very short duration (2 to 8 h) or until 50%of either disc is consumed. Several formulas and ways of denomination arepresented in the literature for calculating the biological effect (Koul, 2005).Nevertheless, the following formula to calculate feeding deterrence providesthe highest accuracy:Feeding deterrence (%) = (C – T)/(C + T) × 100, where C and T arethe consumption of control and treated discs, respectively (Koul, 2005;Pavela et al., 2008).As mentioned above, no-choice assays are most important from the practicalpoint of view, because they are closest in nature to practical application.It is thus advisable to favour such assays.When 90–100% feeding deterrence is obtained in the assays, efficientconcentration (EC 50and EC 95) can be determined in subsequent assays,which is another important parameter for the mutual comparison of extractor substance efficiency. Moreover, it is also of equal importance to determinethe time during which the given insect does not consume food treated withEC 95, as insects may become habituated to antifeedant substances or the biologicallyactive substances may be degraded due to the action of the environment(Koul, 2005). The insects can thus overcome the initial resistance againstfood treated with antifeedant substances, which may cause the products tolose their primary efficiency. However, it must be emphasized that in thisrespect, information on the period during which the insect does not consumefood treated with antifeedant substances is very sporadic, and therefore theexperiments must be completed with such information.6.5 Current Practical Use of Antifeedant SubstancesA long way still remains ahead in order to arrive at the full application potentialoffered by plant allomones in plant protection. No commercial productsbased purely on antifeedant efficiency are yet available. Nevertheless, someproducts that primarily contain substances with an insecticide effect, and atthe same time also show an antifeedant and anti-oviposition effect, are usedat present.Perhaps the most widely known application of antifeedant effects relatesto extracts made of the Indian plant Azadirachta indica A. Juss (syn. Melia azadirachta).A. indica has been well known in India and neighbouring countriesfor more than 2000 years as one of the most versatile medicinal plants havinga wide spectrum of biological activity. A. indica A. Juss and M. azedarach aretwo closely related species of Meliaceae.Extracts made of seeds of this plant contain numerous biologically activesubstances with insecticidal, fungicidal and bactericidal effects, used in manyindustries, ranging from medicine to agriculture. Many works have beenpublished concerning the plant itself, its useful substances, and biological

142 R. Pavelaefficiency including antifeedant effects (Jacobson, 1989; Schmutterer, 1990;Ascher, 1993).In the case of A. indica, substances falling in the group of limonoids(azadirachtin, salanin, nimbin etc.) are responsible for antifeedant activity.Both primary and secondary antifeedant effects have been observed in thecase of azadirachtin (Ascher, 1993). Primary effects include the process ofchemoreception by the organism (e.g. sensory organs on mouthparts whichstimulate the organism to begin feeding), whereas secondary processesare effects such as gut motility disorders due to topical application only(Schmutterer, 1990; Ascher, 1993). Inhibition of feeding behaviour byazadirachtin results from the blockage of input receptors for phagostimulantsor by the stimulation of deterrent receptor cells or both (Mordue andBlackwell, 1993). In a recent study by Yoshida and Toscano (1994), the relativeconsumption rate of Heliothis virescens larvae treated with azadirachtinwas 25% of the control, equivalent to the lowest assimilation efficiency of allnatural insecticides tested. In another study, larvae of Heliothis virescens consumedless food, gained less weight, and were less efficient at convertingingested and digested food into biomass (Barnby and Klocke, 1987). Sensitivitybetween species to the antifeedant effects of azadirachtin is profound.Order Lepidoptera appear most sensitive to azadirachtin’s antifeedanteffects, with Coleoptera, Hemiptera and Homoptera being less sensitive(Mordue and Blackwell, 1993).A whole range of commercial products based on azadirachtin are sold atpresent; however, they utilize another significant effect of azadirachtin typetetranotriterpenoids, namely the growth inhibition effect (Schmutterer, 1990).Extracts from the Indian tree of the Pongamia genus are another exampleof the commercial application of products based on antifeedant andanti-oviposition effects. This genus has one species only, that is Pongamiapinnata L. (syn. P. glabra Vent.; Derris indica Lamk.) which belongs to familyLeguminosae; subfamily Papilionaceae (Kumar and Kalindhar, 2003).P. pinnata is a rich source of flavonoids, the B-ring is either linked to afuran or pyran ring. Some of these flavonoids are known to have biologicalactivity. Antifeedant activities of various extracts of P. pinnata were observedagainst many insect pests of different crops. Under laboratory conditions,0.1% water emulsion of pongam oil (so-called karanj oil) showed antifeedantactivity against Amsacta moorei Butler (Verma and Singh, 1985) or Spodopteralitura F. (Kumar and Kalindhar, 2003). The pongam oil is known to possessstrong repellent activity for egg-laying against many insect pests (Khaire et al.,1993; Kumar and Kalindhar, 2003). The aqueous extracts of seeds and plantsof this species are known to possess ovicidal action against Phthorimaeaoperculella Zell. and Helopeltis theivora Waterh. (Deka et al., 1998).The repellent activities, including host deterrence and anti- oviposition,of pongam oil against the adults of the common greenhouse whitefly Trialeurodesvaporariorum Westwood were tested in greenhouses (Pavela andHerda, 2007a,b). Chrysanthemum plants treated with different concentrations(0.5–2.0%) of water-suspended pongam oil showed relatively longlastinghost deterrent and anti-oviposition effects on the adults of greenhouse

Allelochemicals in Pest Management 143whitefly. Although the repellent effect declined in time and concentration,strong effects on the reduction of oviposition were found, while this effectlasts at least 12 days after application, dependent on concentration.Another example of commercially produced extracts with antifeedanteffects is represented by aromatic plants. For aromatic plants, substancesresponsible for the smell and those that may be isolated using distillationor supercritical extraction have been studied the most. This group of substancesincludes the mono- and di- sesquiterpenes, phenols and some otherhydrocarbons similar in structure (Pavela, 2008a).Many monoterpenes from plant sources have been evaluated as feedingdeterrents against insects (Koul, 1982). However, capillin, capillarin, methyleugenol and ar-curcumene isolated from Artemisia capillaris show promise asantifeedant compounds against cabbage butterfly larvae, Pieris rapae crucivora.The relative strong antifeedant activity of capillin and capillarin suggestthat the C=O carbonyl group instead of CH 2methylene group, a C≡C in aside chain and a lactone ring are some of the many factors that contribute tothe biological activity (Yano, 1987). Various derivatives of these base compoundssuch as methyl eugenol reveal that the 3,4-dimethyl group and1-substituent of 3,4-dimethoxy-1-substituted benzenes contribute to theantifeedant activity (Yano and Kamimura, 1993).Aromatic hydrocarbons show a significant direct insecticide activity,so consequently they have been best studied (Pavela, 2006b, 2008a,b). Nevertheless,antifeedant efficiency is connected not only with aromatic hydrocarbonsin aromatic plants, but also with other polyphenolic substancessynthesized by such plants as part of their defence strategy against diseasesand pests.These polyphenolic substances are currently being studied and becauseof their huge biochemical variety, it is highly probable that an antifeedantactivity will be discovered in the near future that would lead to practicalapplication.Recent results of biological activity studies of substances obtained fromEurasian region plants, too, provide evidence that primary research is necessaryand may lead to commercial application. Very strong, long-lasting antifeedantactivity of seed extracts and root extracts of Leuzea carthamoides plantsagainst the Colorado potato beetle (Leptinotarsa decemlineata) was found(Pavela, 2004a, 2006a) (Fig. 6.2. a,b). This antifeedant efficiency against theabove mentioned pest led to the formulation of a new product based on antifeedantactivity. The research on this aspect, which has been performed bothin our laboratory and in departments around the world, is thus providing itsfirst results.6.6 Prospects for Products Based on Antifeedant SubstancesMany feeding inhibitors from plant sources have so far given excellent resultsin laboratory conditions. In field situations only a few of them are satisfactoryalternatives to traditional pest management. The chemical control is usually

144 R. Pavela(a)(b)Fig. 6.2. Effi ciency of extract obtained from L. carthamoides against larvae Leptinotarsadecemlineata. (a) Control; (b) 15 days after application.with broad-spectrum insecticides, and they have to be broad spectrumby necessity. They have to sell in amounts large enough to accommodatefinancial development, research, and marketing.Nevertheless, the use of antifeedants in pest-management programmeshas enormous appeal. They satisfy the need to protect specific crops while

Allelochemicals in Pest Management 145avoiding damage to non-target organisms so the potential value is great. Infact, insect damage to plants results from feeding or from transmission ofpathogens during feeding; therefore, the chemicals that reduce pest injury byrendering plants unattractive or unpalatable can be considered as potentialsubstitutes for conventional insecticides. The host choice of generalists andto some extent specialists may be modified when feeding inhibitors are used.The range of insect species targeted may be chosen by either the chemicalstructure of the inhibitor or the composition of a mixture of inhibitors, if differentinhibitors are active against different species within the range. Therefore,a multi-component defence strategy of plants themselves could be used,as shown in number of recent studies with non-azadirachtin types of limonoidinhibitors (Koul, 2005) where non-azadirachtin limonoids have two differentmodes of action, such as feeding deterrence and physiological toxicity, whichplay a significant role in the potentiation effect. Moreover some other plantextracts have high potential in commercial application (Pavela, 2007b).For the research to be successful, further assays must be performed andsuitable, new substances with high antifeedant efficiency must be sought.Important research will also concern determining synergism of the effects ofbiologically active substances as such synergism seems to be very significantin increasing the biological activity of substances that have insecticide effects(Pavela, 2008b).Most feeding inhibitors are less stable chemicals than traditional insecticidesand act with lower residual activity and environmental impact. Naturalpredators and parasitoids remain unharmed by feeding deterrents targetingthe herbaceous host insects. As the target sites of antifeedants are different,pesticide-resistant insect populations will still be affected by feeding inhibitors.Multi-component tactics will also slow down the resistance developmentto these new compounds. In fact, lack of resistance is very useful for thepractical application of antifeedants as it is unlikely that oligophagous insectscould develop general resistance to such deterrents, because this wouldresult in a rapid change of their host-plant range, which is determined mainlyby the occurrence of such chemicals in the non-host plants. Different molecularstructures of possible antifeedant compounds could be another advantage.The blend of active constituents might diffuse the selection process,mitigating the development of resistance compared to that expected with asingle active ingredient. This also supports the earlier mentioned contentionthat combination mixtures of antifeedants could be more effective thanindividual compounds.The huge variety of defensive mechanisms of plants, including thesynthesis of allelochemicals, thus provides a research focus in seeking newenvironmentally safe products to provide plant protection against phytophagousinsects. However, such research should be intensified and needsfull cooperation between basic or applied research, manufacturers and theusers. Such cooperation may lead subsequently to an important reduction inthe dependence of agriculture on chemical industries synthesizing toxicinsecticides, as well as increasing the quantity of safe foodstuffs and improvingthe health of this planet’s inhabitants.

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7 Potency of Plant Productsin Control of Virus Diseasesof PlantsH.N. VERMA 1 AND V.K. BARANWAL 21 Jaipur National University, Jaipur, India; 2 Advanced Centre of PlantVirology, New Delhi, IndiaAbstractThe exploitation of the inherent resistance phenomenon and manipulation of inducibledefence in plants is currently receiving much attention by researchers to control virusinfection. Recent advances in the molecular biology of resistance to virus infectionhave presented new approaches for making susceptible crops resistant against virusinfection. These approaches include pathogen-derived resistance to viruses (coatprotein-mediatedresistance, movement-protein-mediated resistance, replicase- andprotease-mediated resistance) and virus resistance through transgenic expression ofantiviral proteins of non-viral origin. Endogenously occurring substances in a fewhigher plants have also been reported to induce systemic resistance in susceptiblehosts against virus infections. Ribosome-inactivating proteins (RIPs) may also play animportant role in the prevention of virus infection.7.1 IntroductionViruses prove to be a menace to humans and the environment because oftheir disease-causing nature. They harm crops and cause economic losses.The recent outbreak of cotton leaf curl virus disease in cotton in the northerncotton-growing region of India has led to a huge yield loss of cotton fibre.Approximately 12,000 hectares of cotton were affected by leaf curl virusdisease during 1996 in Rajasthan alone. An annual loss of US$300 million iscaused by mungbean yellow mosaic virus (MYMV) by reducing the yield ofblack gram, mungbean and soyabean (Varma et al., 1992). The worldwidelosses caused by viral diseases are estimated at about US$60 billionper year.To reduce losses, scientists have explored several strategies to controlvirus infection. However, it is the exploitation of the inherent resistancephenomenon and manipulation of inducible defence in plants that arereceiving much attention from researchers. The common approach for© CAB International 2011. Natural Products in Plant Pest Management(ed. N.K. Dubey) 149

150 H.N. Verma and V.K. Baranwalintroducing resistance against a virus in crops has been achieved throughconventional plant breeding. Limited success has been achieved throughthis method. Recent advances in the molecular biology of resistance tovirus infection have provided new approaches to making susceptiblecrops resistant against virus infection. These approaches includepathogen-derived resistance to viruses (coat-protein-mediated resistance,movement-protein-mediated resistance, replicase- and protease-mediatedresistance) and virus resistance through the transgenic expression ofantiviral proteins of non-viral origin (Baulcombe, 1994).Resistance of plants to virus diseases may be broadly categorized intotwo groups: (i) constitutive; and (ii) induced. Constitutive resistance is heritableand occurs in cultivars, which have gene(s) conferring resistance toviral infection, whereas induced resistance has to be conferred afresh upon asusceptible plant and is normally not heritable. Induced resistance operatesthrough the activation of natural defence mechanisms of the host plant. Thetwo forms of induced resistance are systemic acquired resistance (SAR) andsystemic induced resistance (SIR). In both SAR and SIR, plant defences arepreconditioned by prior infection or treatment that results in resistance (ortolerance) against subsequent challenge by a pathogen or parasite. Greatstrides have been made over the past 20 years in understanding the physiologicaland biochemical basis of SAR and SIR. Much of this knowledge is dueto the identification of a number of chemical and biological elicitors, some ofwhich are commercially available for use in conventional agriculture. However,the effectiveness of these elicitors to induce SAR and SIR as a practicalmeans to control various plant diseases is just being realized.The infection of plants by necrotizing pathogens, including fungi, bacteriaand viruses, induces systemic resistance to subsequent attack by thepathogens. This resistance is called SAR ( Kessman et al., 1994; Ryals et al.,1994). It can also be activated in numerous plants by pre-inoculation withbiotic inducers including pathogens (Sticher et al., 1997). Endogenouslyoccurring substances in a few higher plants have been reported to inducesystemic resistance in susceptible hosts against virus infections. Such plantextracts have been used for protecting economically important crops againstvirus infections (Verma and Baranwal, 1989). Endo genously occurring virusinhibitors may also be ribosome-inactivating proteins (RIPs) (Barbieri andStirpe, 1982; Mansouri et al., 2006; Zhang et al., 2007). Virus infection is preventedif a mixture of RIP and virus is exogenously applied on the leaf surfaceof a susceptible host. RIPs presumably inhibit virus infection by enteringthe cytoplasm along with the virus particle and inhibiting protein synthesison host ribosomes, thus preventing early virus replication (Reddy et al.,1986).On the basis of their antiviral activity, virus inhibitors from plants can begrouped into two categories:1. Plant products that inhibit virus infection by inducing an antiviral stateeither at the site of application (local resistance) and/or at a remote site( systemic resistance) when applied a few minutes or hours prior to virus

Control of Virus Diseases of Plants 151challenge (Verma and Mukerji, 1975; Verma and Awasthi, 1979, 1980; Vermaet al., 1984; Verma et al., 1995a; Prasad et al., 1995; Verma et al., 1996). Suchplant products have been called systemic resistance inducers (SRIs).2. Basic proteins from plants that function by inactivating ribosomes of thehost and have been called RIPs (Barbieri et al., 1993, 2003; Van Damme et al.,2001).7.2 Systemic Resistance Inducers (SRIs)Induction of systemic resistance by plant extracts has been reviewed fromtime to time (Verma, 1985; Verma and Prasad, 1992; Verma et al., 1995a; Vermaet al., 1998). Physico-chemical characteristics of systemic resistance inducersfrom plants such as Boerhaavia diffusa (Verma and Awasthi, 1979, 1980; Srivastava,1995); Mirabilis jalapa (Verma and Kumar, 1980); Cuscuta reflexa(Awasthi, 1981); Clerodendrum aculeatum (Verma et al., 1984; Verma et al., 1996;Kumar et al., 1997; Srivastava et al., 2008); Bougainvillea spectabilis (Vermaet al., 1985; Verma and Dwivedi, 1984; Srivastava, 1995); Pseuderanthemumatropurpureum (Verma et al., 1985) have been studied in some detail (Table7.1).Induced resistance operates through the activation of natural defencemechanisms of the host plant. Extracts from brinjal (Verma and Mukherjee,1975) and a few other higher plants such as Boerhaavia diffusa (Verma et al.,1979), Bougainvillea (Verma and Dwivedi, 1984), Clerodendrum (Vermaet al., 1996; Kumar et al., 1997) and Datura (Verma et al., 1982) induce systemicresistance to viral multiplication in plants. The active products present in theseTable 7.1. Characteristics of systemic resistance inducers obtained from some higher plants.CharacteristicsSRI-yielding plantsBD CA/CI BS PA CR MJSource Root Leaf Leaf Leaf Leaf LeafThermal inactivation 80 90 80 0 70 90points (°C )Nature Glyco-protein Basic Protein Protein 0 Protein ProteinMolecular weight 30 34/29 and 34 28 0 14–18 24(kDa)Active against TMV TMV TMV TMV TMV TMVSHRV SHRV SHRV SHRV SHRV SHRVGMV GMV CGMMV CGMMV GMV PVYPLRV TmYMVBD = Boerhaavia diffusa; CA = Clerodendrum aculeatum; CI = C. inerme; BS = Bougainvillea spectabilis;PA = Pseuderanthemum atropurpureum; CR = Cuscuta refl exa; MJ = Mirabilis jalapa. TMV = Tobaccomosaic virus; SHRV = Sunnhemp rosette virus; GMV = Gomphrena mosaic virus; TRSV = TobaccoRingspot virus; TmYMV = Tomato Yellow Mosaic virus; CGMMV = Cucumber green mottle mosaic virus;PVY = Potato virus Y; PLRV = Papaya leaf reduction virus.

152 H.N. Verma and V.K. Baranwalextracts have no direct effect on viruses; their antiviral activity is mediated byhost cells in which they induce the antiviral state. All groups of plants respondto treatment with extracts of these plants. The active products in plant extractsinducing resistance are mostly small molecular weight proteins which sometimesmay be glycosylated. These proteins are highly thermostable and canwithstand prolonged treatment at alkaline/acidic pH. Plant products fromdifferent species vary in molecular weight and may differ in other characteristicstoo. Incubation of viruses with these antiviral agents has no effect on viralinfectivity, but they exert their antiviral effect by rendering host cells incapableof supporting viral replication. Resistance development is inhibited by actinomycinD and cyclohexamide. This implies that resistance is dependent onDNA-coded information of the cell and the antiviral activity of the antiviralagent is indirect. The antiviral agents showed no host specificity and wereactive against a wide range of viruses (Faccioli and Capponi, 1983).Systemic resistance inducers obtained from plants have been shown tobe effective against a wide range of viruses (Verma and Awasthi, 1979). Leafextract of Clerodendrum aculeatum is active against TMV, SHRV, GMV, TmYMV,(Verma et al., 1984), and an extract of Mirabilis jalapa is effective againstTmYMV, PLRV, CMV, CGMMV (Verma and Kumar, 1980). The extracttreatedplants become resistant to attack by diverse pathogens such as fungi,bacteria and viruses (McIntyre et al., 1981).Mechanism of systemic induced resistance by botanicalsSystemic induced resistance against virus infection by botanicals is not yetfully understood. The botanical resistance inducers themselves do not act onthe virus directly. Verma and Awasthi (1980) demonstrated the de novo synthesisof a virus inhibitory agent (VIA) in untreated leaves of Nicotinia glutinosa,whose basal leaves were treated with root extract of B. diffusa. Theseinduced substances inhibited almost completely tobacco mosaic virus in N.glutinosa, Datura stramonium and D. metel but inhibition of tobacco ring spotvirus or Gomphrena mosaic virus in Chenopodium amaranticolor was lesspronounced. In another study Verma and Dwivedi (1984) found that VIAinduced in C. tetragonoloba inhibited completely the infection of tobamovirusesin all the seven hypersenstive hosts tested. Yet in another study byKhan and Verma (1990), it was observed that VIA produced in C. tetragonolobafollowing treatment with extract of Pseuderanthemum bicolor inhibited completelySHRV, TMV, cucumber green mottle mosaic virus and PVX in theirrespective hypersensitive hosts, namely C. tetragonoloba, D. stramonium,C. amaranticolor and G. globosa. However, the VIA produced by B. spectabilis inN. tabacum cv. Samsun NN, N. glutinosa, and D. stramonium was less effectiveagainst TMV (Verma and Dwivedi, 1984). Thus, it appears that induction ofsystemic resistance by botanicals is non-specific and is effective against abroad spectrum of viruses.The production of VIA is maximum after 24 h of application of B. diffusaroot extract in N. glutinosa. However, B. spectabilis induced maximum VIA

Control of Virus Diseases of Plants 153activity after 48 h of its application in N. glutinosa. Thus, VIA production in ahost is time specific but a general phenomenon. Properties of VIA producedin C. tetragonoloba after application of B. spectabilis leaf extract has been studiedby Verma and Dwivedi (1984). The VIA could be precipitated by ammoniumsulfate and hydrolysed by trypsin but not by ribonulease indicatingthat it was a protein rather than a nucleic acid. The VIA induced by Pseuderanthemumbicolor in C. tetragonoloba was also proteinaceous with a molecularweight of 15 kDa (Khan and Verma et al., 1990). The production of VIA wassensitive to actinomycin D (Verma and Dwivedi, 1984). The mobile signalproduced VIA in the entire plant system. It appears that the putative messengerbecomes active soon after induction and starts producing VIA, reachesa maximal concentration and then starts to decline.Commonly associated with systemic acquired resistance induced bypathogens is the systemic synthesis of several families of serologically distinctlow molecular weight pathogenesis-related (PR) proteins. The localizationand timing of some PR proteins suggested their possible involvement inacquired resistance against viruses. However, definite proof that the inductionof PR proteins causes the acquired resistance has not been given. Plobnerand Leiser (1990) did not find the production of PR proteins during systemicresistance induced by carnation extract in Xanthi-nc tobacco plants. Absenceof PR proteins during induction of SAR by botanicals suggests that anotherbiochemical chain of reaction, other than one operating in pathogen / chemicalinduced SAR, might be operating during botanical induced systemicresistance against virus infection in plants.Durrant et al. (2004) reported that SAR is a mechanism of induced defencethat confers long-lasting protection against a broad spectrum of microorganisms.SAR requires the signal molecule salicylic acid (SA) and is associatedwith accumulation of PR proteins, which are thought to contribute toresistance.Hansen (1989), while reviewing antiviral chemicals for plant diseasecontrol, gave the following characteristics of an ideal antiviral compoundthat will serve all purposes for virus disease management in crops:●●●●Soluble in water or non-phytotoxic solvents.Effective against at least some agriculturally important viruses atnon-phytotoxic concentrations.Easily taken up by plants and distributed throughout the system.Non-toxic by itself and in its catabolic forms to humans, plants andwildlife.Botanical resistance inducers can be classified as ideal virussuppressingagents, as they have all the characteristics of an ideal antiviralcompound. The resistance-inducing proteins from Boerhaavia diffusa andClerodendrum aculeatum can be applied directly by spraying on systemichosts, for management of some commonly occurring virus diseases undergreenhouse conditions or field conditions. The agricultural role of endogenousantiviral substances of plant origin has been reviewed by Vermaet al. (1995).

154 H.N. Verma and V.K. BaranwalThe induced antiviral state in N. glutinosa by SRI from C. aculeatumdecreased considerably after 3 days (Verma and Varsha, 1994). However, theresistance inducing ability of C. aculeatum SRI could be enhanced up to6 days by priming it with certain proteinaceous additives (Verma and Versha,1994). The activity is probably enhanced by modification or enhanced stabilityof proteinaceous inducers by these additives. Thus, one unexploitedapproach to engineering virus resistance is the manipulation of inducibledefences in plants. The production of systemic resistance by the use of botanicalswill be effective against a broad spectrum of viruses and will not breakdown when plants are exposed to high temperatures.Suppression of disease symptoms by true inhibitors may be accomplishedeither by acting on the first stage of the infection process, which is theadsorption of the virus into the host cell, or by blocking or competing withthe virus receptor sites on the leaf surface (Ragetli, 1957; Ragetli andWei ntraub, 1962) or by affecting the susceptibility of the host by altering hostcell metabolism (Verma and Awasthi, 1979). Bozarth and Ross (1964) suggestedthe phenomenon of SAR as a result of the initial infection by which asignal was generated at the site of application and transported throughoutthe plant to respond more effectively to the subsequent infection.Induction of systemic resistance by resistance inducers obtained fromplant extracts was first detailed by Verma and Mukerjee (1975). Extracts froma few plants induce an antiviral state by acting through an actinomycin D(AMD) sensitive mechanism (Verma and Awasthi, 1979; Verma et al., 1984).AMD is an inhibitor of protein synthesis at the transcription level. Concomitantapplication of AMD with SRI reversed the induction of resistance insusceptible plants. However, induction of resistance remains unaffectedwhen AMD is applied 12 h post-treatment. This gives an indication thatplant-extract-induced resistance is a host-mediated response (Verma et al.,1979).The activity at a distance from the point of application might be explainedby the supposition that the SRI present in the plant extract selectively attachesat the surface, and a type of chain reaction starts that elicits the transcriptionof defence-related genes, leading to the production of a new VIA (Verma andAwasthi, 1979).Ribosome inactivating proteins (RIPs)The basic proteins that function by inactivating the ribosome of the host havebeen called RIPs. The RIPs have been shown to be N-glycosidases, whichremove a specific adenine base in a conserved loop of the 28s rRNA ofeukaryotic organisms (Endo and Tsurugi, 1987; Endo et al., 1987) or the 23srRNA of prokaryotes (Hartely et al., 1991). Such damaged ribosomes can nolonger bind the elongation factor-2 (Gessner and Irvin, 1980; Rodes and Irvin,1981). The RIPs damage the ribosome, arrest protein synthesis and cause celldeath. RIPs show antiviral activity against both animal and plant viruses(Barbieri and Stirpe, 1982) and have been classified into two types (Stirpe

Control of Virus Diseases of Plants 155et al., 1992; Barbieri et al., 1993). Type-I RIPs consist of a single polypeptidechain that is enzymatically active. These are scarcely toxic to animals andinhibit protein synthesis in cell-free systems, but have little or no effect onwhole cells. The three well known antiviral proteins PAPs, dianthins andMAP belong to this category. Type-II RIPs contain two types of polypeptidechains. Chain A is linked to chain B through a disulfide bond. Chain B bindsthe toxin to the cell surface and chain A enzymatically inactivates the ribosomes(Olsnes and Pihl, 1982). These are toxic to cells and inhibit proteinsynthesis in intact cells and in cell-free systems. Several similarities existamong the type-I RIPs. They are all basic proteins with a molecular weight inthe range 26–32 kDa. They have an alkaline isoelectric point and are usuallystable, being resistant to denaturing agents and protease. A majority of theRIPs are glycoproteins. Type-I RIPs are strongly immunogenic. Strocchi et al.(1992) established that cross reactivity between RIPs obtained from unrelatedplants was either very weak or absent.Recently cloning and expression of antiviral/ribosome-inactivatingprotein from Bougainvillea xbuttiana was reported by Choudhary et al. (2008).They reported that full-length cDNA encoding ribosome inactivating/ antiviral protein (RIP /AVP) consisted of 1364 nucleotides with an openreading frame (ORF) of 960 nucleotides encoding a 35.49 kDa protein of 319amino acids.The three well known antiviral proteins, namely PAPs (from Phytolaccaamericana), dianthins (from Dianthus caryophyllus) and MAPs (from Mirabilisjalapa) belong to the category of type-I RIPs.Pokeweed antiviral proteins (PAPs)The antiviral protein present in the leaves of Phytolacca americana was purifiedto homogeneity and its molecular weight determined as 29 kDa (Irvin, 1975).This protein, called pokeweed antiviral protein (PAP), had a PI of 8.1 (Irvin,1983). The antiviral effect of PAP was most pronounced when it was coinoculatedwith the virus. PAP also inhibited virus infection when appliedprior to virus challenge. Local lesion formation by TMV on N. tabacum cv.Xanthi-nc was inhibited by nearly 70% even after 48 h of treatment. The virusinhibitory effect of PAP increased with the decrease in the time lapse betweentreatment and challenge inoculation. PAP was less effective in preventingvirus infection when applied a short time after virus inoculation. No inhibitionwas observed when PAP was applied 50 min after virus infection (Chenet al., 1991). PAP reduced infectivity of several mechanically transmitted RNAand DNA viruses when the purified virus or sap from virus-infected plantswas mixed with an equal volume of PAP solution and the mixture rubbed onthe leaves of the local-lesion hosts (N. glutinosa / TMV; Chenopodium quinoa /CMV; C. amaranticolor / TMV, CMV, alfalfa mosaic virus, PVY; Gomphrena globosa/ PVX) or systemic hosts (Brassica campestris / cauliflower mosaic virus;N. benthamiana / African cassava mosaic virus). PAP, thus, appears to be ageneral inhibitor of virus infection (Tomlinson et al., 1974; Stevens et al., 1981;

156 H.N. Verma and V.K. BaranwalChen et al., 1991; Picard et al., 2005). PAP also shows antiviral activity againstseveral animal viruses. It is toxic to cells infected with poliovirus (Usseryet al., 1977) and influenza virus (Tomlinson et al., 1974). It inhibits multiplicationof herpes simplex virus type 1 (Arnon and Irvin, 1980) and humanimmunodeficiency virus (HIV; Zarling et al., 1990). P. americana is now knownto contain three proteins (PAP I, II and III) with similar biological properties.A new insight into the antiviral mechanism of PAP is that PAP depurinationof Brome mosaic virus RNA impedes both RNA replication and subgenomicRNA transcription (Picard et al., 2005).Rajmohan et al. (1999) reported that PAP isoforms PAP-I, PAP-II andPAP-III depurinate RNA of HIV-I. A non-toxic PAP mutant inhibiting pathogeninfection via a novel SA-independent pathway was reported byZoubenko et al. (2000). PAP inhibits translation by depurinating the conservedsarcin/ricin loop of the large ribosomal RNA. Depurinating ribosomesare unable to bind elongation factor 2, and, thus, the translocation stepof the elongation cycle is inhibited. Ribosomal conformation is required fordepurination that leads to subsequent translation inhibition (Mansouri et al.,2006).Carnation antiviral proteins (Dianthins)Sap from carnation leaves shows virus inhibitory activity (Van Kammenet al., 1961; Ragetli et al., 1962). Dianthin 30 and 32 were isolated from theleaves of Dianthus caryophyllus (Ragetli et al., 1962). Local lesion productionby TMV on N. glutinosa was inhibited by 100% when the inhibitor was coinoculatedwith the virus (Stevens et al., 1981). The molecular weights asdetermined by SDS–PAGE are 29.5 and 31.7 kDa, respectively (Stirpe et al.,1981). Immunoelectrophoresis revealed that dianthin 32 is distributed in thegrowing shoots and in the young and old leaves of D. caryophyllus and dianthin30 is distributed throughout the plant (Reisbig and Bruland, 1983). Thetwo are glycoproteins containing mannose and show a weak cross reaction.The nucleotide sequence of cDNA encoding dianthin 30 has been determined(Legname et al., 1991). The carnation proteins are also inducers of systemicresistance (Plobner and Leiser, 1990). Cho et al. (2000) performed isolationand characterization of cDNA encoding ribosome inactivating protein fromDianthus sinensis L.Mirabilis antiviral protein (MAP)The roots, leaves and stem of Mirabilis jalapa show high inhibitory activityagainst plant viruses. The Mirabilis jalapa leaf extract, when used as a foliarspray 24 h prior to virus inoculation, suppressed disease symptoms on a fewsystemic hosts (tomato/tomato yellow mottle virus; Cucumis melo var.momordica/CMV; Cucumis sativa/cucumber green mottle mosaic virus;tomato/tomato yellow mosaic virus; urd/yellow mosaic of urd) (Verma and

Control of Virus Diseases of Plants 157Kumar, 1980). A 50–60% reduction of the virus content in the treated plantswas observed in the infectivity assays. M. jalapa extract was able to check thepopulation of aphids and whiteflies and, thereby, control the natural spreadof a few viruses on the systemic hosts (Verma and Kumar, 1980).MAP isolated from root inhibits mechanical transmission of TMV, PVY,cucumber green mottle mosaic virus, and turnip mosaic virus on local-lesionand systemic hosts and can induce systemic resistance of a low order whenapplied to basal leaves (Kubo et al., 1990). The purified protein consists of asingle polypeptide without a sugar moiety and has a molecular weight of24.2 kDa. It is a basic protein rich in lysine content, with a PI of 9.8 (Kubo etal., 1990). The complete amino acid sequence of MAP has been determined.It consists of 250 amino acids and its molecular weight as determined fromthe sequence is 27,833 kDa. The native MAP was resistant to proteasedigestion (Habuka et al., 1989).MAP produced by M. jalapa cells in suspension culture showed comparablebiological activity with that of the roots and leaves and also reactedpositively with anti-MAP serum (Ikeda et al., 1987). Several nutritional andhormonal factors also affect the formation of MAP by M. jalapa cells in suspensionculture (Ikeda et al., 1987). Bolognesi et al. (2002) reported ribosomeinactivatingand adenine polynucleotide glycosylase activities in M. jalapa L.tissues.Ricinus (RICIN A) antiviral proteinThe ricinus antiviral protein is a type II RIP isolated from Ricinus communisthat has a molecular weight of 65 kDa. Its is a heterodimer consisting of chainA similar to the type-I RIP and chain B linked together by a disulfide bond.Chain B has great similarity to mammalian lectins A. Once inside the cellchain A acts as a type-I RIP and inactivates protein translation machinery.The cDNA for ricin has been cloned (Lamb et al., 1985). Both A and B chainsare encoded by a single gene which has no intron. Detection of ricin andother ribosome-inactivating proteins by an immuno-polymerase chainreaction assay was reported by Lubelli et al. (2006).Role of RIPsThe above examples of RIPs show their important role against both plantviruses and also RNA- and DNA-containing animal viruses (Barbieri et al.1993). The mechanism of antiviral activity suggested for both plant andanimal system involves increased permeability of and easier entry of RIPsinto virus-infected cells, blocking of protein synthesis and reduced virusmultiplication (Barbieri et al., 1993).Virus resistance previously observed in transgenic plants expressing coatprotein genes, and so on, has been specific for the virus from which the genesare derived or for closely related viruses (Beachy et al., 1990) but transgenic

158 H.N. Verma and V.K. Baranwaltobacco and tomato plants expressing the pokeweed antiviral gene are foundto be resistant to a broad spectrum of plant viruses (Lodge et al., 1993).A problem often encountered in using type-I RIPs is the fact that theycannot inhibit protein synthesis in intact cells. However, when coupled totype-II RIP, they can be used effectively. The toxicity of plant materialscontaining type-II RIPs have long been known and has great medicinalpotential. Recently, apoptosis was described in both lymphoid tissue and inthe intestine of abrin- and ricin-poisoned rats. Apoptosis was also observedin tissue culture of cancer cells treated with ricin.The antiviral activity of the RIP could be due to inactivation of ribosomeof the infected plant cell. As compared to the usefulness in the plants, the roleof RIPs in human and animal systems has been much more widely documented.In spite of all these efforts, the biological significance of RIPs innature is not yet known and moreover, their antiviral action does not seem todepend upon the inhibition of host ribosomes (Chen et al., 1993).Great potential exists today in elucidating the possible significance ofRIPs and their exact antiviral role which may not always depend upon inhibitionof host ribosomes. Their co-action with another antiviral mechanismalso needs to be explored, especially their place in the cascade of events followingviral infection up to establishment of resistance, both systemic orlocalized.7.3 Pathogen-induced Systemic Acquired Resistance (SAR)The infection of plants by necrotizing pathogens, including fungi, bacteriaand viruses, induces systemic resistance to subsequent attack by the pathogens.This resistance is called systemic acquired resistance (SAR) (Kessmanet al., 1994; Ryals et al., 1994; Prasad et al., 2001). Also, it can be activated innumerous plants by pre-inoculation with biotic inducers including pathogens(Sticher et al., 1997). One of the prominent features of SAR is that resistanceis expressed against pathogen which can be widely different from theinitial infecting pathogens.In a range of plant species, the development of necrotic lesions in responseto pathogen infection leads to induction of generalized disease resistance inuninfected tissue. Thus, TMV inoculated hypersensitive tobacco cultivardevelops systemic resistance against TMV (virus), Phytophthora parasitica var.nicotinae (fungi) and Pseudomonas tabaci (bacteria). TMV also induced resistanceagainst Peronospora tabacina and reduced reproduction of the aphidMyzus persicae (McIntyre et al., 1981). Thus a single viral agent induced resistancein tobacco against diverse challenges.The first report of virus induced SAR came in 1952. Primary inoculationof the lower leaves of D. barbatus with carnation mosaic virus (CarMV)resulted in the development of fewer lesions on the upper leaves upon challengeinoculation with CarMV (Gilpatrick and Weintraub, 1952). Virusinducedresistance was further substantiated by Ross (1961a, 1961b) andLoebenstein (1963). Cucumber plants infected with tobacco necrosis virus

Control of Virus Diseases of Plants 159(TNV) protected the plant systemically against disease caused by the fungusColletotrichum lagenarium (Jenns and Kuc, 1977). SAR expessed in Vigna plantsfollowing inoculation with TNV against challenge by TNV was not expressedagainst challenge by a CMV which infects the host systemically (Pennazzioand Roggero, 1991). Associated with SAR was the stimulation of ethyleneformingenzyme activity. Infection of ecotype Dijon of Arabidopsis thalianawith turnip crinkle virus (TCV) leads to the resistance against further infectionby TCV or Pseudomonas syringae (Uknes et al., 1993). SAR in cucumberplants against powdery mildew disease, caused by Sphaerotheca fuliginea(Schlechtend Fr.) Pollacci, was induced by localized infection in cucumbercotyledons with TNV (Farrag et al., 2007).The SAR phenomenon is observed both in dicotyledonous and monocotyledonousplants; it provides the third and final line of defence againstpathogens. The first line of defence consists of genetically inherited resistancemechanisms that make plants constitutively resistant to the majority ofpathogens present in the environment. The second line of defence is activatedin the immediate vicinity of the infected or wounded site in an attemptto prevent the spread of pathogens throughout the plant. The local resistanceresponse develops more rapidly than SAR and involves cell-wall and cuticlestrengthening, synthesis of toxins, antifeedants and the production ofdefence-related proteins including the PR proteins. In addition to long-distancesignal molecules, local resistance may be partially mediated throughrelatively immobile endogenous elicitors, which include oligogalacturonidefragments of the plant cell wall (Lamb and Dixon, 1990). Several lines of evidencesuggest that endogenous SA is a signal molecule in SAR. Involvementof SA in SAR came from the discovery that endogenous SA increases by atleast 20-fold in the virus-inoculated leaves of tobacco (Malamy et al., 1990).The increase coincides with the appearance of hypersensitive response (HR)lesions on the inoculated leaves. Accumulation of SA increased with theintensity of HR and was proportional to the dose of virus inoculum (Yalpaniet al., 1991). Tissue accumulation of SA in TMV inoculated xanthi-nc tobaccoparalleled or preceded detectable increase in the levels of PR-1 mRNA inboth inoculated and uninoculated leaves (Malamy et al., 1990).7.4 SAR and the Role of PR ProteinsSAR strongly correlates with the coordinate expression of at least nine familiesof genes (sar genes), of which several encode the PR proteins (Wardet al., 1991). Generally, tissues in which a significant amount of PR protein hasbeen induced are more resistant to infection by pathogen than those that lackPR proteins. PR proteins were first discovered in 1970 in tobacco plants reactinghypersensitively to TMV infection (Gianinazzi et al., 1970; Van Loon andKammen, 1970). PR proteins are produced in plants in response to infectionby viruses, bacteria (Metraux and Boller, 1986), fungi (Gianinazi et al., 1980)and viroids (Conejero et al., 1979) and are also synthesized in response tochemical treatments and specific physiological stresses (Stintzi et al., 1993).

