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Biological Control 21, 274–284 (2001)doi:10.1006/bcon.2001.0934, available online at http://www.idealibrary.com on

Isolation, Identification, and Activity of Mycoherbicidal Pathogensfrom Juvenile Broomrape Plants

Z. Amsellem,* Y. Kleifeld,† Z. Kerenyi,‡ L. Hornok,‡ Y. Goldwasser,† and J. Gressel*,1

*Department of Plant Sciences, Weizmann Institute of Science, Rehovot IL-76100, Israel; †Department of Weed Science, Newe YaarResearch Center, Ramat-Ishay, Israel; and ‡Plant Sciences, Agricultural Biotechnology Center (ABC), Godollo, Hungary

Received August 21, 2000; accepted March 1, 2001; published online May 9, 2001

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Although there are reports of isolation of mycoher-bicidal pathogens attacking the widespread broom-rapes (Orobanche spp.) that parasitize legumes and

egetables, none is in use or available. This is despitehere being no good method of controlling broomrapesn most crops other than by preplant fumigation with

ethyl bromide. Two highly parasitic fungi, Fusariumrthrosporioides strain E4a (CNCM I-164) and F. oxy-porum strain E1d (CNCM I-1622), were isolated fromearly 100 organisms found on diseased, juvenile,merging Orobanche flower stalks. A near-axenic poly-thylene envelope system for culturing broomrape onomato roots was used to ascertain pathogenicity ofhese strains. Both organisms fulfilled Koch’s postu-ates for being primary pathogens. Their DNAs werenalyzed and fingerprinted by restriction fragmentength polymorphism and random amplified polymor-hic DNA, showing that they are indeed different fromach other and from many other Fusarium spp. andther formae speciales of F. oxysporum including atrain that attacks O. cumana on sunflowers. Bothtrains infect O. aegyptiaca, O. cernua, and O. ramosa,ut not O. cumana. They did not infect any of theegetable and legume crops tested and thus seem spe-ific to Orobanche. Tomato plant roots dipped into aungal spore and mycelial suspension and planted inroomrape-infested soil were protected for 6 weeks, asere tomato transplants in pot experiments. About

0% control was also achieved by posttransplant soilrench with fungal suspensions in pot experiments.hese pathogens may be effective as seed, transplant,r soil-drench treatments of high-value vegetable andther crops. © 2001 Academic Press

Key Words: Fusarium arthrosporioides; Fusarium ox-ysporum; mycoherbicides; microbial herbicides; Oro-banche aegyptiaca; parasitic weed control; RAPD;RFLP.

1 To whom correspondence should be addressed. Fax: (1972-8)934-4181. E-mail: Jonathan.Gressel@weizmann.ac.iL.

2741049-9644/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

INTRODUCTION

The parasitic broomrapes (Orobanche spp.) anditchweeds (Striga spp.) are widespread weeds, on av-

erage halving yields on ca. 4% of the world’s crops(Sauerborn, 1991). As these plants attach to crop roots,they cannot be controlled mechanically, except by re-moval of their flower stalks. They are not usually ame-nable to season-long control by selective herbicides,except those used postemergence. Chemical or manualcontrol of flower stalks prevents reseeding, but most ofthe damage to the current crop has been done by thattime, as most systemic herbicides cannot differentiatebetween the crop and the parasitic weed. Repeatedapplications of very low rates of some herbicides havebeen effective (Kleifeld et al., 1996; Hershenhorn et al.,998). The use of biotechnologically derived, herbicide-esistant crops may somewhat alleviate this problemith both Orobanche spp. (Joel et al., 1995; Surov et al.,

1997) and Striga spp. (Abayo et al., 1998) until herbi-cide resistance evolves, which can be rapid to somesystemic herbicides (Gressel et al., 1996).

Biological control could be an effective answer. Theinsect Phytomyza orobanchiae Kalt. (Diptera: Agromy-zidae) consumes broomrape flowers and seeds (Tawfiket al., 1976; Linke et al., 1990), but only after it is toolate to save the current crop. Also, the seed output isreduced by only about 30%, and it has been calculatedthat a biocontrol agent would have to lower seed outputby .95% to have 50% reduction of parasite densitySmith and Webb, 1996).

