Allelochemicals Biologica... - Name

Allelochemicals: Biological Control of Plant Pathogens and Diseases

Disease Management of Fruits and Vegetables 

VOLUME 2 

Series Editor: 

K.G. Mukerji, University of Delhi, Delhi, India

Allelochemicals: Biological 

Control of Plant 

Pathogens and Diseases 

Edited by 

INDERJIT 

University of Delhi, India 

and 

K.G. MUKERJI 

University of Delhi, India

A C.I.P. Catalogue record for this book is available from the Library of Congress. 

ISBN-10 1-4020-4445-3 (HB) 

ISBN-13 978-1-4020-4445-8 (HB) 

ISBN-10 1-4020-4447-X (e-book) 

ISBN-13 978-1-4020-4447-2 (e-book) 

Published by Springer, 

P.O. Box 17, 3300 AA Dordrecht, The Netherlands. 

www.springer.com 

Printed on acid-free paper 

Cover photo: 

Bio Control of powdery mildew pathogen Phyllactinia dalbergae on 

Dalbergia sisoo by hyperparasite Cladosporium spongiosum. 

(Microphotograph taken by Prof. K.G. Mukerji and Mr. S.K. Das) 

All Rights Reserved 

© 2006 Springer 

No part of this work may be reproduced, stored in a retrieval system, or transmitted 

in any form or by any means, electronic, mechanical, photocopying, microfilming, 

recording or otherwise, without written permission from the Publisher, with the 

exception of any material supplied specifically for the purpose of being entered 

and executed on a computer system, for exclusive use by the purchaser of the work. 

Printed in the Netherlands.

CONTENT 

Preface 

1. Discovery and Evaluation of Natural Product-based 

Fungicides for Disease Control of Small Fruits 

David E. Wedge and Barbara J. Smith 

1 

2. Allelochemicals as Biopesticides for Management of 

Plant-parasitic Nematodes 

Nancy Kokalis-Burelle and Rodrigo Rodríguez-Kábana 

15 

3. Allelopathic Organisms and Molecules: Promising 

Bioregulators for the Control of Plant Diseases, 

Weeds, and Other Pests 

Ana Luisa Anaya 

31 

4. The Impact of Pathogens on Plant Interference 

and Allelopathy 

Scott W. Mattner 

79 

5. Allelopathy for Weed Control in Aquatic and 

Wetland Systems 

Ramanathan Kathiresan, Clifford H. Koger , Krishna N. Reddy 

103 

6. Bacterial Root Zone Communities, Beneficial Allelopathies 

and Plant Disease Control 

Antony V. Sturz 

123 

7. The Role of Allelopathic Bacteria in Weed Management 

Robert J. Kremer 

143 

8. The Allelopathic Potential of Ginsenosides 

Mark A. Bernards, Lina F. Yousef, Robert W. Nicol 

157 

9. Antimicrobial and Nematicidal Substances from the 

Root of Chicory (Cichorium intybus) 

Hiroyuki Nishimura and Atsushi Satoh 

177 

10. Disease Resistance in Plants through Mycorrhizal 

Fungi Induced Allelochemicals 

Ren-sen Zeng 

181 

11. Allelochemicals from Ageratum conyzoides L. and 

Oryza sativa L. and their Effects on Related Pathogens 

Chuihua Kong 

193 

Index 207 

List of Contributors 213 

v 

vii

Preface 

Biological control of plant diseases and plant pathogens is of great significance in 

forestry and agriculture. There is great incentive to discover biologically active natural 

products from higher plants that are better than synthetic agrochemicals and are much 

safer, from health and environmental considerations. The development of natural 

products as herbicides, fungicides, and their role in biological control of plant disease 

promises to reduce environmental and health hazards. Allelopathic techniques offer 

real promise in solving several problems linked with biological control of plant pests. 

The allelopathic effect of plants on microorganisms, and microorganisms on 

microorganisms is of great environmental and economic significance. This book is 

organized around the premise that allelochemicals can be employed for biological 

control of plant pathogens and plant diseases. Specifically, this volume focuses on (i) 

discovery and development of natural product based fungicides for agriculture, (ii) 

direct use of allelochemicals as well as indirect effects through cover crops and organic 

amendments for plant parasitic pest control and (iii) application of allelopathy in the 

pest management. 

In an effort to address above points, contributing authors provided up-to-date 

reviews and discussion on allelochemicals-related biological control of plant diseases 

and pathogens. Chapters 1 - 3 discuss discovery and development of allelochmicals 

and their role in the management of plant diseases. Chapter 4 discusses the effects of 

pathogens on the competitiveness and allelopathic ability of their hosts. Chapter 5 

highlights the importance of allelopathy for weed control in aquatic ecosystems. 

Chapters 6-7 deal with bacterial potential in weed management and plant disease 

control. Chapter 8 describes the role of organic compound ginsenosides from 

rhizosphere soil and root exudates of american ginseng plant in control of fungal 

diseases. Antimicrobial and nematicidal substances from the rhizome of chicory has 

been discussed in Chapter 9. The role of allelochemicals induced in mycorrhizal 

plants in imparting disease resistance is given in Chapter 10. The last chapter discusses 

the biocontrol of plant pathogens and diseases by allelochmicals from Ageratum 

conyzoides a weed and rice plants has been highlighted in Chapter 11. 

We are grateful to all authors for providing their valuable work to this volume. 

The articles are original and some have been written for the first time in any book. We 

are indebted to the following referees for their constructive comments and suggestions: 

Ana L. Anaya, Mark Bernards, Nancy Kokalis Burelle, Chester L. Foy, John M. 

Halbrendt, Robert Kremer, Azim Mallik, Susan Meyer, Reid J. Smeda, Tony Sturz, 

David Wedge and Jeff Weidenhamer. The editorial help of Ineke Ravesloot, Publishing 

Department, Springer is sincerely appreciated. It is our hope that this book will serve 

scientific community well, and equally hope that the book will stimulate young students 

to work on biological control of plant pathogens and diseases through natural 

allelochemicals. 

Inderjit and K.G. Mukerji 

October 2005 

vii

DAVID E. WEDGE 1 and BARBARA J. SMITH 2 

DISCOVERY AND EVALUATION OF NATURAL 

PRODUCT-BASED FUNGICIDES FOR DISEASE 

CONTROL OF SMALL FRUITS 

1 

United States Department of Agriculture, Agricultural Research Service, 

Natural Products Utilization Research Unit, The Thad Cochran National 

Center for Natural Products Research, University of Mississippi, University, 

MS 38677, USA; and 2 

Small Fruit Research Station, 306 S. 

High St., Poplarville, MS 39470, USA 

Email : dwedge@olemiss.edu 

Abstract. The continuing development of fungicide resistance in plant and human pathogens necessitates the 

discovery and development of new fungicides. Discovery and evaluation of natural product fungicides is largely 

dependent upon the availability of miniaturized antifungal bioassays. Essentials for natural product bioassays 

include sensitivity to microgram quantities, selectivity to determine optimum target pathogens, and adaptability 

to complex mixtures. Experimental accuracy and precision must be stable between assays over time. These 

assays should be relevant to potential pathogen target sites in the natural infection process of the host and 

applicable to the agrochemical industry. Bioassays should take advantage of current high-throughput technology 

available to evaluate dose-response relationships, commercial fungicides standards, modes of action, and structure 

activity studies. The focus of this chapter is the evaluation of natural product based fungicides for agriculture 

and we will provide a review of bioautography prescreens and microtiter assays (secondary assays). Also presented 

is more detailed information on newer techniques such as the detached leaf assays for evaluating fungicides 

against strawberry anthracnose (Colletotrichum spp.) and field plot trials for gray mold (Botrytis) and anthracnose 

control in strawberry. 

1. INTRODUCTION 

Since the early 1970s, agriculture worldwide has struggled with the evolution of 

pathogen resistance to disease control agents. Increased necessity for repeated chemical 

applications, development of pesticide cross-resistance, and disease resistance management 

strategies have characterized the use of agricultural chemicals to-date. As a 

consequence, producers are currently attempting to control agricultural pests with a 

decreasing arsenal of effective crop protection chemicals. In addition, the desire for 

safer pesticides with less environmental impact has become a major public concern. 

Particularly desirable is the discovery of novel pesticidal agents from new chemical 

classes that are able to operate using different modes of action and, consequently, 

against plant pathogens with resistance to currently used chemistries. In this regard, 

evaluating natural products and extracts as a source of new pesticides is one strategy 

for the discovery of new chemical moieties that have not previously been created by 

synthetic chemists. 

Inderjit and K.G. Mukerji (eds.), 

Allelochemicals: Biological Control of Plant Pathogens and Diseases, 1–14. 

© 2006 Springer. Printed in the Netherlands. 

1

2 

DAVID E. WEDGE 

AND BARBARA J. SMITH 

Antibiotics, antineoplastics, herbicides, and insecticides often originate from plant 

and microbial chemical defense mechanisms (Wedge and Camper, 2000). Secondary 

metabolites, once considered unimportant products, are now thought to mediate chemical 

defense mechanisms by providing chemical barriers against animal and microbial 

predators (Agrios, 1997; Wedge and Camper, 2000). Plants produce numerous chemicals 

for defense and communication, and can elicit their own form of offensive chemical 

warfare by targeting the proliferation of pathogens. These chemicals may have 

general or specific activity against key target sites in bacteria, fungi, and viruses. 

Exploiting the chemical warfare that occurs between plants and their pathogens shows 

promise in providing new natural products for new anti-infectives for human, plant 

and animal health. The successful development of strobilurin fungicides and spinosad 

insecticides has continued the interest in natural products as crop protectants. The 

importance and future of natural product agrochemistry is emphasized by the fact 

that 21 companies have filed 255 patent applications primarily for use of the strobilurin 

class of fungicides (Qo I MET complex 3 inhibitors). 

1.1. Direct Acting Defense Chemicals 

Since the discovery of the vinca alkaloids in 1963, many of the known antitubulin 

agents used in today’s cancer chemotherapy arsenal are products of plant and fungal 

secondary metabolism. Since 1991, 16 of 43 new pharmaceuticals were derived from 

natural products. In certain therapeutic areas 78% of the antibacterials and 74% of 

the anticancer compounds are natural products or have been derived from natural 

products (Roughi, 2003). These “natural products” are probably defense chemicals 

that target and inhibit cell division in invading pathogens (Wedge and Camper, 2000). 

Therefore, it is reasonable to hypothesize that plants and certain fungi can produce 

chemicals, such as resveratrol and strobilurin, that act directly in their defense by 

inhibiting pathogen proliferation, or indirectly by disrupting chemical signal processes 

related to growth and development of pathogens or herbivores (Wedge and 

Camper 2000). 

1.2. Indirect Acting Defense Chemicals 

Plant resistance to pathogens is considered to be systemically induced by some 

endogenous signal molecule produced at the infection site that is then translocated to 

other parts of the plant (Oku, 1994). The search for, and identification of, the putative 

signal molecule(s) is of great interest to many plant scientists because such moieties 

have possible uses as “natural product” disease control agents. However, research 

indicates that no single compound is involved, but rather a complex signal transduction 

pathway, which, in plants, can be mediated by a number of compounds that appears to 

influence octadecanoid metabolism. In response to wounding or pathogen attack, 

fatty acids of the jasmonate cascade are formed from membrane-bound α-linolenic 

acid by lipoxygenase-mediated peroxidation (Vick and Zimmerman, 1984). Analogous 

to the prostaglandin cascade in mammals, α-linolenic acid is thought to participate

NATURAL PRODUCT BASED FUNGICIDES 3 

in a lipid-based signaling system where jasmonates induce the synthesis of a family of 

wound-inducible defensive proteinase inhibitors (Farmer and Ryan, 1992) and low 

and high molecular weight phytoalexins such as flavonoids, alkaloids, and terpenoids 

(Gundlach et al., 1992; Mueller et al., 1993). 

Several plant and bacterial natural products have novel applications as plant 

protectants through the induction of systemic acquired resistance (SAR) processes. 

Commercial products that appear to induce SAR include Messenger® (EDEN 

Biosciences, Inc., Bothell, WA) and the bioprotectant fungicides Serenade® 

(AgraQuest, Davis, CA), Sonata® (AgraQuest, Davis, CA), and Milsana® (KHH 

BioSci, Inc., Raleigh, NC). Messenger is a harpin protein which switches on natural 

plant defenses in response to bacterial leaf spot, wilt, and blight and fungal diseases 

such as Botrytis brunch rot, and powdery mildew. Serenade is a microbial-protectant 

derived from Bacillus subtilis, with SAR activity that controls Botrytis, powdery and 

downy mildews, early blight, and bacterial spot. Sonata is also a microbial-biopesticide 

with activity against Botrytis, downy and powdery mildews, rusts, Sclerotinia blight, 

and rots. Milsana® is an extract from Reynoutria sachalinensis (giant knotweed) 

that induces phytoalexins able to confer resistance to powdery mildew and other 

diseases such as by Botrytis. However, elicitors with no innate antifungal activity 

will not appear active in most current screening high throughput screening systems. 

Many experimental approaches have been used to screen compounds and extracts 

from plants and microorganisms in order to discover new antifungal compounds. 

The focus of this paper is on laboratory methods and field procedures that we use to 

evaluate naturally occurring antifungal compounds produced by plants, pathogens, 

and other terrestrial and marine organisms. As part of a program to discover and 

develop naturally occurring fungicides, several new in vitro detection systems and a 

detached leaf assay were developed to evaluate small amounts of compound. Our 

fungicide field protocol used to test potential lead fungicides using commercial 

strawberry is also presented. 

2.1. Pathogen Production 

2. MATERIAL AND METHODS 

Isolates of Colletotrichum acutatum, C. fragariae and C. gloeosporioides, Phomopsis 

viticola and P. obscurans, Botrytis cinerea and Fusarium oxysporum are maintained 

on silica gel at 4-6 °C. The three Colletotrichum species and P. obscurans strain were 

isolated from strawberry (Fragaria x ananassa Duchesne). Phomopsis viticola and 

B. cinerea were isolated from commercial grape (Vitis vinifera L.) and F. oxysporum 

from orchid (Cynoches sp.). Fungal cultures were grown on potato-dextrose agar 

(PDA, Difco, Detroit, MI) in 9-cm petri dishes and incubated in a growth chamber at 

24 ± 2 °C under cool-white fluorescent lights (55 ± 5 µmols/m 2 /sec) with a 12h photoperiod.

4 

Conidia were harvested from 7-10 day-old cultures by flooding plates with 5 mL 

of sterile distilled water and softly brushing the colonies with an L-shaped glass rod. 

Aqueous conidial suspensions are filtered through sterile Miracloth (Calbiochem- 

Novabiochem Corp., La Jolla CA) to remove hyphae. Conidial concentrations were 

determined photometrically (Espinel-Ingrof and Kekering, 1991; Wedge and Kuhajek, 

1988) from a standard curve based on the absorbance at 625 nm, and suspensions are 

then adjusted with sterile distilled water to a concentration of 1.0x10 6 conidia/mL. 

Standard conidial concentrations are determined from a standard curve for each 

fungal species. Standard turbidity curves were periodically validated using both a 

Bechman/Coulter Z1 (Fullerton, CA) particle counter and hemocytometer counts. 

Conidial and mycelial growth are evaluated using a Packard Spectra Count 

(PerkinElmer Life and Analytical Sciences, Inc., Boston, MA). Conidial growth and 

germ tube development were evaluated using an Olympus IX 70 (Olympus Industrial 

America, Inc., Melville, New York) inverted microscope and recorded with a Olympus 

DP12 digital camera as appropriate for compounds that affect spore germination and 

early germ tube development. 

2.2. Direct Bioautography 



A number of bioautography techniques were used as primary screening systems to 

detect antifungal compounds. Matrix, one-dimensional, and two-dimensional bioautography 

protocols on silica gel thin layer chromatography (TLC) plates and 

Colletotrichum spp. as the test organisms were used to identify the antifungal activity 

according to published methods (Homan and Fuchs, 1970; Moore, 1996; Wedge and 

Nagle, 2000). Matrix bioautography is used to screen large numbers of crude extract 

at 20mg/mL. One-dimensional TLC (1D-TLC) and two-dimensional TLC (2D-TLC) 

are subsequently used to separate and identify the number of antifungal agents in 

extract. Modification of these procedures can be used to visually evaluate natural 

chemical defenses in disease resistant and susceptible plant cultivars (Vincent et al. 

1999). 

A 2D-TLC direct bioautography method was used to evaluate active crude or 

partially purified extracts. This protocol utilizes two sequential TLC runs in which 

the TLC plates are developed once with a polar solvent, turned 90 o , and then developed 

a second time with a non-polar solvent system (Wedge and Nagle, 2000). The 

method takes advantage of the resolving power of 2D-TLC to separate chemically 

diverse mixtures found in crude extracts. Two-dimensional TLC bioautography is 

well suited for resolving extracts containing lipophilic natural products that are difficult 

to separate by single elution TLC. 

Each plate was subsequently sprayed with a spore suspension (10 5 spores/mL) of 

the test fungus and incubated in a moisture chamber for 3 days at 26 ºC with a 12h 

photoperiod. Clear zones of fungal growth inhibition on the TLC plate indicate the 

Mention of trade names or commercial products in this article is solely for the purpose of providing specific 

information and does not imply recommendation or endorsement by the U. S. Department of Agriculture.


presence of antifungal constituents in each extract. Inhibition of fungal growth was 

evaluated 3-4 days after treatment. Antifungal metabolites can be readily located on 

the plates by visually observing clear zones where the active compounds inhibit fungal 

growth (Tellez, 2000,Vincent et al., 1999). The 2-D TLC method eliminates the 

need for the development of large numbers of plates in multiple solvent systems, 

reduces the amount of waste solvents for disposal, and substantially reduces the time 

required to identify active compounds. 

2.3. 96-Well Microbiassay 

The quick discovery and evaluation of natural product fungicides is heavily dependent 

upon miniaturized antifungal bioassay techniques. A reference method [M27- 

A from the National Committee for Clinical Laboratory Standards (NCCLS)] for 

broth-dilution antifungal susceptibility testing of yeast was adapted for evaluation of 

antifungal compounds against sporulating filamentous fungi (Wedge and Kuhajek, 

1998). 

This standardized 96-well microtiter plate assay was developed for the detection 

of natural product fungicidal agents, and can also be used to evaluate purified antifungal 

agents. In our study a 96-well microtiter assay was used to determine sensitivity of C. 

acutatum, C. fragariae, C. gloeosporioides, F. oxysporum, B. cinerea, P. obscurans, 

and P. viticola to the various antifungal agents in comparison with the commercial 

fungicides. Fungicides such as benomyl, azoxystrobin and captan with different 

modes of action were used as standards in these assays. Each fungal species was 

challenged in a dose-response format so that in the final test, compound concentrations 

of 0.3, 3.0, and 30.0 µM were achieved (in duplicate) in the different columns of the 

96-well plates. 

Fungal growth was evaluated by measuring absorbance of each well at 620 nm at 

0, 24, 48, and 72 hr, with the exception of tests with P. obscurans and P. viticola, 

where the data are recorded for up to 120 hr. Treatments are repeated so that mean 

absorbance values and standard errors can be calculated and are used to evaluate 

fungal growth. Differences in spore germination and mycelial growth in each of the 

wells in the 96-well plate demonstrate sensitivity to particular concentrations of 

compound and can indicate fungistatic or fungicidal effects. The microtiter assay 

can also be used to compare the sensitivity of fungal plant pathogens to natural and 

synthetic compounds with industry standards (Wedge et al., 2001). 

A novel application of the microbioassay was also developed for the discovery of 

compounds that inhibit Phytophthora spp. This protocol used the 96-well format for 

high-throughput capability and a standardized method for quantification of initial 

zoospore concentrations for maximum reproducibility. Zoospore suspensions were 

quantifiable between 0.7 and 1.5 × 10 5 zoospores/mL at an absorbance value of 620 

nm. Subsequent growth of mycelia was monitored by measuring absorbance (620 

nm) at 24-hour intervals for 96 hr. Full- and half-strength preparations of each of 

three media (V8 broth, Roswell Park Memorial Institute mycological broth, and mineral 

salts medium), and four zoospore concentrations (10, 100, 1000, and 10,000

6 



zoospores/mL) were evaluated. Both full- and half-strength Roswell Park Memorial 

Institute mycological broth were identified as suitable synthetic media for growing P. 

nicotianae, and 1000 zoospores/mL was established as the optimum initial concentration 

(Kuhajek et al., 2003). 

2.4. Detached Leaf Assay for Fungicide Evaluations In Planta 

Anthracnose susceptible ‘Chandler’ strawberry plants were grown in 10 x 10 cm 

plastic pots in a 1:1 (v/v) mixture of Jiffy-Mix (JPA, West Chicago, IL), and pasteurized 

sand in the greenhouse for a minimum of six weeks before inoculation. The 

plants were grown under standard conditions of a 16-hr day length and 24 o C temperature. 

Growth parameters are varied as needed to accommodate the needs of particular 

studies. 

Whole leaves (petiole and leaflets) were cut from plants no more than four hr 

before treatment or inoculation. Only the second or third youngest leaf on a plant was 

used for the fungicide assay, and only leaves with no visible signs of injury or symptoms 

of disease were collected for testing. Immediately after collection, the leaves 

were placed in a tray lined with moist paper towels and the tray is closed to retained 

near 100% RH and maintained at a cool temperature (~ 12 °C). To test for protective 

fungicide activity, treatment compounds were applied to the upper surface of each of 

the three leaflets on a leaf using a chromatography sprayer. After treatment, the base 

of each leaf stem was inserted into sterile distilled water in a 100 x 10 mm tissue 

culture tube. Each upper surface of each treated leaflet was inoculated with conidia 

from the test fungal isolate within 24 hr of treatment. Inoculated leaves were subsequently 

incubated in a dew chamber for 48 hour at 100% RH, 30 o C and then maintained 

at 25 o C in a moist chamber at 100% RH for 10 days. Sterile distilled water 

was added to each tube as needed to maintain the surface of the water above the base 

of the petiole. If a compound was to be tested for curative activity, the leaflets were 

inoculated 24 hr before the fungicidal compound is applied. 

Experimental compounds were evaluated in a dose-response format. Azoxystrobin 

or other fungicides that have both protective and curative activity were used as a 

standard, and a solvent control was used in every study. The number of disease 

lesions per leaf was used to determine the ability of the test compounds to prevent 

infections. The size of the lesions was used to determine the curative activity of 

compounds. Each fungicide concentration is replicated four times and the experiment 

is repeated at least once. This bioassay does not differentiate between direct 

effects on the fungus and indirect effects through induction of plant defenses. However, 

if a compound is much more active in this in vivo assay than than the in vitro 

microtiter assay, ‘induction’ activity is indicated. 

2.5. 2002-03 Experimental Field Studies at Hammond, Louisiana 

Strawberry plots were established and maintained following standard horticulture 

practices used by commercial strawberry farmers in Lousisana. The following fungi-


cides were applied to strawberries: fenhexamid (Elevate®, Arvesta, San Francisco, 

CA) at 1.6 kg/H, fenhexamid + captan (CaptEvate®, Arvesta, San Francisco, CA) at 

a low rate (3.9 kg/H) and a high rate (5.82 kg/H), azoxystrobin (Quadris®, Syngenta 

Crop Protection, Greensboro, NC) at 0.56 kg/H, cyprodinil + fludioxonil (Switch®, 

Syngenta Crop Protection, Greensboro, NC) at 0.8 kg/H, captan (Micro Flo Company, 

Memphis, TN) at 3.4 kg/H, mylclobutanil (Nova®, Dow AgroSciences, Indianapolis, 

IN) at 0.28 kg/H, pyrimethanil (Scala®, Bayer CropScience, Research Triangle 

Park, NC) at 1.9 kg/H, fenhexamid at 1.68 kg/H + captan at 3.4 kg/H, 

pyraclostrobin (Cabrio®, BASF, Research Triangle Park, NC) at 0.20 kg/H, 

pyraclostrobin + boscalid (Pristine, BASF, Research Triangle Park, NC) at 0.62 kg/ 

H, boscalid (Emerald®, BASF, Research Triangle Park, NC) at 0..59 kg/H, and the 

experimental fungicide, CAY-1 at 0.37 and 0.74 kg/H. Fungicide treatments were 

applied every 7-10 days (or as soon after rainfall as possible) starting at or near fullbloom 

stage and continuing until 1 week before harvesting ceased. 

Berries were harvested twice a week throughout the entire season beginning on 

January 30 and continuing until April 24 in 2003. Total fruit count and weights were 

obtained for both marketable and cull fruits, along with average weight per berry and 

percentages of marketable, physical cull, and diseased cull fruit per acre. Fruit were 

separated into marketable and cull classes, with the culls being further divided into 

physical (deformed or small fruit) culls and diseased culls. Diseases were identified 

and counted on three occasions including berries with gray mold (B. cinerea), anthracnose 

(C. acutatum), leather rot (Phytophthora cactorum), stem end rot (Gnomonia 

comari), and other rots that occurred during the picking season. Plants were rated on 

April 24, 2003 for incidence of foliar disease, number of dead plants due to anthracnose, 

crown rot and plant vigor. Diseases were rated on a scale of 0-5; 0 plants 

showed no leaf symptoms and 5 plants were completely defoliated. Plant vigor was 

rated on a scale of 0-5; 0 were dead plants and 5 were extremely vigorous plants. 

3. RESULTS AND DISCUSSION 

Results from the bioautography studies suggest that chemically different populations 

of microorganisms and plants can easily be distinguished by their characteristic “2D- 

TLC fingerprint” and antifungal zone patterns (Wedge and Nagle, 2000). Thus, this 

system represents a very useful technique for the identification and selection of unique 

chemotypes from microbial isolates (Nagle and Paul, 1999) and plant extract(?) 

collections. 

Chromatographic properties such as relative polarity, UV absorbance, chemical 

reactivity associated with each active metabolite provide valuable information that 

allows for rapid dereplication of known or nuisance compounds. When strains of 

different phytopathogenic fungi with dissimilar fungicide resistance profiles are 

inoculated onto replicate bioautography plates prepared from any given extract 

containing active metabolites, it is possible to visually observe distinct differences in 

the sensitivity of each fungal pathogen to single metabolites. These differences in 

pathogen sensitivity (fungicide resistance) can be observed by direct comparison of

8 



inhibition zone dimensions produced by active metabolites and control standards 

against each pathogenic strain tested. Chemical profiles provide valuable information 

for the rapid selection of specific antifungal metabolites with unique activity against 

fungicide-resistant pathogens and identify new compounds with novel mechanisms 

of action. 

Using modifications of the methods described previously, bioautography 

techniques were successful in allowing us to track the path of naturally occurring 

fungitoxic compounds present in strawberry leaves. Our studies indicated that 

concentrations of fungitoxic compounds vary between anthracnose resistant and 

susceptible cultivars and are present in different amounts in vegetative tissues of 

different ages. Using leaves of the anthracnose-susceptible cultivar Chandler and the 

anthracnose-resistant cultivar Sweet Charlie, we isolated and demonstrated the presence 

of three antifungal compounds. While the mechanism of strawberry anthracnose 

resistance is unknown, results from this study indicate that anthracnose resistance in 

strawberry may depend on the concentration of two constitutive antifungal compounds 

and the elicitation of a third compound in younger leaves. 

These two constitutive antifungal compounds were exhibited in both ‘Chandler’ 

and ‘Sweet Charlie’ plants but ‘Sweet Charlie’ plants produced approximately 15 

times more antifungal activity than ‘Chandler’ plants. Fungal growth inhibition 

associated with extracts from ‘Chandler’ plants appeared to be temporary. A third 

compound, detected exclusively in ‘Sweet Charlie’ plants, was produced only after 

young leaves were sprayed with a commercially available elicitor of antifungal compounds 

(Vincent et al., 1999). 

The antifungal activity of 32 naturally occurring quinones of four major classes: 

1,4-naphthoquinones, 1,2-naphthoquinones, 1,4-benzoquinones, anthraquinones, and 

other miscellaneous compounds from our natural products collection were tested for 

antifungal activity using bioautography. Bioautography allowed for the rapid evaluation 

of quinones which demonstrated good to moderate antifungal activity against 

Colletotrichum spp. Colletotrichum fragariae appeared to be the most sensitive species 

to quinone-based chemistry, C. gloeosporioides of intermediate sensitivity, and C. 

acutatum was the least sensitive species to these naturally occurring compounds 

(Meazza et al., 2003). 

Bioassay-directed isolation of antifungal compounds from an ethyl acetate extract 

of Ruta graveolens leaves yielded two furanocoumarins, one quinoline alkaloid, and 

four quinolone alkaloids, including a novel compound, 1-methyl-2-[6'-(3'’,4'’methylenedioxyphenyl)hexyl]-4-quinolone. 

Antifungal activities of the isolated 

compounds, together with 7-hydroxycoumarin, 4-hydroxycoumarin, and 7methoxycoumarin 

which are known to occur in Rutaceae species, were evaluated 

using bioautography and microbioassay procedures. Four of the alkaloids had moderate 

activity against Colletotrichum species, including a benomyl-resistant C. acutatum. 

These compounds and the furanocoumarins 5- and 8-methoxypsoralen had moderate 

activity against Fusarium oxysporum. The novel quinolone alkaloid was highly active 

against Botrytis cinerea. Phomopsis species were much more sensitive to most of the


compounds tested, with P. viticola being highly sensitive to all of the compounds 

screened (Oliva et al., 2003). 

Hexane and EtOAc phases of MeOH extract of Macaranga monandra demonstrated 

fungal growth inhibition in C. acutatum, C. fragariae, C. gloeosporioides, F. 

oxysporum, B. cinerea, Phomopsis obscurans and P. viticola. Bioassay-guided fractionation 

resulted in the isolation of two active clerodane-type diterpenes that were 

elucidated by spectral methods as kolavenic acid and 2-oxo-kolavenic acid. The 96well 

microbioassay revealed that kolavenic acid and 2-oxo-kolavenic acid produced 

moderate growth inhibition in P. viticola and B. cinerea (Salah et al., 2003). 

The application of the microbioassay to Phytophthora nicotianae was effectively 

used to determine EC 50 values (i.e., effective concentration for 50% growth reduction) 

for eight commercial antifungal compounds (azoxystrobin, fosetyl-aluminum, 

etridiazole, metalaxyl, pentachloronitrobenzene, pimaricin, and propamocarb). These 

EC 50 values were compared to those obtained using conventional plate methods by 

measuring linear growth of mycelia on fungicide-amended medium. The microbioassay 

proved to be a rapid, reproducible, and efficient method for testing the efficacy of 

compounds against P. nicotianae and should be effective for other species of 

Phytophthora as well. The assay requires relatively small amounts of a test compound 

and was suitable for the evaluation of natural product samples (Kuhajek et al., 

2003). 

CAY-1 is a fungicidal steroidal saponin (Mol wt. 1243) isolated and identified 

by DeLucca et al. (2002) from the ground fruit of cayenne pepper (Capsicum 

frutescens). CAY-1 was lethal to germinating conidia of Aspergillus flavus, A. 

fumigatus, A. parasiticus and A. niger. It was also active against agricultural and 

medicinally important fungi and yeast. In vitro dose-response assays with CAY-1 

against plant pathogenic fungi showed that 3.0 µM inhibited growth of C. 

gloeosporioides and C. acutatum by 100% and C. fragariae, P. obscurans, and P. 

viticola by 90%. Sampangine and several alkaloid analogs were isolated from the 

root bark of Cleistopholis patens. These novel broad-spectrum compounds showed 

promising antifungal activity against several serious pathogenic fungi of plants including 

B. cinerea, C. fragariae, C. acutatum, C. gloeosporioides, and F. oxysporium 

(Wedge and Nagle, 2003). 

Detached leaf assays provide us with the opportunity to evaluate new fungicides 

directly on the leaf surface in a dose-response format (Table 1). This assay allowed us 

to benchmark potential lead compounds such as CAY-1 and sampangine with a commercial 

standard (azoxystrobin) of known mode of action (Q o I inhibitor). The number 

of diseased lesions was used to determine effective concentrations needed for 

disease control. Lesion size is used to determine the relative effectiveness of the systemic 

activity that produced curative activity 24 hrs after inoculation. The detached 

leaf assay was also used to establish experimental field rates for future studies. Study 

of ‘protectant’ activity indicated that 1250 ppm. CAY-1 or sampangine appeared to 

be an effective concentration for disease control of anthracnose on the leaf surface, or 

between 100-1000 times the concentration required for in vitro activity (Post, Table 1).

10 



Table 1. Anthracnose disease severity and phytotoxicity (Phyto) scores of detached 

leaves following inoculation with Colletotrichum fragariae isolate CF-75, pre and 

post treatment with commercial and experimental fungicides. 

Azoxystrobin CAY-1 Sampangine 

Level Disease 1 Phyto 1 Disease Phyto Disease Phyto 

Fungicide applied to the upper leaf surface. Leaves not inoculated. 

None 0.29 a 2 0.04 A 0.29 a 0.04 a 0.29 a 0.04 a 

Solvent 0.29 a 0.15 A 0.29 a 0.15 a 0.29 a 0.15 a 

625 0.17 a 0.00 A 0.10 a 0.00 a 0.08 a 0.08 a 

1250 0.19 a 0.08 A 0.21 a 0.04 a 0.13 a 0.04 a 

2500 0.08 a 0.04 A 0.21 a 0.00 a 0.17 a 0.08 a 

lsd 0.34 0.20 0.32 0.16 0.30 0.19 

Pr>F 0.70 0.67 0.75 0.52 0.50 0.80 

Fungicide applied to the upper leaf surface 24 hr before inoculation (Post). 

None 0.75 a 0.33 A 0.75 ab 0.33 a 0.75 a 0.33 a 

Solvent 0.83 a 0.00 B 0.83 a 0.00 b 0.83 a 0.00 b 

625 0.42 ab 0.08 B 0.33 bc 0.00 b 0.25 b 0.00 b 

1250 0.08 b 0.17 ab 0.21 c 0.00 b 0.17 b 0.00 b 

2500 0.42 ab 0.00 b 0.21 c 0.08 b 0.29 b 0.00 b 

lsd 0.54 0.23 0.42 0.17 0.41 0.17 

Pr>F 0.06 0.04 0.02 0.00 0.01 0.00 

Fungicide applied to the upper leaf surface 24 hr after inoculation (Pre). 

None 0.58 a 0.08 b 0.58 abc 0.08 b 0.58 a 0.08 b 

Solvent 0.25 a 0.33 a 0.25 c 0.33 ab 0.25 a 0.33 ab 

625 0.33 a 0.08 b 0.92 ab 0.58 a 0.75 a 0.08 b 

1250 0.83 a 0.08 b 1.00 a 0.25 ab 0.75 a 0.08 b 

2500 0.67 a 0.08 b 0.83 ab 0.08 b 0.58 a 0.50 a 

lsd 0.60 0.23 0.47 0.57 0.36 

Pr>F 0.27 0.14 0.04 0.28 0.36 0.08 

Fungicide applied to the lower leaf surface (translaminar) 24 hr before inoculation. 

625 0.33 a 0.04 a . . . . 0.13 a 0.08 a 

1250 0.08 a 0.04 a . . . . 0.25 a 0.08 a 

2500 0.17 a 0.00 a . . . . 0.29 a 0.08 a 

lsd 0.37 0.09 0.31 0.26 

Pr>F 0.34 0.51 0.46 0.63 

Fungicide applied to the lower leaf surface (translaminar) 24 hr after inoculation. 

625 0.33 a 0.00 a . . . . 0.38 a 0.17 a 

1250 1.21 a 0.25 a . . . . 0.58 a 0.17 a 

2500 0.58 a 0.17 a . . . . 0.67 a 0.17 a 

lsd 1.09 0.31 0.71 0.43 

Pr>F 0.22 0.23 0.64 1.00 

1Disease scored on scale of 0 = no lesions to 3 = severe; phytotoxicity scored on scale of 0 – 5 where, 0 = no 

signs of damage 5 = severe tissue damage. 

2Average values followed by the same letter were not significantly different at the 0.05 level using Least 

Significant Difference at P=0.05.


No systemic or translaminar activity was detected when azoxystrobin, CAY-1, or 

sampangine was applied 24 hrs after inoculation with C. fragariae (Pre, Table 1). 

Field studies indicated that significant differences occurred in marketable yield 

of strawberries as a result of fungicide treatments. The highest marketable yield (17,071 

kg/H) was recorded from plots receiving Pristine fungicide at the rate of 0.62 kg ai/H. 

The next highest yield (16,960 kg/H) was harvested from plants treated with Switch 

fungicide at the rate of 0.8 kg/H. Plants from the untreated control plots yielded more 

fruit than plants from four of the fungicide-treated plots. Plants treated with CAY-1 

produced the lowest yield of cull fruit weighing 1,390 kg/H. compared with the highest 

yield of cull fruit weighing 5,816 kg/H produced on the plots treated with Pristine. 

CAY-1 produced high yields of diseased fruit weighing over 9,400 kg/H. Five fungicide 

treatments resulted in the production of marketable fruit at 60%: Switch® (69.8%), 

Pristine (68.51%), Cabrio® (65.5%), and the two CaptEvate® treatments (5.8 kg. 

and 3.9 kg, 65.8% and 62.7% marketable fruit, respectively). Most treatments that 

were combinations of two fungicides produced the highest marketable yields and 

lowest disease percentage. 

The total number of berries with fruit rot symptoms and the number of berries 

with symptoms of anthracnose or stem end rot from the April 24 harvest time were 

significantly lower from plots treated with the fungicides Switch, Cabrio®, 

CaptEvate®, and Pristine® than from those receiving no fungicide treatment (Table 

2). The most prevalent diseases in the Louisiana field study were anthracnose caused 

Table 2. Fruit rot and field data from April 24, 2003 harvest of fungicide treated 

strawberry plots, Hammond, Louisiana. 

Fungicide Total Rots1 Anthracnose2 Stem end rot2 Plants Dead (%) 3 Foliar disease4 Switch 1.5 f 5 0.8 e 0.0 d 16.3 a 2.8 a 

Cabrio 5.8 f 3.8 e 0.5 d 8.8 cde 2.6 ab 

CaptEvate (High) 6.0 f 5.0 e 0.5 d 5.0 e 1.1 e 

Pristine 7.0 ef 5.0 e 0.3 d 5.0 e 2.5 abc 

CAY-1 (Half) 22.0 def 18.0 de 2.0 cd 15.6 ab 2.8 a 

Captan 27.8 cdef 21.3 cde 3.0 cd 7.5 cde 2.0 bcd 

Quadris 28.3 cdef 20.3 cde 2.8 cd 8.1 cde 1.9 cd 

CaptEvate (Low) 29.0 cdef 23.3 bcde 2.5 cd 7.5 cde 1.8 de 

Captan+Elevate 43.0 cdef 28.8 bcde 7.0 bcd 12.5 abc 2.0 bcd 

CAY-1 (Full* 50.5 bcde 37.8 bcd 7.5 bcd 10.6 abcde 3.1 a 

Scala 59.0 bcd 47.8 bcd 7.0 bcd 9.4 bcde 1.9 cd 

Control 63.5 bcd 45.3 bcd 9.8 abc 5.6 de 2.8 a 

Emerald 70.5 abc 50.3 Bc 7.8 abcd 5.6 de 2.9 a 

Nova 87.8 ab 54.5 Ab 16.0 a 7.5 cde 2.0 bcd 

Elevate 109.5a 84.5 A 14.3 ab 11.9 abcd 2.6 ab 

LSD (0.05) 44.1 31.6 8.4 6.8 0.7 

1Total number of fruit/20 plant plot with any disease symptom. 

2Number of fruit with anthracnose fruit rot symptomn (Colletotrichum acutatum) or Stem-End Rot 

(Gnomonia comari). 

3Percentage of plants/20 plant plot dead from anthracnose crown rot. 

4Foliar disease scored on scale of 0 – 5, where 0 = no disease and 5 = plant defoliated due to foliar disease. 

5Average values followed by the same letter were not significantly different at the 0.05 level using a Least 

Significant Difference.

12 



by the fungus C. acutatum and stem end rot caused by the fungus Gnomonia comari. 

Stem end rot lesions were often invaded by secondary pathogens such as Botrytis and 

Colletotrichum. The combination fungicides such as Elevate® + Captan®, Pristine®, 

and Switch® were effective in controlling this disease complex. The untreated control 

plants and those treated with Emerald® had the most berries affected with stem 

end rot. The incidence of gray mold and anthracnose fruit rot was extremely low. 

Gray mold was controlled by the fungicides Scala®, Elevate® + Captan®, Pristine®, 

Switch®, Emerald®, and Elevate®. 

4. CONCLUSIONS 

Information gained from the examination of thousands of extracts and their associated 

pure compounds have culminated in the development of a variety of standardized 

operating protocols for natural product discovery that are currently being used in our 

laboratory. Successful discovery, evaluation, and development of natural product 

fungicides are totally dependent upon the availability of high quality miniaturized 

antifungal bioassays. Bioassay-directed screening of compounds and extracts is the 

initial step in the discovery process for new agrochemicals and pesticides. 

Standardization of inoculum allows for meaningful comparison of growth 

inhibition between different fungal pathogens, test compounds, and experiments 

repeated in time. Bioautography provides a simple technique to visually follow 

antifungal components through the separation process. The 96-well microbioassay 

allows for the evaluation of microgram quantities, determination of dose-response 

relationships, and comparison of antifungal activity with fungicides with a known 

mode of action. Coupling bioautography techniques with the 96-well microbioassay 

provides us with a discovery protocol that combines the simple and visual nature of 

direct bioautography with the rapid, sensitive, and high throughput capabilities of a 

microtiter system. 

The 96-well microbioassay is accurate and sensitive; as little as 0.1 µM amounts 

of test compound permit discrimination between germination and mycelial growth 

inhibitors and identification of fungicide resistant pathogens. The microbioassay 

utilizes a chemically defined liquid medium with a zwitterion buffer that limits chemical 

interaction with test compounds and controls for pH variations. This new standardized 

method provides high-throughput capability and the capacity to study chemical 

compounds in detail, to perform mode of action studies, and to determine fungicide 

resistance profiles for specific fungal pathogens. 

Detached leaf assays are critical for establishing ‘real world’ activity prior to the 

field testing that agrochemical companies require before investing millions of dollars 

needed to develop a agrochemical. Subsequent efficacy testing in the greenhouse 

ultimately helps determine the potential usefulness of compounds as pest control agents. 

To maximize the detection of natural products, high-throughput bioassay techniques 

must target significant agricultural pests, include relevant commercial pesticide 

standards, and adhere to sound statistical principles.


While the use of SAR inducers is still in its infancy, these compounds will may 

be of use in specialized applications. However, our observations are such that the 

host plant must have a suitable ‘metabolic engine’ - found in many resistant cultivars 

- that is capable of being ‘revved up.’ Susceptible cultivars most often used in fungicide 

evaluations either produce a lower quantity of defense compound and or have a 

longer time lag between pathogen infection and plant symptom development than is 

the case in resistant cultivars. Systemic acquired resistance inducers have been marketed 

(Actigard, Messenger) with limited success and have never recouped their development 

costs. It thus appears unlikely that economically viable SAR products will 

be developed in the near future. 

Even so, allelochemicals and other natural products represent potentially rich 

and new sources of agrochemical plant protectants. The identification of suitable 

natural products coupled with traditional synthesis chemistry should be an effective 

approach for optimizing pesiticide activity and associated chemical properties. New 

requirements for fungicides are stringent, and require that new products must be 

environmentally safe and efficacious at low rates. In addition they must posses low 

mammalian toxicity, operate using novel modes of action, and have a low to moderate 

risk of developing resistance in target pathogens. 

5. REFERENCES 

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Espinel-Ingrof, A., Kerkering, T. M. Spectrophotometric method of inoculum preparation for the in vitro 

susceptibility testing of filamentous fungi. Journal of Clinic Microbiology 1991; 29:393-394. 

De Lucca, A. J., Bland, J. M.,Vigo, C. B., Cushion, M., Selitrennikoff, C. P., Peter, J., Walsh, T. J. CAY-1, a 

fungicidal saponin from Capsicum species. Med Mycol 2002; 40:131-137. 

Farmer, E. E., Ryan, C. A. Octadecanoid precursors of jasmonic acid activate the synthesis of wound-inducible 

proteinase inhibitors. Plant Cell 1992; 4:129-134. 

Gundlach, H., Muller, M. J., Kutchan, T. M., Zenk, M. H. Jasmonic acid is a signal transducer in elicitorinduced 

plant cell cultures. Proc Natl Acad Sci USA 1992; 89:2389-2393. 

Homans, A. L., Fuchs, A. Direct bioautography on thin-layer chromatograms as a method for detecting fungitoxic 

substances. J Chromatogr 1970; 5(2):327-3299. 

Kuhajek, J. M., Jeffers, S. N., Slattery, M., Wedge, D. E. A Rapid Microbioassay for Discovery of Novel Fungicides 

for Phytophthora spp. Phytopathology 2003; 93:46-53. 

Nagle, D. G., Paul, V. J. Production of secondary metabolites by filamentous tropical marine cyanobacteria: 

ecological functions of the compounds. J Phycol 1999; 35 (Suppl. 607):1412-1421. 

Meazza, G., Dayan, F. E., Wedge, D. E. Activity of activity quinones on Colletotrichum spp. J Agric Food 

Chem 2003; 51:3824-3828. 

Moore, R. E. Cyclic peptides and depsipeptides from cyanobacteria: a review. J Ind Microbiol. 1996; 16:134- 

143. 

Mueller, M. J., Brodschelm, W., Spannagl, E., Zenk, M. H. Signaling in the elicitation process is mediated 

through the octadecanoid pathway leading to jasmonic acid. Proc Natl Acad Sci USA 1993; 90:7490- 

7494. 

Oku, H. In: Plant Pathogenesis and Disease Control. Lewis Publishers. Boca Raton, FL 1994; p.64. 

Oliva, A., Meepagala, K. M. Wedge, D. E. Hale, A. L., Harries, D., Aliotta, G., Duke, S.O. Natural Fungicides 

from Ruta graveolens L. leaves, including a new quinolone alkaloid. J Agric Food Chem 2003; 51: 890- 

896 

Pezzuto, J. M. Cancer chemopreventative agents: from plant materials to clinical intervention trials. In: A. D. 

Kinghorn, M. F. Balandrin (eds): Human Medicinal Agents from Plants. ACS Symposium Series No. 

534, American Chemical Society, Washington DC, 1993; 205-215.

14 



Roughi, A. M. Rediscovering natural products. Chemical and Engineering News. October 13, 2003; p. 77-91. 

Salah, A. M., Bedir, E., Ngeh, T. J., Kahn, I. A., Wedge, D.E. Antifungal clerodane diterpenes from Macaranga 

monandra (L.) Muell. Et Arg. (Euphorbiaceae). J Agric Food Chem 2003; 51:7607-7610. 

Tellez, M.R., Dayan, F.E., Schrader, K.K., Wedge, D.E., Duke, S.O. Composition and some biological activities 

of the essential oil of Callicarpa americana L. J Agric Food Chem 2000; 48:3008-3012. 

Vick, B. A., Zimmerman, D. C. Biosynthesis of jasmonic acid by several plant species. Plant Physiol 1984; 75: 

458-461. 

Vincent, A., Dayan, F.E., Maas, J.L., Wedge, D.E. Detection and isolation of antifungal compounds in strawberry 

inhibitory to Colletotrichum fragariae. Adv Strawberry Res 1999; 18:28-36. 

Wedge, D. E., Kuhajek, J. M. A microbioassay for fungicide discovery. SAAS Bull Biochem Biotech 1998; 11: 

1-7. 

Wedge, D. E., Camper, N. D. Connections between agrochemicals and pharmaceuticals. In: Biologically Active 

Natural Products: Agrochemicals and Pharmaceuticals. Cutler, H.G., Cutler, S. J. eds, ACS Symposium 

Series, American Chemical Society, Washington DC, 2000 . 

Wedge. D. E., Nagle, D.G. A new 2D-TLC bioautography method for the discovery of novel antifungal agents 

to control plant pathogens. J Nat Prod 2000; 63:1050-1054. 

Wedge, David E.; Nagle, Dale G. Preparation of sampangine and its analogs as fungicides. U.S. Pat. Appl. 

Publ. 12 pp. CODEN: USXXCO US 2004192721 A1 20040930 CAN 141:273006 AN 2004:803936, 

2004. 

Wedge, D.E., Curry, K.J., Boudreaux, J.E., Pace, P.F., Smith, B.J. In vitro sensitivity and resistance profiles of 

Botrytis cinerea isolates from Louisiana strawberry farms. Proceedings of the 5 th 

North American 

Strawberry Conference, Strawberry Research to 2001; p78-81.

ALLELOCHEMICALS AS BIOPESTICIDES FOR 

MANAGEMENT OF PLANT-PARASITIC 

NEMATODES 

1 

NANCY KOKALIS-BURELLE 1 and RODRIGO 

RODRÍGUEZ-KÁBANA 2 

Research Ecologist, USDA, Agricultural Research Service, U.S. 

Horticultural Research Lab, Fort Pierce, FL, 34945, USA, 2 

Distinguished 

University Professor, Auburn University, Auburn, AL, 36849, USA. 

Email:NBurelle@ushrl.ars.usda.gov 

Abstract. Many allelopathic compounds in their native or processed forms have potential for development as 

viable components of plant-parasitic nematode management strategies. Allelochemicals have been identified 

that possess differing levels of activity against a wide range of plant-parasitic nematodes. In general, these 

compounds are less toxic to nontarget species, and less persistent in soil than chemical nematicides. Operative 

mechanisms for plant-parasitic nematode control with allelopathic compounds include nematicidal activity, 

nematostatic activity, and nematode behavior modification. Allelochemicals are sometimes produced in large 

quantities in plant material or as agricultural waste, making the use of rotation crops, cover crops, and organic 

amendments effective means for production and/or distribution of the active compounds. A greater understanding 

of the effects of soil microbes and environmental conditions on allelopathic compounds is necessary to improve 

their efficacy for control of parasitic nematodes. Use of allelochemicals for nematode control will require that 

growers know specifically what types and population levels of nematodes are present in their production fields. 

Development of improved production and incorporation methods for rotation and green manure crops, and 

appropriate application methods for processed allelochemical compounds, will also enhance the efficacy and 

consistency of these compounds for nematode control. 


For the purpose of this chapter, allelochemicals will be defined as plant metabolites or 

their products that are released into the environment through volatilization, exudation 

from roots, leaching from plants or plant residues, and decomposition of residues 

(Waller, 1985; Putnam and Tang, 1986; Einhellig,1995; Halbrendt, 1996). As such, 

allelochemicals are classified as biopesticides, which are defined as being derived 

from natural materials such as plants and microorganisms and include both substances 

that control pests (biochemical pesticides) and microorganisms that control pests 

(microbial pesticides). Biochemical and microbial pesticides are considered inherently 

less toxic than conventional pesticides and generally affect only the target pest and 

closely related organisms, in contrast to broad spectrum, conventional pesticides that 

may affect organisms as different as birds, insects, and mammals. Often biopesticides 

are effective in small quantities and decompose quickly, resulting in lower exposure 

and fewer pollution problems than conventional pesticides. Also, when used as a 


Allelochemicals: Biological Control of Plant Pathogens and Diseases, 15– 

29. 


15

16 

NANCY KOKALIS-BURELLE AND RODRÍGO RODRIGUEZ-KÁBANA component of Integrated Pest Management (IPM) programs, biopesticides can decrease 

the use of conventional chemical pesticides (Halbrendt, 1996). 

Increased restrictions on use and phase-out of chemical fumigants such as methyl 

bromide and other chemical nematicides for control of plant-parasitic nematodes make 

the discovery of target-specific, environmentally safe, naturally occurring biopesticide 

compounds that suppress nematode populations or modify nematode behavior 

increasingly important. For instance, allelochemicals that affect nematode chemotaxis 

could be invaluable in many different scenarios for nematode control and represents 

an area of research with both great needs for identification of active compounds, and 

great possibilities for their use. The production of chemical cues by plants and 

behavioral responses to these cues by nematodes are critical to successful host location 

and reproduction in nematodes, which are highly dependent on these chemotactic 

stimuli during many stages of their life cycles (Bridge, 1996; Huettel, 1986; Yasuhira 

et al., 1982). 

In order to make effective use of nonchemical or biochemical pest control strategies 

such as allelochemicals for suppression of plant-parasitic nematodes, users need to be 

familiar with the types and quantity of nematodes present in their soil and determine 

the feasibility of growing a cover or rotation crop, using an organic amendment, or a 

formulated biopesticide. Identification of cash crops that produce compounds capable 

of reducing pathogenic nematode populations and that can be incorporated into existing 

production regimes is rare (Gardner et al., 1992; Mojtahedi et al., 1993a; Halbrendt, 

1996). Some notable exceptions to this include commercial production of Crotalaria, 

mustard, African marigold, asparagus, and sesame in India (Bridge, 1996). The level 

to which growers will employ the use of cover crops or rotation crops is ultimately 

dependent on the economic feasibility of this method for nematode control. When 

determining the economic feasibility of this approach, the additional benefits that 

cover crops provide should be considered. These benefits include nitrogen fixation, 

soil stabilization, and weed management (Halbrendt, 1996). 

Many plant constituents and metabolites have been investigated for activity against 

plant-parasitic nematodes. The conditions under which compounds are effective 

against nematodes vary with the compounds (Ferris and Zheng, 1999; Zasada and 

Ferris, 2004). These active compounds, or precursors of active compounds, can often 

be applied to soil as organic amendments, or refined and developed as biopesticide 

compounds. 

Most allelochemicals are short-lived in soil as they are often easily metabolized 

or hydrolyzed, and may require that plants are actively growing and secreting them 

into the rhizosphere in order to be effective (Cheng, 1992). In some cases, breakdown 

products of allelochemicals are the active components against nematodes (Borek 

et al., 1995). Many such compounds are produced upon decomposition of plant material 

that can be useful when incorporated into soil as green manures (Brown and Morra, 

1997; Mojtahedi et al., 1991; 1993a, b; Prot et al., 1992; Zasada and Ferris, 2004). In 

each of these instances, soil physical and chemical conditions, microbial populations, 

and environmental conditions influence the retention, transformation and transport 

of the allelochemicals (Cheng, 1992). Undoubtedly, these physical, microbiological,

ALLELOCHEMICALS : MANAGEMENT OF PLANT-PARASITIC NEMATODES 17 

and environmental factors contribute to the inconsistency of nematode control observed 

in research trials on crop rotation and cover crop systems, biofumigation, and 

biochemical pesticides. It is likely that synthetic chemical nematicides will continue 

to fill short-term nematode control needs while research continues to improve 

nonchemical and biopestide-based approaches, which will eventually become the 

management strategies of choice (Thomas, 1996). In this chapter we will review 

examples of plants known to release allelochemicals into soil while actively growing, 

when incorporated into soil as green manures or organic amendments, and the direct 

application of purified allelochemicals or formulations of biopesticides for plantparasitic 

nematode control. 

2. ROTATION AND COVER CROPS 

There are many examples of crop rotation sequences that passively suppress nematode 

populations which will not be reviewed here. Examples of active nematode suppression 

in crop rotation sequences are typically found with plant species that produce and 

excrete allelopathic compounds. These compounds then affect plant-parasitic 

nematodes in the rhizosphere either directly or indirectly by altering rhizosphere 

microbial populations (Halbrendt, 1996). For the purpose of this chapter, allelopathic 

Table 1. Rotation/cover crops that actively suppress 

parasitic nematode populations in soil. 

Common Name Scientific Name References 

American joint vetch Aeschynomene sp. Rodriguez-Kabana et al., 1991a 

Bahia grass Paspalum spp. Rodríguez-Kábana et al., 1994b 

Castor bean Ricinus communis Rodriguez-Kabana et al., 1991b 

Marigold Tagetes spp. Tyler, 1938 

Steiner, 1941 

Uhlenbroek and Bijloo, 1958, 1959 

Good et al., 1965 

Hairy indigo Indigofera hirsuta Rodriguez-Kabana et al., 1988b 

Horse bean Canavalia ensiformis Rodriguez-Kabana et al., 1992b 

Partridge pea Cassia fasciculata Rodriguez-Kabana et al., 1991a 

Rodriguez-Kabana et al., 1995 

Sesame Sesamum indicum Rodríguez-Kábana et al., 1994a 

Showy clotalaria Crotalaria spectabilis Rodriguez-Kabana et al., 1992b 

Sorghum-sudan grass S. bicolor X S. vulgare var. sudanense Kinloch and Dunavin, 1993 

Sudan grass Sorghum vulgare var. sudanense Mojtahedi et al., 1993a 

Sunn hemp Crotalaria juncea Sipes and Arakaki, 1997 

Robinson et al., 1998 

McSorley et al., 1999 

Wang et al., 2001 

Velvet bean Mucuna deeringiana Rodriguez-Kabana et al., 1992a 

Weaver et al., 1993 

Taylor and Rodriguez-Kabana, 1999 

Vargas-Ayala and Rodriguez-Kabana, 2001 

Vetch Vicia spp. Minton et al., 1966 

Minton and Donnelly, 1967

18 

rotation and cover crops are considered to be those that are not typically incorporated 

into soil in order to realize their allelopathic potential. Green manure crops considered 

to be crops that are most effective for nematode control when incorporated into soil as 

organic amendments. However, there are many examples of effective rotation crops 

that are also incorporated into soil as green manure at the end of the growing-season. 

Plants belonging to 57 families have been shown to possess nematicidal properties 

(Bridge, 1996). Crop rotation (crops planted in sequence) and intercropping (crops 

planted together) have both shown potential for reducing populations of parasitic 

nematodes. Cover crops are a type of rotation employed as an alternative to leaving 

land fallow, and are usually not cash crops. Many plants have been used as rotation 

or cover crops for plant-parasitic nematode control (Table 1). 

The active allelopathic compounds differ with respect to each crop and, in many 

cases, the mode of action for nematode suppression and the effects on nematode 

chemotaxis have not been established. Several examples of rotation crops effective in 

actively suppressing populations or controlling parasitic nematodes are reviewed. 

2.1. Marigold (Tagetes spp.) 

NANCY KOKALIS-BURELLE AND RODRÍGO RODRIGUEZ-KÁBANA Marigold was one of the first plants reported to be highly resistant to root-knot 

nematodes (Meloidogyne spp.) (Tyler, 1938). Soon after resistance was identified in 

marigold, it was reported that root-knot nematode larvae entered the roots but failed 

to develop to sexual maturity (Steiner, 1941). It was later discovered that Tagetes 

spp. produce nematotoxic compounds identified as alpha-terthienyls, which directly 

affect a wide range of nematodes (Uhlenbroek and Bijloo, 1958, 1959; Good et al., 

1965). The evaluation of several marigold species as rotation or cover crops has 

shown Tagetes minuta to be highly resistant to both Meloidogyne incognita and M. 

javanica, and to perform well as a summer cover crop in southern Florida (McSorley, 

1999). The more commonly used T. erecta and T. patula are often used as winter or 

spring bedding plants in Florida, and are not as tolerant of high temperatures as T. 

minuta (McSorley and Frederick, 1994; McSorley, 1999). Because T. minuta is more 

heat- tolerant, it has potential for use as a nematode suppressive summer cover crop 

in Florida during months when fields are often left fallow after fall and spring vegetable 

crop production cycles. Ploeg (1999) found that T. erecta, T. patula, T. signata and a 

hybrid Tagetes reduced galling by M. incognita, M. javanica, M. arenaria, and M. 

hapla in a tomato rotation study. However, all four Meloidogyne species reproduced 

on T. signata ‘Tangerine Gem’. This indicates the potential for substantial variability 

in nematode suppression among similar cultivars of marigold. 