160 H.N. Verma and V.K. BaranwalSeveral classes of the PR proteins either possess direct antimicrobial activityor are closely related to classes of antimicrobial proteins. These include β-1,3-glucanase, chitinase, cysteine-rich proteins related to thaumatin and PR-1proteins. An in vivo role in disease resistance has not been demonstrated forany of the PR proteins. Alexander et al. (1993) demonstrated that constitutivehigh level expression of PR 1-a in transgenic tobacco results in tolerance toinfection by Peronospora tabacina and Phytophthora parasitica var. nicotinae. Onthe other hand, transgenic tobacco plants expressing PR 1-b gene exhibitedno reduction in the severity of TMV symptoms (Cutt et al., 1989). Quite oftenSAR was not correlated with the induction of PR proteins (Kopp et al., 1989; Yeet al., 1989; Cohen et al., 1993; Kessman et al., 1994). Fraser found a poorcorrelation between the levels of PR 1-a protein and also gave evidence forthe occurrence of PR proteins in leaves of healthy tobacco plants duringflowering (Fraser, 1981; 1982). In conclusion, most of the evidence shows thatthe PR proteins are closely associated with, and not necessarily responsiblefor, induced resistance.7.5 Induced Resistance and the Role of Induced AntiviralProteinsVirus-induced new antiviral protein componentsAntiviral substances are formed in plants responding hypersensitively tovirus infection (Verma and Prasad, 1992) and have been recognized as phosphorylatedglycoproteins (Faccioli and Capponi, 1983), glycoproteins (Wieringaand Dekker, 1987), RNA (Kimmins, 1969), traumatic acid (Kato andMisawa, 1976), and protein-like substances (Nienhaus and Babovic, 1978).Chadha and MacNeill (1969) found the formation of an antiviral principle intomato plants systemically infected with TMV. These antiviral compoundsare not generally specific to the plants. Thus, antiviral substances producedin capsicum plants could reduce PVX infection on Gomphrena globosa as wellas Solanum tuberosum (Nagaich and Singh, 1970).The presence of an antiviral factor (AVF) was established in virus- infectedplants which could decrease the number of local lesions produced by TMVand PVY (Sela and Applebaum, 1962). Partially purified AVF from TMVinfected N. glutinosa plants was found to contain both protein and RNA (Selaet al., 1964). It was sensitive to ribonuclease and was resistant to proteolyticenzymes (Sela et al., 1966).Mozes et al. (1978) established that the purified AVF is a phosphorylatedglycoprotein of molecular weight 22 kDa on SDS gels. It is sensitive to pronaseunder conditions suitable for proteolysis of glycoproteins. It remainsactive after treatments with SDS and is stable at pH 2.0. It resembles interferonin many of its properties (Mozes et al., 1978).Previously it was believed that TMV infection was necessary for AVFproduction. But Edelbaum et al. (1983) found that TMV infection could besubstituted by treatment with a mixture of Poly (I), Poly (C), cAMP

Control of Virus Diseases of Plants 161and cGMP for the induction of active AVF in leaves and callus cultures ofN. glutinosa.Inhibitor of virus replicationLoebenstein and Gera (1981) reported, for the first time, an inhibitor of virusreplication (IVR). IVR is released into the medium from TMV-infected cells ofSamsun NN plants and inhibits replication in protoplasts from local-lesionrespondingSamsun plants. IVR is detected as soon as 24 h after inoculationof protoplasts and it is effective when applied up to 18 h after inoculation.IVR is neither host nor virus specific. It inhibits TMV, CMV and also PVX(Gera and Loebenstein, 1983). Gera and Loebenstein (1983) reported that IVRalso inhibited TMV replication in intact leaves when applied to cut stems orwhen used as a spray.Plant-extract-induced virus inhibitory agentIt would be interesting to study the entire cascade of events and properties ofthe virus inhibitory agent (VIA) induced by antiviral agents. Treatment oflower / upper leaves of hypersensitive or systemic hosts of virus withantiviral agents results in the development of resistance throughout the planta few hours later. This is detectable by challenge inoculation with virusesproducing local lesions or systemic symptoms. The lesions are either reducedor totally absent and systemic symptoms are either milder or totally suppressed.Plant extracts or the semi-purified proteins from these plants stimulatethe hosts to produce VIAs that spread to surrounding tissues and otherplant parts (Verma et al., 1996). The VIAs have been isolated from leaves ofplants treated with phytoproteins and they have been shown to inactivatethe viruses in vitro (Verma and Awasthi, 1980; Verma and Dwivedi, 1984;Verma et al., 1996). The production of VIA leading to resistance seems to bean activation of a pre-existing system and hence is easily stimulated. VIA isable to move from one leaf to another through the vascular system of theplant.Therefore, the antiviral agents from plants can be broadly grouped intotwo categories based on their mode of action: (i) those affecting virus in vitro;and (ii) those affecting via host plants.Amongst the latter, we can distinguish (1) those that act by affecting hostsusceptibility and which need to be applied in leaf tissue before or at the timeof virus inoculation, e.g. protein inhibitors from Phytolacca spp., Dianthusspp., Chenopodium spp. and so on, and (2) those acting by inducing the hostresistance mechanism, which can truly be called antiviral agents, and act byobstructing the establishment of virus. They exert their effect when applied afew hours before virus inoculation. Their effect is visible even on non-treatedparts of susceptible plants (Verma and Mukherjee, 1975; Verma and Awasthi,1979; Verma et al., 1979, 1980, 1982, 1985, 1995, 1996; Kumar et al., 1997).

162 H.N. Verma and V.K. BaranwalVerma and Awasthi (1980) reported that the synthesis of VIA is inhibitedif AMD is applied soon after extract treatment. The VIA synthesized is neithervirus specific nor host specific. Extracts containing VIA when incubated withthe virus reduced their infectivity. VIAs from a few hosts have been characterized.The VIA synthesized in the leaves of N. glutinosa, treated with B.diffusa root extract reduced infectivity of TMV on N. glutinosa, Datura stramoniumand D. metel (Verma and Awasthi, 1980). It was, however, less effectivein inhibiting TRSV and GMV on C. amaranticolor. The VIA production wasmaximum after 24 h treatment with the extract. The VIA has a proteinaceousnature (Verma and Awasthi, 1980). VIA synthesized in C. tetragonoloba plantsfollowing treatment with B. spectabilis leaf extract prevented infection oftobamoviruses in seven hypersensitive hosts (Verma and Dwivedi, 1984) andits production was maximum after 48 h treatment. The VIA showed characteristicsof a protein (Verma and Dwivedi, 1984). VIA produced in the samehost upon treatment with Pseuderanthemum bicolor extract completely preventedinfection of SHRV, TMV, CGMMV, and PVX on C. tetragonoloba, D.stramonium, C. amaranticolor and G. globosa, respectively (Khan and Verma,1990).VIA and the antiviral state are induced concomitantly with the systemicresistance induction in host plants, following treatment with certain plantextracts. VIA is an inducible gene product like AVF, IVR and PR proteins. Thephytoproteins occurring in the root / leaf extracts of Boerhaavia diffusa, Clerodendrumaculeatum, Clerodendrum inerme and B. spectabilis etc. possess strongsystemic-resistance-inducing properties (Verma and Prasad, 1992).Clerodendrum aculeatum and Boerhaavia diffusa, the two potentantiviral plantsClerodendrum and Boerhaavia are economically important plants becausemany of their species possess medicinal properties. Almost every part ofthese plants is credited with some medicinal properties and is employed intraditional Indian system of natural therapy.Virus inhibitors from Boerhaavia diffusa and Clerodendrum aculeatuminhibit several plant viruses. These inhibitors modify the susceptibility ofhost plants towards virus infection. The roots of B. diffusa are a rich source ofa basic protein, which has been used for inducing systemic resistance inmany susceptible crops against commonly occurring viruses (Verma andAwasthi, 1979, 1980; Verma et al., 1979; Awasthi et al., 1984, 1985, 1989; Vermaet al., 1995, 1999). This protein or antiviral agent was active against sphericaland tubular viruses in hypersensitive hosts such as Datura metel, Nicotianatabacum var Ky-58, N. glutinosa / TMV, Cyamopsis tetragonoloba / SHRV, Vignasinensis / SHRV, and systemic hosts such as Nicotiana tabacum c.v NP-31 / TMV,Crotolaria juncea / SHRV, N.glutinosa / TRSV, when applied a few hours (17–24h) before virus inoculation or when tested after mixing with virus inoculum(Verma and Awasthi, 1979; Awasthi et al., 1984). The inhibitor is a basic glycoprotein(70–80% protein, 8–13% carbohydrate) with a molecular weight of

Control of Virus Diseases of Plants 16316–20 kDa as determined by gel-filtration chromatography (Verma et al.,1979). The protein has a PI of around 9 and has a molecular weight of 30 kDa(Srivastava, 1995). The RIP was found to be extremely thermostable (Vermaand Awasthi, 1979). Following treatment with the systemic RIP, the host producesa VIA. The VIA shows characteristics of a protein and reduces infectivityof the viruses both in vitro and in vivo (Verma and Awasthi, 1980). Upongel filteration on Sephadex G-25, two active fractions exhibiting proteincharacteristics were recovered (Verma and Awasthi, 1980).The protein occurring in B. diffusa functions as a signal molecule and is ofgreat interest as it has a role in stimulating the defence system of plantsagainst viruses (Singh, 2006; Verma et al., 2006). The VIA is present in bothtreated and untreated leaves (Verma and Awasthi, 1980). Micropropagationof B. diffusa has been carried out for producing the systemic RIP for viraldisease management (Gupta, 1999; Gupta et al., 2004).Susceptible healthy hosts upon treatment with C. aculeatum extractdevelop complete resistance within 4–6 h. The SIR is reversed by the simultaneousapplication of AMD (Verma et al., 1984). Treatment with SRI from C.aculeatum leaves, triggers accumulation of a new defensive VIA in treatedand non-treated leaves of healthy host plants (Verma et al., 1984; Verma et al.,1996, 1999; Srivastava 1999). The SRI in crude sap is resistant to denaturationby organic solvents and extremely thermostable (Verma et al., 1984). Theresistance-inducing activity of C. aculeatum SRI (CA-SRI) is not affected byexogenous application of proteases (Verma et al., 1996). Leaf extract of C. aculeatumis most effective for controlling virus diseases in crop plants (Vermaet al., 1995a; Srivastava 1999; Srivastava et al., 2004). Leaf extract, whensprayed on susceptible host plants, prevents infection of mechanically andwhite fly transmitted viruses in several hosts, e.g. tomato yellow mosaicvirus, tobacco mosaic virus, sunnhemp rosette virus and tobacco leaf curlvirus (Verma et al., 1984; Verma et al., 1995a) (Table 7.2).Table 7.2. Plants containing virus inhibitory activity.Name Family ReferenceAmaranthus albus Amaranthaceae Smookler, 1971Alternanthera brasiliana Amaranthaceae Noronha et al., 1983Acacia arabicaLeguminosaeGupta and Raychauduri, 1971a(Fabaceae)Acer insulera Aceraceae Yoshi et al., 1954Agava americana Agavaceae Simon, 1963Ailanthes excelsa Simaroubaceae Patel and Patel, 1979Alternanthere fi coidea Amaranthaceae Noronha et al., 1983Amaranthus caudatus Amaranthaceae Smooker, 1971Argemone mexicana Papaveraceae Patel and Patel, 1979Beta vulgaris Chenopodiaceae Paliwal and Narinai, 1965Continued

164 H.N. Verma and V.K. BaranwalTable 7.2. Continued.Name Family ReferenceBoerhaavia diffusa Nyctaginaceae Verma and Baranwal, 1988; Mehrotraet al., 2002; Sukhdev, 2006; Singh,2006Bougainvillea spectabilis Nyctaginaceae Verma and Dwivedi, 1983Brassica oleracea Brassicaceae Verma, 1973Callistemon lanceolatus Myrtaceae Gupta and Raychaudhuri, 1971a,b;Carissa edulis Apocynaceae Tolo et al., 2006Celosia plumose Amaranthaceae Patil, 1973Chenopodium amaranticolor Chenopodiaceae Smookler, 1971 Alberghina, 1976Chenopodium ambrisioides Chenopodiaceae Verma and Barranwal, 1983Cinchona ledgeriana Rubiaceae Gupta and Raychauduri, 1971bCitrus medicatimonum Rutaceae Ray, et al., 1979Clerodendrum aculeatum Verbenaceae Verma et al., 1984; Srivastava 1999;Srivastava et al., 2004; Singh, 2006Clerodendrum fragrans Verbenaeceae Verma et al., 1984Cocos nucifera Arecaceae Gendron, 1950Cucurbita maxima Cucurbitaceae Weintrab and Willison, 1983Cuscuta refl exa Cucurbitaceae Awasthi, 1982Datura metel Solanaceae Verma and Awasthi, 1975Datura stramonium Solanaceae Paliwal and Narinai, 1965Dianthus caryophyllus Caryophylaceae Van Kammen et al., 1961Eucalyptus tereticornis Myrtaceae Ray et al., 1979Eugenia jambolana Myrtaceae Verma et al., 1969Euphorbia hitra Euphorbiaceae Weeraratne, 1961Gomphrena globosa Amaranthaceae Grasso and Shephard, 1978Gyandropsis pentaphylla Asteraceae Paliwal and Nariani, 1965Helianthus annus Asteraceae Johari, et al., 1983Hordeum vulgareGramineae Leah, et al., 1991( Poaceae)Mirabilis jalapa Nyctaginaceae Kataoka et al., 1991Opuntia robusta Cactaceae Simons et al., 1963Petunia hybrida Solanaceae Singh, 1972Phyllanthus frafernus Euphorbiaceae Saigopal et al., 1986Phytolacca dodecandra Phytolaccaceae Reddy et al., 1984PseudoranthemumAcanthaceae Verma et al., 1985atropurpureumRumex hastatus Polygonaceae Singh, et al., 1977Solanum nigrum Solanaceae Vasudeva and Nariani, 1952Terminala chebula Combretaceae Gupta and Raychauduri, 1971bTetragonia expansa Tetragoniaceae Benda, 1956Zingiber offi cinale Zingiberaceae Ray et al., 1979The systemic resistance inducer from Clerodendrum aculeatum andClerodendrum inerme have been purified and characterized. A 34-kDa basicprotein was isolated from the leaves of C. aculeatum: 64 μg/ml of proteinprovided complete protection of untreated leaves against TMV infection in

Control of Virus Diseases of Plants 165N. tabacum Samsun NN (Verma et al., 1996). Two basic proteins of 29 and34 kDa (CIP-29 and CIP-34) were isolated from the leaves of C. inerme. Aresistance-inducing protein of 31 kDa was isolated from the same plant(Parveen et al., 2001). The minimum amount of purified proteins required toinduce systemic resistance varied from 16 μg/ml for CIP-29 to 800 μg/mlfor CIP-34 (Prasad et al., 1995). CAP-34, a protein from C. aculeatum, whenapplied to lower leaves of Carica papaya, suppressed the disease caused bypapaya ring spot virus (PRSV). In the control papaya plants the typical diseasesymptoms of PRSV, that is, mosaic to filiformy, appeared in 95% of theplants between 30 and 60 days after virus inoculation. In the CAP-34-treatedpapaya plants the symptoms appeared in only 10% of the plants during thesame period. The presence of PRSV was determined by ELISA and RT–PCR(Srivastava et al., 2009). It appears that protein from C. aculeatum is a goodcandidate for utilization in management of virus diseases. The systemicvirus inhibitory activity of these proteins is due to the host-mediated phenomenonof formation / accumulation of a new virus inhibitory protein inthe treated plants showing systemic resistance. When TMV is mixed withthe induced virus inhibitory protein, the virus is completely inhibited. Thefact that CA-SRI also behaves as an RIP, as it inhibited in vitro protein synthesisin rabbit reticulocyte lysate and wheat germ lysate (Kumar et al.,1997), strongly confirms this belief that antiviral proteins generally have theproperties of RIPs. The sequence of the CA-SRI protein showed varyinghomology (11–54%) with the RIPs from other plant species (Kumar et al.,1997). However, the absence of hybridization between the CA-SRI gene andthe DNA / RNA of Mirabilis, Bougainvillea, rice, pea and tobacco shows thatthe virus inhibitory genes do not react with leaf proteins from Mirabilis,Bougainvillea, Boerhaavia, rice, pea and tobacco. These only indicate thatantiviral proteins from different plants may behave differently and can beused specifically in particular host–virus systems.The pre-inoculation spray of SRIs from C. aculeatum, C. inerme, Boerhaaviadiffusa, B. spectabilis and Pseuderanthemum bicolor modified the susceptibilityof several host plants such as tomato, tobacco, mungbean, urdbean, bhendiand sunnhemp against subsequent infection by viruses. The treatment helpsto protect the susceptible hosts during the vulnerable early stages of development.Induced resistance appears to be a universal process in all susceptiblehosts and can be used to advantage in plant protection. Pre-inoculationsprays (four sprays) of B. spectabilis leaf extract protected plants of Cucumismelo against cucumber green mottle mosaic virus (CGMMV), Crotalaria junceaagainst sunnhemp rosette virus (SRV) and Lycopersicon esculentum againstTMV for 6 days (Verma and Dwivedi, 1983). Since the duration of resistanceconferred by SRIs was up to only 6 days, it was realized that the durabilityof resistance needed to be prolonged under field conditions to achieve betterprotection against virus infection. It has been shown that proteinaceousmodifiers, such as papain, enhanced the activity of CA-SRI and also prolongedthe durability of induced resistance against sunnhemp rosettetobamovirus in Crotalaria juncea up to 12 days (Verma and Varsha, 1995). Inanother study, five weekly sprays of CA-SRI in potted plants of tomato in

166 H.N. Verma and V.K. Baranwalopen fields protected the plants from natural virus infection for more than 2months (unpublished results). However, in another study it was shown thatweekly sprays of leaf extract of C. aculeatum delayed the symptom appearanceof leaf curl virus in tomato and promoted the growth of the plants(Baranwal and Ahmad, 1997). A natural plant compound called NS-83 wasshown to reduce and delay the disease symptoms by TMV, PVX and PVY intobacco and tomato plants under field conditions and the fruit yield intomato was increased by 23.4% (Xin-Yun et al., 1988). Plant extracts from C.inerme and Ocimum sanctum provided a high degree of resistance againsttobacco chlorotic mottle virus in cowpea, possibly by induction of systemicinduced resistance (Mistry et al., 2003). Sumia et al. (2005) demonstratedinhibition of local lesions and systemic infection induced by Tobacco streakvirus in cowpea and French bean by pre-treatment of a proteinaceous substancefrom seeds of Celosia cristata. Foliar sprays with aqueous leaf extractof C. aculeatum plant since the sprouting stage at fortnightly intervals in thefield could significantly protect Amorphophallus campanulatus against infectionby virus. Maximum reduction in disease incidence and symptom severitywas exhibited by plants which received six sprays. Maximum plantgrowth along with considerable increases in corm weight was also observedin such plants (Khan and Awasthi, 2006).It appears that antiviral phytoproteins trigger the host defencemechanism in a specific manner either by signal transduction, as demonstratedin the case of Phytolacca antiviral protein (PAP), and/or by increasedsynthesis of antiviral proteins in host plants treated by systemic resistanceinducers as for C. aculeatum. While success has been achieved in developingtransgenics that have genes for mutant PAP without RIP activity (Smirnovet al., 1997), it remains to be seen how these transgenics can be utilized underfield conditions. On the other hand, SRIs such as CA-SRI show a potential foruse under field conditions. However, a larger quantity of SRIs would berequired for their wider application. Cloning of the genes for CA-SRI andtheir expression has been achieved in an Escherichia coli expression vector.The expressed protein has been shown to inhibit protein synthesis (Kumaret al., 1997). A detailed study is still required to determine whether or nota transgenic with native and mutant CA-SRI genes would be able toshow systemic protection against virus infection. Although the C. aculeatumprotein has been demonstrated to have very high antiviral activity andto be extremely useful as a plant immunizing agent, it has not beencommercialized so far. The proteins from a few other non-host plants havealso been recognized as good defence stimulants, but they have not beendeveloped into products for disease control, because the industry findsit easier to patent newly synthesized compounds than natural plantproducts.Singh (2006) reported the anti-proliferative property of phytoproteinsfrom B. diffusa and C. aculeatum for the retardation of proliferation of humanbreast cancer cell lines as well as the inhibition of activity of the SemlikiForest virus in mice.

Control of Virus Diseases of Plants 1677.6 ConclusionWith the precipitous withdrawal of some of the toxic protectants, it may beprofitable to explore natural plant products as alternatives, particularlyagainst virus diseases where all other methods fail. The phytoproteins ortheir smaller peptides may prove valuable as ‘lead structures’ for the developmentof synthetic compounds. It would pay us to explore this rich sourceof antivirals more thoroughly. The value of these antiviral proteins is unlimitedbecause they are quite safe, non-toxic even after repeated and prolongeduse, and substantially enhance plant growth and yield. Antiviral phytoproteinsmay be useful if they are integrated with other strategies of virus diseasemanagement.Botanical resistance inducers can be classified as ideal virus-suppressingagents, as they encompass all the characteristics of an ideal antiviral compound.Induced resistance operates through the activation of natural defencemechanisms of the host plant and induces systemic resistance to viral multiplicationin plants. The active products present in these extracts have nodirect effect on viruses; their antiviral activity is mediated by host cells inwhich they induce the antiviral state. Systemic resistance inducers obtainedfrom plants have been shown to be effective against a wide range of viruses.Plant extracts or the semi-purified proteins from these plants stimulate thehosts to produce a virus inhibitory agent, which spreads to surroundingtissues and other plant parts.ReferencesAlexander, D., Goodman, R.M., Gut-Rella,M., Glascock, C., Weymann, K., Friedrich,L., Maddox, D., Ahi-Goy, P., Lunz,T., Ward, E. and Ryals, J. (1993) Increasedtolerance of two oomycete pathogensin transgenic tobacco expressingpathogenesis-related protein 1a. Proceedingsof National Academy of Sciences USA90, 7327–7331.Aron, G.M. and Irvin, J.D. (1980) Inhibitionof herpes simplex virus multiplicationby the pokeweed antiviral protein.Antimicrobial Agents Chemotherapy 17,1032–1033.Awasthi, L.P., Chowdhury, B. and Verma,H.N. (1984) Prevention of plant virus diseaseby Boerhaavia diffusa inhibitor. IndianJournal of Tropical Plant Diseases 2, 41–44.Awasthi, L.P., Kluge, S. and Verma, H.N.(1989) Characterstics of antiviral agentsinduced by Boerhaavia diffusa glycoproteinin host plants. Indian Journal of Virology 3,156–169.Awasthi, L.P., Pathak, S.P., Gautam, N.C. andVerma, H.N. (1985) Control of virusdiseases of vegetable crops by a glycoproteinisolated from Boerhaavia diffusa. IndianJournal of PIant Pathology 3, 311–327.Awasthi, L.P. (1981) The purification andnature of an antiviral protein from Cuscutareflexa plants. Archives of Virology 70,215–223.Baranwal, V.K. and Ahmad, N. (1997) Effectof Clerodendrum aculeatum leaf extract ontomato leaf curl virus. Indian Phytopathology50, 297–299.Barbieri, L. and Stirpe, F. (1982) Ribosomeinactivating proteins from plants propertiesand possible uses. Cancer Survey 1,489–520.

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8 Phytochemicals as NaturalFumigants and ContactInsecticides AgainstStored-product InsectsMOSHE KOSTYUKOVSKY AND ELI SHAAYAAgricultural Research Organization, the Volcani Center, IsraelAbstractFor centuries, traditional agriculture in developing countries has used effective methodsof insect pest control using botanicals. In order to make them a cheap and simplemeans of insect control for users, their efficacy and optimal use still need to beassessed. Currently, the measures to control pest infestation in grain, dry stored foodand cut flowers rely heavily on toxic fumigants and contact insecticides. In recentyears, the number of pesticides has declined as health, safety and environmental concernshave prompted authorities to consider restricting the use of toxic chemicals infood. Lately a new field is developing on the use of phytochemicals in insect pestmanagement, such as edible and essential oils and their constituents. The aim herehas been to evaluate the potential use of edible oils obtained from oil seeds andessential oils, and their constituents obtained from aromatic plants, as fumigants andcontact insecticides for the control of the legume pest Callosobruchus maculatusF. (Coleoptera: Bruchidae). The most active edible oils as contact insecticides werecrude oils from rice, maize, cottonseed and palm, and the fatty acids capric acid andundecanoic acid. The essentials oils and their constituents were found to have higheractivity as fumigants than contact insecticides. From our studies, to elucidate themode of action of essential oils, it was possible to postulate that essential oils mayaffect octopaminergic target sites.8.1 IntroductionInsect damage in stored grains and other durable commodities may amountto 10–40% in developing countries, where modern storage technologieshave not been introduced (Raja et al., 2001). Currently, food industries relymainly on fumigation as an effective method for insect pest control in grainand other dry food commodities. At present only two fumigants are still inuse: methyl bromide and phosphine. The first one is mostly being phasedout in developed countries due to its ozone depletion effects (WMO, 1995;© CAB International 2011. Natural Products in Plant Pest Management(ed. N.K. Dubey) 175

176 M. Kostyukovsky and E. ShaayaShaaya and Kostyukovsky, 2006). In addition, there have been repeatedindications that certain insects have developed resistance to phosphine,which is widely used today (Nakakita and Winks, 1981; Mills, 1983; Tyleret al., 1983). It should be mentioned that although effective fumigants andcontact synthesized insecticides are available, there is global concern abouttheir negative effects on non-target organisms, pest resistance and pesticideresidues (Kostyukovsky et al., 2002a; Ogendo et al., 2003). In recent years,attention has been focused on the use of botanicals as possible alternativesto toxic insecticides. Lately, there has been a growing interest in the use ofplant oils and their bioactive chemical constituents for the protection ofagriculture products due to their low mammalian toxicity and low persistencein the environment (Raja et al., 2001; Papachristos and Stamopoulos,2002). Plant oils have repellent and insecticidal (Shaaya et al., 1997;Kostyukovsky et al., 2002a; Papachristos and Stamopoulos, 2002), nematicidal(Oka et al., 2000), antifungal (Paster et al., 1995; Srivastava et al., 2009), antibacterial(Matasyoh et al., 2007), virucidal (Schuhmacher et al., 2003), antifeedantand reproduction inhibitory (Raja et al., 2001; Papachristos andStamopoulos, 2002) effects.Numerous plant species have been reported to have insecticidal propertiescapable of controlling insects (Grainge and Ahmed, 1988; Arnason et al.,1989). The toxicity of a large number of essential oils and their constituentshas been evaluated against a number of stored-product insects. Essential oilsextracted from Pogostemon heyneanus, Ocimum basilicum and Eucaluptusshowed insecticidal activities against Sitophilus oryzae, Stegobium paniceum,Tribolium castaneum and Callosobruchus chinensis (Deshpande et al., 1974;Deshpande and Tipnis, 1977). Toxic effects of the terpnenoids d-limonene,linalool and terpineol were observed on several coleopterans damaging postharvestproducts (Karr and Coats, l988; Coats et al., 1991; Weaver et al., 1991).Fumigant toxic activity and reproductive inhibition induced by a number ofessential oils and their monoterpenenoids were also evaluated against thebean weevil Acanthoscelides obtectus and the moth Sitotroga cerealella ( Klingaufet al., 1983; Regnault-Roger and Hamraoui, 1995). Our earlier investigationson the effectiveness of the essentials oils extracted from aromatic plants,showed great promise for the control of the major stored-product insects.Several of them were found to be active fumigants at low concentrationsagainst these insects (Shaaya et al., 1991, 1993, 1994).The use of edible oils as contact insecticides to protect grains, especiallylegumes, against storage insects is traditional practice in many countries inAsia and Africa. The method is convenient and inexpensive for the protectionof stored seeds in households and in small farms. Many different edibleoils have been studied as stored grain protectants against insects (Oca et al.,1978; Varma and Pandey, 1978; Pandey et al., 1981; Santos et al., 1981; Messinaand Renwick, 1983; Ivbijaro, 1984; Ivbijaro et al., 1984; Pierrard, 1986; Ahmedet al., 1988; Don Pedro, 1989; Pacheco et al., 1995).This chapter evaluates the efficacy of essential and edible oils as fumigantsand contact insecticides to suppress populations of the stored-productinsects, mainly the legume pest insect Callosobruchus maculatus. The pulse

Phytochemicals as Natural Fumigants 177beetle, Callosobruchus maculatus F. (Coleoptera: Bruchidae), is one of the majorpests of stored cowpea, lentils and green and black gram in the tropics(Sharma, 1984; Raja et al., 2007). The infestation often begins in the field onthe dry ripe seeds before harvest. The damage to the seeds can reach up to50% after 6 months of storage (Caswell, 1980) and up to 90% annually, accordingto the International Institute of Tropical Agriculture (IITA, 1989).8.2 Studies with Edible Oils and Fatty Acids: Biological Activityand RepellencyThe biological activity of a number of crude and distilled edible oils and anumber of straight-chain fatty acids, which contain from C5 to C18, was evaluatedin laboratory tests against the common legume pest C. maculatus. Allthe edible oils tested were found to have different degrees of activity at aconcentration of 1 g/kg (= 1 kg/ton) chickpea seeds (Table 8.1).The most active oils were crude oils from rice, maize, cottonseeds andpalm. Of the eggs laid, 90–96% did not develop to larvae and only 0–1% developedto F 1adults in the seeds treated with these oils (Table 8.1). In addition,some of the oils tested were found to prevent oviposition. The most active oilin this regard was the rice crude oil: only 50 eggs were laid on seeds treatedwith 1 g/kg oil compared to 287 eggs on the control seeds (Table 8.1).Table 8.1. Biological activity of various edible oils against Callosobruchus maculatus.OilNumber ofeggs laidEgg mortality(%)Adult emergenceNumber %Crude rice 50 100 0 0Crude maize 137 96 1 1Refi ned maize 150 97 3 2Crude cotton seed 212 99 2 1Refi ned cotton seed 275 89 22 8Crude palm 375 99 4 1Refi ned palm 237 84 38 16Crude soya bean 212 85 21 10Refi ned soya bean 262 96 10 4Crude coconut 125 95 6 5Distilled peanut 150 95 8 5Distilled saffl ower 287 97 9 3Crude olive 175 74 46 26Refi ned olive 300 86 18 6Refi ned sunfl ower 187 82 30 16Distilled kapok 387 88 39 10Control 287 5 270 94Each oil was used at a concentration of 1 g/kg chickpea. The required amount of the oil was first mixedwith acetone (50 ml/kg seeds) and the acetone was evaporated under a hood. Five males and five femaleswere introduced to 5 g seeds. The data are the average of three experiments, each one was in triplicate.