Mycoherbicidal organisms that might be useful forontrol of Orobanche have been isolated (Hodosy andornok, 1983; Al-Menoufy, 1986; Bedi and Donchev,991; Linke et al., 1992; Thomas et al., 1998, 1999),owever, we found that the previously reportedroomrape-attacking pathogens on vegetable cropsere not available (Al-Menoufy, 1986) and other col-

ections (Linke et al., 1992) had perished (J. Sauer-orn, University of Hohenheim, Stuttgart, Germany,

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275MYCOHERBICIDAL PATHOGENS OF BROOMRAPE

personal communication). This suggests that theprevious isolates may not have been sufficiently ac-tive for field use. There has been some progress onbiocontrol with pathogens of O. cumana Wallr. onsunflower, and current progress by groups workingon biocontrol of Orobanche spp. has recently beenreviewed (Thomas et al., 1998, 1999; Amsellem et al.,2000).

As Orobanche spp. are major problems in Israel andthroughout the Mediterranean basin, we decided toascertain whether Orobanche possesses defense mech-anisms active against pathogens that could be over-come to enhance its susceptibility. Our group has pre-viously used this approach to find synergists thatwould enhance the pathogenicity of other mycoherbi-cidal organisms (Sharon et al., 1992; Gressel et al.,997; Watson et al., 2000). We had wished to compare

local isolates with the pathogens isolated elsewhereunder controlled-isolation conditions, to ascertain theirrelative phytotoxicities.

We decided not to use the type of protocols typi-cally used to find pathogens, i.e., from diseased, ma-ture, flowering broomrape stalks, often after seedset, for two reasons: (a) if such pathogens are flower-stalk specific, they will decimate the parasite onlyafter much damage has been inflicted on the crop and(b) it is typical of diseased lesions to contain manysecondary, saprophytic organisms often to the pointwhere the primary pathogen is no longer present oris difficult to isolate. The more mature the lesion, thegreater the proportion of secondary pathogens thatcan be expected. Instead, we chose to isolate poten-tial pathogens from the youngest possible infectedOrobanche shoots (Fig. 1) or tubercles, to isolate thelargest proportion of primary pathogens. Serendipi-tously, our best isolates were found in a melon fieldwhere heavily broomrape-infested crops had previ-ously been cultivated. Melons can be heavily para-sitized by broomrape but the damage is usually min-imal (Jacobsohn et al., 1991). The broomrape that did

arasitize melon plants was probably naturallyeakened by the defense system of the crop, render-

ng the broomrape more susceptible to fungal attack.ven with such procedures, only 2 of nearly 100ingle-organism isolates from these diseased broom-ape plants met the criteria of Koch’s postulates foreing primary pathogens: they infected when inocu-ated as single organisms, they could be reisolated asingle organisms, the reisolates were identical to thearent isolates, and they could reinfect Orobanche.e report here the isolation of these two strains,

ow they could be differentiated from other relatedrganisms, and their use in model greenhouse testso show how the mycoherbicidal pathogens cane used to protect crop transplants from Orobanchepp.

MATERIALS AND METHODS

Selecting Diseased Material

Diseased broomrape plants were collected frommelon, peanut, tomato, and chickpea fields in the northof Israel in 1994. We surveyed crop fields previouslyinfested with broomrape and searched for places wherethe broomrape was smallest or where the just-emergedbroomrape clearly appeared diseased. The two isolatesdescribed below were both isolated in a melon fieldinfested by Orobanche aegyptiaca Pers. (Egyptianroomrape). This field had a very large broomrapeeedbank due to massive broomrape infestations ofrops during previous years. The just-emerging flowertalks on the broomrape plants, which were easily sep-rated from their hosts due to a soft rot near thettachment site (Fig. 1), were collected.

ungal Isolation

Diseased tubercles and juvenile broomrape stalksere cut into 3- to 4-mm pieces, surface-sterilized by

mmersion in 1% sodium hypochlorite solution for 4in, and rinsed three times with sterile glass-distilledater. Three such pieces were placed in each 9-cm-iameter plastic petri dish containing “Original Mar-in’s” medium (Rechcigl, 1978) with 155 mM chloram-henicol added to prevent bacterial growth. Ten daysater, single-organism isolates growing as fungal hy-hae out of the broomrape pieces were subcultured onotato glucose agar (Rechcigl, 1978), also containinghloramphenicol. Single-spore isolates of each strain

FIG. 1. Diseased (right) and healthy (left) young Orobanche sp.lants, at the stage of development where collections for putativeathogens were performed.