There has been little research addressing the extent of allelopathic activity and 

the mechanism involved in nematode suppression with marigold. Sasanelli and DiVito 

(1991) found that aqueous leaf and root extracts and root leachates of two Tagetes sp. 

were nematostatic rather than nematicidal to eggs of the golden nematode Globodera 

rostochiensis. Emergence of juveniles that was suppressed or reduced in the presence


of extracts and diffusates resumed when solutions were removed indicating that 

compounds found in solutions were nematostatic rather than nematicidal. More specific 

information is needed on the range of activity and mechanisms involved in nematode 

suppression with compounds found in marigold. Additional research in these areas 

would increase the potential for successful incorporation of marigold or its active 

allelopathic compounds into production systems for nematode control. 

2.2. Sorghum-sudangrass (Sorghum bicolor X S. sudanense) 

Sorghum has long been recognized for its allelopathic properties toward other plants 

(Guenzi and McCalla, 1966) and more recently to be suppressive to nematodes (Kinlock 

and Dunavin, 1993; Mojtahedi et al., 1993a). It was initially hypothesized that the 

nematicidal compound in sorghum-sudangrass green manures was hydrogen cyanide 

produced by hydrolysis of dhurrin in leaf tissue (Andewusi, 1990). However, Czarnota 

et al. (2003) examined root exudate production and composition of seven genetically 

diverse sorghum accessions, including two sorghum-sudangrass hybrids, and found 

that although variation occurred in exudate constituents among accessions, the 

predominant constituent in all exudates was the phenolic compound sorgoleone 

(Czarnota et al., 2003). 

Suppression of parasitic nematodes in the field with sorghum-sudangrass has 

been inconsistent (MacGuidwin and Layne, 1995). It has been demonstrated that this 

crop is not effective for reducing populations of lesion nematodes (Pratylenchus sp.), 

an important plant-parasitic genus (MacGuidwin and Layne, 1995). Many studies 

have confirmed that sorghum-sudangrass is effective in reducing field populations of 

Meloidogyne spp., but that it cannot be recommended if stubby root nematode, 

Paratrichodorus minor, is present and of concern due to the high reproductive rates 

of P. minor on sorghum-sudangrass (McSorley and Gallaher, 1991; McSorley et al., 

1994a; McSorley and Dickson, 1995). The selective nature of parasitic nematode 

control with this allelopathic crop serves as an example of why it is critical to know 

what nematode species are present in a location when employing cover or rotation 

crops for key nematode pests. Threshold levels for secondary parasitic nematodes 

should be established and susceptibility of potential cover crop known when employing 

these strategies. 

2.3. Sesame (Sesamum indicum) 

Sesame is an important seed and oil crop worldwide. In addition to being a poor host 

for root-knot nematodes (Rodriguez-Kabana et al., 1988a; 1989), sesame is known to 

produce several lignin compounds including sesamin and sesamolin which are 

antioxidants that function as insecticides and insecticidal synergists and are 

hypothesized to be the active allelopathic compounds in sesame (Bedigian and Harlan, 

1986). Research has been conducted in the southern United States during the past 15 

years to evaluate the potential for use of sesame as a profitable rotation crop for root-

20 

NANCY KOKALIS-BURELLE AND RODRÍGO RODRIGUEZ-KÁBANA knot nematode control. Meloidogyne arenaria populations in peanut were reduced 

using a two-year sesame rotation followed by one year of peanut production (Rodriguez- 

Kabana et al., 1994a). Studies by Starr and Black (1995) confirm that sesame can be 

an effective rotation crop for control of M. arenaria and M. incognita but that it is not 

effective in controlling M. javanica. More work is needed to determine the identities 

of the active compounds found in sesame and the nature of their activity with regards 

to parasitic nematodes. 

2.4. Velvetbean (Mucuna deeringiana) 

Velvetbean (Mucuna deeringiana) is a legume commonly used as a cover crop, green 

manure, and forage in many subtropical and tropical regions (Allen and Allen, 1981). 

Velvetbean is known to produce L-DOPA (L-3,4-dihydroxyphenylalanine) which 

inhibits the growth of other plants and also has insecticidal properties (Fujii et al., 

1991; Bell and Janzen, 1971). Vincente and Acosta (1987) demonstrated the 

antagonistic capabilities of velvetbean against plant-parasitic nematodes when used 

as a soil amendment. Vargas-Ayela and Rodriguez-Kabana (2001) found an increase 

in beneficial rhizosphere microorganisms including species of Paecilomyces and 

Burkholderia associated with reduction in disease caused by root-knot nematodes in 

velvetbean- soybean rotations compared to cowpea-soybean rotations. McSorley et al. 

(1994b) found velvetbean to be very successful in reducing populations of M. arenaria, 

M. javanica, and several races of M. incognita. Although the active compounds in 

velvetbean have been identified, little research has been performed to determine how 

to optimize the potential of this crop for nematode suppression. 

2.5. Sunn hemp (Crotalaria juncea) 

Sunn hemp is a legume that has many of the desirable qualities of a good rotation or 

cover crop. In addition to its ability to fix nitrogen, sunn hemp grows rapidly and 

produces a large amount of biomass, increases soil organic matter, sequesters carbon 

(Rotar and Joy, 1983), and suppresses many plant-parasitic nematodes (McSorley et 

al., 1999; Robinson et al., 1998; Wang et al., 2001). Sunn hemp also increases 

populations of important nematode-antagonistic fungi in soil (Quiroga-Madrigal et 

al., 1999; Rodríguez-Kábana and Kloepper, 1998; Wang et al., 2001). Populations of 

microbivorous nematodes, which are involved in soil nutrient cycling, have also been 

shown to increase in sunn hemp-planted soil (Venette et al., 1997; Wang et al., 2003). 

Sunn hemp is well suited for use as a cover crop in subtropical regions (McSorley, 

1999) and did not increase population levels of M. javanica during a test in Hawaii 

(Sipes and Arakaki, 1997). Sunn hemp ‘Tropic Sun’ was highly resistant, but not 

immune, to the Meloidogyne spp. tested (McSorley, 1999). It has been suggested that 

elevated numbers of free-living nematodes, including bacteriovores and fungivores, 

may improve plant tolerance to parasitic nematodes by increasing plant vigor through 

more efficient nutrient cycling, or by increasing numbers of predatory and omnivorous 

nematodes that then feed on plant-parasitic nematodes (Wang et al., 2003).

3.1. Brassicaceae 


3. GREEN MANURES AND ORGANIC AMENDMENTS 

Glucosinolates are compounds produced by many members of the Brassicaceae plant 

family including mustard, rape, canola, and cabbage (Fahey et al., 2001). These 

compounds are β-D-thioglucosides with differing organic side chains, and can be 

aliphatic, aromatic, or indole forms. Enzymatic degradation of glucosinolates produces 

several types of compounds including isothyocyanate (ITC), a well known nematicide 

(Brown and Morra, 1997). 

Glucosinolates are produced in various levels throughout the plant and to different 

degrees among plant species (Fahey et al., 2001). In studies by Potter et al. (1998) the 

nematicidal potential of Brassica spp. leaf and root tissue differed, with leaf tissue 

being far more toxic to P. neglectus than root tissue. However, leaf tissue toxicity was 

not correlated with either total glucosinolate content or with any individual 

glucosinolate, while the suppression achieved with the addition of root tissue was 

highly correlated with levels of 2-phenylethyl glucosinolate within roots (Potter et 

al., 1998). Zasada and Ferris (2004) performed studies where levels of glucosinolates 

in various Brassica spp. were determined and amendments containing equivalent 

amounts of active compounds were compared to determine their efficacy in controlling 

M. javanica and Tylenchulus semipenetrans. They determined that the degradation 

of brassicaceous amendments in soil resulted in a series of biological and chemical 

processes that differed among plant species. It was also determined that consistent 

nematode suppression could be achieved using brassicaceous amendments if the 

chemical composition of the organic material was considered and appropriate levels 

of biomass applied (Zasada and Ferris, 2004). 

3.2. Neem (Azadirachta indica) 

The neem tree is a tropical evergreen tree native to India whose biologically active 

properties have been recognized in Asia for centuries (Thakur et al., 1981). During 

the past 30 years, interest in the extensive variety of biologically active compounds 

produced by neem has increased. Various parts of the neem plant including leaves, 

seeds and bark, produce over 40 active diterpenoid, triterpenoid, limonoid, and 

flavonoid compounds, with a wide range of nematicidal activity (Bhatnagar and 

Goswami, 1987). The most well known and active compounds produced by neem are 

the azadirachtins (Thakur et al., 1981). 

Nematode control has been achieved following the incorporation of neem products 

into soil, and their subsequent decomposition and release of nematicidal compounds 

(Stirling, 1991). Numerous studies have shown that leaves and oilcake of neem are 

effective in controlling M. incognita and increasing growth and yield of vegetable 

crops when used as a soil amendment (Akhtar, 1998; Bhatnagar and Goswami, 1987). 

This effect has also been reported to persist in successive crops (Akhtar and Alam, 

1991; Akhtar and Mahmood, 1996). Neem based products have also shown nematicidal

22 

potential when applied as seed treatments (Akhtar and Mahmood, 1995; 1997) and 

bare-root treatments (Akhtar and Mahmood, 1993; 1994) leading some to conclude 

that compounds found in neem may act as inducers of resistance to some nematodes 

including M. incognita and Rotylenchulus reniformis (Siddiqui and Alam 1988). Other 

nematode-suppressive mechanisms of compounds derived from neem include 

antifeedent, repellent, deterrent, growth disruption, juvenile toxicant, and ovicidal 

properties (Akhtar, 1998). Testing of neem-based products and development of 

application techniques for plant-parasitic nematode control is increasing in western 

countries. There are currently several neem-based pesticides available in the United 

States for use on certain greenhouse and ornamental crops, with many more available 

for use in India as insecticides (Akhtar, 2000). A comprehensive review of the 

nematode suppressive potential of neem products is provided by Akhtar (2000). 

4. ALLELOCHEMICALS AS BIOPESTICIDES 

Extracts of many plants with anthelminthic or antimicrobial properties have been 

proven effective in reducing soil populations of plant-parasitic nematodes (Ferris and 

Zheng, 1999). Experiments which evaluated plant species documented in Chinese 

traditional medicine to be anthelminthic against plant-parasitic nematodes identified 

153 aqueous plant extracts with activity against nematodes (Ferris and Zheng, 1999). 

Within a 24-hour exposure period, seventy-three of the extracts killed either juveniles 

of M. javanica or mixed developmental stages of P. vulnus, or both (Ferris and Zheng, 

1999). Plants containing efficacious components included both annuals and perennials, 

which ranged in type from grasses and herbs to woody trees, representing 46 plant 

families (Ferris and Zheng, 1999). This research illustrates the tremendous potential 

for discovery of new active allelopathic compounds for plant-parasitic nematode 

control. In fact, many of the allelochemicals described below were isolated from 

crops observed to be nematode suppressive as rotation or green manure crops. 

4.1. Glucosinolates 

NANCY KOKALIS-BURELLE AND RODRÍGO RODRIGUEZ-KÁBANA Glucosinolates are compounds primarily found in plants in the family Brassicaceae 

and are described previously in this chapter with respect to green manure crops. 

Enzymatic decomposition of glucosinolates in plant tissue occurs rapidly and is 

primarily attributed to microorganisms in soil (Fenwick et al., 1983). Products of 

glucosinolate degradation include organic cyanides and isothiocyanates which in 

addition to being evaluated as active compounds in green manures and organic 

amendments, have been studied for their their direct toxicity to nematodes as 

biochemical pesticides. Lazzeri et al. (1993) studied the direct effects of purified 

glucosinolates on second-stage juveniles of the sugar beet cyst nematode Heterodera 

schachtii. Compounds were isolated from seeds and plant tissue of brassicaceous 

hosts of the nematode. None of the glucosinolates tested in their native form were 

nematicidal. However, when exposed to the enzyme myrosinase, several compounds 

including sinigrin, gluconapin, glucotropeolin, glucode-hydroerucin, and the entire


group of glucosinolate compounds extracted from rapeseed exhibited various levels 

of nematicidal activity depending on concentration and length of exposure (Lazzeri 

et al., 1993). 

Later studies by Borek et al. (1995) investigated the persistence of glucosinolatederived 

allyl isothiocyanate and allylnitrile in six soils. They found that the two 

compounds differed with respect to the temperature, moisture conditions, and soil 

physical conditions that effected their transformation in soil, and that both compounds 

dissipated from soil at relatively rapid rates. Donkin et al. (1995) studied the toxicity 

of glucosinolates and their enzymatic breakdown products to Caenorhabditis elegans. 

They found that allyl isothyocyanate, one of the decomposition products of the 

glucosinolate sinigrin, was three times more toxic to the nematode C. elegans than 

corresponding glucosinolate itself. 

4.2. Benzaldehyde (benzoic aldehyde) 

Benzaldehyde occurs in seeds of bitter almond (Prunus dulcis). It is found naturally 

in several cyanogenic glucosides and is used in food and fragrances for its almondlike 

aroma and flavor (Harborne and Baxter, 1993). The value of benzaldehyde as a 

fungicide is well established (Flor, 1926), with the nematicidal activity more recently 

demonstrated. Benzaldehyde reduced populations of M. incognita in field microplots 

with no phytotoxicity to cotton at 0.18 -2.14 ml/kg soil (Bauske et al., 1994). The 

combination of chitin and benzaldehyde added to peat-based potting mix improved 

tomato transplant growth and reduced galling by M. incognita in greenhouse trials 

(Kokalis-Burelle et al., 2002). When tested in vitro against M. incognita eggs, 

benzaldehyde was 100% effective as an ovicide for this species of root-knot nematode 

(Kokalis-Burelle et al., 2002). 

The direct effect of benzaldehyde on C. elegans chemotaxis kinetics was analyzed 

by Nuttley et al. (2001). An initial attractive response to 100% benzaldehyde was 

reported, followed by a strong aversion to the chemical. They determined this behavior 

to be mediated by two genetically separable response pathways. Initially, upon 

exposure, the attraction response dominates but eventually gives way to a repulsive 

response. Oka (2001) found that with juveniles of M. javanica, immobilization and 

hatching inhibition in vitro were greater with benzaldehyde and furfural than with 

several other essential oils. Benzaldehyde and furfural also reduced galling on tomato 

in pot experiments where other aldehydes were not effective (Oka, 2001). 

The effects of benzaldehyde combined with several organic amendments on soil 

microbial populations and plant-parasitic and nonparasitic nematodes were investigated 

by Chavarría-Carvajal et al. (2001). They found that benzaldehyde combined with 

most organic amendments reduced damage from parasitic nematodes and selected for 

predominantly gram-positive rhizosphere bacteria. When benzaldehyde was combined 

with root-knot nematode egg parasitizing isolates of the fungus Fusarium solani, 

increasing rates of benzaldehyde in soil reduced nematode penetration and infection 

of the host plant, and resulted in increased parasitism of M. javanica females by the 

fungus (Siddiqui and Shaukat, 2003). However, the increasing rates of benzaldehyde

24 

resulted in lower egg parasitism by the fungus (Siddiqui and Shaukat, 2003). 

4.3. Furfural 

The biological activity of furfural (also known as 2-furancarboxaldehyde, furaldehyde; 

2-furanaldehyde, 2-furfuraldehyde, fural, furfurol) has also been recognized for decades 

(Flor, 1926). Furfural is the aldehyde of pyromucic acid and has properties similar to 

those of benzaldehyde. Furfural is a derivative of furan and is prepared commercially 

by dehydration of pentose sugars obtained from sugarcane, cornstalks and corncobs, 

husks of oat and peanut, and other agricultural waste products (Harborne and Baxter, 

1993). Commercially available products for disease and nematode control are available 

in several countries including the United States. These products include Multiguard 

FFA (furfural (75%) + allyl isothiocynate (25%), Harborchem, Cranford, New Jersey), 

and Multiguard Protect (furfural (50%) + metham sodium (50%), Harborchem, 

Cranford, New Jersey). 

Rajendran et al. (2003) reported improved plant growth and reductions in soil 

populations of M. arenaria and R. reniformis in groundnut with formulations of furfural 

compared to an untreated control, with no effect on free-living nematodes in soil. 

Spaull (1997) also found that free-living nematodes were relatively unaffected by 

furfural application while parasitic nematodes differed in their susceptibility with 

species of Paratichodorus and Xiphinema being more susceptible than Helicotylenchus 

and Tylenchorhynchus. Sipes (1997) found furfural to be as effective as 1,3-D for 

reduction of preplant soil nematode populations in pineapple production. Rodríguez- 

Kábana et al. (1993) found that furfural was an effective nematicide against M. 

arenaria, M. incognita, Heterodera glycines, and Pratylenchus spp. on squash, okra, 

and soybean. Furfural reduced populations of M. incognita in field microplots with 

no phytotoxicity to cotton at 0.18 -2.14 ml/kg soil (Bauske et al., 1994). 

4.4. Thymol 

NANCY KOKALIS-BURELLE AND RODRÍGO RODRIGUEZ-KÁBANA Thymol (isopropyl-m-cresol) is a volatile, phenolic monoterpene produced by several 

plants including thyme (Thymus vulgaris L.) (Baerheim Svendsen and Scheffer, 1985). 

Thymol has well-known antiseptic, antifungal, and anthelminthic properties (Wilson 

et al., 1977) and is also used for food and fragrance applications (Bauer et al., 1990). 

Research by Soler-Serratosa et al. (1996) using combinations of thymol and 

benzaldehyde for root-knot and cyst nematode control on soybeans showed that both 

compounds exhibited wide spectrum nematicidal activity with Meloidogyne spp. and 

Dorylaimid nematodes being more sensitive than cyst nematode and nonparasitic 

nematodes (Soler-Serratosa et al., 1996). In addition to the direct toxicity of these 

compounds to nematodes, it was hyopothesized that stimulation of beneficial microflora 

by the compounds or their products, altered host response, and a deleterious physicochemical 

environment may all contribute to reduced gall formation (Soler-Seratosa et 

al., 1996).

4.5. Citral 


Citral, is the aldehyde of geraniol, and occurs in the volatile oils of lemon grass, 

lemon, orange, limetta, and pimento (Harborne and Baxter, 1993). The flavor of 

lemon oil is largely due to its citral content, and the pure aldehyde may be used to 

increase the flavoring power of commercial samples of that oil (Harborne and Baxter, 

1993). In research trials evaluating the nematicidal potential of citral compared to 

other allelopathic chemicals, citral was less nematicidal against M. incognita juveniles, 

and more phytotoxic to tomato than benzaldehyde in vitro, and when added to a peatbased 

potting mix (Kokalis-Burelle et al., 2002). When tested in vitro against rootknot 

nematode eggs, citral reduced egg viability by 80%, but also decreased tomato 

seed germination and growth in greenhouse trials (Kokalis-Burelle et al., 2002). 

However, when evaluated in soil, citral reduced populations of root-knot nematode 

juveniles, galling on roots, and increased cotton growth when applied at 0.1 -0.5 ml/ 

kg soil in the greenhouse, and at 0.18 -2.14 ml/kg soil in field microplots with no 

phytotoxicity to cotton (Bauske et al., 1994). This difference in phytotoxicity may be 

due to the difference in host plants tested or to the presence of microorganisms in soil 

compared to the relatively uncontaminated conditions that occur in vitro and in potting 

media. 


The use of allelopathic compounds in either their native, degraded, or processed forms 

for plant-parasitic nematode management is receiving increased attention as 

agricultural producers face increasing restrictions on chemical biocides and 

nematicides. Allelochemicals are classified as biochemical pesticides and have been 

shown to possess various levels of activity against a wide range of plant-parasitic 

nematodes, while exhibiting reduced toxicity to nontarget species and reduced 

persistence in soil. The fact that some allelochemicals can be produced in large 

quantities in plant material and incorporated into soil as green manures or organic 

amendments increases their potential for use as components of nematode management 

strategies. The wide array of plant families that produce nematicidal or nematistatic 

compounds provides almost unlimited research opportunities for discovery of novel 

compounds. Determination of the mechanisms responsible for nematode suppression 

with allelopathic compounds also represents a research area with exciting possibilities. 

In order to improve the nematicidal and nematistatic efficacy achieved with allelopathic 

compounds, a greater understanding of the effects of soil microbiology, soil properties 

and environmental conditions on the active compounds is necessary. Currently 

available application methods need to be refined and new and improved methods 

developed to enhance the performance of allelochemicals for nematode control. Further 

investigation is also needed to develop compatible companion applications of 

biochemical pesticides and biological control agents, to determine the effects of 

allelochemicals on nematode nervous systems, and how they act as nematode attractants 

and/or repellents. This research area has enormous potential for discovery of new

26 

NANCY KOKALIS-BURELLE AND RODRÍGO RODRIGUEZ-KÁBANA compounds, for elucidating the direct effects of known compounds on nematode 

behavior, and for the development of new products to serve as components in 

multifaceted approaches to nematode management in the post methyl bromide era. 

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ANA LUISA ANAYA 

ALLELOPATHIC ORGANISMS AND MOLECULES: 

PROMISING BIOREGULATORS FOR THE 

CONTROL OF PLANT DISEASES, WEEDS, 

AND OTHER PESTS 

Laboratorio de Alelopatía, Instituto de Ecología, Universidad Nacional Autónoma 

de México. Circuito Exterior, Ciudad Universitaria. 04510 México, D.F. 

Email: alanaya@miranda.ecologia.unam.mx 

Abstract. Increasing attention has been given to the role and potential of allelopathy as a management strategy 

for crop protection against weeds and other pests. Incorporating allelopathy into natural and agricultural management 

systems may reduce the use of herbicides, insecticides, and other pesticides, reducing environment/soil 

pollution and diminish autotoxicity hazards. There is a great demand for compounds with selective toxicity that 

can be readily degraded by either the plant or by the soil microorganisms. In addition, plant, microorganisms, 

other soil organisms and insects can produce allelochemicals which provide new strategies for maintaining and 

increasing agricultural production in the future. 


AGRICULTURE AND PEST MANAGEMENT SYSTEMS 

Agriculture is one of the world’s largest industries. On a worldwide basis, more people 

are in some way involved in agriculture than in all other occupations combined. 

Agriculture is also United States’ largest industry. This country produces more food 

than any other nation in the world and is the world’s largest exporter of agricultural 

products. According to the 2002 survey from the United States Department of 

Agriculture (USDA) National Agricultural Statistics Service, there are more than 941 

million acres used for farming in the U.S. with the average farm size being 436 acres. 

Agriculture in the United States is becoming more productive. In 1935, there 

were 6.8 million farms in the United States, and the average farmer produced enough 

food to feed 20 people. In 2002, the number of farms was estimated to be 2.16 million, 

and the average U.S. farmer produced enough food each year to feed more than 

100 people. In addition to providing an abundant food supply for domestic markets, 

crops from nearly one-third of U.S. farm acreage are exported to overseas customers 

(IFIC 2004). 

Pest problems and their management vary widely throughout the world, based on 

economical resources, cultural techniques, climate, soil types, and many other 

conditions. As a result, chemical pest control has won a central place in modern 


Allelochemicals: Biological Control of Plant Pathogens and Diseases, 31– 78. 


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agriculture, contributing to the dramatic increase in crop yields achieved in recent 

decades for most major field, fruit, and vegetable crops. 

Farmers must contend with approximately 80,000 plant diseases, 30,000 species 

of weeds, 1,000 species of nematodes, and more than 10,000 species of insects. Today, 

national and international agricultural organizations estimate that as much as 45 

percent of the world’s crops continue to be lost to these types of hazards. In the United 

States alone, about $20 billion worth of crops (one-tenth of production) are lost each 

year (IFIC 2004). 

The word “pesticides” refers to a broad class of crop protection chemicals, 

including four major groups: insecticides, rodenticides, herbicides, and fungicides. 

All pesticides must be toxic, or poisonous, to kill the pests they are intended to control. 

Because pesticides are toxic, they are potentially hazardous to humans and animals. 

Therefore, people who use pesticides or regularly come in contact with them must 

understand the relative toxicity and potential health effects of the products they use 

(Pesticide Education Program. Penn State’s College of Agricultural Sciences, 2004). 

Some pesticides (administered at extremely high dosages) have been found to cause 

cancer in laboratory animals (Agri 21FAO). 

Weeds represent the most serious threat to the sustainability and profitability of 

agricultural production around the world considering these facts: 

Weeds represent the most economically serious pest complex reducing world 

food and fiber production. 

Controlling weeds costs the United States economy more than $15 billion annually, 

surpassing the combined cost of controlling disease and insect pests. U.S. herbicide 

expenditures account for 85% of all pesticide purchases and 62% of the total 

amount of active ingredient applied. 

Nearly all (greater than 95%) of the corn and soybean acreage in the U.S. receives 

herbicide applications. In developing countries, the cost of weed control in terms 

of labor and loss of yields is even greater, proportionally, than in the U.S. 

Worldwide herbicide purchases ($16.9 billion in 1997) constitute 58% of all 

pesticides bought and 53% of the amount of active ingredient applied (1 billion 

kg a.i.) worldwide. 

Crop yield losses from weed competition can be substantial. The degree of loss 

depends on, crop and weed species present; timing and duration of competitive 

interactions; and resource availability (Agri 21 FAO*, IFIC 2004**). 

Worldwide, the competitive effect of weeds causes a 10% loss in agricultural 

production. Yield losses in rice and other grass crops in West Africa have been reported 

to range from 28-100% if weeds such as witchweed (Striga hermonthica) –a parasitic 

weed–are not controlled; the greatest reductions occur on nutrient-poor soils. Left 

unchecked, weeds cause dramatic reductions in food production that eventually can 

* Agri 21 FAO: Agriculture 21. IPM and Weed Management. Food and Agriculture Organization of the 

United Nations (FAO). Agriculture Department. 2004. http://www.fao.org/WAICENT/FAOINFO/AGRICULT/ 

Default.htm. 

** IFIC 2004 : International Food Information Council. 2004. Agriculture & Food Production. Background 

on Agriculture & Food Production. http://www.ific.org/food/agriculture/index.cfm.

ALLEOPATHIC ORGANISMS AND 

MOLECULES 

destabilize economic and social systems. Hence, there is an urgent need to develop 

and refine weed management strategies in crop production that are effective, safe, 

and economically viable. 

Regardless of cropping system or agricultural region of the world, effective weed 

management strategies are needed continually to maintain crop yields and crop quality 

as well as to reduce the negative impact of weeds in future years. This ongoing quest 

to optimize weed management strategies is largely because of the ability of agricultural 

weeds to adapt to many of our most important crop production systems (Agri 21 FAO, 

IFIC 2004). 

Integrated Pest Management (IPM) is a system that works in partnership with 

nature to produce foods efficiently (Upadhyay et al., 1996). The concept began in 

U.S.A. in the 1950s, and recently resurfaced in popularity. Although many definitions 

of IPM have been advanced, two elements are critical: 

using multiple control tactics; 

integrating a knowledge of pest biology into the management system. 

IPM involves the carefully managed use of an array of pest control techniques 

including biological, cultural, and appropriate chemical methods to achieve the best 

results with the least disruption of the environment. With IPM, growers are adopting 

less chemically intensive methods of farming, which may include pest-resistant plant 

varieties, adjustments in planting times, low tillage, and other non-chemical techniques. 

The objectives of IPM are: 

Appreciate the importance of controlling weeds within an integrated pest 

management program. 

Understand the key biological differences that make IPM for weeds more difficult 

than IPM for insects. 

Become familiar with integrated weed management (IWM) strategies. 

The United States Department of Agriculture (USDA) proposed national standards 

for organic farming and handling in 1997.The federal regulations for organic 

standards were finalized in 2001 and began full implementation in 2002. Generally, 

organic food is produced by farmers who emphasize the use of renewable resources 

and the conservation of soil and water to enhance environmental quality for future 

generations (IFIC 2004, Agri 21 FAO). 

Barberi (2002), assessed that despite the serious threat which weeds offer to organic 

crop production, relatively little attention has so far been paid to research on 

weed management in organic agriculture, an issue that is often approached from a 

reductionist perspective. Compared with conventional agriculture, in organic agriculture 

the effects of cultural practices (e.g. fertilization and direct weed control) on 

crop-weed interactions usually manifest themselves more slowly. Weed management 

should be tackled in an extended time domain and needs deep integration with the 

other cultural practices, aiming to optimize the whole cropping system rather than 

weed control per se. Many organic farmers are aware that successful weed management 

implies putting into practice the concept of maximum diversification of their 

cropping system. However, this task is often difficult to achieve, because practical 

solutions have to pass through local filters (soil and climate conditions, availability of 

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and accessibility to external inputs -seeds, crop cultivars, machinery, etc.) and socioeconomic 

constraints (market, tenure status, attitude towards entrepreneurial risk, 

etc.). The use of cover crops and organic amendments, via the promotion of diversity 

in insect, fungal, bacterial or mychorrhyzal communities, may alter antagonist or 

competitive effects to the benefit of crops and to the detriment of weeds. Once factors 

driving these effects are better understood, it might be possible to use this knowledge 

to improve organic weed management systems locally. It would also be helpful to find 

indicators of “functional biodiversity”, where weed species abundance is assessed on 

the role that they have in the agroecosystem (e.g. strong /weak competitors, promoters 

of the presence of beneficial arthropods, etc.). Management of allelopathy is another 

potential tool in the arsenal of the organic farmer (Barberi, 2002). In the United 

States, the rate of increase of organic growers was estimated at 12% in 2000. However, 

many producers are reluctant to undertake the organic transition because of 

uncertainty of how organic production will affect weed population dynamics and 

management. The organic transition has a profound impact on the agroecosystem. 

Changes in soil physical and chemical properties during the transition often impact 

indirectly insect, disease, and weed dynamics. Greater weed species richness is usually 

found in organic farms but total weed density and biomass are often smaller 

under the organic system compared with the conventional system. The improved weed 

suppression of organic agriculture is probably the result of combined effects of several 

factors including weed seed predation by soil microorganisms, seedling predation by 

phytophagus insects, and the physical and allelopathic effects of cover crops (Ngouajio 

and McGiffen, 2002). 

2. ALLELOPATHY 

Increasing attention has been given to the role and potential of allelopathy as a management 

strategy for crop protection against weeds and other pests. Incorporating 

allelopathy into natural and agricultural management systems may reduce the use of 

herbicides, fungicides, nematicides, and insecticides, cause less pollution and diminish 

autotoxicity hazards. There is a great demand for compounds with selective toxicity 

that can be readily degraded by either the plant or by the soil microorganisms. 

Plant, microorganisms, other soil organisms and insects can produce allelochemicals 

which provide new strategies for maintaining and increasing agricultural production 

in the future. Compounds with allelopathic activity may provide novel chemistry for 

the synthesis of herbicides, insecticides, nematicides, and fungicides that are not based 

on the persistent petroleum derived compounds which are such a public health concern 

(Waller and Chou, 1989; Waller, 1999). 

Several crops (some of which can be used as cover crops) have been proved to 

release allelopathic compounds in the soil (Jimenez-Osornio and Gliessman, 1987; 

Blum et al., 1997; Inderjit and Keating, 1999; Anaya, 1999), many of which have 

been chemically characterized (Pereda-Miranda et al., 1996; Inderjit, 1996; Seigler, 

1996; Waller et al., 1999). The idea of exploiting these compounds as natural herbicides 

is therefore very attractive (Putnam, 1988; Weston, 1996; Duke et al., 2000).


MOLECULES 

However, the large majority of the studies carried out on this topic have referred to 

reductionist trials carried out in controlled environments, often with the only aim to 

extract and characterize allelochemicals or, at the most, to test the effect of these 

compounds on the germination of selected sensitive species in bioassays. In the case 

of crop-weed interactions, absolute evidence of the occurrence of allelopathy in the 

field is difficult to obtain, mainly because allelopathic effects are difficult to disentangle 

from resource competition and other biotic effects (Weidenhamer, 1996; Inderjit 

and del Moral, 1997). Additionally, the production and release of allelochemicals 

depend largely upon environmental conditions, usually being higher when plants are 

under stress, e.g. extreme temperatures, drought, soil nutrient deficiency, high pest 

incidence (Einhellig, 1987); also, the range and concentration of chemicals that a 

given species can produce can vary with environment conditions (Anaya, 1999). Other 

effects that need to be examined are allelopathy-mediated weed-weed, weed-crop and 

crop-following (or companion) crop interactions. It is therefore questionable whether 

allelopathy management per se would ever represent a consistently effective weed 

management tool; however, a better understanding of allelopathic occurrence in field 

situations, and of how it is influenced by cultural practices, would make it possible to 

include allelopathic crops in organic cropping systems and use them as a complementary 

tactic in a weed management strategy (Barberi, 2002). 

The extracts of many dominant plants in Taiwan, such as Delonix regia, Digitaria 

decumbens, Leucaena leucocephala, and Vitex negundo, contain allelopathic 

compounds, including phenolic acids, alkaloids, and flavonoids that can be used as 

natural herbicides, fungicides, etc. which are less disruptive of the global ecosystem 

than are synthetic agrochemicals (Chou, 1995). Many important crops, such as rice, 

sugarcane, and mungbean, are affected by their own toxic exudates or by phytotoxins 

produced when their residues decompose in the soil. For example, in Taiwan the yield 

of the second annual rice crop is typically 25% lower than that of the first, due to 

phytotoxins produced during the fallowing period between crops. Autointoxication 

can be minimized by eliminating, or preventing the formation of the phytotoxins 

through field treatments such as crop rotation, water draining, water flooding, and 

the polymerization of phytotoxic phenolics into a humic complex (Chou, 1995). 

Wetland soils provide anoxia-tolerant plants with access to ample light, water, 

and nutrients. Intense competition, involving chemical strategies, ensues among the 

plants. The roots of wetland plants are prime targets for root-eating pests, and the 

wetland rhizosphere is an ideal environment for many other organisms and communities 

because it provides water, oxygen, organic food, and physical protection. Consequently, 

the rhizosphere of wetland plants is densely populated by many specialized 

organisms, which considerably influence its biogeochemical functioning. The roots 

protect themselves against pests and control their rhizosphere organisms by bioactive 

chemicals, which often also have medicinal properties. Anaerobic metabolites, alkaloids, 

phenolics, terpenoids, and steroids are bioactive chemicals abundant in roots 

and rhizospheres in wetlands. Bioactivities include allelopathy, growth regulation, 

extraorganismal enzymatic activities, metal manipulation by phytosiderophores and 

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phytochelatines, various pest-control effects, and poisoning. Complex biological-biochemical 

interactions among roots, rhizosphere organisms, and the rhizosphere solution 

determine the overall biogeochemical processes in the wetland rhizosphere and 

in the vegetated wetlands. In order to comprehend how wetlands really function and 

to understand these interactions it is necessary to implement long-term collaborative 

research (Neori et al., 2000). We can find promising allelochemicals and useful interactions 

in the rich biodiversity of these particular ecoystems, but without doubt, in all 

type of ecosystems. 

3. CHEMISTRY OF ALLELOPATHY 

As we know, plant and microbial compounds are continuously analyzed as potential 

sources of herbicides, pesticides, and pharmaceuticals because they provide a diversity 

of carbon skeletons and there has been success in that a number of compounds 

have shown biological activity. The same bioassays and techniques to reveal mechanisms 

of action apply to the search for herbicides as in the study of allelopathy. Certainly 

there is overlap in goals and compounds studied, but there also is a difference 

in that the starting point in the ‘herbicide search’ might be any natural product, as 

opposed to one identified with allelopathy. Most inhibitors of plants are secondary 

compounds that have their origin in either the shikimate or acetate pathways, or they 

are compounds having skeletal components from both of these origins (Einhellig, 

2002). Waller et al. (1999) listed over twenty classes of secondary metabolites that are 

produced, stored, and released into the rhizosphere where they have biological activity 

as well as undergo microbial transformation and degradation. Einhellig (2002) 

concluded that the 14 categories suggested by Rice (1984) are sufficiently broad to 

still retain validity: water-soluble organic acids, straight-chain alcohols, aliphatic 

aldehydes and ketones, unsatured lactones, long-chain fatty acids and polyacetylenes, 

naphthoquinones, anthraquinones and complex quinones, gallic acids and 

polyphloroglucinols, cinnamic acids, coumarins, flavonoids, tannins, terpenoids, and 

steroids, amino acids, and purines and nucleosides. 

In this chapter some of the main compounds and studies associated with allelopathy 

will be mentioned. 

Terpenoids and phenolics are the most common compounds involved in allelopathic 

interactions. Terpenoids are the largest group of plant chemicals (15,000-20,000) 

with a common biosynthethic origin. The terpenoid pathway generates great structural 

diversity and complexity of compounds, thus generating enormous potential for 

mediating ecological interactions (Duke, 1991; Langenheim, 1994). Terpenoids may 

produce effects on seeds and soil microbiota through volatilization, leaching from 

plants, or decomposition of plant debris. These interactions can significantly affect 

community and ecosystem properties, although studies of plant-plant chemical interactions 

have often been controversial because of difficulty in unambiguously demonstrating 

interference by chemical inhibition rather than through resource competition 

or other mechanisms (Harper, 1977).


MOLECULES 

3.1. Terpenoids and sesquiterpene lactones 

Vokou et al. (2003) compared the potential allelopathic activity of 47 monoterpenoids 

of different chemical groups, by estimating their effect on seed germination and 

subsequent growth of Lactuca sativa seedlings. Apart from individual compounds, 

eleven pairs at different proportions were also tested. As a group, the hydrocarbons, 

except for (+)-3-carene, were the least inhibitory. Of the oxygenated compounds, the 

least inhibitory were the acetates; whenever the free hydroxyl group of an alcohol 

turned into a carboxyl group, the activity of the resulting ester was markedly lower 

(against both germination and seedling growth). Twenty-four compounds were 

extremely active against seedling growth (inhibiting it by more than 85%), but only 

five against seed germination. The compounds that were most active against both 

processes belonged to the groups of ketones and alcohols; they were terpinen-4-ol, 

dihydrocarvone, and two carvone stereoisomers. These authors used a model to 

investigate whether compounds acted independently when applied in pairs. The 

combined effect varied. In half of the cases, it followed the pattern expected under the 

assumption of independence; in the rest, either synergistic or antagonistic interactions 

were found in both germination and elongation. However, even in cases of synergistic 

interactions, the level of inhibition was not comparable to that of a single extremely 

active compound, unless such a compound already participated in the combination. 

The effect of the sesquiterpene cacalol and extracts (water and petroleum ether) 

derived from the roots of Psacalium decompositum (Asteraceae) on the germination 

and radicle growth of two plants, Amaranthus hypochondriacus (Amaranthaceae) 

and Echinochloa crus-galli (Poaceae), and the radial growth of four phytopathogenic 

fungi was described (Anaya et al., 1996). The activity of two cacalol derivatives (methyl 

cacalol and cacalol acetate) was also investigated. Germination of A. hypochondriacus 

was inhibited by almost all the treatments. The extracts and cacalol produced a 

significant inhibition of radicle growth of A. hypochondriacus and E. crus-galli. 

Cacalol acetate showed a specific inhibition on E. crus-galli, and methyl cacalol 

inhibited significantly the growth of A. hypochondriacus. In general, antifungal activity 

depended upon the target fungi and the concentration of each treatment. Cacalol had 

also effects on the morphology and coloration of the fungal mycelium. The bioactivity 

shown by the extracts of Psacalium decompositum on the tested seeds and fungi is 

mainly due to their content in cacalol. 

The allelochemical potential of Callicarpa acuminata (Verbenaceae) was 

investigated using a biodirected fractionation study as part of a long-term project to 

search for bioactive compounds among the rich biodiversity of plant communities in 

the Ecological Reserve El Eden, Quintana Roo, Mexico. Aqueous leachate, chloroformmethanol 

extract, and chromatographic fractions of the leaves of the plant species 

inhibited the root growth of Amaranthus hypochondriacus, Echinochloa crus-galli, 

and tomato (23% , 59%, and 70% respectively). Some of these treatments caused a 

moderate inhibition of the radial growth of two phytopathogenic fungi, 

Helminthosporium longirostratum and Alternaria solani (18% to 31%). The 

chloroform-methanol (1:1) extract prepared from the leaves rendered five compounds: 

37

38 


isopimaric acid, a mixture of two diterpenols: sandaracopimaradien-19-ol and 

akhdarenol, α-amyrin, and the flavone salvigenin. The phytotoxicity exhibited by 

several fractions and the full extract almost disappeared when pure compounds were 

evaluated on the test plants, suggesting a synergistic or additive effect. Akhdarenol, 

α-amyrin and isopimaric acid methyl ether had antifeedant effects on Leptinotarsa 

decemlineata. Alpha-amyrin was most toxic to this insect. No correlation was found 

between antifeedant and toxic effects on this insect, suggesting that different modes 

of action were involved. All the test compounds were cytotoxic to insect Sf9 cells 

while salvigenin, akhdarenol, and isopimaric acid also affected mammalian Chinese 

Hamster Ovary (CHO) cells. Alpha-amyrin showed the strongest selectivity against 

insect cells (Anaya et al., 2003). In this study the authors emphasized that 

allelochemicals involved in allelopathic interactions often have multiple functionality. 

Sesquiterpene lactones (SL) occur in over 15 plant families, predominantly in 

the Asteraceae, and represent with about 3,500 naturally occurring compounds, one 

of the largest groups of natural products. It has been demonstrated that some 

sesquiterpene lactones exhibit a broad spectrum of biological activities including 

phytotoxic and plant growth regulatory properties, cytotoxicity and antitumor 

properties, antimicrobial, insecticidal, molluscicidal and antimalarial activity (Fischer, 

1986). Phytotoxic terpenoids and their possible involvement in allelopathy were covered 

in reviews on mono- and sesquiterpenes (Evenari, 1949; Fischer, 1986, 1991, 1994) 

and biological activities of SL were reviewed by Stevens and Merrill (1985), Picman 

(1986) and Elakovich (1988). Seedlings of Ambrosia cumanensis are inhibited by 

leachates of the adult plants and residues in soils. Some SL of this species have been 

implicated in this autototoxic mechanism (Anaya and del Amo, 1978). In the same 

way, parthenin and coronopilin of Parthenium hysterophorus also exhibited autotoxicity 

toward seedlings and older plants, this fact possibly reveal a mechanism of intraspecific 

population regulation (Picman and Picman, 1984). Axivalin and tomentosin from the 

seeds of Iva axillaris were inhibitory toward the germination and growth of Abutilon 

theophrasti (velvetleaf) (Spencer et al., 1984). The germacranolide-type SL represented 

by dihydrotartridin B significantly inhibited the root growth of Brassica rapa var. 

pervidis (Sashida et al., 1983). The α-methylene-γ-lactone group is present in many 

of the isolated natural sesquiterpene lactones, and has been proposed as one of the 

factors which can determine their allelopathic activity, in particular, as well as their 

biological activity in general. The different spatial arrangements that a molecule of 

SL can adopt is the other factor that has been related with the potential allelopathic 

activity of this type of secondary compounds (Macias et al., 1992). Data of several 

studies on the allelopathic potential of SL clearly demonstrated that they can selectively 

promote or inhibit germination or growth at concentrations as low as 1 µM. It is 

reasonable to assume that rain washes transport SL from the source plant or 

decomposing litter into the soil where they can reach significant concentration levels. 

In the case of isoalantolactone, it has been demonstrated that it can persist in mineral 

and organic soil for 3 months, supporting the assumption that SL play a significant 

role in allelopathic interactions in the environment (del Amo and Anaya, 1976; Stevens 

and Merril, 1985; Picman, 1986; Fischer, 1991).


MOLECULES 

Dehydrozaluzanin C, a natural SL, is a weak plant growth inhibitor with an I 50 

(or IC 50 , the concentration required to inhibit plant growth 50 %) of about 0.5 mM for 

lettuce root growth. It also causes rapid plasma membrane leakage in cucumber 

cotyledon discs. Dehydrozaluzanin C is more active at 50 µM than the same 

concentration of the herbicide acifluorfen. Symptoms include plasmolysis and the 

disruption of membrane integrity is not light dependent. Reversal of its effects on root 

growth was obtained with treatment by various amino acids, with histidine and glycine 

providing ca. 40% reversion. The strong reversal effect obtained with reduced 

glutathione is due to cross-reactivity with DHZ and the formation of mono- and diadducts. 

Photosynthetic, respiratory and mitotic processes, as well as NADH oxidase 

activity appear to be unaffected by this compound. Dehydrozaluzanin C exerts its 

effects on plants through two different mechanisms, only one of which is related to 

the disruption of plasma membrane function (Galindo et al., 1999). 

A structure-activity study to evaluate the effect of the trans,trans-germacranolide 

SL lactones costunolide, parthenolide, and their 1,10-epoxy and 11,13-dihydro 

derivatives (in a range of 100-0.001 µM) on the growth and germination of several 

mono and dicotyledon target species was carried out by Macias et al. (1999). These 

compounds appear to have more selective effects on the radicle growth of 

monocotyledons. Certain factors such as the presence of nucleophile-acceptor groups 

and their accessibility enhance the inhibitory activity. The levels of radicle inhibition 

obtained with some compounds on wheat are totally comparable to those of commercial 

herbicide Logran and allow proposing them as lead compounds. In addition, a structureactivity 

study to evaluate the effect of 17 guaianolide SL (in a range of 100-0.001 

µM) on the growth and germination of several mono- and dicotyledon target species 

was also performed by Macias et al. (2002a). These compounds appear to have deeper 

effects on the growth of either monocots or dicots than the previously tested 

germacranolides. Otherwise, the lactone group seems to be necessary for the activity, 

though it does not necessarily need to be unsaturated. However, the presence of a 

second and easily accessible unsaturated carbonyl system greatly enhances the 

inhibitory activity. Lipophilicity and the stereochemistry of the possible anchoring 

sites are also crucial factors for the activity. 

The dichloromethane extract of dried leaves of Helianthus annuus has yielded, 

in addition to the known SL annuolide E and leptocarpin, and the sesquiterpenes 

heliannuols A,C,D,F,G,H,1, the new bisnorsesquiterpene, annuionone E, and the new 

sesquiterpenes heliannuol L, helibisabonol A and helibisabonol B. Structural 

elucidation was based on extensive spectral (one and two-dimensional NMR 

experiments) and theoretical studies. The sesquiterpenes heliannuol A and 

helibisabonol A and the SL leptocarpin inhibited the growth of etiolated wheat 

coleoptiles (Macias et al., 2002b). In addition to (+)-, (-)- and (±)-heliannuol E, growthinhibitory 

activities of five synthetic chromanes and four tetrahydrobenzo[b]oxepins 

were examined against oat and cress. All heliannuol E isomers exhibited similar 

biological activities against cress, whereas when tested against oat roots, the unnatural 

optical isomer (+) showed no inhibitory activity. Four brominated chromans and two 

39

40 


tetrahydrobenzo[b]oxepins derivatives also showed apparent inhibition against both 

cress and oat (Doi et al., 2004). 

The tremendous impact of parasitic plants on world agriculture has prompted 

much research aimed at preventing infestation. Orobanche and Striga spp. are two 

examples of parasitic weeds that represent a serious threat to agriculture in large 

parts of the world. The life cycle of these parasitic weeds is closely regulated by the 

presence of their hosts, and secondary metabolites that are produced by host plants 

play an important role in this interaction. A special interest has been arising on those 

host-produced stimulants that induce the germination of parasite seeds. Three classes 

of compounds have been described that have germination-stimulating activity: 

dihydrosorgoleone, the strigolactones and SL. Keyes et al. (2001) suggest that 

dihydrosorgoleone is the active stimulant in the root exudates of sorghum and other 

monocotyledonous hosts. However, Butler et al. (1995) and Wigchert et al. (1999) 

suggest that dihydrosorgoleone is less likely to be the germination stimulant in vivo 

because of its low water solubility, and because no correlation between its production 

and the germination of Striga has been found. To date, there is no definite proof that 

the germination of parasitic weed seeds in the field is induced by one single signal 

compound or class of compounds (and indeed such proof will be hard to obtain) 

(Bouwmeester et al., 2003). The capacity of SL, which share some structural features 

with the strigolactones, to induce the germination of S. asiatica has been reported 

(Fischer et al., 1989, 1990). In addition, a decade after the results of Fischer studies, 

Francisco Macías and his group (Pérez de Luque et al., 2000; Galindo et al., 2002) 

performed some studies of the structure-activity relationship (SAR) directed to evaluate 

the effect of several SL as germination stimulants of three Orobanche spp. (O. cumana, 

O. crenata, and O. ramosa). Results are compared with those obtained in the same 

bioassay with an internal standard, the synthetic analogue of strigol GR-24. A high 

specificity in the germination activity of SL on the sunflower parasite O. cumana has 

been observed, and a relationship between such activity and the high sunflower SL 

content is postulated. Molecular properties of the natural and synthetic germination 

stimulants (GR-24, GR-7, and Nijmegen-1) and SL have been studied using MMX 

and PM3 calculations. Consequently, comparative studies among all of them and 

their activities have been made. SL tested present similarities in molecular properties 

such as the volume of the molecule and the spatial disposition of the carbon backbone 

to the natural germination stimulant orobanchol. These properties could be related to 

their biological activity. Considering that the sun-flower–O. cumana interaction is 

highly specific and that sunflower contains many SL, it is tempting to speculate that 

O. cumana has evolved to respond to sesquiterpene lactones (and not or less to 

strigolactones) (Bouwmeester et al., 2003). 

3.2. Phenolics 

In relation with phenolics, Inderjit et al. (1997) conducted a study to understand the 

effects of certain phenolics, terpenoides, and their equimolar mixture through agar 

gel and soil growth bioassays and their recovery from soils. The eight compounds


MOLECULES 

selected for this study were p-hydroxybenzoic acid, ferulic acid, umbelliferone, catechin, 

emodin, 1,8-cineole, carvone, and betulin. Lettuce (Lactuca sativa L.) was used as 

test species for agar gel and soil growth bioassays. Root and shoot growth of lettuce 

was inhibited for all the above except emodin and catechin. However, in soils treated 

with different phenolics and terpenoids, only root growth of lettuce was inhibited, 

whereas shoot growth was promoted. Recovery of p-hydroxybenzoic acid and 

umbelliferone was higher in unautoclaved soils, while that of catechin was lower. 

Nava-Rodriguez et al. (in press) observed the in vitro effects of aqueous leachates 

from fresh and dry, flowering and vegetative stage of Phaseolus species, faba bean, 

alfalfa, vetch, maize, and squash, and weed species on the root growth of selected 

crop and weeds, as well as on two strains of Rhizobium leguminosarum biovar phaseoli 

(CPMex1 and Tlaxcala). Most of the specimens were collected in a traditional 

agricultural drained field (“Camellon”) in Tlaxcala, Mexico where maize, beans, 

squash, alfalfa, faba-beans, and vetch are cultivated in mixed or rotation crops. 

Significant effects of leachates from fresh vegetative and flowering cultivated plants 

and weeds were predominantly stimulatory on the growth of tested crops, being the 

leachates from fresh aerial parts of alfalfa and pinto bean the most stimulatory. 

Nevertheless, aqueous leachates from fresh and dry cultivated legumes (vegetative 

and flowering) inhibited the growth of weeds. In contrast, the aqueous leachates from 

the dry aerial part of almost all plants resulted inhibitory on the root growth of the test 

crops, except maize. Aqueous leachates were also evaluated on the growth of two 

strains of Rhizobium leguminosarum biovar phaseoli. Leachates from some of the 

tested crops significantly stimulated the growth of both Rhizobium strains. The aqueous 

leachates from fresh aerial parts of the weeds Simsia amplexicaulis and Tradescantia 

crassifolia significantly inhibited the growth of CPMex1 Rhizobium strain. On the 

other hand, the aqueous leachates from fresh roots of these same weeds inhibited the 

growth of the Tlaxcala strain. In preliminary chemical tests using thin layer 

chromatography (TLC), phenolics were detected in dry aerial parts of vegetative alfalfa, 

pinto bean, and vetch, and dry aerial part of flowering faba bean suggesting the role 

of these compounds in the allelopathic effects of these legumes. 

Nilsson et al. (1998) reported on the temporal variation of phenolics and a 

dihydrostilbene, batatasin III, in Empetrum hermaphroditum leaves. These authors 

reported that first year shoots produced higher levels of phenolics than older tissues. 

High phenolic concentration was maintained through the second year, but it declined 

afterwards. However, the phytotoxicity of E. hermaphroditum extracts was related 

more to batatasin III than phenolics. 

Hyder et al. (2002) performed a study focused on the presence and distribution of 

secondary phenolic compounds found within creosotebush (Larrea tridentata). Total 

phenolics, condensed tannins and nordihydroguaiaretic acid (NDGA) were measured 

in nine categories of tissue within creosotebush. Total phenolic and condensed tannin 

concentrations were determined using colorimetric methods while NDGA content 

was determined with high performance liquid chromatography (HPLC). Phenolics 

were present throughout the plant with the highest concentrations in green stems 

(40.8 mg/g), leaves (36.2 mg/g), and roots (mean for all root categories=28.6 mg/g). 

41

42 


Condensed tannins were found in all tissues with highest concentrations in flowers 

(1.7 mg/g), seeds (1.1 mg/g), and roots less than 5 mm in diameter (1.1 mg/g). 

Flowers, leaves, green stems and small woody stems (


MOLECULES 

effects on germination and seedling growth of six weeds. Ferulic acid, gallic acid, pcoumaric 

acid, p-hydroxybenzoic acid, vanillic acid, and p-vanillin were bioassayed 

in concentrations of 10, 1, 0.1, and 0.01 mM. Equimolar mixtures containing all 

these phenolics were prepared at the final total concentration of 10, 1, 0.1, and 0.01 

mM to test for possible interactive effects. Chenopodium album, Plantago lanceolata, 

Amaranthus retroflexus, Solanum nigrum, Cirsium sp. and Rumex crispus were the 

selected target weeds. The highest concentration of the compounds inhibited the 

germination of all these weeds, but lower concentrations had no effect or were 

stimulatory. However, effects varied with the weed species, the concentration of the 

compound tested and the compound itself. In assays with the mixture of phenolics 

some additive effects were found (Reigosa et al., 1999). 

Reversible sorption of phenolic acids by soils may provide some protection to 

phenolic acids from microbial degradation. In the absence of microbes, reversible 

sorption 35 days after addition of 0.5-3 mu mol/g of ferulic acid or p-coumaric acid 

was 8-14% in Cecil A(p) horizon and 31-38% in Cecil B-t horizon soil materials. 

The reversibly sorbed/solution ratios (r/s) for ferulic acid or p-coumaric acid ranged 

from 0.12 to 0.25 in A(p) and 0.65 to 0.85 in B-t horizon soil materials. When microbes 

were introduced, the r/s ratio for both the A(p) and B-t horizon soil materials increased 

over time up to 5 and 2, respectively, thereby indicating a more rapid utilization of 

solution phenolic acids over reversibly sorbed phenolic acids. The increase in r/s ratio 

and the overall microbial utilization of ferulic acid and/or p-coumaric acid were much 

more rapid in A(p) than in B-t horizon soil materials. Reversible sorption, however, 

provided protection of phenolic acids from microbial utilization for only very short 

periods of time. Differential soil fixation, microbial production of benzoic acids (e.g., 

vanillic acid and p-hydroxybenzoic acid) from cinnamic acids (e.g., ferulic acid and 

p-coumaric acid, respectively), and the subsequent differential utilization of cinnamic 

and benzoic acids by soil microbes indicated that these processes can substantially 

influence the magnitude and duration of the phytoxicity of individual phenolic acids 

(Blum, 1998). 

Soil solution concentrations of allelopathic agents (e.g., phenolic acids) estimated 

by soil extractions differ with extraction procedure and the activities of the various 

soil sinks (e.g., microbes, clays, organic matter). This led to the hypothesis that root 

uptake of phenolic acids is a better estimator of dose than soil solution concentrations 

based on soil extracts. This hypothesis was tested by determining the inhibition of net 

phosphorus uptake of cucumber seedlings treated for 5 hr with ferulic acid in wholeroot 

and split-root nutrient culture systems. Experiments were conducted with II ferulic 

acid concentrations ranging from 0 to 1 mM, phosphorus concentrations of 0.25, 0.5, 

or 1 mM, and solution pH values of 4.5, 5.5, or 6.5 applied when cucumber seedlings 

were 9, 12, or 15 days old. The uptake or initial solution concentration of ferulic acid 

was regressed on ferulic acid inhibition of net phosphorus uptake. Attempts were 

made to design experiments that would break the collinearity between ferulic acid 

uptake and phosphorus uptake. The original hypothesis was rejected because the initial 

ferulic acid solution concentrations surrounding seedling roots were more frequently 

and consistently related to the inhibition of net phosphorus uptake than to ferulic acid 

43

44 


uptake by these roots. The data suggest that root contact, not uptake, is responsible 

for the inhibitory activity of phenolic acids (Lehman and Blum, 1999). 

Bulk-soil and rhizosphere bacteria are thought to exert considerable influence 

over the types and concentrations of phytotoxins, including phenolic acids that reach 

a root surface. Induction and/or selection of phenolic acid-utilizing (PAU) bacteria 

within the bulk-soil and rhizosphere have been observed when soils are enriched with 

individual phenolic acids at concentrations greater than or equal to 0.25 µmol/g soil. 

However, since field soils frequently contain individual phenolic acids at concentrations 

well below 0.1 µmol/g soil, the actual importance of such induction and/or selection 

remains uncertain. Common bacteriological techniques (e.g., isolation on selective 

media, and plate dilution frequency technique) were used to demonstrate in Cecil Ap 

soil systems: (i) that PAU bacterial communities in the bulk soil and the rhizosphere 

of cucumber seedlings were induced and/or selected by mixtures composed of individual 

phenolic acids at concentrations well below 0.25 µmol/g soil; (ii) that readily available 

carbon sources other than phenolic acids, such as glucose, did not modify induction 

and/or selection of PAU bacteria; (iii) that the resulting bacterial communities readily 

utilize mixtures of phenolic acids as a carbon source; and (iv) that depending on 

conditions (e.g., initial PAU bacterial populations, and phenolic acid concentration) 

there were significant inverse relationships between PAU bacteria in the rhizosphere 

of cucumber seedlings and absolute rates of leaf expansion and/or shoot biomass. The 

decline in seedling growth could not be attributed to resource competition (e.g., 

nitrogen) between the seedlings and the PAU bacteria in these studies. The induced 

and/or selected rhizosphere PAU bacteria, however, reduced the magnitude of growth 

inhibition by phenolic acid mixtures. For a 0.6 µmol/g soil equimolar phenolic acid 

mixture composed of ρ-coumaric acid, ferulic acid, ρ-hydroxybenzoic acid, and vanillic 

acid, modeling indicated that an increase of 500% in rhizosphere PAU bacteria would 

lead to an approximate 5% decrease (e.g., 20-25%) in inhibition of absolute rates of 

leaf expansion (Blum et al., 2000). 

