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Biological Control 21, 182–190 (2001)doi:10.1006/bcon.2001.0933, available online at http://www.idealibrary.com on

Enhancement of Efficacy of Ascochyta caulina to ControlChenopodium album by Use of Phytotoxins and

Reduced Rates of Herbicides1

Maurizio Vurro,*,2 Maria Chiara Zonno,* Antonio Evidente,† Anna Andolfi,† and Pasquale Montemurro‡*Istituto Tossine e Micotossine da Parassiti Vegetali, CNR, viale L. Einaudi 51, 70125 Bari, Italy; †Dipartimento di Scienze Chimico-

Agrarie, Universita di Napoli Federico II, via Universita 100, 80055 Portici, Italy; and ‡Dipartimento di Scienze delle ProduzioniVegetali, Universita degli Studi, via Amendola 165/a, 70122 Bari, Italy

Received August 1, 2000; accepted March 5, 2001

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The use of toxins produced by Ascochyta caulina toenhance the efficacy of this mycoherbicide agent forthe biological control of the noxious weed Chenopo-dium album was investigated. A method of purifica-tion of the fungal culture filtrate was developed toproduce large amounts of a mixture containing threemain toxins: ascaulitoxin, its aglycone, and trans-4-aminoproline. Greenhouse experiments showed thatthe use of toxin solutions (at 1 mg/ml) in conjunctionwith spores of A. caulina (at 106/ml) strongly improvedhe biocontrol efficacy of this fungus by more than0%. Furthermore, the simultaneous application ofoxins or fungal spores, together with low doses oferbicides (metribuzin and rimsulfuron at 1/5 of the

abeled rate), gave better results than single-agentreatments. © 2001 Academic Press

Key Words: Ascochyta caulina; Chenopodium album;weed biocontrol; phytotoxins; mycoherbicide; ascauli-toxin, trans-4-aminoproline; herbicides.

INTRODUCTION

Chenopodium album L. (common lambsquarters orfat-hen) is one of the most successful colonizing species(Allard, 1965) that is able to grow in all inhabited areasexcept in extreme desert climates and on all soil typesover a wide range of pH values (Andreasen et al., 1991).It is a troublesome weed in sugar beets, potatoes,maize, cereals, and vegetables all over the world (Holmet al., 1977). It is currently controlled in most crops byherbicides, but in maize and some vegetables it is rel-atively tolerant or resistant to many herbicides (Myers

1 This paper is dedicated to Professor Antonio Graniti, Universitaegli Studi, Bari, Italy, on the occasion of his 75th birthday.

2 To whom correspondence should be addressed. Fax:139.0805486063. E-mail: ma.vurro@area.ba.cnr.it.

1821049-9644/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

and Harvey, 1993). All this considered, the species is asuitable target for a biocontrol approach.

The perthotrophic fungal species Ascochyta caulina(P. Karst.) v.d. Aa and v. Kest. has been proposed as amycoherbicide against C. album (Kempenaar, 1995).

he postemergence application of fungal pycnidio-pores to young plants causes large necrotic areas oneaves and stems and, depending on the amount ofecrosis developed, plants show retarded growth oreath (Kempenaar et al., 1996).The genus Ascochyta includes several species that

produce a wide array of secondary toxic metaboliteshaving heterogeneous chemical structures and pos-sessing different biological properties (Strange, 1997).On account of the possible use of fungal metabolites asnaturally occurring and safe herbicides, or in combina-tion with pathogens in weed biocontrol (Strobel et al.,1991), it was of interest to ascertain the production oftoxic metabolites by A. caulina and to carry out theirisolation and chemical and biological characterization.Preliminary experiments also had shown the potentialof culture filtrates of this fungus to increase the speedof disease onset (the first phytotoxic effect observed asearly as 1–2 days after treatment) and enhance diseaseseverity (7.2 compared to 3.2), when used in combina-tion with fungal spores (Zonno et al., 1998; Netland etal., 2001).

From the filtrates of stationary fungal culturesgrown in a defined liquid medium, three main phyto-toxic metabolites were purified. The toxins with prom-ising herbicidal properties were trans-4-aminoproline(Evidente et al., 2000), the N2–b-glucopyranoside of theunusual nonproteigenic bis-aminoacid 2,4,7-triamino-5-hydroxyoctandioic acid (named ascaulitoxin) (Evi-dente et al., 1998), and its aglycone (Evidente et al.,001).Exploring new opportunities in integrated weedanagement, studies on the use of pathogenic fungiith low rates of herbicides have shown that combina-

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183EFFICACY ENHANCEMENT OF THE MYCOHERBICIDE Ascochyta caulina

tions of these agents can improve biocontrol efficacy(Christy et al., 1993). Along this line, the present paperdescribes the use of a mixture containing three mainphytotoxins produced by A. caulina, in combinationwith the fungus and reduced rates of chemical herbi-cides (metribuzin and rimsulfuron) to enhance the ef-ficacy of this mycoherbicide.