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276 AMSELLEM ET AL.

were subcultured, and one of the resulting colonies wasused to inoculate several dishes.

Pathogenicity Testing

The pathogenicity of each isolate was tested semiax-enically as follows with the polyethylene envelope sys-tem of Parker and Dixon (1983), as modified for Oro-banche by Daniel M. Joel of the Newe Yaar ResearchCenter (unpublished). A 12 3 28-cm piece of WhatmanGFA glass fiber sheet was placed through an openingflap of 12 3 23 cm, in each 24 3 34-cm, thick-polyeth-ylene file envelope. These were mounted with a file-hanging rod in a frame wrapped with black plastic toprevent light penetration to the roots. Cellulose-con-taining paper cannot be used as the backing and cap-illary source of medium, because cellulose can be de-graded by cellulolytic microorganisms, releasing sug-ars for growth of secondary organisms. Each GFAsheet was wetted with 40 ml of a modified Hoagland’ssolution (final concentrations: 0.5 mM NH4H2PO4, 3mM KNO3, 2 mM Ca (NO3)2, 1 mM MgSO4, 90 mMFeEDTA, 46 mM H3BO3, 9 mM MnCl2, 0.8 mM ZnSO4,0.32 mM CuSO4, 0.016 mM (NH4)6Mo7O24, with the pHadjusted to 5.6).

Seeds of O. aegyptiaca (and in some experiments O.ramosa L. and O. cernua Loeffl.) in small bags formedof Miracloth (Calbiochem, La Jolla, CA) were wettedand surface-sterilized by immersion in 80% ethanol for1 min, followed by 10 min in a mixture of 1% sodiumhypochlorite in 0.01% aqueous Tween 20 (polyoxyeth-ylenesorbitan monolaurate). Seeds were rinsed threetimes in sterile, glass-distilled water and dried over-night at room temperature. About 1500 seeds weredispersed over the wetted GFA sheet in each envelope.The broomrape seeds were allowed to afterripen for6–7 days in the dark at room temperature. Tomatoplants (Lycopersicon esculentum Mill. cv. M82) bearingtwo to three primary leaves grown as transplant plugsin Speedling Insert Trays (kindly provided by HishtilLtd., Ashkelon, Israel) were washed of potting mixture.The plastic flaps on the polyethylene envelopes werethen opened and planted with tomato plants and re-closed.

The plants in the polyethylene file envelopes werekept in a growth chamber at 25°C under 100 mEin/m2/sfluorescent light during a 14-h photoperiod. Thebroomrape seeds germinated, attached to the tomatoroots, and formed small tubercles during the following2 weeks (Fig. 2). These were then sprayed with theputative pathogens as follows. Mycelia with sporeswere scraped with a scalpel from two or three petridishes of each fungal isolate and homogenized in amotor-driven glass/glass conical homogenizer in 10 mlsterile glass-distilled water. The homogenates contain-ing spores and fragmented, viable, hyphal pieces werediluted to 50 ml in 0.02% Tween 20, the flaps of thepolyethylene file envelopes were opened, and 8–12 ml

of this suspension was sprayed on the roots of eachtomato plant with an atomizer (50 mg hyphal piecesand 106 spores/ml). Control plants were mock-sprayedwith sterile glass-distilled water containing 0.02%Tween 20. Some putatively infected tubercles were re-moved; the fungus in them was isolated, retested, re-isolated, and compared to the strain used, according toKoch’s postulates. In experiments in which sporesalone were used, there were four to six replicates ofeach organism, and a glass rod was used to lightlyscrape off spores from the mycelia in the petri dishes.The spores were filtered through sterile Nytex, 100-mm-mesh monofilament–nylon bolting cloth (SwissSilk Bolting Cloth, Zurich, Switzerland), the myceliaon the filters were washed three times with sterileglass-distilled water to force the remaining sporesthrough the filter, and the spores were centrifuged andresuspended in 0.02% Tween 20. Each spore concen-trate was then counted with a hemocytometer anddiluted to the desired concentrations.