178 M. Kostyukovsky and E. ShaayaTo obtain an insight into the nature of the activity of the oils, the activityof straight-chain fatty acids ranging from C5 to C18 at a concentration of4 g/kg was studied against C. maculatus (Table 8.2). The results showed thatC9–C11 acids were the most active in preventing oviposition at this concentration,with the C11 acid the most active: only 13, 13 and 0 eggs were foundon the treated seeds, respectively (Table 8.2).C12–C16 fatty acids were less effective and activity was remarkablydecreased for the lower C5–C7 and higher C17–C18 acids. At lower concentrationsof 1.6, 0.8 and 0.4 g/kg, the C11 acid was found to be the most active;fewer eggs were laid than with C9 or C10 and no eggs developed to adults.The data presented in Table 8.3 show clearly that C9–C11 acids are stronglyrepellent to C. maculatus, but they have no lethal effect on the adults.Field tests using crude palm kernel and rice bran oils showed that bothoils were effective in controlling C. maculatus infestation providing full protectionfor the first 4–5 months of storage at a rate of 1.5–3.0 g/kg seeds. Theypersisted in controlling insect infestation for up to 15 months. The numbers ofadult insects found in the treated seeds after 15 months of storage were onlyabout 10% of that in the control samples (results not shown).8.3 Studies with Essential Oils as Contact InsecticidesThe efficacy of a large number of essential oils has been evaluated for thecontrol of C. maculatus at a concentration of 400 ppm. The various oils testedwere found to have a toxic effect on the eggs laid and on the development ofthe eggs to adults (Table 8.4).Table 8.2. Biological activity of straight-chain fatty acids C5–C18 againstCallosobruchus maculatus.Fatty acidNumber of eggs laidAdult emergenceNumber %Pentanoic(C5) 185 110 59Hexanoic (C6) 130 92 71Heptanoic (C7) 112 66 59Octanoic (C8) 105 22 21Nanonoic (C9) 13 0 0Decanoic (C10) 13 0 0Undecanoic (C11) 0 0 0Dodecanoic (C12) 32 22 69Tridecanoic (C13) 32 20 62Tetradecanoic (C14) 62 45 73Pentadecanoic (C15) 72 56 77Hexadecanoic (C16) 65 37 57Heptadecanoic (C17) 120 88 73Octadecanoic (C18) 140 115 82Control 287 272 95Each fatty acid was used at a concentration of 4 g/kg chickpea. The data are the average of threeexperiments, each of which were in triplicate.

Phytochemicals as Natural Fumigants 179Table 8.3. Toxicology and repellency of the fatty acids C9–C11 tested againstCallosobruchus maculatus.Fatty acidConcentration(g/kg)Number ofeggs laidUndevelopedeggs (%)Adult emergenceNumber %C9 1.6 60 100 0 00.8 67 95 3 40.4 100 72 18 18Control 0 360 3 345 96C10 1.6 65 100 0 00.8 27 92 1 40.4 62 78 7 11Control 0 300 5 280 93C11 1.6 20 100 0 00.8 20 100 0 00.4 22 100 0 0Control 0 300 3 287 96The data are the average of three experiments, each of which were in triplicate.Table 8.4. Contact activity of a number of essential oils on egg laying, egg developmentand F 1of Callosobruchus maculatus.Essential oilNumberEggs laid% of controlvalueEggs developedto larvae (%)No. ofadults F 1Eggs developedto adults (%)Syrian marjoram 171 83 87 0 0Lemon grass 144 70 88 5 3.5Geranium 119 34 14 8 6.7Vistria 157 57 77 15 9.6Basil 51 78 93 23 45Clary sage 40 22 75 22 55Orange 67 34 84 45 67Grapefruit 120 64 77 49 41Lemon 172 88 91 50 29Caraway 97 51 79 62 64Cumin 148 59 74 55 37Thyme 120 69 68 46 38Celery 239 179 79 169 71Thyme leaved savory 98 68 89 75 77Ruta 109 75 76 51 47Rosemary 123 68 79 64 32Peppermint 159 92 83 54 34SEM76 oil 67 28 93 27 17Control 150–350 – 85–95 120–280 80–85Each essential oil was tested at a concentration of 400 ppm and an exposure time of 24 h.Twenty unsexed adults were introduced to the treated and untreated seeds 24 h after treatment.

180 M. Kostyukovsky and E. ShaayaIn the case of seeds treated with Syrian marjoram, the number of eggslaid on the treated seeds was 83% of the control, but 0% of the eggs developedto adults. Other oils were found to cause a reduction in the number ofeggs laid on the treated seeds but were less effective on egg to adult development.In the case of clary sage oil, only 40 eggs were laid on the treated seeds(= 22% of control), but 55% of the eggs developed to adults. In contrast, theessential oil SEM76, obtained from Labiata plant species, was found effectiveon both the reduction of number of eggs laid (28% of control) and on thenumber of eggs that developed to adults (17% of control) (Table 8.4).8.4 Studies with Essential Oils as Fumigants AgainstStored-product InsectsIn order to isolate active essential oils we firstly screened a large number ofessential oils extracted from aromatic plants and isolated their main constituents,using space fumigation (Shaaya et al., 1991, 1993, 1994). The most activecompounds are summarized in Table 8.5.Among the tested compounds, the essential oil SEM76 and its main constituentwere found to be the most potent fumigants. To obtain the LC 90of allinsect species tested at adult stage, SEM76 oil was required at the concentrationof 0.6–1.2 μl/l air (Table 8.5). The main constituent of the oil, which accounts forapproximately 80% of the oil, was also found to have high activity, a little higherthan the oil, as expected (Table 8.5). Space fumigation studies with SEM76against various developmental stages of C. maculatus showed that eggs andyoung larvae before they penetrated into the seeds were the most susceptibleto the compound. A concentration of 0.5 μl/l air was enough to cause 100%mortality of the eggs and the first instar larvae. After the larvae penetratedinside the seeds, they became more tolerant (Table 8.6). Only pupae 1–2 daysbefore adult emergence again became sensitive to the compound (Table 8.6).Table 8.5. Fumigant toxicity of the most active monoterpenes tested on stored-productinsect pests in space fumigation tests.OryzaephilussurinamensisRhizoperthadominicaSitophilusoryzaeTriboliumcastaneumCompoundLC 50LC 90LC 50LC 90LC 50LC 90LC 50LC 901,8-Cineol 3.1 7.3 2.5 4.0 7.2 14.2 7.5 8.5Carvacrol – – >15.0 – >15 – >15.0 –Limonene >15.0 – 6.7 10.3 >15 – 7.6 8.6Linalool 3.0 6.0 6.0 8.5 10.1 19.8 >15 –Pulegone 1.7 2.8 2.8 4.5 0.7 1.4 2.5 3.2SEM-76 –

Phytochemicals as Natural Fumigants 181Table 8.6. Fumigant activity of the essential oil SEM76 extracted from Labiatae species onvarious developmental stages of Callosobruchus maculatus.Stage treatedAgeConcentration(µl/l)No. ofeggsNo. ofadultsF 1Eggs developed toadults (%)Egg 20–24 h 0.5 20 0 0Larvae outside 0–1 day 0.5 20 0 0the seedLarvae inside 2 days 1.0 20 3.5 17the seed1.5 20 4.5 22Larvae 3 days 1.5 20 10.5 533.0 20 8 40Larvae 7 days 1.5 20 14 703.0 20 13 63Larvae 11 days 1.5 20 17.5 883.0 20 16.5 83Pupae4–5 daysbefore1.53.0202012116055emergencePupae1–2 days0.5 20 4 20before1.5 20 1 5emergence 3.0 20 0 0Control 0 20 17 85Twenty unsexed adults were used for each test. The data are an average of three duplicateexperiments. Exposure time was 24 h.8.5 Distribution of a-Terpineol in the Fumigation ChamberMost of the research to study the potential of essential oils and their constituents(terpenes) for the control of insect pests was done using space fumigation,without paying attention to the amount of the fumigant available to thetreated insects. As a model, we studied the distribution of the terpenoidα-terpineol in the fumigation chamber among the air in the chamber space,filter paper and the flask walls. Using concentrations of 3, 5, 10 and 15 mg/lair and stirring at 20°C, only minor differences in the amount of the terpenoidin the chamber space was measured: 0.710, 0.676, 0.782 and 0.897 mg/lair, respectively. The rest was found on the filter paper and the flask walls(Table 8.7). At a higher temperature of 25°C, a higher amount of the terpenoid,50–60%, was recovered in the chamber space compared to the amountrecovered at 20°C (Table 8.7). No pronounced change of the terpenoid in thechamber space was measured at a different intensity of stirring or when nostirring was applied (Table 8.8). It should be mentioned that stirring causeda higher concentration of the terpenoid on the glass walls compared to thefilter paper, and the opposite if no stirring was applied (Table 8.8). Usingstirring at 20°C, we could show that after 2 h of fumigation the air in thefumigation chamber was already saturated (Table 8.9).

182 M. Kostyukovsky and E. ShaayaTable 8.7. The distribution of α-terpineol in the chamber space, fi lter paper and thefumigation chamber walls at different temperatures, using stirring.Concentrationused (mg/l air)Temperature(°C)Amount ofα- terpineolin the chamberspace(mg/l air)Total ofα- terpineolrecovered (%)3 20 0.710 85.0(2.682)5 20 0.676 81(4.265)5 25 1.106 84(4.366)10 20 0.782 105(10.932)10 25 1.165 91(9.53)15 20 0.897 112(17.591)Amount recovered (%)Chamberspace27.8(0.745)16.6(0.710)26.4(1.16)7.4(0.820)12.8(1.22)5.2(0.921)Filterpaper10.7(0.287)18.9(0.805)6.1(0.266)41.1(4.54)32.8(3.13)62(10.89)Flaskwalls61.6(1.65)64.5(2.75)67.4(2.96)51.5(5.68)54.4(5.18)33(5.78)The numbers in brackets are the value of α-terpineol in mg, recovered after 24 h of space fumigation.The volume of the fumigation chamber is 1050 ml. The sensitivity of the GC measurements was ± 15%.Table 8.8. Effect of stirring on the distribution of α-terpineol in the chamber space, fi lterpaper and fumigation chamber walls.Concentrationused (mg/l) airStirring timeand intensityAmount ofα- terpineol inthe chamber Total ofspace α- terpineol(mg/l air) recovered (%)5 24 h moderate 0.965 85.0(4.549)24 h slow 0.856 93(4.265)15 min slow 0.800 99(5.200)0 0.858 103(5.320)15 24 h moderate 0.684 107(16.898)0 0.639 111(17.490)Amount recovered (%)Chamberspace22.7(1.010)16.6(0.710)16.2(0.840)16.9(0.901)4.3(0.718)3.8(0.671)Filterpaper6.7(0.278)18.9(0.805)63.5(3.30)76.7(4.080)51.5(8.70)90.9(15.90)Flaskwalls71(3.160)64.5(2.750)20.4(1.060)6.4(0.339)44.3(7.480)5.3(0.919)The numbers in brackets are the value of α-terpineol in mg, recovered after 24 h of space fumigation.The volume of the fumigation chamber was 1050 ml. The fumigation was conducted at a temperatureof 20˚C. The sensitivity of the GC measurements was ± 15%.

Phytochemicals as Natural Fumigants 183Table 8.9. Amount of α-terpineol measured in the fumigation chamber space at various timeintervals of fumigation, using stirring.Concentrationused (mg/l) airThe amount of α-terpineol (mg) measured in the fumigationchamber space, hours following fumigation2 4 6 243 0.685 0.678 0.865 0.7315 0.877 0.804 – 0.70110 0.900 0.922 – 0.82115 0.838 0.769 0.767 0.857The volume of the fumigation chamber was 1050 ml.The fumigation was conducted at a temperature of 20°C.8.6 Possible Mode of Action of Essential OilsThe essential oil SEM76 and pulegone were found to be highly toxic againstall stored-product insects tested (Shaaya et al., 2001, 2002). The effect of thesetwo compounds on acetylcholinesterase and the octopamine systems ininsects was studied in order to elucidate their mode of action.In our studies with acetylcholinesterase (Greenberg-Levy et al., 1993;Kostyukovsky et al., 2002b), using acetylcholinesterase extracted from Rhizoperthadominica, a stored-product insect, we showed that the inhibitory activityof these two terpenoids on acetylcholinesterase was only evident when highconcentrations of these compounds (10 –3 M) were applied. These doses wereat such high levels that they cannot account for the toxic effects observed invivo by these compounds on the same insect species (Shaaya et al., 2001, 2002),which were obtained at much lower concentration (10 –6 M = 1.5 μl/l of air).The failure of these biologically active compounds to produce a strongerenzyme inhibition indicated that acetylcholinesterase was probably not themain site of action of essential oils.Another possible target for neurotoxicity of essential oils is the octopaminergicsystem in insects. Octopamine is a multifunctional, naturally occurringbiogenic amine that plays a key role as a neurotransmitter, neurohormoneand neuromodulator in invertebrate systems (Evans, 1981; Fig. 8.1), with aphysiological role analogous to that of norepinephrine in vertebrates.The octopaminergic system in insects represents a biorational target forinsecticidal action and has been targeted by various insecticides in the past,e.g., formamidines (Haynes, 1988; Perry et al., 1998). Many of the physiologicalfunctions of octopamine appear to be mediated by a class of octopaminereceptors, specifically linked to a protein coupled to the enzyme adenylatecyclase. These physiological actions have been shown to be associated withelevated levels of cyclic-AMP (Evans, 1984). Using an insect in-vitro cuticulartissue preparation, octopamine was found to induce a significant increase inthe intracellular messenger cyclic AMP (Rafaeli and Gileadi, 1995). SEM76and pulegone directly affect the intracellular response of these tissues bymimicking the octopamine response, thereby increasing the production of

184 M. Kostyukovsky and E. ShaayaNerve impulseHOCHCH 2 NH 2OCH 3 CH 3NCH 3 OCH 3AcetylcholineOHOctopaminePost-synaptic neuronalmembraneMuscularjunctionHemolymphNerve impulseMuscular contraction,other physiological,modulatory functionsFig. 8.1. Neurotransmitters in insect systems as possible sites of action of essentialoil toxicity.Table 8.10. Effect of essential oils in the absence and presence of the octopamineantagonist, phentolamine (10 –5 M), on intracellular cyclic-AMP levels.TreatmentWithoutphentolamineWithphentolamineConcentration(M)Intracellular cyclic – AMP levelspmol/abdominal segmentOctopamine SEM76 Pulegone d-limonene0 0.210 –8 0.39 0.027 0.023 –10 –7 0.58 0.42 0.88 0.020 0.1410 –8 0.18 0.17 – –10 –7 0.05 0.17 0.2 –10 –6 0.11 0.01 – –cyclic-AMP at low physiological concentrations of 10 –8 M (Table 8.10). Moreover,addition of the octopamine antagonist phentolamine strongly antangonizedthe response to octopamine, as well as to the essential oil SEM76 andpulegone, were strongly antagonized and no elevation in cyclic-AMP wasobserved (Shaaya, et al., 2001; Kostyukovsky et al., 2002b). These effects wereobserved at low dilutions of the essential oils (estimated at 10 –7 and 10 –8 M),levels that induced the overt behavioural toxicity responses in vitro (Table8.10; Kostyukovsky et al., 2002b). It should be mentioned that d-limonene,

Phytochemicals as Natural Fumigants 185which was found to have very low toxicity to stored-product insects, did notshow any effect on the production of cyclic-AMP (Table 8.10).Because the essential oil response is strongly insect specific, as is octopamineneurotransmission, the essential oils can mimic the action of octopamineat low doses, and phentolamine, an octopamine inhibitor, has aninhibitory action on the essential oils, we can postulate that the essential oilsmay affect octopamonergic target sites.8.7 DiscussionStudies with edible oils and fatty acids showed that crude oils of rice, maize,cottonseed and palm were found the most potent against C. maculatus comparedto the other oils tested. The purified forms of these oils were found tobe less effective. In field studies, rice and palm crude oils at concentrations of1.5 and 3 g/kg, protected chickpeas completely from insect infestation for aperiod of 4–5 months and partially for up to 15 months. Studies by Khaireet al. (1992) showed that adult emergence of C. chinensis was completely preventedfor up to 100 days in pigeonpea treated with 1% neem oil or karanj oil.In addition, theses oils were found to have no adverse effect on seed germination.Boeke et al. (2004) showed that a number of botanical products mightprovide effective control of C. maculatus in cowpea.The mode of action of edible oils was first attributed to interference withnormal respiration, resulting in suffocation of the insects. The action of theoil, however, is more complex, since insects deprived of oxygen survivedlonger than those treated with oils (Gunther and Jeppson, 1960). From thisstudy and others (Wigglesworth, 1942; Ebeling and Wagner, 1959; Ebeling,1976; Shaaya and Ikan, 1979), it may be postulated that the biological activityof the edible oils is attributed to both their physical and chemical propertiessuch as viscosity, volatility, specific gravity and hydrophobicity.Among the straight-chain fatty acids ranging from C5 to C18 carbonatoms, it was found that C9–C11 acids were the most effective in preventingoviposition of C. maculatus on the treated seeds, but have no lethal effect onthe adults. In earlier studies (Shaaya et al., 1976), we showed that wheat seedstreated with C10 fatty acid, at a concentration of 4 g/kg, repelled Sitophilusoryzae L, but that forced contact of the beetles with the treated seeds wasfound to have no effect on mortality rates of the beetles (Shaaya et al., 1976).It should be mentioned that C. maculatus adults do not consume food andlive approximately 1 week only, whereas S. oryzae adults feed as long as theylive – several months. We postulate, therefore, that fatty acids act as repellentsand the beetles die because of starvation.Essential oils as contact insecticide were found to have low toxicity. Ahigh concentration of 400 ppm of the most active essential oil from Syrianmarjoram was needed to prevent the development from egg to adult. Onthe contrary, essential oils and their constituents as fumigants showedhigh activity in controlling young larvae and pupae prior to emergenceof C. maculatus. The contact and fumigant toxicity of five essential oils,

186 M. Kostyukovsky and E. Shaayacardamom, cinnamon, clove, eucalyptus and neem were also investigatedagainst the cowpea weevil (Mahfuz and Khalequzzaman, 2007). In spacefumigation, at a concentration of 1 μl/l air, Ocimum gratissimum oil and itsconstituent eugenol caused 100% mortality of C. chinensis 24 h after treatment(Ogendo et al., 2008). The essential oils of citrodora and lemongrasswere found to have insecticidal and ovicidal activities against adults andeggs of C. maculatus (Raja and William, 2008). Van Huis (1991), Adabie-Gomez et al. (2006), Henning (2007), Boateng and Kusi (2008) have reportedthe efficacy of Jatropha seed oil as contact insecticide against C. maculatus.Similarly, Kéita et al. (2001) reported that kaolin powder aromatized withpure oil of Ocimum spp. at 6.7 μl/g of pea seeds provided complete protectionover 3 months of storage.In space fumigation studies to evaluate the potency of the essential oilsand their constituents against insect pests, special attention has to be givento not using a higher amount of the fumigant than needed to saturate theair in the fumigation chamber at constant temperature. It should be mentionedthat the amount of the various test materials needed to saturate theair in the fumigation chamber might be different, depending on the physicaland chemical properties of the fumigant. Our findings, as well as resultsof other researchers, suggest that certain essential oils, and particularlytheir terpenoids, are highly selective to insects, since they are probablytargeted to an insect-selective octopaminergic receptor, which is a nonmammaliantarget. The worldwide availability of plant essential oils andtheir terpenoids, and their use as flavouring agents in food and beveragesis a good indication of their relative safety to humans. In our earlier studies,we showed that essential oils have a high potential as alternative fumigantsto methyl bromide for the control of stored-product insects and cut flowers(Shaaya, 1998; Wilson and Shaaya, 1999; Kostyukovsky and Shaaya, 2001;Kostyukovsky et al., 2002). The reduction in the use of conventional synthetictoxic pesticides gives to these bioactive chemicals the potential to beused as a complementary or alternative method in crop production andintegrated pest management.ReferencesAdabie-Gomez, D.A., Monford, K.G., Agyir-Yawson, A., Owusu-Biney, A. and Osae, M.(2006) Evaluation of four local plant speciesfor insecticidal activity againstSithophilus zeamais Motsch. ( Coleoptera:Curculionidae) and Callosobruchus maculatusF. (Coleoptera: Bruchidae). GhanaJournal of Agricultural Science 39, 147–154.Ahmed, K., Khalique, F., Afzal, M., Malik,B.A. and Malik, M.R. (1988) Efficacy ofvegetable oils for protection of greengramfrom attack of bruchid beetle.Pakistan Journal of Agricultural Research 9,413–416.Arnason, E.T., Philogene, B.J.R. and Morand, P.(1989) Insecticides of Plant Origin. AmericanChemical Society Symposium series387, 213.Boateng, B.A. and Kusi, F. (2008) Toxicity ofJatropha seed oil to Callosobruchus maculatus(Coleoptera: Bruchidae) and its parasitoid,Dinarmus basalis (Hymenoptera:Pteromalidae). Journal of Applied SciencesResearch 4, 945–951.

Phytochemicals as Natural Fumigants 187Boeke, S.J., Baumgart, I.R., Van Loon, J.J.A.,van Huis, A., Dicke, M. and Kossou,D.K. (2004) Toxicity and repellence ofAfrican plants traditionally used forthe protection of stored cowpea againstCallosobruchus maculatus. Journal ofStored Products Research 40, 423–438.Caswell, G.H. (1980) A review of the workdone in the Entomology section of theInstitute for Agricultural Research onthe Pests of Stored Grain. Samaru Misc.Paper 99, Zaria, Nigeria.Coats, J.R., Karr, L.L. and Drewes, C.D. (1991)Toxicity and neurotoxic effects of monoterpenoidsin insects and earthworms. In:Heiden, P.A. (ed.) Naturally Occurring PestBioregulators. American Chemical SocietySymposium series, 449, pp. 305–316.Deshpande, R.S. and Tipnis, H.P. (1977) Insecticidalactivity of Ocimum basilicum Linn.Pesticides 11, 11–12.Deshpande, R.S., Adhikary, P.R. and Tipnis,H.P. (1974) Stored grain pest controlagents from Nigella sativa and Pogostemonheyneanus. Bulletin of Grain Technology 12,232–234.Don Pedro, K.N. (1989) Mechanisms of actionof some vegetable oils against Sitophiluszeamais Motsch. (Coleoptera: Curculionidae)on wheat. Journal of Stored ProductsResearch 25, 217–223.Ebeling, W. (1976) Insect integument: a vulnerableorgans system. In: Hepburn, H.R. (ed.)The Insect Integument. Elsevier, Amsterdam,pp. 383–399.Ebeling, W. and Wagner, R.E. (1959). Rapiddesiccation of drywood termites with inertsorptive dusts and other substances. Journalof Economic Entomology 52, 190–207.Evans, P.D. (1981) Multiple receptor types foroctopamine in the locust. Journal of Physiology(London) 318, 99–122.Evans, P.D. (1984) A modulatory octopaminergicneurone increases cyclic nucleotidelevels in locust skeletal muscle. Journal ofPhysiology (London) 348, 307–324.Grainge, M. and Ahmed, S. (1988) Handbook ofPlants with Pest-control Properties. Wileyand Sons, New York, USA.Greenberg-Levy, S., Kostjukovysky, M.,Ravid, U. and Shaaya, E. (1993) Studiesto elucidate the effect of monoterpeneson acetycholinesterase in two storedproductinsects. Acta Horticulturae 344,138–146.Gunther, F.A. and Jeppson, L.R. (1960) ModernInsecticides and World Food Production,Wiley, New York, pp. 284.Haynes, K.F. (1988) Sublethal effects of neurotoxicinsecticides on insect behaviour.Annual Review of Entomology 33, 148–168.Henning, R.K. (2007) Jatropha curcas L. [Internet]Record from Protabase. van derVossen, H.A.M. and Mkamilo, G.S. (eds).PROTA (Plant Resources of TropicalAfrica), Wageningen, Netherlands,available at http://database.prota.org/search.htm.IITA, 1989 International Institute of TropicalAgriculture. Annual Report 1988/89. Ibadan,Nigeria.Ivbijaro, M.F. (1984) Toxic effects of groundnutoil on the rice weevil Sithophilusoryzae (L.). Insect Science and its Application5, 251–252.Ivbijaro, M.F., Ligan, C. and Youdeowei, A.(1984) Comparative effects of vegetableoils as protectants of maize from damageof the rice weevil Sithophilus oryzae (L.).Proceedings of 17th International Congress ofEntomology, Hamburg, Germany, p. 643.Karr, L. and Coats, R.J. (1988) Insecticidalproperties of d-limonene. Journal of PesticideScience 13, 287–290.Keita, S.M., Vincent, C., Schmit, J.P. Arnason,J.T. and Bélanger, A. (2001) Efficacy ofessential oil of Ocimum basilicum L. andO. gratissimum L. applied as an insecticidalfumigant and powder to controlCallosobruchus maculatus (Fab.) (Coleoptera:Bruchidae). Journal of Stored ProductsResearch 37, 339–349.Khaire, V.M., Kachare, B.V. and Mote, U.N.(1992) Efficacy of different vegetable oilsas grains protectants against pulse beetleCallosobruchus chinensis L. in increasingthe storability of pigeonpea. Journal ofStored Products Research 28, 153–156.Klingauf, F., Bestmann, H.J., Vostrowsky, O.and Michaelis, K. (1983) Wirkung vonaltherishcen Olen auf Schadinsekten.Mitteilung Deutsche Gesselschaft fuer

188 M. Kostyukovsky and E. ShaayaAllgemeine und Angewandte Entomologie4, 123–126.Kostyukovsky, M. and Shaaya, E. (2001) Quarantinetreatment on cut flowers by naturalfumigants. Proceedings of the 7th InternationalConference on Controlled Atmosphere andFumigation in Stored Products 29 October to 3November 2000. Fresno, USA, pp. 821–827.Kostyukovsky, M., Ravid, U. and Shaaya, E.(2002a) The potential use of plant volatilesfor the control of stored productinsects and quarantine pests in cut flowers.Acta Horticulturae 576, 347–358.Kostyukovsky, M., Rafaeli, A., Gileadi, C.,Demchenko, N. and Shaaya E. (2002b)Activation of octopaminergic receptorsby essential oil constituents isolated fromaromatic plants: possible mode of activityagainst insect pests. Pest ManagementScience 58, 1–6.Mahfuz, I. and Khalequzzaman, M. (2007)Contact and Fumigant Toxicity of EssentialOils against Callosobruchus maculatus.University Journal of Zoology. RajshahiUniversity 26, 63–66.Matasyoh, L.G., Matasyoh, J.C., Wachira, F.N.,Kinyua, M.G., Muigai, A.T.M. andMukiama, T.K. (2007) Chemical compositionand antimicrobial activity of theessential oil of Ocimum gratissimum L.growing in eastern Kenya. African Journalof Biotechnology 6, 760–765.Messina, F.J. and Renwick, J.A.A. (1983)Effectiveness of oils in protecting storedcowpea from the cowpea weevil (Coleoptera:Bruchidae). Journal of EconomicEntomology 76, 634–635.Mills, K.A. (1983) Resistance to the fumiganthydrogen phosphine in some storedproductspecies associated with repeatedinadequate treatments. MitteilungDeutsche Gesselschaft fuer Allgemine AngewandleEntomologie 4, 98–101.Nakakita, H. and Winks, R.G. (1981) Phosphineresistance in immature stages of alaboratory selected strains of Triboliumcastaneum (Herbst.). Journal of StoredProducts Research 17, 43–52.Oca, G.M., Garcia, F. and Schoonhoven, A.V.(1978) Efecto de cuatro aceites vegetalessobre Sithophilus oryzae y Sitotroga cerealellain maiz, sorgo y trigo almacenados.Revista Colombiana de Entomología 4, 45–49.Ogendo, J.O., Belmain, S.R., Deng, A.L. andWalker, D.J. (2003) Comparison of toxicand repellent effects of Lantana camara L.with Tephrosia vogelii Hook and a syntheticpesticide against Sitophilus zeamaisMotschulsky in maize grain storage. InsectScience and Its Application 23, 127–135.Ogendo, J.O., Kostyukovsky, M., Ravid, U.,Matasyoh, J.C., Deng, A.L., Omolo, E.O.,Kariuki, S.T. and Shaaya, E. (2008) Bioactivityof Ocimum gratissimum L. oil andtwo of its constituents against five insectpests attacking stored food products.Journal of Stored Products Research 44,328–334.Oka, Y., Nacar, S., Putievsky, E., Ravid, U.,Yaniv, Z. and Spiegel, Y. (2000) Nematicidalactivity of essential oils and theirconstituents against the root-knotnematode. Phytopathology 90, 710–715.Pacheco, A.I., De Castro, F., De Paula, D.,Lourencao, A., Bolonhezi, S.and Barbieri,K.M. (1995) Effecacy of soybean and casteroils in the control of Callosobruchusmaculatus (F.) and Callosobruchus phaseoli(Gyllenhal) in stored chickpeas (Cicerarietinum L.). Journal of Stored ProductsResearch 19, 57–62.Pandey, G.P., Doharey, R.B. and Varma, B.K.(1981) Efficacy of some vegetable oils forprotecting greengram against the attackof Callosobruchus maculatus (Fabr.). IndianJournal of Agricaltural Science 51, 910–912.Papachristos, D.P. and Stamopoulos, D.C.(2002) Repellent, toxic and reproductioninhibitory effects of essential oil vapourson Acanthoscelides obtectus (Say) (Coleoptera:Bruchidae). Journal of Stored ProductsResearch 38, 117–128.Paster, N., Menasherov, M., Ravid, U. andJuven, B. (1995) Antifungal activity ofOregano and Thyme essential oils appliedas fumigants against fungi attackingstored grain. Journal of Food Protection 58,81–85.Perry, A.S., Yamamoto, I., Ishaaya, I. and Perry,R. (1998) Insecticides in Agriculture andEnvironment - Retrospects and Prospects.

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9 Prospects of Large-scaleUse of Natural Productsas Alternatives to SyntheticPesticides in DevelopingCountriesD.B. OLUFOLAJIDepartment of Crop, Soil and Pest Management, The Federal Universityof Technology, Akure, NigeriaAbstractThe incidence of pests and diseases on crops in developing countries has caused a lotof food insecurity and negatively affected the agro-allied industries. The use of syntheticpesticides has been the only means of combating the pests and diseases toachieve large-scale food production in developing countries due to lack of appropriatetechnology to formulate their own pest control measures. The use of these chemicalsis associated with a lot of problems. The problems of mammalian toxicity,pollution of the environment and high cost of the few available chemical pesticidesare enormous. The Environmental Protection Agency (EPA) worldwide reacts againstthese problems and encourages research into a better alternative to these syntheticpesticides. The natural products as alternatives are available at all times of the yeardue to their location in the tropical regions, they are easy to formulate, are not toxic tomammals, are less costly and are environmentally friendly. These attributes assist inthe production of cheap and safe food, so the botanicals are assets to developingcountries and encourage their large-scale usage. Extracts from plants such as Azadirachtaindica, and Ocimum spp. are used for the control of various crop pests such ascowpea beetles (Callosobruchus maculatus), and diseases such as wet rot of Amaranthusspp. (Choanephora curcubitarum) and anthracnose of soybean ( Colletotrichumtruncatum).9.1 IntroductionAbout one third of the world’s agricultural produce in terms of plants and animalsare destroyed by a combination of both pests and diseases. In Africa, wheremost of the developing countries are located, most of the food crops are attackedby one pest or another, thereby causing high crop loss (Table 9.1; Ogbalu, 2009).© CAB International 2011. Natural Products in Plant Pest Management(ed. N.K. Dubey) 191

192 D.B. OlufolajiTable 9.1. Estimate of crop losses due to insect attacks in some African countries.Crop Country PestsEstimated croploss (%)CerealsSorghum Chad Busseola fusca 54Nigeria Busseola fusca 47Ethiopia Stemborers 25Rice Nigeria Stemborers / weevils 35–60Ghana Stemborers 25–30Chad Stemborers 35–55Maize Ethiopia Stemborers 32–55Weevils 48–55Nigeria Weevils 68Maruca testulalis 43–68Busseola fusca 24–55Maize tassels Nigeria Thrips 55Ethiopia Stemborers 37Cameroun Stemborers 24LegumesCowpea Nigeria Weevils 30–58Pod borers 60–72Swaziland Pod borers 20–48Ethiopia Hemipterans 38–57Kenya Ophiomyia phaseoli 28–43Chad Pod borers 48–54Roots and tubersCocoyam Nigeria Scale insects 34Cassava Nigeria Zonocerus variegatus 28–31Ghana Zonocerus variegatus 15–27Yam Chad Yam beetles 24–48Nigeria Yam beetles 54Sweet potatoes Nigeria Cylas puncticollis 67–76VegetablesTomatoes Nigeria Dipterous pests 45–90Peppers Nigeria Dipterous / bugs 35–53Okra Nigeria Flea beetles 45–60Bitterleaf Nigeria Lixus cameranus 54–68Diacrisia maculosa 55–87Fluted pumpkin Nigeria Z. variegatus55–82D. maculosaSource: Ogbalu, 2009.Knowledge of natural products or botanicals as pesticides is as old asagriculture and local usage of the plants for both medicinal and pesticidalpurposes also predates history. However, the advent of chemical pesticidesin the mid-20th century has relegated the use of botanicals to the background,the striking negative side effect of these synthetic pesticides notwithstanding (Lale, 2007). Until now the synthetic pesticides still record

Prospects of Large-scale Use of Natural Products 193very formidable success in their control attributes on both pests and diseasesin plants. The discovery of residues of synthetic pesticides in the food chainand their effect on the environment have led to agitation by environmentalists,ecologists and the Environmental Protection Agencies (EPA) worldwideto look inward for a better alternative in order to produce safe food foreverybody, and maintain a friendly environment. The natural productswhich can also be referred to as botanicals and their products have and willcontinue to play important roles in alleviating human suffering and alsocontrol the losses of farm products experienced in most developing countries(Edeoga and Eriata, 2001; Aworinde et al., 2008). Crop production andprotection are being supported by the use of botanicals in developing countriesin Africa and some third-world countries (Lale, 2007; Isman 2008).Researchers in Nigeria and some other African countries have investigatedseveral plant species for their efficacy as pesticides for protecting their cropsagainst pest degradation especially with the storage pests and diseases(Boeke et al., 2001; Ofuya et al., 2007; Olufolaji, 2008).Until recently, conventional pesticides had been used to curb these pestsand, in most cases, quite efficiently. However, the technicalities involved inthe formulation, standardization and distribution of the botanicals as pesticidesto reach the teeming population in the developing countries still remainproblems for their usage (Lale, 2007). There are the attendant problems ofresistance, environmental pollution, and adverse effect on health and climatechange traceable in part to the high levels of chloro-fluoro-carbon being usedas carriers in aerosol pesticides, which is affecting the ozone layer. This led tocaution in the continued use of persistent synthetic pesticides, especiallythose in the class of chlorinated hydrocarbons and the highly toxic organophosphoruspesticides (Olaifa, 2009).9.2 History of Natural Products in the Developing Countriesand their Use by Local Farming CommunitiesDeveloping countries have been endowed by nature with large quantitiesand diverse collections of many such natural products, which are more ofplant origin (botanicals). The population of inhabitants in developingcountries is fed by farm produce from the local peasant farmers. Thus themajor users of botanical pesticides are the local farmers who are less educated,very poor and most of them cannot afford the expensive syntheticpesticides.The tropical forests, characteristic of most developing countries, are theabode of naturally occurring genera and species of most of these botanicals.Over centuries, evolution of these botanicals has occurred to produce variousbiotypes that are in good use today as medicines and pesticides (Table9.2). A survey of the use of the botanicals in local farms revealed that around50% of about 500 peasant farmers in India and Pakistan who stored foodgrains for more than 6 months used neem (Azadirachta indica) leaves to get ridof storage pests, whereas the relatively affluent and better educated farmers

194 D.B. Olufolajiused neem cake for nematode control (Ahmed and Stoll, 1996). These farmersbuy neem cake in the market because they find no synthetic pesticides tobe as equally potent, cheap and readily available. One of the few reportedcases of the use of the botanical pesticidal materials for pest management inNigeria is the application of chilli pepper (Capsicum spp.) and tobacco (Nicotianatabacum) leaf extracts by some farmers in the south western states ofNigeria (Nworgu, 2006), and the use of Chromolaena odorata to solve somepest and disease problems e.g. anthracnose of soybean caused by Colletotrichumtruncatum has also been reported (Ajayi and Olufolaji, 2007). There aremany other plants (Eichhornia crassipes, Vernonia amygdalina, Piper guineense,etc.) used by farmers as botanical pesticides for various pests and diseases,even in their crude form, and they have been found to be very effective (Olufolaji,1999a, 2006, 2008; Ajayi and Olufolaji, 2007). Those farmers haveadopted the use of botanicals in the control of pests and diseases on theircrops due to desperation for survival, and since they are dependent on anagrarian economy, they evolved various method of formulating and usingthe botanicals without sourcing the orthodox expertise and technicalities thatare either not within their reach or expensive to adopt.Table 9.2. Some bioactive plants reported for the control of agricultural pests.Pests Bioactive plants ReferencesMaize weevil Neem seed Ivbijaro, 1983Sitophilus orazae (L.) Eugenia aromatica Lale, 1992, 1994;Aranilewa et al., 2002Callosobruchus maculatus Piper guineenseOlaifa and Erhum,1988Dysdercus superstittiosusOotheca mutabilisRiptortus dentipesCallosobruchus maculatusCallosobruchus maculatusAcrae epomiaDysdercus superstittiosusOotheca mutabilisRiptortus dentipeGrasshopper(Zonocerus variegates)Acrae epomiaSeveral plantsMonodora tenuifoliaZanthoxylum zanthoxyloideAzadirachta indicaOfuya and Dawodu, 2002Adedire and Lajide, 1999Adedire et al., 2003Ogunwolu and Idowu, 1994Lale and Abdulrahman, 1999Lippia adoensis Olaifa et al., 1987Clausena anisataAzadirachta indicaPiper guineenseAzadirachta indicaAzadirachta indicaOkunade and Olaifa, 1987Olaifa and Akingbohungbe,1987aOlaifa and Adenuga,1988a,bOlaifa et al., 1991aTetrapteura tetraptera Azadirachta indica Olaifa et al., 1991bField pests of cowpea, maize, Tephrosia vogelli Adebayo, et al., 2007sorghum, cassava, bananaSource: Olaifa, 2009.