Allelopathy due to humus phenolics is a cause of natural regeneration deficiency 

in subalpine Norway spruce (Picea abies) forests. If inhibition of spruce germination 

and seedling growth due to allelochemicals is generally accepted, in contrast there is 

a lack of knowledge about phenolic effects on mycorrhizal fungi. Thus, Souto et al. 

(2000) tested the effects of a humic solution and its naturally occurring phenolics on 

the growth and respiration of two mycorrhizal fungi: Hymenoscyphus ericae (symbiont 

of Vaccinium myrtillus, the main allelochemical-producing plant) and Hebeloma 

crustuliniforme (symbiont of P. abies, the target plant). Growth and respiration of H. 

crustuliniforme were inhibited by growth medium with the original humic solution (- 

6% and -30%), respectively, whereas the same humic solution did not affect growth 

but decreased respiration of H. ericae (-55%). When naturally occurring phenolics 

(same chemicals and concentrations in the original humic solution) were added to the 

growth medium, growth of H. crustuliniforme was not affected, whereas that of H. 

ericae significantly increased (+10%). These authors concluded that H. ericae is 

better adapted to the allelopathic constraints of this forest soil than H. crustuliniforme


MOLECULES 

and that the dominance of V. myrtillus among understory species could be explained 

in this way. 

Inderjit and Duke (2003) mentioned that the best evidence for allelopathy should 

include some understanding of natural concentrations and rates of allelochemicals. 

For example, (±)-catechin has been isolated from Centaurea maculosa (Bais et al., 

2002), an invasive species in North America for which other lines of evidence suggest 

root allelopathy (Ridenour and Callaway, 2001). The more common enantiomer, (+)catechin, 

has anti-bacterial functions, whereas (–)-catechin has strong allelopathic 

effects on other plants. (±)-catechin is harmless to C. maculosa, but has negative 

effects on other species at concentrations of ˜100 mg L -1 . (±)-catechin is exuded from 

C. maculosa roots creating concentrations from 83.2 to 185 mg L -1 in aqueous solutions. 

Importantly, Bais et al. (2002) found (±)-catechin in extracts from natural soils in 

fields containing C. maculosa in concentrations as far higher than the minimum 

required dose, ranging from 291.6 to 389.8 µg cm -3 . 

In allelopathy studies a central goal is to isolate, identify, and characterize 

allelochemicals from the soil. However, since it is essentially impossible to simulate 

exact field conditions, experiments must be designed with conditions resembling those 

found in natural systems. Inderjit (1996) argued that allelopathic potential of phenolics 

can be appreciated only when we have a good understanding of i) species responses to 

phenolic allelochemicals, ii) methods for extraction and isolation of active phenolic 

allelochemicals, and iii) how abiotic and biotic factors affect phenolic toxicity. 

Duke et al. (2003) summarized the recent research of the Agricultural Research 

Service of United States Department of Agriculture on the use of natural products to 

manage pests. They discussed some studies on the use of both phytochemicals and 

diatomaceous earth to manage insect pests. Chemically characterized compounds, 

such as a saponin from pepper (Capsicum frutescens L), benzaldehyde, chitosan and 

2-deixy-D-glucose are being studied as natural fungicides. Resin glycosides for 

pathogen resistance in sweet potato and residues of semitropical leguminous plants 

for nematode control are also under investigation. Bioassay-guided isolation of 

compounds with potential use as herbicides or herbicide leads is underway at several 

locations. New natural phytotoxin molecular target sites (asparagine synthetase and 

fructose-1,6-bisphosphate aldolase) have been discovered. Weed control in sweet potato 

and rice by allelopathy is under investigation. Molecular approaches to enhance 

allelopathy in sorghum are also being undertaken. The genes for polyketide synthases 

involved in production of pesticidal polyketide compounds in fungi are found to provide 

clues for pesticide discovery. Gene expression profiles in response to fungicides and 

herbicides are being generated as tools to understand more fully the mode of action 

and to rapidly determine the molecular target site of new, natural fungicides and 

herbicides. 

Research on the chemical basis for allelopathy has often been hindered by the 

complexity of plant and soil matrices, making it difficult to track active compounds. 

Recent improvements in the cost and capabilities of bench-top chromatography-mass 

spectrometry instruments make these tools more powerful and more widely available 

45

46 


to assist with molecular studies conducted in today’s expanding field. Such instrumental 

techniques are herein recommended as economically efficient means of advancing 

the rigor of allelopathy research and assisting the development of a better understanding 

of the chemical basis for the allelopathy phenomenon (Haig, 2001). 

4. THE MODE OF ACTION OF ALLELOCHEMICALS 

Allelopathic chemicals alter plant growth and development by a multiplicity of actions 

on physiological processes because there are hundreds of different structures and many 

of the compounds have several phytotoxic effects. Whole plants bioassays and 

physiological tests designed to use a small quantity of compound are keys to strategies 

for elucidating mechanisms of action. Insight into the action of the responsible 

chemicals is critical to a more complete explanation of allelopathy and to its application 

for improving crop production. Unfortunately, we still find that linkages between 

inhibition of growth and the corresponding physiological mechanism are elusive. A 

major part of the problem is the array and diversity of allelochemicals. Several hundred 

different compounds have been identified, and we speculate that many others will be 

eventually being discovered. Most instances of allelopathic inhibition are the result of 

the simultaneous action of several compounds, and often these include compounds 

whose chemistry is divergent (Einhellig, 2002). The visible evidence in bioassays is 

that many phenolics, quinones, sesquiterpene lactones, alkaloids, and others alter 

root morphology. Although the cell membrane is an early interface with 

allelochemicals, relatively little attention has been given to membrane-related effects 

and their molecular targets. 

Effects of a single allelochemical or their mixtures on physiological processes 

include disruption of membrane permeability (Galindo, et al., 1999), ion uptake (Yu 

and Matsui, 1997), inhibition of electron transport in both the photosynthesis and 

respiratory chains (Calera et al., 1995a; Abrahim, et al., 2000), alteration of enzymatic 

activity (Politycka, 1999, Romagni, et al., 2000), and inhibition of cell division (Cruz- 

Ortega et al., 1988; Anaya and Pelayo-Benavides, 1997). In other examples, bioactivitydirected 

fractionation of the methanol extract of the roots of Ratibida mexicana resulted 

in the isolation of two bioactive sesquiterpene lactones, isoalloalantolactone and elema- 

1,3,11-trien-8,12-olide. Both compounds caused a significant inhibition of the radicle 

growth of Amaranthus hypochondriacus and Echinochloa crus-galli, exerted moderate 

cytotoxicity activity against three different solid tumour cell lines and inhibited the 

radial growth of three phytopathogenic fungi. Isoalloalantolactone also caused the 

inhibition of ATP synthesis, proton uptake, and electron transport (basal, 

phosphorylating and uncoupled) from water to methylviologen therefore acting as a 

Hill’s reaction inhibitor.The lactone inhibited only photosystem II (Calera et al., 1995b). 

Other compounds studied by Calera et al., (1995c) were the resin glycoside mixture 

from Ipomoea tricolor. They tested its effect on seedling growth and plasma membrane 

H + -ATPase activity in Echinochloa crus-galli. The resin glycoside mixture as well as 

Tricolorin A, the main compound in the mixture, inhibited the activity of the plasma 

membrane ATPase. In other studies, Cruz-Ortega et al. (1998) observed plasma


MOLECULES 

membrane disruption in root tips and plasmolized cells in the peripheral zone of 

beans and bottle gourd roots treated with the aqueous leachate of S. deppei suggesting 

that the allelopathics of this plant alter some membrane processes. 

4-phenyl coumarins isolated from Exostema caribaeum and Hintonia latiflora 

(Rubiaceae) and some semisynthetic derivatives acted as uncouplers in spinach 

chloroplasts. The glycoside 5-Ο-β-D-glucopyranosyl-7-methoxy-3’,4’-dihidroxy-4phenylcoumarin, 

5,7,3’,4’-tetrahydroxy-4-phenyl-coumarin, and 7-methoxy-5,3’,4’trihydroxy-4-phenylcoumarin 

inhibited ATP synthesis and proton uptake. On the other 

hand, basal and phosphorylating electron transport were enhanced by these compounds. 

The light-activated Mg 2+ -ATPase was slighted stimulated by the last two coumarins. 

In addition, at alkaline pH compound 5,7,3’,4’-tetrahydroxy-4-phenyl-coumarin 

stimulated the basal electron flow from water to methylviologen, but at the pH range 

from 6 to 7.5 the coumarin did not have any enhancing effect. This last compound, 

which possesses four free phenolic hydroxyl groups, was the most active uncoupler 

agent. Probably, the phenolate anions may be the active form responsible for the 

uncoupling behavior of 4-phenylcoumarins (Calera et al., 1996). 

Low molecular weight phenolic compounds were identified in two soils with 

different vegetative cover, Fagus sylvatica and Pinus laricio, and were tested at different 

concentrations on seed germination of Pinus laricio, and on respiratory and oxidative 

pentose phosphate pathway enzymes involved in the first steps of seed germination. 

There are marked differences in the phenolic acid composition of the two investigated 

soils. All the phenolic compounds bioassayed inhibited seed germination and those 

extracted from Pinus laricio soil were particularly inhibitory. Inhibition of germination 

of seeds is strongly correlated to the inhibition of the activities of enzymes of glycolysis 

and the oxidative pentose phosphate pathway (Muscolo et al., 2001). 

Seven-day-old seedlings of cucumber (Cucumis sativus cv. Wisconsin) were treated 

with 0.1 mM solutions of cinnamic acid (ferulic and p-coumaric acids) and benzoic 

acid (hydroxybenzoic and vanillic acids) derivatives as stressors. The content of free 

and glucosylated soluble phenols and the activity of phenylalanine ammonia-lyase 

(E.C.4.3.1.5), phenol-beta-glucosyltransferase (E.C.2.4.1.35.), and beta-glucosidase 

(E.C.3.2.1.21.) in seedling roots as well as their length and fresh weight were examined. 

Changes in glucosylated phenolic content and phenol-beta-glucosyltranspherase 

activity were observed under the influence of all phenolics applied. Treatment with 

ferulic and p-coumaric acids stimulated the increase of phenylalanine ammonia-lyase 

and beta-glucosidase activity and slightly inhibited cucumber root growth (Politycka, 

1998). 

Environmental stresses (biotic and abiotic), including allelochemicals have been 

shown to induce the synthesis of new proteins in plants. These proteins might have 

evolutionary value for survival under adverse environmental situations. Romero- 

Romero et al. (2002) tested the effect of the mixture of toxic allelochemicals from the 

aqueous leachates from Sicyos deppei, Acacia sedillense, Sebastiania adenophora, 

and Lantana camara on the radicle growth and cytoplasmic protein synthesis patterns 

of Zea mays (maize), Phaseolus vulgaris (bean), Cucurbita pepo (squash), and 

Lycopersicon esculentum (tomato). In general, high, medium and low molecular weight 

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cytoplasmic proteins were affected by the different aqueous leachates. Crop plant 

responses were diverse, but in general, an increase in protein synthesis was observed 

in the treated-roots. Maize was the least affected, but both the radicle growth and also 

the protein pattern of tomato were severely inhibited by all allelopathic plants. The 

changes observed on protein expression may indicate a biochemical alteration at the 

cellular level of the tested crop plants. 

Roshchina (2001) discussed the molecular-cellular basis of pollen allelopathy, 

related to possible chemosensory mechanisms. The phenomenon consists of a series 

of events, viz., a) excretion of signalling and regulatory substances from donor cell 

(pollens, pistil stigma); b) recognition of specific signal-stimulus from plant excretions 

by acceptor cell (pollen or pistil stigma); c) transmission of chemical information 

within the acceptor cell (pollen); and d) development of characteristic response in 

acceptor cell. The processes occur in growth, development and normal fertilization. 

In the first stage of interactions, allelochemicals are excreted, which act as chemical 

signals, growth regulators and modulators of cellular metabolism, etc. The 

allelochemicals, acting on fertilization may be, nitrogen-containing substances 

(acetylcholine, histamine, serotonin, dopamine, noradrenaline), phenols [(flavonoids: 

quercetin, kaempferol, rutin), aromatic acids (benzoic, gallic, vanillic)], terpenoids 

(monoterpenes: citral, linalool, cymol), sesquiterpene lactones: azulene and proazulenes 

(desacetylinulicine, inulicine, ledol, artemisinine, grosshemine, gaillardine and 

austricine), and polyacetylenes (capilline) found in flower excretions. These compounds 

were tested in vitro and in vivo on pollen germination of Hippeastrum hybridum. 

Nitrogenous compounds stimulate the growth of pollen tube, whereas, their antagonists 

blocked normal fertilization and thus fruits or seeds did not form. Terpenoids act on 

pollen germination and their stimulatory and inhibitory effects (block fruit formation) 

depend on their concentration. These effects of terpenoids on pollen germination are 

through chemosignalling and possible steps are: a) spreading of information in pollen 

secretions e.g. in olfactory slime; b) binding with special sensors or receptors in 

plasmalemma; and c) transfer of stimulus within the pollen cell to nucleus, where 

spermia appear and a pollen tube starts to grow. Moving from donor cell, 

allelochemicals penetrate the wall of acceptor cell either a) directly (without any 

changes in protoplasmic membrane); or b) after conversions [interaction with foreign 

substance of low or high-molecular weight (enzymes and protectory proteins) secreted 

from donor cells. or compounds of acceptor cell]. Often the second case includes free 

radical processes. The transmission of information within cell is third stage which 

includes participation of secondary messengers (cyclic AMP and GMP, inositol 

triphosphate, Ca ions) and some related enzymatic systems. The final transmission 

occurs in membranes of cellular organelles, which respond to information received 

through changes in enzymatic activity and metabolism. At cellular level, in pollen 

and pistil it may be active excretion, changes in the autofluorescence and membrane 

permeability, regulation of alternative pathways in respiration and photosynthesis 

and switching on free radical processes. 

The phenyl propanoid pathway (PPP) can be stimulated as demonstrated by 

Randhir et al. (2004) in mung bean sprouts through the pentose phosphate and


MOLECULES 

shikimate pathways, by natural elicitors such as fish protein hydrolysates (FPH), 

lactoferrin (LF) and oregano extract (OE). Elicitation significantly improved the 

phenolic, antioxidant and antimicrobial properties of mung bean sprouts. The optimal 

elicitor concentrations were 1 ml/l FPH, 250 ppm LF and 1 ml/l OE for the highest 

phenolic content that was approximately 20, 35 and 18% higher than control, 

respectively, on day 1 of dark germination. The antioxidant activity estimated by Pcarotene 

assay in mung bean sprouts was highest on day 1 of germination for all 

treatments and control. In general, higher antioxidant activity was observed in the 

elicited sprouts compared with control. In the case of 1,1-diphenyl-2-picrythydrazyl 

(DPPH) assay the antioxidant activity for all treatments and control was highest on 

day 2. Among the different elicitor treatments, OE elicited mung bean sprouts showed 

the highest antioxidant activity of 49% DPPH inhibition on day 2. This increased 

activity correlates with high guaiacol peroxidase (GPX) activity indicating that the 

polymerizing phenolics required during lignification with growth have antioxidant 

function. For all elicitor treatments a higher glucose-6-phosphate dehydrogenase 

(G6PDH) activity was observed during early germination following the high phenolic 

content. This is due to the general mobilization of carbohydrates to the growing 

sprouts in response to elicitation. In general the GPX activity steadily increased with 

germination for treatments and control. The higher phenolics produced on day 1 

was utilized for GPX-mediated polymerization to form polymeric phenolics and lignin 

required during germination. The late stage polymerization linked to GPX activity 

preceded stimulation of G6PDH. This indicated that as phenolics were polymerized 

by GPX in late stages, G6PDH linked precursors such as NADPH (2) and sugar 

phosphates were being made available. Antimicrobial activity against Helicobacter 

pylori was observed in the mung bean sprout extract from control, LF and OE 

treatments from the day 1 stage. Both the LF and OE elicited extracts showed high 

antimicrobial activity, which correlated to high antioxidant activity on day 1. The 

higher antimicrobial activity was also observed with the higher stimulation of G6PDH 

and GPX activity during early stages of germination. This leads to the hypothesis 

that enhanced mobilization of carbohydrates (as indicated by G6PDH activity on 

days 2) and 4), enhanced polymerization of simple phenols (as indicated by GPX 

activity on day 3) contributed to high antioxidant activity producing intermediary 

metabolites (day 2). 

5. WEED MANAGEMENT 

Bhowmik and Inderjit (2003) examined some considerable efforts in designing 

alternative weed management strategies due to increase in the number of herbicideresistant 

weeds and environmental concerns in the use of synthetic herbicides. The 

conventional synthetic herbicides are becoming less and less effective against the 

resistant weed biotypes. These authors discussed the role of allelopathic cover crops/ 

crop residues, natural compounds, and allelopathic crop cultivars in natural weed 

management, and gave numerous examples of employing crop residues, cover crops, 

and allelopathic crop cultivars in weed management. They concluded that although 

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we cannot eliminate the use of herbicides, their use can be reduced by exploiting 

allelopathy as an alternate weed management tool for crop production against weeds 

and other pests. 

The use of allelopathy for controlling weeds could be either through directly 

utilizing natural allelopathic interactions, particularly of crop plants, or by using 

allelochemicals as natural herbicides. In the former case, a number of crop plants 

with allelopathic potential can be used as cover, smother, and green manure crops for 

managing weeds by making desired manipulations in the cultural practices and 

cropping patterns. These can be suitably rotated or intercropped with main crops to 

manage the target weeds (including parasitic ones) selectively. Even the crop mulch/ 

residues can also give desirable benefits. The allelochemicals present in the higher 

plants as well as in the microbes can be directly used for weed management along 

with the management of some herbicides (Singh et al., 2003b). 

Singh et al. (2003b) also mentioned that the bioefficacy of allelochemicals can be 

enhanced by structural changes or the synthesis of chemical analogues based on them. 

Further, in order to enhance the potential of allelopathic crops, several improvements 

can be made with the use of biotechnology or genomics and proteomics. In this context 

either the production of allelochemicals can be enhanced or the transgenics with 

foreign genes encoding for a particular weed-suppressing allelochemical could be 

produced. These authors comment that in the former, both conventional breeding and 

molecular genetical techniques are useful. However, with conventional breeding being 

slow and difficult, more emphasis is laid on the use of modern techniques such as 

molecular markers and the selection aided by them. Although the progress in this 

regard is slow, nevertheless some promising results are coming and more are expected 

in future. In this sense, is important to point out that the potential use of transgenic 

plants and other genetically modified organisms (GMO’s) with such or other proposal, 

cause a strong controversial with the principles of organic agriculture defined and 

established by the International Federation of Organic Agriculture Movements 

(IFOAM) founded with the aim to promote an agriculture that is ecologically, 

economically, and socially sustainable. IFOAM is opposed to genetic engineering in 

agriculture, in view of the unprecedented danger it represents for the entire biosphere 

and the particular economic and environmental risks it poses for organic producers 

(IFOAM, 2002) * . 

Using a soil bioassay technique, Conkling et al. (2002) assessed seedling growth 

and incidence of disease of wild mustard (Brassica kaber) and sweet corn (Zea mays) 

in soil from field plots that received either of two treatments: incorporated red clover 

(Trifolium pratense) residue plus application of compost (‘amended soil’), or 

application of ammonium nitrate fertilizer (‘unamended soil’). Soils were analyzed 

for percent moisture, dissolved organic carbon, conductivity, phenolics, and nutrient 

content. A trend toward greater incidence of Pythium spp. infection of wild mustard 

seedlings grown in amended soil was observed during the first 40 days after 

* IFOAM, 2002: International Federation of Organic Agriculture Movements (IFOAM). Position on Genetic 

Engineering and Genetically Modified Organisms. http://www.ifoam.org/pospap/ge_position_0205.html 2002.


MOLECULES 

incorporation (DAI) of red clover and compost, with significant differences (α= 0.05) 

at two out of four sampling dates in 1997, and four out of four sampling dates in 

1998. Incidence of Pythium infection was 10-70% greater in the amended soil treatment 

during that period. Asymptomatic wild mustard seedlings grown in amended soil 

were also on average 2.5 cm shorter (α= 0.05) at 5 DAI than those grown in unamended 

soil in one year out of two. Concentration of phenolic compounds in soil solution was 

correlated with decreased shoot and root growth (r = 0.50, 0.28, respectively) and 

increased incidence of disease (r = 0.48) in wild mustard seedlings in one year out of 

two. Dissolved organic carbon concentration was correlated with increased disease in 

wild mustard seedlings in both years (r = 0.51, 0.33, respectively). Growth of corn 

seedlings did not differ between the two soil treatments, suggesting that red clover 

green manure and compost may selectively reduce density and competitive ability of 

wild mustard in the field. Bioassay results corresponded well with emergence and 

shoot weight results from a related field study, indicating that this technique may be 

useful for screening potential soil treatments prior to field studies. 

Anaya et al. (1987) applied leaves of Alnus firmifolia, Berula erecta and Juncus 

sp., as green manures in corn fields with corn, bean, and squash grown using traditional 

techniques. The growth of weeds during the crop period was decreased by the presence 

of the green manures. At the same time, stimulation of bean root nodulation by 

Rhizobium was obtained with these particular green manure species, nodulation was 

also increased in plots with abundant weed growth. These results suggest that the 

presence of different secondary metabolites liberated by these green manures and by 

some living weeds in the field plots increase the ability of Rhizobium sp. to infect 

bean roots. 

Weed control by rye, crimson clover, subterranean clover, and hairy vetch cover 

crops was evaluated in no-tillage corn during 1992 and 1993 at two North Carolina 

locations (Yenish, et al., 1996). Weed biomass reduction was similar with rye, crimson 

clover, and subterranean clover treatments, ranging between 19 and 95% less biomass 

than a conventional tillage treatment without cover. Weed biomass reduction using 

hairy vetch or no cover in a notillage system was similar averaging between 0 and 

49%, but less than other covers approximately 45 and 90 d after planting. Weed 

biomass was eliminated or nearly eliminated in all cover systems with preplus postherbicide 

treatments. Weed species present varied greatly between years and locations, 

but were predominantly common lambsquarters, smooth pigweed, redroot pigweed, 

and broadleaf signalgrass. Corn grain yield was greatest using pre-herbicides or preplus 

post-herbicides, averaging between 16 to 100% greater than the nontreated control 

across all cover treatments depending on the year and location. 

Studies were conducted by Burgos and Talbert (1996) at the Main Agricultural 

Experiment Station in Fayetteville and the Vegetable Substation in Kibler, Arkansas, 

in 1992 and 1993 on the same plots to evaluate weed suppression by winter cover 

crops alone or in combination with reduced herbicide rates in no-till sweet corn and 

to evaluate cover crop effects on growth and yield of sweet corn. Plots seeded to rye 

plus hairy vetch, rye, or wheat had at least 50% fewer early season weeds than hairy 

vetch alone or no cover crop. None of the cover crops reduced population of yellow 

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nutsedge. Without herbicides, hairy vetch did not suppress weeds 8 wk after cover 

crop desiccation. Half rates of atrazine and metolachlor (1.1 + 1.1 kg al ha (-1)) 

reduced total weed density more effectively in no cover crop than in hairy vetch. Half 

rates of atrazine and metolachlor controlled redroot pigweed, Palmer amaranth, and 

goosegrass regardless of cover crop. Full rates of atrazine and metolachlor [2.2 + 2.2 

kg al ha (-1)] were needed to control large crabgrass in hairy vetch. Control of yellow 

nutsedge in hairy vetch was marginal even with full herbicide rates- Yellow nutsedge 

population increased and control with herbicides declined the second year, particularly 

with half rates of atrazine and metolachlor. All cover crops except hairy vetch alone 

reduced emergence, height, and yield of sweet corn. Sweet corn yields from half rates 

of atrazine and metolachlor equalled the full rates regardless of cover crops. 

It is presently not known what effect wheat root residues have in regulating 

dicotyledonous (dicot) weed emergence in no-till management systems. Past research 

has focused almost entirely on the role of shoot residues, while the role of root residues 

in weed control has been essentially ignored. A field study was designed by Blum et 

al. (2002) to determine the respective effects of wheat shoot and root residues in 

regulating the emergence of three dicotyledonous weed species (morning-glory, pigweed 

and prickly sida). Glyphosate-desiccated wheat plots and fallow plots were surface 

seeded with morning-glory, pigweed and prickly sida during the spring of 1996 and 

1997. Weed seedling emergence was determined for two months during each 

experimental period in plots with or without wheat shoot and/or root residues. The 

resulting data suggested that: a) the closer desiccation of the wheat cover crop occurred 

to the initial emergence of pigweed seedlings, the lower the emergence of that weed, 

b) the effects of wheat shoot and/or root residues on dicot weed seedling emergence 

vary considerably for the different weed species ranging from stimulation to inhibition 

and c) the role of root residues appear to be much more important to regulating weed 

emergence than that of surface shoot residues, Differences in soil moisture and 

temperature associated with the presence or absence of wheat residues could not be 

used to explain the observed treatment effects. 

The growth of four summer season crops, namely Cyamopsis tetragonoloba, 

Sorghum vulgare, Pennisetum americanum and Zea mays, in fields with or without 

residues of the preceding sunflower crop was poor. Crop density, weight of seed or 

grain and total yield were significantly lower in sunflower fields than in the control 

fields (i.e. those without previous sunflower crops). Growth in terms of plant height 

and biomass was drastically reduced after 60 days. The effect was more pronounced 

in the fields where sunflower residues were allowed to decompose than in those where 

residues were completely removed. The soil collected from sunflower fields (both 

with and without residues) was found to be rich in phenolics, which in a laboratory 

bioassay were found to be phytotoxic. The reduced growth and yield of crops can be 

attributed to the release of phytotoxic phenolics from decomposing sunflower residues 

(Batish, et al., 2002). 

John and Narwal (2003) assessed that Leucaena leucocephala is the most 

productive and versatile multipurpose legume tree in tropical agriculture and has 

several uses, thus called ‘miracle tree’. It is a popular choice for intercropping with


MOLECULES 

annuals in hedgerow or alley cropping systems. Its allelopathic effects on oil cereals, 

pulses (peas and beans), oilseeds, vegetables, fodder crops, weeds, trees etc. are reviewed 

in this paper. The foliage and pods of Leucaena contain the toxic amino acid mimosine 

[beta-N-(3-hydroxy-4-pyridone)-alpha-aminopropionic acid] and many other 

phytotoxic compounds. The toxic effects of mimosine oil plants and physiology of its 

action also are discussed. The future areas identified for research in Leucaena are: (a) 

studies on its allelopathic compatibility with different crops to identify sustainable 

agroforestry systems (b) investigations to overcome its adverse allelopathic effects 

and mimosine toxicity and (c) possibility of using the allelopathic compounds in 

Leucaena as natural herbicides. 

Marigold (Tagetes erecta) is another multipurpose crop with ceremonial, 

ornamental, medical and pharmaceutical uses, and reported antimicrobial properties. 

Gómez-Rodríguez et al. (2003) evaluated the effect of marigold intercropped with 

tomato (Lycopersicon esculentum) on Alternaria solani conidia germination in vitro, 

on conidial density and tomato leaf damage in vivo, as well as microclimatic changes, 

compared to tomato intercropped with pigweed (Amaranthus hypochondriacus) and 

monocropped tomato. They found that intercropping with marigold induced a 

significant (P < 0:05) reduction in tomato early blight caused by A. solani, by means 

of three different mechanisms. One was the allelopathic effect of marigold on A. 

solani conidia germination, as it was shown in vitro conditions; while pigweed did 

not have any of this inhibitory effect in conidia germination. The second way was by 

altering the microclimatic conditions around the canopy, particularly by reducing the 

number of hours per day with relative humidity 92%, thus diminishing conidial 

development. The third mechanism was to provide a physical barrier against conidia 

spreading. When intercroppped with tomato, pigweed plants worked also as a physical 

barrier and promoted reductions in the maximum relative humidity surrounding the 

canopy, but to a lesser extent than marigold. 

The allelopathic properties of unburnt (UR) and burnt (BR) residues of Parthenium 

hysterophorus towards the growth of two winter crops-radish and chickpea were 

investigated (Singh et al., 2003c). The extracts prepared from both UR and BR were 

toxic to the seedling length and dry weight of the test crops, those from BR in particular. 

The difference was attributed to the highly alkaline nature of the extracts prepared 

from BR. Growth studies conducted in soil amended with UR and BR extracts and 

residues also revealed phytotoxic effects towards test crops, UR being more active 

than BR unlike crude extracts. These effects were attributed to the presence of phenolics 

rather than to any significant change in pH or conductivity. 

Weston and Duke (2003) focused a review on a variety of weed and crop species 

that establish some form of potent allelopathic interference, either with other crops or 

weeds, in agricultural settings, in the managed landscape, or in naturalized settings. 

They remarked that recent research suggests that allelopathic properties can render 

one species more invasive to native species and thus potentially detrimental to both 

agricultural and naturalized settings. In contrast, allelopathic crops offer strong 

potential for the development of cultivars that are more highly weed suppressive in 

managed settings. Both environmental and genotypic effects impact allelochemical 

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production and release over time. A new challenge that exists for future plant scientists 

is to generate additional information on allelochemical mechanisms of release, 

selectivity and persistence, mode of action, and genetic regulation. In this manner, it 

is possible to further protect plant biodiversity and enhance weed management 

strategies in a variety of ecosystems. 

Ohno et al. (2000) based on previous studies that suggested phenolics from legume 

green manures may contribute to weed control through allelopathy, investigated if 

red clover (Trifolium pratense) residue amended field soils expressed phytotoxicity to 

a weed species, wild mustard (Sinapis arvensis). Field plots involving incorporation 

treatments of wheat (Triticum aestivum) stubble or wheat stubble plus 2530 kg ha(-1) 

red clover residue, were sampled at -12, 8, 21, 30, 41, 63, and 100 days after residue 

incorporation (DAI). Soil-water extracts (1:1, m:v) were analyzed for plant nutrients 

and phenolic content. Phytotoxicity of the extracts was measured using a laboratory 

wild mustard bioassay. There was a 20% reduction of radicle growth in the green 

manure treatment in comparison with the wheat stubble treatment, but only at the 

first sample date after residue incorporation (8 DAI). The radicle growth reduction 

had the highest correlation with the concentration of soluble phenolics in the soil, 

water extracts. Bioassays using aqueous extracts of the clover shoots and roots alone 

predicted a radicle growth reduction of 18% for the quantity of clover amendment 

rate used in the field plots. The close agreement of the predicted and observed root 

growth reduction at 8 DAI further supports clover residue as the source of the 

phytotoxicity. 

The allelopathic influence of sweet potato cultivar ‘Regal’ on purple nutsedge 

was compared to the influence on yellow nutsedge under controlled conditions. Purple 

nutsedge shoot dry weight, total shoot length and tuber numbers were significantly 

lower than the controls (47, 36, and 19% inhibition, respectively). The influence on 

the same parameters for yellow nutsedge (35, 21, and 43% inhibition, respectively) 

was not significantly different from purple nutsedge. Sweet potato shoot dry weight 

was inhibited by purple and yellow nutsedge by 42% and 45%, respectively. The 

major allelopathic substance from ‘Regal’ root periderm tissue was isolated and tested 

in vitro on the two sedges. The I 50 ’s for shoot growth, root number, and root length 

were 118, 62, and 44 µg/ml, respectively, for yellow nutsedge. The I 50 ’s for root number 

and root length were 91 and 85 µg/ml, respectively, for purple nutsedge and the I 50 for 

shoot growth could not be calculated (Peterson and Harrison, 1995). These allelopathic 

substances, the resin glycosides mixture extracted from the periderm tissue of storage 

roots from sweet potato, Ipomoea batatas, was bioassayed for effects on survival, 

development, and fecundity of the diamondback moth, Plutella xylostella. The resin 

glycoside was incorporated into an artificial diet and fed to P. xylostella larvae. First 

instars were placed individually into snap-top centrifuge vials containing artificial 

diet with one of six concentrations of resin glycoside material (0.00. 0.25, 0.50, 1.00, 

1.50, and 2.00 µg/ml). Each replication consisted of 10 individuals per concentration, 

and the experiment was repeated 13 times. Vials were incubated at 25 o C and a 

photoperiod of 14:10 (L:D) h in a growth chamber. After 6 d, surviving larvae were 

weighed and their sex determined, then returned to their vials. Later, surviving pupae


MOLECULES 

were weighed and incubated at 25 o C until moths emerged. Females were fed, mated 

with males from the laboratory colony, and allowed to lay eggs on aluminum foil 

strips. Lifetime fecundity (eggs/female) was measured. There were highly significant 

negative correlations between resin glycoside levels and survival and between glycoside 

levels and larval weight after 0 d of feeding. For larvae that lived at least 6 d, there 

was no additional mortality that could be attributed to the resin glycoside material. 

However, there was a significant positive correlation between glycoside dosages and 

developmental time of larvae (measured as days until pupation). Lifetime fecundity 

also was negatively affected at sublethal doses. Resin glycosides may contribute to the 

resistance in sweet potato breeding lines to soil insect pests (Jackson and Peterson, 

2000). It is important to consider that the use of allelopathic crops or plant residues in 

agricultural management will inevitably affect other crop pests, for example insect 

populations. 

The total resin glycoside content in the periderm of 37 sweetpotato cultivars and 

breeding clones was measured by HPLC and varied greatly among the clones, the 

highest content was 10.02 % of the periderm dry weight and the lowest was 0.05 %. 

Insect damage ratings of the clones and their periderm resin glycoside content were 

negatively correlated and all clones with high resin glycoside content exhibited 

moderate or low injury from insects. Resin glycosides extracted from ‘Regal’ periderm 

and incorporated into potato dextrose agar medium were inhibitory to the growth of 

four fungal species of sweetpotato roots; however, these fungi exhibited variable 

response. These observations provide evidence that sweetpotato resin glycosides 

contribute to the insect and disease resistance in the roots of some sweetpotato-clones 

(Harrison et al., 2003). 

Barazani and Friedman (2001) discussed the impact of allelopathic, nonpathogenic 

bacteria on plant growth in natural and agricultural ecosystems. In some natural 

ecosystems, evidence supports the view that in the vicinity of some allelopathically 

active perennials (e.g., Adenostoma fasciculatum, California), in addition to 

allelochemicals leached from the shrub’s canopy, accumulation of phytotoxic bacteria 

or other allelopathic microorganisms amplify retardation of annuals. In agricultural 

ecosystems allelopathic bacteria may evolve in areas where a single crop is grown 

successively, and the resulting yield decline cannot be restored by application of 

minerals. Transfer of soils from areas where crop suppression had been recorded into 

an unaffected area induced crop retardation without readily apparent symptoms of 

plant disease. Susceptibility of higher plants: to deleterious rhizobacteria is often 

manifested in sandy or so-called skeletal soils. The allelopathic effect may occur directly 

through the release of allelochemicals by a bacterium that affects susceptible plant(s) 

or indirectly through the suppression of an essential symbiont. The process is affected 

by nutritional and other environmental conditions; some may control bacterial density 

and the rate of production of allelochemicals. Allelopathic nonpathogenic bacteria 

include a wide range of genera and secrete a diverse group of plant growth-mediating 

allelochemicals. Although a limited number of plant growth-promoting bacterial 

allelochemicals have been identified, a considerable number of highly diversified 

growth-inhibiting allelochemicals have been isolated and characterized. Some species 

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may produce more than one allelochemical; for example, three different phyotoxins, 

geldanamycin, nigericin, and hydanthocidin, were isolated from Streptomyces 

hygroscopicus. Efforts to introduce naturally produced allelochemicals as plant growthregulating 

agents in agriculture have yielded two commercial herbicides, 

phosphinothricin, a product of Streptomyces viridochromogenes, and bialaphos from 

S. hygroscopicus. Both herbicides have the same mechanism of action. Many species 

of allelopathic bacteria that affect growth of higher plants are not plant specific, but 

some do exhibit specificity; for example, dicotyledonous plants were more susceptible 

to Pseudomonas putida than were monocotyledons. Differential susceptibility of higher 

plants to allelopathic bacteria was noted also in much lower taxonomical categories, 

at the subspecies level, in different cultivars of wheat, or of lettuce. Therefore, when 

test plants are employed to evaluate bacterial allelopathy, final evaluation must include 

those species that are assumed to be suppressed in nature. The release of allelochemicals 

from plant residues in plots of ‘continuous crop cultivation’ or from allelopathic living 

plants may induce the development of specific allelopathic bacteria. 

Striga hermonthica is an obligate root-parasitic flowering plant that severely 

threatens cereal production in sub-Saharan Africa. A potential biological control option 

for reduction of crop yield-loss within the season of application is the use of soilborne 

antagonists of S. hermonthica seed. A study was made (Ahonsi et al., 2002) 

with the aim to select soil-borne fluorescent pseudomonad strains capable of 

suppressing germination of S. hermonthica seeds and consequently reducing parasitism 

and damage to maize. An in vitro screening procedure was developed and was used to 

evaluate 460 fluorescent pseudomonad isolates from naturally suppressive soils. This 

resulted in the identification of 15 Pseudomonas fluorescens/P. putida isolates that 

significantly inhibited germination of S. hermonthica seeds. In a pot experiment using 

steam-sterilized soil, there was a significant reduction in the number of S. hermonthica 

plants on maize grown from seeds that were inoculated with any of the 15 bacterial 

isolates. Inoculation of maize seed with six of these isolates resulted not only in a 

reduced number of S. hermonthica plants, but also in an increased maize shoot biomass 

compared with the check. When soils inoculated with these bacterial isolates were 

left dried for 5 weeks after maize harvest and then planted with a second maize crop, 

no reduction in S. hermonthica parasitism was observed. This suggested that the 

bacteria did not persist in the soil after the first crop of maize. These results suggest 

that saprophytic fluorescent pseudomonads have potential for biological control of S. 

hermonthica in maize and that periodic application of bacteria, perhaps through seed 

treatment, may be necessary for sustained control. 

Chittapur et al. (2001) asserted that integrated weed management systems 

involving catch and trap crops are needed to reduce herbicide use in agriculture and 

to help to control parasitic weed growth. The effective catch crops viz., fodder millet 

(Panicum miliaceum), sorghum (Sorghum bicolor), corn (Zea mays), sudangrass 

(Sorghum sudanense) have been identified for the management of Striga asiatica, 

and the cowpea (Vigna catjang) for S. gesnerioides. Cotton (Gossypium spp.), soybean 

(Glycine max) and peanut (Arachis hypogaea) are important trap crops. Intercropping 

of soybean or peanut with sorghum effectively controls S. hermonthica. Flax


MOLECULES 

(Linum usitatissimum) is a useful trap crop for Orobanche ramosa, O. cernua, O. 

crenata, and O. aegyptica. In India, sunnhemp (Crotolaria juncea), blackgram 

(Phaseolus mungo), greengram (Phaseolus aureus) and sesame (Sesamum indicum) 

have shown good potential for Orobanche control. Rotation of trap crop reduces the 

population of Orobanche and 3 to 4 years long rotation of catch/trap crops provides 

its effective control. Sorghum/maize/paddy (Oryza sativa)-tobacco (Nicotiana tabacum) 

rotation reduces the infestation and weed biomass of Orobanche. Relay cropping of 

tobacco in capsicum (Capsicum annuum), onion (Allium cepa) and peanut also reduces 

the incidence of Orobanche. 

Nagabhushana et al. (2001) remarked that no matter how one may define 

sustainable agriculture, use of soil-conserving cropping practices, less synthetic 

herbicide inputs and better weed control would be compatible components. Previously, 

these components were considered incompatible, since it was widely believed that 

soil-conserving practices required increased pesticide use, including herbicides. 

However, it has been shown that environmental and ecological differences between 

the no-till and conventional tillage can enhance the control of certain weed species in 

no-till cropping systems. With proper choice and manipulation of cover crops and 

residues, it is often possible to reduce the herbicides use. Thus, in eliminating tillage, 

by utilizing the surface mulch and allelochemicals leached from a killed cover crop 

and using most effective herbicides when needed, weed management has become 

much more effective in no-till. In North Carolina, these authors have grown soybean 

(Glycine max), tobacco (Nicotiana tabaccum), corn (Zea mays), sorghum (Sorghum 

bicolor) and sunflower (Helianthus annuus) in killed heavy mulches of rye (Secale 

cereale) without herbicides, other than a non-selective one to kill the rye. Earlyseason 

control of broadleaf weeds such as sicklepod (Cassia obtusifolia), morningglory 

spp. (Ipomoea spp.), cocklebur (Xanthium strumarium), prickly sida (Sida spinosa), 

common purslane (Portulaca oleracea) and pigweed spp. (Amaranthus spp.) has been 

80 to 95%. Rye is the most weed suppressing cover crop among several small grains 

and subterranean clover (Trifolium subterraneum) and crimson clover (Trifolium 

incarnatum) the most suppressive legumes. This approach will still enhance 

agricultural sustainability because: (a) productive top-soil will be conserved, (b) 

herbicide use (especially preemergence herbicides) can be reduced and (c) herbicides 

for cover crop kill and postemergence selective herbicides, even if used, have little 

potential for environmental contamination. 

Staman et al. (2001) stated that in order to demonstrate that allelopathic 

interactions are occurring, one must, among other things, demonstrate that putative 

phytotoxins move from plant residues on or in the soil, the source, through the bulk 

soil to the root surface, a sink, by way of the rhizosphere. These authors hypothesized 

that the incorporation of phytotoxic plant residues into the soil would result in a 

simultaneous inhibition of seedling growth and a stimulation of the rhizosphere 

bacterial community that could utilize the putative phytotoxins as a carbon source. If 

true and consistently expressed, such a relationship would provide a means of 

establishing the transfer of phytotoxins from residue in the soil to the rhizosphere of 

a sensitive species under field conditions, presently, direct evidence for such transfer 

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is lacking. To test this hypothesis, cucumber seedlings were grown in soil containing 

various concentrations of wheat or sunflower tissue. Both tissue types contain phenolic 

acids, which have been implicated as allelopathic phytotoxins. The level of 

phytotoxicity of the plant tissues was determined by the inhibition of pigweed seedling 

emergence and cucumber seedling leaf area expansion. The stimulation of cucumber 

seedling rhizosphere bacterial communities was determined by the plate dilution 

frequency technique using a medium containing phenolic acids as the sole carbon 

source. When sunflower tissue was incorporated into autoclaved soil (to reduce the 

initial microbial populations), a simultaneous inhibition of cucumber seedling growth 

and stimulation of the community of phenolic acid utilizing rhizosphere bacteria 

occurred. Thus, it was possible to observe simultaneous inhibition of cucumber 

seedlings and stimulation of phenolic acid utilizing rhizosphere bacteria, and therefore 

provide indirect evidence of phenolic acid transfer from plant residues in the soil to 

the root surface. However, the simultaneous responses were not sufficiently consistent 

to be used as a field screening tool but were dependent upon the levels of phenolic 

acids and the bulk soil and rhizosphere microbial populations present in the soil. It is 

possible that this screening procedure may be useful for phytotoxins that are more 

unique than phenolic acids. Such an inverse relationship between phytotoxicity and 

the response of rhizosphere bacterial populations was also observed by Blum et al. 

(2000), and such interactions provide indirect evidence for the transfer of 

allelochemicals from the plant root to the rhizosphere. 

In relation with resistance of weeds to herbicides, Duke et al. (2000) mentioned 

that new mechanisms of action for herbicides are highly desirable to fight evolution 

of resistance in weeds, to create or exploit unique market niches, and to cope with 

new regulatory legislation. Comparison of the known molecular target sites of synthetic 

herbicides and natural phytotoxins reveals that there is little redundancy. Comparatively 

little effort has been expended on determination of the sites of action of phytotoxins 

from natural sources, suggesting that intensive study of these molecules will reveal 

many more novel mechanisms of action. These authors gave some examples of natural 

products that inhibit unexploited steps in the amino acid, nucleic acid, and other 

biosynthetic pathways: AAL-toxin, hydantocidin, and various plant-derived terpenoids. 

Natural products have not been utilized as extensively for weed management as 

they have been for insect and plant pathogen management, but there are several notable 

successes such as glufosinate and the natural product-derived triketone herbicides. 

The molecular target sites of these compounds are often unique. Strategies for the 

discovery of these materials and compounds are outlined by Duke et al. (2002a). 

Numerous examples of individual phytotoxins and crude preparations with weed 

management potential are provided by these authors. They described an example of 

research to find a natural product solution of a unique pest management problem 

(blue-green algae in aquaculture), and mentioned the two fundamental approaches to 

the use of natural products for weed management: i) as a herbicide or a lead for a 

synthetic herbicide and ii) use in allelopathic crops or cover crops (Duke et al., 2002b). 

As it was mentioned, crops may be genetically engineered for weed management 

purposes by making them more resistant to herbicides or by improving their ability to


MOLECULES 

interfere with competing weeds. Transgenes for bromoxynil, glyphosate, and 

glufosinate resistance are found in commercially available crops. Other herbicide 

resistance genes are in development. Glyphosate-resistant crops have had a profound 

effect on weed management practices in North America, reducing the cost of weed 

management, while improving flexibility and efficacy. In general, transgenic, herbicideresistant 

crops have reduced the environmental impact of weed management because 

the herbicides with which they are used are generally more environmentally benign 

and have increased the adoption of reduced-tillage agriculture. Crops could be given 

an advantage over weeds by making them more competitive or altering their capacity 

to produce phytotoxins (allelopathy). Strategies for producing allelopathic crops by 

biotechnology are relatively complex and usually involve multiple genes. One can 

choose to enhance production of allelochemicals already present in a crop or to impart 

the production of new compounds. The first strategy involves identification of the 

allelochemical(s), determination of their respective enzymes and the genes that encode 

them, and, the use of genetic engineering to enhance production of the compound(s). 

The latter strategy would alter existing biochemical pathways by inserting transgenes 

to produce new allelochemicals (Duke et al., 2002c).(See controversy between organic 

agriculture and biotechnology - Control of Weeds and Management of Agroecosystems, 

Pag. 18, first paragraph) 

More sophisticated techniques will be used to search for alternative to herbicides 

in agroecosystems. The use of winter cover crops is beneficial to agriculture. Stanislaus 

and Cheng (2002) tried to design a cover crop that self-destructs in response to an 

environmental cue, thereby eliminating the use of herbicides and tillage to remove 

the cover crop in late spring. Here, this novel concept is tested in a model system. The 

onset of summer brings with it elevated temperatures. Using this as the environmental 

cue, a self-destruction cassette was designed and tested in tobacco. A heat-shockresponsive 

promoter was used to direct expression of the ribonuclease Barnase. Because 

Barnase is extremely toxic to cells, it was necessary to coexpress its inhibitor, Barstar, 

whose expression was under the control of the CaMV 35S promoter. The wild-type 

and two mutated Barnase genes, one missense and one translation attenuated, were 

tested. The results indicated that the translation-attenuated version of the Barnase 

gene was most effective in causing heat-shock-regulated plant death. Analysis of the 

T-2 progeny of a transgenic plant carrying this Barnase mutant showed that the Barnase 

gene expression was sixfold higher in heat-shock-treated plants compared with 

untreated plants. This level of Barnase gene expression was sufficient to kill transgenic 

plants. 

Many advances in disciplines such as chemistry, biochemistry, plant breeding, 

genetics, engineering, and others have been applied in a positive manner to improve 

knowledge in weed science. The emerging field of genomics is likely to have a similar 

positive effect on our understanding of weeds and their management in various plant 

agriculture systems. Genomics involves the large-scale use of molecular techniques 

for identification and functional analysis of complete or nearly complete genomic 

complements of genes. Commercial application of genomics has already occurred for 

improvement in certain crop input and output traits, including improved quality 

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characteristics and herbicide and insect resistance. Additional commercial applications 

of genomics in weed science will be identification of genes involved in crop ability. 

Genes controlling early crop root emergence, rapid early-season leaf and root 

development for fast canopy closure, production of allelochemicals for natural weed 

control, identification of novel herbicide target sites, resistance mechanisms, and genes 

for protecting crops against specific herbicides can and will be identified. Successful 

crop improvement in these areas using the tools of genomics will dramatically affect 

weed-crop interactions and improve crop yields while reducing weed problems. In 

relation to improved basic knowledge of weeds and the resulting ability to improve 

our weed management techniques, genomics will offer the weed science community 

many new and exciting research opportunities. Scientists will be able to determine 

the genetic composition of weed populations and how it changes over time in relation 

to agricultural practices, Identification of genes contributing to weediness, perennial 

growth habit, herbicide resistance, seed and vegetative structure dormancy, plant 

architecture and morphology, plant reproductive characters (outcrossing and 

hybridization, introgression), and allelopathy will be identified and utilized with highthroughput 

DNA sequencing and other genomics-based technologies. Using genomics 

to improve our understanding of weed biology by determining which genes function 

to affect the fitness, competitiveness, and adaptation of weeds in agricultural 

environments will allow the development of improved management strategies. 

Information is provided concerning the current state of molecular research in various 

areas of weed science and specific genomic research currently being conducted at 

Purdue University using transfer DNA (TDNA) activation tagging to generate large 

populations of mutated plants that can be screened for genes of importance to weed 

science (Weller et al., 2001). 

6. SUPPRESSIVE SOILS 

When soils are characterized by a very low level of disease development even though 

a virulent pathogen and susceptible host are present, they are known as suppressive 

soils. Biotic and abiotic elements of the soil environment contribute to suppressiveness, 

however most defined systems have identified biological elements as primary factors 

in disease suppression. Many soils possess similarities with regard to microorganisms 

involved in disease suppression, while other attributes are unique to specific pathogensuppressive 

soil systems. The organisms’ operative in pathogen suppression does so 

via diverse mechanisms including competition for nutrients, antibiosis and induction 

of host resistance (Mazzola, 2002). Non-pathogenic Fusarium spp. and fluorescent 

Pseudomonas spp. play a critical role in naturally occurring soils that are suppressive 

to Fusarium wilt. Suppression of take-all of wheat, caused by Gaeumannomyces 

graminis var. tritici, is induced in soil after continuous wheat monoculture and is 

attributed, in part, to selection of fluorescent pseudomonads with capacity to produce 

the antibiotic 2,4-diacetylphloroglucinol. Cultivation of orchard soils with specific 

wheat varieties induces suppressiveness to Rhizoctonia root rot of apple caused by 

Rhizoctonia solani AG 5. Wheat cultivars that stimulate disease suppression enhance


MOLECULES 

populations of specific fluorescent pseudomonad genotypes with antagonistic activity 

toward this pathogen. Methods that transform resident microbial communities in a 

manner which induces natural soil suppressiveness have potential as components of 

environmentally sustainable systems for management of soilborne plant pathogens 

(Mazzola, 2002). 

Actually, agricultural soils suppressive to soilborne plant pathogens occur 

worldwide, and for several of these soils the biological basis of suppressiveness has 

been described. Two classical types of suppressiveness are known. General suppression 

owes its activity to the total microbial biomass in soil and is not transferable between 

soils. Specific suppression owes its activity to the effects of individual or select groups 

of microorganisms and is transferable. The microbial basis of specific suppression to 

four diseases, Fusarium wilts, potato scab, apple replant disease, and take-all, is 

discussed by Weller et al. (2002). One of the best-described examples occurs in takeall 

decline soils. In Washington State, take-all decline results from the buildup of 

fluorescent Pseudomonas spp. that produce the antifungal metabolite 2,4-diacetylphloroglucinol. 

Producers of this metabolite may have a broader role in diseasesuppressive 

soils worldwide. By coupling molecular technologies with traditional 

approaches used in plant pathology and microbiology, it is possible to dissect the 

microbial composition and complex interactions in suppressive soils. 

In three of 12 soils obtained from agricultural fields in California, population 

density development of Meloidogyne incognita under susceptible tomato was 

significantly suppressed when compared to identical but methyl iodide (MI)-fumigated, 

M. incognita re-infested soils. When the 12 soils were infested with second-stage 

juveniles (J2) of M. incognita and the juveniles were extracted after 3 days, significantly 

fewer J2 were recovered from 9 of the 12 non-treated soils than from the MI-fumigated 

equivalents. In one of the 12 soils, infestation 3 weeks before planting resulted in 

lower nematode population densities than infestation at planting in both MI-fumigated 

and non-treated soil. The combination of infestation 3 weeks before planting with 

infestation at planting did not alter the occurrence or degree of root-knot nematode 

suppressiveness (Pyrowolakis et al., 2002). 

Yin et al. (2004) established that for suppressive soils that have a biological 

nature, one of the first steps in understanding them is to identify the organisms 

contributing to this phenomenon. They presented a new approach for identifying 

microorganisms involved in soil suppressiveness. This strategy identifies 

microorganisms that fill a niche similar to that of the pathogen by utilizing substrate 

use assays in soil. To demonstrate this approach, they examined an avocado grove 

where a Phytophthora cinnamomi epidemic created soils in which the pathogen could 

not be detected with baiting techniques, a characteristic common to many soils with 

suppressiveness against P. cinnamomi. Substrate utilization assays were used to identify 

rRNA genes (rDNA) from bacteria that rapidly grew in response to amino acids known 

to attract P. cinnamomi zoospores. Six bacterial rDNA intergenic sequences were 

prevalent in the epidemic soils but uncommon in the non-epidemic soils. These 

sequences belonged to bacteria related to Bacillus mycoides, Renibacterium 

salmoninarum, and Streptococcus pneumoniae. We hypothesize that bacteria such as 

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these, which respond to the same environmental cues that trigger root infection by the 

pathogen, will occupy a niche similar to that of the pathogen and contribute to 

suppressiveness through mechanisms such as nutrient competition and antibiosis. 

A similar experimental approach was developed by Borneman et al. (2004) for 

identifying microorganisms involved in specified functions such as pathogen 

suppressiveness. In this approach, it was postulated that the microorganisms involved 

in pathogen suppressiveness could be discovered by identifying those organisms whose 

populations positively correlate with high levels of suppressiveness. The approach 

has three phases. The first phase is to identify bacterial and fungal rRNA genes (rDNA) 

from soils possessing various levels of suppressiveness. Ribosomal DNA sequences 

that are more abundant in the highly suppressive soils than in the less suppressive 

soils are considered candidate sequences. A method termed oligonucleotide 

fingerprinting of rRNA genes (OFRG) is used to obtain extensive analysis of microbial 

community composition. The second phase of this experimental approach is to verify 

the results obtained from phase one using quantitative PCR. Here, selective PCR 

primers for each of the candidate rDNA sequences are designed. These primers are 

then used to determine the relative amounts of the candidate sequences in soils 

possessing various levels of suppressiveness produced by several different methods 

such as mixing various quantities of suppressive and fumigation-induced nonsuppressive 

soil, biocidal treatments and temperature treatments. In phase three, the 

organisms that consistently correlate with suppressiveness are isolated and amended 

to non-suppressive soils to assess their abilities to produce suppressiveness. The utility 

of this experimental approach was demonstrated by using it to identify microorganisms 

involved in suppressiveness against the plant-parasitic nematode, Heterodera schachtii. 

This general experimental approach should also be useful for the identifying 

microorganisms involved in functions other then pathogen suppressiveness. 

Kloepper et al. (1999) discussed concepts and examples of how naturally occurring 

bacteria (plant-associated bacteria residing in the rhizosphere, phyllosphere, and inside 

tissues of healthy plants –endophytic), and introduced bacteria may contribute to 

management of soilborne and foliar diseases. Some introduced rhizobacteria have 

been found to enhance plant defences, leading to systemic protection against foliar 

pathogens upon seed or root-treatments with the rhizobacteria. In these cases, 

introduction of the rhizobacteria results in reduced damage to multiple pathogens, 

including viruses, fungi and bacteria. An alternative strategy to the introduction of 

specific antagonists is the augmentation of existing antagonists in the root environment. 

This augmentation may result from the use of specific organic, amendments, such as 

chitin, which stimulate populations of antagonists, thereby inducing suppressiveness. 

Intercropping or crop rotation with some tropical legumes, including velvetbean 

(Mucuna deeringiana), lead to management of phytoparasitic nematodes, partly 

through stimulation of antagonistic microorganisms. Some biorational nematicides, 

such as specific botanical aromatic compounds, also appear to induce suppressiveness 

through alterations in the soil microbial community. 

Single isolates of bacterial endophytes, obtained from the nematode antagonistic 

plant species African (Tagetes erecta) and French (T. patula) marigold, were introduced


MOLECULES 

into potatoes (Solanum tuberosum). Several bacterial species possessed activity against 

root-lesion nematodes (Pratylenchus penetrans) in soils around the root zone of 

potatoes, namely: Microbacterium esteraromaticum, Tsukamurella paurometabolum, 

isolate TP6, Pseudomonas chlororaphis, Kocuria varians and K. kristinae. Of these, 

M. esteraromaticum and K. varians depressed the population densities of root-lesion 

nematodes without incurring any yield penalty (tuber wet weight). No significant 

differences were found in the total numbers of P. penetrans nematodes, rhabditid 

nematodes or ‘other’ parasitic nematode species within the root tissues of bacterized 

potato plants compared to the unbacterized check. Overall, tuber fresh weights and 

tuber number were equal to or significantly lower (P < 0.05) in bacterized plants than 

their unbacterized counterpart (Sturz and Kimpinski, 2004). The authors of this study 

conclude that endoroot bacteria from Tagetes spp. can play a role in nematode 

suppression through the attenuation of nematode proliferation, and proposed that 

these nematode control properties are capable of transfer to other crops in a rotation 

as a beneficial ‘residual’ microflora – a form of beneficial microbial allelopathy. 

In relation with this same type of study, Hallman et al. (1998) performed a 

greenhouse experiments with cotton and cucumber to determine the effects of 

inoculation of the parasitic nematode Meloidogyne incognita on population dynamics 

of indigenous bacterial endophytes and introduced endophytic bacterial strains JM22 

(Enterobacter asburiae) and 89B-61 (Pseuedomonas fluorescens) applied as seed 

treatments. Internal communities of endophytic bacteria in roots were generally largest 

in the presence of M. incognita. Recovery of JM22 from cucumber roots was positively, 

but not significantly, associated with soilborne nematode inoculum size, except at 2 

weeks after inoculation. The internal populations of 89B-61 applied to seed also 

increased with nematode applications. The diversity of indigenous bacterial endophytes 

changed within 7 d after M. incognita inoculation. Species richness and diversity of 

endophytic bacteria were slightly, but not significantly, greater for nematode-infested 

plants than for non-infested plants. Alcaligenes piechaudii and Burkholderia pickettii 

occurred only in nematode-infested plants, whereas Bievundimonas vesicularis was 

mainly isolated from nematode-free plants. Agrobacterium radiobacter and 

Pseudomonas spp. were the most common taxa found in both treatments, accounting 

for a total of 41% and 37% of the community for non-inoculated and inoculated 

plants, respectively. JM22 colonized cotton roots internally and was also found in 

high numbers on the root surface around nematode penetration sites and on root galls 

where the root tissue had been disruptured due to gall enlargement. Single cells of 

JM22 were attached to the cuticle of M. incognita juveniles. Sturz et al. (2000) assesses 

that endophytic bacteria and M. incognita form complex associations and an 

understanding of these associations will aid efforts to develop and manage microbial 

communities of endophytic bacteria for practical use as biocontrol agents against 

plant-parasitic nematodes and soil-borne pests and pathogens. 