Measurement of Pathogenicity

Initially, the fungus-treated roots in the polyethyl-ene envelopes were observed at 2-day intervals forvisual changes in tubercle growth and appearance,compared to controls (Fig. 2). In later experiments withclearly pathogenic strains, tubercle growth was moni-tored at 1- to 2-day intervals by measurement of tuber-cle diameter with calipers. Some experiments wereterminated when the tubercles were excised andweighed.

Pathogen Classification

The two pathogenic strains that fulfilled Koch’s pos-tulates were taxonomically classified as Fusarium spp.n the basis of conidial morphology and then comparedith related strains by restriction fragment lengtholymorphism (RFLP) and random amplified polymor-hic DNA (RAPD) at the Institute for Plant Sciences,gricultural Biotechnology Center (ABC), Godollo,

Hungary. Their identifications were made after cultureon potato–sucrose medium and water agar with straw,according to Booth (1971).

The Fusarium strains compared with the above-mentioned strains in RAPD analysis and DNA finger-printing were: F. poae (Peck) Wollenw., TAPO-1, F.oxysporum (Schlecht.: Fr. f.) sp. pisi (J. C. Hall) W. C.Snyder & H. N. Hans., 0294 (ABC isolate), F. oxyspo-rum Schlect.: Fr. f. sp. lycopersici (Sacc.) W. C. Snyder& H. N. Hans, IMI 141 140 (from the InternationalMycological Institute, Egham, England, UK), and F.oxysporum f. sp. glycines, F331 (from the Institute forPlant Genetics, PAS, Poznan, Poland). F. oxysporumSchlecht.: Fr. f. sp. orthoceras Appel & Wollenw. whichis pathogenic to O. cumana attacking sunflowers (Bedi,994) was kindly supplied by J. Sauerborn, Universityf Hohenheim, Stuttgart, Germany.

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277MYCOHERBICIDAL PATHOGENS OF BROOMRAPE

RFLP Probing

Total DNA was extracted as described earlier(Fekete et al., 1995). DNA samples were digested withBglII, EcoRI, and HindIII (Amersham International,Amersham, UK); fragments were separated by electro-phoresis in 0.8% agarose gels. The gels were trans-ferred onto Hybond-N nylon membrane as recom-mended by the manufacturer (Amersham). A 2.3-kbEcoRI–BamHI fragment of the rRNA gene from As-pergillus nidulans R. A. Samson & W. Gams (donatedy A. J. M. Debets, Department of Genetics, Agricul-ural University, Wageningen, The Netherlands) wasabeled with [a32P]dCTP by the Multiprime Labeling

system (Amersham) and used as a probe to revealrestriction fragment length polymorphisms withinrRNA sequences of the Fusarium strains.

RAPD Analyses

Three 10-mer oligonucleotides of a RAPD primer kitobtained from Operon Technologies (Alameda, CA)

FIG. 2. A semiaxenic system for screening putative pathogensected; (B) infected with Fusarium arthrosporioides strain E4a; (C) in the polyethylene envelope system after its roots were washed of potith the pathogenic fungi. The protocols of seeding Orobanche, plantiaterial and Methods.

were chosen. Nucleotide sequences of these primerswere OPE-01 (CCCAAGGTCC), OPE-14 (RGCGGCT-GAG), and OPE-15 (ACGCACAACC). Amplification re-actions were carried out in 25 ml containing 50 ngFusarium DNA, 10 pM primer, 50 mM each dCTP,dGTP, dATP, and dTTP (Boehringer, Mannheim, Ger-many), 1 unit of Taq DNA polymerase (Amersham),and 2.5 ml Amersham buffer under a drop of mineraloil. A Cetus thermal cycler (Perkin-Elmer, Norwalk,CT) was used for amplification. The cycling consisted ofinitial denaturation at 94°C, for 4 min, 35 cycles of 1min at 94°C, 1 min at 38°C, 1 min at 72°C, and a finalincubation at 72°C for 7 min. After polymerase chainreactions (PCR), 8-ml aliquots of the products wereseparated by electrophoresis in 1.2% agarose gels. Gelswere stained with 0.5 mg/ml of ethidium bromide, vi-sualized, and photographed with 312 nm UV illumina-tion. RAPD patterns were assessed by the scoring of 1for the presence and 0 for the absence of the bands thatregularly appeared at the same position in repeatedruns. Similarity coefficients, Sxy values (McDonald and

activity as primary mycoherbicides on tomato seedlings. (A) Unin-cted with F. oxysporum strain E1d. Arrows: (a) tomato plant growng mixture; (b) healthy tubercle; (c) dead tubercles after being sprayedtomatoes, and spraying with fungal suspensions are described under

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278 AMSELLEM ET AL.