Prospects of Large-scale Use of Natural Products 1959.3 Availability of Natural Products in Large and RequiredQuantitiesMost countries in the developing world are favoured by very good climaticconditions and agro-ecosystems that support the growth and developmentof most of the botanicals used for the production of the botanical pesticides.The farmers have a locational advantage in that the botanicals are availablein abundance in their ecosystem and exist throughout the year in both rainyand dry seasons. For example, most of these popular plants grow in the wildand are regarded as weeds, and some of them are not useful for food or rawmaterials for the industries (Table 9.2).However, some are now being cultivated to prevent the extinction ofuseful species. In recent times, most countries in the developing world findit easy to incorporate the botanicals into their agro-forestry programmesand this has increased and maintained the existence of the botanicals inlarge quantities (Ogunnika, 2007). This has further strengthened the largescaleuse of the botanicals as a means of pest and pathogen control in thesub-region. Furthermore, germination of the seeds of most of these botanicalsis not tied down by an unnecessarily long dormant period, which ischaracteristic of some botanicals in developed world (Ogunnika, 2007). Theactivities of the farming population in the tropics have included intercroppingand mix-cropping of main crops with the botanicals. These involveplanting Manihot esculenta (cassava) with Ocimum gratissimum (camphorbasil), Theobromae cacao (cocoa) with Carica papaya (pawpaw) and so on(Ogunnika, 2007). It is worth noting here that some of these botanicals whenmixed or intercropped with the main crop could serve as a control for pestsand diseases in the form of allelopathy. N. tabaccum and some other plantspecies, especially grasses, have been found to possess this attribute( Florentine et al., 2003, Ogunnika, 2007). Eucalyptus camaldulensis and someother botanicals are also used as weed control in farmlands to maximizeyields (Oyun and Agele, 2009). Many farm crops are protected by this meanswithout the need to carry out formulation and application processes for pestand disease control. There is the availability of rain and moisture throughoutthe year and no occurrence of snow or completely dry period in mostdeveloping countries close to the tropics. Thus, plants can grow throughoutthe year and there is no threat of extinction of most of these botanicals, bothwild and cultivated species.The fact that almost half of the world’s forest that houses these botanicalsis in Africa is an indication of the availability of the much needed plants forbotanical pesticides (Owolabi and Olanrewaju, 2007). Farnsworth andSoejarto (1991) estimated that 28% of the total inventories of the world’s plantspecies are used in ethnomedicines. Groombridge (1992), and Lange andSchippmann (1997) reported that over 21,000 plant taxa (including synonyms)are being used worldwide for pesticidal purposes, which amountedto about US$800 million in 1995–1996. If these plants had mainly resided inthe developed world, then developing countries would not benefit immensely

196 D.B. Olufolajifrom this since they are mostly poor. Despite the indiscriminate deforestationin developing countries in which these botanicals exist, rapid and largerecovery of most of these botanicals still occurs. It has been observed thatmost of the botanicals obtained in the tropical forests of developing countriessprout easily and survive through the little water stress experienced duringthe dry season. These attributes support the possibility of large-scale use ofthese botanicals as pesticides.9.4 Formulations of Botanicals in Developing CountriesLocal formulation by peasant farmersMethods adopted by farmers in the developing countries for the formulationand delivery of botanical pesticides vary depending on the parts of plantsused and the target pests. The plant parts could be either pulverized freshwith a pestle and mortar or grinding stones before soaking in water (hot orcold) or sun-dried before pulverizing into powdered form. They are thensoaked and extracted before use. The powdered form could also be used asdust to prevent incursion of pests and diseases on the stored farm produce orfor on-farm dusting for pest and disease control. The formulated botanicalscan be buried near the crop plants such that they can diffuse into the soil andprotect the root or move in a systemic way to control pests and diseases onthe farm. Termites, soil insects and some other soil pathogens such as Pythiumspp. could be controlled through this method.Some botanicals are effective when they are burnt and the fumes aredirected into storage silos or into the soil as in the orthodox fumigationpractice.Scientific formulationsResearchers in most of the developing countries have investigated morethan 100 different species of botanicals for their efficacy in crop protectionagainst pest degradation, especially the storage pests and diseases (Boekeet al., 2001; Ofuya et al., 2007; Olufolaji, 2008). Some researchers have suggestedthat a few botanicals used as pesticides in Nigerian flora withconfirmed pesticidal attributes against stored-product insects merit scientificformulations (Lale, 2001; Ofuya, 2003). However, pesticidal formulationsof most botanicals which have been found effective and may berecommended for use in crop protection in Nigeria are rather simple, andeasy to make in solid and liquid formulations (Ofuya, 2009). Solid formulationsare mainly powders, whereas liquid formulations include oils and thecrude extracts prepared in water and organic solvents. These methods offormulation of the botanicals as pesticides also make their large-scale useeasy and expansive in the developing countries because not muchtechnicality is involved.

Prospects of Large-scale Use of Natural Products 197Methods of formulationFormulations of the botanicals in developing countries are also of immenseimportance in their large-scale use. Due to low-level scientific technicalityand non-availability of sophisticated equipment associated with the productionof synthetic pesticides, the type of formulation associated with the productionof the botanical pesticides in developing countries is the type thatthe majority of the agrarian community and scientists will be able to handleand utilize for effective botanical pesticide formulation.PowdersThese are prepared by harvesting the plant materials, which are then sundriedand pulverized into fine powder. The powders have been investigatedundiluted for stored-product protection against insects and fungi. Therequired quantity of powder is admixed with an appropriate quantity ofcommodity prior to storage. Powders have also been extracted with water(water-extractable powders), filtered and applied as aqueous solutions forprotecting field crops and grains (Table 9.3; Ofuya, 2009).Crude water extractsThese are crude extracts obtained by using water as a solvent, and may beobtained simply by pressing out juices and then diluting in water or throughmaceration (steeping in water for prolong periods). They may also beobtained by infusion (the immersion of plant parts in boiling water for prolongedperiods). Such aqueous extracts or solutions have mostly been investigatedagainst field-crop insect pests and diseases (Table 9.3) (Olufolaji, 2006;Ofuya, 2009).OilsOils are usually extracted from pulverized medicinal plant parts by means oforganic solvents and are used very much like concentrate liquids. OrganicTable 9.3. Some medicinal plants in Nigeria and potential formulations in crop protection.BotanicalAzadirachta indicaPiper guineenseEugenia aromaticaDementia tripetaliaTephrosia vogelliNicotaina tabacumAllium sativumZingiber offi cinaleSource: Ofuya, 2009.Potential formulationDust, water-extractable powder, emulsifi able concentrate, liquidsDust, water-extractable powder, emulsifi able concentrate, liquidsDust, water-extractable powder, emulsifi able concentrate, liquidsDust, water-extractable powder, emulsifi able concentrate, liquidsWater-extractable powder, emulsifi able concentrate, liquidsDust, water-extractable powderWater-extractable powder, emulsifi able concentrate, liquidsWater-extractable powder, emulsifi able concentrate, liquids

198 D.B. Olufolajisolvents, particularly methanol, ethanol, acetone, hexane, petroleum ether,diethyl ether, chloroform or methyl chloride, have been used (Table 9.3)( Olufolaji, 2006; Ofuya, 2009).Mixed formulationsUsing herbal mixtures for crop protection is traditional with some farmers(Kitch et al., 1997). Efficacy of mixed formulations of medicinal plant powdersfor stored-grain protection has been subjected to empirical verification(Ogunwolu and Idowu, 1994; Dawodu and Ofuya, 2000; Emeasor et al., 2007).Overall pesticidal activity of each material was not mitigated by mixing thetwo against the test insects. However, synergistic or additive effects wouldbe desirable to enhance efficacy. Lale (2002) reported that mixing differentessential oils from plants, in some cases, provided much better control thansingle use (Table 9.3) (Ofuya, 2009; Olufolaji, 2006, 2008).Use of adjuvantsOfuya et al. (2007) have demonstrated the possibility of using organicflours from yam, cassava and plantain as diluents in the formulation ofinsecticidal dusts from buds of Eugenia aromatica and dry fruits of Piperguineense. Filtrates that are obtained in the preparation of aqueous neemextracts can be improved after dilution by adding brown sugar, whichimproves the adherence of the filtrate on leaves and other plant parts(Jackai, 1993).9.5 Attributes of the Botanicals Versus ConventionalSynthetic PesticidesThe botanicals have an array of good attributes over the synthetic pesticides,which is encouraging because the low technicality required for use hasenabled the developing world to achieve desirable advantages from theiruse. Ahmed and Stoll (1996) highlighted the various problems caused bysynthetic pesticides including toxicity to non-target organisms such as manand beneficial macro/microbes, pollution in the agro-ecosystem and developmentof resistance by the target organisms. However, the botanical pesticidespossess some special characteristics and may be used as betteralternatives in managing different agricultural pests:●●●The materials used for the production of the botanical pesticides areeasily available since they are usually weeds in the agro-ecosystem.Botanical pesticides are usually inherently less harmful than conventionalpesticides.Botanical pesticides generally affect only the target pest and closelyrelated organisms, in contrast to broad-spectrum conventionalpesticides that may affect organisms as different as birds, insects andmammals.

Prospects of Large-scale Use of Natural Products 199●●Botanical pesticides are often effective in very small quantities andoften decompose quickly, thereby resulting in lower exposure andlargely avoiding the pollution problems caused by conventionalpesticides.When used as a component of integrated pest management (IPM)programmes, botanical pesticides can greatly decrease the use ofconventional pesticides, while crop yields remain high.9.6 The Views of Environmental Protection Agency and the Policyof Most Developing CountriesThe view of the EPA at national and inter national levels is not to supportthe use of synthetic pesticides. These views have made some advancedand developed countries to promulgate laws against the usage of some ofthese synthetic pesticides. In Nigeria, the body vested with the authorityof pesticides registration and control is the National Agency for Food andDrug Adminstration and Control (NAFDAC) (Olaifa, 2009). Despite this,some pesticides such as DDT and some mercuric-based pesticides, whichhad been banned from use due to the residue left on crops for consumptionand because they constitute a health hazard, are still found in pesticidesmarkets. The presence of heavy metals in most pesticides have causeduntold hardship and death to the consumer of the crops on which it has beenused.Most of the developing countries have formulated various policieson the usage of the synthetic pesticides. The policy emanated from theeffect which the pesticides have unleashed on the crops due to theirphyto toxicity, mammalian toxicity and carcinogenic effects on both thecrops and man.In 1994, the Botanical Pesticides and Pollution Prevention Division wasestablished in the Office of Pesticide Programmes to facilitate the registrationof botanical pesticides. This division promotes the use of safer pesticides,including botanical pesticides, as components of IPM programmes. The divisionalso coordinates the Pesticide Environmental Stewardship Programme(PESP).Since botanical pesticides tend to pose fewer risks than conventionalpesticides, the EPA generally requires much less data to register a botanicalpesticide than to register a conventional pesticide. In fact, new botanical pesticidesare often registered in less than a year, compared with an average ofmore than 3 years for conventional pesticides.While botanical pesticides require less data and are registered in lesstime than conventional pesticides, the EPA always conducts rigorous reviewsto ensure that pesticides will not have adverse effects on human health or theenvironment. For the EPA to be sure that a pesticide is safe, the agencyrequires that registrants submit a variety of data about the composition,toxicity, degradation and other characteristics of the pesticide.

200 D.B. Olufolaji9.7 The Attitudes of the Poor Resource Farmers in theDeveloping Countries to the Use of Botanical PesticidesThe poor resource farmers in the developing countries have been relieved bythe use of botanicals as it affords them the advantages of low pest controlcost, easy pest control strategies, minimum pollution in their agro-ecosystemand, ultimately, higher yield and profit. They are now in search of any availableinformation regarding new innovations on botanicals to control thepests on their farm lands. They are also ready to pay for the services becausebotanicals cost less than the conventional synthetic pesticides with all thesafe attributes to food production in the developing world. The farmers arealready trying to combine the research findings of scientists with their agelongpractice to arrive at formidable control measures on their farm lands.Interestingly, the farmers are now adopting the principle of IPM in order tomaximize yields on their farms. Moreover, the botanists who engage in floricultureand horticulture are included in the use of botanicals to preservemost of their important species of plants from the scourge of pests and diseases.The farmers have already formed cooperatives and through themapproach government extension agencies for more enlightenment on the useof botanicals for their benefit.9.8 The Future of the Use of BotanicalsThe future of botanicals as alternatives to synthetic pesticides is bright, in thatmore people are using them due to the availability of the raw materials, accessibilityof the raw materials and easy use of the botanicals, with minimumdemand for specialized application and equipment that is characteristic of syntheticpesticides. Most of the research on pest and disease control in both developedand developing countries are now centred on the use of botanicals asalternatives to synthetic pesticides. The World Health Organization has alsoencouraged the use of botanicals because there is a prevalence of diseases traceableto chemicals used for the control of plant diseases because the consumedplant materials possess high degree of residues that cause carcinogenic diseasesand other health issues. It is also an issue with environmentalists concernedabout the threat of global warming because most of the pollution accredited tocause global warming is traced to the pollutants produced and discharged inthe developing countries (Olufolaji, 2006). The present economic recession haseven made the purchase of synthetic pesticides more expensive and thus difficultfor the poor and even the enlightened farmers in the developing countries,hence the adoption of an alternative, which is the use of botanical pesticides.9.9 ConclusionThe botanicals have solved enormous problems of farmers in the developingworld who are faced with an array of pests and diseases that reduce theiryields and make farming a harder process.

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204 D.B. OlufolajiHumboldt Kellog / 3rd Annual Conferenceof School of Agriculture and AgriculturalTechnology, School of Agriculture andAgricultural Technology, The FederalUniversity of Technology, Akure,Nigeria, pp. 8–13.Oyun, M.B. and Agele, S.O. (2009) Testingof extracts from Eucalyptus camaldulensis,Acacia auriculiformis Gliricidia sepiumfor weed suppression in a maize farm.In: Proceeding of the Humboldt Kellog/5th SAAT Annual Conference of formulationsof Medicinal Plants in Plant andAnimal Production in Nigeria, Schoolof Agriculture and AgriculturalTechnology, The Federal University ofTechnology, Akure. Ondo State, Nigeria,pp. 89–93.

10 Current Status of NaturalProducts in Pest Managementwith Special Reference toBrassica carinata as aBiofumigantMARÍA PORRASDepartment of Crop Protection, IFAPA Centro Las Torres – Tomejil,Sevilla, SpainAbstractThis chapter covers the use of natural products in plant disease management in theform of fungicides and bactericides, and personal experience of the development andoptimization of biofumigation with Brassica carinata and soil solarization as alternativesto the traditional use of chemicals in strawberry production. The potential of biofumigationas a component of the integrated management of soil pathogens has been demonstratedin various agricultural systems. The presence of high amounts of glucosinolates,and of the enzyme myrosinase that catalyses their hydrolysis, linked to the high biocidalactivity of some glucosinolate enzymatic hydrolysis derivative products have beensuggested for a practical use of amending soil with these natural biocidal compoundsthrough cultivation and green manure of selected species of the family Brassicaceae.10.1 IntroductionChemical pesticides such as methyl bromide are being phased out globallybecause of their impact on the ozone layer (European Parliament, 2000).Since 2005, the use of methyl bromide has been banned in European Unioncountries. Chemical, physical and biological alternative methods for pathogencontrol have been evaluated in crop production to replace the compoundslost due to the new registration requirements (Duniway, 2002; Martinand Bull, 2002; Porras et al., 2007b). Natural-product-based pesticides cansometimes be specific to the target species and have unique modes of actionwith little mammalian toxicity (Duke et al., 2003). Modern instrumentationand improved methods should increase interest in natural-product-basedpesticide discovery research (Duke et al., 2002).© CAB International 2011. Natural Products in Plant Pest Management(ed. N.K. Dubey) 205

206 M. PorrasNaturally occurring substances found in fungi, bacteria and plants areimportant sources of molecules with different biological properties. Theymay be developed either as products per se or used as starting points forsynthesis to optimize specific properties. Low mammalian toxicity, low environmentalimpact, low levels of residues in food and compatibility with integratedpest management (IPM) programmes are important considerations inthe election of fungicides for development (Knight et al., 1997).This chapter covers the use of natural products in plant disease management(fungicides and bactericides), and personal experience of Brassicacarinata as a biofumigant in strawberry fields.10.2 Natural Products Used as Fungicides and Bactericides inAgricultureThe natural products used as agrochemicals have been well documented(Knight et al., 1997; Warrior, 2000; Copping and Duke, 2007; Kim and Hwang,2007; Dayan et al., 2009). Microorganisms can synthesize secondary metabolitesof versatile chemical structures with diverse biological activities thatexceed the scope of synthetic organic chemistry (Porter, 1985). Pesticidalpotency is not the only factor for formulating a plant-based product as acommercial pesticide. In-field chemical stability adequate to reduce theapplication times to an economical level, and lowered volatility for sufficientretention on the surface of host plants are also important factors for microbialfungicides to be efficiently applied in agriculture (Kim and Hwang, 2007).Therefore, only a few microbial metabolites have been successfully developedinto commercial fungicides. The excellent fungicidal activity of thesemicrobial metabolites and their potential as lead candidates for further fungicidedevelopment continue to stimulate research and screening for antifungalmicrobial metabolites (Kim and Hwang, 2007; Dubey et al., 2009). Table 10.1summarizes the microbial metabolites, and plant- or animal-derived productsused as fungicides or bactericides in agriculture.Biological control agents (including both bacteriological or fungus origin),plants, natural compounds and preparations have been described withactivity against bacterial or fungal plant pathogens. Furthermore, plants protectthemselves from microbial attacks with both constitutive antimicrobialsand compounds induced by the attacking pathogens (phytoalexins). Naturalproducts have been used to protect plants indirectly from pathogens byinducing systemic acquired resistance (SAR). The SAR-inducing compoundsare termed elicitors. Pathogens cannot evolve resistance directly to the elicitorbecause the activity is indirect, making such products excellent candidatesfor integrated disease management. Nevertheless, elicitors are generallynot as effective as chemical fungicides, partly because the timing of elicitorapplication and threat to the crop by a pathogen is crucial, but difficult tomaximize (Dayan et al., 2009).Disease suppression by biocontrol agents is the sustained manifestationof interactions between the plant, the pathogen, the biocontrol agent, and the

Natural Products as Alternatives to Synthetic Pesticides 207Table 10.1. A summary of natural products used as agrochemicals.Species Products Target pathogens ReferencesBacteriaStreptomycesgriseochromogenesErwinia amylovora(Burrill) WinslowStreptomyceskasugaensisHamad et al.StreptoverticilliumrimofaciensStreptomycesnatalensisStruyk, Hoette, Drost,Waisvisz, Van Eek& HoogerheideS. chattanoogensisBurns & HoltmanBacteria-derivedproductsBlasticidin-SHarpin proteinKasugamycinMildiomycinNatamycinPyricularia oryzaeCavara; perfect stageMagnaporthe grisea(Hebert) BarrXanthomonas campestris(Pammell) DowsonPseudomonas syringaeVan HallPseudomonas solanacearum(Smith) SmithFusarium sp.Phytophthora sp.Magnaporthesalvinii (Cattaneo)Krause & WebsterRhizoctonia solaniKühn (Pelliculariasasakii Ito)Venturia inaequalis(Cooke) WinterErwinia amylovoraBotrytis sp.Guignardia bidwellii (Ellis)Viala & RivasDiplocarpon rosae WolfP. oryzaeCercospora spp.Venturia spp.Cladosporium fulvumErwinia atrosepticaXanthomonas campestrisErysiphe spp.Uncinula necator(Schwein) BurrillPodosphaera spp.Sphaerotheca spp.Fusarium oxysporumSchlechtKim andHwang,2007Copping andDuke, 2007Anon, 2005;Copping andDuke, 2007Copping andDuke, 2007Copping andDuke, 2007Continued

208 M. PorrasTable 10.1. Continued.Species Products Target pathogens ReferencesStreptomyces rimosusSobin et al.Streptomycescacoai var.asoensisIsono et al.OxytetracyclinePolyoxins:polyoxin BErwinia amylovoraPseudomonas spp.Xanthomonas spp.Sphaerotheca spp. andother powdery mildewsBotrytis cinerea Pers.Sclerotinia sclerotiorumDe BaryCorynespora melonis LindauCochliobolus miyabeanus(Ito & Kuribay) Drechslerex DasturAlternaria alternata (Fr.)Keissler and otherAlternaria spp.Copping andDuke, 2007Copping andDuke, 2007Streptomycesgriseus(Krainsky)Waksman & HenriciStreptomyceshygroscopicus(Jensen) Waksman &Henrici var. limoneusPseudomonasfl uorescenspolyoxorim(polyoxin D)StreptomycinR. solaniNectria galligenaBresadola (Diplodiapseudodiplodia Fuckel)Drechslera spp.Bipolaris spp.Curvularia spp.Helminthosporium spp.Bacteria, particularlyeffective against:Xanthomonas oryzaeDowsonX. citri DowsonPseudomonastabaci StevensCopping andDuke, 2007P. lachrymans CarsnerValidamycin Rhizoctonia spp. Copping andDuke, 2007PyrrolinitrinB. cinereaM. grisea(In vitro and in thegreenhouse, not infi eld because ofphotosensitivity)Haas andKeel, 2003;Kim andHwang,2007Continued

Natural Products as Alternatives to Synthetic Pesticides 209Table 10.1. Continued.Species Products Target pathogens ReferencesFungiSaccharomycescerevisiaeMeyer ex HansenStrobilurustenacellusOudemansiellamucidaPlantsCassia tora L.(also known asCassia obtusifolia L.)The two activeingredients arefound in virtually allliving organisms andin their pure formSimmondsiacalifornica Nutt. andS. chinensis Link.Laminariadigitata (Hudson)Fenpiclonil andFludioxonil(Phenylpyrrolesderivates toenhancedphotostability)Fungus-derivedproductsYeast extracthydrolysateStrobilurin AOudemansin AFrom which syntheticstrobilurins such asazoxystrobin andkresoxim-methylhave beendevelopedPlant-derivedproductsCinnamaldehydeL-glutamic acid andγ-aminobutyricacid (Auxien hasregistered thecombination productcontaining bothactive indredients)Fusarium graminearumSchwabeGerlachia nivalisGams & MüllBotrytis spp.Monilinia spp.Sclerotinia spp.niResistance tostrobilurins hasalready evolvedVerticillium spp.Rhizoctonia spp.Pythium spp.Sclerotinia homeocarpaBennettFusarium moniliformevar. subglutinansWollenw. & ReinkingPowdery mildewCopping andDuke, 2007Ishii et al.,2001;Balba, 2007Copping andDuke, 2007Copping andDuke, 2007Jojoba oil Powdery mildew Copping andDuke, 2007LaminarineFungal pathogens ofcereals, particularlyseptoria and powderymildewsCopping andDuke, 2007Continued

210 M. PorrasTable 10.1. Continued.Species Products Target pathogens ReferencesReynoutriasachalinensis(Fr. Schm.) NakaiMacleayacordataR. Br.Rosmarinusoffi cinalisThymusvulgarisAzadirachtaindicaGossypiumhirsutum andAllium sativumExtract of giantknotweedPink plume poppyextractBotrytis spp.Powdery mildewsXanthomonas spp.The target pathogensare those causingfoliar fungal diseases,such as powderymildew, alternarialeaf spotand septoria leaf spotCopping andDuke, 2007Copping andDuke, 2007Rosemary oil ni Dayan et al.,2009Thyme oil ni Dayan et al.,2009Clarifi edniDayan et al.,hydrophobic2009extract ofneem oilCottonseed oil withgarlic extractniDayan et al.,2009AnimalsAnimal-derivedproductsCow Milk Powdery mildews Copping andDuke, 2007Dried, crushedcrustaceanexoskeletonsPoly-D-glucosamineor chitosanPowdery mildewsBotrytis spp.Copping andDuke, 2007ni, information not provided in reference.microbial community on and around the plant and the physical environment(Handelsman and Stabb, 1996). Relevant biocontrol microorganisms of therhizosphere include Pseudomonas spp., Bacillus spp., Streptomyces spp., Trichodermaspp., and non-pathogenic Fusarium spp. (Cook, 1993; Paulitz andBelanger, 2001; Whipps, 2001). Different species of Trichoderma are studiedprimarily for their ability to control plant disease through antagonism, rhizospherecompetence, enzyme production, induction of defence responses inplants, metabolism of germination stimulants, and beneficial growth of thehost following root colonization (Weindling, 1934; Zimand et al., 1996; Baileyand Lumsden, 1998; Howell, 2003; Porras et al., 2007a,b). Determination ofthese effects depends on many interactions that take place in the soil amongTrichoderma spp., other microorganisms, the plant root, and the soil environment(Bailey and Lumsden, 1998).

Natural Products as Alternatives to Synthetic Pesticides 21110.3 Biofumigation with BrassicaThe term biofumigation has been defined as the ‘suppression of soil-bornepathogens and pests by Brassica rotation or green manure crops, throughliberation of isothiocyanates from hydrolysis of the glucosinolates thatcharacterize the Brassicaceae’ (Kirkegaard and Matthiessen, 2004).Biofumigation is based on the action of volatile compounds, especiallyisothiocyanates, produced by the hydrolysis of glucosinolates. Glucosinolatesare secondary metabolites produced by plants belonging to the Capparalesorder (Gimsing and Kirkegaard, 2009). Of the many hundreds of cruciferousspecies investigated, all are able to synthesize glucosinolates, and at least500 species of non-cruciferous dicotyledonous angiosperms have beenreported to contain them. Most glucosinolate-containing genera are clusteredwithin the Brassicaceae, Capparaceae and Caricaceae. Among the Brassicaceae,the genus Brassica contains a large number of the commonly consumablespecies (Fahey et al., 2001). The enzyme myrosinase, produced by all plantsthat produce glucosinolates, is physically separated from the glucosinolatesin the intact plant tissue. Glucosinolates and myrosinase are reported to belocated in vacuoles, and are not colocalized in the same cells (Andréasson etal., 2001) (Fig. 10.1). Upon tissue disruption the glucosinolates and the myrosinasecome into contact (Fig. 10.2) and the myrosinase hydrolyses the glucosinolatesto form hydrolysis products such as isothiocyanate, nitrile andthiocyanate (Gimsing and Kirkegaard, 2009) (Fig. 10.3).The different chemical side-chain structures of the isothiocyanatesdetermine important chemical and physical properties such as hydrophobicityand volatility, and their toxicity (Brown and Morra, 1997). Biofumigationcan be achieved by incorporating fresh plant material (green manure),seed meals (a by-product of seed crushing for oil), or dried plant materialtreated to preserve isothiocyanate activity (Kirkegaard and Matthiessen,2004; Lazzeri et al. 2004; Matthiessen and Kirkegaard, 2006).A field experiment conducted in Huelva (South-west Spain; the mostimportant area of strawberry production in Europe) contributes to the developmentand optimization of biofumigation with Brassica and soil solarizationas alternatives to the traditional use of chemicals in strawberry production(Porras et al., 2008).Solarization is a process that employs solar radiation to heat soil under atransparent plastic film to temperatures that are detrimental to soilbornepathogens. Increased soil temperatures can decrease populations of plantpathogens (Katan, 1981). Solarization can enhance the effectiveness of otherpest management approaches (Katan, 1981; Ben-Yephet et al., 1987; Ramirez-Villapudua and Munnecke, 1988; Porras et al., 2007a,b) and has the additionaladvantages of being a non-chemical alternative method for pathogen control(Batchelor, 2002).The soil in the field experiment had never been previously treated withmethyl bromide, and was naturally infested by Phytophthora spp. Treatmentswere soil solarization (S), biofumigation + solarization (B + S), and theuntreated control (C). Treatments were applied in the same plots each year

212 M. PorrasMyrosinaseGlucosinolateFig. 10.1. Intact vegetal tissue with the myrosinase enzymes and the glucosinolatesphysically separated (within vacuoles).(2005–2006 and 2006–2007). Biofumigation was done with Brassica carinata inthe siliqua stage that showed the major suppressive effect in previous in vitroexperiments (Romero et al., 2008). B. carinata was cut, because upon tissuedisruption the substrate and the enzyme come into contact and the myrosinasehydrolyses the glucosinolates to form hydrolysis products. Thechopped material was incorporated into the soil to a depth of 10 cm using arotary tiller. The soil was covered using clear 50-μm low-density polyethylenemulch, and drip-irrigated from July to September (Fig. 10.4).Plots were covered to avoid the escape of isothiocyanates from the soil byvolatilization and to increase soil temperatures. Drip irrigation was applied toenhance the formation of isothiocyanates in soil (Matthiessen et al. 2004).

Natural Products as Alternatives to Synthetic Pesticides 213MyrosinaseGlucosinolateFig. 10.2. Upon tissue disruption the glucosinolates and the myrosinases come into contactand the myrosinase hydrolyses the glucosinolates to form hydrolysis products.GlucoseSCR NOSO 3 –R N C SR S C NR C NIsothiocyanateThiocyanateNitrileGlucosinolatesFig. 10.3. Glucosinolates and hydrolysis products originated by the activity of the enzymemyrosinase.