MATERIALS AND METHODS

Fungus

A strain of A. caulina (P. Karst) v.d. Aa and v. Kest,isolated from a diseased leaf of C. album, was kindlysupplied by Dr. P. C. Scheepens (Department of Cropand Production Ecology, Wageningen University andResearch Centre, The Netherlands). It was maintainedon potato–dextrose–agar medium as a single-spore cul-ture in the Collection of Istituto Tossine e Micotossineda Parassiti Vegetali, CNR, Bari, Italy (ITEM 1058).

Production of Toxic Culture Filtrates

For the production of toxic metabolites, 1 ml of conid-ial suspension (containing approximately 106 conidia/

l) was added to a 1-liter Roux bottle containing 200l of M-1D medium (Evidente et al., 1998). The cul-

ures were incubated under stationary conditions at5°C in the dark for 4 weeks and then filtered, testedor phytotoxic activity, lyophilized for further purifica-ion, or stored frozen until application.

urification of Metabolite Mixture

Considering the difficulties and the high cost to ob-ain each of the three phytotoxins in pure form, aethod based on ion-exchange chromatography was

et up (Evidente et al., 2000) as the first step of trans-4-aminoproline purification. The lyophilized culture fil-trate of A. caulina (73 g, corresponding to 2 liters) wasdissolved in 75 ml of formic acid (1 M) and applied to aDowex-50 H1 form resin (Fluka, Bush, Switzerland)packed in a chromatographic column (4 3 26 cm). Afterhe lyophilized culture filtrate was loaded, the columnas washed with ultrapure water (1 liter), which re-oved the large amount of saccharose used as carbon

ource in the culture medium together with other non-asic substances. The column was then eluted withH4OH (1 liter, 1 M) and the basic eluate was concen-

trated under reduced pressure in a rotary evaporatorto remove ammonia. The residual solution was lyoph-ilized to give a slightly brown solid residue (700 mg).The residue consisted of a mixture of the phytotoxinstrans-4-amino-D-proline, ascaulitoxin, and its agly-cone, as ascertained by thin-layer (TLC) and high-performance anion exchange (HPAC) chromatographyanalyses (Evidente et al., 2001).

Materials for Treatments

Toxins. The mixture of toxic metabolites, appear-ing as a yellow-brownish, fully water-soluble powder,was weighed and dissolved in distilled water at thedesired concentration. The standard concentrationused was 1 mg/ml, and 10 ml were usually sprayed foreach replication.

Spore suspension. Spores of A. caulina were har-vested from cultures on a wheat bran medium (kindlysupplied by Dr. P. Scheepens), prepared according toKempenaar (1995), by the addition of 1 liter of a Syl-gard solution (silicone-based surfactant; Dow CorningEurope, Belgium) (0.2%) to 80 g of air-dried wheat branculture. After 4 h, the material was filtered throughcheesecloth, obtaining a spore suspension that wasadjusted to 106 spore/ml for application to plants. Allsolutions also contained nutrients (Czapek-Dox broth,1.5 g per liter; yeast extract, 0.3 g per liter). Ten mil-liliters of this suspension was applied for each repli-cate. In the case of treatments that included bothspores and toxins, double-concentrated solutions wereprepared, so that their mixture had the desired finalconcentrations.

Herbicides. Unless stated otherwise, the chosenherbicide was metribuzin (Sencor WG, Bayer, Milano,Italy) which is active against C. album at the recom-

ended rate (105 g a.i./ha). The herbicide was used byhe spraying of 5 ml of a solution containing 0.042 mg.i./ml to a group of four pots or to one tray. Thisorresponded to about 1/5 of the reccomended rate withvolume of 500 liter of water/ha. In the experiment

omparing two different herbicides, as described below,imsulfuron (Titus, Du Pont de Nemours Italiana, Mi-ano, Italy), which is reported to be nearly ineffectivegainst C. album, alone and in combination with me-ribuzin, was also used, at the rate described below.