In addition, Postma et al. (2003) found that compost amended soil has also been 

found to be suppressive against plant diseases in various cropping systems. The level 

and reproducibility of disease suppressive properties of compost might be increased 

by the addition of antagonists. In this study, the establishment and suppressive activity 

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of two fungal antagonists of soil-borne diseases was evaluated after their inoculation 

in potting soil and in compost produced from different types of organic waste and at 

different maturation stages. The fungal antagonists Verticillium biguttatum, a 

mycoparasite of Rhizoctonia solani, and a non-pathogenic isolate of Fusarium 

oxysporum antagonistic to Fusarium wilt, survived at high levels (10 3 -10 5 CFU g -1 ) 

after 3 months incubation at room temperature in green waste compost and in potting 

soil. Their populations faded-out in the organic household waste compost, especially 

in the matured product. In bioassays with R. solani on sugar beet and potato, the 

disease suppressiveness of compost increased or was similar after enrichment with V. 

biguttatum. The largest effects, however, were present in potting soil, which was very 

conducive for the disease as well as the antagonist. Similar results were found in the 

bioassay with F. oxysporum in carnation where enrichment with the antagonistic F. 

oxysporum had a positive or neutral effect. Postma et al. (2003) foresee great potential 

for the application of antagonists in agriculture and horticulture through enrichment 

of compost or potting soil with antagonists or other beneficial micro-organisms. 

All soils are suppressive to phytonematodes to some degree. The degree of 

suppressiveness to them or other soilborne pathogens in a soil can be enhanced not 

only by infesting soil with selected microorganisms, but by the use of appropriate 

cropping systems and the application to soil of specific organic amendments or chemical 

compounds. Conducive cropping systems such as monoculture can reduce soil 

suppressiveness to the point where the soil is not resistant to plant parasitic nematodes 

(Wang et al., 2002). 

Inorganic fertilizers containing ammoniacal nitrogen or formulations releasing 

this form of N in the soil are most effective for suppressing nematode populations. 

Anhydrous ammonia has been shown to reduce soil populations of Tylenchorhynchus 

claytoni, Helicotylenchus dihystera, and Heterodera glycines. The rates required to 

obtain significant suppression of nematode populations are generally in excess of 150 

kg N/ha. Urea also suppresses several nematode species, including Meloidogyne spp., 

when applied at rates above 300 kg N/ha. Additional available carbon must be provided 

with urea to permit soil microorganisms to metabolize excess N and avoid phytotoxic 

effects. There is a direct relation between the amount of “protein” N in organic 

amendments and their effectiveness as nematode population suppressants. Most 

nematicidal amendments are oil cakes, or animal excrements containing 2-7% (w/w) 

N; these materials are effective at rates of 4-10 t/ha. Organic soil amendments 

containing mucopolysaccharides (e.g., mycelial wastes, chitinous matter) are also 

effective nematode suppressants (Rodriguez-Kábana, 1986). 

Vargas-Ayala and Rodriguez-Kábana (2001) established a field microplot trial to 

evaluate nematode population dynamics in a rotation program utilizing nematodesuppressive 

and non-suppressive legumes, and nematode-host and nonhost grass 

species. The rotation treatments consisted of velvetbean (Mucuna deeringiana) or 

cowpea (Vigna unguiculata) during the first year, followed in winter by oat (Avena 

sativa), wheat (Triticum aestivum), rye (Secale cereale), rye grass (Lolium sp.), clover 

(Trifolium sp.), hairy vetch (Vicia villosa), lupine (Lupinus sp.) or fallow. Rotation in


MOLECULES 

the second and third year consisted of soybean (Glycine max). Results showed that 

velvetbean had a generally suppressive effect on populations of root-knot (Meloidogyne 

incognita), cyst (Heterodera glycines), and stunt (Tylenchorhynchus claytoni) 

nematodes in soil and roots. It had little effect on populations of Helicotylenchus 

dihystera. Velvetbean rotations with winter grass species were also effective in reducing 

nematode population densities in soil. Soybean yields were positively correlated with 

velvetbean in rotations with winter grass species. High populations of M. incognita 

were negatively correlated with soybean yields. The use of velvetbean as a rotation 

crop assures reduction of important plant-parasitic nematodes in soil and an 

improvement in soybean yield. 

Wang et al. (2002) made an extensive review on the use of Crotalaria spp. 

(Fabaceae) as a suppressor of agricultural pests, particularly nematodes. These authors 

summarized the knowledge of the efficacy of Crotalaria spp. for plant-parasitic 

nematode management, described the mechanisms of nematode suppression, and 

outline prospects for using this crop effectively. They mentioned that Crotalaria is a 

poor host to many plant-parasitic nematodes including Meloidogyne spp., 

Rotylenchulus reniformis, Radopholus similis, Belonolaimus longicaudatus, and 

Heterodera glycines. It is also a poor or non-host to a large group of other pests and 

pathogens. Besides, Crotalaria is competitive with weeds without becoming a weed, 

grows vigorously to provide good ground coverage for soil erosion control, fixes 

nitrogen, and is a green manure. However, most Crotalaria species are susceptible to 

Pratylenchus spp., Helicotylenchus sp., Scutellonema sp. and Criconemella spp. 

Crotalaria species are used as preplant cover crops, intercrops, or soil amendments. 

When used as cover crops, Crotalaria spp. reduces plant-parasitic nematode 

populations by: i) acting as a nonhost or a poor host, ii) producing allelochemicals 

that are toxic or inhibitory, iii) providing a niche for antagonistic flora and fauna, and 

iv) trapping the nematode. 

A non-host to a nematode species is a plant in which the nematode fails to 

reproduce. A plant is considered as resistant to nematodes when these fail to live 

inside the host or early dead in the host; they decreased the production of eggs; or 

their growth or development are inhibited by the plant (Wang et al., 2002). 

Allelopathic effects of the plant against nematodes were described as a mechanism 

of suppression of nematodes. Soler-Serratosa et al. (1996) evaluated the nematicidal 

activity of thymol, a phenolic monoterpene present in the essential oils of several 

plant families. Thymol was added to soil at rates 25-250 ppm. Initial and final 

population densities of Meloidogyne arenaria, Heterodera glycines, Paratrichodorus 

minor, and Dorylaimoid nematodes, as well as disease incidence, declined sharply 

with increased dosages of thymol. Thymol was also applied at 0, 50, 100, and 150 

ppm to soil in combination with 0, 50, and 100 ppm benzaldehyde, an aromatic 

aldehyde present in nature as a moiety of plant cyanogenic glycosides. Combinations 

in which benzaldehyde was applied at 100 ppm showed synergistic effects in 

suppressing initial and final soil populations of M. arenaria and H. glycines. Significant 

reductions in root galling and cyst formation in soybean were attributable to thymol 

at ≥ 50 ppm. 

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Hallmann and Sikora (1996) confirmed that endophytic fungi isolated from the 

cortical tissue of surface sterilized tomato roots collected from field plots produced 

secondary metabolites in nutrition broth that were highly toxic to Meloidogyne 

incognita. Especially strains of Fusarium oxysporum were highly active with 13 of 15 

strains producing culture filtrates toxic to nematodes. They investigated also the 

mechanism of action of the toxic metabolites produced by the non-pathogenic F. 

oxysporum strain 162 with proven biological control of M. incognita in pot experiments. 

These metabolites reduced M. incognita mobility within 10 min of exposure. After 60 

min, 98% of juveniles were inactivated. Fifty percent of juveniles with exposure of 5 

h were dead, and 24 h exposure resulted in 100% mortality. In a bioassay with lettuce 

seedlings metabolite concentrations >100 mg/l reduced the number of M. incognita 

juveniles on the roots comparing to the water control. The F. oxysporum toxins were 

highly effective towards sedentary parasites and less effective towards migratory 

endoparasites. Non-parasitic nematodes were not influenced at all. Metabolites of 

strain 162 also reduced significantly the growth of Phytophthora cactorum, Pythium 

ultimum and Rhizoctonia solani in vitro. 

Three out of 15 bacterial strains preselected for antagonistic activity in different 

pathosystems showed biocontrol activity towards Meloidogyne incognita on lettuce 

and tomato as described by Hoffmann-Hergarten et al. (1998). They found that seed 

treatment with the rhizobacteria Pseudomonas sp. W34 or Bacillus cereus S18 resulted 

in significant reductions in root galling and enhanced seedling biomass. The yield 

response of M. incognita-infested tomato was tested in a long-term pot experiment 

using three antagonistic bacteria, i.e., Pseudomonas sp. W34, Bacillus cereus S18 

and Bacillus subtilis VM1-32. Significant reduction in M. incognita gall index was 

observed within 18 weeks after inoculation with all three bacterial strains. B. cereus 

S18 caused a 9 % yield increase when compared with the nematode control and thereby 

compensated for the yield loss due to nematode infection. Early maturity of fruits on 

M. incognita-infested tomato plants after inoculation of B. cereus S18 was observed 

when compared with both the nematode and the untreated control. 

Vargas-Ayala et al. (2000) hypothesized that the induction of soil suppressiveness 

to plant parasitic nematodes that occurs following planting of velvetbean (Mucuna 

deeringiana) is associated with the development of an antagonistic microflora in soils 

and rhizospheres. They performed a crop rotation study in microplots, consisting of 

three crop cycles. Cycle 1 involved planting of either velvetbean or cowpea (Vigna 

unguiculata) in the first spring. Cycle 2 during the next fall and winter was fallow or 

cover-cropped with wheat (Triticum aestivum) or crimson clover (Trifolium 

incarnatum). Cycle 3 the next spring was soybean (Glycine max). Rhizosphere fungal 

populations were significantly smaller on velvetbean than on cowpea at the end of 

cycle 1. The use of velvetbean in cycle 1 significantly decreased rhizosphere bacterial 

populations on crops in cycle 2, compared to treatments which had cowpea in cycle 1. 

Velvetbean also influenced bacterial diversity, generally increasing frequency of bacilli, 

Arthrobacter spp. and Burkholderia cepacia, while reducing fluorescent 

pseudomonads. Some of these effects persisted through cycle 3. Fungal diversity was 

influenced in cycle 1 by velvetbean; however, effects generally did not persist through

cycles 2 and 3. The results indicate that the use of velvetbean in a cropping system 

alters the microbial communities of the rhizosphere and soil, and they are consistent 

with the hypothesis that the resulting control of nematodes results from induction of 

soil suppressiveness. 

6.1. Resistence through natural compounds 

Sometimes, natural compounds that confer resistance to a plant against nematode 

infestation could be of foreign origin (Reitz et al., 2000). Recent studies have shown 

that living and heat-killed cells of the rhizobacterium Rhizobium etli strain G12 induce 

in potato roots systemic resistance to infection by the potato cyst nematode Globodera 

pallida. To better understand the mechanisms of induced resistance, Reitz et al. (2000) 

focused on identifying the inducing agent. Since heat-stable bacterial surface 

carbohydrates such as exopolysaccharides (EPS) and lipopolysaccharides (LPS) are 

essential for recognition in the symbiotic interaction between Rhizobium and legumes, 

their role in the R. etli-potato interaction was studied. EPS and LPS were extracted 

from bacterial cultures, applied to potato roots, and tested for activity as an inducer of 

plant resistance to the plant-parasitic nematode. Whereas EPS did not affect G. pallida 

infection, LPS reduced nematode infection significantly in concentrations as low as 1 

and 0.1 mg ml(-1). Split-root experiments, guaranteeing a spatial separation of inducing 

agent and challenging pathogen, showed that soil treatments of one half of the root 

system with LPS resulted in a highly significant (up to 37%) systemic induced reduction 

of G. pallida infection of potato roots in the other half. The results clearly showed 

that LPS of R. etli G12 act as the inducing agent of systemic resistance in potato 

roots. 

In relation with Crotalaria spp. secondary metabolites, it is well known that 

these plants produce pyrrolizidine alkaloids and monocrotaline which have high 

vertebrate toxicity and could potentially be toxic to nematodes; but it is possible also 

that the low C/N ratio of Crotalaria may also contribute to its allelopathic effect 

against nematodes. Materials with very low C/N or high content of ammonia will 

either result in plasmolysis of nematodes, or proliferation of nematophagous fungi 

due to the release of NH4+-N (Rich and Rahi, 1995). 

6.2. Soil Amendments 


MOLECULES 

A study was conducted to determine the effects of combinations of organic amendments 

and benzaldehyde on plant-parasitic and non-parasitic nematode populations, soil 

microbial activity, and plant growth (Chavarria-Carvajal et al., 2001). Pine 

bark, velvetbean and kudzu were applied to soil at rates of 30 g/kg and paper waste at 

40 g/kg alone and in combination with benzaldehyde (300 mul/kg), for control of 

plant-parasitic nematodes. Pre-plant and post-harvest soil and soybean root samples 

were analyzed, and the number of parasitic and non-parasitic nematodes associated 

with soil and roots were determined. Soil samples were taken at 0, 2, and 10 weeks 

after treatment to determine population densities of bacteria and fungi. Treatment 

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effects on microbial composition of the soybean rhizosphere were also determined by 

identifying microorganisms. Bacteria strains were identified rising fatty acid analysis, 

and fungus identification was done rising standard morphological measurements and 

appropriate taxonomic keys. Results showed that most amendments alone or in 

combination with benzaldehyde reduced damage from plant parasitic nematodes. 

Benzaldehyde applied alone or in combination with the amendments exerted a selective 

action on the activity and composition of microbial populations in the soybean 

rhizosphere. In control soils the bacterial flora was predominantly Gram-negative, 

while in soils amended with velvetbean or kudzu alone or with benzaldehyde. Grampositive 

bacteria were dominant. Mycoflora promoted by the different amendments or 

combinations with benzaldehyde included species of Aspergillus, Myrothecium, 

Penicillium, and Trichoderma. 

Calvet et al. (2001) evaluated the survival of two species of plant parasitic 

nematodes: the root-lesion nematode Pratylenchus brachyurus, and the root-knot 

nematode Meloidogyne javanica, in saturated atmospheres of 12 natural chemical 

compounds. The infectivity of two isolates of arbuscular mycorrhizal fungi: Glomus 

mosseae and Glomus intraradices, under identical experimental conditions, was also 

determined. All the compounds tested exerted a highly significant control against M. 

javanica and among them, benzaldehyde, salicilaldehyde, borneol, p-anisaldehyde 

and cinnamaldehyde caused a mortality rate above 50% over P. brachyurus. The 

infectivity of G. intraradices was inhibited by cinnamaldehyde, salicilaldehyde, thymol, 

carvacrol, p-anisaldehyde, and benzaldehyde, while only cinnamaldehyde and thymol 

significantly inhibited mycorrhizal colonization by G. mosseae. 

When soybean plant responses to Meloidogyne incognita infestation were 

compared to resistant (Bryan) and susceptible (Brim) cultivars at 0, 1, 3, 10, 20, and 

34 days after infestation, Qiu and collaborators (1997) observed that the resistant 

cultivar had higher chitinase activity than the susceptible cultivar at every sample 

time beginning at the third day. Results from isoelectric focusing gel electrophoresis 

analyses indicated that three acidic chitinase isozymes with isoelectric points (pIs) of 

4.8, 4.4, and 4.2 accumulated to a greater extent in the resistant compared to the 

susceptible cultivar following challenge. SDS-PAGE analysis of root proteins revealed 

that two proteins with molecular weights of approximately 31 and 46 kD accumulated 

more rapidly and to a higher level in the resistant than in the susceptible cultivar. 

Additionally, three major protein bands (33, 22, and 20 kD) with chitinase activity 

were detected with a modified SDS-PAGE analysis in which glycolchitin was added 

into the gel matrix. These results indicate that higher chitinase activity and early 

induction of specific chitinase isozymes may be associated with resistance to rootknot 

nematode in soybean. 

Antagonists, most likely favored by selected cover crops, include mainly fungal 

egg parasites, trapping fungi, endoparasitic fungi, fungal parasites of females, 

endomycorrhizal fungi, planthealth promoting rhizobacteria, and obligate bacterial 

parasites. There are several hypotheses on how cover crops can enhance nematodeantagonistic 

activities. A series of ecological events may be involved. The decomposing


MOLECULES 

organic material is a significant event because the bacteria which proliferate after 

organic matter incorporation become a food base for microbiovorous nematodes. In 

turn, these nematodes serve as a food source for nematophagous fungi (Wang et al., 

2002). Leguminous crops enhance nematophagous fungi better than other crops. 

Rootknot symptoms were reduced more by alfalfa amendments in a 4-year microplot 

test than by chemical fertilization of plots (Mankau, 1968). Microplots amended with 

alfalfa meal increased nematode-trapping fungal activity of Drechmeria coniospora 

(Van Den Boogert et al., 1994). Pea enhanced the densities and species diversity of 

nematode-trapping fungi more than white mustard or barley. In addition, formation 

of conidial traps of nematode-trapping fungi was more prevalent in the pea rhizosphere 

than in root-free soil (Persmark and Nordbring-Hertz, 1997; Persmark and Jansson, 

1997). 

Being a legume, Crotalaria juncea has characteristics that may make the crop 

useful for nematode antagonism. Plant exudates from Crotalaria spp. were selective 

for microbial species antagonistic to phytopathogenic fungi and nematodes (Rodriguez- 

Kábana and Kloepper, 1998). The changes in soil enzymatic activity was investigated 

by Chavarría and Rodriguez-Kábana (1998) when they incorporated four organic 

amendments (velvetbean, kudzu, pine bark, and urea-N) to the soil to evaluate their 

effects on the root-knot nematode (Meloidogyne incognita). The amendments were 

applied to nematode-infested soil at rates of 0 to 5% and placed in pots planted with 

‘Davis’ soybean (Glycine max). The number of M. incognita juveniles and nonparasitic 

nematodes associated with the soil and root tissues were determined after 8 weeks. 

Soil samples were taken at 0, 2, and 10 weeks after amendment application for 

determination of soil enzyme activities. Most organic amendments were effective in 

reducing root galling and root-knot nematodes and increasing populations of nonparasitic 

nematodes. Catalase and esterase were sharply increased by most rates of 

velvetbean, kudzu, and pine bark. Application of velvetbean, kudzu, and urea to soil 

stimulated urease activity in proportion to the amendment rates. Results suggest that 

complex modes of action operating in amended soils are responsible for suppression 

of M. incognita. 

In relation with nematode-trapping fungi, a major group of nematode antagonists, 

they can be enhanced by incorporation of residues of C. juncea. These fungi have 

been categorized into two groups: parasitic and saprophytic. The saprophytic group 

consists of predators characterized by sticky three-dimensional networks and nonspontaneous 

trap formation. These fungi have a saprophytic and a predatory (trap 

formation) phase. In the presence of nematodes, or even exudates and homogenates 

of nematodes, trap formation is induced. The parasitic group consists of nematodetrapping 

fungi that form constricting rings, adhesive knobs, or adhesive branches. 

These fungi form traps spontaneously, and thus are more effective trappers (Wang, 

2000). 

Among these two groups of nematode trapping fungi, the population densities of 

parasitic fungi are more likely to be enhanced by organic matter due to the rich microbial 

flora and fauna. The nematode trapping by these fungi are not nematode species- or 

trophic group specific, therefore the enhancement of nematode-trapping fungi by 

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organic matter incorporation should lead to increased trapping of plant-parasitic 

nematodes (Wang, 2000). 

Soil amended with C. juncea to give a 1:100 (w:w) concentration, enhanced 

parasitic nematode-trapping fungi, nematode egg parasitic fungi, vermiform stage 

parasites, and bacterivorous nematode population densities more efficiently than soil 

amended with chopped pineapple tissues or non-amended soil. Crotalaria juncea 

amendment enhanced the population densities of nematode-trapping fungi and the 

percentage of eggs parasit-ized by the fungi. Enhancement of nematode-trapping fungi 

was most effective in soils that had not been treated with 1,3-dichloropropene for at 

least 5 months. Suppression of R. reniformis by C. juncea amendment was correlated 

with parasitic nematode-trapping fungi, fungal egg parasites, and bacterivorous 

nematodes. Nematode-trapping fungi population densities were higher in C. juncea 

planted plots than weed fallow plots. However, four months after removal of C. juncea, 

and replacement with pineapple plants, the population densities of nematode-trapping 

fungi greatly decreased (Wang, 2000). 

Suppressive cropping systems rely on the use of precisely defined sequences of 

crops to increase populations and activities of naturally occurring antagonistic 

microorganisms in soil. Some crops such as velvetbean (Mucuna deerengiana) produce 

compounds which are directly toxic to nematodes and stimulate microbial antagonism 

to plant parasitic nematodes. These ‘active’ crops when included in cropping systems 

can increase suppressiveness of the system against nematodes. There are a number of 

active crops throughout the world which can be used in a practical manner to enhance 

naturally occurring biological control of plant parasitic nematodes (Wang, 2000) 

Rich and Rahi (1995) conducted two greenhouse trials to determine the influence 

of ground seed of castor (Ricinus communis), crotalaria (Crotalaria spectabilis), hairy 

indigo (Indigofera hirsuta), and wheat (Triticum aestivum) on tomato (Lycopersicon 

esculentum) growth and egg mass production of Meloidogyne javanica (test 1) or M. 

incognita (test 2). Ground seed from each plant species was individually mixed with 

an air-dried, fine sandy soil at rates of 0, 0.5, 1.0, and 2.0% (w/w). The mixtures were 

placed in one-liter plastic pots, and water was added to bring soil to field capacity. 

After ten days, 0 or 10 000 M. javanica or M. incognita eggs and juveniles were 

added to each pot. A single ‘Homestead’ tomato seedling was transplanted into each 

pot and allowed to grow for 70 days in test 1 and 75 days in test 2. Compared to the 

non-amended control, egg mass production was significantly reduced by all treatments 

except the 0.5% levels of wheat and castor and the 1.0% castor treatment. The 2.0% 

levels of ground seed of Crotalaria and hairy indigo almost completely suppresses 

egg mass production of both M. javanica or M incognita. With the exception of the 

1% Crotalaria treatment in test 2, total plant weight did not differ between treatments 

and the control. 

Morris and Walker (2002) mixed dried ground plant tissues from 20 leguminous 

species with Meloidogyne incognita-infested soil at 1, 2 or 2.5, and 5% (w/w) and 

incubated for 1 week at room temperature (21 to 27 0 C). Tomato (‘Rutgers’) seedlings 

were transplanted into infested soil to determine nematode viability. Most tissues


MOLECULES 

reduced gall numbers below the non-amended controls. The tissue amendments that 

were most effective include: Canavalia ensiformis, Crotalaria retusa, Indigofera 

hirsuta, I. nummularifolia, I. spicata, I. suffruticosa, I. tinctoria, and Tephrosia adunca. 

Although certain tissues reduced the tomato dry weights, particularly at the higher 

amendment rates (5%), some tissues resulted in greater dry weights. These nontraditional 

legumes, known to contain bioactive phytochemicals, may offer considerable 

promise as soil amendments for control of plant-parasitic nematodes. Not only do 

these legumes reduce root-knot nematodes but some of them also enhance plant height 

and dry weight. 

Nematode management is rarely successful in the long term with unitactic 

approaches. It is important to integrate multiple-tactics into a strategy. Crotalaria 

offers the potential to be one of the tactics. Some Crotalaria species are potential 

cover crops for managing several important plant-parasitic nematodes including 

Meloidogyne spp. and R. reniformis. Unfortunately, the residual effects are short term 

(a few months). Crotalaria, a poor host, generally helps reduce nematode population 

densities, but the number of nematodes will resurge on subsequent host crops. The 

damage threshold level, especially on longer-term crops, will often be reached or 

exceeded. This scenario strongly suggests that integrating the Crotalaria rotation 

system with other nematode management strategies is necessary. Among the 

possibilities for integration are crop resistance, enhanced crop tolerance, selection for 

fast growing crop varieties, soil solarization, and biological control. Chemical 

nematicides should be avoided in a cropping system if the objective is to enhance 

nematode-antagonistic microorganisms in the cropping system. Several studies have 

demonstrated the destructive effect of fumigation treatments to nematode antagonistic 

microorganisms. Crotalaria juncea amendments failed to enhance nematode-trapping 

fungi populations in soils that were recently treated with 1,3-dichloropropane. Wang 

et al. (2002) concluded that the major impediment to using Crotalaria is its shortterm 

effect in agricultural production systems, and suggested that integrating other 

pest management strategies with Crotalaria could offer promising nematode 

management approaches. 

7. CONCLUSIONS AND FINAL REMARKS 

In this chapter the different roles that allelopathy can play as a bioregulator tool in 

agriculture are discussed. A wide spectrum of studies are given on allelopathic plants 

and other organisms, the chemistry involved in these studies, the mechanisms of 

action of some allelochemicals, and the use of allelopathy to control weeds, pests 

(nematodes) and diseases. 

Many arguments can be given in favor of organic and sustainable agricultural 

practices as new forms of resources management such as multiple cropping, cover 

crops, organic compost, and biological controls for pests. Allelopathy is an emerging 

tool for a more biorational management of natural resources. However, allelopathy is 

not a simple panacea for the solution of ecological problems in agroecosystems or in 

natural ecosystems. It has not been considered as a universal ecological phenomenon; 

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allelopathy is a challenging and exigent matter of study. At present we have proof 

that secondary metabolites are involved with biotic interactions, and that allelopathic 

effects may restrict or enhance, alone or in relation with other environmental factors 

(light, temperature, humidity and nutrients), the distribution, health and growth of 

species in natural, artificial or managed communities. In the search for application of 

allelopathy knowledge is crucial to understand other biotic interactions (competition, 

defense against herbivory) and also the actual and full significance of a mixture of 

secondary metabolites all together acting in the environment (Anaya, 1999). 

Allelopathy typically operates through the release, modification, and joint action 

of a number of allelochemicals in a particular situation, and transitions through the 

soil add to the complications for explaining the phenomenon. The frontiers in research 

on allelopathy include isolation of additional compounds that may be involved, and 

determining more precisely how allelochemicals production is regulated and how the 

compounds function to inhibit growth. Such information may allow modification of 

crop plants so they have enhanced capability for weed suppression. Alternatively, 

new herbicides, pesticides, and growth regulators may be developed from some of 

plant and microorganisms compounds (Einhellig, 1989). 

In the study of biological interactions mediated by secondary metabolites it is 

very important to perform multidisciplinary investigations in a long term approach in 

order to understand these interactions from an holistic point of view and make use of 

them for beneficial purposes in the management of natural resources in agroecosystems. 

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SCOTT W. MATTNER 

THE IMPACT OF PATHOGENS ON PLANT 

INTERFERENCE AND ALLELOPATHY 

Department of Primary Industries, Knoxfield Centre, Victoria, Private Bag 

15, Ferntree Gully Delivery Centre, 3156, VIC, Australia. 

E-mail : scott.mattner@dpi.vic.gov.au 

Abstract. Pathogenesis can have both detrimental and beneficial impacts on plant fitness. As such, pathogens 

are important forces that influence the structure and dynamics in natural and manipulated plant ecosystems. 

Plant production and numbers within a community are constrained by environmental limitations, which are 

often mediated through plant interference. Competition for resources and allelopathy (chemical interactions) 

are the two most important ways that plants interfere with each other. This chapter reviews the effects of pathogens 

on the competitiveness and allelopathic ability of their hosts. In most cases, pathogens reduce the competitive 

ability of their host, making the host prone to displacement by neighbouring, resistant plants. However, pathogens 

may simultaneously increase the allelopathic ability of their hosts, thereby offsetting their loss in competitiveness 

to varying degrees. Evidence for enhanced allelopathy by infected plants comes in two forms: (i) pathogens 

stimulate the production of secondary metabolites by plants, many of which are implicated in allelopathy (eg 

phenolics), and (ii) field, glasshouse and bioassay studies showing that infected plants may suppress their 

neighbours more than healthy plants, under conditions of low competition. By conferring the benefit of increased 

allelopathy on their hosts, pathogens may maintain a self-advantage through increasing the survival chances of 

their hosts and ultimately themselves. The enhanced allelopathy of infected plants supports the ‘new function’ 

hypothesis, which suggests that pathogens evolve toward a mutualistic relationship with their host through the 

appearance of strains with beneficial effects on the host in addition to their detrimental effects. 


Plant pathogens are disease agents that live in or on their hosts and there obtain 

nutriment to the overall detriment of the plant. Fungal pathogens, which cause about 

70% of all major crop diseases (Deacon, 1997), are often characterised on the basis of 

two extremes in trophism, as necrotrophs or biotrophs (Lutrell, 1974; Parbery 1996). 

Necrotrophic organisms obtain nutriment from necrotic host tissues, which they kill 

prior to colonisation. Consequently, these pathogens although destructive to the host 

have little effect on the physiology of the rest of the plant. Biotrophic pathogens draw 

nutriment directly from living host tissue and can have a critical effect on host 

physiology. Many thorough reviews concerning the impact of infection on host 

physiology already occur in the literature (Goodman et al., 1986; Burdon, 1987; Ayres, 

1991; Sutic and Sinclair, 1991). By example though, pathogens can alter the 

partitioning of assimilates and dry matter; reduce net photosynthesis and increase 

respiration; increase water loss through transpiration and vulnerability to drought; 




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and reduce the overall growth, reproductive capacity and yield of their hosts. 

Consequently, pathogens are important factors that influence the composition and 

dynamics of natural and manipulated plant ecosystems. 

Despite pathogens having an overall detrimental effect on their host, some aspects 

of infection are beneficial (Burdon, 1991; Parbery, 1996). For example, infection 

may promote vegetative growth (Catherall, 1966; Wennström and Ericson, 1991), 

deter or limit grazing by herbivores (Morgan and Parbery, 1980), or increase the 

host’s scavenging ability for nutrients and water (Ahmad et al., 1982; Paul and Ayres, 

1988). By conferring some benefit on their host, these pathogens maintain a selfadvantage 

through increasing the chances of survival of their host and ultimately 

themselves. This is particularly important for obligate biotrophs (e.g. rusts, Uredinales; 

powdery mildews, Erysiphaceae) that require the host for self-preservation. 

In contrast to previous chapters that consider mechanisms for exploiting 

allelopathy for pathogen control, this chapter addresses the reverse situation – the 

impact of pathogens on plant interference and allelopathy. It reviews evidence that 

pathogens decrease the competitiveness and simultaneously increase the allelopathic 

ability of their hosts. The ability of pathogens to increase their host’s allelopathic 

ability may be an important means by which pathogens confer a benefit on their host. 

In so doing, these pathogens encourage the continuation of their host’s genotype into 

proceeding generations. Furthermore, the ability of pathogens to enhance allelopathy 

in their hosts may be one way that obligate biotrophic pathogens are evolving toward 

multualism. 

2. PLANT INTERFERENCE AND ALLELOPATHY 

2.1. Components of Interference 

Harper (1961) introduced the term ‘interference’ to encompass all effects placed on 

an organism by the proximity of neighbours. Plant interference is ‘the response of an 

individual plant to its total environment as this is modified by the presence and/or 

growth of other individuals’ (Begon et al., 1986). Interference may have a positive or 

negative effect on plant growth and may occur between plants of different species, 

between individuals within the same species or even between individual organs on a 

single plant. Some of the important ways plants modify the environment in each 

other’s presence are through: competition, allelopathy, protection (e.g. when an 

unpalatable plant protects a neighbouring palatable plant from grazing), direct 

stimulation (e.g. when nitrogen fixed by a legume becomes available to a non-legume), 

direct contact (e.g. when a thorny plant mechanically abrades neighbouring plants in 

the wind), and non-competitive inhibition (e.g. when a tree provides a rubbing post 

for grazers and so encourages local trampling). Of these mechanisms, competition is 

the most dominant in shaping plant populations (Jolliffe, 1988; Tilman, 1988), but 

research is increasingly demonstrating the importance of allelopathy (Siegler, 1996; 

Wardle et al., 1998). Comprehensive reviews of competition ( Milthorpe, 1961; Tilman,

IMPACT OF PATHOGENS ON PLANT INTERFERENCE AND 

ALLELOPATHY 

81 

1988; Grace and Tilman, 1990; Casper and Jackson, 1997) and allelopathy (Putnam 

and Duke, 1978; Rice, 1984; Inderjit et al., 1995; Anaya, 1999) already occur in the 

literature. 

The view of competition in the present chapter is ‘the interaction between 

individuals brought about by the shared requirements for a resource in limited supply, 

and leading to a reduction in the survivorship, growth and/or reproduction of the 

individuals concerned’ (Begon et al., 1986). Plants mostly compete for the resources 

of light, water and nutrients (Donald, 1963), and less frequently for carbon dioxide, 

oxygen and space (Trenbath, 1974). In the exact sense of the definition, plants do not 

compete so long as these resources are in excess of the needs of both. When plants do 

compete, however, the outcome always reduces the growth of at least one of the 

competitors. The outcome of competition between two plants depends on the ability 

of each species to secure and utilise resources, i.e. their competitiveness. A highly 

competitive plant may be one that has a high rate of uptake of a particular resource or 

a low requirement for that resource (Grace and Tilman, 1990). Plants may differ in 

their ability to compete for individual resources, while environmental differences may 

also influence their overall competitive ability. Differences in competitive ability, in 

turn, help to structure the composition of mixed plant communities. 

2.2. Allelopathy 

Molisch first advanced the term allelopathy in 1937. It derives from the Greek words 

‘allelon’, meaning of each other, and ‘pathos’, meaning to suffer (Mandava, 1985). 

Despite the origin of its root words, Molisch used the term to refer to the chemical 

interactions between all plants (higher plants and microorganisms), including 

stimulatory as well as inhibitory effects. Some authors have considered that the concept 

covers only inhibitory effects (Rice, 1974; Putnam, 1985; Boyette and Abbas, 1995), 

yet others exclude microorganisms from the definition (Putnam and Duke, 1978; 

Putnam, 1985; Pratley, 1996). Many inhibitory chemicals produced by plants, however, 

stimulate growth at low concentrations (Liu and Lovett, 1993; Pratley, 1996), and 

microorganisms can mediate allelopathy (Rice, 1992; Bremner and McCarty, 1993). 

For these reasons, the definition of allelopathy used in this chapter closely follows 

that of Molisch’s, ‘the beneficial and detrimental chemical interaction among [plant] 

organisms including microorganisms’ (Rice, 1984). Although some authors have 

confused allelopathy with competition, the distinguishing feature is that allelopathy 

involves an addition of a chemical to the environment, whereas competition involves 

the shared utilisation of some limited factor required for growth (Muller, 1969; Rice 

1984). 

Any chemical produced by a plant (donor) that stimulates or inhibits the growth 

of a neighbour (receiver or receptor) is broadly termed an allelochemical. Typically, 

allelochemicals are secondary metabolites (Whittaker and Feeney 1971; Rice, 1984; 

Rizvi et al., 1992), produced as by-products of the acetate and shikimic acid pathways. 

They may also form as degradation products from the action of microbial enzymes

82 


(Putnam, 1985). Initially, the reason why plants devote resources to the production of 

these compounds was not understood as they were regarded as functionless waste 

products (Mothes, 1955). It is now increasingly accepted, however, that these 

compounds function as defensive agents against pathogens, insects and neighbouring 

plants (allelopathy). There is an enormous diversity of allelochemicals produced by 

plants (Bansal, 1994), with classification based on chemical structure (Whittaker and 

Feeney, 1971; Mandava, 1985; Putnam 1985) or on origin and chemical properties 

(Rice, 1984). For example, Rice (1984) recognised 14 main categories of 

allelochemicals. Of these groups, however, the phenolics are considered by many as 

the most important (Putnam and Duke, 1979; Mandava, 1985; Inderjit, 1996). 

Putative allelochemicals have been isolated from a variety of different plant organs, 

including shoots, roots, flowers, rhizomes, fruit and seed (Rice, 1984). They are 

stored in cell vacuoles so as not to interfere with the donor plant itself (Chou, 1989). 

Furthermore, secondary metabolites may be bound to sugars as glycosides or occur as 

polymers and crystals rendering them ineffective against the donor (Whittaker and 

Feeney, 1971). The release of allelochemicals into the environment may occur through 

volatilisation, root exudation, leaching or plant residue decomposition (Rice, 1984). 

Allelochemicals induce a wide range of symptoms in receiver plants, ranging 

from sudden wilting and death (e.g. tomato (Lycopersicon esculentum) grown in the 

vicinity of black walnut (Juglans nigra), Hale and Orcutt, 1987), to the more common 

subtle changes in growth. Determination of this reaction depends on the mode of 

action, the concentration, and the susceptibility of the receiver plant to the 

allelochemical. Allelopathic effects may be direct, such as affecting plant metabolism 

and growth, or indirect, such as altering of soil properties and nutrient status (Inderjit 

and Weiner, 2001). Rizvi et al. (1992) explained 12 plant functions that allelochemicals 

may affect, including membrane permeability, stomata function and photosynthesis, 

and cytology and ultrastructure. 

3. THE INFLUENCE OF PATHOGENS ON INTERFERENCE 

AND ALLELOPATHY 

Natural plant populations increase in production and number of individuals until 

constrained by environmental limitations (Burdon, 1987). The constraint of plant 

growth by the environment is often mediated through plant interference. Therefore, 

the ability of plants to interfere with their neighbours is important in determining 

their abundance in a community. Plant pathogens generally reduce the development, 

production and longevity of their hosts. In plant communities, this ‘burden of a 

parasite’ may result in a partly unoccupied niche that resistant plants re-inhabit. 

Overall, pathogens play a significant role in the ecology of plant communities by 

maintaining (Peters and Shaw, 1996) or reducing (Burdon, 1991) species diversity, 

driving succession (Van der Putten et al., 1993), ensuring plants do not establish 

under their parents (Augsberger, 1984) and help determine the long-term composition 

of plant communities (Dobson and Crawley, 1994).


ALLELOPATHY 

3.1. The Impact of Pathogens on Competition 

Several pioneering publications on plant competition suggest that pathogens might 

alter the balance of competition in mixed communities in favour of the resistant 

components (de Wit, 1960; Harper, 1977). Since then, there have been numerous 

reviews and theoretical interpretations of the impact of pathogens in natural 

communities and on competition (Chilvers and Brittain 1971; Burdon, 1982; 1987; 

1991; Dinoor and Eshed, 1984; Gates et al., 1986; Alexander, 1990; Ayres and Paul, 

1990; Clay, 1990; Paul, 1990; Dobson and Crawley, 1994; Jarosz and Davelos, 1995; 

Alexander and Holt, 1998; Mattner 1998), but empirical experimentation is less 

extensive. Empirical studies have usually involved binary mixtures of a host and a 

non-host, grown under optimal conditions of nutrient and water availability, and 

inoculated with a single, copious and homogenous dose of inoculum. With few 

exceptions, these studies show that the influence of a host-specific pathogen reduces 

the vigour of the host, rendering it less able to compete with a neighbouring non-host 

species (Burdon, 1987; Ayres and Paul, 1990; Mattner, 1998). As such, the combination 

of the ‘burden of a parasite’ and competition can have a devastating effect on a plant. 

For example, Groves and Williams (1975) demonstrated that the combined effects of 

competition from subterranean clover (Trifolium subterraneum) and rust infection 

(Puccinia chondrillina) reduced the yield of skeleton weed (Chondrilla juncea) by 

94%. This combined effect was more marked than the action of either the competitor 

(yield was reduced by 70%) or the rust (yield was reduced by 51%) alone. Similarly, 

Friess and Maillet (1996) found that infection by cucumber mosaic virus reduced the 

vegetative yield of its host purslane (Portulaca oleracea), with this effect intensifying 

when infected plants were in competition with healthy plants. In mixtures of common 

lambsquarters (Chenopodium album) and corn (Zea mays) or beetroot (Beta vulgaris), 

foliar infection by Ascochyta caulina reduced the competitiveness of its host 

(lambsquarters) and increased the yield of non-host crops. In corn, infection negated 

the effects of competition from lambsquarters altogether, highlighting its potential as 

a biological control agent (Kempenaar et al., 1996). Despite pathogens reducing the 

competitiveness of their hosts, the effect of infection is often greater on a neighbouring 

non-host than on the host itself. For example, in mixtures of groundsel (Senecio 

vulgaris) and lettuce (Lactuca sativa), rust (Puccinia lagenophorae) increased the 

biomass of lettuce (the non-host) markedly, while only reducing that of its host 

groundsel marginally (Paul and Ayres, 1987a). 

Competitive stress for limited resources intensifies as plant density increases 

(Shinozaki and Kira, 1956). Consequently, the effect of a pathogen on the 

competitiveness of its host is most devastating at high densities. For example, rust 

reduced the competitive ability of groundsel in mixtures with healthy groundsel (Paul 

and Ayres, 1986) or with lettuce (Paul and Ayres, 1987a) more so at high densities 

than at low densities. Similarly, Ditommaso and Watson (1995) demonstrated that 

anthracnose (Colletotrichum coccodes) was more detrimental to the growth of 

velvetleaf (Abutilon theophrasti) in mixtures with soybean (Glycine max) at high 

plant densities. Conversely, the performance of the non-host, relative to the infected 

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host, generally increases as density increases. For example, the ability of lettuce to 

compete with rusted groundsel was greater at high densities than at low densities 

(Paul and Ayres, 1987a). 

Considering that competition occurs for shared resources that are in limited supply, 

it is not surprising that resource availability influences the interaction between 

competition and infection by pathogens. Paul and Ayres (1987b) examined the effect 

of rust on competition between infected and healthy groundsel under conditions of 

drought. They found that rust reduced the competitiveness of groundsel, however, 

this was more so in droughted plots than in well-watered plots. They concluded that 

water stress is important in determining the impact of rust in mixed populations in 

the field. Owing to the effects pathogens can have on nutrient uptake, Paul and Ayres 

(1990) also studied the effect of nutrient supply on the interaction between rust and 

competition. Under high nutrition, groundsel was more competitive than shepherd’s 

purse (Capsella bursa-pastoris). This superiority was lost, however, following the 

inoculation of groundsel with rust. In contrast, shepherd’s purse had a greater 

competitive ability than groundsel under nutrient-poor conditions. Despite this, it 

did not increase its advantage when groundsel was rusted. 

While most studies show that pathogens decrease the competitiveness of their 

hosts and increase that of neighbouring non-hosts, there are several exceptions. For 

example, Catherall (1966) found that for most of the year, barley yellow dwarf virus 

reduced the competitive ability of its host, perennial ryegrass (Lolium perenne), when 

grown with white clover (Trifolium repens). During spring, however, the virus 

stimulated tillering in ryegrass and increased its competitiveness. In another example, 

the rust Puccinia pulsatillae sterilises its host, Pulsatilla pratensis, by inhibiting 

flowering. Yet, infected plants are more vigorous and produce more leaves than 

healthy plants. In a natural community, Wennström and Ericson (1991) found that 

diseased plants had a greater survival rate than healthy plants, which they postulated 

was due to their greater competitiveness. The effects of pathogens on allelopathy may 

also mediate their influence on competition. 

3.2. The Impact of Pathogens on Allelopathy 

The term allelopathy is seldom used in plant pathology (Rice, 1984) even though, by 

definition, allelopathy includes chemical interactions involving microorganisms. 

Furthermore, the same or similar secondary metabolites implicated in allelopathy 

between plants are also important in plant pathology in: enhancing the germination 

of fungal spores (Odunfa, 1978); antibiosis between microorganisms (Di Pietro, 1995); 

regulating fungal growth and development (Calvo et al., 2002); pathogen/host 

recognition (Nicol et al., 2003); the development of disease symptoms (Daly and 

Deverall, 1983); the promotion of infection through the suppression of the host 

(Toussoun and Patrick, 1963); the breaking of fungistasis (Mol, 1995); and host 

resistance to pathogens. Similarly, some authors do not consider chemical interactions 

by microorganisms as part of allelopathy (Putnam and Duke, 1978; Putnam and Tang, 

1986; Pratley, 1996). Rice (1984) explained that this reluctance is due to the chemicals


ALLELOPATHY 

involved not always escaping to the environment. This rationale seems invalid, 

however, because the chemicals involved do enter the environment of the pathogen or 

the plant. Moreover, microorganisms can mediate allelopathy between plants (Rice, 

1992; Bremner and McCartey, 1993). Such difficulties may have hindered the study 

of the impact of pathogens on plant allelopathy in the past. 

Both Rice (1984) and Einhellig (1995) hypothesised that pathogens enhance 

their host’s allelopathic ability, but few studies have observed such a relationship 

(Tang et al., 1995). This is despite several investigations on the effects of pathogens 

on plant competition (Burdon, 1987; Ayres and Paul, 1990; Section 3.1), all of which 

could potentially include allelopathic interactions. However, competition may have 

obscured allelopathy in these experiments (Trenbath, 1974), since competition is 

usually the dominant process of interference (Joliffe, 1988; Tilman, 1988). This is 

particularly so considering that many experiments studying the effect of pathogens 

on competition have utilised high plant densities, where competitive effects are most 

intense. Evidence supporting the hypothesis that pathogens can increase allelopathy 

between plants occurs in at least two forms: (i) pathogens can stimulate secondary 

metabolite production in their hosts, and (ii) field, glasshouse and bioassay experiments 

signifying an increased allelopathic ability of infected plants. 

3.2.1. Effect of Pathogens on Secondary Metabolite Production 

Numerous studies document the ability of plant pathogens to stimulate the metabolic 

activity (Daly, 1976) and increase the production of secondary metabolites (Stoessl, 

1982, 1983; Goodman et al., 1986; Kuc, 1997) by their hosts. Müller and Börger 

(1941) first proposed that plants produce defensive substances called phytoalexins in 

response to infection, which are important to host resistance. Phytoalexins are ‘low 

molecular weight; antimicrobial compounds that are both synthesised by and 

accumulated in plant cells after exposure to microorganisms’ (Paxton, 1981). As such, 

phytoalexins fit the definition of allelochemicals. Like allelochemicals that act against 

plants, phytoalexins are secondary compounds that belong to a wide range of different 

chemical classes (Stoessl, 1982), with more than 300 distinct phytoalexins already 

characterised (Smith, 1996). They are mostly synthesised via the acetate and shikimic 

acid pathways (Bailey, 1982; Bennett and Wallsgrove, 1994), as are allelochemicals 

that act against plants. Notwithstanding their similarities, allelopathy between plants 

and phytoalexin research has developed almost independently. 

Many compounds with phytoalexin activity are also implicated in allelopathy 

between plants (Rice, 1984). For example, isoflavonoids are important phytoalexins 

(Ingham, 1982; Paxton, 1981; Dakora and Phillips, 1996) and allelochemicals (Tamura 

et al. 1967; 1969) from the Leguminosae. Parbery et al. (1984) found that the 

isoflavonoids biochanin A, formononetin and genistein increased in subterranean 

clover by 62%, 123% and 75% respectively following infection by pepper spot 

(Leptosphaerulina trifolii). In comparison, Tamura et al. (1967; 1969) isolated a 

succession of isoflavonoids (including biochanin A, formononetin and genistein) from 

the shoots of red clover (Trifolium pratense) that inhibited its own germination by 

50% at concentrations of 50 ppm. 

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As expected, most studies concerned with the toxicology of phytoalexins 

concentrate on their effects on microorganisms. Increasingly, however, studies show 

that phytoalexins are also toxic to plants. Despite this, they are seldom referred to as 

allelochemicals. Pisatin and phaseollin, pterocarpans produced by pea (Pisum sativum) 

and bean (Phaseolus vulgaris) respectively, were the first phytoalexins characterised 

(Perrin and Bottomly, 1962; Cruickshank and Perrin 1963). Skipp et al. (1977) noted 

that phaseollin inhibited the respiration and growth of cell cultures of bean and tobacco 

(Nicotiana tabacum), eventually causing cell death. Similarly, pisatin reduced the 

growth of callus cultures of pea (Bailey, 1970) and inhibited the root growth of wheat 

(Cruickshank and Perrin, 1961). The phytoalexin rishitin (a sesquiterpene) accumulates 

in potato cells challenged by incompatible isolates of Phytophthora infestans 

(Tomiyama et al., 1968). Studies show that the compound also inhibits pollen 

germination in three Solanum species (Hodgkin and Lyon, 1979); causes lysis of 

potato and tomato protoplasts (Lyon and Mayo, 1978); and cell death in tubers and 

epidermal strips of potato (Ishiguri, et al., 1978; Lyon, 1980). Other studies show that 

phytoaxelins may inhibit seed germination (Chang et al., 1969), growth (Glazener 

and VanEtten, 1978) and cellular metabolism and function (Lyon, 1980; Boydston et 

al., 1983; Kurosaki et al., 1984; Giannini et al., 1990; Spessard et al., 1994) of plants. 

The ability of pathogens to stimulate secondary metabolite production in their hosts 

and for these to affect plant growth provides strong circumstantial evidence for the 

hypothesis that pathogens can increase plant allelopathy. 

Many studies have established that pathogens can increase the production of 

phenolic acids in their hosts, which are perhaps the most important group of plant 

allelochemicals. For example, when challenged by a range of different pathogens, 

concentrations of phenolics often associated in allelopathic interactions have increased 

in a variety plants, including carrot (Daucus carota) (Phan et al., 1991), chickpea 

(Cicer arietinum) (Singh et al., 2002); date palm (Phoenix dactylifera) (Daayf et al., 

2003); potato (Solanum tuberosum) (Kuc et al., 1956); sorghum (Sorghum bicolor) 

(Woodhead, 1981); sugar cane (Saccharum officinarum) (Legaz et al., 1998); sunflower 

(Helianthus anuus) (Spring et al., 1991); and wheat (Triticum aestivum) (Siranidou 

et al., 2002), amongst many others. This increase in phenolics post-infection is one of 

the most important methods of disease resistance in plants. For example, inoculation 

with crown rust (Puccinia coronata f.sp. avenae) induced the production of three 

phenolic compounds (avenalumins I, II and III) by resistant oats (Avena sativa). 

Purified preparations of these chemicals inhibited the germination and germ tube 

growth of crown and stem rust (Puccinia graminis) at concentrations as low as 200 

µg/mL (Mayama, 1981; 1982). Similarly, Mandavia et al. (2000) found that varieties 

of cumin (Cuminum cyminum) tolerant of Fusarium wilt (Fusarium oxysporum f.sp. 

cumini) contained higher concentrations of salicylic acid, hydroquinone and 

umbelliferone in their root, stem and leaf tissues than susceptible varieties. These 

phenolics inhibited fungal spore germination and mycelial growth of F. oxysporum. 

However, to this author’s knowledge, the effect of the increased phenolic concentrations 

in infected plants on plant allelopathy has not been investigated empirically.


ALLELOPATHY 

The production of glucosinolates by plants in the Capareles and their subsequent 

hydrolysis to toxic isothiocyanates, thiocyanates, and nitriles has been one of the 

most intensively studied systems in allelopathy, due partly to the similarity of these 

breakdown products to some synthetically produced soil fumigants (and therefore 

termed biofumigation; Kirkegaard et al., 2000). Glucosinolates are important in plant 

defence against insects, pathogens and nematodes. Jay et al. (1999) showed that 

infection of Brassica napus with beet western yellows virus increased glucosinolate 

concentration in tissues by 14%. Similarly, Li et al. (1999) found that infection of B. 

napus by Sclerotinia sclerotiorum increased glucosinolate content in resistant, but 

not in susceptible varieties. Exposure to the pathogenic bacterium, Erwinia carotovora, 

triggered the production of glucosinolates in Arabidopsis thaliana (Brader et al., 

2001). The hydrolysis products from these glucosinolates inhibited the growth of E. 

carotovora in culture. Furthermore, Tierens et al. (2001) demonstrated that a range 

of pathogens were more aggressive in infecting an A. thaliana mutant that did not 

produce glucosinolates than the wild type, suggesting the importance of glucosinolates 

in protecting against infection. However, the role of glucosinolates in disease resistance 

may be species specific, since Andreasson et al. (2001) found that infection of B. 

napus by Leptosphaeria maculans had no effect on glucosinolate concentration in the 

resistant or susceptible host. Despite the ability for at least some pathogens to increase 

glucosinolate production in the Capareles, no one has investigated whether this directly 

translates to an increased allelopathic effect against neighbouring plants. 

3.2.2. The Effect of Rust on Ryegrass Allelopathy 

Perennial ryegrass (Lolium perenne) and white clover (Trifolium repens) are important 

components of improved pastures grown in temperate regions worldwide. The ability 

of ryegrass to become dominant in pastures has led to numerous investigations that 

demonstrate its potential to interfere with companion plants through allelopathy (Naqvi, 

1972; Naqvi and Muller, 1975; Newman and Rovira, 1975; Newman and Miller, 

1977; Gussin and Lynch, 1980; Buta et al., 1987; Quigley et al., 1990; Sutherland 

and Hoglund, 1990; Wardle et al., 1991; Prestidge et al., 1992; Chung and Miller, 

1995; Mattner, 1998; Mattner and Parbery, 2001). For example, in a study investigating 

the allelopathic ability of nine pasture species, Takahashi et al. (1988) found that 

leachate from soil surrounding ryegrass was the most inhibitory to the growth of the 

target species, including clover. In a subsequent experiment, they circulated nutrient 

solution between the roots of ryegrass and clover to eliminate competition effects. 

They found that the growth of clover declined in the system, particularly when the 

proportion of ryegrass was high. When they incorporated XAD-4 resin into the system 

(which selectively traps organic hydrophobic compounds) clover grew normally. 

Moreover, ryegrass became yellow and stunted when grown alone in the system, but 

this was prevented by the presence of the resin or clover (Takahashi et al., 1991). In 

a further experiment, root exudate from ryegrass not only inhibited the growth of 

clover, but also lettuce seedlings. The phytotoxic fraction of the extract contained pmethoxybenzoic, 

lauric, myristic, pentadecanoic, palmitoleic, palmitic, oleic and stearic 

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acids. Sodium salts of myristic, palmitic, oleic and stearic acids suppressed the growth 

of clover at concentrations as low as 5 ppm (Takahashi et al., 1993). Similarly, in a 

study examining the allelopathic effects of a number of crop and pasture species, 

Halsall et al. (1995) found that aqueous extract from the dried shoots of perennial 

ryegrass suppressed the germination, radicle elongation, nodulation and seedling root 

elongation of subterranean and white clover. The magnitude of this inhibition increased 

as the concentration of the extract increased. 

Crown rust, caused by Puccinia coronata f.sp. lolii, is the most devastating fungal 

disease of ryegrass, with epidemics regularly occurring between spring and autumn 

in temperate regions worldwide (Mattner and Parbery, 2001). Severe epidemics reduce 

ryegrass tillering by 20-38% (Lancashire and Latch, 1966; Mattner, 1998), leaf 

emergence by 60%, leaf area by 62%, root growth by 75% (Mattner, 1998), and increase 

the rate of leaf senescence by up to 184% (Lancashire and Latch, 1966; Trorey, 1979; 

Plummer et al., 1990; Mattner, 1998). Losses of herbage yield in ryegrass from rust 

have been as great as 94% (Critchett, 1991), with seed yield losses ranging from 12- 

36% (Hampton, 1986; Mattner 1998). Furthermore, rust infection reduces forage 

quality (Isawa et al., 1974; Trorey, 1979; Potter, 1987) and palatability to grazers 

(Cruickshank, 1957; Heard and Roberts, 1975). 

As would be expected by the devastating effect that rust has on ryegrass growth, 

most studies show that rust reduces the competitiveness of ryegrass with non-host 

plants such as clover. For example, in mixed swards of ryegrass and clover, Lancashire 

and Latch (1970) found that rust reduced ryegrass yield by 84% and increased the 

yield of clover by 87%. Furthermore, the proportion of clover in the rusted sward 

increased from 24% at the beginning to 80% at the termination of their experiment. 

Thus, their study pointed to a lowered competitiveness of rusted ryegrass. In mixtures 

of rust resistant and susceptible ryegrass, Potter (1987) found that rust reduced the 

yield of the susceptible cultivar and increased that of the resistant one, concluding 

that rust reduced ryegrass competitiveness. Similarly, crown rust infection in swards 

of ryegrass and cocksfoot (Dactylis glomerata) reduced ryegrass composition from 

30% to 15%, and was more marked in rust susceptible than resistant cultivars (Trorey, 

1979). However, in a series of experiments, Mattner (1998) reported an anomaly to 

the results of this previous research. 

In pot studies consisting of 50:50 mixtures of ryegrass and clover grown over a 

range of plant densities, Mattner (1998) found that rust reduced the yield of ryegrass 

by an average of 41%. However, interference from rusted ryegrass suppressed clover 

biomass by up to 47% compared with interference from the more productive, healthy 

ryegrass. The onset of the suppression of clover by rusted ryegrass was rapid, occurring 

as early as 6-13 days after inoculation, which according to some growth parameters 

was earlier than the effects of rust on ryegrass itself. The suppression of clover by 

rusted ryegrass was greatest at low plant densities and diminished or disappeared as 

density increased. In a separate trial, rusted ryegrass again suppressed clover growth, 

even after the removal of infected tissue by cutting and after the death of the ryegrass. 

In this instance, ryegrass killed by infection, with a competitive ability of virtually 

zero, inhibited the growth of clover more than living plants of healthy ryegrass. In


ALLELOPATHY 

further trials, high rust severity and high soil moisture contents increased the 

suppression of clover by rusted ryegrass. 

The ability of rust to directly inhibit or infect clover could not explain the results 

from these studies, since clover is a non-host of P. coronata and inoculation with this 

rust did not reduce the yield of clover monocultures. Under some conditions, infection 

by rusts can increase the scavenging ability of some hosts for water and nutrient 

resources (Ahmad et al., 1982; Paul and Ayres, 1988), potentially increasing their 

competitive ability. For this to explain the results from Mattner’s studies, however, 

the suppression of clover by rusted ryegrass should have been greatest at high plant 

densities where competition for resources was most intense. Instead, the suppression 

of clover by rusted ryegrass was greatest at low densities where resources were plentiful 

and competition was low. Furthermore, the ability of rusted ryegrass to suppress 

clover continued even beyond the death of the plant, when it had no capacity to scavenge 

resources. For these reasons, Mattner (1998) posed the hypothesis that rust reduces 

the competitiveness of ryegrass, while simultaneously increasing its allelopathic ability. 

In this way, it was expected that the expression of allelopathy by rusted ryegrass was 

greatest at low densities, where there was little competition and the effects of rust in 

reducing ryegrass competitiveness did not obscure its effects on increasing allelopathy. 