Martinez, 1991), were calculated between the recentlyisolated E1D Orobanche-attacking strain of F. oxyspo-rum and the other reference strains of this fungus.

Isolation Greenhouse Experiments

Small pot experiments. A soil mixture of peat:tuff-rock:loam (1:1:1) was uniformly mixed with 5000 sur-face-sterilized O. aegyptiaca seeds per liter of soil andput into 150-ml pots, four replicates per treatment.Fungal biomass was prepared as described earlier(Amsellem et al., 1999), and suspensions were pre-

ared for both fungal strains containing 10 g of myce-ia, homogenized for 60 s at high speed with an Ultra-urrax homogenizer (Janke & Kunkel, Staufen I,reisgau), and 109 spores in 1 liter 0.02% Tween 80. A

mixture was also prepared containing 5 g per litermycelia and 0.5 3 109 spores of each fungal strain. Aolution of 0.02% Tween 80 was used as a control.omato transplant plugs were directly pretreated withhe pathogens by dipping the whole plug in the sus-ension of the pathogens and planting in the O. aegyp-iaca-infested pots. Two more drenches of 50 ml of thebove suspensions were applied to each pot, 2 and 3eeks after transplanting, each followed by heavy wa-

ering. The pots were held in the greenhouse at 25°Cay (14 h) and 22°C night (10 h) for 2 months.Large pot experiments. Clay–loam soil (58% clay,

3% silt, 19% sand, 2% organic matter) was uniformlyixed with O. aegyptiaca seeds as above and put into

0-liter pails, five replicates per treatment. Each potas drenched with a 250-ml suspension (concentra-

ions as above), 3 weeks after transplantation of plugsrom Speedling Insert Trays (kindly supplied by Hish-il Ltd., Ashkelon, Israel) of celery (Apium graveolens. cv. Tender Crisp), pepper (Capsicum annuum L. cv.ehava), and tomato (Lycopersicon esculentum cv. Bri-

gade) or direct seeding of carrot (Daucus carota L. cv.Nantes), chickpea (Cicer arietinum L. cv. Ayelet),melon (Cucumis melo L. cv. Galia), sunflower (Heli-anthus annuus L. cv. Iregi), and vetch (Vicia sativa L.cv. Yovel). Drenching was followed by heavy watering.The pots were held in a heated greenhouse, 25°C dayand 22°C night for 3 months.

TAB

Suppression of Broomrape Tubercl

Added growth per tubercle (ml 6 SE/tube

Control FARTHb

7 days 115.9 6 32.5 12.1 6 7.514 days 126.1 6 33.0 27.9 6 5.6

a The tubercle volumes just before spraying were 66.1 6 13.0 ml peerfect spheres at that stage. Averages (6SE) of infestation on fives appearing solid and white and not discolored or black. Control pl, and 14 days, respectively. The experiment was repeated twice wi

b FARTH, Fusarium arthrosporioides E4a; FOXY, F. oxysporum E

RESULTS AND DISCUSSION

Isolation and Taxonomic Classification

Nearly 100 single-organism isolates were obtainedfrom diseased juvenile broomrape plants (Fig. 1) andtheir pathogenicity was ascertained. Only two of thesestrains (Fig. 2) proved to be pathogenic to Orobanche,meeting Koch’s postulates for being primary patho-gens. They were identified as Fusarium oxysporumSchlecht.:Fr. strain E1d (FOXY) and F. arthrospori-oides Sherb. strain E4a (FARTH), based on spore andother characteristics. These organisms strongly af-fected broomrape growth on tomato roots, killing 50–100% of the broomrape tubercles in different experi-ments over a 5-year period and severely inhibiting thegrowth of most of the remaining tubercles (Table 1).The pathogens had no visible effect on the tomatoplants. To the best of our knowledge, this is the firstreport of F. arthrosporioides attacking broomrapes.Many strains of F. oxysporum have been reported pre-viously to attack O. crenata Forsk. (Al-Menoufy, 1986;Linke et al., 1992), whereas F. oxysporum f. sp. ortho-ceras has been reported to attack O. cumana and therelated O. cernua (Bedi and Donchev, 1991; Bedi, 1994;

homas et al., 1998, 1999).Strains of both F. arthrosporioides strain E4a and F.