Plants. For greenhouse experiments, plants wererown in pots (four plants in each pot) in a greenhouseo the fourth-leaf stage at 20–25°C and under naturalight conditions. For each treatment, four replicationsfour pots, each containing 4 plants) were used. For theemi-field experiment, plants were grown in a green-ouse in seedling trays (228 plants in each tray) andransferred to the field 1 day before treatment, whenhey were at the two- to four-leaf stage. This allowed aigh percentage of seed germination and a very uni-orm plant height. Three trays were used for eachreatment.

pplication Procedures

Application of the solutions/suspensions was madeith a hand sprayer. For treatments including botherbicide and toxins and/or spore applications, the her-icide solutions were applied first and allowed to dryor 15–30 min, followed by the spore/toxins application.

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184 VURRO ET AL.

For greenhouse experiments, plants were treated andkept under high-moisture conditions (85–95% relativehumidity) overnight and then kept under greenhouseconditions as described above.

Greenhouse and Semi-Field Experiments

Comparison of herbicidal activity of culture filtrate/mixture of metabolites. Efficacy of the culture filtratewas compared to that of the purified mixture of toxins,both alone and with the spore suspension. There werea total of six treatments. Fresh and dry weights ofepigeous plant parts were measured 7 days after treat-ment.

Use of different doses of toxins. The mixture of tox-ns was used at the standard concentration (1 mg/ml)nd at two lower concentrations (1/5 and 1/10), corre-ponding to 0.2 and 0.1 mg/ml, in combination withpore suspension and the herbicide at the low dose.here were 13 treatments. Fresh and dry weights ofpigeous plant parts were determined 7 days afterreatment.

Use of toxins with different herbicide treatments.he influence of toxins and spores with different re-uced-rate herbicide treatments was determined in areenhouse experiment. Three herbicidal treatmentsere used at a 1/5 rate with respect to the labeled rate:

imsulfuron (2.5 g a.i./ha), metribuzin 1 rimsulfuron(10.5 1 1.25 g/ha), and metribuzin (21 g/ha). Labeledrates were also applied, for a total of 19 treatments.Five milliliters of the herbicidal solution and/or 10 mlof the toxins solution/spore suspension were applied toeach treatment. Plant stems were cut at the soil sur-face and fresh and dry weights were determined.

Influence of time of treatment on herbicidal efficacy oftoxins. The potential for synergistic effects of the dif-ferent components of treatments and the influence ofplant age on efficacy were determined by the testing ofthe mixture of toxins and the herbicide at the reducedrate simultaneously on plants at the four-leaf stage(time zero) and by the application of one agent 3 or 6days after the application of the other. One week afterthe last treatment, fresh and dry weights of epigeousplant parts were recorded.

Semi-field Experiment 1. The plants in trays (threefor each treatment) were sprayed with spore suspen-sion, toxins, and the herbicide at the reduced rate(metribuzin at the concentration of 21 g a.i./ha) for atotal of eight treatments. The application was carriedout late in the afternoon to exploit the higher humidityduring the night. Trays were watered as needed afterthe first day and kept in the field, and 2 weeks aftertreatments a visual estimation of the herbicidal effectwas scored, according to the standard of the EuropeanWeed Research Sociey (1 5 no effect; 9 5 maximumefficacy). Plants were then cut at the soil surface andfresh and dry weights were determined.

Semi-field Experiment 2. Considering the quite lowactivity of the toxin at the standard dose in the firstsemi-field experiment, a second experiment was car-ried out, using the toxins at a concentration five timeshigher, with two different herbicide treatments at areduced rate. The plants in trays (four for each treat-ment) were sprayed with toxins (5 mg/ml) and twoherbicides at a 1/5 rate with respect to the labeled rate:metribuzin (21 g a.i./ha) and rimsulfuron (2.5 g a.i./ha),with a total of six treatments. Five milliliters of theherbicidal solution and/or 10 ml of the toxins solutionwere applied to each treatment. The application wascarried out late in the afternoon to exploit the higherhumidity during the night. Trays were watered asneeded after the first day and kept in the field. Twoweeks after treatments, the plants were cut at the soilsurface and fresh and dry weights were determined.

Statistical Analysis

The data were analyzed by analysis of variance andthe means were compared by Duncan’s multiple rangetest.