Furthermore, high plant densities may detoxify or dilute the action of allelochemicals 

(Thijs et al., 1994). 

To test the validity of this hypothesis and to separate the effects of competition 

and allelopathy, four bioassays for allelopathy were conducted (Mattner, 1998; Mattner 

and Parbery, 2001). Each bioassay highlighted the potential for extracts, leachate, or 

residues from ryegrass to inhibit the yield of clover through allelopathy, and for rust 

to enhance this potential. For example, soil previously growing rusted ryegrass 

suppressed clover biomass by 36% compared with soil previously growing healthy 

ryegrass. Similarly, leachate from soil supporting rusted ryegrass suppressed clover 

biomass by 27% compared with that from healthy ryegrass (Mattner and Parbery, 

2001). Although some bioassays have confounding and interpretational problems 

(Stowe, 1979; Inderjit and Weston, 2000), the conformity of results between these 

different bioassays provides strong evidence for the hypothesis that rust infection 

increases the allelopathic ability of ryegrass. Furthermore, in the field, the proportion 

of prickly lettuce (Lactuca serriola) reduced in areas of a depleted pasture dominated 

by rusted ryegrass, compared with areas dominated by non-rusted ryegrass (Mattner, 

1998). Further bioassays provided evidence that this association potentially related 

to an enhanced allelopathic ability of ryegrass rather than to differences in soil 

chemistry. Thus, this study highlighted the potential for rust to increase ryegrass 

allelopathy in the field. 

Mattner (1998) suggested two mechanisms by which rust may increase ryegrass 

allelopathy. Firstly, rust may directly stimulate the production of allelochemicals by 

ryegrass in a defensive response to infection. Alternatively, or additionally, the increased 

rate of tissue senescence in ryegrass induced by rust may result in a higher concentration 

of plant residues reaching the soil. These residues may then form an allelochemical 

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source – either directly or following their decomposition. Most evidence gathered 

from his studies supported the first hypothesis. For example, when ryegrass residues 

were incorporated into soil, their ability to suppress clover growing in that soil depended 

on the residues being rusted and not their overall concentration. Furthermore, the 

knowledge that pathogens stimulate secondary metabolite formation in their hosts 

and the rapid effect rusted ryegrass had in suppressing clover, supported rust directly 

stimulating allelochemical production in ryegrass. 

Mattner’s findings appear to contradict those of Lancashire and Latch (1970) 

who studied the identical biological system in the field, and found that the proportion 

of clover in the sward more than doubled following infection of the ryegrass component 

by rust. However, Lancashire and Latch (1970) conducted their study under conditions 

that favoured the expression of the reduced competitiveness rusted ryegrass, i.e. at 

higher plant densities (2150 plants/m 2 ) than those of Mattner (1998) (57 plants/m 2 ). 

More importantly, however, their results only occurred in the highly susceptible ryegrass 

cultivar, Ruanui. In a more resistant cultivar, Ariki, rust reduced ryegrass yield by 

18%, but clover was unable to take advantage of this reduction, producing the same 

dry weight when grown with rusted ryegrass as when grown with healthy ryegrass. 

Furthermore, rusted Ariki ryegrass actually suppressed the growth of clover at some 

harvests. Perhaps rust infection increased the production of defensive chemicals by 

Ariki, thereby increasing its resistance to rust and its allelopathic ability against clover. 

Lancashire and Latch (1970) disregarded these results because, overall, the yield of 

Ariki was abnormally poor in their experiment compared with several previous studies. 

Nonetheless, the poor yield of Ariki ryegrass occurred in both rusted and non-rusted 

treatments and does not explain the inability of clover to compensate for the reduced 

yield of rusted ryegrass, or the suppression of clover by rusted ryegrass. Rather, the 

hypothesis that rust increases the allelopathic response of ryegrass while also reducing 

its competitiveness fits their results. 

Although there is strong circumstantial evidence from field, glasshouse and 

bioassay studies that rust increases the allelopathic ability of ryegrass, many questions 

remain. An important next step is to identify the allelochemicals concerned. Also, 

do rusted plants simply produce allelochemicals in higher concentrations or do they 

produce entirely different allelochemicals to healthy plants? Also, what is the 

allelochemical source in the plant and the mechanism of release to the environment? 

This information will provide a clearer understanding of the influence of pathogens 

on allelopathy. 

3.2.3. The Effect of Neotyphodium lolii on Ryegrass Allelopathy 

The endophytic fungus Neotyphodium lolii commonly infects perennial ryegrass 

forming a mutualistic relationship with its host. Apart from obtaining nutriment, the 

host provides the endophyte with a relatively exclusive niche and a vehicle for its 

transmittance through infected seed (Clay, 1987). The host benefits in several ways, 

including: (i) increased seed germination, dry matter production and tillering compared 

with non-infected ryegrass (Latch et al., 1985; Quigley, 2000); (ii) resistance to plant


ALLELOPATHY 

diseases caused by nematodes (Stewart et al., 1993), fungi (Latch, 1993) and viruses 

(Lewis and Day, 1993); (iii) increased tolerance of drought stress (Ravel et al., 1997), 

(iv) increased nitrogen use efficiency (Arachevaleta et al., 1989); and (v) increased 

competitiveness (Sutherland and Hoglund, 1989). Additionally, the fungus produces 

various alkaloids (e.g. peramine, ergovaline, and lolitrem) that protect the host against 

herbivory (Clay, 1996). 

There is much speculation as to the role of the endophyte on the allelopathic 

ability of its host. This speculation originated with the observation that endophyteinfected 

ryegrass suppressed the growth of companion clovers to a greater extent than 

endophyte-free ryegrass (Stevens and Hickey, 1990). In order to explore several 

hypotheses on how this relationship might arise, Sutherland and Hoglund (1989) 

grew swards of endophyte-infected and endophyte-free perennial ryegrass in plots 

with white clover. Endophyte-infected ryegrass produced 16% more dry matter and 

suppressed the yield of clover by 72% compared with endophyte-free ryegrass. Neither 

mowing nor grazing by sheep affected the relationship. This demonstrated that selective 

grazing pressure on clover following a decline in the palatability of endophyte-infected 

ryegrass was not responsible for the reduction in clover yield. Rather, they suggested 

that the suppression was due to an increased competitiveness and allelopathic ability 

of endophyte-infected ryegrass. They implicated allelopathy in this effect because 

endophyte-infected ryegrass of a comparable yield to endophyte-free ryegrass, 

suppressed the yield of clover to a greater extent than endophyte-free ryegrass. 

In a subsequent experiment, Sutherland and Hoglund (1990) surrounded individual 

plants of white clover with zero to seven endophyte-infected or endophyte-free perennial 

ryegrass plants. This experiment failed to demonstrate that endophyte-infected ryegrass 

suppressed clover more than endophyte-free ryegrass. They explained that this might 

be due to the cutting regime imposed on ryegrass and the non-return of these clippings 

to the soil, which possibly contained allelochemicals. A bioassay experiment, which 

showed that aqueous extracts from the shoots of endophyte-infected ryegrass inhibited 

the shoot growth of clover seedlings by 8%, supported their explanation. In a similar 

experiment, Quigley et al. (1990) found that aqueous extracts from endophyte-infected 

ryegrass depressed the root length of four germinating legumes (including white 

clover), by an average of 10% compared with extract taken from endophyte-free 

ryegrass. In contrast, after conducting several bioassays for allelopathy, Prestidge et 

al. (1992) found little evidence to suggest that the presence of endophyte in ryegrass 

enhanced its allelopathic effect. Field trials also failed to show that clover yield 

declined when grown with endophyte-infected ryegrass. Similarly, Watson et al. (1993) 

and Mattner (1998) failed to substantiate an increased allelopathic effect of endophyteinfected 

ryegrass. Furthermore, there was no apparent interaction between endophyte 

and rust infection in increasing the allelopathic ability of ryegrass (Mattner, 1998). 

Applebee et al. (1999) found that the toxicity of tall fescue (Festuca arundinacea) 

infected with Neotyphodium coenophialum increased at elevated concentrations of 

CO 2 in the atmosphere. In a bioassay study conducted in sterile sand, Sutherland et 

al. (1999) applied aqueous extracts from ryegrass to potted clover seedlings. Extracts 

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from three ryegrass cultivars infected with three different strains of endophyte, all 

inhibited the growth of clover, by up to 27% compared with extracts from endophytefree 

ryegrass. Both ryegrass cultivar and endophyte strain influenced the degree that 

ryegrass extracts inhibited clover, but this did not relate to the type of alkaloids produced 

by the different endophyte strains. These studies suggest that both environmental 

and genetic influences may moderate the triggers for enhanced allelopathy by endophyte 

infected grasses, and this may explain the discrepancy in results between individual 

studies. Nonetheless, the majority of evidence suggests that endophyte infection has 

the capacity to increase ryegrass allelopathy, which is a further added benefit conferred 

by this mutualist to its host. Although the endophyte is a non-pathogenic organism, 

its ability to stimulate plant allelopathy adds further weight to the hypothesis that 

infection increases the allelopahtic ability of host plants. 

3.2.4. Other Systems 

The ability of rusts to stimulate allelopathy in their hosts may not be limited to ryegrass, 

as the rusts Puccinia hordei and Uromyces troflii-repentis increased the suppression 

of white clover by barley grass (Hordeum leporinum) and subterranean clover (Trifolium 

subterraneum), respectively (Mattner, 1998). Yet, the effect was not universal since 

Puccinia coronata in wild oat (Avena fatua), Puccinia graminis in cocksfoot (Dactylis 

glomerata) and Puccinia recondita in soft brome (Bromus mollis) all failed to increase 

their host’s allelopathic ability. 

Kong et al. (2002) studied the allelopathic potential of goatweed (Ageratum 

conyzoides) under different environmental stresses, including infection by powdery 

mildew (Erysiphe cichoracearum). Infection stimulated the production of 17 of the 

24 volatile chemicals produced by goatweed that they investigated, with total volatile 

production increasing by 50%. Exposure to the volatiles released by infected goatweed 

stimulated the growth of peanut (Arachis hypogaea), redroot amaranth (Amaranthus 

retroflexus), Italian ryegrass (Lolium multiflorum) and cucumber (Cucumis sativus) 

compared with volatiles from healthy goatweed. In contrast, volatiles from infected 

plants inhibited the growth of three fungal pathogens (Rhizoctonia solani, Botrytis 

cinerea, and Sclerotinia sclerotiorum). For this reason, they postulated that the 

allelochemicals stimulated by fungal infection in goatweed are more important in the 

defence of the plant against infection, rather than against competition from 

neighbouring plants. Nonetheless, this study currently provides the clearest 

demonstration of the ability of pathogens to influence the allelopathic ability of plants. 

4. IMPLICATIONS FOR PATHOGEN 

AGGRESSIVENESS AND EVOLUTION 

Plant/pathogen interactions in natural populations are often explained in terms of coevolution, 

which is ‘the joint evolution of two (or more) taxa that have close ecological 

relationships but do not exchange genes, and in which reciprocal selective pressures 

operate to make the evolution of either taxon partially dependent upon the evolution


ALLELOPATHY 

of the other’ (Pianka, 1978). Burdon (1987) explained the co-evolution of plants and 

their pathogens in terms of the gene-for-gene concept of resistance and virulence, 

using the following model: 

(a) A uniform host population possessing a single gene for resistance is challenged 

by a uniform pathogen population with the complementary virulence gene, 

resulting in pathogenesis. 

(b) Under the above conditions, chance mutation favours the appearance of a novel 

resistance gene preventing pathogenesis through enhanced resistance. These 

resistant individuals have a greater fitness than their susceptible neighbours and, 

consequently, increase in frequency within the population. 

(c) As the frequency of the resistant genotype increases, selective pressure favours 

the appearance of a pathogen race with a novel virulence gene capable of attacking 

the resistant host. 

(d) As resistance is broken down, the advantage of the previously resistant host 

genotype in terms of plant fitness is lost, and its frequency within the population 

falls. 

(e) Under these conditions the appearance of yet another resistant gene is favoured 

and the cycle continues. 

Although Burdon’s model provides a good example of the concept of co-evolution, 

it is important to note that it does not account for the cost of resistance and virulence 

to the host and pathogen (Parker and Gilbert, 2004), host tolerance to disease (Roy 

and Kirchner, 2000), or the specificity of a pathogen to its host (Kirchner and Roy, 

2002). 

The importance of virulence (the degree or measure of pathogenicity) in 

determining the ability of a pathogen to infect a particular host is central to the concept 

of co-evolution of plants and pathogens. Despite this, its importance has often been 

emphasised to the exclusion of another component of pathogen fitness – aggressiveness 

(Burdon, 1987). In plant pathology, the term aggressiveness has been defined 

inconsistently (Jarosz and Davelos, 1995; Kirchner and Roy, 2002) or considered 

synonymous with virulence (Parker and Gilbert, 2004), but here is considered the 

negative effect of infection on plant fitness (Jarosz and Davelos, 1995). The role of 

aggressiveness in interactions between plants and biotrophic pathogens is represented 

by two seemingly opposing views. Harper (1977) suggested that biotrophic pathogens, 

which need living tissue for their survival, evolve towards minimising their 

aggressiveness, with evolutionary equilibrium occurring when a pathogen attains 

commensalistic relationship with its host. On the other hand, less aggressive pathogens 

are prone to displacement by more aggressive strains (Jarosz and Davelos, 1995), 

implying that pathogens should evolve towards increased aggressiveness. Despite 

these arguments portraying the role of aggressiveness differently, they need not be 

mutually exclusive. 

As an adjunct to the concept of co-evolution, the New Function Hypothesis 

proposed by Clay (1988) suggests that pathogens may evolve towards a mutualistic 

relationship with their hosts through the appearance of pathogen strains with beneficial 

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effects on the host in addition to their detrimental effects. In this way pathogens 

minimise their aggressiveness through the acquisition of ‘new functions’ that increase 

host fitness and, at the same time, are less prone to displacement by more aggressive 

strains by maintaining their original capacity for disease. A ‘new function’ suggested 

in this chapter is the potential for pathogens to increase the allelopathic ability of 

their host, which to varying degrees offsets the infected hosts’s loss in competitiveness. 

By conferring some benefit on its host, the pathogen maintains a self-advantage through 

increasing the survival chances of its host and ultimately itself. 

Since biotrophic parasites, such as the rusts, are heavily dependent on the 

continuity of their host’s genotype into succeeding generations, the evolution of 

interactions that enhance the chances of host survival are important. It is well 

established that infections of fodder species by biotrophic pathogens can create 

conditions that either limit the grazing of their hosts, limit the number of grazing 

animals, or both. Morgan and Parbery (1980) found that infection by Pseudopeziza 

medicagnis lowered protein content, digestibility and palatability of lucerne as well 

as increasing its oestrogenic activity. Similarly, rust reduces the digestibility and 

quality of ryegrass (Isawa et al., 1974; Trorey, 1979; Potter, 1987). For this reason 

ruminants preferentially graze healthy ryegrass rather than rusted ryegrass 

(Cruickshank, 1957; Heard and Roberts, 1975), indirectly benefiting rusted ryegrass. 

Furthermore, evidence presented in this chapter supports the ability of rust to add a 

further benefit to ryegrass, that of increased allelopathy with neighbouring plants. In 

a similar manner to crown rust, amongst other benefits, the mutualistic endophyte N. 

lolli reduces ryegrass palatability to ruminants (Fletcher and Sutherland, 1993) and 

increases its allelopathic ability (Sutherland and Hoglund, 1990; Quigley et al., 1990; 

Sutherland et al., 1999). The parallels between these two systems suggest that the 

pathogenic relationship between crown rust and ryegrass is evolving toward mutualism. 


Pathogens are important forces that influence the structure and dynamics of plant 

communities. Although there are numerous interpretations and studies on the impact 

of pathogens on plant competition, few studies have considered their effect on 

allelopathy. Currently, however, most evidence suggests that pathogens may 

simultaneously decrease the competitiveness and increase the allelopathic ability of 

their hosts. By conferring the benefit of increased allelopathy on their hosts, pathogens 

may offset their host’s loss in competitiveness. In so doing, these pathogens make 

their hosts less prone to displacement by resistant components of the plant ecosystem 

and encourage the continuation of their host’s genotype into proceeding generations, 

and ultimately their own. Finally, the similarities in the ability of the ryegrass pathogen, 

P. coronata, and the ryegrass mutualist, N. lolii, to increase their host’s allelopathic 

capacity suggests that this ‘new function’ may be one way that the rust pathogen is 

evolving toward mutualism.


ALLELOPATHY 

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101

RAMANATHAN KATHIRESAN 1 , CLIFFORD H.KOGER 2 

AND KRISHNA N. REDDY 3 

ALLELOPATHY FOR WEED CONTROL IN 

AQUATIC AND WETLAND SYSTEMS 

1 Professor, Department of Agronomy, Annamalai University, 

Annamalainagar, Tamilnadu 608 002, India. 2 Weed Ecologist, Southern 

Weed Science Research Unit, USDA-ARS, P. O. Box 350, Stoneville, 

Mississippi 38776, USA. 3 Corresponding author. Plant Physiologist, 

Southern Weed Science Research Unit, USDA-ARS, P. O. Box 350, 

Stoneville, Mississippi 38776, USA 

E-mail : kreddy@ars.usda.gov 

Abstract. Allelochemicals offer ample scope for ecologically safe and effective weed control in aquatic and 

wetland systems. This could be attributed to the absence of soil interface in aquatic habitats that contributes 

largely for rapid degradation of allelochemicals. Simpler strategies involving allelopathy especially for small 

holder farms, low input agriculture and aquatic environments with appreciable results have been reported. Such 

strategies include use of allelopathic cultivars, organic manures and plant products. Though allelopathic 

suppression of weeds could not be construed as an alternate to replace synthetic herbicides, it can fit in an 

integrated weed management program very well as a prime component. Such strategies are reviewed. Further, a 

specific case study for the use of plant product along with insect agents for controlling water hyacinth in India 

and different steps involved in selecting allelopathic plant products for aquatic weed control are discussed. 


Many of the compounds produced by green plants that are not involved in primary 

plant metabolism are observed to function as chemical warfare agents against 

competing plants and pests. Many such natural compounds have the potential to be 

exploited as herbicides or as leads for discovery of new herbicides (Duke, 1986; 

Hoagland, 2001). Allelopathy simply refers to chemical warfare between different 

plants, in which the bio-chemicals from one plant impair germination, growth, survival, 

reproduction, and behavior of other plants. These allelopathic chemicals are produced 

by a ‘donor’ and transmitted to a ‘receiver’ that can either be ‘injured’ or ‘stimulated’. 

Allelochemicals act through direct interference with physiological functions of 

‘receiver’ such as seed germination, root growth, shoot growth, stem growth, symbiotic 

effectiveness or act indirectly through additive or synergistic impact along with 

pathological infections, insect injury and/or environmental stress. Though many of 

these allelochemicals exhibit inhibitory response on various morpho-physiological 

functions of receiver plants and such responses being observed to be dose dependant 

in a linear fashion, their concentrations required for control of weeds on a field scale 

are impracticably higher. Further, the degree of selectivity is often a factor limiting 




103

104 

RAMANATHAN KATHIRESAN, CLIFFORD H. KOGER , KRISHNA N. REDDY 

for their widespread commercial use. The soil-interface in agricultural field conditions 

also affects the effectiveness of these allelochemicals. However, allelochemicals provide 

ample opportunity for safe, selective and eco-friendly weed control in aquatic and 

wetland systems as resistance offered by soil-interface is largely circumvented by the 

water-interface. Under aquatic environment, the transport of allelochemical to the 

receiver plant is much more rapid, thus reducing the need for higher doses of 

allelochemicals for effective weed suppression. 

2. ALLELOPATHIC PLANT MATERIALS 

In spite of the widespread apprehension that applications of allelopathy in agriculture 

need to undergo an extensive refinement and techno-commercial perfections, simpler 

means especially for small holder farms with appreciable results have been reported. 

Strategies that involve the mechanism of allelopathy to an appreciable magnitude 

include use of allelopathic cultivars, mulches, organic manures, and plant products in 

wetlands. In aquatic systems, strategies include use of allelopathic plant products and 

plant species capable of aggressively replacing invasive undesirable weed species. 

2.1. Allelopathic Crop Cultivars in Wetlands 

Apparent allelopathic activity in rice accessions against the weed duck salad 

[Heteranthera limosa (Sw.) Willd] was first observed at USDA-ARS in Arkansas, 

during 1985-86. Within the next five years, 412 rice accessions that imparted 

allelopathic suppression of duck salad with in a radius of 10 cm were identified by 

USDA-ARS from their germplasm collection comprising 16,000 rice accessions from 

99 countries. The allelopathic suppression of the weed red stem (Ammania coccinea 

Rottb) over an area of 10 cm radius from the rice plant was observed with 145 accessions 

(Dilday et al., 1994). A hybrid between P1 338046 (allelopathic) and Katy (nonallelopathic) 

was shown to possess superior agronomic traits in green house studies 

and quantitative inheritance was indicated for the allelopathic activity in hybrid rice 

(Dilday et al., 1998). In Egypt, more than 30 rice varieties were shown to be allelopathic 

on barnyardgrass [Echinochloa crusgalli (L.) Beau.] and 10 rice varieties were observed 

to suppress smallflower umbrella sedge (Cyperus difformis L.). These allelopathic 

rice varieties inhibited the root development and emergence of first or second leaf of 

both weeds (Hassan et al., 1995). Chemical composition of ethyl acetate extracts from 

different rice cultivars allelopathic to barnyardgrass in South Korea consisted mainly 

fatty acid esters, unsaturated ketones, polycyclic aromatic compounds, and alkaloids 

(Kim and Shin, 1998). Gas chromatography/mass spectrometry (GC/MS) analysis of 

extracts of water from wetland with allelopathic rice plants showed significant levels 

of 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 4-hydroxy cinnamic acid and 3, 4dihydroxy 

hydrocinnamic acid (Mattice et al., 1998). Many studies have indicated 

that the active compounds involved in rice allelopathy are phenolic compounds (Rice, 

1987; Chou et al., 1991; Inderjit, 1996; Mattice et al., 1998). The GC/MS analysis of 

Kouketsumochi, a potential allelopathic rice variety identified several compounds

ALLELOPATHY FOR WEED CONTROL 105 

such as sterols, benzaldehydes, benzene derivatives, long chain fatty acids, esters, 

aldehydes, ketones, and amines as to have been biologically active (Kim et al., 2000). 

Kim and Shin (2003) observed that more than one allelochemical is likely to be involved 

in rice allelopathy and that rice allelopathy is not due to one specific phytotoxin. Such 

leads in rice allelopathy research offer scope for incorporation of this self defense 

mechanism in improved rice varieties, through breeding programs. A very important 

element needed for achieving this goal is understanding of the structure of the genetic 

background of the trait. 

However, today’s knowledge on allelopathy genetics is limited and this restricts 

the efforts for intentional breeding to genetically improve allelopathic potential of 

crops. A valuable milestone in this direction has been reached with the contributions 

of Jensen et al. (2001). Quantitative Trait Loci (QTL) mapping through 142 DNA 

markers were located in 142 recombinant inbred lines derived from a cross between a 

japonica upland strain with strong allelopathic traits and an indica irrigated strain 

with weak allelopathic traits. Three main loci, independently contributing for 10% of 

the promotion of allelochemical synthesis were localized to rice chromosomes 2 and 

3. The two QTL traits on chromosome 3 were closely linked, offering scope for easy 

manipulation. Three different approaches have been attempted recently to produce 

allelopathic crops. Traditional breeding methods are suggested to be a reasonable 

alternative by Courtois and Olofsdotter (1998). The first approach involves crossing 

of two parents with contrasting behavior and derivation of recombinant inbred lines 

(RILs) through single – seed descent (SSD). The second approach involves introduction 

of allelopathic traits in to an elite restorer line in developing three – line hybrid rice. 

Simultaneous back crossing and selfing methods of breeding were attempted to develop 

hybrid rice with allelopathic activity and its counterpart isogenic hybrid rice with a 

non-allelopathic effect on weeds. Three lines of rice, Kouketsumochi, Rexmont and 

IR-24 were used as allelopathic donor, non-allelopathic, and restoring genes, 

respectively (Lin et al., 2000). Analysis and monitoring of allelopotential and heterotic 

performance in laboratory and green house indicated a positive and significant 

allelopathy in this hybrid rice. A third approach suggested is molecular that could be 

achieved by the regulation of gene expression related to allelochemical bio-synthesis 

and insertion of allelochemical regulating genes into non-allelopathic crops to induce 

the synthesis of allelochemicals (Duke et al., 2001). 

2.2. Crop Residues and Organic Manures for Allelopathic Suppression of Weeds 

The residues of crops grown during preceding seasons or tree components of the farm 

are incorporated in the field before raising field crops to serve as manures. However, 

such crop residues also offer other environmental benefits that include protection 

from soil erosion, increase in biological diversity including beneficial organisms and 

suppression of pests and weeds (Sustainable Agriculture Network, 1998). The residues 

of such crops can suppress weeds by releasing allelochemicals (Teasdale, 2003). Living 

mulches, intercrops or smother crops may provide physical weed suppression but 

their effects in part depend on allelopathy (Bond, 2002). Cover crops suppress weeds

106 


during early crop season, but they do not provide full-season weed suppression (Reddy, 

2001; Reddy et al., 2003; Teasdale, 1996). Selective activity of tree allelochemicals 

from Leucaena leucocephala (Lam.) Dewit and Eucalyptus species on different crops 

and weed species have been reported by Ferguson and Rathinasabapathi (2003). Field 

studies at the Experimental Farm, Annamalai University, India assessed the impact 

of different crop residues applied as green leaf manure to both rice nurseries and 

wetland (transplanted) rice fields (Parthiban and Kathiresan, 2002; Kathiresan, 2004). 

The green leaves of Eucalyptus globulus L., L. leucocephala and Calotropis gigantea 

L. were applied at 5 t ha -1 . Green leaves were soil incorporated at the time of final 

land preparation, flooded with water, and allowed to decompose. After 7 days, the 

land was leveled and used as nursery to raise rice seedlings. Similarly, in wetland 

rice fields, green leaves were incorporated and rice seedlings were transplanted into 

puddled soil. The results showed that Eucalyptus and Leucaena leaves significantly 

reduced the density of Echinochloa spp. and Cyperus rotundus L. in the nursery. In 

wetland rice fields, the densities of C. rotundus, Cyperus littoralis Gaud., C. difformis 

and Sphenoclea zeylanica Gaertn. were drastically reduced by these two crop residues. 

The weed suppression was even better than the rice herbicide, butachlor. However, 

use of these crop residues in the nursery reduced rice populations. Laboratory studies 

supported this observation and clearly showed a direct inhibitory response of higher 

magnitude on rice seed and Echinochloa seed by the leaf leachates whose 

concentrations corroborate with field doses. The crop management strategy of applying 

green leaf manures of Eucalyptus and Leucaena could compliment weed suppression 

(comparable to that of a single application of pre-emergence herbicide) in wetland 

transplanted rice. The risk of inhibition of rice seed germination restricts the application 

of this technique to rice nursery. However, it is possible to include these green leaves 

as component of an integrated weed management strategy in wetland rice production 

as rice seedlings are less sensitive to these crop residues. An array of compounds such 

as cineol, pinene, caffeic acid, gallic acid, eucalyptin, hyperoside, and rutin, which 

constitute primary allelochemicals in Eucalyptus, might have suppressed the 

germination of weed seed as well as rice in the nursery as observed in other studies 

(Rao et al., 1994; Sivagurunathan et al., 1997). Aqueous extracts of leaves, seeds and 

litter of Leucaena were shown to be significantly phytotoxic on many test species in 

South Korea (Chou, 1990) and the chemicals isolated include mimosine, quercetin 

and gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic and p-coumaric acids. 

Recuperation of nutrients absorbed by the crops in small holder farms of developing 

countries has been traditionally taken care of through the incorporation of several 

kinds of bulky organic manures. Besides crop residues, other farm wastes such as 

cattle manure and some post harvest byproducts of crops are frequently used for 

manuring the crops. Filter pressmud, a bi-product of sugar factories, that crystallizes 

sugar from cane juice is the precipitated impurity from cane juice. Pressmud from 

cane juice accounts for about 3% and all sugar factories accumulate enormous quantities 

of pressmud. This bi-product is widely used in South Asian countries as organic 

manure. This pressmud upon incorporation in the soil, prior to transplanting of rice 

in wetlands, releases allelopathic metabolites that contribute to weed suppression.


Allelopathic suppression of weeds by the application of pressmud as organic manure 

in wetland agriculture has been included as a vital weed management component in 

Integrated Systems of Farming (Arulchezian and Kathiresan, 1990; Kathiresan, 2004a). 

2.3. Invasive Weeds with Allelopathic Potential 

Invasive species, particularly those non-indigenous to terrestrial, aquatic, wetland, 

and wildlife habitat, cause extensive environmental and economical damage to native 

ecosystems. Invasive plant species alone cause losses in excess of $35 billion annually 

in the U.S.A. (Pimentel et al., 2000). Non-indigenous weeds in the U.S.A. are estimated 

to invade 700,000 hectares of wildlife habitat per year (Babbitt, 1998). Invasive aquatic 

weeds are a significant problem in the U.S.A. Aquatic weeds choke waterways, reduce 

recreational use of lakes and rivers, and alter aquatic animal species (Pimentel et al., 

2000). In the U.S.A. alone, an estimated $100 million is spent annually for managing 

aquatic weed species (OTA, 1993). An estimated $15 million is spent on managing 

the aquatic weed species hydrilla [Hyrdilla verticillata (L.F.) Royle] in the state of 

Florida, U.S.A. (Center et al., 1997). Invasive weeds in terrestrial ecosystems such as 

pastures cause estimated losses of $2 billion annually in the U.S.A. (Pimentel, 1991). 

Examples of invasive weeds in pastures are cogongrass [Imperata cylindrica (L.) 

Beauv.], yellow starthistle (Centaurea solstitialis L.), and purple loosestrife (Lythium 

salicaria L.). Specifically, cogongrass is a C 4 , rhizomatous, perennial monocot that 

has become an invasive weed in many gulf-states of the southeastern U.S.A. since its 

introduction to the U.S.A. in the late 19 th and early 20 th centuries (Byrd and Bryson, 

1999; Dickens, 1974; Dickens and Buchanan, 1971; Elmore, 1986). Cogongrass grows 

in tropical, subtropical, and some temperate regions of the world (Akobundu and 

Agyakwa, 1998; Bryson and Carter, 1993), and is found on all continents except 

Antarctica (Holm et al., 1977). Cogongrass is among the most troublesome weeds 

worldwide (Falvey, 1981; Holm et al., 1977). It is extremely competitive with crops 

and neighboring plant communities (Eussen and Wirjahardja, 1973). It has been 

reported to reduce corn (Zea mays L.) grain yield by 80 to 100% (Koch et al., 1990; 

Udensi et al., 1999). Koch et al. (1990) also reported > 90% yield reduction for 

intercropped corn and cassava (Manihot esculenta Crantz) grown in cogongrass 

infested fields. 

Cogongrass competes with other plant species commonly found in similar 

ecosystems (roadways, pastures, mining areas, parks, and other natural and recreational 

areas) via allelopathic-type mechanisms. Residues and liquid exudates of cogongrass 

foliage and root residues have been shown to reduce germination and shoot and root 

growth of monocot and dicot weed and crop species (Koger and Bryson 2004; Koger 

et al., 2004). Germination of bermudagrass [Cynodon dactylon (L.) Pers.] and Italian 

ryegrasses (Lolium multiflorum Lam.) was reduced by as much as 97%, and shoot and 

root growth by as much as 96% at the highest concentrations. However, cogongrass 

had no allelopathic effect on hemp sesbania [Sesbania exaltata (Raf.) Rydb. Ex A. W. 

Hill] (Koger and Bryson, 2004). Phenolic compounds present in foliage and roots of 

cogongrass may be responsible for the allelopathic inhibition of germination and

108 


seedling development of other species. Inderjit and Dakshini (1991) reported several 

phenolic compounds extracted from leachates of cogongrass foliage and roots/rhizomes 

reduced germination and shoot and root length of mustard [Brassica juncea (L.) 

Czern and Coss.] and tomato (Lycopersicon esculentum Mill.). Inderjit and Dakshini 

(1991) also found phenolic compounds in leachates of soil collected near the 

rhizosphere of cogongrass as well as up to 3 m away that were not present in control 

soils. These reports suggest that allelopathic effect of cogongrass is species-specific. 

Thus, allelochemicals from cogongrass may serve as potential leads in discovery of 

new selective herbicides. 

2.4. Plant Products for Allelopathic Control of Weeds 

Presence of abundant moisture in wetlands allow faster transport of allelochemicals 

from the applied plant products to the target weeds. Aquatic systems have the advantage 

of no soil-interface that restricts the activity of allelochemicals on susceptible flora. 

As a result, use of plant products for allelopathic control of weeds has more potential 

in wetlands and aquatic systems than soil containing terrestrial systems. Carrot grass 

(Parthenium hysterophorus L.), an invasive obnoxious weed originated from Mexico 

has shown to be allelopathic on another introduced invasive aquatic weed water 

hyacinth [Eichhornia crassipes (Mart.) Solms. Laubach] in India. Dried residues of 

leaves and flower of carrot grass applied in to the water at 0.5% (w/v) killed water 

hyacinth within one month. Residues of leaves of carrot grass showed the highest 

biological activity followed by flowers, stem and roots. The flowers and leaves of 

carrot grass also had higher total phenolic acid levels in the medium which was 

responsible for the inhibition (Pandey et al., 1993). Dry powder of an epiphytic plant 

Cassytha sp dissolved in water infested with water hyacinth at 1-2% (w/v) resulted in 

complete desiccation of leaves of water hyacinth with in 15 days and drastic reduction 

in biomass (Kauraw and Bhan, 1994). Parthenin, a sesquiterpene lactone that is one 

of the major toxins in the weed, proved lethal to water lettuce (Pistia stratiotes Linn.) 

and duck weed (Lemna perpusilla Torr.) at 50 ppm concentration and to water hyacinth 

at 100 ppm concentration. The mechanism of allelopathic inhibition by parthenin 

was identified to be associated with decline in water use, root dysfunction, excessive 

leakage of solutes resulting in massive damage to cellular membranes, loss of 

dehydrogenase activity in the roots and destruction of chlorophyll in the leaves (Pandey, 

1996a). 

Among several allelochemicals (p-hydroxybenzoic acid, anisic acid, salicylic acid, 

coumaric acid, fumaric acid, tannic acid, gallic acid, chlorogenic acid, vanillic acid, 

caffeic acid and ferulic acid), p-hydroxybenzoic acid had highest phytotoxicity on 

aquatic weeds and was lethal at 50 ppm to all weeds tested, including floating aquatic 

weeds like salvinia (Salvinia molesta Mitchell), azolla (Azolla nilotica Decne. ex 

Mett.), spirodella (Spirodella polyrhiza (L.) Schieiden) and lemna (Lemna 

paucicostaha Hegelm.), and submerged weeds like hydrilla (H. verticilata), 

ceratophyllum (Ceratophyllum demersum L.) and najas (Najas graminea Del.). 

However, p-hydroxybenzoic acid was lethal for water hyacinth and water lettuce only


at 100 ppm. Water hyacinth (E. crassipes), water lettuce (P. stratiotes) and water fern 

(S. molesta Baker.) were relatively more tolerant to allelochemicals except for phydroxybenzoic 

acid compared to other floating or submerged weeds (Pandey, 1996b). 

An Indian medicinal herb Coleus amboinicus Lour., commonly used for curing 

cold, flu and other such ailments upon raw consumption, showed remarkable activity 

on water hyacinth among different weeds and herbs tried for their allelopathy on 

water hyacinth. Dried leaves of this medicinal herb C. amboinicus were ground to 

powder and applied to the water system as a suspension (30 g L -1 ). Death of water 

hyacinth occurred within 24 h and nearly 100% reduction in biomass was achieved 

within 9 days. However, spraying C. amboinicus over the foliage of water hyacinth at 

100 g L -1 proved ineffective due to lack of penetration and absorption of allelochemicals 

from dried powder into the plant system. Applied as a suspension in water, the natural 

product was active, killing the weed even at lower dosages of 12.5 g L -1 within 14 

days (Kathiresan and Kannan, 1998). In the Philippines, 57 different compounds, 

including α humulene, carvacrol, thymol, α-pinene and α-terpine were identified 

from C. amboinicus. Some of these compounds showed a high degree of biological 

activity, proving lethal to several micro organisms, insects and snails (Vasquez et al., 

1999). C. amboinicus powder was shown to inhibit algal growth in static-water systems 

(Kathiresan, 1998). 

Graded dosages of the natural products of C. amboinicus were evaluated for their 

inhibitory effect on water hyacinth (Kathiresan, 1999) through specific bio-assay 

methods, involving whole plants and cut leaves, separately. The study indicated that 

dry powder of C. amboinicus is a candidate plant product for the control of floating 

aquatic weeds water hyacinth, water lettuce and duck weed with rapid biological 

activity. The biological activity is due not only to mere water loss but also physiological 

effects (as compared to sodium chloride at 40 g L -1 , the standard desiccant with 25% 

weed biomass reduction after 9 days). The data also implies a higher magnitude of 

inhibition on root system (Kathiresan, 2000). 

The lowest dosage of 1% concentration (10 g L -1 ) may seem high, but is similar 

to that widely used for therapeutic purposes. Earlier studies have shown that water 

hyacinth was relatively tolerant to allelochemicals, as 100 ppm of p-hydroxybenzoic 

acid was required to cause death, whereas 50 ppm concentration was sufficient for 

other weeds (Pandey, 1996b). Similarly, fungal oxalates and pure oxalic acid that 

caused considerable and severe chlorosis in other aquatic weeds induced only slight 

chlorosis in water hyacinth (Charudattan and Lin, 1974). Under these circumstances, 

a crude extract of a safe medicinal herb C. amboinicus, causing death of water hyacinth 

within 9 days, might offer an effective control strategy. Further, a stable inhibitory 

response caused by C. amboinicus powder applied to cut leaves of water hyacinth 

under controlled conditions in static water at dosages ranging from 30 g L -1 down to 

1.0 g L -1 reduced fresh weight from 61 to 49% respectively, suggesting that C. 

amboinicus dry powder could exert adequate allelopathy at low dosages. The lowest 

dosage of 0.1 g L -1 (100 ppm) of C. amboinicus caused 24% fresh weight reduction. 

Apparently, the dosage of C. amboinicus powder lethal to water hyacinth could be 

reduced drastically, if either the natural product or the active principle is formulated

110 


to enhance absorption through foliage. In India, when the sources of irrigation water 

recede during the summer, the smaller volume of water is more accessible for treating 

with C. amboinicus at 10 g L -1 (1% w/v). C. amboinicus also hold promise for biocontrol 

of water hyacinth on small farms. In another aquatic habitat, Myriophyllum 

spicatum L. has been shown to be allelopathic on submerged macrophytes and the 

compound that was identified as major active allelochemical Tellimagrandin II (Gross 

et al., 1996) also interferes with photosystem II and photosynthetic oxygen evolution 

in Anabaena sp. (Leu et al., 2002). 

Barnyardgrass is a problem weed in wetland transplanted rice. The weed 

infestation begins with use of contaminated rice seeds. Morphology of barnyardgrass 

closely resembles rice during seedling stage. The seedlings are pulled along with rice 

seedlings from the nursery and transplanted in the main field. Transplanted seedlings 

of barnyardgrass may escape control with pre-emergence herbicides in the main field. 

Allelopathy of rice seeds is shown to be stimulatory to the germination of barnyardgrass. 

Farmers in developing countries normally soak the gunny bags with rice seeds harvested 

in the previous season, in water canals overnight prior to seeding. The allelochemiclas 

similar to gibberellin released from the rice seeds help break the dormancy of 

barnyardgrass and stimulate germination (Kathiresan and Gurusamy, 1995). To 

overcome this allelopathic stimulation of barnyardgrass, it is recommended that small 

farmers soak the rice seeds in open containers, skim the floating barnyardgrass seeds 

(due to differential specific gravity) as a preventive weed control measure. However, 

in other studies rice hull extracts have been shown to exert allelopathic inhibition of 

germination and growth in barnyardgrass (Ahn and Chung, 2000). 

3. ALLELOPATHIC MICROORGANISMS 

Microbes play a vital role in designing and implementing allelopathic control of aquatic 

weeds and certain weeds of wetland eco-systems. They serve as agents for classical 

bio-control in several parts of the world with a simple idea of locating highly host 

specific agents from the native range of the weed and introducing them in new regions 

requiring control where the weed has established, after rigorous experimental 

evaluation. The microbial toxins either in their parental form or as metabolites also 

offer scope to serve as effective herbicides for weed control. Some of the microbial 

toxins exhibit a new mode of action with novel target site that could stimulate discovery 

of new herbicidal chemistry. 

3.1. Pathogens for Weed Control 

Bio-control using pathogens holds promise mostly in non-cropland situations because 

of the slow pace of control of weeds and the wider window for control as compared 

with the shorter window of the cropping season and associated disturbances under 

cropland situations. However, in aquatic systems they appear to be more realistic, due 

to the absence of such cropping barriers and enhanced mode of dispersal in water. 

Among the wetland weeds, Echinochloa crusgalli and Cyperus rotundus are being


evaluated for biological control using the fungi Exserohilum monocerus and Dactylaria 

higginsi (Luttrell) MB Ellis, respectively (Charudattan and Dinoor, 2000). Fungal 

pathogens of aquatic weeds have been identified from Neotropical regions since the 

late 1970s. Alligator weed (Alternanthera philoxeroides (Mart.) Griseb) introduced 

from Brazil, became a serious invader of aquatic systems in Australia, Asia and North 

America. A preliminary survey of fungal pathogens on alligator weed in Brazil has 

yielded two species Nimbya alternantherae (Holocomb and Antanopoulos) Simmons 

and Alcorn and Cercospora alternantherae Ellis and Langois (Barreto and Torres, 

1999). Both caused leaf spots on the alligator weed and their virulence varied with 

geographic range and altitude. Aquatic ferns such as Salvinia molesta and Salvinia 

auriculata JB Aublet., are regarded as invasive aquatic weeds in many of the water 

habitats in Neotropics, Africa and Asia. Necrotic spots caused by the fungus Drecshlera 

sp. on S. auriculata and the inoculum was easily produced and pathogenic (Muchovej 

and Kushalappa, 1979). Cattail (Typha domingensis Pers.,) native to Neotropics has 

also evolved to be invasive in aquatic systems. The fungal pathogen Phoma typhae – 

domingensis is regarded as a potential candidate for the development of mycoherbicide 

for cattail (Barreto and Evans, 1996). Egeria densa Planch, a worse submerged aquatic 

weed interrupts hydro electricity generation, damages grids, and causes substantial 

economic loss to hydroelectric companies. Among eight fungal isolates that have 

shown promise, Fusarium graminearum Schw. was pathogenic to E. densa causing 

chlorosis followed by necrosis and complete tissue disintegration (Barreto et al., 2000). 

Three different fungi (Chaetomella raphigera Swift, Cercospora sp. and 

Mycosphaerella sp.) caused severe blight in parrot’s feather (Myriophyllum aquaticum 

(Vell.) Verdc), an invasive aquatic weed (Barreto et al., 2000). Water hyacinth, 

recognized as the world’s worst aquatic weed (Holm et al., 1977), has been the prime 

target weed for bio-control. One of the first pathogens to be patented as a mycoherbicide 

was Cercospora piaropi Tharp. (Charudattan, 1996). Other promising pathogens are 

Uredo eichhorniae Fragosco and Ciferri, Myrothecium roridum Tode ex Fr. Alternaria 

eichhorniae NagRaj and Ponnappa (Barreto et al., 2000). 

3.2. Microbial Toxins as Leads for Herbicides 

The unique relationship between plants and their pathogens suggest that 

microorganisms may be a better source of future herbicides than allelochemicals 

produced by higher plants (Duke, 1986). The major constraint with most 

allelochemicals from higher plants is that their range of selectivity is narrow and they 

are often autotoxic. Perhaps, this may be one reason that breeding for crop plants to 

produce higher levels of allelochemicals has not been aggressively pursued. In contrast, 

many microbial phytotoxins are both selective and efficacious at low rates. Bialaphos 

[L-2-amino-4-(hydroxyl) (methyl) o-phosphinyl)-butyryl-L-alanyl-L-alanine], a 

tripeptide extract from Streptomyces viridichromogens Schulz. Freiburg. marketed 

as Herbiace®, tentoxin, a cyclic tetrapeptide produced by Alternaria alternata (Fr.) 

Keissl., rhizobitoxine produced by Rhizobium japonicum Kirchner and an analogue 

of cystathionine are a few examples. Several non-host selective phytotoxins produced 

by microorganisms have been reported (Duke, 1986; Hoagland, 2001).

112 


4. ALLELOPATHY – LEAD FOR NOVEL HERBICIDAL CHEMISTRY 

Allelochemicals can provide potential leads in discovery of new herbicides with novel 

molecular target sites of action. Hydantocidin from Streptomyces hygroscopicus B. 

Straubinger is an analogue of the allelochemical phosphinothricin and inhibits 

adenylosuccinate synthetase. This analogue mimics the substrate inositol 

monophosphate (IMP) and binds the enzyme 1000 fold tighter than IMP, thereby 

forming a dead-end complex. Hadacidin and alanosine, both microbial products are 

also inhibitors of this enzyme (Duke et al., 2000). To date, bialaphos and glufosinate 

derived from microbial compounds are the two most successfully commercialized 

herbicides (Hoagland, 2001). Glufosinate is the synthetic version of phosphinothricin, 

a breakdown product of bialaphos (Hoagland, 2001; Lydon and Duke, 1999). Bialaphos 

sold as herbicide in Japan is derived from Streptomyces species is a proherbicide that 

is metabolically degraded to phosphinohricin by target plant in order to be herbicidally 

active. Glufosinate is the only commercial herbicide that inhibits glutamine synthetase 

despite plethora of natural and synthetic products known to inhibit glutamine synthetase 

(Lydon and Duke, 1999). A gene coding for an enzyme that acetylates the active acid 

of phosphinothricin rendering it non-phytotoxic, phosphinothricin acetyl transferase 

(Pat) gene has been isolated from S. hygroscopius. This gene has been used in truncated 

form to transform crops such as maize to impart tolerance to glufosinate (Copping, 

2002). Other examples of herbicides derived from natural product chemistry are 

triketones from Syngenta and cinmethlin from BASF. Another potential herbicide of 

microbial origin is AAL-toxin, a natural metabolite from Alternaria alternata f sp. 

lycopersici. Monocots were generally immune to the effects of AAL-toxin, whereas 

several broad leaved species are susceptible. Abbas et al. (1995) proposed that the 

selective weed control could be possible through AAL-toxin. 

5. SCREENING OF ALLELOPATHIC PLANT MATERIALS 

Allelopathy is essentially a chemical defense mechanism used by plants to keep other 

plants out of their space. Though allelopathy forms a part of network of chemical 

communication between plants, it is part of plant interference along with competition 

for resources. Competition involves the removal or a diminution of shared resources, 

while allelopathy involves the addition of a chemical compound to the environment 

through different processes (Rice, 1984; Putnam, 1985). Allelopathic chemicals in 

general affect seed germination, root growth, shoot growth and/or seedling vigor in 

the early stages of the receiver’s growth and may interfere with metabolic functions 

like photosynthesis, membrane permeability, biosynthesis of enzymes, lipids, protein, 

etc. as the receiver progress in growth processes. In aquatic systems and wetlands, 

screening of allelopathic plant materials for biological efficiency is relatively easier 

as the allelochemicals are frequently absorbed through the roots of the receiver and 

transported from the donor directly through water without much of resistance from 

the soil-interface. However, screening processes have different phases involving either 

larger size or larger population of target weeds in different environments. Screening


is used mainly to confirm the biological efficiency of the substances to a magnitude 

which would at least serve as component of integrated weed management though not 

as a holistic weed control measure. Such a rigorous or repeated experimentation with 

different sets or modes of bio-assay becomes imperative as many of the allelochemicals 

exhibit inhibitory response on seedling germination and establishment but seldom 

lethal on large sized receiver plants. Screening techniques for allelopathy in aquatic 

systems or allelochemicals which are transported mainly through water body should 

include some critical points raised by Willis (1985), Putnam and Tang (1986), and 

Cheng (1992). They are: 

• Pattern of inhibition of one species by another must be shown using suitable 

controls, describing the symptoms and quantitative growth reduction; 

• The putative aggressor plant must produce a toxin; 

• There must be a mode of toxin release from the plant to the environment and thus 

the target plant; 

• The afflicted plant must have some means of toxin uptake, be exposed to the 

chemical in sufficient quantities and time to cause damage, and to notice similar 

symptoms; 

• The observed pattern of inhibition should not be explained solely by physical 

factors or other biotic factors, especially competition. 

The first phase or initial stage of screening include bio-assays. Those plant 

materials that are confirmed to possess biological activity through bio-assay need to 

be further studied for their dose response pattern under controlled environment. Plant 

materials elucidating appreciable response even at minimal doses could be further 

evaluated for their allelopathic potential under natural habitats. 

5.1. Bio-Assays 

Bioassays are an integral part in all studies of allelopathy. They have multiple uses 

such as evaluating allelopathic potential of different plant material, tracing activity 

during extraction, purification, and identification of bioactive compounds. The 

techniques used vary greatly and one has to standardize the procedure independently, 

and modify to suit the occasion and conditions. According to Rice (1984) and Putnam 

and Tang (1986), seed germination is used as a test in most bioassays. Though different 

types of bioassays are used, all of them in general include seed placed on substrate 

saturated with the test solution. Germination, as indicated by the emergence of the 

radical 2 mm beyond the seed coat is scored over a period of time. The factors that 

need to be kept constant are oxygen availability, osmotic potential of the test solution, 

pH, and temperature. The elongation of the hypocotyls or coleoptiles are often observed 

along with germination. Dry biomass which is easier to measure could be used as a 

measure of growth (Bhowmik and Doll, 1984). Sensitivity is normally higher in growth 

bioassays than in germination bioassays. When the quantity of allelochemicals is 

limited, agar cultures can be used. Pre-germinated seeds can be exposed to the agar 

cultures containing test solution.

114 


Bioassay for searching of allelopathy in aquatic weeds is comparatively easier 

and this could be designed under both laboratory or greenhouse conditions using 

either part or whole plant of the aquatic weed (Kathiresan, 2000; Kannan, 2002). 

Whole plants of floating or submerged aquatic weeds targeted for allelopathic control 

can be grown in suitable containers with water containing standardized nutrients. 

The powder of candidate allelopathic substances or plant products are added to water 

either on w/v or v/v basis with appropriate untreated controls. Periodic observations 

at designated intervals could include reduction in root length and mass, reduction in 

shoot length and mass, desiccation, scorching and bleaching or mortality score similar 

to herbicide injury. Based on the screening data, plant products could be classified in 

to highly allelopathic, moderately allelopathic, and less allelopathic. For example, 55 

different plant products were screened for allelopathic inhibition of water hyacinth 

involving whole plants as well as cut leaves at Annamalai University. In bio-assays 

involving whole plants, ten of them including C. amboinicus, P. hysterophorus and 

L. leucocephala were highly allelopathic based on fresh weight reduction (>30%) of 

water hyacinth within 48 hr after treatment. Another 12 including Acalypha indica 

Linn., Trianthema portulacastum L. and Sesbania grandiflora (L.) Pers. showed 

moderate allelopathy, fresh weight reduction of water hyacinth was 15-30% within 

48 hr after treatment. Twelve other plant products including Croton sparsiflorus 

Morong, Cleome viscosa L. and Eclipta alba L. showed less allelopathy, fresh weight 

reduction of water hyacinth was less than 15% within 48 hr after treatment (Table 1). 

The remaining 21 plant species including Leucas aspera Spreng. Curcuma longa L. 

and Euphorbia hirta L. did not show any allelopathic effect on water hyacinth (Kannan, 

2002). To measure dose dependant responses of allelopathic substances more precisely 

Table 1 : Percentage reduction in fresh weight of water hyacinth (Eichhornia 

crassipes.) due to various plant products. (Kannan, 2002) a . 

Treatments Days After Treatment 

plant products @ 30 g L-1 2 4 6 8 

Coleus amboinicus 56.57 (69.66) 90.00 (100.00) 90.00 (100.00) 90.00 (100.00) 

Acalypha indica 29.98 (24.97) 44.24 (48.68) 90.00 (100.00) 90.00 (100.00) 

Leucas aspera -b - - - 

Croton sparsiflorus 20.53 (12.30) 34.51 (32.11) 45.37 (50.66) 90.00 (100.00) 

Curcuma longa - - - - 

Trianthema portulacastum 27.06 (20.70) 45.09 (50.17) 90.00 (100.00) 90.00 (100.00) 

Cleome viscosa 21.10 (12.96) 35.77 (34.17) 45.66 (51.16) 90.00 (100.00) 

Leucaena leucocephala 34.84 (32.65) 90.00 (100.00) 90.00 (100.00) 90.00 (100.00) 

Sesbania grandiflora 27.17 (20.86) 44.51 (49.16) 90.00 (100.00) 90.00 (100.00) 

Parthenium hysterophorous 36.92 (36.10) 90.00 (100.00) 90.00 (100.00) 90.00 (100.00) 

Euphorbia hirta - - - - 

Eclipta alba 21.27 (13.17) 33.93 (31.17) 44.94 (49.91) 90.00 (100.00) 

Control - - - - 

SED CD (P = 0.05) 

2.40 

4.80 

2.34 

4.69 

1.25 

2.51 

- 

- 

a Figures in parenthesis are original values before arc-sine transformation. 

b No allelopathic effect.


and to detect the sensitivity of the target weed to minute quantities of allelopathic 

substances, incised plant parts of aquatic weeds would serve better than whole plants. 

For screening of different allelopathic substances for inhibition of water hyacinth, a 

specific bio-assay method was designed with cut leaves of water hyacinth (Kathiresan, 

2000; Kannan, 2002). This bioassay involved exposure of cut leaves of water hyacinth 

to graded doses of plant materials to be tested (dissolved in water). The leaves of 

water hyacinth plants (with healthy leaves submerged in water) were detached by 

cutting the petiole with a razor blade with care to retain the incision point below 

water level. The detached portions of leaf with a part of petiole intact was kept 

submerged in water for 90 seconds to ensure that no air was trapped internally. Then 

these leaves were transferred to scintillation vials with water where in different plant 

products were dissolved and individually compared with an untreated control. The 

percent fresh weight reduction of the cut leaves was calculated using the formula 

Initial weight of the cut leaves – weight after 24 hr of treatment 

X 100 

Initial weight of cut leaves 

5.2. Dose Response Studies 

Even if a plant product proves appreciably allelopathic on aquatic weeds in bio-assay, 

tracing the pattern of allelopathic inhibition with graded doses of the plant product 

becomes vital as aquatic systems requires enormous quantities of plant product due to 

the quantum of water body (dilution), which in many cases is not practically feasible. 

Furthermore, this dose response has to be plotted for differing morpho-physiological 

states of the weed that occurs in common, as the quantity of allelochemical required 

for a knock down effect is less with a small statured weed compared to that of larger 

sized weed. For this purpose, before screening of allelopathic plant products on water 

hyacinth, the latter was classified in to different morphological stages (large, medium 

and small) prevalent in the state of Tamilnadu, India using discriminant analysis 

(Kannan and Kathiresan, 1998). Both whole plant and cut leaf bio-assays were done 

on each of the three stages of water hyacinth. Higher (10 to 30 g L -1 ) doses were used 

in whole plants to impart a near lethal effect on the whole plant and lower doses were 

used in cut leaf bio-assays to cause some allelopathic injury, if not lethal. The doses 

used for water hyacinth cut leaf bio-assay were 30, 25, 20, 15 10, 5 2.5, 1, 0.5, 0.25, 

and 0.1 g L -1 . The dose response data revealed that cut leaf-bioassay was superior to 

whole plant assay. For example, C. amboinicus at as low as 0.1 g L -1 dose caused 24% 

fresh weight reduction in water hyacinth within a week of exposure (Kathiresan, 

2000). Therefore, cut leaf bio-assay could be useful to detect allelopathic potential of 

plant products which otherwise may have been missed if whole plant assay was used. 

In contrast, the dose response study data revealed that lethal doses for large, medium, 

and small plants of water hyacinth were relatively closer. C. amboinicus at low (10 g 

L -1 ) dose caused death of the water hyacinth after 20 days whereas at high (25 g L -1 ) 

dose caused death within one week.

116 


5.3. Field Testing of Allelopathic Substances 

In many instances, the materials or plant substances that prove to be allelopathic in 

laboratory or pot culture experiments may not elucidate similar magnitude of 

allelopathic response on aquatic weeds in aquatic environments, watersheds, or 

wetlands. Hence, it is imperative to confirm plant products for their allelopathic 

potential on weeds in their own natural habitat. A search was made on allelopathic 

plant products for use in water hyacinth control programs at Department of Agronomy, 

Annamalai University. Ten of 55 different plant products that showed allelopathic 

suppression of water hyacinth within 48 h of treatment were selected and tested for 

their efficacy in natural habitats. The field testing was done in a two tier model. First, 

the ten plant products were tested in microponds (simulated natural habitat). Second, 

the plant products that confirmed to be allelopathic in microponds were further 

evaluated in natural watersheds. 

5.4. Preparation of Microponds 

Small (microponds) ponds of 1m x 1m x 1m dimension were dug in the field and 

canals with running water on both sides of the pond were maintained to minimize 

seepage loss of water from the ponds. Initially, the microponds were conditioned by 

flooding and maintaining water as needed for 20 days. After 20 days, healthy water 

hyacinth plants were introduced and allowed to grow for 2-3 days. The plant products 

to be tested were dissolved in the water of microponds, separately. Allelopathic potential 

of the plant products was evaluated in comparison with untreated control using data 

on fresh weight, number of healthy leaves, and chlorophyll content of the water 

hyacinth. 

5.5. Evaluation in Natural Habitats 

Top three best performing plant products in the micropond study were selected for 

realistic confirmation of their allelopathic potential in natural habitats. To this end, 

three watersheds of 168, 123, and 492 m 2 area were selected at three different locations 

within a radius of 50 km from Annamalai University. Water hyacinth was allowed to 

establish in watersheds and the surface area of water was completely covered by weed. 

Each watershed was divided in to three equal strips using polyethylene sheets stretched 

between bamboo poles running down the entire depth of water, with the poles anchored 

to the bottom of the watershed. The plant products were applied to water individually 

to one of the strips in each watershed. The data were collected on fresh weight, number 

of healthy leaves, and chlorophyll content at five days interval. Results proved that 

the plant products showing higher magnitude of allelopathic inhibition on water 

hyacinth in initial bio-assays like C. amboinicus continued to retain and exhibit the 

same magnitude of inhibition on water hyacinth with out any dissipation in natural 

watersheds or aquatic environments.