xysporum strain E1d had somewhat atypical sporesased on the full description below. The F. oxysporum1d was deposited with the Collection Nationale deultures de Microorganismes (CNCM), Institut Pas-

eur, Paris, under Accession No. I-1622, and the F.rthrosporioides E4a was deposited with the CNCMnder Accession No. I-1621. F. arthrosporioides Strain

E4a (FARTH) produces fast-growing colonies reaching8.0 cm in diameter on potato–sucrose agar after 4 days.The aerial hyphae are reddish-brown, and a deep redpigment diffuses into the agar. Sporulation is abun-dant, with two types of conidia present: microconidia 0to 1-septate, pyriform or slightly curved, with an aver-age size of 10–20 3 3.5–4.0 mm; macroconidia arefusoid, slightly curved with a F. gibbosum Wollenw.-like apical cell, 3- to 5-septate, and 25–50 3 4 mm insize. Pionnotal-like sporodochia are absent. This strain

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) Appearance of tubercles (% healthy)

OXYb Control FARTH FOXY

.8 6 5.9 100 6.3 56.3

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ubercle. The volumes were calculated assuming that tubercles wereix infested tomato plants are shown. Healthy tubercles are defineds bore an average of 3 6 1, 3 6 1, and 2 6 1 healthy tubercles at 0,imilar results. The data are from one experiment.

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279MYCOHERBICIDAL PATHOGENS OF BROOMRAPE

was thus identified as F. arthrosporioides. F. oxyspo-rum Strain E1d (FOXY) grows less rapidly; the colonydiameter after 4 days on potato–sucrose agar was 4.5cm. The aerial hyphae are pale beige. A grayish bluepigment diffuses into the agar after prolonged cultur-ing. Mostly microconidia are formed, in false heads,and these are oval, 0-septate, and 5–12 3 3.0 mm insize. Very few macroconidia are formed on tapwateragar with straw, and they are 3-septate and 25 3 3.5mm in size. The apical cells are rounded; chlamydos-pores are abundant, rough-walled, and globose, withan average diameter of 10 mm. This strain was thusidentified as a F. oxysporum.

Molecular Characterization of the Pathogens

The three oligonucleotide primers used for RAPDanalyses, OPE1, OPE14, and OPE15, clearly distin-guished between the two Orobanche-pathogenic Fusar-ium isolates and F. poae and from other formae spe-ciales of F. oxysporum (Fig. 3). F. arthrosporioides E4aand the reference isolate of F. poae both exhibitedstrikingly unique patterns, whereas the four strains ofF. oxysporum had some common and some uniquebands. Altogether, 65 RAPD markers were identified in

FIG. 3. RAPD differentiation of six Fusarium isolates with prim, F. poae TAPO-1; lane 4, F. oxysporum E1d; lane 5, F. oxysporum

phage DNA size markers are shown in lane 1 on left.

the four strains of F. oxysporum and their sizes wereestimated with a PstI-digested l phage ladder. TheseRAPD markers of these strains were grouped into amatrix, and the similarity indices (Sxy) were calculated(Table 2). The Sxy values determined for F. oxysporumE1d and three other formae speciales of this speciesranged from 0.68 to 0.74, demonstrating that the Oro-anche pathogen isolate E1D is only distantly relatedo the crop pathogenic strains of F. oxysporum. In a

separate experiment there was a small but clear dif-ference between F. oxysporum E1D and F. oxysporum f.sp. orthoceras. The Southern hybridization showedthree bands for f. sp. orthoceras and two bands forstrain E1D (Sxy 5 0.82), indicating that they are dif-erent, yet closely related (data not shown).

