1st NeemWorkshop - GDeffune (2001) Semiochemicals in Agriculture the Use of Pheromones and Alelochemics in Plant Systemic Resistance

Geraldo Deffune

Neem (Azadirachta indica A. Juss) is a member of Meliaceae family, a fast-growing tropical evergreen plant whose products were found effective against economically important insect pests and diseases. All parts of this plant particularly leaf, bark, and root extracts have the biopesticidal activities. Azadirachtin, a biopesticide obtained from neem extract, can be used for con-trolling various insect pests in agriculture. It acts on insects by repelling them, by inhibiting feeding, and by disrupting their growth, and reproduction. Neem-based formulations do not usually kill insects directly, but they can alter their behavior in significant ways to reduce pest damage to crops and reduce their reproductive potential. The neem is considered as an eas-ily accessible, eco-friendly, biodegradable, cheap, and non-toxic biopesticide which control the target pests. Thus, this re-view highlighted the extract, byproducts and roles of neem that can be used as potential biopesticide in agriculture.

Consequence upon the geometrically rising world population and the increasing pressure on food items, it has become increasingly necessary to increase food production from the present level. The possibility of achieving this is not only to increase production but also to protect the crops cultivated. Crop protection can be achieved through several means. One of such is the use of pesticides. This paper therefore reviews the use of neem extracts as bio-pesticides among other plant species with inherent pesticidal activities. It is no doubt that the chemical pesticides or insecticides possess inherent toxic substances that endangers the ecological environment, operators of application equipment, soil microbiota and consumers of the agricultural products. It is therefore important that we encourage the use of biological pesticides as they affect only target pest, are easily biodegradable, increase farm land fertility, environmentally friendly, cost effective and ease of availability. It is also important that because of the low cost of production of bio-pesticides it should be encouraged as an option in African countries especially Nigeria in agricultural practices. The practice of farmers making their own neem-based products for pest control would reduce their dependence on external inputs for agriculture. It would also reduce their cost of pest control to almost zero, leaving only labour as a potential expenditure item. Pests can also be controlled without the use of toxic chemical pesticides, which will reduce the harm posed to humans and the environment alike. There is wide scope for innovation in developing neem as an efficient bio-pesticide. There is enough information to encourage the use of different neem extracts. With the increasing trend of using bio fertilizers, insecticides and pesticides, neem should be increasingly cultivated and grown all over the world to get active ingredient-azadirachtin, responsible for stopping the growth cycle of pests. Neem is also assuming a lot of importance in crop management. Considering the fact that neem is not only a cheaper, naturally occurring product and an effective method to control pests and insects, but also has no side effects on plants or other living beings especially soil micro biota.

Neem plant is considered as the most useful traditional plant in India. The various properties of different parts of neem tree are used mainly as insecticide, fertilizer, manure, soil conditioner, urea coating agent, fumigant etc. in recent era the major challenge is to increase the food production without harming the environment and can control the pest. Since, last decades pesticides have become an integral component in sustainable agriculture and the modern cultural practices like use of chemical pesticides and fertilizers are in eliminable. The natural pesticides from Azadirachta indica are considered as less harmful, biodegradable, least persistence, less toxic to non-target organism and also economic. Fruitful results of application of formulated neem based products in agriculture will provide a cost effective technology to the farming community.

Prof. Dr. R. Soares de Faria and Dr. H. Kleeberg (Eds.) Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control Abstracts of the 1. Workshop „Neem and Pheromones“ University of Uberaba, Brazil, March 29. – 30. 2001 Held under the auspices of: Prof. Dr. Márcio Augusto de Sousa Nogueira Director of the INSTITUTE OF ENVIRONMENTAL SCIENCES AND TECHNOLOGY and Prof. Dr. Marcelo Palmério Rector of the U NIVERSITY OF U BERABA Organised by: With the support of: GTZ, Eschborn, Germany, Bioexton & Quinabra, Brazil Printed by: Trifolio-M GmbH, Sonnenstrasse 22, D-35633 Lahnau, Germany · Printed 2001 in Germany All rights of reproduction, in whole or in part, e.g. in print or by film, radio, television, or any other media are reserved. Contents: Modern Developments of Methods for the Control of Plant Pests and Ectoparasites in Agriculture 3 Hubertus Kleeberg 3 Possible Uses of neem – Traditional Methods of India and Modern Methods of Pest Control 5 Hubertus Kleeberg 5 NEEM IN BRAZIL - PLANTATIONS, EXTRACTS, RESEARCH AND UTILIZATION 8 Sueli S. Martinez, 8 Properties of NeemAzal -T/S – experiences and possibilities in biological plant protection 10 Edmund Hummel and Hubertus Kleeberg Main Pests which Can Potentially Be Controlled with Techniques based on Neem Products in Brazil Sueli S. Martinez Processing of Neem for plant protection – simple and sophisticated, standardized extracts Beate Ruch and Reinhard Wolf Quality control of Neem material Beate Ruch 10 23 23 25 25 28 28 NeemAzal-T/S – Estimation of residue data based on the analytics of the leading compound Azadirachtin A 31 Beate Ruch 31 Semiochemicals in Agriculture: the Use of Pheromones and Alelochemics in Plant Systemic Resistance 34 Geraldo Deffune Possibilities of the Use of Pheromones for Pest Control in Brazil Armin Kratt & Edmund Hummel 34 43 43 PotenTial of Use, Production of Extracts and Development of Products of Neem based Medicaments and Uses in Agriculture 45 Mauro Luiz Begnini 45 Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Modern Developments of Methods for the Control of Plant Pests and Ectoparasites in Agriculture Hubertus Kleeberg Trifolio-M GmbH, Sonnenstrasse 22, D-35633 Lahnau, Germany e-mail: info@trifolio-m.de; www.trifolio-m.de O n behalf of different reasons research for the development of new active ingredients for the control of insect pests is going on in chemical companies, universities, research institutions an small enterprises. In addition to commercial reasons the development of resistancies of the pest insects against relatively simple synthetic substances is a driving force for new developments. Furthermore the minimisation of toxicological and ecotoxicological side-effects for pest control products is a declared aim. Methods for biological pest control have the additional advantage, that their production is achieved by a minimal input of fossile energy and that the products usually are highly specific, easily biodegradable and produced with minimal risk to environmental pollution. In many countries integrated methods for pest and resistancy management are state of the art. The ongoing discussions concerning the quality of agricultural production are leading to higher levels of consciousness in the public, increased awareness on the usage of synthetic chemicals vs. biological means and marketing conceptions which favour agricultural produce which is produced according to IPM or organic farming guidelines. In this connection the concern of the consumer is not the philosophy behind the production but the demand for produce free of residues. Although the legal requirements for the official registration of products for biological pest control are the same or even higher (due to the complexity of the nature of the biological products) as for synthetic chemicals, their specificity is usually much higher; consequently their potential market volume and hence their economic profit chance is small. Due to the enormous – and increasing - expenditure for the development and registration of pest control agents (currently several hundred million of DM) only concerted efforts of environmentally and socioeconomically concerned groups may lead to new products in addition to political decisions. In addition to the obviously favourable use of beneficial insects in agriculture the intensification of the application of pheromones, different plant extracts, products of microbiological origin and different mineral products seems possible. The potential uses of pheromones in the frame-work of monitoring, mass trapping or mating disruption strategies are obvious and need further introduction into the practice. Due to the very high specificity of the pheromones this seems to be a laborious and consequently expensive process. Research for active substances originating from or containing microorganisms (including viruses) is going on world-wide. In the European Union more than 20 microbiological products for the control of pest insects or fungi and plant diseases are under practical evaluation. -3- Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Mineral products are still widely used but may in specific cases exhibit undesired side-effects especially with respect to beneficial insects. In comparison to vegetable oils some extracts of different parts of plants may have a highly specific potential for pest control. Among these, extracts of Chrysanthemum flowers (containing pyrethrins), Bitterwood (containing quassin) and Neem seeds (containing azadirachtins) are used traditionally and seem to have a practical potential in future. The official registration procedure and the legislative control bodies take care that the properties of commercial products are within the accepted limits. It is obvious that uncontrolled and usually nonstandardised traditional extracts of the above mentioned plants may vary considerably in quality. This may lead to severe failures of the application in practice and consequently serious economic losses of farmers as well as damage to the environment. For this reason the standardisation of means for biological is indispensable for modern products for biological plant protection. -4- Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil POSSIBLE U SES OF NEEM – TRADITIONAL M ETHODS OF I NDIA AND M ODERN M ETHODS OF PEST CONTROL Hubertus Kleeberg Trifolio-M GmbH, Sonnenstrasse 22, D-35633 Lahnau, Germany e-mail: info@trifolio-m.de; www.trifolio-m.de The Neem tree Leaves, seed kernels, bark and roots of the tropical Neem tree Azadirachta indica A. Juss are used in India since times immemorial for curing many diseases. In a holistic perception the protection of plants and animals against diseases and illness is a medical issue as well. The