RESULTS

The method used to purify the mixture of toxins wasrelatively easy to use and inexpensive, and it quicklyyielded large amounts of the metabolites. The lyophi-lized material, containing three main metabolites inalmost pure form, was a highly water-soluble, yellow-ish powder that could be easily sprayed as a solution.This mixture contained ascaulitoxin, its aglycone, and

FIG. 1. Herbicidal effect of Ascochyta caulina culture filtrate andof purified toxins alone or in combination with the fungus on thefresh weight of Chenopodium album shoots. Control, untreatedplants; F, fungal spores (106/ml); T, toxins (1 mg/ml); CF, culturefiltrate. Plants were treated at the fourth-leaf stage. Values denotedby different letters are significantly different at P 5 0.01.

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185EFFICACY ENHANCEMENT OF THE MYCOHERBICIDE Ascochyta caulina

trans-4-aminoproline approximately in a 6:8:1 ratio byweight.

Comparison of Herbicidal Activities of CultureFiltrate/Mixture of Metabolites

All treatments caused significant effects compared tothe control (Fig. 1). In particular, the pure toxins andthe culture filtrate strongly enhanced the herbicidalefficacy of the fungus. The effect of the toxins wascomparable to that of the culture filtrate, when used

FIG. 2. Effect of application of a mixture of toxins (1 mg/ml) on C.album plants grown in greenhouse in pots (right). Control plants areon the left. The picture was taken 7 days after the plants weresprayed.

FIG. 3. Influence of purified toxins at different doses, in combalbum growth. Control, untreated plants; T, toxins (1, 1 mg/ml; 2educed rate (0.042 mg a.i./ml). Plants were treated at the fourifferent at P 5 0.01.

alone or in combination with the fungus. The advan-tages of use of the purified toxins instead of the culturefiltrates include easier handling, possibility of use ofprecise doses, complete water solubility, and an abilityfor longer storage. For these reasons, all subsequentexperiments were carried out with the mixture of me-tabolites, at the same concentration usually found inthe culture filtrate.

Use of Different Doses of Toxins

Best results were achieved with the toxins, at thestandard dose (1 mg/ml) (Fig. 2) or at a lower dose (0.2mg/ml) in combination with a reduced dose of metribu-zin (1/5 of the labeled rate) (Fig. 3). Compared to theuntreated control, the toxins used at a lower concen-tration with respect to the standard dose caused about66% fresh weight reduction, herbicide alone causedaround 70% reduction, and the combined treatmentcaused more than 90% reduction (Fig. 3).

Use of Toxins with Different Herbicide Treatments

Reductions in fresh weight were seen when fungalspores and/or toxins were used in combination withrimsulfuron at the reduced rate of 2.5 g a.i./ha. In fact,rimsulfuron is weakly effective on C. album, as shownby the treatment at the labeled rate, which reduced theplants’ fresh weight by about 60% compared to that ofthe untreated control (Fig. 4). Otherwise, when thisherbicide was used at the reduced, ineffective rate in

tion with fungal spores and a herbicide at a reduced rate, on C.2 mg/ml; 3, 0.1 mg/ml); F, fungal spores (106/ml); H, herbicide atleaf stage. Values denoted by different letters are significantly

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186 VURRO ET AL.

combination with the toxins or the fungus, an effect asgood as or better than the labeled rate was achieved.An analogous result was observed when rimsulfuronand metribuzin were used simultaneously in combina-tion with toxins or fungus.

Influence of Time of Treatment on Herbicidal Efficacyof Toxins

Maximum growth reduction (fresh weight reduction)was obtained when the toxins and the herbicide atreduced rate (21 g a.i./ha were used (Fig. 5). This treat-ment gave better growth reductions than the toxins orthe herbicide alone. Treatment of plants with singleagents 3 or 6 days after the time zero, when the plantswere larger, resulted in low efficacy, with negligiblereductions in plant growth compared to the control.Conversely, if at least one of the two agents (toxins orherbicide at low dose) were sprayed at time zero, theefficacy of the treatment was comparable to the bestresult obtained (toxin 1 herbicide at time zero).

emi-Field Experiment 1

In this experiment, all treatments except the tox-ns alone were significantly different from the controlFig. 6). Toxins increased the efficacy of the herbicide21 g a.i./ha), causing a reduction of about 10% of

FIG. 4. Herbicidal effect of A. caulina toxins, fungal spores, andControl, untreated plants; T, toxins (1 mg/ml); F, fungal spores ((10.5 1 1.25 g a.i./ha); H3, Metribuzin (21 g/ha). H1t, H2t, and H36.25, and 105 g/ha, respectively). Plants were treated at the foudifferent at P 5 0.01.

plant weight compared to the control. Plant growthwas substantially reduced when the fungus was ap-plied alone or in combination with herbicide or tox-ins, although the best results were obtained when allthree ingredients were applied in combination. Thereduced efficacy of combined treatments, with re-spect to the single fungal application, could be due tothe weather conditions that were very favorable forthe fungus, whose activity may have concealed thoseof the other agents. Analogous results have beenobtained by visual estimation (data not shown), withthe best herbicidal activity obtained using a combi-nation of all three agents (Fig. 7).