Total DNA samples from F. arthrosporioides E4a, F.oae TAPO-1, and three strains of F. oxysporum, E1d,331, and 0294, were digested with restriction endo-

ucleases, separated, blotted, and probed with a frag-ent of the rRNA gene from A. nidulans. Characteris-

tic RFLP fingerprint patterns for each strain were ob-tained in the BglII-digested samples (Fig. 4). Thisprovided further evidence for the genetic distinctnessof the two Orobanche pathogen strains from other

OPE-1, OPE-14, and OPE-15. Lane 2, F. arthrosporioides E4a; lane141 140; lane 6, F. oxysporum F 331; lane 7, F. oxysporum 0294; l

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280 AMSELLEM ET AL.

Fusarium species and formae speciales, as rRNA genesequences are considered to be among the most con-served regions of the genome. The conservative natureof this sequence was further supported by the findingof digestions with two other frequently used restrictionenzymes, EcoRI and HindIII. Still, as they revealed nopolymorphisms, they are of little use to ascertainsmall, interstrain differences.

Applications in Polyethylene Envelopes

Experiments were performed to ascertain the level ofspores (without mycelia) required to suppress broom-rape. Spore suspensions were sprayed on the attachedbroomrape tubercles ca. 12 days after broomrape at-tachment. Results measured 10 days later showed acontinuous dose–response curve with no apparentthreshold, which is atypical for fungal infections (Fig.5). Freshly prepared, chopped mycelia infected Oro-banche more quickly than conidia, with significant dif-ferences apparent after 1 day in the polyethylene en-velope system (Fig. 6). The maximum level of controlwas also much greater with chopped mycelia. In for-mulation experiments using dry Stabileze micropellets(Quimby et al., 1999) the chopped mycelia remainedviable much longer than spores from suspension cul-tures, allowing long-term storage of mycelia (Amsellemet al., 1999).

Washed roots of tomato transplants were dipped intoa spore and mycelial homogenate before being placedinto the polyethylene envelopes, to ascertain whetherthe fungus would continue to grow along the roots. Themaximum root length at that time was 7 cm. Oro-banche seeds that germinated and attached to roots 2weeks later and then formed tubercles were eventuallykilled by the fungus. Tubercles attached to roots as faras 15 cm from the original transplant plugs werekilled, suggesting that the fungus grew along the sur-face of the tomato roots, attacking later-infecting Oro-banche. This was validated by excision of root pieces ofyoung roots .20 cm from the area of inoculation, plat-ing of the pieces and culturing of the fungus on amedium, and demonstration that the fungus waspresent at those distances (data not shown). Addition-ally, germinating Orobanche seedlings were killed bythe fungus growing along the rhizoplane before attach-ing to young tomato roots (Fig. 7). The viable rootlets inthe controls were hyaline and hard to see and theinfected roots and the seeds were blackened by thefungi.

Experiments with Plants in Soil

Transplant plugs were dipped in a suspension ofmycelia and spores and planted in pots containing Oro-banche seeds. Additionally, control treatment (withoutspores) or chopped mycelial suspensions (with spores)were drenched into pots 2 and 3 weeks after planting.The predipping of the roots in a mycelial suspension

TABLE 2

RAPD Scores and Similarity Coefficients for Four Strainsof F. oxysporum

Primer RAPD marker

Fusarium oxysporum

E1d IMI 141 140 F331 0294

PE 14260 1 1 1 12780 1 0 1 12700 1 1 0 02480 1 0 0 02120 1 1 0 01970 1 0 1 01790 1 0 1 11630 1 0 1 01350 1 0 0 11220 1 1 1 11180 1 1 1 11050 1 0 0 0940 1 1 1 1910 0 1 0 0760 0 1 0 0650 0 1 1 1600 1 0 0 0490 1 0 0 0370 0 0 0 1270 1 1 1 1PE141190 0 0 1 12070 1 1 1 11630 1 1 1 01570 0 0 1 01500 0 1 0 01440 1 0 0 01380 1 1 1 11300 1 1 0 11220 1 1 0 01010 1 1 1 1860 1 1 1 1850 1 1 1 1790 1 1 1 1720 1 1 1 1690 0 1 0 1660 1 0 1 0610 1 1 1 1570 1 1 1 1500 0 0 1 0440 1 1 1 1380 1 0 0 0PE152630 0 0 1 02170 1 0 1 12070 1 1 1 01870 0 1 0 01710 1 0 0 01630 1 1 0 11470 0 1 0 11320 0 1 0 11180 0 1 0 01050 0 0 0 11010 1 1 1 0940 1 1 1 1850 1 0 0 1820 1 0 1 1760 1 1 1 1740 1 1 1 1690 1 1 1 1600 1 0 0 0540 1 1 1 1490 1 1 1 1470 1 1 1 1380 1 1 1 0370 0 0 0 1310 0 0 1 0

xy* — 0.69 0.74 0.68

* Pairwise similarity coefficients were calculated between F. oxys-orum E1d and F. oxysporum IMI 141 140, F. oxysporum F 331, and. oxysporum 0294.