leaves and especially the seed kernels of the Neem tree and their extracts have been used for the control of various insect pests in India. O ur research has combined the experience of the thousands years old Indian experience and modern demands for plant protection products. The result of our development is the Azadirachtin concentrate “NeemAzalTM“ and its formulations like NeemAzalTM-T/S. What is NeemAzal-T/S? NeemAzal-T/S is a formulation of the highly concentrated active ingredients of the Neem tree, namely the Azadirachtins. This concentrate – named “NeemAzal” – contains in an average 34% AzadirachtinA, about 20% other Azadirachtins and 46% of inert ingredients like lipids, oligosaccharides, hydrate water. NeemAzal may for example be formulated with the help of surfactants (produced from renewable resources) and/or plant oils to obtain a concentrate with sufficient shelf life for practical application. Physico-chemistry and degradation NeemAzal-formulations usually have a shelf life of more than 2 years if stored below 20íC in a dark place. They spread easily for example on leaf surfaces and show good penetration of the fur of animals. An octanol-water distribution coefficient below 10 indicates a low potential for accumulation in fatty tissue and hence in the food chain. Azadirachtins are not much adsorbed by soil and thus leach rapidly. However, the degradation is very fast, so that a risk of contamination of ground water can be excluded. In water NeemAzal is transformed very rapidly by light. After spray application to leaves and fruits AzadirachtinA is degraded rapidly with a half life of the order of very few days. Toxicology NeemAzal and the formulation NeemAzal-T/S has been investigated thoroughly with respect to possible toxicological impacts to mammals. Neither acute nor subchronic or chronic studies indicate the presence of important risks for humans or mammals. This is especially established with respect to carcinogenicity, teratogenicity, reproduction etc. In this connection it is important to state that these “non-toxic” properties refer only to the concentrate NeemAzal and its standardised formulation and not to other “Neemproducts” since these may have considerably different compositions. -5- Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Ecotoxicology NeemAzal-T/S has been studied carefully with respect to possible side effects on the environment. The high “No O bservable Effect Concentrations” NO EC indicate an extremely low risk to aquatic organisms; this is true especially in view of the low concentrations of AzadirachtinA which are necessary for efficient applications in practice (i.e. of the order of 15 to 30 ppm AzadirachtinA in typical spraying solutions). Beneficials are generally not influenced to a meaningful extent by NeemAzal-T/S applications - with the exception of thin skinned species (like syrphids). Acute as well as reproduction studies with honey bees show that no adverse effects may be considered after application of NeemAzal-T/S. Studies on chicken as well as field observations do not show any significant effects with respect to birds. Mode of action After the treatment with NeemAzalTM-T/S larvae react with feeding and moulting inhibition and mortality; adult (beetle) show feeding inhibition, infertility and to a lesser degree mortality. As a result of this comparatively slow „insectistatic“ mode of action of NeemAzalTM-T/S a final assessment of the treatment should be done 7-10 days after application under practical conditions. The number of dead pest insects is not necessarily a good evaluation criterion. For the assessment the following criteria are appropriate: loss of leaf mass, damage to plants, formation of honey dew, crop yield, development of the pest population, positive effects on beneficials. The success of the application with NeemAzalTM-T/S depends on the progress of the pest infestation and adequate timing of the treatment. In the case of a temporary infestation and synchronous development of pest populations one application per generation or season is generally sufficient (under European climatic conditions, usually one or two generations, for example: appearance of fundatrices of the Rosy Apple Aphid Dysaphis plantaginea, first adults of Elder Bush Aphid Aphis sambuci (Hom., Aphididae), first young larvae of Colorado Beetle Leptinotarsa decemlineata, beginning of flight of Cockchafers (Melolontha sp.)). In case of a permanent infestation (several generations like Aphids, Thrips, White Flies, Spider Mites etc.) repetitive applications are required. The interval between treatments is usually 7 to 14 days and depends on climatic conditions and infestation pressure. NeemAzalTM -T/S is harmless to most beneficials - they are an important factor in the control of the remainder of the pest population. NeemAzalTM -T/S can favourably be combined with the use of beneficials in plant protection conceptions. Phytotoxicity information NeemAzalTM -T/S was tested with many plants under outdoor and greenhouse conditions and shows generally good plant compatibility during the warm season. The compatibility of NeemAzalTM -T/S depends -6- Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil on the variety and species of plants. Some ornamental varieties react with leaf and blossom damages. Some pear varieties react with strong leaf necrosis. In the case of plant species that normally react insensitive, individual varieties can exhibit incompatibilities and it is proposed to perform sensitivity tests with a few plants or some leaves in the respective stadium of growth 3 to 5 days before treatment of larger areas. -7- Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil NEEM IN BRAZIL - PLANTATIONS, EXTRACTS, RESEARCH AND UTILIZATION Sueli S. Martinez, IAPAR - Instituto Agronomico do Parana - Plant Protection, C. P. 481, 86001-970 Londrina PR, Brazil. suemart@onda.com.br During the decade of 1930, Brazil used to extract rotenone from several vegetal species, which were used in the country and exported mainly to USA. Also pyrethrum and nicotine were extracted from Chrysanthemum cinerariaefolium and Nicotiana tabaci. This market was replaced by the commercialization of synthetic molecules and, after 1950, Brazil started to import and apply chemical products in agriculture, with their notorious consequences to vertebrates and to the environment. In Brazil, several conditions aggravate this picture, like: high costs of the products, incorrect use, low level of instruction of the growers, low surveillance by the government and climatic inadequacy of the protective clothes and consequent low use. This situation demands new methods of pest control alternative the chemical insecticides, less toxic to men, less pollutant, with lower residues, cheap and which can be produced in the farm. Changes in the profile of producers and consumers are the most recent tendencies: consumers are more concerned to the possible risks caused to health by food produced conventionally, producers are interested in less pollutant technology, besides the fact that the organic products can achieve better prices in the market and their area has considerably increased. Neem and its products fit quite well in this expectance due to the nature of the compounds, mode of action, very low toxicity, among others. In Brazil, neem research started in IAPAR, Londrina County – PR, with the introduction of neem seeds from Philippines, in 1986. Although the neem tree is typically tropical, the material introduced from Philippines grew well and produced fruits in small quantities. The next step was to evaluate the adaptation of neem biotypes from different origin in several localities in Brazil. For this purpose, seeds were imported from Dominican Republic, Nicaragua and India (Pune) and planted in Londrina and Paranavai (Parana State), Jaboticabal (Sao Paulo State) and Brasilia (Federal District). Although Brasilia had the best conditions to grow the neem tree, the work was interrupted by difficulties in conducting the experiments. Similar development was observed among plants from different origin. The genetic variability was very high and trees with very diverse characteristics were obtained from the same material. The best development took place under the highest temperature and sandy soil, which happened in Paranavai. In all regions, significant fruit production occurred after about four years, reaching 7 to 8 kg fresh fruit/plant. Flowering goes from December to April and fruiting happens from March to May. From these findings came the interest of growers to plant neem tree, mainly in Central Region of Brazil, where the tropical climate is more favorable. At the moment, there are near 150,000 neem trees distributed in different states, mainly: Federal District, Goias, Para, Parana, Sao Paulo and Tocantins. These plantations are young, most of them not more than three to four year old. They were established to produce seeds, seedlings, leaves, fruits and wood. However, this production still finds limitations, like lack of adapted varieties and of technologies to cultivate and harvest, high costs of labor and marked not yet clarified. -8- Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Several products start to be available in the market, like: emulsified oil, dry leaves, grounded seeds, antiseptic cream and shampoos for animal, soaps, among others. However, these products still need to have their technology of production and stability improved, need to be registered, besides recommendations provided by research on the organisms they can control, doses, methods of application, etc. The scientific research with neem in Brazil has spread out more recently, as an answer to these needs. In IAPAR the action of neem extracts and neem oil has been studied on pests of economic importance, mainly coffee pests and also on natural enemies. Development, consumption, reproduction, mortality in different life stages and repellency were evaluated. Neem oil or leaf and fruit extracts were efficient on the following species: Leucoptera coffeella, Diabrotica speciosa, Bemisia tabaci, Spodoptora frugiperda, S. littoralis, Alabama argillacea and on the mites Brevipalpus phoenicis, Phyllocoptruta oleivora, Tetranychus urticae and Polyphagotarsonemus latus. The predator Cycloneda sanguinea was proved less susceptible to neem oil. O ther research institutes and universities have included nim in their investigations, like: Universidade Federal de Viçosa/MG, Universidade de Goias/Goias, Embrapa Arroz e Feijão/Goias, Escola Superior de Agricultura Luiz de Queirós/SP, Universidade Estadual de Londrina /PR, Embrapa Tabuleiros Costeiros, Embrapa Milho e Sorgo/MG, Universidade Federal Rural de Pernambuco/PE, Faculdade de Ciencias Agricolas e Veterinaria de Jaboticabal -UNESP/Sao Paulo. Some of the results are here described. Neem oil caused larval mortality of Plutella xylostella in Brassicae (Maranhão et al., 1997) and reduced egg viability and caused mortality of the mite Mononychellus tanajoa (Gonçalves, 2000); leaf extracts caused larval mortality of S. frugiperda in corn (Viana et al., 2000); neem oil caused mortality and prevented the development of populations of Calosobruchus maculatus in stored beans (Oliveira et al., 1998); neem extracts are also being studied to control ticks and Hematobia irritans in cattle (H. Amorim). IAPAR develops research with the tree, aiming to obtain plants more adapted to subtropical conditions and to test the adaptation of neem grafted on Melia azedarach, more adapted in subtropical conditions. Besides, evaluates azadirachtin content along plant phenology and within extracts stored under different conditions. Brazilian experiences and tendencies till the moment indicate a great potential of production and use of neem based technologies in the country, mainly due to the wide areas with climate favorable to plant neem trees, to the interest of growers, and to the possibility to develop technology to produce and harvest neem and to industrialize products, besides the wide potential market. -9- Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil PROPERTIES OF NEEM AZAL-T/S – EXPERIENCES AND POSSIBILITIES IN BIOLOGICAL PLANT PROTECTION Edmund Hummel and Hubertus Kleeberg Trifolio-M GmbH, Sonnenstr. 