Semi-Field Experiment 2

In this experiment toxins alone applied at 5 mg/mlwere able to reduce plant weight by 30%, whereas bothherbicides at reduced rate were ineffective. Plantweight reduction of more than double that caused bythe toxins alone was obtained when the toxins weresprayed in combination with metribuzin or rimsulfu-ron at a 1/5 of the labeled rate (Fig. 8).

DISCUSSION

The use of compounds that could weaken physicaland biochemical defences of the plant, or increase the

duced rates of herbicides on C. album plants grown in greenhouse.6/ml); H1, rimsulfuron (2.5 g/ha); H2, metribuzin 1 rimsulfuronefer to reccomended labeled doses of the treatments (12.5, 52.5 1-leaf stage. Values denoted by different letters are significantly

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187EFFICACY ENHANCEMENT OF THE MYCOHERBICIDE Ascochyta caulina

FIG. 5. Influence of treatment time on herbicidal efficacy of fungal toxins in combination with a chemical herbicide at a reduced rate.ontrol, untreated plants; T, toxin (1 mg/ml); H, herbicide (metribuzin at the reduced rate of 0.042 mg a.i./ml). 3d and 6d, mean applicationsand 6 days after the beginning of the experiment. Plants were treated at the fourth-leaf stage. Values denoted by different letters are

ignificantly different at P 5 0.01.

FIG. 6. Growth of C. album plants exposed to different treatments with fungal toxins, spores, and herbicide at a reduced rate in asemi-field experiment. Plants were seeded and grown in transplanting trays (228 plants in each tray) and kept in a greenhouse until theiruse. Control, untreated plants; T, toxin (1 mg/ml); F, fungal spores (106/ml); H, herbicide (metribuzin at the reduced rate of 21 g a.i./ha).Plants were treated at the fourth-leaf stage. Values denoted by different letters are significantly different at P 5 0.01.

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188 VURRO ET AL.

aggressiveness of the pathogen, as a tactic to increasethe efficacy of pathogens used for weed control hasalready been considered. Among these, the applicationof sublethal doses of herbicides to enhance the efficacyof biocontrol agents has been widely considered. Forexample, low doses of imazaquin in combination withAlternaria zinniae Pape ex M.B. Ellis increased theefficacy of the fungus to control Noogoora burr (Xan-thium occidentale Bertol.) and restricted the plant’sability to recover after fungal application due to theherbicide’s ability to interfere with protein synthesis(Auld et al., 1997). A soil-borne fungus, Fusarium so-ani (Mart.) App. & Wr. f. sp. cucurbitae combined with

trifluralin controlled Texas gourd [Cucurbita texana(Scheele) Gray] better than single treatments with thefungus or the herbicide, showing a synergistic activity(Weidemann and Templeton, 1988). In our experi-ments, a synergistic effect of spores with the herbicideseemed to occur. In fact, metribuzin sprayed at a sub-lethal dose improved fungal efficacy in terms of reduc-tion in plant growth. Also, A. caulina toxins used incombination with the pathogen had a positive effect onfungal activity. As already observed (Zonno et al.,1998), usually the first symptoms appeared on plantsafter only 1–2 days in the case of simultaneous appli-cations, whereas they appeared more slowly when thepathogen was used alone. This faster colonization ofplant tissues by the pathogen could also render thepathogen less dependent on environmental conditionsthat are usually limiting factors to the practical use ofmycoherbicides. Furthermore, as already described for

with fungal toxins and herbicides at reduced rate, in a semi-fieldlants in each tray) and kept in a greenhouse until their use. Control,g a.i./ha); H2, rimsulfuron at reduced rate (2.5 g a.i./ha). Plants weresignificantly different at P 5 0.05.

FIG. 7. Effect of combined treatment of toxins, fungal spores,and a chemical herbicide (metribuzin) at the reduced rate of 21 ga.i./ha on plants grown in transplanting trays in a greenhouse. Aftertreatment, the trays were kept outside the greenhouse under fieldconditions. Treated tray is at the bottom and the control is at the top.