214 M. Porras(a)(b)(c)(d)Fig. 10.4. Brassica carinata (a) used as biofumigant was chopped (b), buried (c),and covered and drip-irrigated (d).Biofumigation and solarization increased foliar surface and strawberryyield the most each year, and differences were observed relative tosolarization alone and the untreated control (Tables 10.2 and 10.3).Biofumigation and solarization reduced Phytophthora spp. soil populationrelative to the untreated control, but it did not totally eliminate thepathogen. According to previous experiments (Porras, 2005; Porras et al.,2007a), the results indicate that solarization may serve as a component inan integrated, sustainable approach to the management of Phytophthoraspp. in strawberry.The presence of high amounts of glucosinolates, and of the enzymemyrosinase that catalyses their hydrolysis, linked to the high biocidalactivity of some glucosinolate enzymatic hydrolysis derivative products(mainly isothiocyanates and nitriles) have suggested the practicalpossibility of amending soil with these natural biocidal compoundsby the cultivation and green manure of selected species of the familyBrassicaceae (Lazzeri et al., 2004). The potential of biofumigation as a

Natural Products as Alternatives to Synthetic Pesticides 215Table 10.2. Foliar surface (cm 2 ) in December and January 2005–2006 and 2006–2007.2005–2006 2006–200716th December 13th January 19th December 9th JanuaryB + S 295.90 a 502.49 a 367.04 a 435.14 aS 229.91 b 413.58 b 301.07 b 346.31 bC 204.74 b 350.57 c 208.32 c 228.03 cF 2,37429.00* 20.83* 36.10* 53.48*Letters indicate a signifi cant difference, Tukey HSD test; *, P

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11 Fungal Endophytes:an Alternative Sourceof Bioactive Compoundsfor Plant ProtectionR.N. KHARWAR 1 AND GARY STROBEL 21 Department of Botany, Banaras Hindu University, India; 2 Department ofPlant Sciences, Montana State University, Bozeman, USAAbstractEndophytes are a group of microorganisms that represent an abundant anddependable source of bioactive and chemically novel compounds with potentialfor exploitation in a wide variety of applications. The mechanisms through whichendophytes exist and respond to their surroundings must be better understood inorder to be more predictive about which higher plants to seek, study and employin isolating their microfloral components. This may facilitate the natural productdiscovery process. Endophytic fungi are now attracting great interest fromresearchers for an alternative way of controlling plant pathogens.11.1 IntroductionThe bioactive compounds of natural origin have been under ever-increasingdemand to solve various problems related to human health and agriculture.Plants have provided humans with resources for healing purposes for millennia.Some representative and well known medicines derived from plantsare quinine, digitalin, taxol, aspirin, reserpine and many more (Wani et al.,1971; Hoffman et al., 1998). Some plants have been under great threat as aresult of the enormous pressure brought upon them by virtue of their healingand biocontrol properties.In some cases, plant-associated fungi are able to make the same bioactivecompounds as the host plant itself and one of the best examples of thisis the discovery of the gibberellins in Fusarium fujikuroi in the early 1930s(Borrow et al., 1955). Eventually it was learnt that the gibberellins are one ofonly five classes of phytohormones that are to be found in virtually allplants. Endophytic (endon = inner; phyton = plants) fungi are a groupof microorganisms that reside in healthy and functional inner tissues ofthe plants without causing any detectable symptoms (Petrini, 1991).© CAB International 2011. Natural Products in Plant Pest Management218 (ed. N.K. Dubey)

Fungal Endophytes 219The observation that fungi make the same compounds as their host ledStierle et al. (1993) to examine the prospect that endophytic fungi associatedwith Taxus brevifolia may also produce taxol, the most promising naturalbioactive molecule discovered against cancer. This significant discoveryestablished that the endophytic fungi which reside in the living healthy tissuesof the plant, may produce one or more bioactive principles or naturalproducts with either medicinal/therapeutic or biocontrol potential whichwere previously known to be produced by the host plant (Wani et al., 1971;Stierle et al., 1993; Dimetry et al., 1995; Hoffman et al., 1998; Gahukar, 2005).Taxol itself is the world’s first billion dollar anticancer drug and its mainsource is Taxus spp. Potentially, a fungal source of taxol would reduce itsprice and save the plant, in some areas, from extinction. The success of findingfungal taxol has produced a paradigm for other bioactive compoundsto be found in endophytic fungi. This might have happened during thecourse of evolution of symbiosis between fungus and host plant, wherereciprocal gene transfer has occurred. As observed in the case of Agrobacteriumtumefaciens, the t-DNA of the bacterial plasmid was transferred andincorporated into the genome of the host plant. This is a fine example of thetransaction of genetic materials between prokaryote and eukaryote. However,in case of the fungal endophyte–host relationship, it is a eukaryote toeukaryote transaction.As the fungi are a less studied group of microbes, only a few studiescan be exemplified. Alkaloids synthesized by Neotyphodium sp. in its grasshosts have been implicated in fescue toxicosis in rangeland animals. Thechemistry and biology of this and other grass endophytes are reviewedelsewhere. Unfortunately, because this work was so comprehensive, onemay be led to the conclusion that endophytes only produce toxic compoundsin their respective hosts and that they hold no promise for anymedicinal applications. It turns out that this is simply not the case. Endophytesexamined from a plethora of sources show that an overwhelmingnumber of them produce natural products with promising potential formany applications.Bacon and White (2000) gave an inclusive and widely accepted definitionof endophytes: ‘Microbes that colonize living, internal tissues of plantswithout causing any immediate, overt negative effects’ (Fig. 11.1). While theusual lack of adverse effect nature of endophyte occupation in plant tissuehas prompted a focus on symbiotic or mutualistic relationships betweenendophytes and their hosts, the observed biodiversity of endophytes suggeststhey can also be aggressive saprophytes or opportunistic pathogens(Promputtha et al., 2007).Both fungi and bacteria are the most common microbes existing as endophytes.It would seem that other microbial forms most certainly exist inplants as endophytes such as mycoplasmas, rickettsia, streptomycetes andarchebacteria. In fact, it may be the case that the majority of microbes existingin plants are not culturable with common laboratory techniques, makingtheir presence and role in plants even more intriguing. The most frequentlyisolated endophytes are the fungi (Redlin and Carris, 1996). However, at the

220 R.N. Kharwar and G. StrobelFig. 11.1. A prominent endophytic fungal mycelium lying between the cellular spaces.outset, it is important to note that the vast majority of plants have not beenstudied for any endophytic association. Thus, enormous opportunities existfor the recovery of novel fungal forms, including genera, biotypes, as well asspecies in the myriad of plants yet to be studied. Hawksworth and Rossman(1987) estimated there may be as many as 1.5 million different fungal species,yet only about 100,000 have been described. As more evidence accumulates,estimates keep rising as to the actual number of fungal species. For instance,Dreyfuss and Chapela (1994) have estimated at least 1 million species ofendophytic fungi alone. It seems obvious that endophytes are a rich andreliable source of genetic diversity and may represent many previouslyundescribed species.Out of 300,000 higher plants that exist on the earth, only a handful,primarily grass species, have been studied relative to their endophyticbiology. Consequently, there is enormous opportunity to find new andinteresting endophytic fungi amongst the myriad of plants in different settingsand ecosystems. The intent of this chapter is to provide insights intothe occurrence of endophytes in nature, the products that they make, andhow some of these organisms show their potential for human and plantuse. The chapter discusses the rationale for study, methods used, andexamples of some of the endophytes isolated and studied over the courseof time. This chapter, however, also includes some specific examples thatillustrate the work done in this emerging field of bioprospecting andsourcing the endophytic fungi.

Fungal Endophytes 22111.2 Diversity in EndophytesOf the myriad of ecosystems on Planet Earth, those having the greatest generalbiodiversity of life seem to be the ones that also have the greatest numberand most diverse endophytes. Tropical and temperate rainforests are themost biologically diverse terrestrial ecosystems. The most threatened of thesespots cover only 1.44% of the land’s surface, yet they harbour over 60% of theworld’s terrestrial biodiversity. In addition, each of the 20–25 areas identifiedas supporting the world’s greatest biodiversity also support unusually highlevels of plant endemism (Mittermeier, 1999). As such, one would expect,that with high plant endemism, there should also exist specific endophytesthat may have evolved with the endemic plant species. Biological diversityimplies chemical diversity because of the constant chemical innovation thatis required to survive in ecosystems where the evolutionary race to surviveis most active. Tropical rainforests are a remarkable example of this type ofenvironment. Competition is great, resources are limited and selection pressureis at its peak. This gives rise to a high probability that rainforests are asource of novel molecular structures and biologically active compounds(Redell and Gordon, 2000).Bills et al. (2002) describe a metabolic distinction between tropical andtemperate endophytes through statistical data which compares the numberof bioactive natural products isolated from endophytes of tropical regions tothe number of those isolated from endophytes of temperate origin. Not onlydid they find that tropical endophytes provide more active natural productsthan temperate endophytes, but they also noted that a significantly highernumber of tropical endophytes produced a larger number of active secondarymetabolites than did fungi from other substrata. This observation suggeststhe importance of the host plant as well as the ecosystem in influencingthe general metabolism of endophytic microbes.11.3 Techniques for Isolation, Preservation and Storage ofEndophytesCultures for product isolationDetailed techniques for the isolation of microbial endophytes are outlined ina number of reviews and technical articles (Strobel et al., 1993; Strobel, 2002;Strobel and Daisy, 2003; Strobel et al., 2004; Castillo et al., 2005). If endophytesare being obtained from plants growing in polar regions, the dry tropics, orsome temperate areas of the world, one can expect to acquire from none toonly one or two endophytic cultures per plant sample (0.5–10.0 cm limbpiece). However, from the wet tropics this number may rise to 20–30 or evenmore microbes per plant piece. Given limited fermentation capabilities, it iscritical to label and store cultures of freshly isolated microbes for work in thefuture and for patent and publication purposes.

222 R.N. Kharwar and G. StrobelGenerally, preservation in an aqueous 15% glycerol solution at –70°C isan exceedingly good procedure for saving cultures until work on them canproceed at a later date (Strobel et al., 1993; Strobel, 2002; Strobel and Daisy,2003; Strobel et al., 2004).Growing fungal cultures on sterilized barley or other grains withsubsequent storage at –70°C is an effective alternative to the glycerol storagesolution.Plant selection norms and ethicsIt is important to understand that the methods and rationale used to guideplant selection seem to provide the best opportunities to isolate novel endophyticmicroorganisms at the genus, species or biotype level. Since the numberof plant species in the world is so great, creative and imaginative strategiesmust be used to quickly narrow the search for endophytes displayingbioactivity (Mittermeier et al., 1999).A specific rationale for the collection of each plant for endophyte isolationand natural product discovery is used. Several hypotheses govern thisplant selection strategy:1. Plants from unique environmental settings, especially those with anunusual biology, and possessing novel strategies for survival are seriouslyconsidered for study.2. Plants that have an ethnobotanical history (use by indigenous peoples)that are related to the specific uses or applications of interest are selected forstudy. These plants are chosen either by direct contact with local people orvia local literature. This approach may also provide benefits to the local/tribal people by documenting their help under a participatory research programme.The ethics of science restrict the researcher drawing the monetaryand intellectual benefits alone, especially when knowledge of local people isused. However, it often happens that at the time of documentation, scientistsdon’t give proper importance to the knowledge source or the local people,and this must be checked to maintain the sanctity of the research community.Ultimately, it may be learned that the healing powers of the botanical source,in fact, may have nothing to do with the natural products of the plant, but ofan endophyte inhabiting the plant.3. Plants that are endemic, having an unusual longevity, or that haveoccupied a certain ancient land mass, such as Gondwanaland, are also morelikely to lodge endophytes with active natural products than other plants.4. Plants growing in areas of great biodiversity, it follows, also have theprospect of housing endophytes with great biodiversity.Distinct environmental settings are therefore considered to be a promisingsource of novel endophytes and their compounds, and so too are plantswith an unconventional biology. For example, an aquatic plant, Rhyncholacispenicillata, was collected from a river system in south-west Venezuela wherethat plant faced constant beating by virtue of rushing waters, debris, and

Fungal Endophytes 223tumbling rocks and pebbles. These environmental insults created manyportals through which common phytopathogenic oomycetes could enter theplant. Still, the plant population appeared to be healthy, possibly due to theprotection provided by an endophytic product. This was the environmentalbiological clue used to choose this plant for a comprehensive study of itsendophytes.An example of plants with an ethnobotanical history is the snakevineKennedia nigriscans, from the Northern Territory of Australia, which wasselected for study since its sap has traditionally been used as a bush medicine.In fact, this area was selected for plant sampling as it has been hometo the world’s longest standing human civilization, the Australian Aborigines.The snakevine is harvested, crushed and heated in an aqueous brewby local Aborigines in South-west Arnhemland to treat cuts, wounds andinfections. As it turned out, the plant contains an entire suite of streptomycetes(Castillo et al., 2005). One in particular, designated Streptomyces NRRL30562, has unique partial 16S rDNA sequences when compared to those inGenBank (Castillo et al., 2002). It produces a series of actinomycins includingactinomycins D, Xo ß, and X 2. It produces novel, broad-spectrum peptideantibiotics called munumbicins. This seems to be an excellent example illustratingthe potential benefits of knowledge of indigenous peoples andresearchers.Furthermore, some plants generate bioactive natural products and haveassociated endophytes that produce the same natural products. Such is thecase with taxol, a highly functionalized diterpenoid and famed anticanceragent that is found in Taxus brevifolia and other yew species (Taxus spp.) (Waniet al., 1971). In 1993, a novel taxol-producing fungus Taxomyces andreanae wasisolated and characterized from Taxus brevifolia (Strobel et al., 1993).While combinatorial synthesis produces compounds at random, secondarymetabolites, defined as low molecular weight compounds notrequired for growth in pure culture, are produced as an adaptation forspecific functions in nature (Demain, 1981). Schulz (2001) points out thatcertain microbial metabolites seem to be characteristic of certain biotopes,both at an environmental as well as an organism level. Accordingly, itappears that the search for novel secondary metabolites should centre onorganisms that inhabit unique biotopes. Thus, it behoves the investigator tocarefully study and select the biological source before proceeding, ratherthan to take a totally random approach in selecting the source material.Careful study also indicates that organisms and their biotopes that are subjectedto constant metabolic and environmental interactions should produceeven more secondary metabolites (Schulz at al., 2002). Endophytes aremicrobes that inhabit such biotopes, namely higher plants, which is whythey are currently considered as a wellspring of novel secondary metabolitesoffering the potential for exploitation of their medical benefits (Tanand Zou, 2001). Moreover, novel compound-screening experiments havealready proved that endophytes are considerably ahead of soil isolates,with 51% new structure compounds compared with 38%, respectively(Schulz et al., 2002).

224 R.N. Kharwar and G. StrobelMolecular techniques provided unexpected and thrilling results withPestalotiopsis microsporaIt is of some compelling interest how the genes for taxol production may havebeen acquired by endophytes such as Pestalotiopsis microspora (Long et al.,1998). Although the complete answer to this question is not at hand, someother relevant genetic studies have been performed on this organism.P. microspora Ne 32 is one of the most easily genetically transformable fungithat has been studied to date. In vivo addition of telomeric repeats to foreignDNA generates chromosomal DNAs in this fungus (Long et al., 1998). Repeatsof the telomeric sequence 5′-TTAGGG-3′ were appended to non-telomerictransformingDNA termini. The new DNAs, carrying foreign genes and thetelomeric repeats, replicated independently of the chromosome and expressedthe information carried by the foreign genes. The addition of telomeric repeatsto foreign DNA is unusual among fungi. This finding may have importantimplications in the biology of P. microspora Ne 32 since it explains at least onemechanism as to how new DNA can be captured by this organism and eventuallyexpressed and replicated. Such a mechanism may begin to explain howthe enormous biochemical variation may have arisen in this fungus. Also, thisinitial work represents a framework to aid in the understanding of how thisfungus may adapt itself to the environment of its plant hosts and suggeststhat the uptake of plant DNA into its own genome may occur. In addition, thetelomeric repeats have the same sequence as human telomeres which pointsto the possibility that P. microspora may serve as a means to make artificialhuman chromosomes, a totally unexpected result.11.4 Natural Products and Phytochemicals from EndophytesIt has been suggested that the reason some endophytes produce certain phytochemicals,originally characteristic of the host, might be related to a geneticcombination of the endophyte with the host that occurred in evolutionarytime (Tan and Zou, 2001). This is a concept that was originally proposed as amechanism to explain why Taxomyces andreanae may be producing taxol (Stierleet al., 1993). All aspects of the biology and interrelationship of endophyteswith their respective hosts is a vastly under-investigated and exciting field(Strobel, 2002; Strobel and Daisy, 2003; Strobel et al., 2004). Thus, more backgroundinformation on a given plant species and its microorganismal biologywould be exceedingly helpful in directing the search for bioactiveproducts (Lane et al., 2000). Presently, no one is quite certain of the role ofendophytes in nature and what appears to be their relationship to varioushost plant species. While some endophytic fungi appear to be ubiquitous(e.g. Fusarium spp., Pestalotiopsis spp., Aspergillus spp. and Xylaria spp.), onecannot definitively state that endophytes are truly host specific or even systemicwithin plants any more than assume that their associations are chanceencounters (Promputtha et al., 2007). Frequently, many endophytes of thesame species are isolated from the same plant, and only one or a few biotypes

Fungal Endophytes 225of a given fungus will produce a highly biologically active compound inculture. A great deal of uncertainty also exists about what an endophyte producesin culture and what it may produce in nature. It does seem possiblethat the production of certain bioactive compounds by the endophyte in situmay facilitate the domination of its biological niche within the plant or evenprovide protection to the plant from harmful invading pathogens, insectsand environmental stresses (Omacini, et al., 2001; Rodriguez, et al., 2008). Furthermore,little information exists on the biochemistry and physiology of theinteractions of the endophyte with its host plant. It would seem that manyfactors changing in the host depending on the season, and other factorsincluding age, environment, and location, may influence the biology of theendophyte. Indeed, further research at the molecular level must be conductedin the field to study endophyte interactions and ecology. All of these interactionsare probably chemically mediated for some purpose in nature. An ecologicalawareness of the role these organisms play in nature will provide thebest clues for targeting particular types of endophytic bioactivity with thegreatest potential for bioprospecting.There is a general call for new antibacterial, antifungal, antiviral and chemotherapeuticagents that are highly effective and possess low toxicity. Thisnew approach of search is driven by the development of resistance in infectiousmicroorganisms (e.g. Staphylococcus, Mycobacterium, Streptococcus, Bacillus,Aspergillus spp.) to existing drugs and by the menacing increase ofnaturally resistant organisms. The ingress to the human population of disease-causingagents such as AIDS, Ebola and SARS requires the discoveryand development of new drugs to combat them. Not only diseases such asAIDS require drugs that target them specifically, but new therapies areneeded for treating ancillary infections that are a consequence of a weakenedimmune system. Furthermore, others who are immunocompromised (e.g.cancer and organ-transplant patients) are at high risk of infection by opportunisticpathogens, such as Aspergillus, Cryptococcus and Candida sp., that arenot normally major problems in the human population. In addition, moredrugs are needed to treat efficiently parasitic protozoan and nematodal infectionssuch as malaria, leishmaniasis, trypanomiasis, incephalitis and filariasis.Malaria alone is more dangerous in causing human death each year thanany other single infectious agent with the exception of AIDS and tuberculosis(NIAID, 2001). However, the enteric diseases as a group claim the most liveseach year of any other disease complex and, unfortunately, the victims aremostly children (NIAID, 2001).Endophytic fungi are now attracting great interest from researchers as analternative source in controlling plant pathogens. The control of late blight,caused by Phytophthora infestans, is very difficult by chemical means, becauseof the high virulence of the pathogen and increasing resistance to availablefungicides (Griffith et al., 1992). The fermentation broths of 52.3% of endophyticfungi displayed growth inhibition of at least one pathogenic fungus,such as Neurospora sp., Trichoderma sp. and Fusarium sp. (Huang et al., 2001).In a similar study, fermentation broths of nine (4.8%) out of the 187 endophyticfungi isolated from mainly woody plants were highly active against

226 R.N. Kharwar and G. StrobelP. infestans in tomato plants (Park et al., 2005). Induced resistance wasgenerated in tomato plant against fusarium wilt by endophytic Fusariumoxysporum (Duijff et al., 1998). Sclerotinia sclerotiorum is a common root-,crown- and stem-rot-causing pathogen in cabbage, common bean, citrus, celery,coriander, melon, squash, soybean, tomato, lettuce, cucumber and so on.Cyclosporine is characterized as a major antifungal substance against S. sclerotiorumfrom the fermentation broth of F. oxysporum S6 (Rodriguez et al.,2006). F. oxysporum strain EF119, isolated from roots of red pepper, showedthe most potent in vivo anti-oomycete activity against tomato late blight andin vitro anti-oomycete activity against several oomycete pathogens (Kimet al., 2007). Out of 510 endophytic fungi screened, 64 strains exhibited antifungalactivities against Candida albicans, Candida glabrata, Candida krusei,Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus flavus, Rhizopusoryzae, Trichophyton rubrum and Microsporum canis (Anke et al., 2003).Narisawa et al. (2000) found that the root endophytic hyphomycete Heteroconiumchaetospira suppressed Verticillium yellows in Chinese cabbage inthe field. Verticillium wilt is one of the most destructive diseases of aubergine.Eleven out of 123 isolates of endophytic fungi, especially Heteroconiumchaetospira, Phialocephala fortinii, Fusarium, Penicillium, Trichoderma and myceliumradicis atrovirens (MRA), after being inoculated onto axenically rearedaubergine seedlings, almost completely suppressed the pathogenic effects ofa post-inoculated, virulent strain of Verticillium dahliae (Narisawa et al., 2000).Out of 39 endophytes of Artemisia annua investigated, 21 showed in vitro antifungalactivity against crop-threatening fungi Gaeumannomyces graminis var.tritici, Rhizoctonia cerealis, Helminthosporium sativum, Fusarium graminearum,Gerlachia nivalis and Phytophthora capsici (Liu et al., 2001). The extracts ofendophytic Alternaria sp., isolated from medicinal plants of Western Ghats ofIndia, inhibited the growth of C. albicans (Raviraja et al., 2006). About 41.2%of all the isolated endophytic fungi from rice plants showed antagonismagainst Magnaporthe grisea, Rhizoctonia solani and Fusarium moniliforme. Colletotrichumgloeosporioides (Penz) Penz & Sacc. was isolated as an endophytefrom healthy leaves of Cryptocarya mandioccana and had antifungal activityagainst phytopathogenic fungi Cladosporium cladosporioides and Cladosporiumsphaerospermum (Inacio et al., 2006). Fungal endophytes Chaetomium spp.,Phoma sp., isolated from asymptomatic leaves of wheat, reduced the numberof pustules and the area of pustules of Puccinia recondita f. sp. Tritici (Dingleand Mcgee, 2003). An in vitro study showed 40%, 65% and 27% antagonisticinteraction by endophytic morphospecies against cacao pathogens Moniliophthoraroreri, Phytophthora palmivora and Crinipellis perniciosa, respectively,while in-field endophytic C. gloeosporioides produced a significant decrease inpod loss (Mejia et al., 2008).For novel natural products, exciting possibilities exist for those who arewilling to venture into the wild and unexplored territories of the world toexperience the excitement and thrill of engaging in the discovery of endophytes,their biology, and potential usefulness. It is evident that naturalproduct-basedcompounds have an immense impact on modern medicine.For instance, about 40% of prescription drugs are based on them, and well

Fungal Endophytes 227over 50% of the new chemical products registered by the US Food and DrugAdministration (FDA) as anticancer agents, antimigraine agents and antihypertensiveagents from 1981 to 2002 are natural products or their derivatives(Newman et al., 2003). Excluding biologics, between 1989 and 1995, 60% ofapproved drugs and pre-new-drug-application (NDA) candidates were ofnatural origin. From 1983 to 1994, over 60% of all approved and pre-NDAstage cancer drugs were of natural origin, as were 78% of all newly approvedantibacterial agents (Concepcion et al., 2001). The discovery and developmentof taxol is a modern example of a natural product that has made anenormous impact on medicine (Wani et al., 1971; Suffness, 1995; Schulz andChristine, 2005).Antibiotics from endophytic fungiGenerally, the most commonly isolated endophytic fungi are in the group ofFungi Imperfecti or Deuteromycotina. Also, it is quite common to isolateendophytes that produce no fruiting structures whatsoever such as Mycelia-Sterilia.Cryptosporiopsis cf. quercina is the imperfect stage of Pezicula cinnamomea,a fungus commonly associated with hardwood species in Europe. It was isolatedas an endophyte from Tripterigeum wilfordii, a medicinal plant native toEurasia (Strobel et al., 1999). On Petri plates, C. quercina demonstrates excellentantifungal activity against some important human fungal pathogensincluding C. albicans and Trichophyton spp. Cryptocandin, a unique peptideantimycotic related to the echinocandins and the pneumocandins (Walsh,1992), was isolated and characterized from C. quercina (Strobel et al., 1999).This compound contains a number of peculiar hydroxylated amino acidsand a novel amino acid, 3-hydroxy-4-hydroxymethyl proline (Fig. 11.2). It isgenerally true that not one but several bioactive and related compounds areproduced by an endophytic microbe. So it is that other antifungal agentsrelated to cryptocandin are also produced by C. quercina. Cryptocandin isalso active against a number of plant pathogenic fungi including S. sclerotiorumand Botrytis cinerea. Cryptocandin and its related compounds are currentlybeing considered for use against a number of fungi causing diseases ofthe skin and nails.Cryptocin, a unique tetramic acid, is also produced by C. quercina(Fig. 11.3) (Li et al., 2000). This unusual compound possesses potent activityagainst Pyricularia oryzae, the causal organism of ‘rice blast’, one of the worstplant diseases in the world, as well as a number of other plant pathogenicfungi (Li et al., 2000). The compound was generally ineffective against anarray of human and plant pathogenic fungi. Nevertheless, with minimuminhibitory concentrations against P. oryzae at 0.39 μg/ml, this compound isbeing examined as a natural chemical control agent for rice blast and is beingused as a model to synthesize other antifungal compounds.Pestelotiopsis microspora is a common rainforest endophyte (Strobel, 2002;Strobel and Daisy, 2003). It turns out that enormous biochemical diversity

228 R.N. Kharwar and G. StrobelHOOH3,4-Dihydroxy-homotryosineGlutamineOHH 2 NONOOHNONHOO4-HydroxyprolineOHNHO3-Hydroxy-4-hydroxymethyl-prolineHNOHONHOHThreonineOH4,5-DihydroxyornithineONH(CH 2 ) 14Palmitic acidFig. 11.2 Cryptocandin A, an antifungal lipopeptide obtained from the endophytic fungusCryptosporiopsis cf. quercina.CH 3ONHOHOHCH 3CH 3OCH 3H 3 CHFig. 11.3 Cryptocin, a tetramic acid antifungal compound found in Cryptosporiopsiscf. quercina.does exist in this endophytic fungus and many secondary metabolites areproduced by various strains of this widely dispersed organism. One suchsecondary metabolite is ambuic acid, an antifungal agent, which has beenrecently described from several isolates of P. microspora found as representative

Fungal Endophytes 229isolates in many of the world’s rainforests (Fig. 11.4; Li et al., 2001). As aninteresting spin-off to endophyte studies, ambuic acid and another endophyteproduct, terrein, have been used as models to develop new solid-stateNMR tensor methods to assist in the characterization of molecularstereochemistry of organic molecules (Harper et al., 2001).A strain of P. microspora was isolated from the endangered tree Torreyataxifolia and produces several compounds that have antifungal activity,including pestaloside, an aromatic β-glucoside, and two pyrones, pestalopyroneand hydroxypestalopyrone (Lee et al., 1995a,b). These compounds alsopossess phytotoxic properties. Other newly isolated secondary productsobtained from P. microspora (endophytic on Taxus brevifolia) include two newcaryophyllene sesquiterpenes, pestalotiopsins A and B (Pulici et al., 1996a,b),2α-hydroxydrimeninol and a highly functionalized humulane. Variation inthe amount and types of products found in this fungus depends on both theculture conditions and the original plant source from which it was isolated.Pestalotiopsis jesteri is a newly described endophytic fungal species fromthe Sepik river area of Papua New Guinea, and it produces the highly functionalizedcyclohexenone epoxides, jesterone and hydroxy-jesterone, whichexhibit antifungal activity against a variety of plant pathogenic fungi (Li andStrobel, 2001). Jesterone has subsequently been synthesized with completeretention of biological activity (Fig. 11.5; Hu et al., 2001). Jesterone is one of18HO H HCH 317H 3 C1516 141312HHH118199OH107OH56H4HO3H2COOH1Fig. 11.4. Ambuic acid, a highly functionalized cyclohexenone epoxide produced by anumber of isolates of Pestalotiopsis microspora found in rainforests around the world.OHHOHOHHHFig. 11.5. Jesterone, a cyclohexenone epoxide from Pestaliotiopsis jesteri, hasantioomycete activity.

230 R.N. Kharwar and G. Strobelonly a few products from endophytic microbes for which total synthesis hasbeen successfully accomplished.Phomopsichalasin, a metabolite from an endophytic Phomopsis sp., representsthe first cytochalasin-type compound with a three-ring system replacingthe cytochalasin macrolide ring. This metabolite exhibits antibacterialactivity in disc diffusion assays (at a concentration of 4 μg/disc) against Bacillussubtilis, Salmonella gallinarum and Staphylococcus aureus. It also displays amoderate activity against the yeast Candida tropicalis (Horn et al., 1995).An endophytic Fusarium sp. isolated from Selaginella pallescens collectedin the Guanacaste Conservation Area of Costa Rica was screened for antifungalactivity. A new pentaketide antifungal agent, CR377, was isolatedfrom the culture broth of the fungus and showed potent activity againstC. albicans in agar diffusion assays (Brady and Clardy, 2000). Colletotricacid, a metabolite of Colletotrichum gloeosporioides, an endophytic fungus isolatedfrom Artemisia mongolica, displays antibacterial activity against bacteriaas well as against the fungus Helminthsporium sativum. Antimicrobialproducts have been identified from another Colletotrichum sp. isolated fromArtemisia annua, a traditional Chinese herb that is well recognized for itssynthesis of artemi sinin (an antimalarial drug) and its ability to inhabitmany geographically different areas. Not only did the Colletotrichum sp.found in A. annua produce metabolites with activity against human pathogenicfungi and bacteria, but also metabolites that were fungistatic to plantpathogenic fungi (Lu et al., 2000).A novel antibacterial agent, guignardic acid, was isolated from the endophyticfungus Guignardia sp.; the organism was obtained from the medicinalplant Spondias mombin of the tropical plant family Anacardiaceae found inBrazil. The compound was isolated by UV-guided fractionation of the fermentationproducts of this fungus and is the first member of a novel class ofnatural compounds containing a dioxolanone moiety formed by the fusionof 2-oxo-3-phenylpropanoic acid and 3-methyl-2-oxobutanoic acid, whichare products of the oxidative deamination of phenylalanine and valine,respectively (Fig. 11.6; Rodriguez-Heerklotz et al., 2001).Another antibacterial compound javanicin is produced by endophyticfungus Chloridium sp., under liquid- and solid-media culture conditionsOOOH 3 CO –CH 3OFig. 11.6. Guignardic acid from Guignardia sp. obtained from Spondias mombin, anAnacardiaceaeous plant in Brazil.

Fungal Endophytes 231(Kharwar et al., 2009). This highly functionalized naphthaquinone exhibitsstrong antibacterial activity against Pseudomonas spp., representing pathogensto both humans and plants. The compound was crystallized and thestructure was elucidated using X-ray crystallography. The X-ray structureconfirms the previously elucidated structure of the compound that was donethrough standard spectroscopic methods (Fig. 11.7).Volatile antibiotics from fungal endophytesMuscodor albus is a newly described endophytic fungus obtained from smalllimbs of Cinnamomum zeylanicum (Worapong et al., 2001). This xylariaceaous(non-spore-producing) fungus effectively inhibits and kills certain other fungiand bacteria by producing a mixture of volatile compounds (Strobel et al.,2001). The majority of these compounds have been identified by gas chromatography/ mass spectrometry (GC/MS), synthesized or acquired, and thenultimately formulated into an artificial mixture that mimicked the antibioticeffects of the volatile compounds produced by the fungus (Strobel et al., 2001).Individually, each of the five classes of volatile compounds produced by thefungus had some antimicrobial effects against the test fungi and bacteria butnone was lethal. However, collectively they acted synergistically to causedeath in a broad range of plant and human pathogenic fungi and bacteria.The most effective class of inhibitory compounds was the esters, of whichisoamyl acetate was the most biologically active; however, in order to be lethalit needs to be combined with other volatiles (Strobel et al., 2001). The compositionof the medium on which M. albus is grown dramatically influences thekind of volatile compounds that are produced (Ezra and Strobel, 2003).The ecological implications and potential practical benefits of the ‘mycofumigation’effects of M. albus are very promising given the fact that soilfumigation utilizing methyl bromide will soon be illegal in the USA. Methylbromide is not only a hazard to human health but it has been implicated incausing destruction of the world’s ozone layer. The potential use of mycofumigationto treat soil, seeds, and plants will soon be a reality as AgraQuest ofDavis, California, has EPA approval to release this organism for agriculturaluses. The artificial mixture of volatile compounds may also have usefulnessOOHCH 3CH 3H 3COOOHOFig. 11.7. Javanicin, an antibacterial compound isolated from endophytic fungusChloridium sp.

232 R.N. Kharwar and G. Strobelin treating seeds, fruits and plant parts in storage and while being transported.In addition, M. albus is already in a limited market for the treatmentof human wastes. Its gases have both inhibitory and lethal effects on suchfaecal-inhabiting organisms as Escherichia coli and Vibrio cholera. It will beused for this purpose in coming years. Studies are underway that show itspromise to fumigate buildings, thus removing the potential for fungi tocontaminate building surfaces and cause health risks.Using M. albus as a screening tool, it has now been possible to isolate otherendophytic fungi producing volatile antibiotics. The newly described M. roseuswas twice obtained from tree species found in the Northern Territory ofAustralia. This fungus is just as effective in causing inhibition and death oftest microbes in the laboratory as M. albus (Worapong et al., 2002). Other interestingM. albus isolates have been obtained from several plant species growingin the Northern Territory of Australia and the jungles of the Tesso Nilo area ofSumatra, Indonesia (Ezra et al., 2004; Atmosukarto et al., 2005).A non-muscodor species (Gliocladium sp.) has also been discovered toproduce volatile antibiotics. The volatile components of this organism aretotally different from those of either M. albus or M. roseus. In fact, the mostabundant volatile inhibitor is [8]-annulene, formerly used as a rocket fueland discovered here for the first time as a natural product. However, thebioactivity of the volatiles of this Gliocladium sp. is not as good or as comprehensiveas that of the Muscodor spp. (Stinson et al., 2003). Due to the volatileantibiotic producing properties of these fungi, they could be used againstseveral soil plant pathogens to reduce their inoculum. The latter, G. roseum,has already shown its potential through the production of a series of hydrocarbonsand hydrocarbon derivatives as mycodiesel (Strobel et al., 2008).Interestingly, another novel species of Muscodor sp., M. crispans, has beenreported to produce a mixture of strong volatile compounds (VOCs) thatwere effective against a wide range of plant pathogens, including the fungiPythium ultimum, Phytophthora cinnamomi, S. sclerotiorum and Mycosphaerellafijiensis (the black sigatoka pathogen of bananas), and the serious bacterialpathogen of citrus, Xanthomonas axonopodis pv. citri (Mitchell et al., 2010).Antiviral compounds from endophytic fungiAnother fascinating use of products from endophytic fungi is the inhibitionof viruses. Two novel human cytomegalovirus (hCMV) protease inhibitors,cytonic acids A and B, have been isolated from solid-state fermentation of theendophytic fungus Cytonaema sp. Their structures were elucidated asp-tridepside isomers using MS and NMR methods (Guo et al., 2000). It isapparent that the potential for the discovery of compounds having antiviralactivity from endophytes is in its infancy. The fact, however, that some compoundshave been found already is promising. The main limitation to compounddiscovery to date is probably related to the absence of commonantiviral screening systems in most compound discovery programs. Since noantiviral compound isolated from endophytes has been tested against

Fungal Endophytes 233virulent plant pathogens, an intensive exploration is therefore needed toidentify some potential antiviral compounds which could be very effectiveagainst variety of pathogenic plant viruses.Antioxidants from fungal endophytesTwo compounds, pestacin and isopestacin, have been obtained from culturefluids of Pestalotiopsis microspora, an endophyte isolated from a combretaceaousplant, Terminalia morobensis, growing in the Sepik River drainage systemof Papua New Guinea (Strobel et al., 2002; Harper et al., 2003). Bothpestacin and isopestacin display antimicrobial as well as antioxidant activity.Isopestacin was attributed with antioxidant activity based on its structuralsimilarity to the flavonoids (Fig. 11.8). Electron spin resonance spectroscopymeasurements confirmed this antioxidant activity showing that the compoundis able to scavenge superoxide and hydroxyl free radicals in solution(Strobel et al., 2002). Pestacin occurs naturally as a racemic mixture and alsopossesses potent antioxidant activity, at least one order of magnitude morepotent than that of Trolox, a vitamin E derivative (Harper et al., 2003). Theantioxidant activity of pestacin arises primarily via cleavage of an unusuallyreactive C–H bond and, to a lesser extent, through O–H abstraction (Harperet al., 2003). Endophytes also help plants to keep fit through balancing thegeneration and elimination of reactive oxygen species (Rodriguez andRedman, 2005). The use of chemical antioxidants is being slowly prohibiteddue to their small carcinogenic effect, and thus the demand for naturalOOOOHONHOOOHOHOOOHFig. 11.8. Isopestacin, an antioxidant produced by an endophytic Pestalotiopsis microsporastrain, isolated from Terminalia morobensis growing on the north coast of Papua New Guinea.