22, 35633 Lahnau, Germany e-mail: info@trifolio-m.de; www.trifolio-m.de During the period 1994-2000 we have optimised the commercial formulation NeemAzal-T/S with respect to its application by field and laboratory trials. The efficacy was tested against more 140 species of mites (Acari) and insects from Coleoptera, Diptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera and Thysanoptera. The results (see table) show, that NeemAzal-T/S is effective against a large variety of free feeding sucking and biting pests. In the phytotoxiticy experiments it was observed, that the formulation may be toxic to certain varieties of pear-trees and certain varieties of ornamentals in greenhouses. In order to obtain reliable results with respect to efficacy and phytotoxicity many tests have to be performed under various practical conditions. In O ctober 1998 we obtained the registration for the use of NeemAzal-T/S in Germany for the control of sucking insects, white flies, leaf miners and spider mites ornamentals in greenhouses and in 2000 for Rosy Apple Aphid, Colorado Potato Beetle, Winter Moth and other pest for field-application. All results show that the application of NeemAzal-T/S is efficient with respect to the registered target pests and does not bear special risks to humans and the environment. Due to these favourable properties work for the expansion of the registration for plant protection is in progress. Formulations which may efficiently be used for the control of human or animal ectoparasites have been developed and are currently tested. From the various results the effects of the NeemAzal application can be summarised in the term „Insectistatic“. The main aspects of this mode of action are: Phenomenon Timing Description Assessment Feeding inhibition after hours reduced food uptake reduction of: plant damage, faeces, honey dew Inactivity after days to 1-2 over all reduction of: weeks fitness, molting inhibition, starvation mortality Fertility reduction after weeks (next generation) reduction of population reduction of progeny - 10 - the next Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Summary of results with NeemAzal-T/S Index* Species Pest Number of results: positive undecisive negative - - 2 Acari, Tetrapodili 0 Eriophyes rubi, E. gracilis Blackberry Gallmite Acari, Tetranychidae +++ Oliconychus coffeae Red Spider Mite 10 - - +++ Panonychus ulmi - 2 1- - +++ Tetranychus cinnabarinus Spider Mite 1 - - +++ Tetranychus urticae Spider Mite 15 4 1 - 2 - - + + + Criocerus asparagi, C.duodec. - 2 - - + + + Chrysolina varians - 1 - - + + + Galerucella nymphaeae - 2 - - Coleoptera 0 Raphidodopalpa foveicollis Coleoptera, Chrysomelidae ++ Dicladispa armigera Rice Hispa 2 2 1 0 Gastroidae viridula - - 1 - +++ Leptinotarsa decemlineata Color. Pot. Beetle 26 - 1 +++ Oulema melanopus - 1 - - +++ Phaedon cochleariae - 1 - - 0 Phyllotreta sp. - 2 3 3 0 Psylliodes od. Phyllotreta sp. - - 2 - - 11 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Coleoptera, Coccinellidae ++ Epilachna vagintioctopunctata Epilachna Beetle 1 - - ++ Henosepilachna vigintioctp. . Afr. Mel. Ladybird 3 1 1 Coleoptera, Curculionidae 0 Anthonomus pomorum - 1 - 1 - Ceuthorrhynchus assimilis, C.napi - - - 4 - Curculio nucum - - - 1 0 Coenorhinus aeguatus - - 1 - + Hylobius abietis - 2 - - 0 Otiorhynchus sulcatus - 1 1 - 0 Phyllobius sp. - - - 1 0 Phynchites bachus - - 1 - - - - 1 Coleoptera, Nitidulidae 0 Meligethes aeneus Coleoptera, Scarabaeidae +++ Melolontha hippocastani Cockchafer 8 2 2 +++ Melolontha melolontha Cockchafer 6 - 1 Index* Species Pest Number of results: positive undecisive negative - 1 - Heteroptera, Pentatomidae 0 Antestiopsis orbitalis Coffee Bug - 12 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Heteroptera,Miridae 0 Lygus pabulinus - 8 1 2 0 Plesiocoris rugicollis - 1 1 - Banana-spotting 1 - - Heteroptera,Coreidae 0 Amblypelta lutescens Diptera +++ Agromyzidae - 2 +++ Sciaridae - 5 3 - Leaf Miners 8 1 1 Phytomyza sp. Leaf Miner 1 - - Napomyza gymnostoma Leaf Miner 1 - - Cabbage Maggot 2 1 2 Carrot Fly - - 1 Diptera, Agromyzidae +++ Liriomyza huidrobrensis, L. trifolii +++ ++ Diptera, Anthomyiidae 0 Delia brassicae, D. floralis Diptera, Psilidae 0 Psila rosae Diptera, Cecidomyiidae 0 Contarinia tritici - - - 1 0 Dasineura brassicae, D. mali - 1 - 1 0 Orseolia oryzae Rice Gall Midge 1 - - - 1 - - Diptera, Muscidae 0 Musca domestica Diptera, Trypetidae - 13 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil 0 Platyparea poeciloptera - - - 1 0 Rhagoletis cerasi Cherry Fly 2 4 - Mealy Bug 2 - - Homoptera ++ Coccidae Homoptera, Adelgidae 0 Pineus pini/orientalis - - 1 - 0 Dreyfusia nordmannianae - 1 - - Homoptera, Aleyrodidae +++ Aleurocanthus spiniferus White Fly 1 - - +++ Aleurothrixus floccosus White Fly - 1 - +++ Bemisia tabaci Cotton White Fly 12 4 - +++ Dialeurodes kirkaldyi White Fly 1 - - +++ Trialeurodes vaporariorum White Fly 20 7 1 Index* Species Pest Number of results: positive undecisive negative Homoptera, Aphididae +++ Acyrthosiphon pisum, A. scariolae Green Pea Aphid 2 - 2 + Acyrthosiphon scariolae Salad Aphid - - 2 - 14 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil +++ Aphis fabae - 7 - 1 +++ Aphis gossypii Cotton Aphid 11 8 3 +++ Aphis nasturtii - 2 - - Aphis pomi Green Apple Aphid 3 2 2 +++ Aphis sambuci Elder Bush Aphid 5 1 1 +++ Aulacorthum circumflexum - 1 - - +++ Aulacorthum solani - 4 - 1 Brachycaudus helichrysi - 1 2 - Cabbage Aphid 4 11 2 0 + + + + 9 Brevicoryne brassicae + Cavariella aegopodii - 2 - 2 + Cryptomyzus ribis - 1 - - 0 Drepanosiphum platanoides - 1 - - +++ Dysaphis devecta - 3 1 - +++ Dysaphis plantaginea Rosy Apple Aphid 41 - 1 1 - - ++ Dysaphis pyri - Hyalopterus pruni - - 1 - +++ Macrosiphoniella sanborni Black Aphid 2 1 - +++ Macrosiphum euphorbiae - 7 1 - +++ Macrosiphum rosae Green Rose Aphid 5 - 1 Macrosiphum rosaeformis Aphid 1 - - Megoura viciae Vetch Aphid 2 - - 0 Metopolophium dirhodum Rosy Grain Aphid 1 - - 0 Myzus nicotianae Aphid 1 - - Myzus persicae - 8 - - 0 0 +++ +++ - 15 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil 0 ++ 0 Nasonovia ribi snigri - - 2 6 Phorodon humuli Hop Aphid 4 - 1 Rhopalosiphum insertum - 1 1 4 - 1 - - - 1 - - - - - 1 Homoptera, Ortheziidae 0 Orthezia tillanssidae Homoptera, Chaitophoridae 0 Chaitophorus capreae Homoptera, Eriosomatidae 0 Eriosoma lanigerum Homoptera, Cicadoidae ++ Amrasca biguttula Leaf Hopper 9 2 2 ++ Empoasca flavescens (vitis) Grape Leaf Hopper - 1 1 ++ Idiocerus niveosparsus Mango Hopper 1 - - Index* Species Pest Number of results: positive undecisive negative Homoptera, Cicadellidae ++ Eupterus melissae Leaf Hopper 1 - - ++ Nephotettix virescens Green Leaf Hopper 2 2 - 0 Nilaparvata lugens Leaf Hopper - 1 - 0 Schapoideus titanus Leaf Hopper 1 - - 0 Idiocerus niveosparsus Leaf Hopper 1 - - Sogatella fucifera White Plant Hopper 1 - - ++ - 16 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Homoptera, Coccidae + Coccus hesperidium - - 2 - + Neopulvinaria imeretina - 1 - - Brown Plant Hopper 6 1 - - - 1 - Homoptera, Delphacidae ++ Nilaparvata lugens Homoptera, Diaspididae 0 Lepidosaphes ulmi Homoptera, Lachnidae 0 Eulachnus agilis - - - 1 0 Schizolachnus obscurus - - 1 - Dactulosphaera vitifoliae - 2 - - 0 Homoptera, Pseudococcidae Planococcus citri Citrus Mealy Bug - 1 - 0 Planococcus lilacinus Mealy Bug - 2 - 0 Pseudococcus longispinus Mealy Bug - 1 - Agonoscena targionii - 1 - - Psylla pyri - 1 - - Homoptera, Phylloxeridaea +++ Homoptera, Psyllidae 0 +++ Hymenoptera, Diprionidae 0 Diprion sp. - 1 - - 0 Hymenoptera, Tenthredinidae Hoplocampa testudinea - 2 2 1 Lepidoptera - 17 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil 0 Achaea janata Rose L. Caterpillar 1 - - 0 Ascotis selenaria Giant Looper - - 1 0 Hellula sp. Cab. Head Borer 2 - - 0 Hymenia recurvalis Leaf Caterpillar 1 - - 0 Leucinodes orbonalis Shoot&Fruit Borer 2 1 4 Fall Webworm 2 - - Potato Tuber Moth - 1 - Lepidoptera, Arctiidae +++ Hyphantria cunea Lepidoptera, Gelechiidae 0 Phthorimaea operculella Index* Species Pest Number of results: positive undecisive negative Lepidoptera, Geometridae +++ Operophthera brumata Winter Moth 17 2 - +++ Bupalus piniarus Pine Looper 1 - - Lepidoptera, Gracillariidae +++ Phyllocnistis citrella - - 50 - +++ Cameraria ohridella - 2 - - +++ Lithocolletis leucographelle - 2 1 - - 2 - - Lepidoptera, Lasiocampidae 0 Dendrolimus pini Lepidoptera, Lymantriidae - 18 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil +++ Euproctis chrysorrhoea Brown Tail Moth - - 1 +++ Lymantria dispar. Gypsy Moth 3 - - +++ Lymantria monacha Nun Moth 2 - - Apple Leaf Miner- - 1 - Lepidoptera, Lyonetiidae +++ Leucoptera malifoliella Lepidoptera, Noctuidae ++ Earias vittella Fruit & Shoot Borer 1 - - ++ Heliothis armigera „Amer.