6. ALLELOPATHY AND INTEGRATED WEED MANAGEMENT 

Because of limited resources, an average farmer in a developing country can neither 

afford to take big economic risks nor opt for technologies associated with a lot of 

external inputs. As a result, research on vegetation management strategies capable of 

minimizing weed infestation and simultaneously favoring sustainable crop production 

that are economical and eco-friendly needs attention (Akobundu, 2000). Allelopathy 

fits in to this approach as one of the integral principles in any such cropping systems 

involving crop rotation, inter-cropping, cover crop, and off-season land management 

(such as raising green manures and ploughing in situ). Linking similar integrated 

farming approaches to integrated weed management and integrated pest management 

helps to address bio-diversity concerns with a simultaneous reduction in agrochemical 

use especially in low input agriculture and small hold farms. A 3-yr study of weed 

management in wetland transplanted rice, rice - mung bean cropping sequence with 

treatments assigned to the same plots every season at Annamalai University revealed 

that lowland weeds like C. difformis was drastically reduced by the introduction of a 

relay crop of mung bean in the sequence (Kathiresan, 2002). Raising a green manure 

crop of Sesbania aculeata Poir in the off-season (May - July) and ploughing it in situ 

at the age of 45 days, before the cultivation of rice in the first (August - January) as 

well as second (January - April) season, helped in reducing weed competition in both 

the rice crops (Gnanavel and Kathiresan, 2002). Off-season land management such 

as raising green manure crop significantly reduced the weed seed reserves in the soil 

through allelopathic interference, whereas rotation of an upland crop like mung bean 

with rice interrupted the weed flora in lowland through mung bean residues. 

Integrated weed management assumes significance in managing aquatic systems. 

Use of herbicides are constrained with drastic reduction in water quality and ultimate 

ill effect on associated non-target organisms. In countries like India, herbicides are 

yet to get registered for use in aquatic systems. Under these conditions managing 

infestations of water hyacinth, water fern, and water lettuce is challenging. In one of 

the recreational lakes with tourist attraction in a hill resort in Ooty, in the state of 

Tamilnadu, India, the public authority has spent heavily (Indian Rupees 1.25 crores, 

about US $200,000) for manual clearing of water hyacinth for one time. Similarly, 

thousands of army personnel were used for clearing water hyacinth in a lake in 

Bangalore, the capital city of Karnataka State, India. Classical biological control is 

the only option available and that too is difficult in situations where the water body 

dries off in the peak summer, leaving the released insects to starve and die due to 

interrupted host range. Accordingly, integration of short term control measures with 

classical biocontrol might offer excellent results. Allelopathy reinforced classical biocontrol 

research has been targeted and taken up at Department of Agronomy, 

Annamalai University through National Agricultural Technology Project funded by 

Indian Council of Agricultural Research. This project originated from the basic concept 

of allelopathic inhibition of water hyacinth by C.amboinicus as mentioned earlier. 

However, the requirement of plant product for treating larger watersheds might pose 

practical difficulties. Previous results also indicated that if absorbed in to plant through

118 


foliage, the plant product could be very effective even under very low doses (0.1 g 

L -1 ). The only hurdle faced for application of the plant product on the foliage is retention 

of plant product due to the repulsion by leaf cuticle. Any rupture and/or damage to 

leaf cuticle could potentially enhance absorption of plant product. To this end, the 

well established insect bio-control agents in India, Neochetina bruchii Hustache / 

eichhorniae Warner were chosen for the study to serve as a component of integrated 

weed management. These weevils normally scrape on the leaves of water hyacinth. 

An attempt was made to integrate both these bio-control tools viz. classical biocontrol 

using N. bruchii / eichhorniae and application of the plant product C. 

amboinicus. Integrating both the tools are possible with two different sequences. 

Treating the water body first with plant product at a lesser dose with the expectation 

that it will reduce the vigor of the weed, predisposing it for faster and rapid destruction 

by the insect agents that are to be released later is one possibility. Whereas releasing 

the insect agents first on the weed host, allowing them to make leaf scrapings that 

might help foliar uptake of plant product that could be sprayed later is another. Both 

these sequences were compared in the study. It was observed that treating the water 

body first with plant product followed by the release of insect agents on the weed 

showed an antagonistic interaction, as the insects migrated from treated, partially 

killed plants to healthy plants. The second sequence of releasing the insect agents 

first followed by spraying of the plant product on the weed foliage produced an additive 

or synergistic response with rapid and complete weed control with in a single season. 

The optimum inoculation loads of insect agents, concentration of the spray fluid of 

plant product required, length of interlude between the release of insect agents and 

spraying of plant product were standardized for all three different growth stages of 

the weed, and the success of this integrated approach was demonstrated at three different 

watershed environments in the state of Tamilnadu, India. The plant product was also 

shown to be safe for the insect agents with out inducing migratory behavior and 

without causing any histo-pathological injury on different tissues of the insects like 

salivary gland, gut, cutin, testis, and brain. Further, the integrated approach also 

proved safe for non-target organisms and water quality (Kathiresan, 2004b). 


Plants can interfere with each other through allelopathy or competition for resources. 

Allelopathy can be used in weed management in several ways including cover crops, 

smother crops, green manure crops, breeding for allelopathic crop cultivars, mulching 

and crop residue management. Allelopathic suppression of weeds will not replace 

synthetic herbicides which are the dominant method of weed control in many countries 

nor will it be economically competitive with herbicides. However, allelopathy can fit 

in an integrated weed management strategy very well as a vital component. This 

approach could reduce the sole dependence on synthetic herbicides for solving many 

complex weed problems. The examples discussed herein include an aggressive rice 

cultivar for complimenting weed control in direct seeded rice and plant productreinforced 

classical bio-control (through weevils) of water hyacinth.

Disclaimer 


Mention of trade names or commercial products in this publication is solely for the 

purpose of providing specific information and does not imply recommendation or 

endorsement by the United States Department of Agriculture. 

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ANTONY V. STURZ 

BACTERIAL ROOT ZONE COMMUNITIES, 

BENEFICIAL ALLELOPATHIES AND 

PLANT DISEASE CONTROL 

Prince Edward Island Department of Agriculture, Fisheries, Aquaculture 

and Forestry, P.O. Box 1600, Charlottetown, P.E.I., C1A 7N3, Canada. 

Tel: 902-368-5664, FAX: 902-368-5661 

E-mail:avsturz@gov.pe.ca 

Abstract. The release of root exudates from plants encourages the formation of beneficial bacterial communities 

in the root zone capable of generating secondary metabolites that improve plant health and crop yield. Metabolites 

with antibiotic or lytic action have been identified , while others are known to induce systemic disease resistance 

in the host plant, or interfere with the nutritional requirements of phytopathogens. However, despite existing 

positive relationships between bacterial communities and their plant hosts, man-made attempts at applying 

bacteria for biocontrol purposes have met with limited success. Inconsistent performance of biocontrol bacteria 

in the field may be due to the variable expression of genes involved in the biocontrol action, or simply the 

resistance of established soil communities to a sudden and inundative influx of adventive bacterial species or 

strains. Regardless of the inherent capacity of ‘naturally occurring’ soil microbial ecosystems to buffer 

anthropogenic interference, crop management systems are regularly used to distort agro-ecosystems through, 

for example, the use of tillage operations, alternate cropping systems, monoculture, crop rotation length, fertilizer 

and organic amendments, and various crop protection chemistries. The management of soil microbial communities 

for disease control appears to involve, in part, the creation of short term chaos in the microbial community 

through the application of such perturbation stresses. While hope remains that bacterial communities with 

biocontrol activity will one day be used as an adjunct to, or replacement for, agrichemical crop protectants, 

reliable biological controls that moderate pathogen attack remain elusive. In the interim, disease suppressive 

soils may be encouraged to form through the use of modest perturbation stresses that promote microflora 

species’ diversity and functionalities underpinning natural bioantagonism. 


In its broadest sense allelopathy has been defined as “...any process involving secondary 

metabolites produced by plants, microorganisms, viruses and fungi that influence the 

growth and development of biological systems...” (IAS, 1998). In the bacterial 

kingdom, the production of secondary metabolites (allelochemicals) can result in the 

development of mutualistic, beneficial or antagonistic relationships (Smith and 

Goodman, 1999; Boller, 1995) amongst bacteria, or between bacteria and other living 

organisms. As such, bacterial secondary metabolites can act at the trophic level, directly 

affecting nutrient uptake or metabolism, or at the informational level, being recognized 

as signals by appropriate chemoperception systems (Dusenbery, 1992; Boller, 1995). 

Consequently, allelopathy may be viewed as an ecological phenomenon (Romeo, 2000) 

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© 2006 Springer. Printed in the Netherlands.

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capable of regulating ecosystem health and biodiversity (Wardle et al., 1997; Mallik, 

2000). 

Many secondary metabolites can act at both the trophic and the informational 

level, becoming attractants or repellents, toxins or growth stimulants, depending upon 

the microbial partnerships involved (Dusenbery, 1992). Accordingly, microbially 

produced allelochemicals have been reported to express themselves as lytic agents or 

enzymes (Fridlender et al., 1993; Jacobson et al., 1994; Glick et al., 1999), antibiotics 

(Lynch, 1976; Bender et al., 1999), siderophores (Buysens et al., 1994; Marschener 

and Crowley, 1997) auxins (Patten and Glick, 1996; Glickman et al., 1998; Glick et 

al., 1999), volatile compounds (Claydon et al., 1987; Bakker and Schippers, 1987) 

and phytotoxic substances (Hoagland and Cutler, 2000). 

An intimacy is seen to extend between plants and microbes, in as much as plants 

appear able to influence the composition of the microbial community around their 

root systems by leaking specific carbohydrates, carboxylic and amino acids (Grayston 

et al., 1998) into the root zone, as well as through the ‘carbon-loading’ that occurs as 

root cell material is sloughed off during root growth (Hawes and Brigham, 1992). 

Hawes et al., 1998). In turn, rhizobacteria appear able to induce root exudation 

responses in plants (Bolton et al., 1993; Merharg and Killham, 1995). The result is a 

circular allelopathic cascade initiated by plant root exudates that trigger a positive 

microbial ‘allelopathic feedback’ in which the final receptor organism is also the 

initiator. 

It is this allelochemical interaction amongst soil microbial communities and the 

way in which their relationships subsequently influence plant health and disease 

development that will form the emphasis of the present chapter. 

2. MICROBE-MICROBE INTERACTIONS 

2.1. Commensal Relationships, Protocooperative Assemblages and Plant Pathogens 

A large proportion of successful biocontrol events in the infection court can be attributed 

to the positive outcome of multiple allelopathic episodes governed by the interplay 

among bacterial populations. In the root zone - that region characterized as occurring 

outside the host plant, but within the sphere of influence of the root system - bacterial 

populations often form protocooperative assemblages (loose associations) that are 

mutually beneficial (though not obligatory). By contrast, commensal relationships 

where the microorganism (commensal) benefits, while the host (plant) is neither 

harmed nor helped, are a feature of the closer physical juxtaposition of bacteria and 

host plant, often marked by the sharing of the same food resource. This latter 

relationship can be a feature of bacteria inhabiting the root surface (rhizoplane), but 

can also be extended to include those bacterial colonists found within plants 

(endophytes)-specifically in the endoroot tissues. However, not withstanding the above, 

distinctions between commensal or protocooperative assemblages become equivocal

BACTERIAL ROOT ZONE COMMUNITIES, BENEFICIAL ALLELOPATHIES AND PLANT DISEASE CONTROL 

in the face of community antagonism to pathogen invasion, and should, perhaps, be 

viewed independently of any plant health benefits. 

While pathogen antagonism has been collectively termed biological control and 

defined by Baker (1987) as ‘... a decrease of inoculum or the disease-producing activity 

of a pathogen accomplished through one or more organisms, including the host plant...’, 

it can also be argued that antagonism, at the communal level, is not necessarily directed 

at phytopathogens specifically, but at any group of organisms ‘invading’ the trophic 

level of an established community. In this sense, interactions amongst autochthonous 

(established) consortia (functional groupings) of microflora can be classified as 

beneficial where they promote plant health or inhibit phytopathogen attack (Mukerji 

et al., 1999). 

Accordingly, any exochthonous latecomer (colonist or pathogen) is likely to 

provoke a negative response from an established community, since its arrival will 

upset any balance amongst the community members (Atlas, 1986). In this respect, 

the resilience of a soil microbial community, when expressed in terms of its ability to 

inhibit invasions (colonization) by ‘non-community’ species will, in part, define its 

stability. 

As such, exochthonous species, enter the niche environment by chance but cannot 

maintain themselves in an active condition (Cooke and Rayner 1984). By contrast, 

indigenous communities may be subdivided into the slow growing autochthonous 

groups and the fast growing transient zymogenous groups - the former surviving on 

refractory substrates, and so, for the most part, remaining constantly active, while the 

latter only become active when a suitable food resource presents itself, and so are 

otherwise quiescent (Cooke and Rayner 1984). 

2.2. ‘Self-awareness’ in Bacterial Communities 

It is believed that population density of a consortium component species mediates 

population function through ‘self-awareness’ mechanisms such as ‘quorum sensing’, 

that enable bacteria to communicate among and between species in a consortium 

(Miller and Bassler, 2001). 

The degree to which bacterial consortia behave as commensal, protocooperative 

or pathogenic assemblages is dictated (in part) by the component populations’ density, 

which will vary according to the prevailing abiotic conditions affecting secondary 

metabolite production - including those with antibiotic properties (Grimwood et al., 

1989; Tateda et al., 2001). Key abiotic factors include, among others, pH, temperature, 

moisture, salinity, oxygen concentration and carbon availability (Duffy and Défago, 

1999; Gaballa et al., 1997; Gutterson et al., 1988; Nakata et al., 1999; Shanahan et 

al., 1992; Slininger and Jackson, 1992; Slininger and Sheawilbur, 1995) 

A population’s ability to identify ‘itself’ through the recognition of diffusible 

signaling molecules (autoinducers) - generally acylated homoserine lactones (acyl- 

HSLs) for gram-negative bacteria, and oligopeptides for gram-positive bacteria - elicits 

the modulation of gene expression, that can alter bacterial function in ways that may 

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ultimately destabilize cooperative behaviour (Salmond et al., 1995; Albus et al., 1997; 

Surette and Bassler, 1998). 

Paradoxically, the ability to exchange low molecular weight diffusible signal 

molecules suggests that certain species specific traits are repressed to allow individual 

bacteria to form consortia, so enabling them to survive within a specific habitat until 

such time as it benefits that species population to dissolve the co-operative group. 

This event is often catalyzed by the build up of sufficient amounts of signal molecule 

to activate receptor proteins that trigger changes in gene expression. 

Quorum sensors in bacteria have been implicated in regulating a range of 

physiological activities including conjugal transfer (Piper et al., 1993), swarming 

responses (Givskov et al., 1997), sporulation (Dworkin and Kaiser, 1985; Hoch, 1995), 

biofilm formation (Davies et al., 1998), and, most importantly, the regulation of 

virulence genes that initiate extracellular polysaccharides, enzymes, surfactants and 

antibiotics prior to and during pathogen attack (Beck von Bodman and Farrand, 1995; 

Fuqua, et al., 2001; Parsek et al., 1999; Whithers et al., 2001). 

Thus, for example, the causal agent of potato soft rot, Erwinia carotovora, will 

only initiate exoenzyme secretion - involving cellulases and various pectinases - when 

cell density levels reach a certain threshold (Pirhonen et al., 1993); presumably to 

ensure that invading bacteria do not prematurely elicit a host-defence response prior 

to achieving sufficient bacterial cell numbers to mount a successful infection. 

Interestingly, E. carotovora exoenzymes are produced in tandem with the antibiotic 

carbapenem, which it is believed inhibits other competitor bacteria in the infection 

court (Bainton et al., 1992). Consequently, it appears that some pathogen’s attack 

and self-defence measures are co-ordinated during attempts at plant infection. 

2.3. Community Self-regulation 

The biological basis of community homeostasis is generally believed to result from 

the dynamic balance which member populations in the community must exert to 

inhibit the recognition of ‘population self’ at the expense of the ‘community self’. 

Where complex regulatory cascades control gene expression of colonization and 

pathogenicity, gene induction is generally modulated by the rate at which a population’s 

signal molecule strength is diluted away. Accordingly, any situation that allows for 

signal molecule build-up - such as the close proximity of cells, or an environment 

limited diffusion rate - will result in the greater the likelihood that population selfrecognition 

and gene expression will be triggered. 

To-date, compounds reported to regulate population density dependent behaviours 

include N-acyl-homoserine lactones, among plant associated Proteobacteria (Eberl, 

1999; Fuqua et al., 2001), ã-butryolactone in the Streptomyces (Yamada and Nihira, 

1998), oligopeptides in a variety of gram-positive species (Dunny and Winans, 1999; 

Kleerebezem and Quadri, 2001), cyclic dipeptides in some gram-negative species 

(Holden et al., 1999) and fatty acid and butyrolactone derivatives in the plant pathogenic 

bacteria Xanthomonas campestris and Ralstonia solanacearum (Barber et al., 1997).


2.4. Microbe-microbe Disruption of Quorum Sensing Mechanisms 

It should now be apparent that quorum sensing plays a significant role in the biology 

and regulation of both plant-microbe and microbe-microbe interactions. And while 

pathogenic bacteria depend significantly on quorum sensing regulation to coordinate 

the saprophytic and parasitic phases of their life cycles, plants and their adherent root 

zone microbial communities have evolved mechanisms by which to disrupt this strategy. 

For example, Variovorax paradoxus, a relatively common soil organism, is able 

to utilize (degrade) acyl-HSL signaling molecules as an energy source (Leadbetter 

and Greenberg, 2000) with the effect that those classes of bacteria relying on acyl- 

HSLs for cell-to-cell signaling molecules will be kept ‘unaware’ of their own presence 

and population density. Pierson et al. (1998) demonstrated that approximately 8% of 

rhizobacteria recovered at random from the surfaces of wheat roots could specifically 

stimulate quorum sensing gene regulated expression in adjacent P. aureofaciens 

bacteria. Thus different bacteria appear able to exchange quorum sensing signals, 

with the possibility of forming functional mixed communities (Bauer and Robinson, 

2002). 

Several instances have been reported of soil bacteria in possession of enzymes 

designed to degrade or inactivate acyl-HSLs (Bauer and Robinson, 2002; Dong et al., 

2001, 2002; Leadbetter, 2001; Whitehead et al., 2001). A case in point is the lactonase 

enzyme, AiiA, from Bacillus cereus, which, it is believed, opens the lactone ring in 

acyl-HSLs, thereby reducing signal strength in the order of a 1000 fold (Dong et al., 

2000). In circumstances where B. cereus and E. carotovora co-exist as commensals 

in field soils, AiiA is able to inactivate the acyl-HSL autoinducer in E. carotovora 

rendering the pathogen avirulent. 

Clearly, understanding the mechanism of signal synthesis, and being able to 

identify signal synthesis inhibitors, has implications for developing quorum sensingtargeted 

antivirulence molecules, or engineering beneficial communities which utilize 

acyl-HSL signals as an energy source and so inhibit pathogenic trait expression. In 

the former case, AHL signal-inactivating molecules (the so-called ‘quorum quashing’ 

moieties) - namely AHL-lactonases and AHL-acylases - have already been identified 

in a Ralstonia sp. isolated from a mixed-species biofilm (Lin et al., 2003). 

However, while selection pressures upon component bacterial populations in a 

community will include biological pressures created by microbially mediated 

moderation of habitats, external abiotic pressures created by environmental 

perturbations - such as climate or crop management practices - also exist. As a result, 

even though “... microorganisms are potentially everywhere, [the] environment 

selects...” (Alexander, 1997). 

3. MICROBIAL ANTAGONISM AND DISEASE CONTROL 

3.1. Microbially Induced Biological Control in Soils 

Since every living soil sample will yield organisms with antagonistic activity to some 

other organism, or group of organisms, it has almost become axiomatic that 

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“...antagonistic potential resides in every soil microorganism...” (Baker and Cook, 

1974). Consequently, it is generally held that most soils possess the biological 

propensity to inhibit or reduce their soil microflora’s tendency toward disease, and so 

can be considered disease suppressive to some extent (Hornby, 1983). As a result, 

there exists in the literature a vast array of a terms used to describe soils that are 

inhospitable to plant pathogens. For example, i) soils where plant pathogens fail to 

become established have been referred to as resistant (Walker and Snyder, 1933), 

long-life, immune, intolerant, or antagonistic (Baker and Cook, 1974; Huber and 

Schneider, 1982), ii) soils where pathogens become established but fail to produce 

disease have also been termed suppressive (Schroth and Hancock, 1982); while iii) 

soils where disease incidence diminishes with continued monoculture have been termed 

decline soils (Shipton, 1975; Hornby, 1979, 1983). 

Attempts to simplify the biological basis for disease suppression in agricultural 

soils have reduced this concept to two broad mechanisms; namely that of i) a “general 

suppression” based upon the activity of the total microbial biomass that is not 

transferable between soils, and ii) a “specific suppression” that depends upon the 

activity of specific groups of microorganisms (Weller et al., 2002). 

Whether a bacterial population behaves pathogenically or not will be a function 

of that component species’ genetics, the restraints which other members in the 

community are able to impose, and the result of any overriding selection pressures 

dictated by environmental factors governing habitat type and host predisposition to 

disease. 

Environmentally mediated host predisposition to disease have been linked with 

obligate pathogen performance, and included exposure to cold (Schulz and Bateman, 

1969), low light intensity, or short day lengths (Foster and Walker, 1947), salinity 

stress (MacDonald, 1982), high temperature (Edmunds, 1964), and drought or moisture 

stress (Boyer, 1995; Duniway, 1977). 

In contrast, factors predisposing host plants to attack by rogue members of a 

commensal community, or protocooperative assemblage, are less well understood, 

though it appears likely that any dramatic change in the niche environment can provide 

an ecological advantage that benefits some community members at the expense of 

others. During the resulting population increase of the favoured community population, 

cell density dependent pathogenesis is triggered. 

3.2. Disease Suppression and Pathogen Evasion 

The wide array of nomenclature used to describe disease suppression in agricultural 

soils, is matched by an equally wide variety of individual microbial mechanisms 

postulated to explain these phenomena. However, it should be noted that these 

mechanisms are fairly presumptive, and, if they occur in vivo, are likely to operate in 

parallel with each other (Figure 1). 

In general, microbial biocontrol mechanisms have been classified according to 

effect (Baker, 1968) and have included such actions as parasitism/predation, niche 

competition, antibiosis and systemic induced resistance - the latter three falling


Induced Systemic 

Resistance 

ROOT – SOIL 

INTERFACE 

Niche competition/ 

exclusion 

Lytic enzyme 

production 

Microbe-microbe disruption 

Quorum sensing and 

community self-awareness 

Root camouflage 

Direct antibiosis action 

ENDOROOT EXOROOT 

Figure 1. Parallel action of disease suppression mechanisms operating within the host plant 

(endoroot) and in the surrounding soil (exoroot). 

reasonably comfortably within the ambit of allelopathy (see Keel and Défago, 1997; 

Mukerji et al., 1999; Weller, 1988). 

Five common mechanisms are usually cited, namely: 

i) Resource(niche) competition (or exploitation) (O’Sullivan and O’Gara, 1992; 

Stephens et al., 1993; Whipps, 2001) - for example, siderophore (chelator)producing 

bacteria with high affinities for, and capable of sequestering, specific 

mineral elements, can inhibit phytopathogens with the same requirement if that 

mineral is limited in the soil (Schroth and Hancock, 1982; Dowling et al, 1996; 

Loper and Henkels, 1997, 1999). 

ii) Antibiosis action - through the production of specific or non-specific microbial 

metabolites with antibacterial, antifungal and anti-nematode activity (Levy and 

Carmelli, 1995; Fujimoto et al., 1995; Raaijmakers et al., 2002; Thomashow et 

al., 1997). To-date, several antibiotic substances have been identified of which 

those produced by the pseudomonads have been particularly well characterized. 

Of these, antibiotics identified with biocontrol properties include the 

phloroglucinols, phenazine derivatives, pyoluteorin, pyrrolnitrin, cyclic 

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c

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lipopeptides and hydrogen cyanide (Haas and Keel, 2003). Among the other 

antibiotics characterized are agrocin 84 (Agrobacterium sp.), herbicolin A 

(Erwinia sp.), iturin A, surfactin, and zwittermicin A (Bacillus sp.) and 

xanthobacin (Stenotrophomonas sp.) (Hashidoko et al., 1999; He et al., 1994; 

Sayre and Starr, 1988; Thomashow et al., 1997; Silo-Suh et al., 1994). A comprehensive 

account of bacterially produced antibiotics may be found in Raaijmakers 

et al. (2002). 

iii) Lytic enzyme action - a feature of several bacteria with proven biocontrol ability, 

and generally involves the direct degradation of pathogen cell wall material, or 

the disruption of a particular developmental stage. Thus, for example, chitinase 

production by Serratia plymuthica has been reported to inhibit spore germination 

and germ-tube elongation in Botrytis cinerea (Frankowski et al., 2001), while ß- 

1,3-glucanase synthesized by Paenibacillus sp. and Streptomyces sp. can lyse 

fungal cell walls of Fusarium oxysporum f. sp. cucumerinum (Singh et al., 1999). 

Other enzymes produced by bacteria with biocontrol activity include hydrolase 

(Chernin and Chet, 2002), laminarinase (Lim et al., 1991) and protease (Kamensky 

et al., 2003). 

iv) Induced systemic resistance (ISR) in plants (Wei et al., 1991; Tuzun and Kloepper, 

1994) - whereby non-pathogenic rhizobacterial stimulation of defence-related 

genes is elicited through the encoded production of jasmonate (van Wees et al., 

1999), peroxidase (Jetiyanon et al., 1997) or enzymes involved in the synthesis 

of phytoalexins (van Peer et al., 1991). Though no specific ISR-eliciting signal 

has been identified, thus far, evidence for the involvement of lipopolysaccharides, 

siderophores and phloroglucinols has been submitted (Hoffland, et al., 1995; 

Leeman et al., 1995, 1996; Maurhofer et al., 1994; van Wees et al., 1997), and, 

v) Root camouflage (Gilbert et al., 1994) - proposed as a mechanism to explain the 

observation that certain rhizobacterial populations in disease resistant cultivars 

are able to minimize the ‘attractive’ nature of the host’s root system so masking 

its presence to potential plant pathogens by restricting local population density 

development. Such microbial systems may operate in tandem with those that 

desensitize the chemoperception systems of microorganisms in the root zone, 

through the over production of chemical stimulii (Armitage, 1992; Dusenbery, 

1992). 

The parallel operation of all these biocontrol mechanisms in a four dimensional 

soil space makes their action and interaction difficult to follow. Biocontrol strains 

only occupy a small fraction of the root surface, in microcolonies spread out unevenly 

along the root surface (Bowen and Rovira, 1976; Normander et al., 1999). Disease 

suppression, when it occurs through antibiosis, is most likely restricted to local action 

only, and most probably at sub-inhibitory levels. Even so, antibiotics can cause intense 

physiological effects upon neighbouring organisms at subinhibitory concentrations. 

Quinolone and macrolide antibiotics have been reported to block cell- to cell signaling, 

and the production of virulence factors in P. aeruginosa (Grimwood et al., 1989; 

Tateda et al., 2001). Similarly, subinhibitory concentrations of antibiotics can suppress 

adherence mechanisms in bacteria (Breines and Burnham, 1994), and the production


of extracellular virulence factors in bacteria (Herbert et al., 2001). Accordingly, 

secondary metabolites can impact soil microbial ecosystems in a variety of ways, and 

at a variety of levels (Haas and Keel, 2003). 

3.3. Partitioning of disease suppressive bacteria in the endo and exoroot 

Most research into soil bacterial communities has been restricted to the exoroot - that 

fraction of the microfloral community found in the rhizoplane, plant rhizosphere or 

root zone soil (Haas and Keel, 2003). Endoroot bacteria have largely been ignored, 

despite plant-bacteria interactions extending into the endoroot of all plants (Conn et 

al., 1997; Bensalim et al., 1998). The frequent recovery of communities of endophytic 

bacteria, in the absence of any pathological condition (Chanway, 1996) and the finding 

that bacterial endoplant communities are capable of mediating against phytopathogen 

invasion (Benhamou et al., 1996) has led to the suggestion that plants may have coopted 

bacteria as part of a disease suppressive response to phytopathogen attack. 

Several instances have been reported of endophytic bacteria as effective biocontrol 

agents (van Buren et al., 1993; Brooks et al., 1994). van Peer et al. (1990) found that 

endo- and exoroot bacteria from the same genera formed discrete sub-populations, 

each suited to colonizing their respective environmental niches. Tissue-specific 

relationships can form between communities of bacterial endophytes and their host 

plant, and endobacteria have been shown to adapt functionally to certain tissue sites 

and among certain tissue types (Sturz et al., 1999). Unfortunately, the population 

densities of endophytic bacteria tend to be highly variable among plant tissues and so 

may be of little practical value in terms of affording plants a comprehensive first line 

of defence to pathogen attack. 

4. ‘ENGINEERING’ BENEFICIAL MICROBIAL ALLELOPATHIES 

4.1. Anthropogenic intrusions 

The premise underlying most anthropogenic biocontrol systems is the notion that it is 

possible to encourage the occurrence and development of beneficial rhizobacterial 

allelopathies in the root zone, primarily through the direct application of specific 

biocontrol agents, and or soil conditioning amendments (Sturz and Christie, 2003). 

It must be acknowledged at the outset that such systems have had varying degrees of 

success. 

4.2. Inundative approaches in biological control 

In general, anthropogenic attempts to import biological control agents into the field 

have been through the inundative application of super quantities of a few key biocontrol 

or plant growth promoting agents. These have been applied directly as drenches or 

sprays, or alternatively as a cell suspensions incorporated in mulches or compost 

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material. Carriers such as granular peat formulations, mineral soils (Chao and 

Alexander, 1984), bacterial encapsulations within polymer gels (Bashan, 1986) or in 

natural gum or talc mixtures (Kloepper and Schroth, 1981) have also been tried. 

Perhaps, in hindsight, it is not surprising that such inundative approaches have 

been relatively unsuccessful, with most biocontrol agents failing to fulfill their initial 

promise. Such failures have usually been attributed to poor competence of biocontrol 

agents in the infection court and the difficulties associated with the instability of 

biocontrol agents in culture (Schroth et al., 1984; Weller, 1988); not least because in 

the case of bacterial agents, the accumulation of extracellular signalling molecules 

within large population densities of individual strains, can modulate a diverse range 

of metabolic processes, some of which are incompatible with the goals of 

phytoprotection (see above). 

However, the use of single antagonists may itself be an inappropriate strategy, 

arising from the belief that a given plant disease can be attributed to a single pathogen 

only (Baker, 1987). Such one-on-one syndrome concepts follow from the belief that 

successful control of a pathogen is achievable with a single fungicide, or by singlefactor 

resistance, coupled with the observation that single antagonists have often 

provided effective control in presumptive tests for antagonists in in vitro studies, or as 

biocontrol agents applied to sterilized soil (Baker, 1987) 

Needless to say, the very practice of applying massive quantities of a single bacterial 

species to the infection court will not only alter the putative biocontrol agent’s 

physiology, but also its niche behaviour. Perhaps, more intriguingly, it may also 

garner a general and antagonistic response from the resident population. 

Several attempts at engineering bacterial strains with more reliable biocontrol 

performance have been tried, whereby biosynthetic genes for various antibiotics have 

been designed to be constitutively over-expressed. On occasion, such engineered 

strains have provided improved plant protection in the soil microcosm (Delany et al., 

2001; Ligon, et al., 2000; Timms-Wilson et al., 2000). However, in long term field 

evaluations, engineered derivatives have also lacked consistency; loss of stable 

performance and lack of superiority to wild-type strains being cited as the principal 

reasons for failure (Bakker et al., 2002). 

Although it would be premature to generalize the findings of such studies, it 

appears that the engineering of a single trait (antibiotic production) in a single 

biocontrol strain can not overcome the problem of inconsistent performance in the 

field, given the multi-factor nature of biocontrol mechanisms and the potential for 

interaction with wild-type species in the soil microbial community. 

While one-on-one antagonism may indeed be the sole operating mechanism 

involved in microbial disease suppression, an equally valid interpretation might be 

that the inundative addition of biocontrol agents can stimulate a general antagonistic 

response from the autochthonous microbial population to the ‘invader’ (inundating) 

species. In this circumstance and irrespective of any inconsistencies in the field 

performance of biocontrol agents attributed to unfavourable edaphic factors - such as 

temperature, soil moisture, pH, clay content, soil type - biocontrol success or failure 

may simply be due to the resident community’s antagonistic response following the


inundative insertion of a non-indigenous species. Consequently, both pathogen and 

biocontrol agent are inhibited in collateral fashion and to various degrees; a scenario 

that is congruent with the defensive mutualism theory proposed by Clay (1988). 

4.3. Modifying Soil Agro-ecosystems 

The extent to which producers can develop beneficial root zone allelopathies amongst 

microbial communities will depend largely upon the resilience of the soil in question 

(Szabolcs, 1994) and the type of crop management and tillage systems being practised 

(Sturz and Christie, 2003). Plant species are known to apply a selective and specific 

influence on microbial diversity in the rhizosphere through their differential root 

exudate spectra (Grayston et al., 1998), and the plastic nature of the relationship 

between resident microbial communities. Thus the level of disease suppressiveness in 

a soil is eminently amenable to deformation through the use of selected cultural 

practices. This regardless of the inherent capacity of ‘natural’ soil microbial ecosystems 

to buffer anthropogenic interference. 

Crop management systems are regularly used to distort agro-ecosystems through, 

for example, the use of tillage operations, alternate cropping systems, monoculture, 

crop rotation length, fertilizer and organic amendments, and various crop protection 

chemistries. The management of soil microbial communities for crop yield 

maximization appears to involve, in part, the creation of short term chaos in the 

microbial community through the application of a plethora of perturbation stresses 

(Odum et al., 1979). Moderate levels of ‘input perturbation’ are considered to improve 

ecosystem performance, while higher levels of perturbation stress result in performance 

loss. 

Input perturbations have commonly been used to modify soil microbial agroecosystems 

at the expense of pathogen populations. The subsequent variation in 

habitat and increase in niche heterogeneity - though on a microscale and at multiple 

sites along the root - is believed to encourage microbial biodiversity and consequently 

increase the potential for root zone competition (Smucker, 1993; Andrews and Harris, 

2000). Thus, for example, increasing soil acidity (Davis and Callihan, 1974, Sturz et 

al., 2003), applying irrigation soon after tuber initiation (Lapwood et al., 1973; 

Oestergaard and Nielsen, 1979) and the addition of soil amendments, green manures 

and mulches (Tremblay and Beauchamp, 1998) have all been relatively successful in 

reducing the development common scab on potatoes. 

Disease suppression has also been achieved against a wide range of pathogens by 

incorporating green manures (plough-down crops) (Tu and Findlay, 1986), animal 

manures (Gorodecki and Hadar, 1990) and composts (including organic solid wastes) 

(Nelson and Hoitink, 1983; Cohen et al., 1998) into field soils. All these amendments 

can encourage aggressive competition among microbial communities (Hoitink and 

Boehm, 1999; Hoitink and Fahy, 1986; Hoitink et al., 1997), with the added effect 

that manure and compost decomposition can release both volatile and non-volatile 

toxic compounds that inhibit phytopathogenic nematodes (Sayre et al., 1965; Abawi 

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134 


and Widmer, 2000) and reduce the survival rates of pathogenic microbes (De Brito et 

al., 1995; Chen et al., 1987 a, b). 

Though time consuming, unfashionable and often slow to show effect, traditional 

crop production practices that involve environmentally sustainable practices, such as 

conservation tillage (Sturz et al., 1997; Bockhus and Shroyer, 1998), ‘creative’ 

fallowing options (Sturz et al., 2001), manuring (Hoitink and Boehm, 1999), long 

term crop rotations (Peters et al., 2003) and compatible cropping systems (Sturz et 

al., 2003), can yield plant health and crop yield benefits. Whether such knowledgebased, 

time-intensive management practices can be made more popular remains the 

challenge. 

5. CONCLUSION 

The close relationships formed between plant root systems and their respective 

rhizobacterial communities can lead to profoundly positive allelopathies that improve 

plant health and crop yield. The selective release of root exudates and plant leachates 

activates and sustains specific beneficial rhizobacterial communities in the root zone 

(endo- and exoroot). In turn these bacterial communities are able to generate a wide 

array of secondary metabolites which can improve plant health, either directly through 

biological control mechanisms, or by the modulation of phytopathogen populations 

in root zone. Simultaneously, certain root zone bacteria can induce systemic disease 

resistance in their plant host. Certain microbial communities are also able to interact 

functionally with each other through a variety of sensing mechanisms and gene 

expression triggers that are prompted by bacterial secondary metabolites with multiple 

activity. 

Despite the natural occurrence of strong positive relationships that can develop 

between bacteria and plants, anthropocentric attempts at applying bacteria for 

biocontrol purposes have had limited commercial success, notwithstanding advances 

in our understanding of the molecular mechanisms governing biocontrol interactions 

in the rhizosphere. The inconsistent performance of biocontrol bacteria in the field 

could be due to variable expression of genes involved in biocontrol, or merely the 

resistance of established soil communities to a sudden and inundative influx of 

adventive bacterial species or strains. 

While the hope remains that bacteria with biocontrol activity will one day be 

used as an adjunct to, or replacement for, commercial chemical fungicides, it will be 

necessary to better understand the influence on, within and between those microbial 

communities resident in the soil agro-ecosystems. In the interim, more traditional 

‘hit and miss’ methods for encouraging serendipitous beneficial allelopathies to arise 

in root zone communities - through, for example, the application of perturbation 

stresses - still have a role to play in modern systems of crop management, since they 

encourage the development of microflora species’ diversity and functionalities that 

underpin the antagonism systems promoting biological control in disease suppressive 

soils.


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ROBERT J. KREMER 

THE ROLE OF ALLELOPATHIC BACTERIA IN 

WEED MANAGEMENT 

U.S.D.A., Agricultural Research Service, Cropping Systems & Water Quality 

Unit, University of Missouri, Columbia, Missouri, U.S.A. 

Email: KremerR@missouri.edu 

Abstract. Allelopathic bacteria encompass those rhizobacteria that colonize the surfaces of plant roots, produce, 

and release phytotoxic metabolites, similar to allelochemicals, that detrimentally affect growth of plants. Practical 

application of this group of bacteria to agriculture could contribute to biological weed management systems that 

have less impact on the environment than conventional systems by reducing inputs of herbicides. Allelopathic 

bacteria have been investigated for potential as inundative-type biological control agents on several weeds. 

Because allelopathic bacteria generally do not attack specific biochemical sites within the plant, unlike 

conventional herbicides, they offer a means to control weeds without causing direct selective pressure on the 

weed population, therefore, development of resistance is not a major consideration. Additionally, the use of 

allelopathic bacteria appears to be environmentally benign relative to herbicides. These characteristics make 

allelopathic bacteria an attractive approach for managing crop weeds in a sustainable manner, even within the 

boundaries of conventional agriculture systems. However, recent evidence suggests that indigenous allelopathic 

bacteria might be exploited under certain crop and soil management practices that are inherently part of sustainable 

agricultural systems. The development of “weed-suppressive” soils in diverse sustainable systems is encouraging 

because indigenous populations of allelopathic bacteria may develop in several soils and environments using 

similar practices. The recent demonstrations of apparent weed-suppressive soils may lead to development of 

specific management strategies for the establishment and persistence of native allelopathic bacteria directly in 

soils conducive to annual weed infestations. 


Biological weed management is a system that incorporates the use of diverse biological 

organisms and biologically-based approaches including allelopathy, crop competition, 

and other cultural practices to significantly reduce weed densities in a manner that is 

similar to use of chemical herbicides alone (Cardina, 1995). Interest in developing 

effective biological weed management systems continues to increase because of a 

growing awareness of problems associated with the constant and intensive use of 

chemical herbicides, which include surface- and groundwater contamination, 

detrimental impacts on nontarget organisms, development of weeds resistant to 

herbicides (including those that are used in transgenic herbicide-resistant crops), and 

consumer concerns for residues on food (Gliessman, 2002). A component of biological 

weed management involves biological control, the intentional use of living organisms 

(insects, nematodes, fungi, and bacteria) to reduce the vigor, reproductive capacity, 

density, or impact of weeds (Quimby and Birdsall, 1995). A number of reviews are 

143 




144 


available that discuss the various strategies of biological control and the numerous 

organisms that are involved as biological agents within these strategies (TeBeest, 

1991; Harley and Forno, 1992; Boland and Kuykendall, 1998). The focus of the present 

paper will be a broad group of bacteria that are associated with seeds and seedlings, 

which can be developed to suppress the establishment and growth of weeds in 

agroecosystems. 

Unlike many herbicides and biological agents that have been developed to attack 

growing weeds established in crops, most bacterial agents target weed seeds residing 

in soil and the roots of developing weed seedlings. The microenvironments consisting 

of the zones of soil surrounding the seed (spermosphere) and root (rhizosphere) provide 

organic substrates that are readily available for soil microorganisms in contrast with 

the nutrient-limiting condition of the bulk soil (Kennedy, 2005). The spermosphere 

and rhizosphere, therefore, are ideal sites for establishing selected bacteria able to 

suppress weed seed germination and seedling growth because of the continuous supply 

of carbon and energy sources released from germinating seeds and developing root 

systems (Zahir et al., 2004). Soil borne bacteria adapted to competitive colonization 

of the spermosphere, rhizosphere, and the root can be grouped under the general term 

rhizobacteria (Schroth and Hancock, 1982). Although the spermosphere is defined 

with reference to a seed prior to root emergence, the zone of soil contains substances 

exuded from the seed that rapidly attracts and regulates a microbial community, which 

often, if the seed survives, establishes the dominant microbial communities of the 

longer-lived rhizosphere environment (Nelson, 2004). A component of the 

rhizobacteria group in the spermosphere and rhizosphere that inhibit plant growth 

without causing obvious disease symptoms are known as deleterious rhizobacteria. 

Deleterious rhizobacteria are predominantly saprophytic bacteria that live on or in 

plant seeds and roots, surviving on organic compounds released by plant seed root 

cells (Schippers et al., 1987). These bacteria do not parasitize the plant or penetrate 

the stele like major or true pathogens, but may colonize seed tissues or root hairs and 

the root tip as well as in the intercellular spaces of root cortical cells (Schippers et al., 

1987), in which case they may be characterized as “endorhizal bacteria.” Because 

many of these deleterious rhizobacteria release phytotoxic metabolites that are also 

considered allelochemicals that influence the growth of plants, it has been suggested 

that term “allelopathic bacteria” may more accurately describe these bacteria (Barazani 

and Friedman, 1999; Sturz and Christie, 2003). Therefore, rhizobacteria that 

detrimentally affect seed germination and seedling development of weeds through the 

production of allelochemical substances will be referred to as allelopathic bacteria 

(AB) in this paper. 

The use of rhizobacteria in weed management has typically involved an inoculative 

approach whereby selected AB are applied at high rates to establish critical population 

densities in soil or on vegetative residues to achieve rapid initiation of growth-inhibitory 

activity (Kremer and Kennedy, 1996). The goal is not complete kill or eradication of 

the weed population but the reduction of the competitive ability of the weeds growing 

with the crop. Recent investigations of AB suggest that sustainable agricultural

ALLELOPATHIC BACTERIA IN WEED MANAGEMENT 145 

practices for many crops may be linked to management of the indigenous bacterial 

communities for biological control of weeds, thus reducing dependence on selected 

AB as inoculative biocontrol agents. Development of agroecosystems with the capacity 

to suppress weeds using naturally-occurring soil bacteria-weed interactions has received 

very little attention (Gallandt et al., 1999). Therefore, weed management strategies 

involving AB should also consider the development of beneficial, indigenous soil 

bacteria similar to a conservation biological control approach (Newman et al., 1998), 

which conceivably would address sustainable weed management with reduced or no 

herbicide inputs (Parr et al., 1992). 

The objectives of this chapter are to demonstrate how both introduced and 

indigenous AB contribute to weed management in cropping systems and to identify 

crop and soil management strategies that promote the proliferation of AB. 

2. ALLELOPATHIC BACTERIA AND INTERACTIONS IN 

AGROECOSYSTEMS 

Biological weed control using introduced agents including selected AB has often 

been limited by inconsistency of efficacy when placed into practical field use. This 

suggests that standard screening bioassays conducted under laboratory conditions 

poorly represent actual environmental situations where biological activity must be 

expressed. For individual AB agents, therefore, selection, bioassays, and inoculum 

development should be based on ecological principles, accounting for characteristics 

of the environment in which the agents are to be introduced. The principles should 

further be applied to potential strategies for enhancing indigenous AB in the soil 

environment. The ecological interactions associated with biological control of weed 

Soil Environment 

Allelopathic Bacteria 

Introduced | Indigenous 

Cultural 

Practices 

Weed Crop 

Figure 1. Environmental-interaction diagram associated with allelopathic 

bacteria in agroecosystems.

146 


seeds and seedlings with AB can be expressed graphically (Figure 1) to emphasize 

the following key areas for consideration: a) biology and ecology of weeds; b) growth 

of the crop within the established cultivation system; c) characterization of AB, 

including those that are selected and introduced as biological control agents and those 

that are indigenous to the soil environment; and d) the wide range of cultural practices 

available for implementation of biological control within the agroecosystems. 

After recognizing the need for consideration of ecological interactions, a program 

for development of selected AB or management of the indigenous bacterial 

population can be undertaken 

3. CHARACTERIZATION OF ALLELOPATHIC BACTERIA 

Kremer et al. (1990) identified colonizing ability, chemotactic response, and mode of 

action to be vital characteristics for the successful development of rhizobacteria as 

weed biocontrol agents. Bacteria that can rapidly colonize the root will likely be a 

successful biocontrol agent. Migration towards the seed or root is the first step in 

colonization, illustrated by movement of rhizobacterial isolates through 2-cm of soil 

towards velvetleaf seeds (Begonia and Kremer, 1994). As the seedling develops, 

movement of bacteria along roots and within the rhizosphere is influenced by root 

binding sites, amounts of organic material present, type of root (i.e., seminal vs. 

nodal roots), water movement through soil and along roots, and soil texture (Bolton 

et al., 1993). Compared to other bacterial groups present in non-rhizosphere soil, 

gram-negative bacteria readily colonize the rhizosphere, partly due to their metabolic 

diversity (Nehl et al., 1997). Pseudomonads are particularly adapted for rhizosphere 

colonization because of the ability to utilize diverse carbon sources present in root 

exudates. Observations with scanning electron microscopy reveal the intimate 

relationship of rhizoplane colonization by selected AB (Figure 2). 

Figure 2. Root surface of a two-week old velvetleaf root colonized by Pseudomonas 

fluorescens strain 239 cells (arrow) aligned in intercellular spaces of root epidermal cells. 

Magnification is X 6,000. Scanning electron micrograph from Begonia et al. (1990).


Successful competition of bacteria living in the rhizosphere depends on several 

factors, including rapid growth on multiple substrates, antibiotic production, and 

downward growth with the root. A major factor contributing to successful competition 

of rhizobacteria over other microorganisms is the growth stimulation by exuded organic 

compounds and sloughed-off root hair and epidermal cell materials (De Weger et al., 

1995). The ability to efficiently compete for these available resources and to produce 

siderophores for obtaining iron is important in establishment, colonization, and 

persistence of rhizobacteria in the rhizosphere. 

A characteristic of many AB is the high specificity toward their weed host(s) 

with no detrimental effects on growth of nonweedy plant species (Cherrington and 

Elliott, 1987; Elliott and Lynch, 1985; Kennedy et al., 1991; 2001). Although effects 

on plants are subtle (Kremer and Kennedy, 1996), AB may be as significant as 

traditional bacterial pathogens in affecting plant growth (Schroth and Hancock, 1982; 

Suslow and Schroth, 1982). Because AB attack the seed and/or seedling rather than 

the growing plant, weed seed or vegetative propagule production is suppressed, a key 

to any weed management program, which reduces the need for repeated postemergence 

herbicide applications and increases the chances of success for control of a growing, 

competitive weed (Aldrich and Kremer, 1997). 

4. MODES OF ACTION OF ALLELOPATHIC BACTERIA 

Many AB strains produce secondary metabolites that are inhibitory to plants, including 

phytotoxins and antibiotics, which can be considered allelopathic. Phytotoxins from 

fluorescent Pseudomonas spp., a diverse group of plant pathogenic bacteria abundant 

in the soil and rhizosphere, have been well studied (Mitchell, 1991). There are fewer 

reports on phytotoxins from AB and many have not been extensively studied. 

A phytotoxin from Pseudomonas fluorescens strain D7 was shown to be 

responsible for root growth inhibition of downy brome (Bromus tectorum) (Tranel et 

al., 1993). Further characterization revealed that the active fraction was a complex of 

chromopeptides, other peptides and fatty acid esters in a lipopolysaccharide matrix 

(Gurusiddaiah et al., 1994). Secondary metabolites isolated from Pseudomonas 

syringae strain 3366 inhibitory to downy brome consisted of phenazine-1-carboxylic 

acid, 2-aminophenoxazone and 2-aminophenol (Gealy et al., 1996). Gealy et al. 

(1996) showed that phenazine-type antibiotics of Pseudomonas fluorescens also 

inhibited downy brome root growth. Electron microscopy of AB colonizing the 

rhizoplane and endorhizal cells of leafy spurge (Euphorbia esula) revealed disruption 

of plant cell walls and membranes apparently due to production of phytotoxins and/or 

enzymes by the bacteria, which consequently inhibited seedling growth (Souissi et 

al., 1997). AB may also produce “phytotoxic antibiotics” that affect plant growth 

such as the broad-spectrum antibiotic, 2,4-diacetylphloroglucinol, released by P. 

fluorescens strain CHA0, which suppressed soilborne fungal plant pathogens but was 

also highly phytotoxic to seedlings of several plant species (Keel et al., 1992). 

Plant-inhibitory effects of some AB are auxin-mediated, illustrated by direct uptake 

of bacterially produced indoleacetic acid (IAA). Plant response to microbially

148 


synthesized auxins is related to the concentration released into the rhizosphere. Growth 

inhibition of several weed and crop species was correlated with elevated IAA levels 

produced by AB (Sarwar and Kremer, 1995). Similar responses were observed when 

tryptophan, an IAA precursor, was added to soil (Sarwar and Frankenberger, 1994; 

Sarwar and Kremer, 1995). Presumably, the presence of extra tryptophan in the soil 

provided rhizobacteria with additional substrates for auxin biosynthesis. 

The production of hydrogen cyanide (HCN), a volatile metabolite that negatively 

affects root metabolism and root growth by inhibiting cytochrome oxidase respiration, 

is common among rhizosphere pseudomonads (Schippers et al., 1990). The rate of 

HCN synthesis is affected by the availability of precursors such as glycine, methionine, 

proline and cyanogenic glucosides (Knowles and Bunch, 1986; Schippers et al., 1990). 

The amino acid composition of root exudates as well as environmental factors affecting 

root exudation (i.e, light intensity, soil water potential, nutrients) may be important 

as well (Schippers et al., 1990). Two strains of cyanogenic pseudomonads, 

Pseudomonas putida and Acidovorax delafieldii, significantly inhibited velvetleaf 

(Abutilon theophrasti) growth, but did not reduce corn growth in the presence of 

supplemental glycine (Owen and Zdor, 2001). Cyanide production by several 

rhizobacterial strains was a major factor in the inhibition of seedling growth of several 

weed species and was suggested as a trait for consideration in selecting AB as potential 

weed biological control agents (Kremer and Souissi, 2001). 

5. ROLES OF ALLELOPATHIC BACTERIA IN WEED 

MANAGEMENT STRATEGIES 

Strategies for the potential use of allelopathic bacteria for weed management include 

application of selected cultures as bioherbicides, integration of bioherbicides with 

other crop and soil management practices, use of AB in sustainable agricultural systems 

with other non-chemical means of weed control, and management of soils to enhance 

populations and activity of indigenous AB as a conservation biological control strategy. 

5.1. Bioherbicides 

The high host specificity of AB is a disadvantage for their use as a biocontrol strategy 

in most agroecosystems that are typically infested with multiple weed species. However, 

AB may have the greatest impact for management of specific weed problems in certain 

cropping systems. Isolates of AB specific for management of downy brome in wheat 

in the Pacific Northwest are under development as bioherbicides (Kennedy et al., 

1991; 2001). In Kansas, selected AB suppressed early seedling growth of downy 

brome and Japanese brome (Bromus japonicus) in soils under winter wheat production 

(Harris and Stahlman, 1996). Several strains of rhizobacteria from over 2000 accessions 

isolated from prairie soils in Canada have been selected for inhibition of downy brome 

and green foxtail (Setaria viridis) seed germination and seedling growth (Boyetchko, 

1999). AB that were formulated in a starch-based granular carrier and applied to soil


in leafy spurge-infested sites in South Dakota suppressed growth by decreasing root 

weight and root carbohydrate content (Brinkman et al., 1999). 

Most of the weeds targeted for biocontrol by AB infest cereal and row crops, but 

a few are perennial weeds of rangeland and forest ecosystems (Kremer, 2002). Selected 

AB are intended for soil application, however, some cultures might be effective when 

applied directly to growing weeds in a postemergence control strategy. Selected AB 

might also be applied directly to growing weeds as a postemergence control strategy. 

For example, cultures and cell-free supernatants of AB strains sprayed on common 

chickweed (Stellaria media), common lambsquarters (Chenopodium album), and field 

pennycress (Thlaspi arvense) in the greenhouse and field reduced plant biomass and 

survival (Weissmann and Gerhardson, 2001). Preliminary results suggest AB cultures 

might be used for selective weed control in growing crops through a one-time foliar 

application or as a follow-up to soil-incorporation of the same cultures. 

5.2. Integrated Weed Management 

Integrated weed management systems rely on a number of available strategies including 

tillage, cultural practices, herbicides, allelopathy, and biological control to reduce the 

weed seedbank, prevent weed emergence, and minimize competition from weeds 

growing with the crop (Aldrich and Kremer, 1997). These systems may be most suitable 

for implementing bioherbicides based on AB to counteract their limited weed host 

specificity. Like chemical herbicides, such bioherbicides may be most effective as a 

component in a multi-faceted management program rather than as a single tactic 

approach (Hatcher and Melander, 2003). Effective weed management offers several 

opportunities for integration of selected AB at the critical stages of weed development: 

as seeds in soil, as growing and competitive plants, and during seed production (Aldrich 

and Kremer, 1997). This may be the most promising situation for AB to be considered 

as practical management options in cropping systems. 

To broaden the limited spectrum of activity of AB, several tactics have been 

proposed for integration of these organisms with other weed management methods. 

Weed growth suppression by AB combined with herbicides applied at sublethal rates 

has met with some success (Greaves and Sargent, 1986). AB inhibitory to downy 

brome and jointed goatgrass suppressed growth to a greater extent when combined 

with metribuzin and/or diclofop at less than label rates (Kremer and Kennedy, 1996). 

An understanding of the mechanisms of herbicide-AB interactions will lead to 

strategies where AB selected for activity toward a weed can be paired with a specific 

chemical that increases the susceptibility of that weed to the AB (Kremer, 1998). Use 

of AB in this manner may develop into a systems management approach that involves 

integration of bioherbicides and herbicides on a physiological basis to control 

economically important weeds in corn and other crops. This is currently under intensive 

evaluation as a potential integrated management system in Europe (Müller-Schärer 

et al., 2000). Successful development of these integrated strategies will increase efficacy 

of AB agents, reduce herbicide inputs for weed control, and decrease potential 

environmental contamination.

150 


Application of selected AB with cultural practices such as tillage may be effective 

in integrated weed management. Greater proportions of indigenous rhizobacteria 

inhibitory to downy brome and jointed goatgrass were detected under either 

conventional or reduced tillage compared to no-till (Kremer and Kennedy, 1996). 

Vegetative residues at or near the soil surface may serve as substrates for production 

of weed-suppressive chemicals by AB applied as bioherbicides directly to the residues. 

Kremer (1998) suggested that application of AB to surface vegetative residues to 

promote phytotoxin production might suppress weed growth prior to planting the 

crop, similar to a preemergence herbicide tactic. Crop rotation may also be manipulated 

to encourage development of specific inhibitory bacteria on weed roots. A “rotation 

effect” in corn, due partly to certain rhizobacteria specifically associated with corn 

roots, illustrates the potential for using AB to achieve suppression of weeds in crop 

rotation systems (Turco et al., 1990). Crop rotation may be manipulated to encourage 

development of specific inhibitory bacteria on weed roots. Thus, weed seeds and 

seedlings might be attacked by selected AB directly inoculated onto crop seeds or by 

promoting colonization of crop roots by microbial agents applied at planting (Skipper 

et al., 1996). Crop roots may deliver AB to adjacent roots of weeds and also maintain 

or even enhance AB numbers for attack of seedlings emerging later in the season. 

These observations suggest that combination of biocontrol agents, including selected 

AB, with cultural practices enhance weed growth suppression (Kremer and Kennedy, 

1996) and may be effective in controlling weeds that escape cultural control methods 

(Hatcher and Melander, 2003). 

5.3. Sustainable Agricultural Systems 

Sustainable agricultural systems involve a range of technological and management 

options to reduce costs, protect health and environmental quality, and enhance 

beneficial biological interactions and natural processes. Sustainable agricultural 

systems offer the greatest opportunities to study and refine nonchemical weed 

management (Liebman and Gallandt, 1997) and yield valuable information useful in 

developing improved bioherbicides, including AB, and advancing their use in broader 

biologically based weed management systems. 

High inputs of organic amendments and green manure (cover crop residues) in 

sustainable agricultural systems promotes the ability of crops to compete more 

vigorously with weeds, which intuitively suggests that the efficacy of bioherbicides 

would also be enhanced when used with these amendments (Gallandt et al., 1999). 

Addition of organic matter to soil is one of the most effective ways to change soil 

environment and favor increases in populations of beneficial soil organisms. Organic 

amendments subjected to decomposition in soils release compounds that suppress 

pathogens and provide substrates for other organisms, indigenous or those added to 

the amendments, that may also produce compounds that inhibit pathogens and/or 

weed seedling growth. Inoculation of organic materials (manures, composts, mulches) 

has been suggested as a means for assuring establishment and efficacy of the selected


biocontrol agents, however, field performance to date has been inconsistent (Sturz 

and Christie, 2003). 

Cover crops and mulches as components of sustainable management systems 

may be used for integrating bioherbicides by delivering the agents on seeds and 

promoting their establishment in soils for attack of weeds and seedlings prior to 

planting. Recent research demonstrated that several cover crop species inoculated 

with selected AB at planting established and maintained the selected bacterial 

populations on their roots and in adjacent soil. When giant foxtail (Setaria faberi) 

emerged later in the season, the selected bacteria colonized seedling roots after the 

cover crop was terminated (Kremer, 2000). The selected AB and allelopathic cover 

crop residues acted synergistically to suppress the growth of the weeds. 