234 R.N. Kharwar and G. Strobelantioxidant compounds from natural resources has increased, so that theshelf life of stored crop produce and plant health could be enhanced to caterfor a hungry population, and endophytes may be one of the novel naturalresources to achieve this goal (Tejesvi et al., 2008).Immunosuppressive compounds from fungal endophytesImmunosuppressive drugs are used today to prevent allograft rejection intransplant patients and in the future they could be used to treat autoimmunediseases such as rheumatoid arthritis and insulin-dependent diabetes. Theendophytic fungus, Fusarium subglutinans, isolated from Tripterygium wilfordii,produces the immunosuppressive, but non-cytotoxic diterpene pyrones,subglutinols A and B (Lee et al., 1995). Subglutinols A and B are equipotent inthe mixed lymphocyte reaction (MLR) assay and thymocyte proliferation(TP) assay with an IC 50of 0.1 μM. The famed immunosuppressant drugcyclosporin A, also a fungal metabolite, was roughly as potent in the MLRassay and 10 4 more potent in the TP assay. However, the lack of toxicity associatedwith subglutinols A and B suggests that they should be explored ingreater detail as potential immunosuppressants (Lee et al., 1995).11.5 ConclusionGeographical, floristic and significant seasonal variations that exist in differentparts of globe provide conducive/or adverse conditions for the luxuriantgrowth of microbes in a wide range of different habitats, including livingtissues of higher plants where they grow as endophytes. Due to great variationin plant biodiversity and seasonal changes in tropical and subtropicalregions, there is a need to collect/isolate various types of promising endophyticfungi, especially from rainforests and mangrove swamps, which maybe able to produce an enormous variety of potential bioactive natural compounds.The fungi, as a group, hold enormous potential as sources of antimicrobials.The observations prove that this group of organisms resides insidethe healthy plants tissue, as endophytes, without causing any detectablesymptoms. Therefore, we strongly feel that there is a need to accelerate andfocus the research to exploit the maximum potential of the promising endophytesfor natural-product discovery, which could at least facilitate someexisting problems of the huge population.The past history of endophytic research in India especially with fungi isnot so encouraging. It seems that workers who have started this research inIndia are still actively involved in advancing their research manifesto withthis ‘under-studied’ group of microbes, and have not advanced to the fieldsand forests of the countryside looking for novel microbe–plant associations.Prof. T.S. Suryanarayanan and his group (Chennai) have initiated biodiversityand distribution patterns of fungal endophytes with some medicinal plantsin India and have published several papers along this line. They have also

Fungal Endophytes 235isolated some bioactive compounds and melanin from endophytic fungi(Suryanarayanan et al., 2004). To date, the overall situation in India has notdrastically improved, as is the case in other countries such as China, Japanand Brazil (Verma et al., 2009). However, several research groups have startedpaying more attention to various aspects of endophytic fungi. No more thana dozen research groups at different places in India are vigorously involvedeither with biodiversity or in natural-product discovery from this untappedand alternative resource. It has become obvious to many workers throughoutthe world that endophytic microbes have enormous potential for solvingmany of humankind’s problems. Thus, with the discovery of new compounds,we can protect our interests in agricultural and medical industriesas well as plant health (Omacini et al., 2001; Gunatilaka, 2006; Gimenez et al.,2007). Currently, some focused research on the different aspects of endophytesis being carried out and valuable results have been published (Shankar et al.,2003; Seena and Sridhar, 2004; Amna et al., 2006; Verma et al., 2006, 2007, 2008,2009; Gond et al., 2007; Tejesvi et al., 2007; Gangadevi and Muthumarry, 2008;Kharwar et al., 2008, 2009).It is important for all involved in this work to realize the importance ofacquiring the necessary permits from governmental, local, and other sourcesto collect and transport plant materials (especially from abroad) from whichendophytes are to be eventually isolated. In addition to this aspect of thework is the added activity of producing the necessary agreements andfinancial-sharing arrangements with indigenous people or governments incase a product does develop an income stream.Another concern is that if endophytic fungi may produce a natural productof host origin, a host plant may also acquire the ability to produce some‘mycotoxins’ or other secondary metabolites of fungal origin. The need fornew and useful compounds to provide assistance and relief in all aspects ofthe human condition is ever growing. Drug resistance in bacteria, the appearanceof new life-threatening viruses, the recurrent problems of diseases inpeople with organ transplants, a variety of severe diseases affecting plantsand the tremendous increase in the incidence of fungal infections in theworld’s population all underscore our inadequacy to cope with these problems.Environmental degradation, loss of biodiversity, and spoilage of landand water also add to problems facing us, and each of these in turn can havehealth- and hunger-related consequences.The researchers who are interested in exploiting endophytes should haveaccess to, or have some expertise in, microbial taxonomy and this includesmodern molecular techniques involving sequence analyses of 16S and 18SrDNA. Currently, endophytes are viewed as an outstanding source of bioactivenatural products because there are so many of them occupying literallymillions of unique biological niches (higher plants) growing in so manyunusual environments. Thus, it would appear that a myriad of biotopicalfactors associated with plants can be important in the selection of a plant forstudy. It may be the case that these factors govern which microbes are presentin the plant as well as the biological activity of the products associated withthese organisms.

236 R.N. Kharwar and G. StrobelCertainly, one of the major problems facing the future of endophyte biologyand natural-product discovery is the rapidly diminishing rainforests,which hold the greatest possible resource for acquiring novel microorganismsand their products. The total land mass of the world that currently supportsrainforests is about equal to the area of the USA (Mittermeier et al.,1999). Each year, an area the size of Vermont or greater is lost to clearing, harvesting,fire, agricultural development, mining or other human-orientedactivities (Mittermeier et al., 1999). Presently, it is estimated that only a smallfraction (10–20%) of what were the original rainforests existing 1000–2000years ago are currently present on the earth (Mittermeier et al., 1999). Theadvent of major negative pressures on them from these human-related activitiesappears to be eliminating entire mega-life forms at an alarming rate. Fewhave ever expressed information or opinions about what is happening to thepotential loss of microbial diversity as entire plant species disappear. It canonly be guessed that this microbial diversity loss is also happening, perhapswith the same frequency as the loss of mega-life forms, especially since certainmicroorganisms may have developed unique symbiotic relationshipswith their plant hosts. Thus, when a plant species disappears, so too does itsentire suite of associated endophytes. Consequently all of the capabilities thatthe endophytes might possess to provide natural products with medicinalpotential are also lost. Multi-step processes are needed now to secure informationand life forms before they continue to be lost. Areas of the planet thatrepresent unique places housing biodiversity need immediate preservation.Countries need to establish information bases of their biodiversity and at thesame time begin to make national collections of microorganisms that live inthese areas. Endophytes are only one example of a life form source that holdsenormous promise to impact many aspects of human existence. The problemof the loss of biodiversity should be one of concern to the entire world.AcknowledgementsThe authors express appreciation to the NSF, USDA, NIH, the R&C Board ofthe State of Montana and the Montana Agricultural Experiment Station forproviding financial support for some of the work reviewed in this report.RNK expresses his sincere thanks to DST, New Delhi, for financialassistance.ReferencesAmna, T., Khajuria, R.K., Puri, S.C., Verma, V.and Qazi, G.N. (2006) Determination andquantification of Camptothecin in anendo phytic fungus by liquid chromatography-positivemode electrospray ionizationtandem mass spectrometry.Current Science 91, 208–211.Anke, H., Weber, R.W., Heil, N., Pauls, S.,Teinert, M., Pauluat, T., Kuenzel, E.,Huth, F., Eckard, P. and Kappe, R. (2003)Antifungal compounds from endophyticfungi. Interscience Conference on AntimicrobialAgents and Chemotherapy. 43,Sep 14–17, 2003; abstract no. F-1236.

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12 Suppressive Effectsof Compost Teaon PhytopathogensMILA SANTOS, FERNANDO DIÁNEZAND FRANCISCO CARRETEROPlant Production Department, University of Almería, Almería, SpainAbstractSuppression of soilborne and airborne diseases of horticultural crops by compost hasbeen attributed to the activities of antagonistic microorganisms. A great diversity ofbiological control agents naturally colonize compost. The knowledge of mechanismsfor biological control through the action of compost or its water extracts (compost tea)is necessary in order to increase the efficiency of the suppressing power. Modes ofaction of biocontrol agents include: inhibition of the pathogen by antimicrobial compounds(antibiosis), competition for iron through production of siderophores, competitionfor colonization sites and nutrients supplied by seeds and roots, induction ofplant resistance mechanisms, inactivation of pathogen germination factors present inseed or root exudates, degradation of pathogenicity factors of the pathogen such astoxins, parasitism that may involve production of extracellular cell wall-degradingenzymes, for example, chitinase and β-1,3 glucanase that can cause lysis of pathogencell walls. None of the mechanisms are necessarily mutually exclusive and frequentlyseveral modes of action are exhibited by a single biocontrol agent. Indeed, for somebiocontrol agents, different mechanisms or combinations of mechanisms may beinvolved in the suppression of different plant diseases. The healthy development ofplants, as well as the biological control of soilborne fungi, originates from manymicroorganisms, partly due to the production of siderophores under iron-restrictingconditions. The siderophores are natural chelators that keep iron available for plantsin soil. Iron also plays a major role in nutrient competition among pathogens andbeneficial microorganisms in infection sites.12.1 IntroductionSuppressive soils or suppressive substrates are characterized by a very lowlevel of disease development even though a virulent pathogen and susceptiblehost are present. Biotic and abiotic elements of the soil environment contributeto suppressiveness; however, most defined systems have identified biologicalelements as primary factors in disease suppression. Many soils possess© CAB International 2011. Natural Products in Plant Pest Management242 (ed. N.K. Dubey)

Suppressive Effects of Compost Tea on Phytopathogens 243similarities with regard to microorganisms involved in disease suppression,while other attributes are unique to specific pathogen-suppressive soil systems.Modes of action of biocontrol agents include: inhibition of the pathogenby antimicrobial compounds (antibiosis); competition for iron throughthe production of siderophores; competition for colonization sites and nutrientssupplied by seeds and roots; induction of plant-resistance mechanisms;inactivation of pathogen germination factors present in seed or root exudates;degradation of pathogenicity factors of the pathogen such as toxins; parasitismthat may involve production of extracellular cell-wall-degrading enzymes,for example, chitinase and β-1,3 glucanase that can cause lysis to pathogencell walls (Keel and Défago, 1997; Whipps, 1997). None of the mechanismsare necessarily mutually exclusive and frequently several mode of action areexhibited by a single biocontrol agent. Indeed, for some biocontrol agents,different mechanisms or combinations of mechanisms may be involved inthe suppression of different plant diseases (Whipps, 2001). So the organismoperatives in pathogen suppression do so via diverse mechanisms includingcompetition for nutrients, antibiosis and induction of host resistance. NonpathogenicFusarium spp. and fluorescent Pseudomonas spp. play a crucialrole in naturally occurring soils that are suppressive to Fusarium wilt. Suppressionof take-all of wheat (Triticum aestivum), caused by Gaeumannomycesgraminis var. tritici, is induced in soil after continuous wheat monocultureand is attributed, in part, to selection of fluorescent Pseudomonads with thecapacity to produce the antibiotic 2,4-diacetylphloroglucinol. Cultivation oforchard soils with specific wheat varieties induces suppressiveness to Rhizoctoniaroot rot of apple (Malus domestica) caused by Rhizoctonia solani AG 5.Wheat cultivars that stimulate disease suppression enhance populations ofspecific fluorescent pseudomonad genotypes with antagonistic activity towardthis pathogen. Methods that transform resident microbial communities in amanner that induces natural soil suppressiveness have the potential ascomponents of environmentally sustainable systems for the management ofsoilborne plant pathogens (Mazzola, 2002).During the past decades, compost prepared from a variety of organicwaste has been utilized successfully in container media for suppressing soilbornediseases (Table 12.1). Examples are composts prepared from tree barks,liquorice (Glycyrrhina glabra) root waste, grape pomace, separated cattlemanure, and municipal sewage sludge (Kuter et al., 1998). Suppressive soilsor substrates are characterized by a very low level of disease developmenteven though a virulent pathogen and susceptible host are present. Diseasesthat have been shown to be effectively suppressed by compost use includethose caused by Fusarium, Phytophthora, Pythium and R. solani. Diseasesuppression in compost has been attributed mainly to elevated levels ofmicrobial activity (Chen et al., 1988b, Santos et al., 2008). There are severalmechanisms involved in the suppression: the competition among microbialpopulations for available carbon or nitrogen, iron competition, mycoparasitism,production of inhibitors or hydrolytic enzymes by microorganisms andinteractions with some saprophytes. Since 1991, several reports have shownthat some rhizosphere microorganisms can induce systemic resistance in

244 M. Santos et al.Table 12.1. Different composted material suppressive to soilborne disease.Compostedmaterial Pathogens ReferencesTree bark Phytophthora cinnamomi Hoitink et al., 1977; Sivasithamparam, 1981;Spencer and Benson, 1981, 1982;Blaker and MacDonald, 1983;Hardy and Sivasithamparam, 1991Phytophthora citricola Spencer and Benson, 1981;Hardy and Sivasithamparam, 1991Phytophthora drechleri Hardy and Sivasithamparam, 1991Phytophthora nicotianae Hardy and Sivasithamparam, 1991Pythium ultimum Daft et al., 1979; Chen et al., 1987;Chen et al., 1988 a,bRhizoctonia solani Daft et al., 1979; Stephens et al., 1981;Nelson and Hoitink,1983Fusarium oxysporum Chef et al., 1983; Cebolla and Pera, 1983;Trillas-Gay et al., 1986Nematodes Malek and Gartner, 1975;Mc Sorley and Gallaher, 1995Pine bark Phytophthora spp. Sivasithamparam, 1981;Spencer and Benson, 1981, 1982Pythium spp. Gugino et al., 1973; Zhang et al., 1996Fusarium oxysporum Chef et al., 1983; Pera and Calvet, 1989Grape marc Pythium aphanidermatum Mandelbaum et al., 1988Rhizoctonia solani Gorodecki and Hadar, 1990Sclerotium rolfsii Gorodecki and Hadar, 1990;Hadar and Gorodecki, 1991Olive marc Fusarium oxysporum f. sp. Pera and Calvet, 1989dianthiMunicipal Pythium spp. Lumsden et al., 1983sewage sludgeMunicipal solid Rhizoctonia spp. Mathot, 1987residuesCattle manure Pythium aphanidermatum Mandelbaun et al., 1988Phytophthora nicotianae Szczech et al., 1993Rhizoctonia solani Gorodecki and Hadar, 1990Sclerotium rolfsii Gorodecki and Hadar, 1990Fusarium oxysporum Garibaldi, 1988;Szczech et al., 1993Beer industry Pythium graminicola Craft and Nelson, 1996sludgeLiquorice roots Pythium aphanidermatum Hadar and Mandelbaun, 1986plants to root and foliar disease (Han et al., 2000). This systemic resistancewas found in compost by Zhang et al. (1996).Most of the composts are naturally suppressive against root rot diseasecaused by Phytophthora and Pythium (Santos et al., 2008), nearly 20% are naturallysuppressive against damping off caused by Rhizoctonia (Hoitink and

Suppressive Effects of Compost Tea on Phytopathogens 245Boehm, 1999), less than 10% of the compost induced systemic resistance inplants (Zhang et al., 1996, 1998; Hoitink and Boehm, 1999).There are two primary mechanisms by which the colonies of biocontrolorganisms in compost combat disease, general suppression and specificsuppression.General suppression occurs when a high-microbial activity environmentis created in which the germination of pathogen propagules is inhibited.General suppression occurs when many different organisms compete withpathogens for nutrients (as root or seed exudates) and/or produce generalantibiotics that reduce pathogen survival and growth. In compost there is aslow release of nutrients which supports beneficial activity of this microbiotic.Biocontrol agents that colonize composts include bacteria such asBacillus, Enterobacter, Flavobacterium balusstinum, and Pseudomonas; actinomycetessuch as Streptomyces; and fungi such as Trichoderma and Gliocladium(Hoitink et al., 1991b).General suppression owes its activity to the total microbial biomass inthe soil or substrate and is not transferable between them. Whether soilorganic matter can support biological control depends on its decompositionlevel and the types of biocontrol agents present on the substrate (Hoitink andBoehm, 1999). The carrying capacity of organic matter in the substrate limitssuppressiveness to pathogens that depend on exogenous sources of nutrients(root exudates) for germination and infection. Excessively stabilized organicmatter such as dark peat has a limited ability to sustain activity of the generalmicrobial biomass in soil (Hoitink et al., 1993). Dark decomposed sphagnumpeat is consistently conductive to Pythium root rot, whereas light sphagnumpeat harvested from the surface of peat bogs is less decomposed and has ahigher microbial activity (Hoitink et al., 1991a). Light peat moss is suppressiveagainst Pythium for a short time (6–7 weeks).Specific suppression involves the action of one or a few specific microbialagents in suppressing a specific pathogen (Hoitink, 1993). They exert hyperparasitismon the pathogen or induce systemic resistance in the plant to specificpathogens. Specific suppression owes its activity to the effects ofindividual or select groups of microorganisms and is transferable. This canbe achieved by inoculating the compost with the desired microbial agent(Hoitink, 1993).The suppression effect on pathogens is linked to the type of pathogens.Those pathogens that have a small propagule size, such as Pythium and Phytophthoraspecies, are susceptible to general suppression (‘nutrient-dependent’pathogens). They have small nutrient reserves and need to rely on an externalcarbon source and other nutrients. Pathogens with a large propagule size,such as Sclerotium rolfsii and R. solani, are susceptible to specific suppression.Structures like sclerotia are less susceptible to microbial competition but specifichyperparasites such as Trichoderma species will colonize the sclerotia.Composting conditions as well as the materials that are composted arecritical, with the type of the material impacting on the sort of active microflora.Thus the composting of lignocellulosic wastes will induce a specificsuppression of Rhizoctonia by Trichoderma species, while Penicillium fungi

246 M. Santos et al.predominate in grape pomace (high sugar, low cellulose) in suppressingS. rolfsii. In this medium, Trichoderma spp. were absent and would have beenineffective even if applied. Rhizoctonia (a common nursery pathogen) is alsoa very active saprophyte in fresh wastes but not in low-cellulose mature composts.Trichoderma, by contrast, colonizes both. However, the hyperparasiticactivity and antibiotic production by Trichoderma is repressed in fresh compostbecause of the high glucose level. Saline composts (for instance overfertilizedmedia) enhance Pythium and Phytophthora, whereas composts withlow C/N ratios that release nitrogen, especially as ammonia, promotes thegrowth of Fusarium on host species. The appropriate control of the pottingmedium with a pH greater than 5.0 and moisture content (oven dried)maintained between 40 and 50% is also important (Hoitink et al., 1997).12.2 Competition Among Microbial PopulationsMicrobiostasisThere is a competition for nutrients in exudates such as sugars and aminoacids which leak out of seeds during germination or out of root tips as plantsgrow through the soil. Pathogens that grow or swim to these sources of nutrientsmust compete with the beneficial microflora at the infection site on thesurface of the seed or root (Hoitink and Changa, 2004). This type of competitionplays a major role in general suppression and with ‘nutrient-dependent’pathogens such as Pythium and Phytophthora species. The competition includesmicrobial competition for nutrients and competition for infection sites androot colonization. Limited nutrient availability to the germinating spores orinvading hyphae is the most common explanation given for the microbialcause of fungistasis. On the other hand, fungistasis has been attributed to thepresence of antifungal compounds of microbiological origin. The productionof antifungal compounds is correlated with available carbon, which is notsurprising, as microbial production of antibiotics can be induced by interspecificcompetition for substrates (Ellis et al., 2000; Slattery et al., 2001). The sensitivityof plant pathogenic fungi to fungistasis is thought to protect themfrom germinating and initiating growth under unfavourable conditions (e.g.,the absence of a host plant) (Garrett, 1970; Lockwood, 1977). However, thisprotection comes at a cost, since the viability of resting structures decreasesduring prolonged incubation in soils (Lockwood, 1986). The negative effectsof fungistasis on the inoculum density of plant pathogenic fungi has beensuggested as a mechanism to explain the commonly found correlationbetween fungistasis and disease suppressiveness (Lockwood, 1977; Hornby,1983; Larkin et al., 1996; Knudsen et al., 1999).Iron competition also plays a major role in nutrient competition amongpathogens and beneficial microorganisms in infection sites (Hoitink andChanga, 2004). Ionic forms of iron, especially iron (III), its most common state,are very insoluble, especially when plants are grown on calcareous soils orsoils with high pH. Iron is only available to organisms at concentrations at or

Suppressive Effects of Compost Tea on Phytopathogens 247below 10 –18 M in soil solutions at neutral pH (Handelsman and Stabb, 1996).Iron salts that have low solubility such as iron oxides, carbonates, phosphates,hydroxides and some forms of insoluble chelates are created in certain soiltypes that make iron not readily available to plants. To circumvent the solubilityproblem, many microorganisms synthesize and utilize very specificlow molecular weight (500–1000 Da) iron chelators called siderophores (thatcan bind iron and are sent out by the cells to absorb iron, as well as being usedinside the cell to store it). When grown under iron-deficient conditions, manymicrobes will synthesize and excrete siderophores in excess of their own drycell weight to kidnap and solubilize iron and made it unavailable to the pathogens.The typical system involves a siderophore, which is an iron-bindingligand, and an uptake protein, which transports the siderophore into the cell.The fluorescent pseudomonads produce a class of siderophore known as thepseudobactins, which are structurally complex iron-binding molecules. Analysesof mutants lacking the ability to produce siderophores suggest that theycontribute to the suppression of certain fungal and oomycete diseases (Duijffet al., 1994; Buysens et al., 1996). An interesting aspect of siderophore biologyis that diverse organisms can use the same type of siderophore. Microorganismsmay use each other’s siderophores if they contain the appropriate uptakeprotein (Koster et al., 1993; Raaijmakers et al., 1995), and plants can evenacquire iron from certain pseudobactins (Duijff et al., 1994).Microorganisms in compost produce siderophores that keep iron in anavailable form for plants in the soil, even at high pH. Compost also produceswater-soluble humic substances, including fulvic acids, which keep iron, zinc,manganese and other trace elements in solution (Chen and Inbar, 1993).AntibiosisAntibiosis is defined as antagonism mediated by specific or non-specificmetabolites of microbial origin, by lytic agents, enzymes, volatile compoundsor other toxic substances (Jackson, 1965; Fravel, 1988). Antibiotic productionappears to be important to the survival of microorganisms through the eliminationof microbial competition for food sources, which are usually verylimited in soil (Ellis et al., 2000; Slattery et al., 2001). Antibiotic production isvery common among soil-dwelling bacteria and fungi. Inhibition in the Petridish may be the result of antibiosis, but it is not easy to show that this antibiosisis actually responsible for disease suppression. First, the antibioticmust be extracted, purified and identified chemically. Then it is necessary toshow that the microorganism grows in the microhabitat of the pathogen, andthat the antibiotic is produced in the right place, at the right time, and in sufficientamounts to control disease. It is also necessary to demonstrate that thepathogen is sensitive to the antibiotic. Genetic analyses have been particularlyinformative in determining the role of antibiotics in biocontrol, in partbecause mutants can be screened easily in vitro for changes in antibioticaccumulation, providing the means to conduct thorough genetic analyses(Handelsman and Stabb, 1996).

248 M. Santos et al.Antibiotic production by strains of the bacteria Pseudomonas and Bacillushas been shown to be important to the successful biocontrol of several cropdiseases. The antibiotics zwittermicin A and kanosamine, produced by thebiocontrol agent Bacillus cereus UW85, appear to be important in the biocontrolof oomycetes such as Phytophthora (Silo-Suh et al., 1994; Milner et al.,1996). The antibiotic phenazine derivatives, produced by Pseudomonas fluorescensstrain 2-79 and P. aureofaciens strain 30-48, control take-all of wheat(Weller and Cook, 1983; Brisbane and Rovira, 1988).Trichoderma and Gliocladium are closely related fungal biocontrol agents.Each one produces antimicrobial compounds and suppresses disease bydiverse mechanisms, including the production of the structurally complexantibiotics gliovirin and gliotoxin (Howell et al., 1993). Mutants withincreased or decreased antibiotic production show a corresponding effect onbiocontrol (Howell and Stipanovic, 1983; Handelsman and Stabb, 1996).HyperparasitismHyperparasitism is parasitism on another parasite. The mycelium andresting spores (oospores), hyphae or sclerotia of several pathogenic soilfungi such as Pythium, Phytophthora, Verticillium, Rhizoctonia, Sclerotinia andBotrytis (Fig. 12.1) are invaded and parasitized (mycoparasitism) or are lysed(mycolysis) by several non-pathogenic microbes.Fig. 12.1. Microscopic photography (¥ 400) of Trichoderma saturnisporum catching Botrytiscinerea.

Suppressive Effects of Compost Tea on Phytopathogens 249Mycoparasitism is parasitism of a pathogenic fungus by another fungus.These events require specific interactions between the parasite and fungalhost. The process involves direct contact between the fungi, resulting in deathof the plant pathogen and nutrient absorption by the parasite. Trichodermaspp. parasitize fungal plant pathogens. The parasite extends hyphal branchestoward the target host, coils around and attaches to it with appressorium-likebodies, and punctures its mycelium (Chet et al., 1981; Goldman et al., 1994).Mycoparasites produce cell-wall-degrading enzymes, which allow them tobore holes into other fungi and extract nutrients for their own growth. Butmany so-called mycoparasites also produce antibiotics, which may firstweaken the fungi they parasitize. So the digestion of host cell walls is accomplishedby a battery of excreted enzymes, including proteases, chitinases andglucanases. These enzymes often have antifungal activity individually andare synergistic in mixtures or with antibiotics (Di Pietro et al., 1993; Loritoet al., 1993, 1994; Handelsman and Stabb, 1996). By manipulating their activitythrough the construction of ‘overproducing’ mutants, enzyme-negativemutants or even transgenic plants expressing the enzyme, a role for theirproduction in biocontrol has been implied (Whipps, 2001).The best documented examples of hyperparasitism involve the mycoparasiticTrichoderma spp. on R. solani. Highly competitive as a saprophyte,R. solani, can utilize cellulose and colonize fresh bark but cannot colonize thelow-in-cellulose mature bark compost. However, isolates of Trichoderma thatfunction as biological agents for R. solani are capable of colonizing maturecompost. Biological control does not occur in fresh, undecomposted organicmatter because both fungi grow as saprophytes and R. solani remains capableof causing disease. In mature compost, on the other hand, sclerotia of R. solaniare killed by the hyperparasites and biological control prevails (Hoitink andFahy, 1986; Chung and Hoitink, 1990). One way to ensure specific suppressionis to inoculate the compost with the appropriate beneficial microorganisms,fungus like Trichoderma sp., Talaromyces flavus, non-pathogenic Fusariumoxysporum, or bacterial like Bacillus subtilis and Streptomyces griseoviride,which leads to increased levels of disease suppressiveness.Induced resistanceThe natural resistance of plants to pathogens is based on the combined effectsof preformed barriers and induced mechanisms. In both cases, plants usephysical and antimicrobial defences against the invaders. In contrast to constitutiveresistance, induced resistance relies on recognition of an invaderand subsequent signal transduction events leading to the activation ofdefences (Mauch-Mani and Métraux, 1998). Plants possess active defencemechanisms against pathogen attack; some biotic and abiotic stimuli increasetheir tolerance to infection by a pathogen, by activation of these active defencemechanisms. This phenomenon is known as induced resistance. Inducedresistance was defined by Kloepper et al. (1992) as ‘the process of active resistancedependent on the host plant’s physical or chemical barriers, activated

250 M. Santos et al.by biotic or abiotic agents’. Induced defence responses are regulated by anetwork of interconnecting signal transduction pathways in which thehormonal signals salicylic acid (SA), jasmonic acid (JA) and ethylene (ET)play a major role (Pieterse and Van Loon, 1999; Glazebrook, 2001), and otherhormones such as brassinosteroids and abscisic acid can also be involved(Nakashita et al., 2003; Ton and Mauch-Mani, 2004). The phenotypic effects ofactivated defences are quite similar when the stimuli are biotic or abioticagents, but the biochemical and mechanistic changes appear to be subtlydifferent (Van Loon et al., 1998; Whipps, 2001). This has resulted in the terminduced systemic resistance (ISR) for bacterially induced resistance andsystemic acquired resistance (SAR) for that induced by abiotic agents ormicroorganisms that cause localized damage (Pieterse et al., 1996).SAR confers long-lasting protection against a broad spectrum of microorganisms.SAR requires the signal molecule SA and is associated with theaccumulation of pathogenesis-related (PR) proteins such as chitinase, β-1-3-glucanases or proteinase inhibitor, which are thought to contribute to resistance(Durrant and Dong, 2004). One of the major differences between SARand ISR is that PR proteins are not universally associated with bacteriallyinduced resistance (ISR) (Hoffland et al., 1995) and SA is not always involvedin the expression of ISR.Several reports suggest that compost and compost-amended soil mayalter the resistance of a plant to disease. This was observed in airborne diseasessuch as powdery mildew of wheat and barley (Tränkner, 1992), earlyblight and bacterial spot of tomato (Lycopersicum esculentum) (Roe et al., 1993)or Anthracnose and Pythium root rot in cucumber (Cucumis sativus) (Zhanget al., 1996). A select few beneficial microorganisms of compost can inducemechanisms of ISR. These microorganisms activate biochemical pathways inplants leading to ISR to root as well as some foliar diseases. This mechanismhelps explain the often heard statement that plants raised on healthy organicsoils are more able to resist diseases (Zhang et al., 1998). Plants produced in compost-amendedmixes harbouring biocontrol agents that induce systemic resistancehave higher concentrations of proteins related to host defence mechanisms(Zhang et al., 1998). The activation of plant defence produces changes in cellwallcomposition, de novo production of PR proteins such as chitinases andglucanases, and synthesis of phytoalexins, although further defensive compoundsare likely to exist but remain to be identified (Heil and Bostock, 2002).Zhang et al. (1996, 1998) showed that composted pine-bark-amendedpotting mix provide Pythium root rot and Anthracnose control in cucumber byinducing systemic resistance (reported as SAR) utilizing split-root techniques.Experiments showed that when only some of the roots of a plant arein compost-amended soil, while the other roots are in diseased soil, the entireplant can still acquire resistance to the disease. On those plants, the systemicprotection induced by compost was accompanied by increased peroxidaseactivity in leaf tissue. Acid peroxidase was previous reported as a putativemolecular marker of SAR in cucumber.ISR plays a role in the suppression of plant pathogens that colonize aerialand soil parts (Pharand et al., 2002). Krause et al. (2003) demonstrated that

Suppressive Effects of Compost Tea on Phytopathogens 251less than 2% of 80 different batches of compost tested induced systemic resistancein radish (Raphanus sativus) against bacterial leaf spot. The effect wasdue to the activity of specific biocontrol agents in the batches of compost thatsuppressed bacterial leaf spot. They identified Trichoderma hamatum 382(Bonord.) Bainer (T 382) as the most active inducer of ISR in radish (Khan et al.,2003). Another species of the genera Trichoderma (Trichoderma harzianum RifaiT-203) was reported as an inducer of ISR in pepper (Capsicum annum) seedlingsagainst Phytophthora capsici when the seeds were previously treated with thisbiological agent (Ahmed et al., 2000). Khan et al. (2003) report how Phytophthoraroot and crown rot of cucumber caused by P. capsici was suppressed significantlyin cucumber transplants produced in a composted cow-manureamendedmix compared with those in a dark-sphagnum-peat mix. In split-rootbioassays, Trichoderma hamatum 382 (T 382) inoculated into the compostamendedpotting mix significantly reduced the severity of Phytophthora rootand crown rot of cucumber caused by P. capsici on paired roots in the peatmix. This effect did not differ significantly from that provided by a drenchwith benzothiadiazole (BTH) or mefenoxam (Subdue MAXX).12.3 Suppression of Plant Pathogens by Compost TeaThe disease-suppressive characteristic of organic tea was reported as early as1973 by Hunt et al. immobilizing the sting nematode (Belonolaimus longicaudatus).Compost tea or compost water extract can be defined as a wateryextract of compost produced through a deliberate process. The goal is toenhance populations of beneficial microbes that can then exert a biologicalcontrol over pathogens. The terminology used is not clear because there areseveral different methods used for making compost teas, those where tea isaerated during the production of the tea, and where no aeration is used andcompost is just passively steeped with little agitation. The review of Scheuerelland Mahaffee (2002a) clarified the numerous terms that have been used todescribe composts’ fermentation; many are synonymous or easy confusedwith other concepts. Terms used include compost tea, aerated compost tea,organic tea, compost extracts, watery fermented compost extract, amendedextracts, steepages and slurries. Compost tea is produced by mixing compostwith water and culturing for a defined period, either actively aerating (aeratedcompost tea; ACT) or not (non-aerated compost tea; NCT) and with orwithout additives that are intended to increase microbial population densitiesduring production (Scheuerell and Mahaffee, 2002a). These teas havebeen shown to act as a natural fungicide; they contain populations of variousbiofungicidal microbes and organic chelators Teas can be used as a foliarspray to inhibit late blight caused by Phytophthora infestans on tomatoesand potatoes, the suppressive effect of organic teas are of a living microbialnature and the sterilized or micron-filtered tea had little ability to impact onpathogens (Weltzien and Ketterer, 1986; Weltzien, 1989).There are several reports on the control of plant pathogens or plant diseaseswith organic teas including airborne and soilborne diseases; compost

252 M. Santos et al.teas coat plant surfaces (foliar application) or roots (liquid drench application)with living microorganisms and provide food for beneficial microbes.Plasmopara viticola or downy mildew of grape leaves (Weltzien and Ketterer,1986), Botrytis cinerea or grey mould (Elad and Shtienberg, 1994; Diánez, 2005;Koné et al., 2010), Phytophthora cinnamomi (Hoitink et al., 1977), Fusariumoxysporum f.sp pisi or Fusarium wilt of peas (Khalifa, 1965), Fusarium oxysporumf.sp cucumerinum or Fusarium wilt of cucumber (Ma et al., 1999) Pythiumultimum or damping-off in pea (Pisum sativum) (Tränkner, 1992).Multiple modes of activity are involved in suppressing plant diseasewith NCT, whereas no studies have determined the mechanism involvedwith ACT (Scheuerell and Mahaffee, 2002a). The microbes in compost teascan suppress diseases in several ways: induced resistance, antibiosis andcompetition (Brinton, 1995; Scheuerell and Mahaffee, 2002a), and directdestruction of pathogens structures (Ma et al., 2001).The microbiotic of NCT (Weltzien, 1991) and ACT (Ingham, 2003) hadbeen described as being dominated by bacteria. It is important to know howthe manipulation of the compost tea production process enriches and/orselects for individual microbe populations. Scheuerell and Mahaffee (2002b)studied the use of ACT and NCT produced with and without nutrient additives,to drench peat-perlite growing media that was inoculated withPythium ultimum and planted with cucumber seeds. They used differentnutrient additives, fungal nutrients (soluble kelp, humic acids and rockdust) and bacterial nutrients (molasses-based nutrients solution). The mostconsistent compost tea formula for suppression damping-off in cucumberwas ACT produced with the fungal nutrients, whereas the disease was notsuppressed with ACT produced with the bacterial nutrients and withoutnutrients.12.4 Grape Marc CompostGrape marc is the waste from wine production; once the juice has beenextracted, the skin 7% (w/w), stalks 5% (w/w) and seeds 4% (w/w) are allredundant. In total, 15–20% of wine production is waste, comprising thousandsof tonnes. The amount of marc that is generated from fruit is known tovary for a number of reasons, including whether the grapes have been irrigatedand the type of equipment used to press the grapes (Jordan, 2002).Spanish production of grapes for wine production is near 5 million tonnes;up to 700,000 tonnes of marc is estimated to have been generated throughoutSpain. The marc, if not treated effectively, can cause a number of environmentalhazards ranging from surface and groundwater pollution to foulsmells. In the European Union all by-products of wine production (grapemarc and wine lees) are obliged to be distilled; the legal basis for distillationmeasures in the EU is given in chapter II of title III, Art.27 of regulation (EC)N°1493/1999. In European countries grape marc is first distilled to recoveralcohol, followed by washing for tartrate recovery, then seed separation andfinally the remainder is either burnt for energy recovery or composted along