“ Bullworm 15 4 - +++ Mamestra brassicae Cabbage. Army Worm 11 2 - ++ Mythimna albistigma Cutworm 1 - - ++ Spodoptera littoralis Eg. Cott. Leafworm 1 - - Spodoptera litura Leaf Catapillar 4 5 - + Lepidoptera, Pieridae +++ Eurema blanda - 1 - - +++ Pieris brassicae, P. rapae Cabbage. Butterfly 6 - 3 Lepidoptera, Pyralidae 0 Cnaphalocrocis medinalis Rice Leaf Folder 3 5 - 0 Diaphania hyalinata - - - 1 0 Eurema blanda - 1 - - 0 Evergestis forficalis Garden Pebble Moth 1 - - 0 Leucinodes orbonalis Shoot&Fruit Borer 2 - - 0 Scirpophaga incertulus Yellow Stem Borer 2 - - - Tryporiza incertulus Rice Stem Borer 1 6 5 Lepidoptera, Tineidae - 19 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Tineola bisselliella Cloth Moth 3 - - +++ Lepidoptera, Thaumetopoeidae Thaumetopoea pityocampa - 2 - - +++ Thaumetopoea processionea Brown Tail Moth 8 2 - ++ Index* Species Pest Number of results: positive undecisive negative Lepidoptera, Tortricidae ++ Adoxophyes orana Sum. Tortrix Moth 4 - 1 0 Cydia leucostoma Tea Flushworm 1 - - - Cydia pomonella Codling Moth - - 2 0 Eupoecilia ambiguella Grape Berry Moth - 1 1 0 Lobesia botrana Europ. Grape Moth - 2 4 0 Pandemis heperana Sommer Fruit Moth 1 - - Tortrix viridana Green O ak Moth - 1 - 14 6 - 7 - - +++ Lepidoptera, Yponomeutidae +++ Plutella xylostella +++ Yponomeuta malinellus, DBM Yponomeuta padellus Thysanoptera, Thripidae +++ Chloethrips oryzae Thrips - 1 - +++ Frankliniella occidentalis Thrips 12 4 1 +++ Parthinothrips dracaenae Thrips - 1 - +++ Scirtothrips sp. Tea Thrips 1 - - +++ Taeniothrips sp. Tea Thrips 1 - - - 20 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil +++ * Index: Thrips tabaci, T. meridionalis Thrips 3 5 2 + + + : efficient control established; + + : efficient control possible; + : control may be possible after optimisation of the method of application; 0 : undecided, further tests necessary; - : efficient control not possible Efficacies depend on: i). the time of application. ii). the concentration, and iii). the sensitivity of the target insect with respect to the different effects of the mode of action. Hence the optimisation of the application method of NeemAzal-T/S is decisive for the efficacy and the certainty of the success of the application in practice. Taking into account the short half life of the active ingredient it seems useful to define operationally two groups of infestation: 1. batchwise appearance of the pest: with a good timing one application is sufficient for the control of one generation of the pest (like for: colorado potato beetle (1. to 2. larval instar); cockshafer (ratio male : female adults ~ 1 : 1); rosy apple aphid (appearance of fundatrices); 2. persistent infestation: at the presence of young developmental stages the application should be repeated at intervals of 1 - 3 weeks (like: thrips, white fly, or green rose aphid). The results of practical applications show that application methods can be worked out which permit an efficient control of many different pests. These efforts seem to be worth while in order to find new solutions of pest control for biological as well as for integrated farming with a toxicologically and ecotoxicologically safe product like NeemAzal-T/S. Phytotoxicity information NeemAzal-T/S was tested with many plants under outdoor and greenhouse conditions and shows generally good plant compatibility during the warm season. The compatibility of NeemAzal-T/S depends on the variety and species of plant. Some ornamental varieties react with leaf and blossom damages. Some pear varieties react with strong leaf necrosis already from spray drift. It can not be excluded that damage can occur in cases of plants with known good compatibility. In ornamentals the following plants react on NeemAzal-T/S-treatment with: good leaf and blossom compatibility - Antirrhinum majus, Acalypha hispida, Argyranthemum frutescens, Astericus, Begonia-hybrids, Bidens ferulifolius, Brachycome, chrysanthema (Merced, Bronze Arola, Kory), Celosia cristata, Convolvulus sabatius, Coreopsis (girls eye), Dendranthema grandiflorum, D. indicum, Diascia, Euryops chrysanthemoides, Fuchsia, F.-hybrids, Gazania splendens, Gerbera jamesonii, Glechoma, Helichrysum petiolare, Kalanchoe (Boston), Lantana-Camara-hybrids, Lobelia, L speciosa, Manettia bicolor, Mentha, Carnations (Aristo), Slipperwort, Pelargonien, Petunia, Pilea microphylla, Roses (Komet), - 21 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Rudbeckia, Sanvitalia procumbens, Scaevola, Sutera, African marigold (yellow), Torenia fournieri, Verbena (Tapien blue) (Sunvop (P), good leaf compatibility - Agerathum houstonianum, Alonsoa, Alyssum, Amaranthus, Calceolaria hybrids, Callistephus chinensis, Calocephalus brownii, Centaurea, Cestrum, Clarkia, Cleome, Coleus, Cosmos, Cuphea, Cynara scolymus, Dahlia, Dianthus barbatus, Dimorphoteca, Eucalyptus, Eustoma grandiflorum, Ficus, Felicia, Gazania, Gnaphalium, Helianthus, Heliotropium arborescens, Iresine lindenii, I. herbstii, Kochia, Lavatera, Limonium, Lotus, Lysimachia, Melampodium paludosum, Mesembryanthemum crystallinum, Nicotiana, Nigellia, Pennisetum, Penstemon, Plectranthus fruticosus, Polygonium, Portulaca, Ricinus, Salvia farinacea, Saintpaulia (Miho io), Senecio, Serenoa, Streptocarpus, Tanacetum, Tithonia, Trachelium, Viola, Veronica, Zinnia, blossom damages - Begonia semperflorens hybrids, Chrysanthema (Deep luv), Euphorbia pulcherrima (Peter star, Cortez), Gerbera (Pretty red, Sigma, Luciana), Impatiens Neu-Guinea hybrids, Impatiens walleriana, Pelargonium-Peltatum-hybrid, P.-Zonale-hybrids, Solanum rantonnetti, Saintpaulia (Miho io), African marigold, Verbenen (individual sorts), leaf damages - Abutilon hybrids, Cestrum, Datura, Euphorbia pulcherrima, Impatiens Neu-Guinea hybrids, Impatiens walleriana, passion flower, Solanum rantonnetti, Roses (Papa Meilland, White Noblesse, Saphir, Ducat, Eveline, Alina, Baronesse, Lola, Black Magic, Noblesse, Roulette, Funcky Jazz, Arabia). In orchards serious plant toxicity has been observed in the case of pear varieties ‘Conference’, ‘Alexander Lukas’, ‘Bristol Cross’, ‘Comice’, 'Guyot’, ‘HW 606’, ‘Illinois 13 bars 83 Maxi’, ‘Lectier’, ‘Trevoux’, ‘Winter dechant’. In the case of plant species that normally react insensitive, individual varieties can exhibit incompatibilities and it is proposed to perform sensitivity tests with a few plants or some leaves in the respective stadium of growth 3 to 5 days before treatment of larger areas. - 22 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil M AIN PESTS WHICH CAN POTENTIALLY BE CONTROLLED WITH TECHNIQUES BASED ON NEEM PRODUCTS IN BRAZIL Sueli S. Martinez IAPAR – Instituto Agronomico do Parana / Plant Protection, C. P. 481, 86001-970 Londrina PR, Brazil. suemart@onda.com.br The neem tree, Azadirachta indica A. Juss, contains terpenoids with repellent and deterrent properties to insects and which can also cause growth disruption, reduce fertility and fecundity and kill. They also present acaricide, nematicide and fungicide properties. The main compound is azadirachtin, which occurs in variable concentrations in aqueous and alcoholic extract prepared with leaves and fruits, oil, dry leaves and grounded fruits, fruit cake, among others. Neem effects were proved on more than 400 species of insects and many of the genera present in the literature occur in Brazil and have economic importance. However, most of the studies were run in laboratory conditions and further information need to be included before they can be recommended. Studies run at IAPAR, in 1991 demonstrated that aqueous leaf extract added to diet caused larval mortality of Spodoptera frugiperda in laboratory. When sprayed on bean plants the same extract reduced egg laying of Bemisia tabaci in greenhouse. Azadirachtin sprayed on bean leaves reduced leaf consumption of Diabrotica speciosa, in a dose dependent manner. It was also shown to delay the development of Spodoptora littoralis (Martinez & van Emden, 1999), to reduce food intake, weigh gain and growth and to affect digestibility. It caused dose dependent mortality, crescent along S. littoralis development, and produced abnormalities which can be considered with mortality. Azadirachtin reduced fertility and prolonged life cycle, impairing population growth. It was more efficient when ingested than when sprayed and more lethal when administered to larvae in the final instar, producing larva-pupa intermediates (Martinez & van Emden, 2001). Neem oil was efficient against mites, which are difficult to be controlled with conventional acaricides. Oranges were sprayed with neem kernel extracts at 15 and 30% (p/v) and infested with mites. Mortalidade over 80% was observed in Brevipalpus phoenicis, Phyllocoptruta oleivora, Tetranychus urticae, Polyphagotarsonemus latus, after 24h in laboratory. In the field, neem kernel extract at 20% (p/v) caused mortality of B. phoenicis in citrus above 70% during 28 days, when 85% died (Meneguim & Martinez, 1998). In tests with and without choice, neem oil reduced egg laying in Leucoptera coffeella when coffee seedlings were sprayed (Martinez & Meneguim, 1999). Besides, neem oil caused egg mortality above 70% for those deposited on treated leaves, reducing even more the infestation potential of L. coffeella in coffee plantations. The action of neem oil was less evident with the predator Cycloneda sanguinea. Sprays of neem oil at 0,5% straight on adults did not affect survival. Larval mortality was higher but no reduction in aphids consumption was observed (Martinez & Meneguim - IAPAR). Neem extracts are being tested on ticks and on Hematobia Irrirans,in cattle (H. Amorin, FCAVUNESP/Jaboticabal-SP). Good control is obtained for both organisms when fresh leaves are added to food or leaf extracts are sprayed on the back of the animal. Spray on feces shows efficiency for H. irritans. Several species of soil and airborne fungi which occur in Brazil were affected by neem extracts: Rhizoctonia solani (found in beans, potato, soybean) and Fusarium oxysporium (found in beans, soybean), - 23 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Helminthosporium nodulosum, Alternaria tenuis, Colletotricum. Although neem extracts have not controlled fungi species which infect harvested fruits, they delay fruit rotting, so preventing the early contamination with fungi, like H. nodulosum. Neem cake was proved efficient against nematodes. Neem prevents egg development of Pratylenchus sp. e Meloydogine incognita and reduces larval ability to penetrate the roots. Neem and its extracts might not be taken simply as insecticides. They possess multiple effects which can be added, affecting not only the population in test but also the new generations which develop from that. By this reason, its effects deserve to be well evaluated in laboratory conditions to better understand the field results. A most profound study on neem action on most of Brazilian pest species under laboratory and field conditions is still necessary. However we can already conclude that neem has a high potential to be used in IPM programs and in O rganic Farming, by its wide range of action, multiple effects, good potential of association with biological control, special characteristics which make resistance difficult, besides the very low toxicity and quick degradation in the field. - 24 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil PROCESSING OF NEEM FOR PLANT PROTECTION – SIMPLE AND SOPHISTICATED , STANDARDIZED EXTRACTS Beate Ruch and Reinhard Wolf Trifolio-M GmbH, Lahnau, Germany The neem tree (Azadirachta indica) is native to South Asia and grows best along the tropical belt. The areas of origin are mainly India and Myanmar. Due to the multitude of possible uses the tree has been spread throughout the world, presumably by Indian immigrants with extensive knowledge about these possibilities. The neem tree is undemanding in view of ecological aspects (soil and water) is fast growing, has the advantage to protect areas against erosion (i.e. Sahel zone) and to halt desertification. The global occurrence and abundance is estimated and presented in the table below. Neem occurrence in million trees World Asia/ Oceania Africa Caribbean/ Latin America Industrialised Countries 60-90 27-39 31-45 5,5-6,5 approx. 