5.4. Suppressive Soils and Conservation Biological Control 

Soils under sustainable management may develop antagonistic microbial populations 

in rhizospheres of selected weeds that suppress their growth. This occurrence is similar 

to natural disease-suppressive soils in which indigenous soil microorganisms effectively 

protect crop plants from soilborne plant pathogens (Weller et al., 2002). Diseasesuppressive 

soils may be defined as soils in which a pathogen does not establish or 

persist, establishes but causes little or no damage, or establishes and causes disease 

for a short time but thereafter the disease is less important even though the pathogen 

may persist in the soil (Baker and Cook, 1974). Suppression is due primarily to 

antagonistic microorganisms, however, soil physical and chemical factors also may 

be involved (Weller et al., 2002). Similarly, weed-suppressive soils may be defined as 

soils in which certain weeds do not establish or persist, or establish and grow with the 

crop but cause little interference due to suppressed growth and vigor caused by native 

AB. Native and desirable plants may also in stimulate high populations of AB in their 

rhizospheres that reduce growth of invasive weed species, suggesting that plant-soil 

interactions are also involved in development of weed-suppressive soils (Kulmatiski 

et al., 2004). 

Evidence of apparent weed-suppressive soils has been reported for a variety of 

sustainable cropping systems. A study of crop management practices on claypan soils 

(Epiaqualfs) that involved reduced tillage, maintenance of high soil organic matter, 

and limited inputs of agrichemicals found increased levels of AB associated with 

weed seedlings that likely contributed to natural weed suppression (Li and Kremer, 

2000). It was reported that agronomic practices that resulted in relatively high organic 

matter, such as uncultivated prairie, organic farming and integrated cropping systems, 

supported higher proportions of weed AB. Compost-amended soils planted to winter 

wheat showed 29 and 78% reductions in broadleaf and grassy weed densities, 

respectively, compared to soils amended with inorganic fertilizers only (Carpenter- 

Boggs et al., 2000). Organic amendments (composts and cover crops) increased soil 

microbial biomass and decreased the seedbank density and emergence of shepherd’s

152 


purse (Capsella bursa-pastoris) and burning nettle (Urtica urens) in a California 

vegetable production field (Fennimore and Jackson, 2003). The natural soil 

suppressiveness of the parasitic weed Striga hermonthica in Nigeria appears to be 

related to soils under rotations of cereal and leguminous crops that promote antagonist 

microbial populations that destroys Striga seeds before germination or kills the 

germinated seedlings (Berner et al., 1996; Dashiel et al., 1991). Each of the above 

systems strongly suggests that AB growth can be exploited as a sustainable weed 

control strategy using relatively simple management practices. 

Tactics and approaches for manipulating the field environment to enhance survival, 

physiological behavior, and performance of AB might easily be incorporated into 

diverse sustainable crop production systems. Such a strategy for natural weed 

suppression, also known as conservation biological control (Newman et al., 1998) or 

endemic soil-based control (Kulmatiski et al., 2004) relies on establishment of 

populations of indigenous or endemic, weed-suppressive microorganisms in soil. As 

demonstrated previously, many of these indigenous microorganisms are AB (Kremer 

and Li, 2003; Li and Kremer, 2000). Management practices including tillage, crop 

rotation, residue manipulation, and organic amendments enhance or induce favorable 

factors in the habitat for sustaining effective populations of natural AB. Crop 

management practices that involve reduced tillage, maintain high soil organic matter, 

and limit inputs of agrichemicals increased levels of deleterious rhizobacteria associated 

with weed seedlings and contribute to natural weed suppression (Li and Kremer, 

2000). Deliberate use of management practices that benefit natural weed-antagonistic 

AB can adversely affect weed population dynamics in production fields through seed 

and seedling mortality and growth suppression. 

6. SUMMARY 

The future use of AB to manage weeds in both conventional and sustainable agriculture 

seems promising. Because AB generally do not attack specific biochemical sites within 

the plant, unlike conventional herbicides, they offer a means to control weeds without 

causing direct selective pressure on the weed population, therefore, development of 

resistance is not a major consideration. Additionally, the use of AB appears to be 

environmentally benign relative to herbicides. These characteristics make AB an 

attractive approach for managing crop weeds in a sustainable manner, even within 

the boundaries of conventional agriculture systems. The recent demonstrations of 

apparent weed-suppressive soils may lead to development of specific management 

strategies for the establishment and persistence of native AB directly in soils conducive 

to annual weed infestations. 

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MARK A. BERNARDS 1 , LINA F. YOUSEF 1 , ROBERT W. NICOL 2 

THE ALLELOPATHIC POTENTIAL 

OF GINSENOSIDES 

1 

Department of Biology, University of Western Ontario, London, 

2 

ON, Canada, N6A 5B7 

NovoBiotic Pharmaceuticals, Cambridge MA, 02138, USA 

Email: bernards@uwo.ca 

Abstract. American ginseng (Panax quinquefolius L.) is a perennial herb valued for the medicinal properties 

of its large, fleshy tap root. These medicinal properties are purported to be due to the triterpenoid saponins, or 

ginsenosides, that accumulate to 3-6% of the root dry weight. We asked the question: what is the ecological role 

of ginsenosides in Panax species? In addressing this question, we have determined that ginsenosides, like other 

saponins, possess fungitoxic properties, although different fungi and oomycotan organisms appear to be 

differentially affected by them in vitro. In order to play an allelopathic role, however, ginsenosides must be 

present in the soil at biologically active (i.e., ecologically relevant) concentrations. Results to date support the 

hypothesis that ginsenosides are phytoanticipins and serve as host resistance factors. The success of certain 

pathogens (e.g., Pythium cactorum, Pythium irregulare, Cylindrocarpon destructans) on ginseng may arise 

from their ability to detoxify or otherwise utilize ginsenosides as a nutritive source or growth stimulating factor, 

while other soil borne organisms appear susceptible to their fungitoxic properties. Ginsenosides have been 

isolated from rhizosphere soil and root exudates suggesting that these compounds are involved in allelopathic 

interactions between the host plant and soil fungi. Ultimately this allelopathic interaction may influence the 

fungal diseases of ginseng. 


American ginseng (Panax quinquefolius L.) produces triterpenoid saponins called 

ginsenosides, which are slowly released into the rhizosphere. Ginsenosides, like other 

saponins, are fungitoxic, and as such may modify the balance of microorganisms in 

the soil; i.e., they are potentially allelopathic. From a disease management point of 

view, the extent to which ginsenosides alter the soil microbial community may have 

profound consequences, especially when disease causing organisms are favoured in 

the new balance. Understanding how ginsenosides affect soilborne microbes is important 

in understanding disease cycles in this crop. 

2.1. Saponins 

2. SAPONINS 

Saponins, are glycosylated natural products with surfactant and soap-like properties 

that tend to froth in aqueous solution, even at low concentration (Dewick, 1997). 

157 




158 

MARK A. BERNARDS, LINA F. YOUSEF, ROBERT W. NICOL 

They also cause haemolysis, though are generally non-toxic when taken orally. Saponins 

are broadly categorised as being either triterpenoid- or steroid-derived, despite the 

common triterpenoid origin of both. Triterpenoid saponins are widely distributed in 

many dicot families, and are generally based on pentacyclic triterpene parent carbon 

skeletons (e.g., α- and β-amyrin), or tetracyclic parent compounds (e.g., the 

dammaranes). By contrast, steroidal saponins are commonly found in monocots, 

and are characterised by a spiroketal moiety at C-22. Plants of the Solanaceae 

contain steroidal glycoalkaloid saponins, which are nitrogen analogues of steroidal 

saponins. 

In general, saponins are glycosylated at the C-3 hydroxyl group, typically with a 

glucose molecule. However, there are other potential glycosylation sites on the parent 

carbon skeletons, and multi-site glycosylation can occur. Accordingly, saponins are 

subdivided into monodesmosidic (one glycosylation site) and bisdesmosidic 

(glycosylation sites at both ends of the compound) categories. The majority of saponins, 

however, are monodesmosidic. While glucose is the most common sugar in 

saponins, arabinose, galactose, rhamnose and xylose, as well as sugar acids are also 

present. The glycosylation patterns of saponins can lead to small families of compounds 

based on the same parental carbon skeleton (i.e., sapogenin or aglycone), 

differing only in the composition and arrangement of sugars. 

2.2. Saponins as Fungitoxic and Plant Defense Compounds 

There are numerous reports in the literature that describe the anti-fungal activity of 

saponins (Zimmer et al., 1967; Levy et al., 1986; 1989; Marston et al., 1988; Takechi 

and Tanaka, 1990; Ohtani et al., 1993; Ouf et al., 1994; Favel et al., 1994; Escalante 

et al., 2002; Nicol et al., 2002). These reports coupled with molecular data (Bowyer et 

al., 1995; Papadopoulou et al., 1999) suggest that these phytochemicals are constitutive 

plant defenses against fungi (Osbourn, 1996; 2003), that is phytoanticipins (Van 

Etten et al., 1994). For example, the role that the triterpenoid saponin avenacin A-1 

plays in conferring resistance to “take-all disease”, caused by Gaeumannomyces 

graminis in oats, is well established (Turner, 1956; Burkhardt et al., 1964; Maizel et 

al., 1964; Bowyer et al., 1995; Papadopoulou et al., 1999). 

While the molecular mechanism of saponin fungitoxicity is not known, avenacin 

A-1 as well as steroidal glycoalkaloid saponins such as α-tomatine and solanine have 

been demonstrated to form complexes with membrane sterols, thereby causing a reduction 

in membrane integrity (Roddick, 1979; Steel and Drysdale, 1988; Keukens et 

al., 1992; 1995; Armah et al., 1999). Two models have been proposed to explain the 

consequences of saponin-sterol aggregation in target membranes (Morrissey and 

Osbourn, 1999). One suggests that the deleterious effects of saponins are related to 

the formation of transmembrane pores (Armah et al., 1999), whereas the other suggests 

that membrane integrity is compromised due to the extraction of sterols (Keukens 

et al., 1992; 1995). Regardless of the exact mechanism, saponins probably disrupts 

fungal membranes through complexation with ergosterol, the major membrane sterol 

in higher fungi (Evans and Gealt 1985; Weete 1989; Griffiths et al., 2003).

THE ALLELOPATHIC POTENTIAL OF GINSENOSIDES 159 

Saponins may act as host chemical defenses, but because fungi successfully attack 

plants containing these defenses, there must be means of tolerating or avoiding 

saponin toxicity. Fungal resistance to defensive chemicals in general can either involve 

enzymatic detoxification of antifungal compounds or the more ambiguous “innate 

resistance” (Morrissey and Osbourn, 1999). Some fungi are able to detoxify 

phytoalexins (Van Etten et al., 1995) as well as phytoanticipins such as saponins 

(Osbourn et al., 1995a; Van Etten et al., 1995; Weltring et al., 1997). Although 

saponin detoxification generally occurs via enzymatic cleavage of the saccharides to 

form the aglycone parent compound, different fungi employ different strategies and 

enzymes. For example, fungal detoxification of α-tomatine by Botrytis cinerea (Quidde 

et al., 1998) Septoria lycopersici (Arneson and Durbin, 1967) and Verticillium alboatrum 

(Pegg and Woodward, 1986) involves cleavage of one terminal monosaccharide 

from the tetrasaccharide chain at carbon three of the sapogenin, whereas Fusarium 

oxysporum f. sp. lycopersici removes the entire tetrasaccharide (Ford et al., 1977). 

The saponin-detoxifying enzymes tomatinase from S. lycopersici and avenacinase 

from G. graminis exhibit high sequence similarity (Osbourn et al., 1995b), whereas 

the tomatinase from F. oxysporum f. sp. lycopersici appears to be more related to a 

different family of glycosyl hydrolases (Roldán-Arjona et al., 1999). The ability to 

efficiently detoxify host chemical defenses determines virulence and host range in 

fungal pathogens such as G. graminis, Rhizoctonia solani and Phoma lingam (Bowyer 

et al., 1995; Pedras et al., 2000a,b). Efficient detoxification is taken an extra step by 

S. lycopersici as the hydrolysis products of the saponin-based defense subsequently 

inhibit inducible defenses of the host by interfering with signal transduction pathways 

(Bouarab et al., 2002). 

Transformation of secondary metabolites (e.g., cleaving sugars from saponins) 

has been suggested as a way of providing fungi with a carbon source in addition to 

achieving detoxification (Van Etten et al., 1995; Roldán-Arjona et al., 1999). Since 

some fungal detoxification enzymes are repressed by glucose (Straney and Van Etten, 

1994; Roldán-Arjona et al., 1999) they may have a dual function. This would be 

analogous to non-pathogenic fungi that obtain carbon from phenolic monomers produced 

during lignin decomposition (Henderson and Farmer, 1955; Rahouti et al., 

1989). Presumably it would be advantageous for pathogens to circumvent host defenses 

and simultaneously derive nutrition. 

Pathogens in the Pythiaceae (Oomycota) appear to possess an innate resistance to 

the toxic effects of saponins. Members in this family are reported to be relatively 

unaffected by aescin (Olsen, 1971) and other saponins (Assa et al., 1972) in vitro, and 

probably the lack of ergosterol in these species (Olsen, 1973a; Weete, 1989) allows 

them to avoid saponin toxicity (Arneson and Durbin 1968; Olsen, 1971; Weltring, 

1997). Although Pythium spp. appear to be unaffected by saponins, saponin toxicity 

can be induced through the addition of sterols to the growth medium (Olsen, 1973b; 

Steel and Drysdale, 1988). Members of the Pythiaceae can incorporate sterols by the 

binding and transport action of small protein carriers called elicitins (Mikes et al., 

1997; Panabières et al., 1997; Capasso et al., 2001) and this uptake of exogenous 

sterols could be the cause of the observed increase in the deleterious effects of saponins 

on these organisms.

160 


2.3. Criteria for Saponins as Allelochemicals 

Allelopathy is the study of those interactions between and among plants and microbes 

that are mediated by secondary compounds i.e., allelochemicals (Rice, 1984). The 

concept of allelopathy is most frequently applied within the context of plant-plant 

chemical interactions. Allelochemicals can act as a form of chemical interference 

between competing species (Rice, 1984) as well as conspecific plants (Singh et al., 

1999). However, it has recently been argued that allelochemicals are unlikely to 

reach phytotoxic levels in the soil and therefore plant-microbe allelopathy may be 

more likely to occur (Schmidt and Ley, 1999). It has been suggested that slow diffusion 

rates and complexation reactions in soil, coupled with degradation/utilization by 

microbes would generally prevent allelochemicals from accumulating to phytotoxic 

concentrations (Schmidt and Ley, 1999). Microbes are expected to metabolize 

allelochemicals because soil is generally considered to be low in available carbon 

(Sparling et al., 1981; Scow, 1997) and these organisms are known to utilize a wide 

range of molecules as carbon sources (Henderson and Farmer, 1955; Black and Dix, 

1976; Campbell et al., 1997). 

Two related but unintegrated lines of evidence suggest that plants do in fact 

influence specific soil microbes via secondary chemicals. First, both plant species 

and genotype are known to affect the rhizosphere species composition of mycorrhizal 

fungi (Johnson et al., 1992) and actinomycetes and some bacteria (Azad et al., 1987; 

Miller et al., 1989; Larkin et al., 1993). Recently, results obtained using molecular 

(Miethling et al., 2000) and physiological (Grayston et al., 2001) methods have confirmed 

the primary importance of plant species on the composition of soil microbial 

communities. Second, of the carbon fixed by photosynthesis in plants, an estimated 

10 to 20% is released into the rhizosphere (Bowen and Rovira, 1991) and in some 

instances this amount may exceed 20% (Shepherd, 1994). The potential effects of 

soil-deposited carbon may extend a distance from the root, as carbon fixed by the 

aerial portions of maize plants has been found over 3 cm away from the roots (Helal 

and Sauerbeck, 1984). A diverse array of organic secondary compounds from plants 

(e.g., alkaloids, phenolics, quinones, saponins, stilbenes) are potent antifungal agents 

(Grayer and Harborne, 1994), and various species/pathovars of fungi can be differently 

susceptible to these chemicals (Zimmer et al., 1967; Arneson and Durbin, 1968; 

Suleman et al., 1996; Sandrock and Van Etten, 1998). It then follows that if secondary 

compounds are present in the rhizosphere, they could influence the growth and/or 

species composition of the soil microbial community and therefore have to be considered 

allelochemicals. However, with the exception of the flavonoids identified in treemycorrhizal 

interactions (Bécard et al., 1992; Lagrange et al., 2001) and legumenodulating 

bacteria interactions (D’Arcy Lameta and Jay, 1987; Peters and Long, 

1988) and the determination of chemicals in Arabidopsis thaliana root exudates 

(Narasimhan et al., 2003), these “root exudates” are not well characterized. In order 

to establish whether specific compounds or groups of compounds such as saponins 

are allelochemicals, therefore, they first must be shown to be present in the rhizosphere 

(i.e., to determine their ecologically relevant concentration), and second, be


shown to be biologically active at their ecologically relevant concentration. Lastly, 

the allelopathic role of the compounds has to be demonstrated at the field level. 

3.1. The Host Plant 

3. GINSENG AND GINSENG SAPONINS 

American ginseng (Panax quinquefolius L.) is a native North American member of 

the Araliaceae, a family whose more than 800 species are found mostly in the tropics 

(Lawrence, 1951). Panax quinquefolius is a perennial understory herb that is associated 

with deciduous forests (Fountain, 1986; Anderson et al., 1993) and ranges from 

Ontario and Quebec, south to northern Florida and west to Minnesota (Small and 

Catling, 1999). The aboveground tissues senesce at the end of each growing season 

and estimates of the maximum age of this plant are 23-30 yr (Anderson et al., 1993) 

to >50 yr (Lewis and Zenger, 1982). Ginseng typically has one aerial stem, with 

three to five palmately compound leaves and an umbelliferous inflorescence. The 

main pollinators of the small greenish-white to greenish-yellow flowers are bees 

(Catling and Spicer, 1995) and the mature red fruits usually contain two seeds, but 

range from one to three seeds (Anderson et al., 1993; Schlessman, 1985). Before 

germinating, the seeds of American ginseng require an after-ripening period of one 

to two winters (Lewis and Zenger, 1982; Anderson et al., 1993). Panax quinquefolius 

is threatened in Canada (Small and Catling, 1999), probably due to over-collection of 

the root for economic gain. 

The main commercial product from ginseng is the taproot, which is sought for its 

purported medicinal properties. Commercially, the roots are harvested after 3-5 yr of 

intensive cultivation. American ginseng has been commercially cultivated in Canada 

since the late nineteenth century (Proctor and Bailey, 1987), under artificial shade or 

in interplanted woodlands. In 2002 Ontario, which is one of Canada’s largest provincial 

producers, exported over one thousand metric tons of ginseng root representing a 

value of almost $ 40 million (OMAF, 2003). However, commercial productivity is 

hindered by susceptibility to several fungal diseases of the leaves, stem, fruit and 

roots. 

3.2. Ginsenoside Structure and Biosynthetic Origins 

Ginsenosides are triterpenoid saponins primarily based on two tetracyclic dammarane 

ring structures: (20S)-protopanaxadiol and (20S)-protopanaxatriol (Figure 1), with 

one representative (Ro) derived from oleanoic acid. While more than 25 ginsenoside 

structures have been identified in Panax spp. (Fuzzati et al., 1999), six are considered 

major: the (20S)-protopanaxadiol-derived Rb 1 , Rb 2 , Rc and Rd and the (20S)protopanaxatriol-derived 

Re and Rg 1 . With the exception of a few monodesmosidic 

compounds (e.g., Rh 2 , Rg 3 , Rh 1 , Rf and Rg 2 ), ginsenosides are generally bisdesmosidic. 

(20S)-Protopanaxadiols are 3-O and 20-O diglycosides, while (20S)-protopanaxatriols

162 


are 6-O and 20-O diglycosides (Figure 1). Ginsenosides can represent approximately 

3-7% dry weight of the root (Court et al., 1996; Li et al., 1996; Nicol et al., 2002) and 

are found in the foliage at lower levels (Chen and Staba, 1978). In Asian ginseng, 

Panax ginseng C.A. Meyer, ginsenosides are found in the outer cortex and periderm 

of roots (Tani et al., 1981). 

Figure 1. The Major Ginsenosides of Panax spp. The three main triterpenoid aglycones 

(20S)-protopanaxadiol, (20S)-protopanaxatriol, oleanoic acid), as well as the six major 

ginsenosides of Panax spp. are shown. Ginsenosides are named according to their migration 

properties on TLC, with no regard for whether they are (20S)-protopanaxadiol-, (20S)protopanaxatriol- 

or oleanoic acid-derived. Ginsenoside Rf (not shown) is a major 

ginsenoside of P. ginseng but is not found in P. quinquefolius.


Triterpenoids are derived from the cytosolic mevalonate pathway via the common 

30-carbon intermediate squalene (Dewick, 1997; Haralampidis et al., 2002). Briefly, 

oxidation of squalene to 2,3-oxidosqualene generates an intermediate that can be 

folded and cyclised (by 2,3-oxidosqualene cyclases) into any one of the different classes 

of triterpenes found in plants. That is, if the 2,3-oxidosqualene is folded into a chairboat-chair-boat 

configuration, protonation of the 2,3-epoxide (which ultimately 

generates the 3-OH common to all triterpenoids and steroids) results in the concerted 

cyclisation of 2,3-oxidosqualene into a protosteryl cation which upon protonation 

yields either cycloartenol (plants) or lanosterol (animals, fungi), both of which are 

precursors to the steroidal saponins and steroids. Alternative folding into a chairchair-chair-boat 

configuration gives rise to a dammarenyl cation (Figure 2), which 

Figure 2. Hypothetical Biosynthesis of Ginsenoside Aglycones. The biosynthesis of ginsenoside 

aglycones begins with the formation of squalene-2,3-oxide by a flavoprotein-containing epoxide 

synthase (1). A putative dammarane synthase (2a-c) is thought to bind squalene-2,3oxide 

and act as a template for folding it into a chair-chair-chair-boat configuration (2a). 

Protonation of the epoxide initiates a concerted series of ring closures (2b), terminating in a 

dammarenyl cation with the appropriate stereochemistry. In Panax spp., the dammarenyl cation 

is expected to be quenched by water (2c) yielding 3,20-dihydroxydammarene. Subsequent 

hydroxylation reactions would yield the protopanaxadiol and protopanaxatriol parent carbon 

skeletons of the common ginsenosides. Two potential hydroxylation pathways are shown (see 

text for details). All enzymes involved in the production of the tetracyclic ginsenosides are 

hypothetical for Panax spp. (based on Dewick, 1997; Haralampidis et al., 2002).

164 


can undergo subsequent 1,2-hydride and/or 1,2 alkyl shifts and further annulation. 

Loss of a proton yields pentacyclic saponin precursors such as α- or β- amyrin or 

lupeol. 

In ginseng, however, only one relatively low abundant ginsenoside (Ro) has a 

pentacyclic parent structure similar to most other non-ginseng triterpenoid saponins. 

Instead, most ginsenosides are based on the (20S)-protopanaxadiol and (20S)protopanaxatriol 

parent carbon skeletons derived from the dammarenediol structure 

that results from the quenching of the dammarenyl cation by water. This reaction 

(i.e., chair-chair-chair-boat template folding, concerted annulation and final cation 

quenching) is hypothesized to be catalysed by dammarenediol synthase (Dewick, 1997; 

Haralampidis et al., 2002), although this has not been proven. Other enzymes involved 

in triterpenoid biosynthesis have been identified in Asian ginseng (P. ginseng) including 

an oxidosqualene cyclase (â-amyrin synthase, Kushiro et al., 1998) as well as other 

candidate oxidosqualene cyclases and enzymes involved in modification of the 

sapogenin (Jung et al., 2003). Further hydroxylation reactions to yield the (20S)protopanaxadiol 

and (20S)-protopanaxatriol parent carbon skeletons from 3,20dihydroxydammarene 

have not been described. However, two routes are theoretically 

possible (Figure 2). In the first, 3,20-dihydroxydammarene is hydroxylated at C-12 to 

yield the (20S)-protopanaxadiol skeleton directly. Subsequent hydroxylation at C-6 

yields the (20S)-protopanaxatriol skeleton. Alternatively, the (20S)-protopanaxatriol 

skeleton could arise independent of the (20S)-protopanaxadiol skeleton via 

hydroxylation at C-6 first, followed by C-12 hydroxylation. In either case, the 

assumption is made that the parent carbon skeletons are assembled first, before 

glycosylation. No details are yet available with respect to the glycosylation of (20S)protopanaxadiol 

and (20S)-protopanaxatriol parent carbon skeletons. Clearly 

glycosylation of the unique C-20 hydroxyl group, which is glycosylated in all 

ginsenosides except two (20S)-protopanaxadiols (Rh 2 , Rg 3 ) and Ro, represents a novel 

glycosyl transferase activity, as does glycosylation of C-6 of the (20S)-protopanaxatriols. 

4. GINSENOSIDE FUNGITOXICITY IN VITRO 

4.1. Differential Response of Fungi and Oomycetes to the Addition of Ginsenosides 

to Culture Media 

In commercial gardens, ginseng is attacked by the foliar pathogens Alternaria panax, 

A. alternata, and Botrytis cinerea, and the root pathogens Cylindrocarpon destructans, 

Rhizoctonia solani, P. cactorum, Py. irregulare, Py. ultimum and several species of 

Fusarium including F. oxysporum and F. solani (Brammall, 1994a, b; Reeleder and 

Brammall, 1995; Punja, 1997). Although the pathogens in the Pythiaceae (i.e., 

Phytophthora cactorum, Pythium irregulare, Py. ultimum) are superficially similar, 

and share the same nutritional mode as the other pathogens, they are not true fungi, 

but rather belong in the kingdom Straminipila (Burnett, 2003). Most other ginseng


pathogens, are ascomycetes. The activity of ginsenosides against these pathogens, as 

well as saprotrophic Trichoderma spp., was evaluated in vitro (Nicol et al., 2002; 

unpublished data). The Trichoderma spp. are potential antagonists towards soilborne 

pathogens, evident by the greater levels of Trichoderma spp. found in healthy ginseng 

fields than in replanted ginseng fields with high disease incidence (Shin and Lee, 

1986). In vitro anti-fungal bioassays were completed by adding ginsenosides to growth 

media and comparing the relative growth to that of controls (i.e., with no ginsenoside 

addition to the growth media). Under these conditions, the growth of six of the nine 

tested organisms was inhibited. The highest growth inhibition was observed in the 

saprotrophs T. harzianum and T. hamatum followed by the leaf pathogen A. panax 

and T. viride (Table 1). 

Table 1. Growth Response of Selected Fungal and Oomycotan Species to 

Ginsenosides. The relative growth of the microorganisms was compared in vitro with 

and without the addition of ginsenosides isolated from Panax quinquefolius roots. 

Growth was standardized to that of controls (i.e., no ginsenosides added) to allow 

comparisons across species, even though different assay conditions were used for 

different organisms. Growth data is shown as a mean ± standard deviation. With the 

exception of F. oxysporum, the growth of all organisms in the presence of ginsenosides 

was significantly different from the mean growth of the respective controls (data not 

shown). Data compiled from Nicol et al. 2002, 2003 and unpublished data. 

Fungal Species % Growth relative to control 

Trichoderma harzianum -26.4 ± 0.9 

Trichoderma hamatum -22.2 ± 2.8 

Trichoderma viride -9.4 ± 1.1 

Alternaria panax -17.1 ± 3.8 

Fusarium solani -3.3 ± 0.6 

Fusarium oxysporum -3.0 ± 0.8 

Cylindrocarpon destructans +7.6 ± 2.9 

Phytophthora cactorum +324.9 ± 1.0 

Pythium irregulare +392.8 ± 0.5 

The growth of the two Fusarium species, F. oxysporum and F. solani, was consistently 

found to be slightly lower, but not always significantly different from control 

(Table 1). Conversely, the growth of the causal organisms of some of the most devastating 

diseases in the North American ginseng industry (i.e., C. destructans, P. 

cactorum and Py. irregulare) was significantly stimulated over that of control. When 

analysed as a group, significantly different growth responses to the ginsenosides were 

observed across the fungal and oomycotan species tested (Table 1). That is, the organisms 

tested were generally inhibited (Trichoderma spp. and A. panax), unaffected 

(Fusarium spp.) or stimulated (C. destructans, P. cactorum and Py. irregulare) by 

ginseng saponins. By comparison, greater antifungal activity was found with aescin, 

a mixture of saponins from the horse chestnut tree (Nicol et al. 2002) and consequently, 

ginsenosides can only be considered to be mildly antifungal.

166 


4.2. “Detoxification” of Ginsenosides by Oomycetes 

As noted in Section 1.2., two mechanisms of resistance have been proposed to explain 

the insensitivity of Oomycetes to saponins, (i) innate resistance, due to little or no 

sterols in the membranes of Oomycete species and (ii) detoxification, via an enzymatic 

degradation of saponins into less bioactive derivatives. With respect to the 

observed growth stimulation of the Oomycetes Py. irregulare and P. cactorum in the 

presence of ginsenosides in vitro, it is not clear which of these mechanisms may be 

involved. However, preliminary results suggest that the “detoxification” of ginsenosides 

(exemplified by Py. irregulare) is the likely mechanism. For example, we initially 

hypothesized that if the fungitoxicity of ginsenosides resulted from their interaction 

with sterols, then the addition of ergosterol to the growth medium of Pythium spp. 

and Phytophthora spp. would render these organism more susceptible (as has been 

demonstrated with aescin, e.g., Olsen, 1973b). However, we have found that the 

addition of ergosterol and ginsenosides to the growth medium of the two ginseng 

pathogens, Py. irregulare and P. cactorum resulted in a cumulative biomass increase 

(~ 200% and 150% compared to control colony weights) relative to cultures grown 

with either ergosterol or ginsenosides alone (Nicol et al., 2003). This observation 

suggests that the resistance of Pythium spp. and Phytophthora spp. to ginsenosides 

cannot simply be due to a lack of sterol content in their membranes. 

Recently, a glycoside hydrolase (BGX1) was described for P. infestans (Brunner 

et al., 2002), the first glycosidase of its kind for an oomycotan organism. With the 

assumptions that (i) the pathogenesis mechanisms of Oomycetes are similar to those 

of fungi (Latijnhouwers et al., 2003), and (ii) ginsenosides act as preformed defense 

compounds (Nicol et al., 2002), then the production of glycosidases by members of 

the Pythiaceae could be an important factor in explaining their pathogenicity to American 

ginseng. Current attempts to rationalize the effect of ginsenosides on the growth 

of Py. irregulare involve exploring the ability of this organism to modify ginsenosides 

in vitro. Thus, when ginsenosides were re-extracted from spent broth that had been 

supplemented with 0.1% ginsenosides prior to inoculation with Py. irregulare, only 

the (20S)-protopanaxatriol-derived ginsenosides Re and Rg 1 were recovered (Figure 

3, lower trace). That is, the (20S)-protopanaxadiol ginsenosides (Rb 1 , Rb 2 , Rc, Rd) 

were all substantially, if not completely depleted from the medium compared to the 

initial supplement (compare Figure 3 upper trace with Figure 3 lower trace). 

Moreover, the decline in (20S)-protopanaxadiol ginsenosides in the spent broth 

of Py. irregulare was linear over the five day culture period (data not shown). 

The disappearance of protopanaxadiol ginsenoside peaks from HPLC chromatograms 

was coincident with the appearance of an unidentified peak eluting at approximately 

45 minutes (Figure 3, lower trace). It is tempting to speculate that the 45 

minute peak is the (20S)-protopanaxadiol aglycone resulting from the de-glycosylation 

of ginsenosides by Py. irregulare, but this requires experimental verification. By contrast, 

the profile of ginsenosides recovered from broth inoculated with T. hamatum, (a 

control organism sensitive to ginsenosides; Table 1) was unaltered relative to that of 

broth in which no organism had been cultured (compare Figure 3 upper trace with


Figure 3. HPLC Analysis of Ginsenosides. Ginsenosides were isolated from spent 

broth in which either no organism (upper trace), Trichoderma hamatum (middle trace) 

or Pythium irregulare (lower trace) had been cultured for five days at 25 o C in the 

dark. Ginsenosides were chromatographed on a Microsorb-MV C-18 column (150 x 

4.6 mm, 5 mm) using an acetonitrile:H 2 O gradient (Nicol et al., 2002), and detected 

at 203 nm. The * in the lower trace indicates the unknown metabolite found in the 

spent broth of Py. irregulare.

168 

Figure 3 middle trace). Therefore, the differential response of these two organisms to 

the presence of ginsenosides in their growth medium is coincident with differences in 

the profile of ginsenosides that can be recovered from their spent medium. 

Interestingly, preliminary observations also show that the recovery of ginsenosides 

from the spent broth of Py. irregulare is dependent on the presence of sucrose in the 

original culture medium, since significantly smaller amounts of ginsenosides were 

recovered from a spent broth lacking sucrose (Yousef and Bernards, unpublished 

data). This observation implies that Py. irregulare is using ginsenosides as a source 

for carbon. However, the additional observation that mineral broth supplemented with 

a concentration of glucose equimolar to that expected to be released into the medium 

by ginsenoside de-glycosylation, does not support the same degree of growth increase 

in Py. irregulare biomass observed when the same broth is supplemented with 

ginsenosides (Yousef and Bernards, unpublished data), argues against a simple carbon 

source mechanism. Since Pythium spp. have been reported to incorporate sterols into 

their membranes (Olsen, 1973a) and because sterols are known to mediate growth 

(Nes, 1987), we now hypothesize that Py. irregulare secretes saponinases to (partially) 

deglycosylate ginsenosides, the latter of which are then incorporated as “sterol” 

triterpenoids into its membrane. It is further assumed that under the experimental 

conditions employed, this incorporation favours growth. 

5.1. RETS and Soil Analysis 


5. GINSENOSIDES IN THE RHIZOSPHERE 

When the soil associated with three-year-old ginseng roots was extracted and analysed 

by HPLC, six major ginsenosides were tentatively identified by co-elution with 

standards. While much of the soil chemical HPLC profile remains unidentified, HPLC- 

MS analysis confirmed the presence of the six major ginsenosides (Rb 1 , Rb 2 , Rc, Rd, 

Re and Rg 1 ) plus pseudoginsenoside F 11 and another protopanaxadiol ginsenoside in 

the soil extracts (Nicol et al., 2003). The amount of ginsenosides as percent weight of 

dry soil was calculated to range from 0.02% to 0.098% (average 0.06%). 

In order to confirm that ginsenosides were present in the exudate of intact ginseng 

roots, (and not isolated from residual root tissue in our soil preparations) root exudates 

were collected from pot-grown ginseng plants using a root exudate trapping system, 

or RETS (Tang and Young, 1982). HPLC analysis of the trapped exudate revealed 

the presence of peaks that had the same retention times as the ginsenoside standards. 

These peaks were not present in the exudate collected from control pots (no ginseng 

plants) and were taken as evidence of ginsenosides in the exudate of pots containing 

ginseng plants. HPLC-MS analysis of the trapped exudate confirmed the presence of 

the same suite of ginsenosides as found in the soil (Nicol et al., 2003). After 

quantification of the ginsenoside content of the root exudates, the individual ginseng 

plants were determined to be losing approximately 25 µg of ginsenosides per day 

(i.e., amount of recovered ginsenoside / number of plants / number of days the 

experiment ran).


5.2. Bioactivity of Ginsenosides at Ecologically Relevant Concentrations 

As emphasized in Section 1.3, before ginsenosides can be considered allelochemicals, 

it has to be demonstrated that they are in fact present in the rhizosphere soil at biologically 

active concentrations. Therefore, after demonstrating the presence of 

ginsenosides in the soil, in vitro bioassays were conducted using ginsenosides at an 

ecologically relevant concentration of 0.06%. At this level, ginsenosides were shown 

to remain bioactive. That is, the growth of Py. irregulare was significantly greater 

than control, while that of T. hamatum remained unchanged (Nicol et al., 2003). 

5.3. Plant-Fungal Allelopathy in Ginseng Gardens and Implications for Disease 

The discovery of ginsenosides in ginseng root exudates and rhizosphere soil at biologically 

active concentrations suggests that plant-fungal allelopathy could occur within 

this crop. This in turn could influence disease levels as the ginseng secondary chemicals 

potentially, and differentially, influence the growth of different groups of soilborne 

organisms (i.e., pathogens and antagonistic saprotrophs). 

Naturally-occurring soilborne antagonists can play a role in preventing or reducing 

disease levels in crops (Azad et al., 1987; Miller et al., 1989; Larkin et al., 1993). 

However, the applied use of Trichoderma spp. as a biocontrol agent often does not 

exert the desired level of disease control. One reason is that isolates of these biocontrol 

fungi often suffer from poor rhizosphere competence (Papavizas, 1982; Chao et al., 

1986). Consequently, poor (natural) Trichoderma spp. competence in the rhizosphere 

of ginseng could lead to increased disease levels in the plant. Interestingly, in an 

attempt to address the issue of the lack of Trichoderma spp. rhizosphere competence, 

it was found that exudates from healthy plant roots did not support the growth of T. 

harzianum, but did in fact support that of Py. ultimum (Green et al., 2001). More 

evidence for the involvement of a soil factor in preventing successful biocontrol by 

Trichoderma spp. is found in the work of Hong et al. (2000) where several Trichoderma 

isolates were shown to inhibit C. destructans in vitro, but this biocontrol effect 

failed to materialize when applied to potted ginseng plants. Again, the specific mechanism 

involved in both of these reports was not identified, but our results suggest that 

plant-fungal allelopathy is one possible explanation. 

Ginsenoside-mediated stimulation of several major pathogens is likely to be 

involved in the eventual establishment of ginseng diseases. Three important root 

pathogens were repeatedly observed to grow better after the addition of ginsenosides 

to their growth medium (i.e., C. destructans, P. cactorum and Py. irregulare). 

Therefore, it can be hypothesized that the chemical environment of the ginseng 

rhizosphere favours fungal root pathogens over potential antagonists of these pathogens 

and that this has a direct consequence on the year-to-year disease levels in ginseng 

gardens. We are still working on the exact mechanism of growth stimulation in 

pathogens, but our preliminary results lead us to believe that in one case (i.e., Py. 

irregulare) the activity is extra-nutritional. For Py. irregulare, increased growth may 

be due to the ginsenosides or transformed ginsenosides acting as sterol analogs. Also,

170 


rhizosphere secondary chemicals in general may be serving as signal molecules for 

plant pathogens in a manner analogous to Rhizobium-legume and tree-mycorrhizae 

symbioses (Peters and Long, 1988; Larange et al., 2001). The ability of rhizosphere 

phytochemicals to fulfill a molecular need for the pathogen or to induce cellular 

pathways important for pathogenesis or virulence is a relatively unexplored avenue of 

plant-microbe allelopathy, even though this has obvious implications for diseases in 

economically important plants. 

Anecdotal evidence suggests that ginseng crops suffer from a replant syndrome 

and a common farming practice is to avoid planting consecutive ginseng gardens in 

the same area. Although there is no data on the cause of the replant problem in 

ginseng, it has been suggested that pathogens could be involved (Reeleder et al., 

1999). In fact the ginseng replant problem bears a similarity to the situation in apple 

orchards. Using our results, a replant syndrome in ginseng gardens could be postulated 

to have the same basic ingredients (i.e., phytochemicals, pathogens and antagonists) 

present in the model of the apple replant syndrome. For example, it is well 

established that the “dominant causal agents” (Mazzola, 1998) of the apple replant 

syndrome are a complex of soilborne fungal pathogens, especially species in the genera 

Cylindrocarpon, Fusarium, Phytophthora and Pythium (Mazzola 1999; Isutsa 

and Merwin, 2000) including C. destructans, P. cactorum and Py. irregulare (Braun, 

1991; 1995; Mazzola, 1998). Although pathogens play a primary role, antagonists 

have also been implicated through results obtained from their application as biocontrol 

microbes (Utkhede et al., 2001) or surveys of orchard soil (Mazzola, 1999). Finally, 

it has been suggested that an important component of apple replant syndrome is the 

induction of virulence in microbes by root exudates (Szabo and Wittenmayer, 2000). 

Similarly, unidentified allelochemicals may also be involved in the asparagus replant 

syndrome (Peirce and Colby, 1987; Hartung and Stephens, 1983; Pedersen et al., 

1991). Therefore, a ginseng replant syndrome, like that of apple and asparagus, 

could be due to a phytochemical-mediated alteration in the soil microbial population 

resulting in the stimulation of a complex of pathogens coupled with an inhibition of 

beneficial microbes. In other words, allelopathy may play a role in year-to-year soilborne 

crop disease cycles as well as replant syndromes. 

6. CONCLUDING REMARKS 

Plant disease researchers often focus on causal agents in relative isolation. Our research 

further demonstrates that other host-derived factors may also play an important role 

in the etiology and severity of plant diseases and replant syndromes. That is, allelopathy 

may contribute to plant diseases through the ability of certain microbes to grow and/ 

or survive preferentially in the phytochemical profile of the rhizosphere surrounding 

specific crop roots. Because pathogenicity is a common nutritional mode in microbes, 

it is not surprising that pathogens are adapted to the chemical environment of their 

host plants. Manipulation of rhizosphere chemistry, therefore, either through genetic 

modification of plants, identification and use of microbe-influencing genotypes or the


planting of mixed crops, represents a potential, but as yet underdeveloped, opportunity 

to reduce disease levels in economically important plants. 

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HIROYUKI NISHIMURA and ATSUSHI SATOH 

ANTIMICROBIAL AND NEMATICIDAL 

SUBSTANCES FROM THE ROOT OF CHICORY 

(Cichorium intybus) 

Department of Biosciences and Technology, School of Engineering, 

Hokkaido Tokai University, Sapporo 005-8601, Japan 

Email: nishimura@db.htokai.ac.jp 

Abstract. Antimicrobial sesquiterpenoids, 8a-angeloyloxycichoralexin and guaianolides such as cichoralexin 

and 10a-hydroxycichopumilide were isolated and identified from the root of chicory (Cichorium intybus). These 

sesquiterpenoids exhibited antifungal activities against Pyricularia oryzae, Pellicularia sasaki and Alternaria 

kikuchiana. Ether soluble phenolics from the chicory root were found to exhibit nematicidal activity. The 

addition of dry chicory root powder to noodles, a boiled fish paste, and cocoa- and coffeecakes, during food 

processing, provided some protection from food spoilage organisms, and this product may have value as a 

natural food preservative. 


Green plants produce numerous secondary compounds, that are not involved in primary 

metabolism (Kaur et al., 2000). The essential role that secondary metabolites, 

such as terpenoid and phenolic compounds, play in complex interactions among living 

organisms in the natural environment is gradually being determined. Although 

some of these natural products have been found to be pollinator or feeding attractants, 

many of them seem to function as chemical weapons against insects, pathogenic organisms, 

and competing plants (Whittaker and Feeny, 1971; Seister, 1977; Berenhaum, 

1995; Inderjit and Duke, 2003). Such secondary metabolites may take part in plantanimal, 

plant-microorganism and plant-plant interactions, and are generally termed 

allelochemicals. 

Chicory (Cichorium intybus) is cultivated in cool regions such as Northern Europe. 

Recently, this vegetable has arisen out of claims that it is able to promote “good 

health” since no pesticides are used to cultivate chicory in the field, while the plant 

remains noticeably free from herbivore and microbial attack. The bitter substances, 

lactupicrin, 8-deoxylactucin and some phenolics had previously been shown to pos- 

sess insect antifeedant properties in chicory (Rees and Harborne, 1985). Specifically, 

sesquiterpenoid lactones from chicory leaves, such as 8-deoxylactucin and lactupicrin 

(Figure 1), were identified as insect antifeedants against desert locust, Schistocerca 

gregaria. Similarly, we found some biologically active secondary metabolites in the 

177 




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HIROYUKI NISHIMURA AND ATSUSHI SATOH 

chicory roots and reported their activity against microorganisms and small animals 

living in soil (Nishimura et al., 2000). In addition, chicory dry-powder has a potential 

application in preserving processed foods. 

HO 

a : R = H, 8-deoxylactucin 

b : R = OCOCH 2 C 6 H 4 OH(p), lactupicrin 

2. ANTIPATHOGENIC SUBSTANCES 

2.1. Antimicrobial Substances in Chicory Root 

O 

H 

O 

Figure 1. Insect antifeedants in chicory leaves. Source: Nishimura et al. (2000). Reproduced 

after permission from Kluwer Academic Publisher (Springer). 

White shoots germinated from cultivated chicory roots have been used for French and 

Italian dishes for a long time. We noticed that fungi were not observed on the chicory 

root in spite of being placed in moist conditions favorable for the development of rots. 

We found that chicory root extracts, recovered with hexane or ether, exhibited antifungal 

activities against Cladosporium herbarum, Pyricularia oryzae, Pellicularia sasaki 

and Alternaria kikuchiana. The ether extract was fractionated by SiO 2 column 

chromatography. Rechromatography in a SiO 2 column and HPLC with a CHCl 3 -MeOH 

solvent system revealed three active compounds. Two of these were sesquiterpenoids 

(C-1 and C-2) identified as cichoralexin (Monde et al., 1990) and 10αhydroxycichopumilide, 

respectively, by the interpretation of spectral data. From the 

interpretation of its spectral data, the third compound (C-3) was identified as 8αangeloyloxy-cichoralexin 

(Figure 2) (Nishimura et al., 2000). These three 

sesquiterpenoids, cichoralexin, 10α-hydroxycichopumilide and 8αangeloyloxycichoralexin 

from the chicory root were found to have significant 

antimicrobial properties. 

O 

H 

R

ANTIMICROBIAL AND NEMATICIDAL SUBSTANCES FROM CHICORY ROOT 179 

1.97 (dd) 

O 

1.11 (d) 

2.15 (m) 

H3C 1.79 (m) 

H H 

H 

2.16 (m) 

H 

5.20 (dt) 

O 

5.95 (s) H 

4.00 (t) 

H 

H 

O 

H 

H 2.18 (ddd) 

2.28 (s) H3C 2.87 (dd) 

O 

H 2.60 (dq) 

1.40 (d) 

2.2. Nematicidal Substances in Chicory Root 

O 

We found that the ether and ethyl acetate extracts of chicory roots exhibited the best 

nematicidal activities. The extracts were separated according to their acidity to give 

organo-acidic, phenolic, basic and neutral fractions. The phenolic fraction was found 

to have the highest activity. 

Figure 3. The relative survival ratios (%) of the golden nematode (Globodera rostochiensis) 

following exposure to chicory plant extracts recovered from the skin, inner part, wounded part 

and hairy roots of chicory and from the acidic fraction of neighboring soil. 

Source: Nishimura et al. (2000). Reproduced with permission from Kluwer Academic Publisher 

(Springer). 

CH 3 

CH 3 

CH 3 

H 

1.91 (m) 

2.01 (dq) 

6.17 (qq) 

Figure 2. Chemical structure of 8α-angeloyloxycichoralexin. Source: Nishimura et al. (2000). 

Reproduced after permission from Kluwer Academic Publisher (Springer). 

Hairy roots 

R. S. R. =91.0% 

Acidic fraction 

from rhizoplane 

soil 

R. S. R. =53.5% 

Acidic fraction from 

bulk field soil 

R. S. R. =100.0% 

R. S. R. = Relative Survival Ratio 

Inner part 

R. S. R. =41.7% 

Wounded part 

R. S. R. =78.6% 

Rhizome skin 

R. S. R. =37.5%

180 

HIROYUKI NISHIMURA AND ATSUSHI SATOH 

The nematicidal activities of extracts from different parts of a chicory root were 

examined. The relative survival ratios (R. S. R.) at the root skin, inner root tissue, 

wounded root tissue and hairy roots were measured (Figure 3). The extract from the 

root skin exhibited the highest nematicidal activity. Interestingly, the nematicidal 

activity of an acidic fraction from rhizoplane soil was much higher than that from 

bulk field soil. We found that some phenolics from the root also exhibited nematicidal 

activity. Thus, it seems that secondary metabolites such as terpenoids and phenolics 

can play an important role in chicory defense. 

3. UTILIZATION OF ALLELOCHEMICALS IN PROCESSED FOODS 

Traditional Japanese foods - noodles, a boiled fish paste, and cocoa- and coffee cakes 

- were cooked without chicory dry powder. After five days, food spoilage fungi such 

as Mucor and Rhizopus species were observed growing on the test foods. Interestingly, 

when 0.1 – 5% (w/w) of the dried chicory powder was added to the processed 

foods, no fungi were observed even after ten days storage. It seems that antimicrobial 

sesquiterpenoids in chicory root can inhibit the growth of spoilage fungi. 

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REN-SEN ZENG 

DISEASE RESISTANCE IN PLANTS THROUGH 

MYCORRHIZAL FUNGI INDUCED 

ALLELOCHEMICALS 

Chemical Ecology Lab., Institute of Tropical & Subtropical Ecology, 

Agricultural College, South China Agricultural University Wushan, Tianhe 

District, Guangzhou, 510642 P.R. China 

Email: rszeng@scau.edu.cn; zengrs8@hotmail.com 

Abstract. Allelochemals induced in mycorrhizal plants play an important role in disease resistance. Mycorrhizal 

associations are the most important symbiosis systems in terrestrial ecosystems and offer many benefits to the 

host plant. Arbuscular mycorrhizal associations can reduce damage caused by soil and root - borne pathogens. 


As plants are non-motile orgnaisms and lack the sophisticated immune system that 

animals have, they had to develop their own defence system against pathogens and 

predators, and systems to lure motile creatures for fertilization and dissemination. 

Plants are capable of synthesizing an overwhelming variety of low-molecular-weight 

organic compounds, called secondary methbolities, many with very unique and complex 

structures. More than 100,000 different secondary metabolites have been found from 

plants (Buckingham, 1998; Dixon, 2001), with many more yet to be discovered; 

estimated of the total number in plants exceed 500,000 (Mendelsohn and Balick, 

1995). Many of these compounds are differentially distributed among limited taxonomic 

groups within the plant kingdom and, conversely, each plant species has a distinct 

profile of secondary metabolites. 

Many of these secondary products are bactericidal, fungicidal, repellent, or even 

poisonous to herbivores. Plant secondary metabolites have for thousands of years 

played an important role in medicine, crop disease and weed control. Nowadays, they 

are used either directly or after chemical modification. Examples of these compounds 

include flavonoids, phenols and phenolic glycosides, unsaturated lactones, sulphur 

compounds, saponins, cyanogenic glycosides and glucosinolates (Go’mez Garibay et 

al., 1990; Bennett and Wallsgrove, 1994; Grayer and Harborne, 1994). 

Plant diseases cause serious losses in crop production. Pesticide application is 

currently the primary way to control crop disease, but it has raised an array of 

environmental problems. Achieving sustainable agriculture will require avoiding a 

181 




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REN-SEN ZENG 

heavy reliance on pesticides. Enhancing crop resistance against diseases and herbivores 

is an ideal approach to reduce pesticide application. 

Plants possess both constitutive and inducible chemical defense mechanisms. 

Before pathogen infection, plants may contain significant amounts of constitutive 

secondary metabolites including phenolics, terpenoids, and steroids, which are toxic 

to invading organisms (Levin, 1976; Mauch-Mani and Metraux, 1998). Plants may 

also activate their production of certain defensive chemicals after pathogen infection. 

These inducible defense compounds are usually only produced and accumulated after 

specific recognition of the invading organism, and are known as phytoalexins (Dixon, 

1986; Hammerschmidt, 1999). Plants can acquire induced resistance to pathogens 

after infection with necrotrophic attackers, nonpathogenic root-colonizing 

pseudomonads, salicylic acid, beta-aminobutyric acid and many other natural or 

synthetic compounds (Conrath et al., 2002; Benhamou, 1996). 

Mycorrhizal fungi provide an effective alternative method of disease control 

especially for those pathogens which effect the below ground plant parts. In mycorrhizal 

fungi lies enormous potential for use as biological control agent for soil-borne diseases 

as the root diseases are of the most difficult to manage and lead to losses in disturbing 

proportions. 

The mycorrhizal symbiosis substantially influence plant growth under a variety 

of stressful conditions and their role in biological control of soil/root - borne pathogens 

is of immense importance both in the agricultural system as well as in the forestry 

(Linderman, 1994). 

Mycorrhizal associations increase growth and yield of many crop plants by 

enhancing nutrient uptake, resistance to drought and salinity and increases tolerance 

to pathogens (Gianinazzi-Pearson, 1996; Mukerji, 1999; Singh et al., 2000; Ludwig- 

Müller, 2004). Of the seven types of mycorrhiza known (Srivastava et al., 1996; Mukerji 

et al., 1997; Raina et al., 2000; Redecker et al., 2000), ectomycorrhiza and vesiculararbuscular 

mycorrhiza (VAM) are more important in agriculture and forestry. 

Ecotmyocrrhizal associations are more prevalent in temperate and sub-temperate 

regions, while VAM/AM associations are common features of tropical and substropical 

regions of the world. During colonization, distinct structures are formed by the 

arbuscular mycorrhizal (AM) fungi, with in the host roots - internal hyphae, arbuscules 

and vesicles (Walker, 1992). The complex cellular relationship between host roots 

and AM fungi requires a continuous exchange of signals, for proper development of 

mycorrhiza in the roots of a host plant (Gianinazzi-Pearson, 1996). Plant hormones 

may be a suitable candidate for the regulation of such a symbiosis. There is little 

information about the function of plant harmones during the colonization process 

although there is evidence that they are involved in signaling events between AM 

fungi and host plants (Barker and Tagu, 2000; Ludwig - Müller, 2000a,b). In addition, 

it has been suggested that phytohormones, such as IAA and cytokinins, released by 

mycorrhizal fungi may also contribute to the enhancement of plant growth. 

(Frankenberger Jr. and Arsad, 1995). 

This review describes the role of mycorrhizal associations in disease control.


ALLELOCHEMICALS 183 

2. MYCORRIZA IN DISEASE RESISTANCE 

Safir (1968) found that inoculation of onion with Glomus mosseae could significantly 

reduce pink root disease due to Pyrenochaeta terrestris. Later studies indicate that 

AM fungi can induce resistance or increase tolerance to some root-borne pathogens 

(Azcon - Aguilar and Barea, 1996, 1997; Caron et al., 1986; Caron, 1989; Cordier et 

al., 1997; Dehne, 1982; Hooker et al., 1994; Trotta et al., 1996). Glomus mosseae 

protected peanut plants from infection by pod rot fungal pathogens Fusarium solani 

and Rhizoctonia solani (Abdalla and Abdel-Fateh, 2000). 

The Glomus intraradices increased P uptake and reduced disease development 

of Aphanomyces euteiches in pea roots (Bodker et al., 1998) Mycorrhization with 

Glomus mosseae and G. intraradices induced local or systemic resistance to 

Phytophthora parasitica in tomato roots (Cordier et al., 1996, 1998; Pozo et al., 

2002). Decreased pathogen development in mycorrhizal and non-mycorrhizal parts 

of inoculated roots is associated with accumulation of phenolics and plant cell defense 

response. The protective effects induced by AM fungi against a phytoplasma is reported 

in tomato (Lingua et al., 2002). 

AM protects an annual grass from root pathogenic fungi in the field (Newsham 

et al., 1995). Inoculation of onion with Glomus sp. Zac-19 delayed onion white rot 

epidemics caused by Sclerotium cepivorum Berk by two weeks and increased the 

yield by 22% under field conditions (Torres-Barragan et al., 1996). Diospyros lotus 

inoculated with Glomus mosseae, Glomus intraradices, Glomus versiforme 

significantly increased the plant growth and decreased the disease caused by Cercospora 

kaki under field conditions. The AM fungal inoculum even suppressed postharvest 

disease of potato dry rot (Fusarium sambucinum) in prenuclear minitubers (Niemira 

et al., 1996). 

Root rot caused by Fusarium solani significantly contributes to crop yield decline, 

up to 50%. The inoculation of common bean (Phaseolus vulgaris) with Glomus 

mosseae, besides decreasing propagule number of F. solani in the rhizosphere, 

decreased root rot by 34 to 77% (Dar et al., 1997). In the presence of the root nodulating 

symbiont Rhizobium leguminosarum, mycorrhizal inoculated plants were more tolerant 

to the fungal root pathogen. This indicates that interactions between mycorrhizal 

fungi and other rhizosphere microbes might have greater effects on soil-borne 

pathogens than mycorrhizal fungi alone. Davis and Menge (1980) found that Glomus 

fasciculatum reduced Phytophthora root rot of citrus at low level of soil phosphorus 

but had no effect in high phosphorus soil. The VAM fungi has also been employed as 

biocontrol agents for Macrophomina root rot of cowpea and Fusarium wilt of tomato 

(Ramaraj et al., 1988). The understanding of the mechanisms of plant disease resistance 

in mycorrhizal plants would provide better directions towards the development of 

efficient crop production and sustainable agriculture. 

3. MECHANISM OF DISEASE CONTROL BY MYCORRHIZAL FUNGI 

The mycorrhizal symbiosis involves several mechanisms in control of plant diseases. 

(i) Creating a mechanical barrier for the pathogen penetration and subsequent spread

184 

REN-SEN ZENG 

as in the case of sheathing mycorrhiza. In ectomycorrhizal associations forming highly 

interwoven mycelial network cover on the root (fungal mantle) and internally with 

cortical cells whose cell walls are surrounded by fungal hyphae i.e., hartigs’ net 

(Ingham, 1991; Maronek, 1981). 

(ii) Thickening of cell wall through lignifications and production of other 

polysaccharides which in turn hinder the entry of root pathogen (Dehne and Schonbeck, 

1979a,b). 

(iii) Stimulating the host roots to produce and accumulate sufficient concentrations 

of metabolities (terpenes, phenols etc.) which impart resistance to the host tissue 

against pathogen invasion (Krupa et al., 1973; Sampangi, 1989). 

(iv) Stimulating flavonolic wall infusions as in the case of Laccaria bicolor which 

prevented lesion formation by the pathogen Fusarium oxgsporum in roots of Douglas 

fir (Strobel and Sinclair, 1991). 

(v) Increasing the concentraton of orthodihydroxy phenols in roots which deter 

the activity of pathogens (Krishna and Bagyarj, 1984). 

(vi) Producing antifungal and antibacterial antibiotics and toxins that act against 

pathogenic organisms (Marx, 1972). 

(vii) Competing with the pathogens for the uptake of essential nutrients in the 

rhizosphere and the root surface (Reid, 1990). 

(viii) Stimulating the microbial activity and competitions in the root zone 

(rhizosphere, rhizoplane) and thus preventing the pathogen to get access to the roots 

(Rambelli, 1973; Singh and Mukerji, 2005). Roots colonized by VAM/AM fungi may 

also harbour more actinomycetes antagonistic to root pathogens (Secilia and Bagyaraj, 

1987; Dixon, 2000; Bansal et al., 2000; Singh et al., 2000; Mukerji, 2002). 

(ix) Compensating the nutrient absorption system from damage to roots by 

pathogens (Simth and Reid, 1997). 