FIG. 8. Growth of C. album plants exposed to different treatmentsexperiment. Plants were seeded and grown in transplanting trays (228 puntreated plants; T, toxin (5 mg/ml); H1, metribuzin at reduced rate (21treated at the fourth-leaf stage. Values denoted by different letters are

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189EFFICACY ENHANCEMENT OF THE MYCOHERBICIDE Ascochyta caulina

A. caulina (Kempenaar, 1995), plant age plays an im-portant role in toxin efficacy. In fact, the application ofthe fungus and the toxins on plants over the two- tofour-leaf stage results in a reduction of their efficacy.

Secondary plant metabolism (phenylpropanoid path-way) is a major biochemical pathway related to severaldefence processes in plants. Increased activity of phe-nylalanine ammonia-lyase (PAL), a key enzyme of thispathway, in response to pathogen attack, was demon-strated by Hoagland (1990). Recently, Vurro and Ellis(1997) showed that some fungal toxins, applied at con-centrations causing no macroscopic toxic effects, wereable to suppress the induction of PAL. Thus, a possiblemechanism of action of the A. caulina toxins could bethe interference with or the inhibition of PAL induc-tion, causing an increased susceptibility of plants tothe pathogen. This could lead to a new strategy in useof toxins, for example at low doses, mixed with othermycoherbicides to help their herbicidal properties. Inthis regard, interesting results have been achievedwith the chemical herbicide glyphosate, which wasshown to greatly help Alternaria cassiae Jurair &Khan in causing disease on Senna obtusifolia (L.) Irwin& Barnaby, when used in combination with the patho-gen (Sharon et al., 1992). In fact, this herbicide sup-presses plant defence by lowering phytoalexin produc-tion, which is otherwise stimulated by the pathogenduring its infection (Sharon and Gressel, 1991).

The availability of each of the three toxins of A.caulina would permit better determination of their usein biological and integrated management strategies. Infact, previous observations, using a leaf-puncture as-say, have shown that ascaulitoxin is active at differentlevels on a wide array of plant species (Evidente et al.,1998), whereas trans-4-aminoproline is ineffective onmonocots (Evidente et al., 2000) and the aglycone was

ore active than its analog (A. Evidente et al., unpub-ished). In preliminary experiments, when the mixturef toxins was applied on different weed and crop spe-ies, a diverse range of activity, from no toxicity (e.g.,orn and beet) to very high activity (e.g., Senecio sp.),as seen (M. Vurro et al., unpublished).With regard to the relationship of the fungus or

ungal toxins with herbicides, the herbicidal mecha-ism of action does not appear to be crucial for im-rovement in activity. In fact, the toxins were effectivehen used with metribuzin, which acts as an inhibitorf photosynthesis at the level of photosystem II, andith rimsulfuron, which is an aceto-lactate synthase

nhibitor. The strong improvement of the efficacy ofimsulfuron, which is nearly ineffective against C. al-um when used at the labeled concentration, couldave an interesting practical application in terms ofanagement of herbicide resistance. Furthermore, ex-

loration of toxin activity could expand the action spec-rum of herbicides or biocontrol agents.

Considering that these toxins were able to promoteisease development even when used at a concentra-ion 20 times lower than the standard dose (1 mg/ml),t should be possible to optimize the toxin concentra-ion in the spray solution, reducing the amount needednd thus increasing efficacy. Also, studies to optimizehe coformulation of the toxins with living agentsould contribute to increased control efficacy.The amount of toxins used in the experiments is

uite low (around 500 g/ha) and comparable to theoses of some synthetic chemical herbicides currentlyn use. This is further support to the practical use ofoxins to supplement biocontrol agents. The use of aigher concentration of the toxin (e.g., 2500 g/ha) couldnsure a better effect in the field. Another importantspect for an eventual practical use of the toxins isheir production in large amounts at low cost. Accord-ngly, studies are in progress both to improve microbi-logical production with different strains/fermentationystems and to chemically synthesize the phytotoxins.

ACKNOWLEDGMENTS

This investigation was supported in part by grants from the Eu-ropean Project FAIR5-CT97-3525 entitled “Optimizing BiologicalControl of a Dominant Weed in Major Crops” and in part by grantsfrom the Italian Ministry of University and Scientific TechnologicalResearch. The authors thank Mr. Cesare Lasorella and Mr. MarianoFracchiolla, University of Bari, for technical support and statisticalanalysis, respectively.

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