0,5 The neem tree has numerous potential uses and nearly every part of the tree can be used. Details are presented in the following table. Part of the tree Usage Seeds Pesticides, O il Extraction, etc. O il Soap, Pesticides, Cosmetics, etc. Cake Plant Protection, Fertiliser, Animal Fodder Fruits Food, Medicine, Oil Extraction Leaves Medicine, Cosmetics, etc. Part of the tree Usage Twigs Dental Hygiene Wood Firewood, Construction Material, etc. - 25 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Bark Toothpaste, Medicine Roots Medicine Neem is abundant in many developing countries with low technological possibilities and financial constraints hence, farmers in their countries have to rely on simple processing techniques. The requirements for the preparation of aqueous neem seed kernel (NSK) extracts are: - harvest or collect the fruits - remove pulp - dry seeds - grind seeds - mix aqueous extract - sieve aqueous extract - apply aqueous extract Following this procedure 10 to 20 kg neem seeds per hectare treated area are needed. The advantages of aqueous extracts are that every farmer can learn how to prepare the extract (no specialists knowledge is necessary) and that no expensive machinery is involved in the production. Q uite often the aqueous NSK extracts give satisfying results and the cost/benefit ratio seems acceptable but there are some disadvantages which have to be discussed. The above mentioned requirements for the production of NSK extracts are extremely labour intensive and the collecting or harvesting of the fruits takes place at a time where other crops have the highest demands. As a result the farmer can invest less time for the crops which are necessary for his income. Furthermore the proper treatment of the neem fruits and seeds requires in fact specialist knowledge. O therwise unwanted by-products like mycotoxins may be formed! The most important disadvantage is, that the aqueous extracts do not have a standardised content of active principles. This results in unknown quantities of active ingredients applied to the crop. This may lead to either an insufficient or an excess amount of a.i. for effective pest control. This in turn leads either to a failure of crop protection or suboptimal use of available a.i., which is in both cases a reason for increasing costs. In the worst case the farmer will lose confidence in biological pest control and return to synthetic pesticides. - 26 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil There are other possibilities for biological pest control i.e. “Ready To Use” neem products such as NeemAzal-T/S. This EC formulation is made from neem seeds of controlled quality and it is produced in a resource saving technical process where most of the input is recycled. The content of active principles is monitored per batch and adjusted in the EC formulation to a standard value. The concentration of mycotoxins is monitored and controlled to a level below the threshold declared safe for food (4 µg/kg German legislation). A large number of studies have been performed to determine the necessary amount of NeemAzal-T/S to be applied per hectare for pest control. Because of the reliable quality of NeemAzal-T/S and the standardized concentration of a.i. the farmer can apply NeemAzal-T/S equally exactly. There will be neither losses of crop due to inadequate amounts of a.i. nor financial losses due to excess amounts of a.i. applied. NeemAzal-T/S is an easy to use formulation: that means no additional labour is required and the farmer can concentrate on his value crops. Another advantage is the high storage stability for a minimum of 2 years without significant loss of a.i. NeemAzal-T/S can be used in a fashion similar to other conventional pesticides with the benefit of environmental safety. - 27 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Q UALITY CONTROL OF NEEM MATERIAL Beate Ruch Trifolio-M GmbH, Lahnau, Germany For pest control purposes the most interesting part of the Neem tree is neem seed material which comprises the insecticial ingredient Azadirachtin A. Furthermore there are other Azadirachtins (Azadirachtin B, Azadirachtin H, etc.) as well as Nimbines and Salannins. Depending on the quality there are 2 to 9 grams acitve ingredients per kilogram seed material. In this context quality control of neem material is mainly the analysis of active ingredients of the Neem seed kernels, Neemoil and Neem formulations. The analytical method is the high performance liquid chromatography – HPLC. Best results are obtained with a UV detection of 214 nm, columns filled with reversed phase C18 or C12 material, and eluents which consits of H 2O mixtures with CH 3 O H or CH 3CN. A HPLC chromatogram of available Neem standard solutions is presented in the following diagram. Standard solution mixture approx. 0,01 mg/ml each 100 DN mExt DS N S 50 AzA AzH AzB 0 5 10 15 20 Time (min) AzH: Azadirachtin H; AzB: Azadirachtin B; AzA: Azadirachtin A DN: Desacetyl-Nimbin; DS: Desacetyl-Salannin; N: Nimbin; S: Salannin The most important Neem ingredient is Azadirachtin A. It is the major compound for insect control for plant protection purposes as well as for control of human and animal ectoparasites. - 28 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil For comparison of analytical results the GTZ Pesicide Service Project (Germany) initiated a collaboration of Neem experts to develop a standard method for sample preparation and analysis. The results are published as CIPAC method no. 4042 “High performance liquid chromatographic method for the analysis of Azadirachtin in Neem kernels, Neemoil and formulated prodcuts”. Experimental aspects of this method: Generally the determination of Azadirachtin A in an analysis solution should be performed as soon as possible after sample preparation. If this is not possible the sample has to be stored in a refrigeratior with T < -15°C. Each sample has to be analysed in duplicate and at least two independent analysis solutions from each sample have to be prepared. Sample preparation Neemoil Approx. 250 mg of Neemoil plus 10 drops of Tween 85 are dissolved in a 50 ml flask with the chromatographic eluent HPLC analysis Neem formulation Approx. 50 mg of formulated product is dissolved in the HPLC eluent and filled up to the final volume of 100 ml HPLC analysis Neem kernels1) Weight loss of Neem kernels by drying A container with approx. 5 g kernels (shell has to be removed) has to be placed in an oven with 103 ± 2 °C for 17 ± 1 h. After drying the material has to be cooled down in a desiccator for 30-45 min. The rel. humidity in the lab should not exceed 70%. 2) Extraction of Azadirachtin A of Neem kernels Approx. 3 g kernels in 30 ml methanol have to be homogenised with a tissue mixer for 3 minutes. After filtration of the extract this procedure has to be repeated twice. The filtrates have to be combined in a 100 ml flask and filled up to the mark with methanol. An aliquot of this solution is diluted in HPLC eluent (1:10 v/v) HPLC analysis Calibration At least 5 standard solution should cover a concentration range of 1 to 50 µg/ml for setting up a calibration curve. For quantification of the Azadirachtin A content the mean response factors (Azadirachtin A content / peak area of the calibrant) or the regression parameters of the calibration curve (y = ax + b) have to be taken into account. - 29 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Calculation of the Azadirachtin A (AzA) content Neem kernels: AzASample = AzAdil * V * DF * 100 W * (100 − MC) AzASample AzA content of the sample [mg/g dry matter] AzAdil AzA content in the dilution [mg/ml] V total volume of extract DF dilution factor W fresh weight of the sample MC moisture content – mean weight loss on drying [%] The factor (100-MC) has to be neglected for calculations of Azadirachtin A in Neemoils and Neem formulations. - 30 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil NEEM AZAL-T/S – ESTIMATION OF RESIDUE DATA BASED ON THE ANALYTICS OF THE LEADING COMPOUND AZADIRACHTIN A Beate Ruch Trifolio-M GmbH, Sonnenstr. 22, D-35633 Lahnau, Germany Registration of a plant protective agent in Germany requires submission of reports for different subjects, e.g. toxicology, efficacy. Residue behaviour of plant protective agents is a very important aspect for registration purposes. Azadirachtin A is established as a leading compound for analytical purposes in the active ingredient NeemAzal and its formulation NeemAzal-T/S. Before starting with residue studies a lot of preliminary work has to be done. First a method for a special problem (i.e. analysis of Azadirachtin A in soil) has to be developed. This includes the extraction of Azadirachtin A from the matrix, purification of the extract and finally HPLC-analysis. This development is followed by the validation of the method where the limit of detection and quantification, the recovery rate, accuray and precision as well as specifity and selectivity have to be determined. Residue studies in water are important for the control of the concentrations of NeemAzal and NeemAzalT/S in tests of toxicological and ecotoxicological relevance. It is important to keep in mind, that the halflife-time of Azadirachtin A in water is dependent strongly on the pH. pH Temperature [°C] half-life time [d] 4 20 49,9 7 20 19,5 8 20 4,4 Method for residue analysis in water An extraction procedure is not necessary. The water samples have to be concentrated, dependent on the expected concentration of Azadirachtin A in the sample. It is strongly recommended to perform a solid phase extraction for the concentration, because the Azadirachtin A will degrade during evoparation of the water at higher temperatures. Residue analysis in soil is an important tool for monitoring and controlling of the concentrations of NeemAzal and NeemAzal-T/S in tests of ecotoxicological and environmental relevance. These tests are adsorption/desorption studies, leaching activity, degradation in soil and side effects on soil micro flora (earthworms). - 31 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Method for residue analysis in soil Approx. 