Suppressive Effects of Compost Tea on Phytopathogens 253with sludge from distillation and tartrate recovery to produce fertilizers foragricultural use (Johnston, 2001).In general, grape-marc compost (GMC) has low nutrient status and conductivity;this compost has low water-holding capacities. It has a high contentof lignin and cellulose and a low content of water-soluble carbohydrates.There are a few reports about the disease-suppressive effects of GMC.Oka and Yermiyahu (2002) tested in pot and in vitro experiments the suppressiveeffects of GMC, on the root-knot nematode Meloidogyne javanica. Veryfew root-galls were found on tomato roots grown in soil containing 50%grape marc compost. Significant reductions in galling index were also foundin tomato plants grown in soils containing smaller concentrations of thiscompost. The water extract of GMC showed weak nematicidal activity to thejuveniles and eggs.GMC and its water extracts have been reported to suppress fungal diseasessuch as B. cinerea on tomato and pepper (Elad and Shtienberg, 1994).GMC suppressed the soilborne disease caused by R. solani and S. rolfsii(Gorodecki and Hadar, 1990; Hadar and Gorodecki, 1991). Hadar andGorodecki (1991) found an inhibitory effect of GMC on sclerotial germinationand viability, and associated this effect with high numbers of Penicilliumisolated from sclerotia. Penicillium and Aspergillus spp. have been reported tocolonize GMC; Trichoderma spp. hyperparasites of R. solani were not recoveredfrom this compost (Gorodecki and Hadar, 1990; Hadar and Gorodecki,1991). The age of the composted grape marc had a major effect on suppression;immature GMC (3 months of composting) failed to inhibit sclerotialgermination (Hadar et al., 1992). Suppression of Pythium aphanidermatum wasreported on grape marc compost, the beneficial effect of the compost wasnegated when the medium was autoclaved, and restored when compost thathad not been autoclaved was mixed with the sterile one (Hadar et al., 1992).Compost can provide natural biological control of diseases of roots aswell as the foliage of plants. Its water extracts (compost tea) has been proposedas a substitute for synthetic fungicides (Zhang et al., 1998). Most of thepapers published on the control of pathogens by means of compost tea havestudied pathogens from the aerial part of the plants, the number of trials thatuse NTC being higher. Research into the control of soilborne pathogens bymeans of compost tea has been lower, although this practice is common inecological agriculture (Scheuerell and Mahaffee, 2002a).Our research focuses on soilborne disease suppression by GMC. Thegrape marc compost was produced in the University of Sevilla. It was compostedin 40 m 3 windrows and turned each week. Composting took 5 months.During composting, the grape marc pile was fertilized with ammoniumnitrate, superphosphate, iron sulfate and magnesium sulfate. Microbiologicalanalysis of our grape marc was performed using the dilution plate technique(Wakelin et al., 1998) on different agar growth media: water agar (WA)pH11 (actynomicetes), tryptose soy agar (TSA) 1/10 (bacteria), glucose peptonemedium (GP) (yeast) and malt extract agar (MEA) (fungi). All the plateswere incubated at 25°C, and WA and TSA plates were also incubated at 40°C,for detection of thermophylic bacteria and actynomicetes. Two analyses of

254 M. Santos et al.Thermophilicactinomycete13 %Fungi 5%Yeast 2%Thermophilicactinomycete25%Fungi 3%Yeast 1%Mesophilicactinomycete24%Mesophilicbacteria 31%Mesophilicactinomycete9%Mesophilicbacteria 30%Thermophilicbacteria 25%Thermophilicbacteria 32%Fig. 12.2. Distribution of the different morphologies found in the fi rst (left) and second (right)analysis of grape marc compost microfl ora.the same compost were done, 6 months apart. Microbiological analysis ofGMC showed a high number of microbial morphologies in the compost. Inthe first analysis, 192 different morphologies were found, and 240 in the second,6 months later. Most of the morphologies present in the first and secondanalyses were bacteria, with the average percentages being 31% mesophylicbacteria, 28% thermophylic, 16% mesophylic actinomycetes and 20% thermophylicactinomycetes, and only a few moulds 4% and yeasts 1%; thedistribution of morphologies found in both assays is shown in Fig. 12.2.Most microorganisms, both aerobic and anaerobic, respond to conditionsthat restrict iron ions due to the production of siderophores, whereby ionsare kidnapped or assayed thus preventing their availability. Siderophorescan act as growth factors and some as powerful antibiotics (Neilands, 1981).Many studies have described suppressiveness in agricultural soil due to siderophores.Thus, the addition of different species of Pseudomonas to a conductivesoil infected with Fusarium oxysporum f. sp. lini makes this soilsuppressive, preventing the development of the disease. The addition ofFe-EDTA reverses this situation (Kloepper et al., 1980). This principle alsoapplies to Gaeumannomyces graminis var. tritici, and several species of Pythium(Becker and Cook, 1984; Weller et al., 1986).The presence of siderophores produced by the microorganisms presentin the grape marc aerated compost tea and their involvement in the developmentof eight phytopathogenic fungi in the soil and one mycopathogenicfungus was studied.The results obtained from the in vitro analysis of the inhibiting effect ofACT on fungal development are shown in Fig. 12.3. The effect of the incubationtime of ACT on the efficiency of the in vitro fungal suppression of thenine fungi tested highlighted an increase in inhibition percentages as thetime of incubation of the compost increased (1, 7 or 14 days) being 80–100%for extract F (Filtered), in extractions after 1 and 7 days, and 100% after

Suppressive Effects of Compost Tea on Phytopathogens 25514 days, for all the cases examined. In the case of extract C (microfiltered), theresults vary depending on the fungus, with the same inhibition tendencypercentage increasing as the incubation time and the extract concentrationadded to the medium increase, reaching 100% inhibition after 14 days of trialfor most of the fungi, except for the two races of Fusarium oxysporum f. sp.lycopersici (Fig. 12.3). From the results obtained, we need to highlight theabrupt change in inhibition percentages for the fungi R. solani and Pythiumaphanidermatum, using the microfiltered tea after 7 and 14 days’ incubation.This effect is the result of measuring the radial growth of the fungus, but it1201001 day ACT 1 week ACT 2 weeks ACTPhytophthora parasitica (8)% Inhibition806040200Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8 Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8 Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8120100+ FeCl 380% Inhibition6040200Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8 Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8 Fa8 Fb8 Fc8 Ca8 Cb8 Cc8 Ea8 Eb8 Ec8*Vertical T bars represent standard errorsTreatmentFig. 12.3. In vitro analysis of the inhibiting effect of ACT on fungal development.

256 M. Santos et al.does not reflect the effect on the density of the mycelium, which can only beobserved under a microscope with a clear inhibition by the compost extracts,although this cannot be expressed in the graph.The sterilization of compost water extracts annuls the suppressing effecton fungal growth shown by non-thermally treated extracts, except forVerticillium dahliae and V. fungicola, for which inhibition values reach 60%,in compost tea incubated for 1 day at concentration levels of 10 and 15%.The detection of siderophores on GMC tea, which can affect the developmentof the fungal mycelium, occurred equally in the extract obtained simplyby filtering (F), in those that were microfiltered (C ) and in those that weresterilized (E), with the addition of FeCl 3to the medium. In these, the biologicalcomponent has not been eliminated, so besides the potential inhibitionthat siderophores can cause, we must bear in mind other antagonistic effectssuch as competition for nutrients, space, and so on.These results suggest that the microorganisms present in GMC producesiderophores, which grow outside the cells and kidnap iron, stopping itsavailability, thus preventing the in vitro development of the phytopathogensstudied.This study confirms the in vitro inhibition of the growth of eight pathogensand one mycopathogen, as well as the important role of siderophores inthis suppression. In our previous studies, the inhibiting power of GMC hadbeen verified against the nine fungi tested. As can be expected, the suppressivenessshown by GMC extracts is a combination of various factors, such ascompetition for nutrients, antibiosis, and production of lytic enzymes outsidethe cells and of low molecular weight molecules that are capable ofdegrading the fungus wall.The difference between the use of soil-applied composts and waterycompost extracts perhaps is best summarized as that the teas give immediatebut very short-term control of surface-spreading pathogens, while soil compostacts more slowly over a longer period of time and requires much largeramounts (York and Brinton, 1996).12.5 ConclusionDiseases that have been shown to be effectively suppressed by compost useinclude those caused by Fusarium, Phytophthora, Pythium and R. solani. Severalreports suggest that compost and compost-amended soil may alter theresistance of plants to disease. The microbes in compost teas can suppressdiseases in several ways: induced resistance, antibiosis and competition, anddirect destruction of pathogens structures. The effect is due to the activity ofspecific biocontrol agents in the batches of compost that suppress the disease.There are several reports on the control of plant pathogens or plant diseaseswith organic teas including airborne and soilborne diseases. GMC and itswater extracts have been reported to suppress fungal diseases. Compost canprovide natural biological control of diseases of roots as well as the foliage ofplants. Its water extracts (compost tea) have been proposed as substitutes for

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13 Biotechnology: a Tool forNatural Product SynthesisSANATH HETTIARACHIDepartment of Botany, University of Ruhuna, Matara, Sri LankaAbstractMolecular biology is arguably the fastest growing field in all biological sciences. Newtechniques are discovered and they soon find applications. The search for naturalproducts has a long history, as illustrated by the dependence of traditional medicineon botanicals. With the finding that microorganisms can produce useful productssuch as penicillin, scientists took the challenge to explore the microbial world for newnatural products. Although the search continues with increasing intensity, findingnew and more useful products would not have matched with the effort without thesupport of biotechnology. Although the most powerful approach is genetic manipulation,other techniques such as mutagenesis, breeding and protoplast fusion and therelatively old biotechnology of plant tissue culture are very useful. These also includeeven more simple approaches such as optimizing culture conditions and design offermenters. The combination of technologies together with innovative ideas hasalready increased the production level of already existing natural products andexpanded the diversity of products obtainable from biological sources. In addition toharvesting products from living organisms either in the wild or in cultivation, thedevelopments in metagenomics have also paved the way to harness the bioproductformingability of unculturable microorganisms.13.1 IntroductionBy perusing the various definitions given by different authorities and indictionaries, it becomes apparent that natural products are predominantlychemical compounds of biological origin and are extracted from plants andanimals that produce them during secondary metabolism. None the less,naturally occurring mineral compounds may also be categorized as naturalproducts. Among the myriad of natural compounds, many have pharmaceuticaland other similar applications such as in pest and disease control inagriculture. Furthermore, natural antioxidants, bioflavours, biopreservatives,© CAB International 2011. Natural Products in Plant Pest Management(ed. N.K. Dubey) 263

264 S. Hettiarachinatural colourings, fragrances and microbial polysaccharides are becomingthe choice to replace artificial chemicals in the food and cosmetic industries.Several contemporary approaches have widened the usefulness of naturalcompounds. For example, a new move employing rhizosphere metabolamicsfor bioremediation of polychlorinated biphenyls is suggested (Narasimhanet al., 2003), where plants exuding high levels of phenylpropanoids haveenhanced rhizosphere populations capable of degrading pollutants.The study of the chemistry and extraction of natural products is soimportant that an entire discipline of chemistry is termed natural productchemistry. Many natural products have quite complex chemical structures,including stereochemistry, and hence their chemical synthesis is difficult. Yetsome simpler compounds of natural origin are made by total synthesis andthese are also treated as natural compounds. Scientists are also keen to elucidatethe biochemical pathways of natural product formation. This informationwill lead to the development of technologies for the overproduction ofalready existing natural products as well as producing entirely new productsthrough biological systems.Plants and animals have coexisted with their pests, pathogens,grazers/predators and other competitors throughout evolution. In order forthem to succeed in such a hostile atmosphere, coevolution has gifted theorganisms with defence mechanisms of different natures, chemical defencebeing one of them. Therefore living beings are natural factories of chemicalwarfare. Scientific investigations guided by traditional knowledge have led tothe identification of useful natural compounds for the benefit of humankindin medicine, veterinary medicine and in agriculture. Members of all classes ofliving beings from bacteria to higher plants and animals have been identifiedas sources of natural products. According to the accepted definitions of biotechnology,exploiting these organisms either from the wild or by cultivationfor the production and extraction of products comes under biotechnology.This exploitation has already threatened some valuable plants and animalswith local or even global extinction due to over-exploitation, for example formedicinal uses (Kala, 2005; Maundu et al., 2006). The use of new biotechnologicaltools would certainly ease the pressure on natural populations.Although biotechnology plays an important role in non-biologicalnatural products, these are not included in this discussion.13.2 Biotechnology in Genetic Diversity Revelation andConservationIt is well known that different varieties / races of the same species have differentchemical properties and hence their level of importance as sources ofnatural products may be different. Until recent times, the taxonomy andidentification of plants, animals and microorganisms were based on morphological(vegetative and reproductive) and anatomical characters. However,these features usually cannot differentiate between two closely relatedindividuals and hence cannot identify those individuals with the same

Biotechnology and Natural Product Synthesis 265morphology, but differences in their genetic makeup. Molecular biologicaltools, based on DNA, protein or secondary metabolites, provide additionaltaxonomic markers to supplement information gathered through classicaltaxonomical methods for further characterization. This enables the identificationof variants not only at species or subspecies levels, but tracing downto variations between two individuals. Such biotechnological approachesthus enable the identification of more useful genotypes for exploitation innatural product synthesis. Identification of more useful genotypes is obviouslyvery useful in the conservation of genetic diversity. Systematic studiescan indicate which genomes to search, sample and study for useful products.Systematics also provides answers to questions relating to the evolution ofchemical and physical structures and their synthesis or ontogeny.The biotechnological tools available for systematics are protein profiles,polysaccharides, plasmids, DNA–DNA hybridization, various PCR-basedtechniques and DNA sequencing and alignment. With the assistance of bioinformatics,the molecular data are processed and phylogenetic relationshipsare generated. This facilitates the search for a better performer withinproducers of a known product or the search for new products, by narrowingdown the scope of the search. Certain traits have evolved only once andtherefore will only be present within a single clade or very closely relatedclades, rather than being present randomly across diverse organisms. For anexample in bioproducts, taxol is known to be present in the yew family,Taxaceae. Therefore if one is interested in discovering other organismsproducing taxol, the most logical approach would be to start the quest fortaxol and other taxanes in plants closely related to Taxaceae family. Theother sister family most closely related is Podocarpaceae and taxane hasbeen found in Podocarpus gracilior for the first time outside Taxaceae ( Stahlhutet al., 1999). The story of mustard oil or mustard glucosinolates is adifferent one. With the exception of the genus Drypetes (a member ofMalpighiales), all the other mustard oil producers are in families belongingto one ordinal clade, Brasicales. As order Brasicales and genus Drypetes arephylogentically distant, one can assume that mustard oil biosynthesisevolved twice (Rodman et al., 1993). This is confirmed by the presence oftwo different biosynthetic pathways. Therefore, although the final productis the same, the evolution of the biosynthetic pathway is different. Thisinformation is extremely important in the search for alternative pathwaysfor the synthesis of natural products (i.e. one has to search in distantlyrelated clades).Molecular tools are extremely sensitive in revealing minor differences inclosely related genotypes within a species. As discussed elsewhere in thischapter, identifying genotypes of interest in bioproduct formation is particularlyimportant for exploitation, but not restricted to that. This is equally ormore important in conservation strategies. A genotype once lost shall not beable to be reconstituted. Exploring possibilities of making use of other biotechnologicaltools such as plant-cell and organ culture also help preservethe plants in the field. In addition, micropropagation using tissue culturetechniques is useful in this context.

266 S. HettiarachiRecombinant gene technology in natural productsRecombinant gene technology is perhaps the most wonderful tool a biotechnologistever had. Conventional breeding of plants and animals canbring the genes of two different but closely related individuals togetherthrough the fusion of two gametes in sexual reproduction. The nuclearDNA of eukaryotic organisms is organized into chromosomes and theincompatibility in chromosome numbers and chromosome morphology isthe main barrier for bringing genes together from different parents to producea hybrid. However, today’s technology is such that the identificationof DNA controlling a particular trait, isolation of that particular DNA, invitro modification of that DNA, inserting the modified DNA into a desiredorganism to express the trait in the new organism, have become possiblein an appropriately equipped laboratory. This is a straightforwardapproach when the final product is a peptide because engineering the basesequence in an open reading frame under proper control units is sufficient.It is a different matter when it comes to more complex molecules, such assecondary metabolites, which are products of a series of biochemical reactionseach mediated by a specific enzyme. The maturation of the moleculemay also involve chemical modifications within the environment of theproducer cell. In order to make breakthroughs in the production of naturalproducts, particularly those that are not peptides, using biotechnology, anunderstanding of the regulation of the secondary metabolite pathwaysinvolved at the levels of products, enzymes and genes, including aspectsof transport and compartmentation in eukaryotic cells, are of paramountimportance.Antibiotics are natural products of microbial origin. However, these arenaturally produced only in minute quantities that are not even detectable innature. Overproducing individuals are present in nature and screening largepopulations can always yield better producers. Appropriate modifications toculture medium and culture conditions also result in higher production.Generating mutants artificially and screening increase the chances of findingsuch overproducers. A big leap in this direction was possible due to the newbiotechnological tools such as genetic engineering. This possibility wasalready under consideration for more than two decades (Chater, 1990). Therehad been a slow progress as antibiotic synthesis occurs as a combination ofthe action of several genes.While antibiotics are extremely useful in medicine and veterinary medicine,the development of resistance by the target pathogens is an increasingproblem. Therefore heavy investments are made into research leading to thediscovery of new antibiotics. However, the natural antibiotics belong to a fewfamilies and it may not be possible to isolate new and therapeuticallyimportant antibiotics belonging to new families. Once resistance is developedit is possible this resistance extends easily for that particular family ofantibiotics. Therefore the modification of already known antibiotics to generatesemisynthetic antibiotics, either chemically or enzymatically, may bemore appealing.

Biotechnology and Natural Product Synthesis 267As microorganisms can be cultivated in fermenters much more easily thanplant cells and they are more amenable to genetic alterations via mutagenesisand genetic engineering, they have become factories of products originallyproduced by other organisms. This is a case of producing natural products inan ‘unnatural’ producer. The development of biotechnological approaches forenhanced production of such natural products is hindered by the complexityinvolving unknown regulatory genes and enzymes (Oksman-Caldentey andInzé, 2004). In this context gene technology can help in overexpression, constitutiveexpression, breaking tissue specificity and so on, rather than expressionin a different organism. This is possible when control mechanisms of expressionare identified. It may be traced down to a particular molecule whicheither keeps the entire process switched off (suppressor) or required for switchingon the system (inducer). Once the suppressor is knocked off or the induceris constitutively expressed without tissue specificity, complex metabolitesmay be produced with a higher yield in the producer itself. One major limitationin plant cell suspension cultures is the tissue specificity of the metabolites.Therefore breaking tissue specificity should be given more attention in orderto exploit the full potential of this technology.Although animal cells can also be genetically manipulated, other problemsassociated with animal-cell and tissue culture are still limiting the formationof new products in animals. Nevertheless pharming, a technologystill in its infancy, to produce pharmaceuticals in farm animals is a focal pointin natural product formation with animals. What is basically done in pharmingis to transform animals with genes coding for useful bioproducts, andallow expression and harvesting, for example by milking. Pharming hasyielded drugs such as growth hormone, blood components such as haemoglobin,and large quantities of certain proteins needed for research. Althoughpharming is a novel production platform, it is yet to break through manyhurdles such as regulatory barriers, consumer acceptance, environmentalissues and ethical concerns. The term pharming is not restricted to productionthrough animals, but also includes pharmaceutical production in geneticallymodified plants. Ramessar et al. (2008) reviewed the progress made inmaize plants, the first plants used in pharming, as a platform for effectiveand safe molecular pharming.Human insulin is the first mammalian protein expressed in bacteria andlater became a huge contributor to the pharmaceutical industry. All humaninsulin preparations available in pharmacies are of microbial origin, basedon two technologies using Escherichia coli as the expression system and differenttechnology using yeasts to secrete the precursor of insulin (Petrides et al.,1995). Another industrial success is the production of rennet or chymosinenzyme, which is important in making cheese. The original source was thestomach of calves. As the amount extracted from a single stomach is verylow, production could not meet demand and hence the cost was very high.This was first cloned and expressed in E. coli (Nishimori et al., 1982). Recombinantchymosin is now produced by two fungi, namely Kluyveromyces lactisand Aspergillus niger, in addition to E. coli, by different manufacturers(Neelakanthan et al., 1999).

268 S. HettiarachiA comprehensive list of plant proteins that have been expressed in E. coliand in two yeasts, namely Saccharomyces cerevisiae and Pichia pastoris, is providedby Yesilirmak and Sayers (2009).Whole plants can serve as factories of desired products – not only thosethat are present naturally, but also products that are engineered into them.Edible vaccine production is one such example. Antigenic components ofpathogens are expressed in plants, and when eaten they can act as antigensto trigger antibody production, thereby developing immunity against thepathogen in question. Neither purification of the antigen nor formulationinto vaccine is necessary. In engineering Hepatitis B edible vaccine into lupin(Lupinus luteus) and lettuce (Lactuca sativa), the gene coding for a viral envelopesurface protein has been transferred from the viral genome to the plantgenome with necessary modifications for expression in plants. When tissuesof transgenic plants were fed to mice and human, they developed HepatitisB virus specific antibodies to the plant-derived protein (Kapusta et al., 1999).A fusion protein of components from two naturally occurring proteins,cholera toxin B subunit and insulin, has been expressed in potato tubers. Thisprotein, when ingested with the potato tuber, can enter into the gut- associatedlymphoid tissues, as it is linked to the carboxy-terminal sequence of thecholera toxin B subunit. When mice, having a diabetic mellitus condition dueto insulin autoimmune disease, were fed with transgenic potato, a substantialreduction in pancreatic islet inflammation and a delay in the progressionof clinical diabetes were observed (Arakawa et al., 1998).Nevertheless, the production of secondary metabolites depends mainlyon pathway engineering / metabolic engineering, rather than engineeringthe gene responsible for the final product. Due to the complex nature andspecificity of reactions at strain level rather than at species or broader taxonomiclevel, the progress has been slow. Transfer of the putrescine:SAMN-methyltransferase (PMT) gene from Nicotiana tabacum under the controlof the CaM V 35S promoter into a Duboisia hybrid has increased theN- methylputrescine concentration in hairy root cultures. The increase hasbeen reported as 2–4-fold in transgenic versus wild type. The enzyme catalysesthe N-methylation of the diamine putrescine to N-methylputrescine,which is the first specific precursor of both tropane and pyridine-type alkaloids.Both of these types are present in Duboisia roots, however the concentrationsof these alkaloids did not increase in the transgenic hairy root culture,despite higher levels of the precursor (Moyano et al., 2002). Although thefinal product formation was not enhanced, this is a step forward. Workingalong similar lines it was also possible to engineer the enzyme hyoscyamine6-hydroxylase from Hyoscyamus niger, under the regulation of the CaMV 35Spromoter into a Duboisia hybrid and found one of their transgenic lines produced74 mg/l scopolamine (Palazóna et al., 2003). The enzyme was knownto catalyse two steps in scopolamine biosynthesis.In the above section the importance of modifications, either enzymaticor chemical, to natural products was mentioned. These ‘unnatural’ modificationsto natural products can be done in a more ‘natural’ manner bycombinatorial alterations by engineering genes relevant for the enzymes. An

Biotechnology and Natural Product Synthesis 269example of this approach is reported by McDaniel et al. (1999) who engineerederythromycin polyketide synthase to produce a library of over 50macrolid antibiotics. Kantola et al. (2003) provided an analysis of availableliterature on this subject giving details of the gene clusters involved. Hopwoodet al. (1985) was the first group to produce ‘hybrid’ antibiotics by transferringgenes encoding enzymes making a set of antibiotics in one strain toanother. This results in new antibiotics depending on the substrate specificityof the enzymes. Thus the possibility of finding more efficient antibiotics ismade possible.Metabolic engineering or combinatorial synthesis of traits frombacteria to plants has also been successful. For example, a bacterial geneencoding p-hydroxycinnamoyl-CoA hydratase/lyase (HCHL) has beenexpressed in Beta vulgaris. One line was able to accumulate the glucoseester of p-hydroxybenzoic acid (pHBA) at a rate of 14% of dry weight(Rahman et al., 2009).Combinations of different approaches are of course possible and sometrials have already given positive results. The effect of combining metabolicengineering with providing elicitors in the culture medium has been testedby Zhang et al. (2009) in flavonoid production by hairy root cultures of Glycyrrhizauralensis. In metabolic engineering, overexpression of chalcone isomerasewas achieved by Agrobacterium-mediated transformation with a 150%increase of total flavonoid over the wild type. When PEG8000 and yeastextract was added to the medium, the engineered line produced over 300%total flavonoids.Plant tissue and cell culture in natural productsMany higher plants produce economically important organic compoundssuch as oils, resins, tannins, natural rubber, gums, waxes, dyes, flavours andfragrances, pharmaceuticals, and pesticides. Plants have been identified sinceancient times as a source of remedies for ailments. Medicinal plants, eventoday, are given the highest importance in the pharmaceutical industry,despite the fact that some of these chemicals, which were first identified asbotanicals, are synthesized chemically. It is a general rule in nature that if onehas continued enthusiasm, patience and the right approach, one shall encountera better performer of the function of interest. Natural product formationis no exception. Screening of natural plant populations has taken placethroughout civilization and will continue. This is now happening in a morerigorous manner at the level of genetic diversity and identity with the aid ofmolecular biology and biotechnology tools. On the other hand, it is also possibleto use the biotechnological tools for the generation of new varieties withmore desirable traits, as discussed in the above section with regard to microorganisms.Once a better performer is identified, the next concern would behow to maintain the genetic constitution so that it would be used for a longperiod without genetic variation. Tissue culture is the first option availablefor the clonal propagation of plants within a short period of time in a small

270 S. Hettiarachiarea. Similarly regenerated plants through micropropagation are extremelyimportant to relieve the pressure on the wild plants exerted by overexploitation.Depending on the tissue specificity of the product, the harvesting maybe destructive as vital parts, such as roots or bark, of the plant may have tobe removed in harvesting, imposing additional heavy pressure on wildpopulations.As mentioned in the review by Chaturavedi et al. (2007), the micropropagationand field establishment of Dioscorea floribandu in India has been a successstory. They calculate the number of plantlets that could be obtained within ayear starting from a single nodal cutting as 2,560,000 in comparison to a maximumof 10 plants from a single tuber which can be obtained after 3 years ofgrowth in the field. Thus there cannot be any doubt about the power of micropropagation,especially for those plants that cannot be rapidly propagatedotherwise. Even with those plants, the advantage of having propagation oftrue-to-type plants cannot be underestimated. The report criticizes the progressmade in India in the micropropagation of medicinal plants, despite thefact that experiments in various institutes have come up with protocols endingup in the field of a long list of medicinal plants. This fact is mentioned here inorder to highlight the inefficiency of technology transfer.The extraction of natural products from wild or field-grown plants, however,suffers from many impediments such as low production, and no uniformityof production from place to place, from time to time and from one plantvariety to another. Riker and Hildebrandt (1958) were probably the first scientistswho had vision of the power of plant tissue culture in natural productsynthesis. As tissue and cell cultures can be maintained under desirable cultureconditions, once culture media and culture conditions for optimumgrowth and optimum production have been worked out, the above problemscan be mitigated. When tissue is collected from cultivated or wild plants,there is a long waiting time from plantlet formation to product formationas extraction may be possible only after reaching maturity or some otherstatus.Among several other scientists working in the late 1970s and early 1980s,Berlin (1984) visualized plant tissue culture as a future potential process fornatural product synthesis and stressed that more research was needed for theelucidation of regulatory controls in biosynthetic pathways. Alfermann andPetersen (1995) demonstrated the potential of plant tissue and cell culturesfor natural product synthesis by referring to research by many scientists whowere able to produce various metabolites at the laboratory scale.It has been possible to manipulate biosynthesis through genetic manipulation,and overexpression and expression of new products has been possiblein cultured cells and organs. Organogenesis may be required for generatingcertain tissue-specific substances (Rout et al., 2000). The primary purpose ofplant tissue culture was the exploitation of the totipotency of the plant cellfor mass-scale propagation of useful varieties of plants. The plants obtainedare expected to be genetically identical and hence form a clone. However,variations occur within the clone due to somaclonal variations as a result ofreplication errors in mitotic cell divisions. While it seems undesirable, the

Biotechnology and Natural Product Synthesis 271generation of somaclonal variations is intentionally done for the furtherimprovement of the selected plant variety. The screening can be done inmuch the same way as for microorganisms – in Petri dishes rather than in agreenhouse or a field. Therefore overproducers and producers of new metabolitescan be easily and rapidly selected. The generation of variants can alsobe achieved by somatic fusion of two protoplasts of cells from two differentparents. The parents may be closely or distantly related. This can bringgenetic information required, for example, for synthesis of one metaboliteand an enzyme that can further transform it into a more useful product orproducts. This section of science is called combinatorial biosynthesis. Thevariants made by either method can usually be regenerated into whole plantsor, if desired, can be maintained in cell or organ cultures.Certain metabolites are tissue specific. Even for metabolites that are nottissue specific, the higher yield is given in compacted tissues rather than inloose-cell suspensions. For this, organogenesis is necessary. A very goodsolution is available as plant tissues transformed by Agrobacterium rhizogenescan grow indefinitely as roots in appropriate culture media. Once transformed,the cells can grow as hairy roots without the bacterium. When productsare formed and secreted or made to secrete into the medium, productrecovery is easy as it is not contaminated by cells. Further, continuousremoval is also possible. While all these facts are fascinating and encouraging,the published literature shows only two plant metabolites that areproduced on a commercial scale. Shikonin is the first botanical produced ona commercial scale using plant cell cultures of Lithospermum erythrorhizon.The specific productivity of shikonin has been increased 25-fold by simultaneousin situ extraction using n-hexadecane added before 15 days and immobilizationof cells in algenate beads, in comparison with production in cellsuspension culture (Kim and Chang, 2004).Immobilized cells have shown to be better producers in general. Whencomparing yields of scopadulcic acid B, a diterpene that has antiviral andanti-tumour activity, production by Scoparia dulcis in suspension culture andcells immobilized in Luffa sponge, Mathew and Jayachandran (2009) noted350.57 mg/g of cells by the 19th day by immobilized cells in contrast to 50.85mg/g of cells after 30 days of incubation in suspension culture. This is asevenfold increase with a shorter time of incubation. Continuous removalof products and inhibitory substances is an added advantage of usingimmobilized cells.The only other compound produced using plant cell culture that hassuccessfully entered the market is ginseng saponin originating from Panaxginseng. Further improvements to the yield have been shown in hairy rootcultures fed with specific nitrogen and phosphorous sources (Jeong andPark, 2006).Catharanthus roseus is one of the plants that showed success in the productionof alkaloids in cell suspension culture. These are indole alkaloids, ofwhich the bisindole alkaloids vinblastine and vincristine are antineoplasticmedicines and the monoindole alkaloids (ajmalicine and serpentine) are antihypertensiondrugs. Since the first results published 30 years ago (Kurz et al.,

272 S. Hettiarachi1980), continuous efforts have been made experimenting with various cellsuspension culture techniques with modifications of culture conditions inorder to maximize the production of desired compounds. The research focusingon improvements on both cell culture and bioreactor aspects is the subjectof the review by Zhao and Verpoorte (2007). Nevertheless, further research isnecessary to bring products of C. roseus to the commercial scale. Berberine, anisoquinolene alkaloid, has been produced in cell suspension culture of Tinosporacordifolia at a concentration of 5.5 mg g −1 dry wt in 24 days (Rao et al.,2008). By screening eight cell lines, the authors were able to find one line thataccumulated 13.9 mg g −1 dry weight of berberine. This is a 5–14-fold increasein product formation compared to that of the intact plant. This is an exampledemonstrating the potential of plant cell cultures and somaclonal variationfor the natural product industry. A tenfold increase brings down the cost ofthe product in the market by at least the same magnitude.Cyclotides are small cyclic peptides stabilized by disulfide bonds betweensix conserved cystine residues. Their biological activities include anti-HIV,antimicrobial and insecticidal actions, and hence they are important botanicalsin biocontrol. In addition, due to their high stability, cyclo tides are verygood candidates for the development of drug delivery systems. Dörnenburget al. (2008) developed techniques to produce cyclotides using callus, suspensionculture and hydroponic cultures of Oldenlandia affinis and evaluated themfor Kalata B1 accumulation. In vitro culture produced only up to 15% of KalataB1 in comparison with plants grown in hydroponics. Further improvement isprobable by manipulating culture conditions and the same group reported ahigher rate of cell multiplication in a 25-l photobioreactor (Seydel et al., 2009).They claim that this approach for harvesting Kalata B1 is more profitable thanother methods, such as field cultivation and chemical synthesis.Cell suspension culture is also useful in the synthesis of volatile oils. Bymanipulating culture conditions, Ishikura et al. (1984) obtained a yield of0.005% to 0.01% of volatile oils of the fresh weight of the cells of Cryptomeriajaponica cell suspension.The progress of research in the development of hairy root technology formetabolite production has been reviewed by Guillon et al. in 2006. The delicatenature of the hairy roots is one of the major problems in maintaininghairy root cultures. Srivastava and Srivastava (2007) reviewed the problemsand different technologies available to overcome them. The basic types ofreactors are either liquid phase or gas phase or even a combination of the two.The nature, applications, perspectives and scale up are discussed therein.The hairy root growth, production of the desired substance(s) and secretionmay require different culture conditions. The production can be enhancedby providing the precursors at the right time and the right concentration. Theaddition of cadaverine to hairy root cultures of Nicotiana rustica shifted thealkaloid production in considerable favour of anabasine with a concomitantdiminution of nicotine (Walton et al., 1988), whereas the addition of mentholor geraniol at 25 mg l −1 to hairy root cultures of Anethum graveolens did nothave an impact on the growth and resulted in the transformation of thecompounds to volatile products (Faria et al., 2009). In a recent experiment,

Biotechnology and Natural Product Synthesis 273Hernandez-Vazquez et al. (2010) were able to increase the production ofcentellosides in cell cultures of Centella asiatica by feeding with α-amyrintogether with DMSO to assist the penetration of α-amyrin in to the cells.The secretion can be enhanced by manipulating the culture mediumeither by increasing the leakiness of tissues or by introducing a trappingsystem to trap the secreted metabolite. For example, Rudrappa et al. (2004)reported an increased recovery of betalaines (red natural pigment) productionby hairy roots of B. vulgaris up to 97.2% by using alumina:silica (1:1) inthe culture medium. The use of elicitors is a useful approach to enhance theproductivity as some secondary metabolites are naturally produced inresponse to a signal. This has been shown practically by incorporating bothlive and autoclaved bacteria in the conversion of hyoscyamine to scopolamineby hairy root cultures of Scopolia parviflora (Jung et al., 2003).Podophyllotoxin is a useful botanical extracted from Podophyllum hexandrumfor treating general warts. Chattopadhyay et al. (2002) used a 3-l bioreactorfor the cell suspension culture of P. hexandrum and successfullyrecovered podophyllotoxin. It has been noted that providing coniferin as aprecursor in the culture medium increases the productivity, but this is economicallyprohibitive. An innovative method to circumvent this problem isto co-cultivate P. hexandrum cell suspension with hairy roots of Linum flavum,which produces coniferin. This approach has increased the final productformation by 240% (Lin et al., 2003).A further step in bringing the substrate and enzymes from differentsources has been made possible through combinatorial biosynthesis. Here,rather than cocultivating the two individual plants in vitro, the genetic traitscontrolling the production of one compound, which will be the precursor ofthe final product, and the enzyme required for the transformation are broughttogether into one cell. A somatic hybrid of two Solanum species producingsolanidine and solanthrene (S. tuberosum) and tomatidine (S. brevidens) producedall three steroidal glycoalkaloid aglycones. In addition all hybrids alsoproduced a totally new compound, demissidine (Laurila et al., 1996). Theseauthors propose a hypothesis to describe this which explains the productionof demissidine by hydrogenating the double bond at position 5 of solanidineof S. tuberosum by a hydrogenase enzyme of S. brevidens.13.3 Expanding Natural Product DiversityIn the quest for natural products for the benefit of humankind, it can often bethought that the existing natural product diversity is not sufficient. The generationof mutants is a tool available for increasing natural product diversity.Random mutagenesis is stimulated by treating with chemical mutagens orradiation. Although these are useful and have been employed for makingvariants and subsequent screening, with the better understanding of DNAand development of new biotechnological tools, specific, site-directedmutagenesis is possible. This has reduced the effort in screening a largenumber of mutants for the expected outcome.