(x) Changing the amount and type of plant root exudates. Pathogens dependent 

on certain exudates will be at a disadvantage as the exudates change (Matsubara et 

al., 1995; Newsham et al., 1995). 

4. PLANT DEFERENCE REACTIONS 

There is considerable evidence for the role of VAM/AM fungi in control of root 

pathogens (St-Arnaud et al., 1995, 1997; Mukerji, 1999). In AM associations some 

plant resistance marker molecules are formed in the tissue which interfere with the 

pathogens to attack (Gianinazzi-Pearson et al., 1996). These are phytoalexins, callose, 

peroxidase, chitinase, β-1,3 glucanase, PR-1 protein. Plants which constitutively overexpress 

defence-related genes provide interesting material for determing how AM 

fungi contend with plant defence, or their modifications may occur in the expression 

of other genes in such plants is still unclear. 

4.1. Mycorrhiza induced secondary Metabolites 

In AM plants, accumulation of secondary metabolites has been reviewed (Azcon- 

Aguilar and Barea, 1996; Morandi, 1996; Vierheilig et al., 1998; Mukerji, 1999).



Phytoalexins are produced de novo in plants in response to infection or attempted 

invasion by microbial pathogens or treatment with abiotic agents (Harborne and 

Ingham, 1978). They are low molecular weight, anti-microbial compounds that are 

both synthesized by and accumlated in plants after contact (exposure) with microbial 

pathogens (Paxton, 1981). Enhancement of disease resistance in mycorrhizal plants 

is due to accumulation of soluble antimicrobial phytoalexins. Rishitin and solavetivone 

are well-known phytoalexins produced by potato plants in response to pathogen 

infection (Engström et al., 1999). Mycorrhizal colonization of potato by Glomus 

etunicatum stimulated the accumulation of the phytoalexins rishitin and solavetivone 

in the roots of plantlets challenged with Rhizoctonia solani but did not change 

phytoalexin levels in non-challenged plantlet roots (Yao et al., 2002, 2003). The 

phytoalexin concentrations in mycorrhizal roots were significantly higher than those 

roots only challenged with Rhizoctonia solani. The result shows that mycorrhization 

can amplify phytoalexin production. Accumulation of phenolpropanoid compounds 

such as phytoalexins, and hydroxyproline-rich glycoproteins, which play important 

roles in plant defense, have been shown to increase either temporarily during 

mycorrhiza formation (Lambais, 2000; Garcia- Garrido and Ocampo, 2002). The 

major phytoalexin in alfalfa (Medicago truncatula) is the isoflavonoid (-)-medicarpin. 

Mycorrhizal colonization resulted in medicarpin accumulation between 7 and l3 days 

(Harrison and Dixon, 1993). The isoflavonoids glyceollin and coumestrol which are 

fungitoxic and nematostatic, respectively, accumulated in the mycorrhizal roots 

(Morandi and Le Querre, 1991). Isoflavonoids also has been showed to accumulate in 

mycorrhizal soybean roots (Morandi, 1984). 

Consistent increases in formononetin levels and transient increases in medicarpin- 

3-0-glycoside and formononetin conjugates were found in inoculated alfafa roots when 

mycorrrizal colonization began (Volpin et al., 1995). It is interesting that concentrations 

of formononetin increases when an AM fungus was in the rhizosphere, even when 

the plants growing there were not yet colonized by the fungus (Volpin and Kapulnik, 

1994). Formononetin has not been shown to have any antimicrobial activities, but is 

a precursor of isoflavonoid phytoalexin produced in alfalfa in response to microbial 

infections. 

Mycorrhizal roots of many plants develop so-called ‘yellow pigment’ , which 

has been used as an indicator to estimate the degree of mycorrhizal colonization 

(Fyson and Oaks, 1992). The chromophore of the ‘yellow pigment’ is an acyclic C 14 

carotenoid-derived polyene called mycorradicin (Klingner et al., 1995). Other 

components of the highly complex mixture of apocarotenoids accumulating in 

mycorrhizal roots are glycosylated C 13 cyclohexenone derivatives (Peipp et al., 1997). 

Mycorrhizal roots of barley, wheat, rye and oat were found to accumulate the blumenina 

terpenoid glycoside (Maier et al., 1995). The level of the compound was directly 

related to the degree of root colonization. Upon colonization by AM fungi, roots of 

many plant families accumulate certain apocarotenoids (Fester et al., 1999, 2002; 

Maier et al., 1995; 1998, 1999, 2000; Walter et al., 2000). Colonization of barley, 

wheat and maize roots by different arbuscular mycorrhizal fungi, i.e. 

185

186 

REN-SEN ZENG 

Glomus intraradices, Glomus mosseae, and Gigaspora rosea leads to the accumulation 

of similar cyclohexenone derivatives (Vierheilig et al., 2000). However, no fungusspecific 

induction of these compounds are known. Pathogens and endophyte did not 

induce the formation of cyclohexenone derivatives in barley roots (Maier et al., 1997). 

The role of cyclohexenone derivatives in disease resistance is unknown. 

In response to pathogen attack, plants activate an array of inducible defense 

reactions, many of which involve the transcriptional activation of the corresponding 

defense genes, including genes that encode enzymes involved in the synthesis of lignin 

and phytoalexins (Dixon et al., 1984; Dixon and Harrison 1990). Transcript levels of 

some pivotal enzymes of defense response of plants significantly increase after 

mycorrhizal fungal infection (Harrison and Dixon, 1993; 1994). Several inducible 

defence-related genes, including those encoding isoflavonoid phytoalexins such as 

phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase 

(CHI) and for the cell wall structural protein HRGP, have been reported to be induced 

during mycorrhiz establishment (Tagu and Martin, 1996). Mycorrhization resulted 

in a local and systemic induction of plant defence-related enzymes chitinase, 

chitosanase and beta-1,3-glucanase, as well as superoxide dismutase in tomato plants 

(Pozo et al., 2002). Chalcone isomerase (CHI) and chitinase activities increased in 

inoculated roots prior to mycorrhizal colonization (Volpin et al., l994, Kapulink et 

al., 1996), whereas the increase in PAL activity coincided with colonization. Production 

of some new compounds, and increase in the activity of the enzymes peroxidase and 

polyphenol oxidase, was observed following inoculation with AM fungi (Charitha 

Devi and Reddy, 2002). Dumas-Gaudot et al. (1992a,b) found new chitinase isoforms 

that were specifically induced in several AM associations and were different from 

those elicited by root fungal pathogens, indicating a different pattern of plant response 

to pathogenic and mutualistic fungi. Glomus mosseae induced new chitinase isoforms 

in tomato roots (Pozo et al., 1996). Expression of genes encoding enzymes that 

synthesize phenolpropanoid compounds has been detected in mycorrhizal roots (Garcia- 

Garrido and Ocampo, 2002). Other defense related genes shown to be upregulated in 

mycorrhizal symbioses include: genes involved in metabolism of reactive oxygen 

species, chitinase and beta I ,3-glucanase, and genes involved in senescence, including 

glutathione-S-transferase. Mycorrhiza also induced changes in PR protein expression 

in tobacco leaves (Shaul et al., 1999). 

Colonization of barley, wheat and maize and rice roots by Glomus intraradices 

resulted in strong induction of transcript levels of the pivotal enzymes of 

methylerythritol phosphate pathway of isoprenoid biosynthes i.e., 1 -deoxy-D-xylulose 

5-phosphate synthase (DXS) and 1 -deoxy-D-xylulose 5-phosphate reductoisomerase 

(DXR) (Walter et al., 2000). At the same time six cyclohexenone derivatives were 

characterized from mycorrhizal wheat and maize roots. DXS2 transcript levels are 

low in most tissues but are strongly stimulated in roots upon colonization by 

mycorrhizal fungi, correlated with accumulation of carotenoids and apocarotenoids 

(Walter et al., 2002). Some reports show that the AM symbiosis may cause an increase, 

decrease, or no change in the plant defense reactions (Guenoune et al., 2001; Mohr et 

al., 1998).



DIMBOA (2,4-dihydroxy-7-methoxy-1 ,4-benzoxazin-3-one) play an important 

role in the chemical defense of cereals against insects, pathogenic fungi and bacteria 

(Niemeyer, 1988). Researches with maize indicate that mycorrizal colonization induced 

accumulation of DIMBOA and increase in transcript levels of Bx9. Concentrations of 

DIMBOA in maize roots inoculated both G. mosseae and Rhizoctonia solani were 

significantly higher than those roots inoculated separately with G. mosseae or 

Rhizoctonia solani. 

Phenolic compounds in plants play a role in disease resistance (Morandi, 1996). 

Mycorrhizal plants of maize accumulated more phenolic compounds p-hydrocinnamic 

acid and ferulic acid in roots than non-mycorrhizal plants (Huang, 2003). Mycorrhizal 

Ri T-DNA transformed carrot roots accumulated more phenolic compounds when 

challenged by Fusarium oxysporum (Benhamou et al., 1994). All cultivars of pea 

colonized with G. mosseae accumulated more phenolic acids, and total phenolic 

accumulation were closely correlated to disease intensity (Singh et al., 2004). 

Many studies show that AM fungi initiate a host defence response which is 

subsequently suppressed (Lambais and Mehdy, 1993; Volpin et al. 1994, 1995). The 

decreases were accompanied by differential reductions in the levels of mRNAs encoding 

for different endochitinase and endoglucanase isoforms. But the activation of specific 

plant defence reactions by AM fungi could predispose the plant to an early response 

to attack by a root pathogen (Gianinazzi-Pearson et al., 1994). 

Although studies on growth inhibition of fungi by isolated plant compounds 

suggest a role in plant defense, such in vitro tests may not always give an accurate 

indication of the significance of these compounds in restricting fungal growth in the 

plant. Despite increasing efforts in research on metabolic changes in mycorrhizal 

plants, the precise understanding of the mechanisms is poorly understood, and the 

role of secondary metabolites induced by AM fungi in disease resistance is still obscure. 


Mycorrhizal fungi protect plant roots from diseases in several ways : (i) by providing 

a physical barrier to the invading pathogens. Physical protection is more likely to 

exclude soil insects and nematode than bacteria or fungi in ectomycorrhizal plants. 

However some nematodes can penetrate the fungal mantle, (ii) by competing with the 

pathogen; (iii) by producing allelopathic chemicals like secondary metabolites, 

antagonistic chemicals like antibiotics, toxins etc., and amount and type of the root 

exudates. 

For effective and persistent disease management and biocontrol the need is to 

evaluate the mycorrhizal symbionts in the natural system under field conditions. The 

use of mixed inoculum of mycorrhizal symbionts can be more effective and give 

better results than use of a single species. Selection of superior indigenous mycorrhizal 

symbionts may have an adaptive advantage to the soils and environment in 

which pathogen and host co-occur as compared to non-indigenous mycorrhizal 

symbionts. 

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REN-SEN ZENG 

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CHUIHUA KONG 

ALLELOCHEMICALS FROM Ageratum conyzoides L. 

AND Oryza sativa L. AND THEIR EFFECTS 

ON RELATED PATHOGENS 

Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, 

China, and South China Agricultural University, Guangzhou 510642, China. 

E-mail: kongch@mail.edu.cn 

Abstract. Allelochemicals play an important role in biological control of plant pathogens and diseases. Weed 

Ageratum conyzoides L. and food crop Oryza sativa L. can produce and release many kinds of allelochemicals 

participating in their defense against pathogens. The essential oil from A. conyzoides has been found to have 

significant negative effects on several plant pathogens. In the A. conyzoides intercropped citrus orchard, A. 

conyzoides released allelopathic flavones and agreatochromene into the soil to reduce the populations of soil 

pathogenic fungi Phytophthora citrophthora, Pythium aphanidermatum and Fusarium solani. Further research 

revealed that ageratochromene underwent a reversible transformation in the soils, that is, ageratochromene 

released from A. conyzoides plants was transformed into its dimers, and the dimers can be remonomerized in the 

soils. The reversible transformation between ageratochromene and its dimers in the A. conyzoides intercropped 

citrus orchard soil can be an important mechanism maintaining bioactive allelochemicals at an effective 

concentration, thus, sustaining the inhibition of pathogenic fungi in soil. Many kinds of allelochemicals in rice 

were identified. Among them, alkylresorcinols, flavone and cyclohexenone had high antifungal activities on 

Pyricularia oryzae and Rhizoctonia solani. Furthermore, these antifungal allelochemicals formed by rice can 

be triggered by a large number of abiotic and biotic factors. Antifungal allelochemicals from rice mainly involved 

two types of diterpenes and flavones, including momilactones A and B, oryzalexins A-F and S, phytocassanes 

A-E and sakuranetin. These compounds help rice establishing its own pathogen defense mechanism. However, 

it remains obscure which allelochemicals in rice are predominantly involved in defense mechanisms against the 

pathogens. Therefore, further clarification of the resistance mechanism and multiple functions of these compounds 

on rice are warranted. 


Plants produce many kinds of low-molecular-mass secondary metabolites that are 

generally non-essential for the basic metabolic processes of the plant. Among these 

secondary plant metabolites, some are known as allelochemicals that improved defense 

against other plant competition, microbial attack or insect/animal predation. Plants 

cannot move to escape pathogens. However, plants have evolved to successfully 

withstand infection by a vast majority of pathogens that attack them (Stuiver and 

Custers, 2001). It was found that plants biosynthesize phytoalexins as soon after 

pathogenic attack (Dangl and Jones, 2001). The concept of phytoalexins as induced 

anti-microbial allelochemicals in plant was first developed in 1940 (Muller and Borger, 

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1940; Bailey and Mansfield, 1982). Anti-microbial allelochemicals or phytoalexins 

have been extensively investigated and play important roles in plant defense (Dixon, 

2001). Most of anti-microbial allelochemicals have relatively broad-spectrum activity 

on pathogens and specificity is often occurred in cropping systems. Accordingly, an 

understanding of the interactions between allelochemicals and pathogenic organisms 

is essential in disease control in agro-ecosystems. 

The structures and sources of many allelochemicals with anti-microbial activity 

have been documented (Dixon, 2001; Grayer and Harborne, 1994; Harborne, 1999). 

In this chapter, evidence for anti-microbial functions of allelochemicals from weed 

Ageratum conyzoides L. and food crop Oryza sativa L. has been reviewed. Effects of 

these allelochemicals on related pathogen management in the A. conyzoides 

intercropped citrus orchard and the paddy ecosystem were discussed. 

2. ALLELOCHEMICALS OF A. conyzoides AND THEIR EFFECT ON 

RELATED PATHOGENS 

2.1. Ageratum conyzoides L. 

Ageratum conyzoides of the family Compositae (Asteraceae) is native to Central 

America (Kossmann and Groth, 1993) Caribbean and Florida (USA). It has spread to 

West Africa, Southeast Asia, South China, India, Australia and South America 

(Okunade, 2002; Stadler et al., 1998). A. conyzoides is an annual erect, branched 

herb growing 15 to 100 cm tall. Its stem is covered with fine white hairs, leaves are 

opposite, pubescent with long petioles and include glandular trichomes. It has a shallow 

tap root system. The inflorescence contains 30 to 50 pink or purple flowers arranged 

in a corymb and are self-incompatible (Jhansi and Ramanujam, 1987). The fruit is an 

achene with an aristate pappus and is easily dispersed by wind and animals fir. Seeds 

are positively photoblastic and remain viable upto 12 months (Okunade, 2002). The 

seeds germinate between 20-25°C. It prefers a moist, well drained soil but may tolerate 

dry conditions (Ladeira et al., 1987). This species has great morphological variations 

and appears highly adaptable to varying ecological conditions (Hu and Kong, 2002a). 

It is a pioneer plant growing in ruined sites and cultivated fields and often becomes 

dominant and forms a stand in natural community and is resistant to common insects 

or diseases (Liang and Hunag, 1994). Although it is harmful to crops and invades 

cultivated fields and interferes with the natural community compositions, it has been 

used as folk medicine in several countries and it has anti-microbial, insecticidal and 

nematicidal properties (Ming, 1999; Okunade, 2002). In Central America A. 

conyzoides has been bred for many colours of flowers (Stadler et al., 1998). In South 

China A. conyzoides is traditionally used as green manure in fields to increase the 

crop yields, and usually is intercropped as understory in citrus orchards to suppress 

weeds and control other pests (Liang and Hunag, 1994; Kong et al., 2004b). This 

species appears to be a valuable agricultural resource (Ming, 1999).

ALLELOCHEMICALS FROM AGERATUM CONYZOIDES AND ORYZA SATIVA 

2.2 The essential oil of A. conyzoides and their biological activities on related 

pathogens 

A. conyzoides has a wide range of secondary metabolites including flavonoids, 

chromenes, benzofurans and terpenoids. Among these secondary metabolites, some 

are allelochemicals inhibiting the growth of other organisms (Okunade, 2002; Pari et 

al., 1998). Usually, A. conyzoides can produce and release volatile chemicals into the 

environment. The concentration of its released volatiles is so high that the unpleasant 

odor can be smelled in the fields. Therefore, most investigations have focused on 

chemical components of its essential oil (Albersberg and Singh, 1991; Ekundayo et 

al., 1988; Menut et al., 1993; Wandji et al., 1996). It was found that ageratochromenes 

and their derivatives, monoterpenes and sesquiterpenes were the major components 

of the essential oil from A. conyzoides (Kong et al., 1999; 2002a; Pari et al., 1998). 

The allelopathic potential of volatile allelochemicals from A. conyzoides has been 

reported in our previous papers (Kong et al., 1999; 2002a; 2004a). Anti-microbial 

effects of the essential oil from A. conyzoides have been confirmed for a long time 

(Biond et al., 1993; Dixit et al., 1995; Rao et al., 1996). Table 1 showed that several 

fungal pathogens, such as Rhizoctonia solani, Botrytis cinerea, Sclerotinia sclerotiorum, 

were significantly inhibited by the essential oils of A. conyzoides (Kong et al., 2001; 

2002a). The quantity and variety of allelochemical produced by A. conyzoides varies 

depending on its growth stages and habitats and so do their growth inhibitory effects 

on the pathogens (Kong et al., 2002a, 2004a). A. conyzoides produces different volatiles 

in larger quantities when infected with Erysiphe cichoracearum (Kong et al., 2002a). 

Table 1. Inhibitory effects of essential oil from the A. congzoides collected from 

different growth stages and habitats on fungal pathogens. 

The essential oil collected Pathogens 

from R. solani B. cinerea S. sclerotiorum 

Growth stages 

4-leaf 23.8±3.8a 19.6±5.3a 39.8±7.6a Pre-flowering 56.3±10.1b 49.7±9.1b 60.2±7.9b Peak-flowering 100c 82.3±11.5c 100c Mature 60.6±8.8b 58.3±7.4b 40.8±9.5a Habitats 

Cultivated field 79.6±10.2d 83.5±6.3c 93.8±7.7c Under citrus canopy 58.4±4.3b 45.6±6.1b 66.2±5.5b Roadside 100 c 100d 100c Control 

50% Carbendazin 100c 59.8±5.2c 92.3±7.9c Test concentrations of the essential oil were 100 µg/ml. All data are inhibitory percentage of spore germination 

of fungal pathogens tested and mean of 3 replicates with standard error. Data in a column not followed by the 

same letter are significantly different, p=0.05, ANVOA with Ducan’s multiple range test. 

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CHUIHUA KONG 

2.3. Allelochemicals and pathogen management in the A. conyzoides intercropped 

citrus orchard 

Besides the volatiles, A. conyzoides can biosynthesize and release non-volatile 

allelochemicals into the soil, thus, inhibiting the growth of other plants and 

microorganisms in soils. Polymethoxyflavones, ageratochromene and its analogues 

are rare in natural products but they have been found in A. conyzoides (Adesogan and 

Okunade, 1979; Gonzalez et al., 1991; Horie, et al 1993; Okunade, 2002). These 

compounds have obvious anti-microbial activity and have been used in managed 

ecosystem. 

In South China A. conyzoides is often intercropped in citrus orchards as an 

understory plant that quickly becomes dominant in citrus orchards. In addition, 

intercropping A. conyzoides makes the citrus orchard ecosystem more favorable for 

predatory mites (Amblyseius spp.). These mites are effective natural enemies of the 

citrus red mite (Panonychus citri). Further investigations showed that the pathogenic 

fungi Phytophthora citrophthora, Pythium aphanidermatum and Fusarium solani were 

isolated from both the A. conyzoides intercropped and non-intercropped citrus orchards 

soils. However, populations of these fungi were lower in the A. conyzoides intercropped 

citrus orchard than in the non-intercropped citrus orchard (Figure 1), indicating that 

intercropping with A. conyzoides in citrus orchards markedly decreased the population 

of soil pathogenic fungi. It may have resulted from the phytotoxins in the A. conyzoides 

intercropped citrus orchard soil (Kong et al., 2004c). 

P. citrophthora P. aphanidermatum F. solani 

Figure 1. Population of major pathogenic fungi in the A. conyzoides intercropped and nonintercropped 

citrus orchards soils. Population density was mean individual in per gram soil.

ALLELOCHEMICALS FROM AGERATUM CONYZOIDES AND ORYZA SATIVA 197 

Several flavones, ageratochromene and its two dimers were isolated and identified 

from the A. conyzoides intercropped citrus orchard soil (Figures 2 and 3), their amounts 

ranged from 11 to 93µg g -1 in the air dried soil. However, these chemicals could not be 

found in the non-intercropped citrus orchard soil. The results showed that these 

chemicals were primarily released from ground cover plants of A. conyzoides and 

accumulated in the soil year by year. 

A: R 1 =R 2 =OCH 3 ; B: R 1 =H, R 2 =OCH 3 ; C: R 1 =OCH 3 , R 2 =H 

D: R 1 =R 3 =OCH 3 , R 2 =H; E: R 1 =R 2 =R 3 =OCH 3 ; 

F: R 1 =R 3 =H, R 2 =OCH 3 ; G: R 1 =H, R 2 =R 3 =OCH 3 ; 

H: R 1 =R 3 =OCH 3 , R 2 =OH; I: R 1 =R 3 =OCH 3 , R 2 =α- rhamnosyl. 

Figure 2. Flavones produced and released from the A. conyzoides 

intercropped citrus orchard soil. 

Bioassays showed that ageratochoromene and flavones could significantly inhibit 

spore germination of the pathogenic fungi P. citrophthora, P. aphanidermatum and F. 

solani, but two dimers of ageratochromene had no inhibitory effects on them (Table 

2). Thus, the flavones and ageratochromene could be one of the key factors that A. 

conyzoides plants are able to reduce the populations of soil pathogenic fungi in the 

citrus orchard. Two dimers, though not biologically active, may be the products of 

ageratochromene transformation in soil. 

Further studies revealed that ageratochromene underwent a reversible 

transformation in the soils, that is, ageratochromene released from ground A. 

conyzoides plants was transformed into its dimers, and the dimers can be 

remonomerized in the soils (Kong et al., 2004c). However, this dynamic transformation 

did not occur in the soil with low organic matter and fertility (Figure 3). The reversible 

transformation between ageratochromene and its dimers in the A. conyzoides 

intercropped citrus orchard soil can be an important mechanism maintaining bioactive

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CHUIHUA KONG 

Table 2. Inhibitiory effects of allelochemicals isolated from the A. conyzoides 

intercropped citrus orchard soil on major pathogenic fungi. 

Chemicals P. citrophthora P. aphanidermatum F. solani 

Flavone A 100 a 90.6±8.6 a 72.1±6.5 a 

Flavone B 90.5±7.6 b 75.9±8.1 b 39.9±5.8 b 

Flavone C 91.2±6.3 b 76.3±6.5 b 35.6±7.1 b 

Flavone D 100 a 90.5±8.7 a 59.6±7.1 c 

Flavone E 100 a 93.2±9.2 a 60.2±7.3 c 

Flavone f 87.5±7.9 b 80.5±7.2 b 38.9±4.5 b 

Flavone G 77.3±6.1 c 74.2±6.2 b 40.2±5.3 b 

Flavone H 98.6±8.0 b 80.3±6.6 b 57.7±4.1 c 

Flavone I 39.8±2.1 d 39.7±7.5 c 35.3±5.0 b 

Ageratochromene 43.5±6.4 d 37.6±5.4 c 60.8±9.9 c 

Dimer A 6.1±1.1 e 0.9±0.3 d 1.8±0.6 d 

Dimer B 5.0±0.9 e 1.6±0.3 d 7.2±1.6 d 

50% Carbendazin 100 a 74.8±6.1 b 58.3±3.2 c 

Test concentrations of the essential oil were 100 µg/ml. All data are inhibitory percentage and 

mean of 3 replicates with standard error. Data in a column not followed by the same letter are 

significantly different, p=0.05, ANVOA with Ducan’s multiple range test. 

Figure 3. Transformation and degrading of ageratochormene in the soil. 

Ageratochormene transformation was in soil with high organic matter content and high fertility. 

Transformation occurred from step 1 to step 3, only in soil with low organic content and fertility. 

allelochemicals at an effective concentration, thus, sustaining the inhibition of 

pathogenic fungi in soil. 

Therefore, A. conyzoides has been advocated for intercropping in citrus orchards 

and utilized on more than 150,000 ha of citrus orchards in South China and gave 

substantial ecological and economic benefits (Liang and Huang, 1994). It is an excellent 

example of applied aspects of allelopathy in agro-ecosystem.


3.1. Rice antifungal allelochemicals 

3. RICE ALLELOCHEMICALS AND THEIR 

EFFECTS ON RELATED PATHOGENS 

Rice (Oryza sativa L.) is one of the principal food crops in the world. Its production is 

characterized by heavy use of herbicides and fungicides that may cause environmental 

problems in the paddy ecosystem (Kim and Shin, 2000). Accordingly, rice 

allelochemicals can potentially be used to improve weed and pathogen management 

in rice production. Therefore, search for allelochemicals from rice has been extensively 

studied (Chung et al., 2001; Kong et al., 2002b; Mattice et al., 1998; Rimando et al., 

2001). A range of phenolic acids was identified as potent allelochemicals from rice 

tissues and root exudates (Chung et al., 2001; Mattice et al., 1998; Rimando et al., 

2001). However, these phenolic acids are unlikely to explain the allelopathy of rice 

since their soil concentrations never reach phytotoxic levels (Olofsdotter et al., 2002). 

More recently, an increasing number of studies have shown that a few flavones, 

diterpenoids and other types of compounds are also the potent allelochemicals from 

rice (Kato-Noguchi et al., 2002; 2003; Kong et al., 2002b; 2004d,e; Lee et al., 1999). 

These allelochemicals can be biosynthesized in rice seedlings and then released into 

their surroundings at ecologically relevant concentrations to inhibit the germination 

and growth of associated weeds. Similarly, rice allelochemicals may participate in the 

defense mechanisms of rice against pathogens. 

In our laboratory, a flavone (5,7,4.-trihydroxy-3,5.-dimethoxyflavone), a 

cyclohexenone (3-isopropyl-5-acetoxycyclohexene-2-one-1) and a liquid mixture of 

low polarity, containing long-chain and cyclic hydrocarbons (Table 3), were isolated 

and identified from leaves of allelopathic rice accession PI 312777 (Kong et al., 2004e). 

Both the flavone and cyclohexenone significantly inhibited spore germination of 

Pyricularia oryzae and Rhizoctonia solani, but the mixture containing low polarity 

constituents did not show any inhibitory effect on them, even at high concentrations 

(Figure 4a,b). The IC 50 values of the flavone on spore germination of P. oryzae and R. 

solani were ca 50 and 70.g. g -1 , while the cyclohexenone were ca 75 (P. oryzae) and 

95 (R. solani), respectively. At all concentrations tested, the inhibitory activity of the 

flavone on pathogens was slightly higher than that of the cyclohexenone. The complete 

inhibition of both compounds on spore germination of the pathogens was observed at 

250.g. g -1 . (Figure 5a, b). 

a. b. 

5,7,4¡Ç-trihydroxy-3¡Ç,5¡Ç-dimethoxyflavone 3-isopropyl-5-acetoxycyclohexene-2-one-1 

Figure 4. a. Flavone; b. Cyclohexenone.

200 

CHUIHUA KONG 

(a) (b) 

Figure 5. Inhibitory activities of flavone, cyclohexnone and mixture with low polarity against 

spore germination of (a) P. oryzae and (b) R. solani at different concentrations. 

Table 3. Chemical constituents of the mixture with low 

polarity isolated from rice leaves 

Retention time (min) Chemical constituents Relative amount (%) 

1.89 2-methylhexane 8.77 

5.40 4,6-dimethylundecane 2.48 

6.28 2,2,6-trimethyldecane 2.35 

7.26 2-hexyl-1-decanol 2.11 

7.58 1-butyl-2-propylcyclopentane 8.45 

8.19 2,6-dimethyl-decahydronaphthalene 22.45 

8.40 trans, trans-1,10-dimethylspiro[4,5]decane 8.03 

8.48 2,3-dimethyl-decahydronaphthalene 3.37 

8.63 trans, cis-1,8-dimethylspiro[4,5]decane 8.19 

8.78 1,2-dimethyldecahydronaphthalene 11.49 

9.10 cis, cis-1,1-dimethylspiro[4,5]decane 11.60 

10.18 1,4,6-trimethyl-1,2,3,4-tetrahydronapthalene 1.77 

14.35 hexadecanoic acid, methyl ester 1.85 

- Other unknown components* 7.09 

*A total of 11 components and their relative amounts was less than 1% of the mixture with low 

polarity 

Alkylresorcinols (Figure 6) are another kind of antifungal agent that were isolated 

and identified from etiolated rice seedlings (Suzuki et al., 1996; 1998). Antifungal 

activities of alkylresorcinol against spore germination of P. oryzae were correlated 

with their concentration and structure, but complete inhibition of their spore 

germination occurred only at a concentration of over 100.g.ml -1 .


R=-(CH 2 ) 12 CH 3 , -(CH 2 ) 14 CH 3 , -(CH 2 ) 16 CH 3 , -(CH 2 ) 7 CH=CH(CH 2 ) 5 CH 3 , - 

(CH 2 ) 7 CH=CH(CH 2 ) 7 CH 3 , -(CH 2 ) 7 CH=CHCH 2 CH=CH(CH 2 ) 4 CH 3 

Figure 6. Alkylresorcinols. 

3.2. Diterpene and flavone phytoalexins from rice 

Allelochemicals play an important role in rice disease resistance (Bailey and Mansfield, 

1982). Antifungal allelochemicala, phytoalexins produced by rice in response to injury, 

physiological stimuli or in the presence of infectious agents (Hammerschmidt, 1999). 

In particular, phytoalexins can be induced and accumulated by rice after fungal 

infection. Phytoalexins from rice mainly involve two types of diterpenes and flavones, 

including momilactones A and B, oryzalexins A-F and S, phytocassanes A-E and 

sakuranetin (Figures 7, 8). 

Momilactone A Momilactone B Sakuranetin 

Oryzalexin A: R 1 =OH, R 2 =CH 3 Oryzalexin E: R=CH 3 Oryzalexin S 

Oryzalexin B: R 1 =O, R 2 =OH Oryzalexin F: R=CH 2 OH 

Oryzalexin C: R 1 =O, R 2 =O 

Oryzalexin D: R 1 =OH, R 2 =O 

Figure 7. Typical diterpene and flavone phytoalexins from rice.

202 

CHUIHUA KONG 

Momilactones A and B are the first phytoalexins characterized from any member of 

the Gramineae. They were isolated initially as plant inhibitors from rice husk (Kato et al., 

1977), and were subsequently found to be produced by rice in response to either 

infection by the blast disease that caused by the pathogen Pyricularia oryzae or 

irradiation with UV light (Cartwright et al., 1977; 1981). They had significant 

phytoalexin-like activity in P. oryzae and Helminthosporium oryzae. More recently, 

momilactones A and B have been found in rice root exudates, which have been found 

to participate in defense against weeds (Kato-Noguchi et al., 2002; 2003; Kong et al., 

2004d; Lee et al., 1999). 

Phytocassane B Phytocassane C 

(ED 50 =20ì g/mg) (ED 50 =4ì g/mg) (ED 50 =7ì g/mg) 

Phytocassane D Phytocassane E 

(ED 50 =25ì g/mg) (ED 50 =6ì g/mg) 

Figure 8. Structures of phytocassanes A-E and their antifungal activities (the ED 50 

values) on spore germination of Magnaporthe grisea. 

Oryzalexins A-F and S, phytocassanes A-E and sakuranetin were isolated from 

rice leaves infected with blast fungus, Magnaporthe grisea (Akatsuka et al., 1985; 

Kato et al., 1993; 1994; Kodama et al., 1992; Koga et al., 1995; 1997; Nakazato Y et 

al., 2000). Oryzalexins A-C strongly inhibited the spore germination of P. oryzae. 

Their ED 50 values were 130, 68 and 136 ppm, respectively. Complete inhibition of P. 

oryzae spore germination was observed at 200 ppm. Oryzalexins A-C also strongly 

suppressed the germ tube elongation of P. oryzae. Their ED 50 values on P. oryzae 

germ tube elongation were 35, 18 and 35 ppm, respectively (Akatsuka et al., 1985). 

Similarly, oryzalexins D-F and S significantly inhibited spore germination of the rice 

blast fungus (Kato et al., 1993; 1994; Kodama et al., 1992). Noteworthy, oryzalexins 

E had slightly lower antifungal activity than oryzalexins D, but higher than Oryzalexins 

A-C (Kato et al., 1993). Phytocassanes A-E are the diterpenes with a cassane skeleton


(Figure 8). They were produced in rice leaves and stems with M. grisea and R. solani. 

Phytocassanes A-E had high antifungal activity against the pathogenic fungi, M. 

grisea and R. solani (Koga et al., 1995; 1997). Their ED 50 values in prevention of 

spore germination and germ tube growth of M. grisea were very low (Figure 8). 

Sakuranetin is a flavonoid-type phytoalexin that was identified from rice plants. It 

had high antifungal activity and a large amount of accumulation in rice leaves 

(Nakazato et al., 2000). 

It has been shown in host-pathogen interactions that resistance reactions can be 

triggered by a large number of abiotic and biotic factors. Among the chemical factors, 

macromolecules of microbial origin are very important. Plant defense responses are 

stimulated by very low concentrations of these molecules (Darvill and Albersheim, 

1984). It was confirmed that a chemical for plant disease control might function by 

activating the natural resistance mechanisms of the host, for example, 2,2-dichloro- 

3,3-dimethyl cyclopropane carboxylic acid may exert its systemic fungicidal activity 

against the rice blast disease caused by P. oryzae (Cartwright et al., 1977). The 

application of methionine on wounded rice leaves induced the production of rice 

phytoalexins, sakuranetin and momilactone A. In the paddy field, methionine treatment 

has been demonstrated to reduce rice blast (Nakazato et al., 2000). 

An increasing number of studies have shown that rice phytoalexins are induced 

by elicitor that are produced by pathogenic microorganisms and make field disease 

control by inducing the pathogen defense mechanism in rice (Schaffrath et al., 1995; 

Tamogami et al., 1997a,b). Elicitors have been investigated extensively. It has been 

shown that jasmonic acid and its related compounds play important roles as the 

signaling molecules that elicit the production of phytoalexins in rice. Sakuranetin 

production may be elicited by exogenously applied jasmonic acid in rice leaves. 

Furthermore, sakuranetin production by exogenously applied jasmonic acid was 

significantly counteracted by amino acid, cytokinin, kinetin and zeatin (Tamogami et 

al., 1997a,b). 

4. CONCLUSION 

Many interactions between plant pathogens and their hosts are allelopathic. 

Allelochemicals can be applied in biological control of weeds and plant diseases (Rice, 

1995). Our research suggested that allelochemicals produced and released from A. 

conyzoides intercropping in citrus orchard did play important roles in integrated pest 

management. Many kinds of allelochemicals in rice not only inhibit the germination 

and growth of weeds, but also participate in the defense against pathogens. However, 

it remains unclear which allelochemicals in rice are predominantly involved in defense 

mechanisms against the pathogens. Therefore, further clarification of the resistance 

mechanism and multiple functions of rice allelochemicals are warranted. 

Acknowledgments : The author thanks Dr. Inderjit and anonymous reviewers for 

thoughtful criticisms and correcting English on earlier versions of the manuscript. The 

work was supported by National Natural Science Foundation of China (NSFC 

No.30170182; 30430460) and Hundreds-Talent Program of Chinese Academy of Sciences.

204 

CHUIHUA KONG 

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Index 

A 

Abutilon theophrasti, 38, 83, 148 

Acacia sedillense, 47 

Acalypha indica, 114 

Acidovorax delafieldii, 148 

Adenostoma fasciculatum, 55 

Aeschynomene sp., 17 

Ageratum conyzoides, 42, 92, 194, 195, 196, 197, 

198, 203 

Aggressiveness, 93 , 94 

Agrobacterium radiobacter, 63 

Agrobacterium sp. 130 

Alcaligenes piechaudii, 63 

Allium cepa, 57 

Alnus firmifolia, 51 

Alternanthera philoxeroides, 111 

Alternaria alternata, 111, 164 

Alternaria alternata f sp. lycopersici, 112 

Alternaria eichhorniae, 111 

Alternaria kikuchiana, 178 

Alternaria panax, 164, 165 

Alternaria solani, 37, 53 

Amaranthus hypochondriacus, 37, 46, 53 

Amaranthus retroflexus, 43, 92 

Amaranthus spp., 57 

Amblyseius spp, 196 

Ambrosia cumanensis, 38 

Ammania coccinea, 104 

Anabaena sp, 110 

antifungal, 165, 178, 200, 203 

Aphanomyces euteiches, 183 

Arabidopsis thaliana, 87, 160 

Arachis hypogaea, 56, 92 

Arthrobacter spp., 66 

Ascochyta caulina, 83 

Aspergillus flavus, 9 

Aspergillus fumigatus, 9 

Aspergillus niger, 9 

Aspergillus parasiticus, 9 

Aspergillus sp., 68 

Avena fatua, 92 

Avena sativa, 64, 86 

Azadirachta indica, 21 

Azolla nilotica, 108 

B 

Bacillus cereus, 66, 127 

Bacillus mycoides, 61 

Bacillus subtilis, 3, 66 

Bacillus sp., 130 

Belonolaimus longicaudatus, 65 

207 

Berula erecta, 51 

Beta vulgaris, 83 

Bievundimonas vesicularis, 63 

biocontrol , 66, 110, 111, 118, 124, 128, 129, 130, 

131, 132, 133, 134, 145, 146, 148, 149, 150, 

169 , 170, 183, 187 

bioherbicides, 148, 149, 150, 151 

biological control, 25, 56, 70, 71, 111, 125, 131, 134, 

143, 145, 146, 148, 151, 152, 182 

biopesticide, 16, 15, 17 

biotrophic, 80, 93, 94 

biotrophs, 79 

Botrytis cinerea, 3, 5, 7, 8, 9, 92, 130, 159, 164, 

195 

Botrytis sp., 3, 12 

Brassica juncea, 42, 108 

Brassica kaber, 50 

Brassica napus, 87 

Brassica rapa var. pervidis, 38 

Brassica spp., 21 

Bromus japonicus, 148 

Bromus mollis, 92 

Bromus tectorum, 147 

Burkholderia cepacia, 66 

Burkholderia pickettii, 63 

Burkholderia sp., 20 

C 

Caenorhabditis elegans, 23 

Callicarpa acuminata, 37 

Calotropis gigantea, 106 

Canavalia ensiformis, 17, 71 

Capsella bursa-pastoris, 84, 152 

Capsicum annuum, 42, 57 

Capsicum frutescens, 9, 45 

Cassia fasciculata, 17 

Cassia obtusifolia, 57 

Cassytha sp., 108 

Centaurea solstitialis, 107 

Ceratophyllum demersum, 108 

Cercospora alternantherae, 111 

Cercospora amboinicus,114 

Cercospora kaki, 183 

Cercospora sp., 111 

Chaetomella raphigera, 111 

chemotaxis, 18 

Chenopodium album, 43, 83, 149 

Chondrilla juncea, 83 

Cicer arietinum, 86 

Cichorium intybus, 177 

Cirsium maculosa, 45

208 

Cirsium sp., 43 

Cladosporium herbarum, 178 

Cleistopholis acutatum, 12 

Cleistopholis fragariae, 11 

Cleistopholis patens, 9 

Cleome viscosa, 114 

Coleus amboinicus, 109, 114 , 115, 116, 117 

Colletotrichum acutatum, 3, 5, 7, 8, 9, 12 

Colletotrichum coccodes, 83 

Colletotrichum fragariae , 3, 5, 8 , 9, 11 

Colletotrichum gloeosporioides, 3, 5, 8, 9, 10 

Colletotrichum sp., 3, 4, 8, 12 

commensal, 124, 125, 127, 128 

compatible, 57 

contamination, 143, 149 

Criconemella sp., 65 

Crotalaria juncea, 17, 20, 57, 69, 70, 71 

Crotalaria retusa, 71 

Crotalaria spectabilis, 17, 70 

Crotalaria sp.,16, 65, 67, 69, 71 

Croton sparsiflorus, 114 

Cucumis sativus cv. Wisconsin, 47 

Cucumis sativus, 92 

Cucurbita pepo, 47 

Cuminum cyminum, 86 

Curcuma longa, 114 

Cyamopsis tetragonoloba, 52 

Cylindrocarpon destructans, 164, 165, 169, 170 

Cylindrocarpon sp., 170 

Cynoches sp., 3 

Cynodon dactylon, 107 

Cyperus difformis,104, 106, 117 

Cyperus littoralis, 106 

Cyperus rotundus, 106, 111 

D 

Dactylaria higginsi, 111 

Dactylis glomerata, 88, 92 

Daucus carota, 86 

Delonix regia, 35 

Digitaria decumbens, 35 

Diospyros lotus, 183 

Drechmeria coniospora, 69 

Drecshlera sp., 111 

E 

Echinochloa crus-galli, 37, 46, 104, 111 

Echinochloa spp, 106 

Eclipta alba, 114 

Egeria densa, 111 

Eichhornia crassipes, 108, 109, 114 

elicitors, 203 

Empetrum hermaphroditum, 41 

Enterobacter asburiae, 63 

epidemics, 88 

INDEX 

Erwinia carotovora, 87, 126, 127 

Erwinia sp, 130 

Erysiphe cichoracearum, 92, 195 

Eucalyptus globulus, 106 

Eucalyptus sp., 106 

Euphorbia esula, 147 

Euphorbia hirta, 114 

Exostema caribaeum, 47 

Exserohilum monocerus, 111 

exudate, 19, 35, 40, 69, 87, 107, 124, 148, 160, 168, 

170, 184, 199 

F 

Fagus sylvatica, 47 

Festuca arundinacea, 91 

fungistasis, 84 

fungitoxic, 157, 158, 185 

Fusarium graminearum, 111 

Fusarium oxysporum f. sp. cucumerinum, 130 

Fusarium oxysporum f.sp. cumini, 86 

Fusarium oxysporum f. sp. lycopersici, 159 

Fusarium oxysporum, 3, 5, 8, 9, 64, 66, 86, 164, 

165, 187 

Fusarium sambucinum, 183 

Fusarium solani, 23, 164, 165 , 183, 196, 197 

Fusarium sp., 60, 61, 165, 170, 183 

G 

Gaeumannomyces graminis var. tritici, 60 

Gaeumannomyces graminis, 158, 159 

Gigaspora rosea, 186 

Globodera pallida, 67 

Globodera rostochiensis, 18 

Glomus etunicatum, 185 

Glomus fasciculatum, 183 

Glomus intraradices, 68, 183, 186 

Glomus mosseae, 68, 183, 186, 187 

Glomus versiforme, 183 

Glomus sp., 183 

Glycine max, 56, 65, 66, 69, 83 

Gnomonia comari, 7, 12 

Gossypium spp., 56 

H 

Hebeloma crustuliniforme, 44 

Helianthus annuus, 39, 57, 86 

Helicobacter pylori, 49 

Helicotylenchus dihystera, 64, 65 

Helicotylenchus sp.,24, 65 

Helminthosporium longirostratum, 37 

Helminthosporium oryzae, 202 

Heteranthera limosa, 104 

Heterodera glycines, 24, 64, 65 

Heterodera schachtii, 22, 62 

Hintonia latiflora, 47

Hippeastrum hybridum, 48 

Hordeum leporinum, 92 

Hymenoscyphus ericae, 44 

Hyrdilla verticilata, 107, 108 

I 

Imperata cylindrica, 107 

Indigofera hirsuta, 17, 70, 71 

Indigofera nummularifolia, 71 

Indigofera spicata, 71 

Indigofera suffruticosa, 71 

Indigofera tinctoria, 71 

inoculum, 12, 63, 83, 111, 125, 145, 183, 187 

Ipomoea batatas, 54 

Ipomoea tricolor, 46 

Ipomoea sp., 57 

Iva axillaris, 38 

J 

Juglans nigra, 82 

Juncus sp., 51 

K 

Kocuria kristinae, 63 

Kocuria varians, 63 

L 

Laccaria bicolor, 184 

Lactuca sativa, 37, 41, 83 

Lactuca serriola, 89 

Lantana camara, 47 

Larrea tridentata, 41 

Lemna leucocephala, 114 

Lemna paucicostaha, 108 

Lemna perpusilla, 108 

Leptinotarsa decemlineata, 37 

Leptosphaeria maculans, 87 

Leptosphaerulina trifolii, 85 

Leucaena leucocephala, 35, 52, 106, 114 

Leucaena sp., 53, 106 

Leucas aspera, 114 

Linum usitatissimum, 57 

Lolium multiflorum, 92, 107 

Lolium perenne, 84, 87 

Lolium sp., 64 

Lycopersicon esculentum, 47, 53, 70, 82, 108 

Lythium salicaria, 107 

M 

Macaranga monandra, 9 

Macrophomina sp., 183 

Magnaporthe grisea, 202, 203 

Manihot esculenta, 107 

Medicago truncatula, 185 

Meloidogyne arenaria, 18, 20, 24, 65 

Meloidogyne hapla, 18 

INDEX 209 

Meloidogyne incognita, 18, 20, 21, 23, 24, 25, 61, 

63, 65, 66, 68, 69, 70 

Meloidogyne javanica, 18, 20, 21, 22, 23, 68, 70 

Meloidogyne spp, 19, 18, 24, 64, 65, 71 

Microbacterium esteraromaticum, 63 

Mucuna deeringiana, 17, 20, 62, 64, 66, 70 

mycoherbicide, 111 

Mycosphaerella sp, 111 

Myriophyllum aquaticum, 111 

Myriophyllum spicatum, 110 

Myrothecium roridum, 111 

Myrothecium sp., 68 

N 

Najas graminea, 108 

necrotrophic, 182 

necrotrophs, 79 

nematophagous, 67, 69 

Neochetina bruchii, 118 

Neotyphodium coenophialum, 91 

Neotyphodium lolii, 90, 94 

Nicotiana tabaccum, 57, 86 

Nimbya alternantherae, 111 

O 

Orobanche aegyptica, 57 

Orobanche cernua, 57, 40 

Orobanche crenata, 40, 57 

Orobanche cumana, 40 

Orobanche ramosa, 40, 57 

Orobanche sp., 40, 57 

Oryza sativa, 57, 194, 199 

P 

Paecilomyces sp., 20 

Paenibacillus sp, 130 

Panax cactorum, 164 

Panax ginseng, 162 

Panax quinquefolius, 157, 161 

Panax spp, 161 

Panicum miliaceum, 56 

Panonychus citri, 196 

Paratrichodorus minor, 19, 65 

Paratichodorus sp., 24 

Parthenium hysterophorous, 38, 53, 108, 114 

Paspalum sp., 17 

Pellicularia sasaki, 178 

Penicillium sp., 68 

Pennisetum americanum, 52 

Phaseolus aureus, 57 

Phaseolus mungo, 57 

Phaseolus vulgaris, 47, 86, 183 

Phaseolus sp., 41 

Phoenix dactylifera, 86 

Phoma lingam, 159

210 

Phoma typhae-domingensis, 111 

Phomopsis obscurans, 3, 5, 9 

Phomopsis viticola, 3, 5, 9 

Phomopsis sp., 8 

phyllosphere, 62 

phytoalexins, 3, 56, 85, 86, 159, 182, 184, 185, 186, 

193, 194, 201, 202, 203 

phytophagus, 34 

Phytophthora cactorum, 7, 66, 164,165,166, 169, 

170 

Phytophthora cinnamomi, 61 

Phytophthora citrophthora, 196, 197 

Phytophthora infestans, 86, 166 

Phytophthora nicotianae, 6, 9 

Phytophthora parasitica, 183 

Phytophthora sp., 5, 9, 166, 170, 183 

phytoprotection, 132 

phytotoxic, 25, 46, 52, 57, 64, 87, 106, 124, 160, 

199 

phytotoxin, 35, 42, 44, 45, 56, 57, 58, 59, 105, 111, 

147, 150 

Picea abies, 44 

Pinus laricio, 47 

Pistia stratiotes, 108, 109 

Pisum sativa, 86 

Plantago lanceolata, 43 

Pluchea lanceolata, 42 

Plutella xylostella, 54 

Portulaca oleracea, 57, 83 

Pratylenchus brachyurus, 68 

Pratylenchus neglectus, 21 

Pratylenchus penetrans, 63 

Pratylenchus vulnus, 22 

Pratylenchus sp., 19, 24, 65 

predators, 69, 181 

propagule, 147, 183 

Prunus dulcis, 23 

Psacalium decompositum, 37 

Pseudomonas aeruginosa, 130 

Pseudomonas aureofaciens, 127 

Pseudomonas chlororaphis, 63 

Pseudomonas fluorescens, 56, 63, 147 

Pseudomonas putida, 56, 148 

Pseudomonas syringae, 147 

Pseudomonas sp., 60, 61, 63, 66, 147 

Pseudopeziza coronata, 94 

Pseudopeziza medicagnis, 94 

Puccinia chondrillina, 83 

Puccinia coronata f.sp. avenae, 86 

Puccinia coronata f.sp. lolii, 88 

Puccinia coronata, 89, 92 

Puccinia graminis, 86, 92 

Puccinia hordei, 92 

Puccinia lagenophorae, 83 

INDEX 

Puccinia pulsatillae, 84 

Puccinia recondita, 92 

Pusatilla pratensis, 84 

Pyrenochaeta terrestris, 183 

Pyricularia oryzae, 178, 199, 200, 202 

Pythium aphanidermatum, 196, 197 

Pythium irregulare, 164, 165, 166,168,169, 170 

Pythium ultimum, 66, 164, 169 

Pythium sp., 50, 159, 166, 168, 170 

R 

Radopholus similis, 65 

Ralstonia solanacearum, 126 

Ralastonia sp., 127 

Ratibida mexicana, 46 

Renibacterium salmoninarum, 61 

Reynoutria sachalinensis, 3 

Rhizobium etli, 67 

Rhizobium japonicum, 111 

Rhizobium leguminosarum biovar phaseoli, 41 

Rhizobium leguminosarum, 183 

Rhizobium sp., 41, 51, 67, 170 

Rhizoctonia solani, 60, 64, 66, 92, 159, 164, 183, 

185, 187, 203,195,199 

rhizoplane, 108, 124, 131, 146, 147, 160, 169, 180, 

184 

rhizosphere, 17, 20, 23, 35, 36, 42, 44, 57, 58, 62, 

67, 68, 108, 131, 133, 134, 144, 146, 147, 148, 

151, 157, 169, 170, 183, 184, 185 

Ricinus communis, 17, 70 

rotation, 16, 17, 18, 19, 20, 22, 35, 41, 64, 66, 71, 

117, 134, 150, 152 

Rotylenchulus reniformis, 22, 24, 65, 70, 71 

Rumex crispus, 43 

Ruta graveolens, 8 

S 

Saccharum officinarum, 86 

Salvinia auriculata, 111 

Salvinia molesta, 108,109, 111 

Schistocerca gregaria, 177 

Sclerotinia sclerotiorum, 87, 92, 195 

Sclerotinia sp., 3 

Sclerotium cepivorum, 183 

Scutellonema sp., 65 

Sebastiania adenophora, 47 

Secale cereale, 57, 64 

Senecio vulgaris, 83 

Septoria lycopersici, 159 

Serratia plymuthica, 130 

Sesamum bicolor, 17 

Sesamum indicum, 17, 19, 57 

Sesbania aculeata, 117 

Sesbania exaltata, 107 

Sesbania grandiflora, 114

Setaria faberi, 151 

Setaria virdis, 148 

Sicyos deppei, 47 

Sida spinosa, 57 

Simsia amplexicaulis, 41 

Sinapis arvensis, 54 

Solanum nigrum, 43 

Solanum tuberosum, 62, 86 

Solanum, 86 

Sorghum bicolor,17, 19, 56, 57, 86 

Sorghum sudanense, 56 

Sorghum vulgare var, sudanense,17 

Sorghum vulgare, 17 , 52 

spermosphere, 144 

Sphenoclea zeylanica, 106 

Spirodella polyrhiza, 108 

Stellaria media, 149 

Stenotrophomonas sp., 130 

Streptococcus pneumoniae, 61 

Streptomyces hygroscopicus, 56, 112 

Streptomyces viridichromogens, 56, 111 

Streptomyces sp.,112, 130 

Striga asiatica, 40, 56 

Striga gesnerioides, 56 

Striga hermonthica, 32, 56, 152 

Striga sp., 40, 132 

suppression, 17, 18, 20, 21, 34, 55, 60, 61, 70, 72, 

84, 88, 89, 90, 91, 104, 105, 106, 107, 118, 

128, 132, 133, 149, 150, 151, 152 

susceptible, 4, 8, 13, 55, 68, 86, 88, 90, 93, 108, 

112, 160 

synergistic, 103, 118 

systemic acquired resistance, 13 

T 

Tagetes erecta, 18, 53, 62 

Tagetes patula, 18, 62 

Tagetes signata, 18 

Tagetes spp., 17, 63 

Tephrosia adunca, 71 

Thlaspi arvense, 149 

Thymus vulgaris, 24 

INDEX 211 

Tradescantia crassifolia, 41 

Trianthema portulacastum, 114 

Trichoderma hamatum, 165, 166, 169 

Trichoderma harzianum, 165 

Trichoderma viride, 165 

Trichoderma sp., 68, 165, 169 

Trifolium incarnatum, 57, 66 

Trifolium pratense, 50, 54, 85 

Trifolium repens, 84, 87 

Trifolium subterraneum, 57, 83, 92 

Trifolium sp., 64 

Triticum aestivum, 42, 54, 64, 66, 70, 86 

Tsukamurella paurometabolum, 63 

Tylenchorhynchus claytoni, 64, 65 

Tylenchulus semipenetrans, 21 

Tylenchorhynchus sp., 24 

Typha domingensis, 111 

U 

Uredo eichhorniae, 111 

Uromyces troflii-repentis, 92 

Urtica urens, 132 

V 

Vaccinium myrtillus, 44 

Variovorax paradoxus, 127 

Verticillium albo-atrum, 159 

Verticillium biguttatum, 64 

Vicia villosa, 64 

Vicia sp., 17 

Vigna catjang, 56 

Vigna unguiculata, 64, 66 

virulence, 93, 170 

Vitex negundo, 35 

Vitis vinifera, 3 

X 

Xanthium strumarium, 57 

Xanthomonas campestris, 126 

Xiphinema sp., 24 

Z 

Zea mays, 47, 50, 52, 56, 57, 83, 107

List of Contributors 

Ana Luisa Anaya 

Laboratorio de Alelopatía, Instituto de Ecología, Universidad Nacional 

Autónoma de México. Circuito Exterior, Ciudad Universitaria. 04510 México, 

D.F. Email: alanaya@miranda.ecologia.unam.mx 

Mark A. Bernards 

Department of Biology, University of Western Ontario, London, ON, Canada, 

N6A 5B7, Email: bernards@uwo.ca 

Ramanathan Kathiresan 

Professor, Department of Agronomy, Annamalai University, Annamalainagar, 

Tamilnadu 608 002, India. E-mail : rm.kathiresan@sify.com 

Cilfford H. Koger 

Weed Ecologist, Southern Weed Science Research Unit, USDA-ARS, 

P. O. Box 350, Stoneville, Mississippi 38776, USA. 

Nancy Kokalis-Burelle 

Research Ecologist, USDA, Agricultural Research Service, U.S. Horticultural 

Research Lab, Fort Pierce, FL, 34945, USA 

Email:NBurelle@ushrl.ars.usda.gov 

Chuihua Kong 

Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, 

China, and South China Agricultural University, Guangzhou 510642, China. 

E-mail: kongch@mail.edu.cnw 

Robert J. Kremer 

U.S.D.A., Agricultural Research Service, Cropping Systems & 

Water Quality Unit, University of Missouri, Columbia, Missouri, U.S.A. 

Email: KremerR@missouri.edu 

Scott W. Mattner 

Department of Primary Industries, Knoxfield Centre, Victoria, 

Private Bag 15, Ferntree Gully Delivery Centre, 3156, VIC, Australia, 

scott.mattner@dpi.vic.gov.au 

Robert W. Nicol 

NovoBiotic Pharmaceuticals, Cambridge MA, 02138, USA 

Email: bernards@uwo.ca 

Hiroyuki Nishimura 

Department of Biosciences and Technology, School of Engineering, Hokkaido 

Tokai University, Sapporo 005-8601, Japan 

Email:nishimura@db.htokai.ac.jp 

213

214 

LIST OF CONTRIBUTORS 

Krishna N. Reddy 

Plant Physiologist, Southern Weed Science Research Unit, USDA-ARS, 

P. O. Box 350, Stoneville, Mississippi 38776, USA; 

Email: kreddy@ars.usda.gov 

Rodrigo Rodríguez-Kábana 

Distinguished University Professor, Auburn University, Auburn, 

AL, 36849, USA. Email : NBurelle@ushrl.ars.usda.gov 

Atsushi Satoh 

Department of Biosciences and Technology, School of Engineering, Hokkaido 

Tokai University, Sapporo 005-8601, Japan Email:nishimura@db.htokai.ac.jp 

Barbara J. Smith 

Small Fruit Research Station, 306 S. High St., Poplarville, MS 39470. 

Email : dwedge@olemiss.edu 

Antony V. Sturz 

Prince Edward Island Department of Agriculture, Fisheries, Aquaculture and 

Forestry, P.O. Box 1600, Charlottetown, P.E.I., C1A 7N3, Canada. 

Tel: 902-368-5664, FAX: 902-368-5661, E-mail:avsturz@gov.pe.ca 

David E. Wedge 

United States Department of Agriculture, Agricultural Research Service, 

Natural Products Utilization Research Unit, The Thad Cochran National 

Center for Natural Products Research, University of Mississippi, University, 

MS 38677, USA Email : dwedge@olemiss.edu 

Lina F. Yousef 

Department of Biology, University of Western Ontario, London, ON, Canada, 

N6A 5B7 

Ren-Sen Zeng 

Chemical Ecology Lab., Institute of Tropical & Subtropical Ecology, 

Agricultural College, South China Agricultural University Wushan, 

Tianhe District, Guangzhou, 510642 P.R. China 

Email: rszeng@scau.edu.cn, zengrs8@hotmail.com

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