50 g soil has to be extracted with 50 ml CH 3O H followed by filtration and washing. Afterwards a solid phase extraction for purification has to performed. It may be necessary to make use of different solid phase sorbents to obtain a sufficient purificated extract. Residue analysis in plant material is necessary for the evaluation of MRL values (Maximum Residue Levels) and ADI values (Acceptable Daily Intake) by the authorities. These values are important for the evaluation of waiting periods after the last treatment of plant material with NeemAzal-T/S. The aim of this work is protection of the consumer. Method for residue analysis in plant material First step is an extraction procedure where the plant material has to be homogenized thoroughly. The amount of plant material and the choice of the solvent is dependet on the plant matrix. At least two solid phase extraction are necessary (polar and nonpolar sorbents). Up to now it is not possible to present a general method for extraction and sample preparation of different plant materials. Results of residue studies have shown that there is a possiblily to divide plant material in two groups: - leafy vegetables (lettuce, spinach, etc.) - fruity vegetables and fruits (tomatoes, apples, etc.) A comparison of the decrease of Azadirachtin A on/in tomato leaves (representative for leafy vegetables) and tomatoes is presented in the following diagrams. ppm AzA 40,1 30,1 20,1 10,1 0,1 0 50 100 150 time [h] 200 250 Azadirachtin A on/in tomato leaves after treatment with the tenfold recommended concentration (5% NeemAzal-T/S ) - 32 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil 0,5 ppm AzA 0,4 0,3 0,2 Azadirachtin A on/in tomateo after treatment with the tenfold recommended concentration 0,1 0,0 0 50 100 time [h] 150 (5% NeemAzal-T/S ) Aside from the different half-life-times in tomato leaves and tomatoes the concentration of Azadirachtin A directly after treatment is different as well. O ther residue studies show the same tendency - the leafy vegetables show a higher initial concentration of Azadirachtin A (approx. 3 mg Azadirachtin A per kg) whereas the fruity vegetables and fruits show an initial concentration of approx. 0,1 mg Azadirachtin A per kg. Those results help us to propose waiting periods for the estimation of residues in plant material. O n basis of the “Diätverordnung” which is the strictest limit of the German authorities regarding residues in food (it demands that less than 0,01 mg residue per kg is existing) we have to wait for 8-9 days after treatment with the consum of leafy vegetabels and 9-12 days for fruity vegetables and fruits respectively. - 33 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil SEMIOCHEMICALS IN AGRICULTURE: THE U SE OF PHEROMONES AND ALELOCHEMICS IN PLANT SYSTEMIC RESISTANCE Geraldo Deffune Instituto de Ciêcias e Tecnologia do Ambiente - Universidade de Uberaba, Av. Nenê Sabino, 1801 - Bairro Universitário 38.055-500 Uberaba MG Brazil. e-mail: gdeffune@ig.com.br 1. Introduction: Chemical Mediators in the Organic Agricultural context Besides the undeniable knowledge and yield (per unit area) achievements of Agronomic Science in the 20th Century, its progresses have brought with them a paradoxical increase in both the incidence and number de species of pests e diseases on world crops (Altieri, 1995; Paschoal, 1994 & 1995). Parallel to this, a marked decrease on food quality was recorded, both due to losses of nutritional qualities as to food and environmental contamination by agrochemicals, generating the so-called ”agricologenic diseases”, i.e., primarily caused by agricultural practices and techniques (Deffune, 2000; Hodges & Scofield, 1983). The not-so-recent advances in the research on chemical mediators (Nordlund et al., 1981) of decisive importance on both intra and interspecific (e.g.; between plants, plant-microbe and plant-insect) interactions, show that while it is surely impossible to totally eradicate a micro-organism, worm or arthropod which happen to be adapted to favourable conditions in agricultural systems, it seems well achievable to develop strategies which can ”fool” or deviate these highly specialized and biologically programmed organisms (in relation to their respective food chains and webs) from crops which can be, on their turn, selected, strengthened and in a large extent immunized against economic levels of damage. In this case, a systemic strategy or holistic approach must especially combine the factors (among others) of soil fertility management with the chemical and dynamic mediation mechanisms between organisms in the agro-ecosystems, as suggested by the very founders of O rganic Agriculture (Steiner, 2000/1924; Howard, 1940a&b; Chaboussou, 1972, 1980 & 1985). 2. Plant Resistance Mechanisms The importance of acquired and induced resistance mechanisms in plants has been known for quite a long time, since the works of Chester (1933) and Gaumann (1946). The natural protection of plants against pathogenic agents and herbivores (e.g. insects, mites, molluscs) is given by several mechanisms. It is partly based on a variety of constitutional barriers already present in the plant tissues prior to any attack or infection. The combined effect of all these barriers is called Constitutional Resistance. Additionally, through stress or inoculation, plants can activate or be stimulated to produce a variety of biochemical protection mechanisms called Acquired or Induced Resistance Mechanisms. As these stimuli or signals can translocate or communicate from the primary stress or inoculation loci, toward reasonably distant tissues, promoting systemic defence reactions, the term ”Systemic” has been added both to Acquired and Induced Resistance (Sticher et al., 1997). These last two mechanisms, SAR and ISR, are distinguished from each other by the nature of either the induction method or the inducing agent (or elicitor) which provokes, or, to be more precise, evokes the reaction, as follows: 1. When the induction is unintentional and/or the elicitor is a pathogenic agent or parasite, the reaction is called Systemic Acquired Resistance (SAR). - 34 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil 2. When the induction is intentionally promoted and/or the elicitor is a beneficial, symbiotic or neutral agent (i.e., neither a pathogenic nor a parasitic organism), or it is abiotic (e.g. mineral, apneumonic, dead), the reaction is called Induced Systemic Resistance (ISR). Since both mechanisms depend on signal cascades of the biochemical or biodynamic type (for being properties of living, autopoietic or self-regulated organisms), which cannot be isolated from the plant’s general metabolism, it can be deduced that both SAR and ISR are linked to other induced metabolic adaptations in plants, which can effectively influence parameters like growth, biomass, reproduction, yield and quality (Abele, 1973; Spiess, 1978; Samaras, 1977; Margulis, 1995; Koepf, 1993 & Koepf et al., 1996). 3. Chemical Mediators, Applied Allelopathy and Metabolic-Dynamic Induction This induction of different metabolic adaptations in plants, animals and microbes is regulated by chemical mediators or ”Mediochemicals” which are classified according to their functional role (Nordlund et al., 1981; Deffune, 2000a), as shown in Figure 1. Apneumones can constitute a separate group of compounds, independent of allelochemics, which would include abiotic elicitors and biodynamic preparations (Nordlund, 1981; Deffune, 2000a). Class C H E M I C A L M E D I A T O R S Sub-Class Functional Category 1.1. Plant: Auxins, Cytokinins, Gibberelins, Abscisic Acid, etc 1. Hormones: 1.2. Animal: Ecdysone, Juvenile and others not present in humans Intra-individual 1.3. Human: Adrenalin, Insulin, Thyroxin, ADH, TSH, FSH, LH, etc chemical messengers (produced inside a single organism) 2.1.1. Sexual 2.1. Pheromones: 2.1.2. Alarm Intraspecific Interactions 2.1.3. Epideictic (spacing) 2. Semiochemicals: 2.1.4. Attractive, Aggregating, etc Inter-individual chemical messengers, 2.2. Allelochemics: 2.2.1. Allomones: active between Interspecific Between Beneficial for Emitter different organisms Interactions Living Detrimental for Receiver Organisms 2.2.2. Kairomones: Beneficial for Receiver Detrimental for Emitter 2.2.3. Synomones: Beneficial for Both Effects produced by Non-Living Substances on Organisms 2.2.4. Apneumones: Stimulatory or Inhibitory Figure 1. Schematic classification of Chemical Mediators. According to Rice (1984), the antagonistic allelopathic agents or Allomones (item 2.2.1 in Figure 1) functionally subdivide themselves in four classes: 3.1. Antibiotic: a chemical produced by a micro-organism and effective against another micro-organism. 3.2. Koline: chemical produced by a higher plant and effective against another higher plant. - 35 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil 3.3. Marasmine: a compound produced by a micro-organism, which is active against a higher plant, term proposed by Gaumann (1946). 3.4. Phytoncide: proposed by Waksman (1952) for an agent produced by a higher plant, which is effective against a micro-organism. Nevertheless the term can be extended to plant agents with detrimental effects on arthropods and herbivores in general. This classification sheds new light on the so-called allelopathic interactions in agroecosystems, offering new forms of crop management (e.g.; companion plants and weed suppressors) and the development of techniques which constitute the new face of Applied Allelopathy (Rice, 1995; Deffune, 2000). Phytoalexins, or plant chemical defence substances are today understood in the wider context of Plant Systemic Resistance and Semiochemicals (Deverall, 1977; Nordlund, 1981). According to the functional schematic classification of Semiochemicals (mediators chemical between different individual organisms), phytoalexins are specifically defined as Phytoncidal Allomones, i.e., Allelochemicals (interspecific semiochemicals) with beneficial effects for the emitting plants and detrimental for phytopathogenic microorganisms (Waksman, 1952; Rice, 1984; Deffune, 2000). Thus, natural pesticides like Neem (Azadirachta indica) extracts can be classified as insecticidal Phytoncides (Allomones) or even as Apneumones. From the knowledge of the forms of action of different chemical mediators, one can study the possible mechanisms and applications involved, which can be due to mass-action dependant biochemical reactions (in relation to the participating constituent elements), or to dynamic or energetic ”pulses” evoked by ultrahigh dilutions (Endler & Schulte, 1994). 