274 S. HettiarachiIn an attempt to restore the ability to produce taxol, an anticancer drug,to an endophytic fungus, Tubercularia sp., mutagenesis and genome shufflingin protoplasts resulted in no restoration, but the production of new metabolites(Wang et al., 2010). One mutant produced three new and one alreadyknown sequiterpenoids, whereas another one made 18 novel compoundsbelonging to different classes and 10 already known compounds which werenot present in the wild-type strain.Altering the culture condition alone can induce the production of differentnatural products by an individual organism. If the mechanism of controlof a metabolic pathway is understood, then breaking silence in expressionshould be simple. Sometimes the presence of another organism in theneighbourhood may influence the induction of a pathway leading to a previouslyunseen product. Therefore mixed fermentation can result in anincreased concentration of already expressed or undetected or unexpressedmetabolites in crude extract, and the production of new analogues of knownmetabolites due to combined or extended pathways (Pettit, 2009).Each and every step of biochemical pathways are supposed be catalysedby a specific enzyme. Nevertheless, the size of the total genome, as unravelledby genome-sequencing projects, cannot account for the large number ofmetabolites present in organisms. Post-transcriptional modifications leadingto the formation of isozymes can be partly responsible for the high diversityof metabolites, but this has now been mainly attributed to the low specificityof some enzymes, which means that one enzyme can convert several similarprecursors or intermediates into several products causing ramifications inthe next step, and finally ending up with several end products. The numberof end products shall be much higher if there was no compartmentalizationof substrates and enzymes within a cell and in different cells. Bringing a naturalsubstrate in one cell in contact with an enzyme from another cell mayresult in entirely new ‘natural’ products. This is possible through breeding ofsexually compatible individuals, or through protoplast fusion, which is possiblebetween rather distant relatives. This technology is known as combinatorialbiosynthesis or metabolic engineering or heterologous expression, andhas been applied to microorganisms as well as to plants.Polyketides are highly diverse natural products, including those importantas antibiotics and other pharmaceuticals such as anticancer agents andimmunosuppressors. The most important producers are actinomycetes, butother bacteria, fungi, plants and marine animals also contain polyketides.They are formed by the sequential polymerization of carboxylic acids, and assuch carboxylic acid monomers become a source of variation in polyketides.The diversity has been possible due the modular nature of the polyketidesynthases (PKSs). Once transcribed, the peptides assemble to make the functionalenzyme consisting of different number of polypetides, each havingtwo modules. The large protein adds one monomer at each module to thegrowing chain. Mutagenesis in each module is possible and the outcome ofeach mutation and combinations of those can cause a great variety ofpolyketides (Kosla, 1997). The first polypeptide in the assembly of themegasynthase initiates polymerization and is the minimal PKS containing

Biotechnology and Natural Product Synthesis 275two enzymatic domains (ketosynthase (KS)_malonyl-CoA:ACP transferase(MAT) didomain) and an acyl-carrier protein (ACP) domain. Zhang et al.(2008) removed the minimal PKS from Gibberella fujikuroi in two formats andengineered it in E. coli – cyclized polyketide production was observed. In thefungus cyclization is C2–C7, whereas in E. coli cyclization of C9–C14 andC7–C12 were predominant. By expressing one of the two minimal PKSs(PKS_WJ) and downstream processing enzymes in E. coli they were able tosynthesis nonaketide anthraquinone SEK26 in good amounts. This work alsoencourages scientists to investigate further the generation of variety throughheterologous expression.A very good example is the engineering of a new metabolic pathway inE. coli by a combination of mixing genes and modifying catalytic functions byin vitro evolution. In this experiment, phytoene desaturases were assembledby shuffling modules from different bacteria and expressing them in E. coli.The different strains generated were screened for new carotenoid pigments.One such chimeric enzyme had introduced six, in place of four, double bondsinto phytoene. Another set of chimeric lycopene cyclases had also been madeand engineered into the cells expressing the above novel phytoene, thusextending that pathway to produce a variety of coloured compounds. Onesuch combination produced cyclic carotenoid torulene in E. coli. Thisexperiment reported by Schmidt-Dannert (2000) suggests the unlimitedpossibilities of obtaining novel natural products by rational design.Apart from the usual ribosomal polymerization of amino acids intopeptides, a mechanism of non-ribosomal enzymatic polymerization of aminoacids similar to that of polyketide synthesis is also present in living systems.For example, the well known hepatotoxin, microsystine produced by Microsystisspp., is formed by a microcystin synthetase complex consisting ofpeptide synthetases, polyketide synthases and hybrid enzymes (Dittmannet al., 2001). Accordingly, heterologous expression in a similar way to PKS ispossible in view of introducing new variants of microsystins in the quest fornovel natural products.A great proportion of microorganisms cannot be cultivated and hence noinformation on their potential for natural product formation is available.This portion reaches an astounding 90% or 99% of the total populations insoil. They belong to bacteria and archaea and also viruses. This also indicatesthe presence of an extremely large, yet untouched resource. Although theorganisms are unculturable, their DNA can be isolated from environmentalsamples and amplified using PCR techniques. The isolation, amplificationand study of DNA from a total community is referred to as metagenomics, orenvironmental or community genomics. Once DNA is amplified, purificationand sequencing is straightforward. Sequencing helps in ‘seeing’ what ispresent, and at least how close they are to culturable, known members of thecommunity. On the other hand, a functional approach, in which the DNA isexpressed in surrogate hosts, is also very useful in finding out what they cando. When analysing clones of recombinant surrogate hosts for new products,there is a big chance of finding new bioproducts, perhaps some of which maybe in entirely new families of compounds.

276 S. HettiarachiFragments of DNA can be cloned using artificial bacterial chromosome(BAC) vectors. These are designed to carry larger fragments than those producedby PCR techniques. Rondon (2000) used this technique and revealed agreat deal of information on phylogenetic, physical and functional propertiesof the metagenome. Some of the clones showed antibacterial and nucleaseactivity and the DNA sequences governing these characteristics were foundto be different from those already known. This directly supports the idea thatunculturable organisms provide a source of novel genes. Other functionsobserved were the production of amylase and lipase and hemolysis.13.4 ConclusionThe search for natural products has been rekindled with realization of thevast biological diversity and genetic diversity that make biological systemsan unlimited resource. Furthermore, the global society is demanding naturalproducts and rejecting synthetic chemicals for all possible uses, includingfood and additives, drugs, cosmetics and so on. Although plants and animalshad been a source of such products from ancient times, the horizons nowhave widened and continue to expand, particularly with the development ofmodern biotechnological tools. Man was unaware that he was exploitingmicroorganisms as they are invisible to the naked eye. Some products becomeredundant over time, as is the case for antibiotics for which resistance hasdeveloped in target (and non-target) organisms. Further new diseasesemerge, new lifestyles demand new products, and biotechnology is neededto satisfy those demands. As new tools in biotechnology have been found,one can expect that the trend will continue. Exploring and exploiting newresources will be feasible in the future, as evidenced by metagenomics innew product formation which harness the biosynthetic capability ofunculturable microorganisms. However, with all of these developments, thenumber of bioproducts that have already conquered the market is not encouraging.Therefore, there is a need for more intensive research on optimizingproduction of already identified bioproducts, with simultaneous researchefforts on new product formation. It can also be noted that technology transferis not occurring effectively, because the already available technologiesremain in the laboratories where they were generated without reaching theindustrial fermenters or industrial-scale farms.ReferencesAlfermann, A.W. and Petersen, M. (1995)Natural product formation by plant cellbiotechnology: results and perspectives.Plant Cell, Tissue and Organ Culture 43,199–205.Arakawa T., Yu, J., Chong, D.K.X., Hough, J.,Engen, P.C. and Langridg, W.H.R. (1998)A plant-based cholera toxin B subunit–insulin fusion protein protects againstthe development of autoimmune diabetes.Nature Biotechnology 16, 934–938.Berlin, J. (1984) Plant cell cultures—a futuresource of natural products? Endeavour 8,5–8.

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IndexPage numbers in italic indicate figures, those in bold indicate tables.active components for antifungalactivity 95–96antifungal active methanolinvestigations 96antimicrobial compounds 95secondary metabolites 95useful antimicrobial phytochemicals95active packaging 35–37advantages of preservative foodpackagingfilms 35–36alternative to modified atmospherepackaging (MAP) 36innovative concept 35effect of MAP packaging tested36–37mustard essential oil 36oil in liquid phase 37use of wax-coated paper and board36aerated compost tea (ACT) 251, 252inhibiting effect on fungaldevelopment 255aflatoxin secretion inhibitors 15aflatoxins 2produced by species in Aspergillus 24Africacrop losses 191, 192forest 195agriculture, in developing countries 1–2agrochemical industry 81aliphatics 60allelochemicals 135–136allomones 135apneumones 135kairomones 135synomones 135allomones 136–137plant metabolites with negativeeffect on insect behaviour136antifeedants 136–137anti-ovipositants 136repellents 136substances meeting requirementsfor new botanicalinsecticides 137allyl isothiocyanate (AITC) 10, 10, 36–37stored in plants as a glucosinolate10α-terpineol 181, 182, 183α-terthienyl 11, 11ambuic acid 228–229, 229antibiosis 247–248antibioticsdevelopment of resistance to 266281

282 Indexantibiotics continuedmodification 266natural products of microbial origin266antifeedant action mechanism 138–141biological assays 139–140, 140general procedure adopted140–141feeding deterrents 138food choice 138formula to calculate feedingdeterrence 141leaf discs 139products can lose primary efficiency141use of bioassay techniques 139antifeedant research 137–138antifeedant efficiency 138history of research 137recent emphasis 137–138studies showing efficacy 137antifeedant substances 141–143aromatic hydrocarbons 143aromatic plants 143Colorado beetle 143, 144monoterpenes as feeding deterrents143no antifeedant efficiency incommercial products 141Pongamia genus 142antifeeding activities 142repellent activities 142–143present study of poyphenolicsubstances 143prospects for products based on143–145multi-component tactics 145non-azadirachtin liminoids 145use in pest managementprogrammes 144–145antifeedants 12, 136–137deter phytophagous insects 136prospects for practical use 136–137antifungal active component (plants)96–106antifungal agents 22antimicrobial peptidesbiopesticidal peptides andinhibition 112generation of antimicrobialpeptides 112antimicrobial peptides from biocontrolmicroorganisms 112–113,113–114classified into groups of linear andcyclic peptides 112–113efficacy 113, 113–114peptide synthesis 112–113antimicrobial peptides from plants114–115plant defensinsinhibitory activity 114–115range of biological functions 115antimicrobial productsapplied to foods, research in 27–34from plant extracts, current status71–72Carvone 71Milsana ® 71new products 71plant essential oils as organicfungicides 71new approach to microbial diseasecontrol via SAR 72antimicrobials of plant origin 92aromatic compounds 53–59, 60–61alkaloids, examples 59, 59non-phenolic aromatic compounds53, 54OH groups absent 53tetrapyrrole goup 53, 54phenolic compounds 55–59flavonoids 57–59, 57, 61simple phenols all monomeric55–57, 55aromatic plant compounds 12Azadirachta indica see neem (Azadirachtaindica)azadirachtin 7–8commercial products 142considered non-toxic to mammals7–8highly oxidised triterpenoid 7, 7primary and secondary antifeedantactivities 142behavioural insect control 13benzoquinine 70β-Asarone 13, 14bioactive compounds 218bioactive substances 44

Index 283biocontrol agents 206, 210biodeterioration 91–92biofumigation with Brassica 211–215biological activity and repellency177–178activity of fatty acids 178, 178C9—C11 acids 178, 179, 185activity of some edible oils 177, 177biological diversity 221biotechnology in genetic diversity264–273molecular biological tools andidentification of variants265minor differences in closelyrelated genotypes 265molecular tools and conservationstrategies 265plant tissue and cell culture 269–273recombinant gene technology266–269antibiotics 266edible vaccine production 268genetic alteration 267hepatitis B 268human insulin preparations267importance of modifications268–269metabolic engineering 269more complex molecules 266peptide as final product 266pharming 267production of rennet(chymosin) 267systematics, uses of 265biotechnological tools available265story of mustard oilbiosynthesis 265botanical pesticides 4attitudes of resource-poor farmersin developing countries to200now adopting IPM principles200willing to pay for the services200environmentally safe 5–10, 11–12replacement by synthetic pesticides5botanical products, historical use 4–5plant diseases and disease controlmethods 4–5botanical resistance inducers 153botanicalsin developing countries 196–198local formulation by peasantfarmers 196burnt and fumes used 196methods of formulation197–198crude water extracts 197,197mixed formulations 197,198oils 197–198, 197powders 197, 197use of adjuvants 198scientific formulations 196simple to make, easy to use196future use 200alternative to syntheticpesticides 200WHO 200mechanisms of systemic 152–154use in third world countries193–194versus conventional pesticides198–199alternative management198–199brassinosteroids 51, 51, 80, 81carvone 10, 10non-toxic botanical insecticide 10cereals 27–33mycotoxins 27chemicals, synthetic 3coevolution and coexistence 264combinatorial biosynthesis 269, 271, 273compost tea 242–257aerated compost tea (ACT) 254–256competition among microbialpopulations 246–251antibiosis 247–248hyperparasitism 248–249induced resistance 249–251microbiostasis 246–247grape marc compost 252–256

284 Indexcompost tea continuedmicrobes in compost teas 256–257suppression of plant pathogens251–252aerated compost tea (ACT) 251,252clarification of terms 251compost tea described 251macrobiotic of NCT and ACT252non-aerated compost tea 251,252reports on control of plantpathogens/diseases251–252cotton leaf curl virus disease 149crop protectants, non-synthetic 47cyclosporin 226defensive plant mechanisms 135deforestation, and botanicals 195–196developing countriescrop protection a necessity 2destruction of food in storage 2farmers/researchers seekingalternatives to syntheticpesticides 43food industries rely mainly onfumigants 175–176formulations of botanicals in 196–198history of natural products and useby local farmingcommunities 193–194Nigeria 194tropical forest botanicals 193,194importance 1–2insect damage in stored grains 175use of natural products 48eco-friendly control measures 2–4edible oils 176edible oils and fatty acids 177,177–8, 178, 179crude oils against C. maculata185no adverse effect on seedgermination 185mode of action complex 185elicitors 119, 120–122, 206SAR-inducing compounds 206endophyte biologydiminishing rainforests a majorproblem 236microbial diversity loss 236endophytes 220definition of 219diversity in 221fungi and bacteria 219–220metabolic distinction 221molecular techniques 224addition of telomeric repeats224genes for taxol production 224plant selection norms and ethics222–223collection of Rhyncholacispenicillata 222–223distinct environmental settings222–223hypotheses governing plantselection strategy 222secondary metabolites 223snakevine Kennedia nigriscans223taxol and Taxus brevifolia 223researchers 235techniques for isolation,preservation and storage221–224cultures for product isolation221–2storage solutions 222endophytic fungias alternative sources 225–226examples 226Taxus brevifolia and taxol 219antibiotics from endophytic fungi227–231artemisinin 230colletotric acid 230Cryptocandin 227, 228Cryptocin 227, 228guignardic acid 230, 230javanicin 230–231Pestalotiopsis jesteri, jesteroneand hydroxy-jesterone229–230, 229Pestelotiopsis microspora227–229, 229

Index 285phomopsichalasin 230antioxidants from endophytic fungi233–234isopestacin 233, 233compounds from naturalresources 233–234P. microspora, compounds,pestacin and isopestacinfrom 233pestacin 233antiviral from endophytic fungi232–233call for new effective agentswith low toxicity 225discovery in its infancy 232–233fungi 234immunosuppressivecompounds 234F. subglutinans and subglutinolsA and B 234natural-based compounds226–227problem of resistant organisms225production of certain bioactivecompounds 225volatile antibiotics from fungalendophytes 231–232Gliocladium sp. 232M. albus 232M. albus 231M. crispans 232M. albus 231–232Environmental Protection Agency (EPA)and developing countries 199DDT 199establishment of BotanicalPesticides and PollutionPrevention Division 199register botanical pesticides 199use of synthetic pesticides 199views of 199enzymes, proteinase inhibitors, lectinsand PR proteins 115–119lectins 117naturally occurring insecticides117lytic enzymes 115–117lysozymes 116–117other applications for chinolyticenzymes 116potential for plant-diseasemanagement 116use on plant pathogens bybiocontrol agents 115PR proteins 118–119families 118–119‘inducible defence-relatedproteins’ 118proteinase (protease) 117–118active against plant pathogenicnematodes 117effective against phytopathogens in vitro andin vivo 117in the green consumerizationcontext 118used to engineer virusresistance in transgenicplants 118essential oils 12–14, 28, 176advantage of bioactivity in thevapour phase 34antifungal effects and antiaflatoxigenicafficiency25antimicrobial additives 23chemosterilant activity 13–14, 14cinnamon and clove 24as contact insecticides andfumigants 185–186difference in anti-fungal andaflatoxin efficacy 25–26and edible oils 176–177efficacy in inhibiting aflatoxinproduction 24–26clove, mountain thyme andpoleo antifungal effect25C. camphora and A. galanga oils25P. graveolens and A. parasiticus25zimmu extract and inhibition ofAFB1 26encapsulation 14as fumigants against stored-productinsects 180, 180, 181space fumigation with SEM76essential oil 180, 181space fumigation studies/tests180, 180, 186

286 Indexessential oils continuedimportant role in plant protection12insect repellants 13in vitro assays againstmycotoxigenic fungi 24in vitro screening of 37 oils 24as inhibitors of fungal growth andaflatoxin production 15lemongrass oil and mycotoxigenicfungi on maize 27–29assayed on irradiated maize 29mustard essential oil 36AITC, fungistatic or fungicidal36–37octopamine 14plant essential oils 71, 186possible mode of action 183–185d-limonene 184Push-Pull (stimulo-deterrentdiversionary) strategy 13semio-chemicals 13and synthetic pesticides 14therapeutic use of 26Ethiopian subsistence farmingsystems 48fescue toxicosis 219flavonoids 57–8, 57, 61glycosides 61, 61kinones 58–9, 58tannin, hydrolysable andcondensed 58, 58food, loss during storage 92fruits 33–34blue mould in apples and pears 33AITC treatments in pears 33disease incidence 33pepperfruit 34cinnamon essential oil 34volatile eugenol 33–34fumigants and contact synthesizedinsecticidesbotanicals as possible alternativesto 176global concern over negative effects176pest resistance and pesticideresidues 176fungal endophytes 218–236fungal toxins, controlled by naturalplant products 15fungigroup of microbes 219plant associated 218fungicides, chemical 42pressure to reduce in foods 3–4resistance to accelerating 3fungicides, natural 47fungistasis 246genotypes 265gibberellins 218glucosinolates 10, 211and myrosinaseco-located in same cells 211, 212formation of hydrolysisproducts 211, 213tissue disruption 211, 213secondary metabolites 211grape marc compost 252–256age and suppression 253disease-suppressive effects 253environmental hazards 252inhibition of sclerotial germination253soilborne suppression and diseases253–256treatment in Europe 252–3waste from wine production 252hairy root technology 272–273hyperparasitism 248–9India 234–235induced resistance 249–251induced antiviral proteins 160–166C. aculeatum and B. diffusa 162–166antiviral proteins 165, 166efficacy of foliar leaf sprays 166medicinal properties 162plant viruses 162–163pre-inoculation spray of SRIs165protein occurring in B. diffusa163proteinaceous modifiers165–166

Index 287systemic resistance inducer164–165treatment with SRI from C.aculeatum leaves 163virus inhibitory activity163–164inhibitor of virus replication 161Samsun plants 161plant-extract-induced virusinhibitory agent (VIA)161–162antiviral agents 161hypersensitivity to virusinfections 160purified AVF 160two categories 161VIAs 162virus-induced new antiviral proteincomponents 160–161induced systemic resistance (ISR)250–251insect growth reguatory chemicals(IGRS) 11–12insect pests 2insect repellentsfrom rainforest plants 11volatile terpenoids 12insecticide effects 135insecticides, toxicity of 2–3integrated pest management (IPM) 43,68, 200development 4in organic agriculture 45pest control in conventionalcropping systems 44juvabione 11–12, 12lemongrass essential oil 27–29, 32limonene 51, 51limonoids 142non-azadirachtin 145maize 27–31aflatoxin accumulation 27essential oils tested for fungalcontrol/mycotoxinprevention 29–31A. asperrima and A.striate 30dried basil leaves 30anise, boldus, mountain thyme,clove and poleo 29–30effects of cinnamon, clove,oregano and palma roseoils studied 30herb oils 29onion and garlic 29oregano oil 30lemongrass 27–29, 28potential prevention ofbiodeterioration 96–101active compound 98, 98in vitro evaluation antifungalactivity 99–100test fungi for antifungalactivity assay 99use of poisoned foodtechnique 99–100in vivo evaluation to preventbiodeterioration100–101results 100–101isolation and characterizationof antifungal activecomponent 98–9molecular structure ofbioactive compound99, 99P. corylifolia 96–98, 97melaninantimelanogenic compounds 127infectivity of plant pathogenic fungi127metagenomics 275, 276methyl bromide (fumigant) 65–66, 205microbial population, competitionamong 246–51antibiosis 247–8antibiotic production 248defined 247and inhibition in the Petri dish247Trichoderma and Gliocladium248hyperparasitism 248–249Trichoderma spp. 249mycoparasites 249parasitism on another parasite248, 248

288 Indexmicrobial population, competitionamong continuedinduced resistance 249–51compost/compost-amendedsoils 250defined 249–50ISR role in suppression of plantpathogens 250–251ISR and SAR 250microbiostasis 246–247competition for nutrients inexudates 246fungistasis 246iron competition 246–247iron salts, synthesis and use ofsiderophores 247and pseudobactins 247mungbean yellow mosaic virus (MYMV)149mycotoxicosis 22mycotoxigenic fungi, in vitro assaysagainst 23–24aqueous extract of Adenocalymmasativum 23–24extracts of Cynara cardunculus 23zimmu 23mycotoxigenic fungi and mycotoxins infoodsnatural preservatives 22plant products 22–27defence mechanisms againstpathogens evolved 33mycotoxins 2in foods 21–22naturally occurring secondarymetabolites 21significant impact oneconomics 22production 24–27aflatoxins 24myrosinase 211, 212, 213natural bio-herbicides 72–77allelopathydefined 73, 195release of protective chemicals73weed management programmes73augmentation biocontrol 73biological approach to weed control72classical biocontrol (inoculativebiocontrol) 73current status of natural herbicides76–77commercialized for weedcontrol in organicagriculture 77ecologically more favoured 76inundative biocontrol 72–73need to control weeds 72plants with herbicidal properties74–76allelochemicals inhibiting seedgermination andgrowth 75–76bioactivities of plant extracts75buckwheat 75essential oils used for weedcontrol 76extracts of lucerne cultivars 74potential of W. shinensis 74research into allelopathicactivity of plant extracts75natural compoundsfrom plants with bio-stimulatorypotential 78–81brassino steroids 80, 81, 81ComCat 80–81, 81effect of liquid seaweed extracton improving yield ofcanola 79products developed 78properties in plant extracts 79prototype product 80from wild plants 46–48plant allelochemicals 78replacing artificial chemicals infood 263–264secondary metabolites 78usefulness 264natural pest controls using botanicals 16natural plant compoundsbiocidal effect 111for controlling plant pathogens andpests 110–111with microbial activity 47non-biocidal suppressors 111

Index 289natural plant extractspotential to use as plant growthregulators/naturalherbicides 44–45use to control pathogens 44natural plant productsaims of research 47biodegradable and eco-friendly 82contribution of biochemicals 44final application to food products37inhibitory action on fungal cells 26use in agriculture 46–47natural product chemistry 264natural product diversity, expansionof 273–276attempts to restore taxol-producingability to an endophyticfungus 274combinatorial biosynthesis 274E. coli, engineering new metabolicpathway in 275fragments of DNA can be clones276metagenomics 275nonribosomal enzymaticpolymerization of aminoacids 275polyketides, formation of 274–275mutagenesis 274site-directed mutagenesis 273natural product research 60natural product synthesis 263–276natural productsavailability 195–196cultivation within agro-forestryprogrammes 195growth throughout the year195inter-cropping and mixcropping195use as weed control infarmlands 195biofumigation with Brassica211–215importance in third-world countries193and phytochemicals fromendophytes 224–234plant tissue and cell culture in269–273and ailments 269biosynthesis manipulation 270combinatorial biosynthesis 271,273cyclotides (small cyclicpeptides) 272development of hairy roottechnology 272–273immobilized cells 271indole alkaloids 271–272organogensis 270, 271plant tissues culture andcomaclonal variations270–271podophyllotoxin 273regeneration throughmicropropagation 270shikonin 271tissue culture 269use as fungicides and bactericidesin agriculture 206–210factors in formulating plantbasedproducts aspesticides 106plant protection from microbialattack 206summary of natural productsused as agrochemicals206, 207–210use of 47–48see also botanicals; natural plantproductsnatural substances in fungi, bacteria andplants, pesticidal properties 215neem (Azadirachta indica) 6–8, 15, 25,47and integrated pest management7–8biopesticides for pest control 6insect growth regulators (IGRs) 7leaf extracts 26limonoids 142organic farming 7–8potential alternative to chemicalbasedpesticides 6seed extracts 141–142use of leaves 193use of neem cake 194nematodes 11New Zealand 74nicotine 4, 5

290 Indexnuts 35controlling aflatoxin infection 35effect of fumingation with essentialoil of mustard 35inhibition of germination of fungi 35use of protectants 35octopaminebiological role in insects 14as neurotransmitter 183, 184paddy 101–106active principle 102, 102antifungal assay 101comparative efficiency studies 102,104complete inhibition of test fungiobserved 102, 103D. hamiltonii 101, 101, 105–106evaluation of the bioactivecompounds 105, 105in vitro antifungal activity assay 102pathogen control 215pathogen suppression 243pesticides 2aerosol 193eco-friendly and natural 106essential-oil-based 13natural-product-based 205persistence in biological systems 3pesticides, chemicalbeing phased out globally 205benefits to crop producers 43and creation of greenconsumerization 110downside of 192–193hazards to environment 43long-term effect of environmentalcontamination 46novel compounds in place of 110–11results of persistent andindiscriminate use 92undesirable impacts andenvironmental risks 110phenolic acids 75phenols, simple 55–57all monomeric 55, 55formation of coumarin glycosides56, 56development as naturalherbicide 56–57as natural herbicides 55hydrokinone 55salicylic acid 55phenyl propanoidscoumaric acid and caffeic acid55–56, 56potential for development 56synthesized from p-coumaricacid 56synthesized from phenylalanine 55–56phytopathogens 110plant diseases, problems andconsequences of 42–43plant endemism 221plant extractsantimicrobial compounds 59–62chemical defences 59constitutive and inducedcompounds 59–60flavonoids and saponins61–62intensive plant screeningprogrammes forbioactivity 60antimicrobial properties of 59–72aqueous extracts 93–94current status 71–72in vitro evaluation for antifungalactivity 93–95plants with antibacterial properties69–71plants with antifungal properties62–69secondary metabolites withantimicrobial properties60–61solvent extracts 94–95methanolic and ethanolic barkextracts 94petroleum ether and methanolicextract of E. ayapanatested 94testing for antifungal activity94–95plant oils, effects of 176plant pathogens, biocontrol of 111–125antimicrobial peptides produced112–115

Index 291enzymes, proteinase inhibitors,lectins and PR proteins115–119green consumerization 119–125proteinaeous compounds 111–112proteinic inducers of plantresistance 119–125plant productsantimicrobial activity studies anddemonstrated 23disc diffusion test 23as pesticides, current worldwideuse 5–10use in active packaging 35–37plant species and insecticidalproperties 176plant–insect relationships 134–135a ‘food’–‘consumer’ relationship135plantsisolation and identification 96–106problems in managing protection109–110serious efforts to screen forbiological activities 92under exploited for plant healthneeds 106use of bioactivity-directedfractionation and isolation(BDFI) 96with antibacterial properties 69–71Hedera helis extract and fireblight on pome fruits70–71large-scale screeningprogrammes 69testing of in vitro growthinhibition of E.amylovora 69–70with antifungal properties 62–69active secondary leaf extractmetabolites 62antifungal potential 64antimicrobial activity 65antimicrobial efficacy of plantextracts 68–69effective control of grapedowny mildew 63–64significant inhibition ofspore germination64effects of some essential oilsagainst R. solani 67evaluation of potential of plantextracts to be applied inIPM programmes 68exploiting indigenousknowledge of medicinalplants 64grapefruit extract extract usedto control Phytophthoraspp. 64–5inhibitionof mycelial growth ofC. cladosporioides 62of spore germination ofsome fungi byAjoene 62investigation of phenolcomposition of twocarnation cultivars 65methanol extract of C. emoryi62natural plant extracts andsoil-borne pathogens65–6neem tree extracts 66plant seeds 67–68saponins 63South African plants withfungitoxic properties 63PR proteins 118–119, 159–160precocenes 12, 12proteinic inducers of plant resistance119–125Chitozars 120cold shock protein (CspD) 120–122harpins 120induction of plant resistance topathogens 119active defence mechanismsinitiated 119general and proteinaceouselicitors 119MF3 protein 122–125MF3 integration with chitosan124MF3 polypeptide chain 123peptidyl-prolyl cis/transisomerases (PPIases)122plant FKBPs 122

292 Indexproteinic inducers of plant resistancecontinuedMF3 protein continuedPPIase interaction with pathogenproteins 122–123PPIases and plant—pathogenrelations 122–123rape lines with mf3 insertion124systemic character of resistance124Psoralea corylifolia 96–101, 97Push-Pull (stimulo-deterrentdiversionary) strategy 13pyrethrum 8, 9, 46–47as insecticide 8low doses 8pyrethroids versus pyrethrin 8quassin and neo-quassin 11rainforests 221repellents/repellency 136ribosome inactivating proteins (RIPs)150, 154–155rice 32–33plant extracts 32–33rotenone 4, 5used in commercial insecticides 4ryanodine 4, 5sabadilla (cevadilla) 8–10saponins 52, 52, 61–62, 63biological activity 61secondary metabolites 21, 49–59, 78, 95,223alkaloids 59with antimicrobial properties 60–61aromatic components 53–9non-phenolic aromaticcompounds 53, 54phenolic aromatic components55–59isoprenoid components 49–53brassinosteroids 51, 51, 52, 80,81, 81diterpenes 50, 51hemiterpenes 49, 50isoprene units and repeatingunits 49, 49monoterpentes 50, 50saponins 52, 52sesquiterpenes 50, 50sterols 51–52, 51, 52tetraterpenes 53, 53triterpenes 51semio-chemicals 13siderophores 247, 254, 256, 257soilborne suppression 253–256detection of siderophobes 256microbiological analysis 254, 254production of compost 253production of siderophores 254results from in vitro analysis 254–6,255soil-applied composts and wateryextracts 256solarization 211sorghum 32South Africa 13Blyde River Canyon 63determination of antifungalpotential of plants 63search for plant species withfungitoxic properties 63statins 125–128ergosterol 125and fungal melanin 127lovastatin and compactin in plantprotection 126natural inhibitors of sterolbiosynthesis 125sterols 125stored food commodities 15strychnine 11, 11subsistence farmers 46suppressive soils/suppressivesubstratesbiological elements 242–243composting conditions andmaterials 245–246general suppression 245modes of action of biocontrol agents243soilborne disease 243–245, 244specific suppression 245sustainable agriculture 4synthetic chemicalsconsumer resistance to 46

Index 293increased production 81systemic acquired resistance (SAR) 206endogenously occurring virusinhibitors 150induced by infection of plants 150pathogen-induced 158–159dicotyledonous andmonocotyledonousplants 159endogenous SA 159first report of virus-inducedSAR 158pre-inocculation with bioticinducers 158and role of PR proteins 159–160systemic induced resistance (SIR) 150,163systemic resistance inducers (SRIs)151–158, 163, 167carnation antiviral proteins(dianthins) 156characteristics 151induced resistance 151–152mechanisms of systemic inducedresistance by botanicals152–154absence of PR proteins 153production of VIA sensitive toactinomycin D 153suppression of diseasesymptoms 154systemic synthesis of some PRproteins 153VIA production in hosts 153virus inhibitory agents (VIAs)152mirabilis antiviral protein (MAP) 156–157leaf extract 156–157purified protein 157obtained from plants 152pokeweed antiviral proteins (PAPs)155–156pre-inoculation spray 165resistance development inhibitedby actinomycin D andcyclohexamide 152ribosome inactivating proteins(RIPs) 154–155PAPs, dianthins and MAPsbelong to type-I RIPs155show antivral activity againstboth animal and plantviruses 154–155shown to be N-glucosidases154Type-I RIPs 155Type-II RIPs 155ricinus (RICIN A) antiviral protein157A and B chains 157type II RIP 157role of RIPs 157–158virus resistance 157–158biological significance of RIPs158problem using type-I RIPs 158taxol 265anticancer drug 219, 227fungal source 219terpenoids 7, 7, 12, 60, 186Trichoderma 210virus disease resistanceconstitutive resistance 150induced resistance 150virus infection 150virus inhibitors from plants 150–151plant products using local orsystemic resistance 150–151ribosome inactivating proteins(RIPs) 151viruses 149wheat 31–32A. speciosa root isolates 31–32resveratrol 31wild plants 44, 46world agriculture 46

natural-products-in-plant-pest-management

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