4. Processes involved in ISR and SAR The respected French researcher Francis Chaboussou (1980 & 1985) ha already anticipated the ISR and SAR mechanisms as he suggested that conventionally cultivated plants were sick because agrochemicals (pesticides and fertilizers) would deviate secondary metabolic processes from the balanced production of defence structures and substances, generating an excess of free amino-acids and soluble nutrients (especially nitrogen). This unbalance would leave plant cells excessively turgid and weak, promoting the proliferation of pests and diseases. O n the other hand, Chaboussou also understood that the elimination of positive natural stress factors - for example, contact with a soil rich in organic matter and microbes, would leave plants less resistant to parasite attacks. Contrarily, he mentioned that traditional treatments like Bordeaux Mixture seemed to stimulate a natural reaction of the plant and he’s foreseen in this nonantagonistic approach of plant health a true ”agronomic revolution” (Chaboussou, 1980 & 1985). In this sense, research has accumulated evidences on the effectiveness of apneumonic substances containing eliciting agents for SAR (e.g., alkaloids, flavonoids, terpenoids, cumarins, sulphites, glucosídios, tannins, purines, organic fatty acids) frequently present in compost and organic manures in general, as well as in allelochemical and biodynamic preparations (Doubrava et al., 1988; Koepf, 1993). The ubiquitous jasmonates, for example, which act in very low concentrations both as allomones (i.e., antagonistic metabolites) against insects and inducers of SAR, are biosynthesized (probably through lipoxygenase action) from linolenic acid, which is present in the chloroplasts' thylakoids (the grana and stroma lamellae) of most plant species (Sticher et al., 1997; Salisbury & Ross, 1992). - 36 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Therefore, both the action and elicitation of phytoalexins are intimately related to wider SAR and ISR mechanisms, as shown in the review by Sticher et al. (1997) on their general effectiveness and on the biochemical pathways through which fatty acids (e.g.; arachidonic, linolenic, linoleic, oleic), peptides (systemin, elicitin), salicylates, jasmonates, ethylene and even electrical signals are produced and act either directly or as signalling mediators for SAR and ISR. Probably the most common signalling molecule for SAR is salicylic acid (SA), biosynthesized from the amino acid phenylalanine. SA is today recognized as a phytohormone, besides its well known antipyretic and analgesic effects in animals (Raskin, 1992). However, its most important roles in plant organisms seem to be as both an endogenous signal and an external elicitor of SAR and ISR through the induction of PR genes (PRs stands ffor ”pathogenesis related proteins”). Although these processes seem limited to temperatures under 32º C, Quarles (1996) reports that either aspirin or willow bark extracts (Salix alba, S. vitellina), not only promote resistance in garden plants but also attract useful predator mites for biological control.. However, there are different ISR and SAR elicitation pathways, independent from SA accumulation (Rice, 1984 & 1995; Sticher et al., 1997). 4. Abiotic and Biological Elicitors Elemental abiotic elicitores like Si, Cu, Ag, Hg (Rouxel et al., 1989 & 1991) and even and synthetic agents like polyacrylic acid also follow SA independent ISR pathways, as their effects still take place at temperatures above 32º C (Sticher et al., 1997). Sulphur is an essential part of many active volatile and allelopathic compounds like (Hengel & Kirkby, 1982): 1. Mustard oils, glucosides or glucosinolates, present mainly in members of the Cruciferae; e.g.; sinigrin (in Brassica nigra), gluconasturtin (in Nasturtium officinale), glucobarbarin (in Reseda luteola), glucosinalbin (in Sinapis alba), glucotropaeolin (in Tropaeolum majus, Tropaeolaceae). 2. Sulphoxides, e.g.; the lachrymatory factor in onion and the odour of garlic, which contains allicin - both a molluscicide and an insecticide (Singh et al., 1995). 3. Elicitins, which are small hydrophilic cystein-rich proteins. 4. Also for the production of ethylene from the amino acid methionine. An example of sulphur's importance in plant resistance processes is the effectiveness of metabolized elemental S, identified in resistant genotypes of cocoa (Theobroma cacao L.) to verticillium wilt (Verticillium dahliae Kleb.; Resende et al., 1996). Plant promoting rhizobacteria (PGPR) and vesicular-arbuscular mycorrhiza (VA) present in soil organic matter and plant communities also effectively promote ISR and are most probably the cause of observed crop protection using diversification and induced resistance in low-input cereal/legume cropping systems (Cooke, 1996; Sticher et al., 1997). - 37 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil Last but not least, the importance of Silicon (Si) in the plant systemic resistance context is being rediscovered (Epstein, 1994) – given that Silicon was already mentioned by Liebig (1842) in the very beginnings of Agricultural Chemistry and recommended in biodynamic treatments by Steiner (2000) in 1924; not only as an efficient resistance elicitor, but also as a general metabolic regulator and essential nutrient element for both plants and animals (Simpson & Volcani, 1981; Salisbury & Ross, 1992; El Behairy, 1994). 5. Allelodynamics: the A,B,C of Biodynamics. As the processes which elicit the biochemical signal cascades involved in ISR e SAR neither take into account nor are directed to the plant’s cosmic connections presupposed and aimed at by the BiologicalDynamic agricultural system, it was suggested that the observed effects in the activity of ultra-high dilutions in biological systems (Endler & Schulte, 1994) be defined as allelodynamic, i.e., dynamic effects of highly diluted allelopathic substances (Deffune, 2000a). Thus, Biological-Dynamic Agriculture - with its original concept of healthy internal relationships in ”agricultural organisms or individualities” (Scofield, 1986) and systematic application of apneumonic extracts on crops, was also the modern precursor of Applied Allelopathy - considering that the management of general allelopathic interactions was already known in times as early as Theophrastus' (ca. 374-287 BC), Aristotle's favourite disciple and successor (Keynes, 1929; Rice, 1984; Deffune, 1990 & 2000). Therefore, it is interesting to note that the old criticism and discredit by the agrochemical establishment toward concepts of ”natural control” for pests and diseases based on the stimulation of the plants’ natural resistance in the O rganic and Biodynamic systems, are largely due to a lack of information on the recent advances in the fields of ISR and SAR (Pio et al.,1984; Deffune et al.,1994; 1996; Langer, 1995; Cooke, 1996; Sticher et al., 1997). O n the other hand, the supposed efficacy (which has already been submitted to research without positive confirmation) of the so-called ”natural inputs” - somewhat complex organo-chemical recipes and microbial inoculi (e.g.; ”Agroplus ®”, ”Super-8”, ”Super-Magro”, ”EM”) recently popularised in the organic agricultural circles, may well be due to simple effects of either abiotic (e.g.; the sulphur and copper present in most of these recipes) or biological elicitors (organic metabolites and/or saprophytic microbes). These effects can in principle be obtained through simpler and cheaper management strategies and preparations, like Silica suspensions (ground quartz or Diatomaceous earth), mature Compost extracts, or 0,1% diluted lime-sulphur and Bordeaux mixtures. Similarly, part of the experimentally confirmed effects of Biodynamic Preparations, are also due to elicitation processes and signal cascades at the genetic, hormonal and biochemical levels (Raupp & König, 1996; Deffune 1981, 1990, 1999 e 2000). Referencias Bibliográficas 1. Abele, U. (1973) ”Vergleichende Untersuchungen zum konventionellen und biologish-dynamischen Pflanzenbau unter besonderer Berücksichtigung von Saatzeit und Entitäten”. PhD Thesis, Univ. of Gießen, pp. 1-119. - 38 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil 2. Altieri, M. A. (1995) Agroecology: the science of sustainable agriculture; pp. 179-199 (Organic Farming) and pp. 367-379 (Toward sustainable agriculture). Westview/IT Publications, London. 3. Bailey, J.A. and Mansfield, J.W.; Eds. (1982) Phytoalexins, pp. 1-16, 253-282, 289-312 & 319-322. Blackie, London. 4. Chaboussou, F. (1972) ”La Trophobiose et la protection de la Plante”. In Revue des Questions Scientifiques 143, pp. 27-47 & 175-208; Bruxelles. 5. Chaboussou, F. (1980) Les Plantes Malades des Pesticides - Bases nouvelles d’une prévention contre maladies et parasites; pp. 22-75. Éditions Debard, Paris. 6. Chaboussou, F. (1985) Santé des cultures, une revolution agronomique. Ed. Flammarion, Paris; pp. 192. 7. Chester, K.S. (1933) "The problem of acquired physiological immunity in plants". In Q. Rev. Biol. 8, pp. 275-324. 8. Cooke, B.M. (1996) ”Crop Protection Using Diversification and Induced Resistance in Low-Input Cereal/Legume Cropping Systems” Monograph of the Dept. of Environmental Resource Management. EC CAMAR. 9. Deffune, G. (1981) Agricultura Ecológica II: Deter as Pragas e Conservar o Solo, dois grandes desafios (Ecological Agriculture II: Pest Control and Soil Conservation, two great challenges) in Planeta August 1981, n o 107, pp. 19-24. Edit. Três Ltda, SP, Brazil. 10. Deffune, G. (1990) Effects of humic acids and three bio-dynamic preparations on the growth of wheat seedlings. MSc Thesis, 34 pages (including 6 figures in appendix), Wye College, University of London. Deffune, G. (1993) Adubo de Resíduos Orgânicos - como fazer e usar Compostagem. Vídeo técnico VHS, Agrodata Vídeos, Curitiba-Pr, Brazil. 11. Deffune, G. (1998) 1o Relatório Técnico de Consultadoria em Agricultura Biológica. Projecto Piloto na Serra da Peneda – Desenvolvimento Rural Sustentável. ADERE-Peneda-Gerês, Arcos de Valdevez, Portugal. 12. Deffune, G.; Packter, T.; Konzen, R.W. and Fröhlich, G. (1992a) Como Produzir Hortaliças sem Agrotóxicos - Um Exemplo de Agricultura Sustentável - Parte 1. Vídeo técnico VHS, Agrodata Vídeos, Curitiba-Pr, Brazil. 13. Deffune, G.; Packter, T.; Konzen, R.W. and Fröhlich, G. (1992b) Como Produzir Hortaliças sem Agrotóxicos - Parte 2. VHS Agrodata Vídeos, Curitiba-Pr, Brazil. 14. Deffune, G.; Šimunek, P.; Scofield, A.M.; Lee, H.C. and López, L. 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(1981) ”Semiochemicals: a Review of the Terminology”. In Semiochemicals - their role in pest control (Nordlund, Jones & Lewis, Eds.), pp. 13-28. John Wiley & Sons, N.York. 38. Paschoal, A.D. (1994) Produção Orgânica de Alimentos – agricultura sustentável para os séculos XX e XXI. PCLQ, ESALQ-USP, Piracicaba-SP, 191 pgs. 39. Paschoal, A.D. (1995) ”Modêlos Sustentáveis de Agricultura”. In Agricultura Sustentável 2:1, pp. 11-16. CNPMA-EMBRAPA (Braz. Agric. Res. Enterprise), Brasil. 40. Pio D.M.; Hoffman, M.A. & Deffune, G. (1984) "Neue Biologisch-Dynamische Entwicklung in Brasilien" in Lebendige Erde 6, 269-274. 41. Quarles, W. (1996) ”Protect your garden with aspirin and salicylate”. In Common Sense Pest Control XII (2): 16-19. Box 7414, Berkeley, CA 94707. 42. Raskin, I. (1992) ”Salicylate, new plant hormone”. In Plant Physiol. 99: 799-803. - 41 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil 43. Raupp, J. and König, U.J. (1996) ”Biodynamic preparations cause opposite yield effects depending upon yield levels”. In Biological Agriculture & Horticulture 13: pp. 175-188. AB Acad. Publs., UK. 44. Resende, M.L.V.; Flood, J.; Ramsden, J.D.; Rowan, M.G.; Beale, M.H. and Cooper, R.M. (1996) ”Novel phytoalexins including elemental sulphur in the resistance of cocoa (Theobroma cacao L.) to Verticillium wilt (Verticillium dahliae Kleb.)”. In Phys. and Mol. Plant Path. 48: pp.347-359. 45. Rice, E.L. (1984) Allelopathy. Academic Press, Orlando, 422 pp. 46. Rice, E.L. (1995) Biological Control of Weeds and Plant Diseases: Advances in Applied Allelopathy. Univ. of Oklahoma Press, USA; 439 pp. 47. Rouxel, T.; Kollmann, A.; Boulidard, L. and Mithen, R. (1991) ”Abiotic elicitation of indole phytoalexins and resistance to Leptosphaeria maculans within Brassiceae”. In Planta 184, pp. 271-278. 48. Rouxel, T.; Sarniguet, A.; Kollmann, A. and Bousquet, J.F. (1989) ”Accumulation of a phytoalexin in Brassica spp in relation to a hypersensitive reaction to Leptosphaeria maculans”. In Phys. and Mol. Plant Path. 34: 507-517. 49. Salisbury, F.B. and Ross, C.W. (1992) Plant Physiology. pp. 3-135, 207-213, 329-441, 464-485, 531548, 601-619. Wadsworth, Belmont, CA; 682 pages. 50. Samaras, I. (1977) Das Nachernteverhalten unterschiedlich gedüngter Gemüsearten mit besonderer Berücksichtigung physiologischer und mikrobiologischer Parameter. PhD Thesis, Univ. of Gieβen, pp. 4198. 51. Scofield, A.M. (1986) "Editorial: Organic Farming - the Origin of the Name”. In Biological Agriculture & Horticulture 4, pp.1-5. AB Academic Publishers, UK. 52. Simpson, T.L. and Volcani, B.E.; Eds. (1981) Silicon and siliceous structures in biological systems. Springer-Verlag New York; pp. 3-12 (Introduction), 555 pages. 53. Spiess, H. (1978) Konventionelle und biologisch-dynamische Verfahren zur Steigerung der Bodenfruchtbarkeit. PhD Thesis, University of Gieβen, Germany; pp. 1-112. 54. Steiner, R. (2000) Fundamentos da agricultura biodinâmica – vida nova para a terra (curso de oito conferências de 7 a 16 Junho de 1924) 2 a edição traduzida por G. Bannwart, pp. 28-41 & 169-183. Ed. Antroposófica, 235 pp. 55. Sticher, L.; Mauch-Mani, B. and Métraux, J.P. (1997) ”Systemic Acquired Resistance”. In Annu. Rev. Phytopathol. 35, pp. 235-70; Annual Reviews Inc. 56. Waksman, S.A. (1952) Soil Microbiology. John Wiley & Sons; pp. 5-152. - 42 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil POSSIBILITIES OF THE U SE OF PHEROMONES FOR PEST CONTROL IN BRAZIL Armin Kratt & Edmund Hummel Trifolio-M GmbH, Sonnenstr. 22, 35633 Lahnau, Germany e-mail: info@trifolio-m.de; www.trifolio-m.de What are pheromones? Pheromones are chemical substances that are used in the animal world to transmit information from one individual to another. Mainly insects, first of all moths but also beetles are using this transmission to communicate. Amongst the different types of pheromones, like aggregation, alarm and tracing pheromones, the sex pheromones play the most important role in the field of plant protection. Sex pheromones are released by female insects signalling their readiness for copulation and allowing the males to track them down. The pheromones, which are carried by the motion of air over great distances, are detected by the antennae of the males. These antennae are very sensitive instruments and in some cases able to recognize even single molecules. What are they good for? Pheromones can be synthesized synthetically and are used in pheromone traps as bait. The most important part of the trap is the so called dispenser which contains a small amount of the attractant. It mimics a female insect, misleading and attracting the males to be caught in the trap. Pheromones are basically species specific and the males are attracted by the substances that are released by their own species only. Therefor traps can be built, attracting the target moth or beetle only. Monitoring The most common use of pheromone traps. A few traps are placed in the area of interest. By counting the number of insects caught over a certain period, information about the presence of a insect pest, its flight activity and the pest population density is easily available (flight curve). Based on this information appropriate measures can be taken. Certified Dispensers ensure, that the results obtained during one season can be compared with those of the next year. They are produced from the same batch of pheromone and are continuously tested in the laboratory and in the field. Advantage Information about the flight activity of a particular pest is easily available. No time consuming examination of the plants is necessary and no expert knowledge is needed. Appropriate proceeding is possible according to the outcome of the count. - 43 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil No catches of the target pest are observed: It is most likely that no damage from this pest has to be expected. As a consequence no pesticide treatment is necessary. This saves work and money and keeps your plantation free of nature burdening pesticides. Target insects are caught in the traps: This means some damage has to be considered caused by the larvae hatching from the eggs. In consequence control measures are necessary. The flight curves indicate the optimum time period for pesticide application. Spraying throughout the whole season can be avoided. Mass trapping For several pests like bark beetles or palm weevils the usage of a greater number of traps has proven to be effective. Especially in areas where the target insect population is low the number of insects can be reduced markedly. Thus mass trapping can be sufficient to reduce the damage, they or their breed respectively may cause, to an acceptable level. Advantage: Plant protection without the need of a pesticide and without polluting the environment. Due to the specificity of pheromones no other harmless or beneficial insects are killed. Attract & kill All methods mentioned above are using any kind of trap to keep the once attracted insects inside. Attract & kill works different. Using pheromones the target insect is lured to a place where it gets into contact with a poison (widely used is Permethrine). Males contacting poisoned area die within hours. Thus, reproduction is reduced. Mating disruption An additional and in its effect different way of using pheromones for plant protection. Many dispensers releasing a large amount of pheromone are placed all over the area that has to be protected (150 to 1000 dispensers per hectare). A high and permanent concentration of pheromone is generated which prevents the meeting between individuals of the opposite sex by masking the natural pheromone. Insects are not able to locate their females which remain not impregnated and cannot reproduce themselves. Using this technique vineyards and orchards are protected successfully from damage caused by grape berry moth, grapevine moth and codling moth without using any pesticide. Mating disruption of Pink Bollworm in cotton is successfully used in Egypt. The method is used and tested against a variety of other insects. - 44 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil POTENTIAL OF U SE, PRODUCTION OF EXTRACTS AND D EVELOPMENT OF PRODUCTS OF NEEM BASED M EDICAMENTS AND U SES IN AGRICULTURE Mauro Luiz Begnini Instituto de Ciências Biológicas e da Saúde - Universidade de Uberaba, Av. Nenê Sabino, 1801 - Bairro Universitário 38.055500 Uberaba MG Brazil.m_begnini@yahoo.com In the medicinal chemistry, original products of plants increase in the therapeutics, creating a growing trend for in research of new pharmaceuticals and active substances with medicinal properties. In this context the plant Nim Azadirachta indica A. Juss was introduced in Brazil in 1986 in the Agronomic Institute of Paraná (IAPAR), it has been waking up the interest of Researchers in Brazil, in studying its use as natural insecticide for the control of ectoparasites. Nim is a plant of Asian origin belonging to the family of the meliáceas, natural of Burma and of the arid areas of the Indian sub-continent, being considered a plant with important insecticidal properties. It is very resistant and of fast growth and reaches usually 10 to 15 m of height and depending on the soil type can reach up to 25 meters in height. Its red, lasting and resistant wood is a succedaneum of the mogno, with the advantage of faster growth. O f the tree Neem, or " tree of the life " as it is known in India all parts are used. Seeds, fruits, bark and roots are used as raw material in the production of insecticides, fungicides, carrapaticides and fertilizers in the combat of dangerous plagues. Medicinal products are also produced, such as vermifuge, healing, antibacterial and further on products for personal hygiene, as soap, shampoo, dental cream. For the use as a botanical insecticides several aspects should be taken into consideration: extraction, conservation of the extracts, efficient dose, stability, toxicity, cost. All these aspects are understood when the main substances contained in this insecticide is identified. Recently a work was developed in the University of Uberaba, through the comparative study of the physical-chemical properties of the oil of the seeds of Nim of different areas. Three samples of oil of the seeds of Nim were studied comparatively. Those oil samples were obtained through an exhaustive extraction in an extractor of the type soxhlet using n-hexane as extraction solvent. After the extraction of the oil of the seeds, the solvent was evaporated and they were used in comparative studies with samples of oil of different areas. The Nim oil samples came from the Dominican Republic, another from Fortaleza (Ceará-Brazil) and another from the National Center of Researches of Rice and Bean - EMBRAPA (Goiás-Brazil). Some comparative analyses of these oils were accomplished, such as determination of the peroxide index, determination of the iodine index and of the saponification index. That study of the physical-chemical - 45 - Practice Oriented Results on Use and Production of Plant Extracts and Pheromones in Integrated and Biological Pest Control; 1. workshop: March 29. – 30. 2001; Uberaba, Brazil analyses of the oil of the seeds of the nim cultivated in different areas is fundamental to demonstrate some differences which may exist in the chemical composition of the oils of these seeds. Through the analysis of the results, it was possible to observe that the samples of the oil of the Nim seeds of different places presented the same results basically in relation to the peroxides indexes and of iodine. In relation to the peroxide index, it was observed that the oils of the nim seeds present a very small index, demonstrating an additional stability when compared to a sample of sunflower, Heliantus annus oil in similar conditions. However, for the saponification index for the three samples, was observed that the oil sample originating from the Dominican Republic presented an index of saponification superior to the anothers. In that way, some differences in the constitution of the oils, of quantitative or qualitative greatness were observed. That allows to say, that the composition of the active substances of the plant depends on the place of cultivation of the same, possessing different concentrations of the active ingredients between one place and another. - 46 -

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