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281MYCOHERBICIDAL PATHOGENS OF BROOMRAPE

significantly reduced the number of tubercles (Fig. 8A)and the biomass of tubercles (Fig. 8B) compared to thecontrols. The predip plus drenches were far more effec-

FIG. 4. RFLP differentiation of F. oxysporum strains. An EcoRI–o total DNA samples from four strains of F. oxysporum, digested witel is presented on the left; a Southern blot of this gel is shown on t, F. oxysporum E1d; lane 4, F. oxysporum F 331; lane 5, F. oxyspor

FIG. 5. Effect of fungal dose of Fusarium arthrosporioides strain4a on Orobanche tubercle development. Infested tomato plant rootsere sprayed to runoff with spore suspensions of F. arthrosporioides

train E4a (four plants per treatment). Tubercle death was measuredfter 10 days. During this period there was a natural 50% self-thinningf tubercles on the controls. This amount of self-thinning was thusubtracted from all treatments prior to the plotting of the data. Theontrol plants bore an average of 7 6 2 live tubercles per plant. Thexperiment was repeated twice. The data are from one trial.

tive than the predip treatments, reducing the numberof live tubercles to less than 20% of the control (Fig. 8).There was no gain in activity (nor antagonism) when amixture of both organisms was used. Plants were re-moved from the pots and the soil was washed away 8weeks after planting, when the tomato plants werealready flowering and setting fruit. No large tuberclesformed on transplants treated with the fungi. The to-

mHI fragment of the rRNA gene from A. nidulans were hybridizedglII, EcoRI, and HindIII, respectively. An ethidium bromide-stainedight. Lane 1, F. arthrosporioides E4a; lane 2, F. poae TAPO-1; lane0294; l phage DNA size markers are shown on the left.

FIG. 6. Effect of mycelial and spore inocula on the infection oftubercles. Broomrape-infected tomato plants roots were sprayed torunoff with 10 ml of 106/ml spore suspension or 106 propagules/ml of

omogenized mycelial pieces in 0.02% Tween 20. A solution of 0.02%ween 20 was used as a control. Data are expressed as the averagef dead tubercles. The experiment was repeated twice with similaresults.

Bah Bhe r

282 AMSELLEM ET AL.

mato plants without the fungal treatment were in-fested with large and healthy Orobanche tubercles.

Host Range Studies

Before field experimentation was to be initiated, itwas imperative to clarify three aspects of pathogenicityof these fungi: (a) which Orobanche species are con-trolled by each of these fungi, (b) whether the fungi arepathogenic to any of the commonly cultivated crop spe-cies, and (c) whether they will control Orobanche onany crops other than melon and tomatoes.

The polyethylene envelope system was used to ascer-tain the pathogenicity of three Fusarium strains ondifferent Orobanche species. The FARTH and FOXYwere compared with the F. oxysporum f. sp. orthoceras.The data on pathogenicity of these strains on O.cumana were graciously supplied by J. Sauerborn. Ourstrains of FARTH and FOXY were pathogenic to thethree Orobanche species that commonly attack le-gumes and vegetables in the Middle East, yet wasavirulent against O. cumana, which almost specificallyattacks sunflowers, among crop species (Table 3). Con-versely, F. oxysporum f. sp. orthoceras was pathogeniconly on O. cumana and not pathogenic on any of theOrobanche spp. that attack vegetables and legumes.

FIG. 7. Growth and phytotoxicity to Orobanche germlings of Findicate alive germling in the control, or dead germlings germinatinas seen by the blackening of the rootlet and seed before attachment

FIG. 8. Enhanced O. aegyptiaca control on tomato by two postplplanting. (A) Number of live tubercles per pot, above and below grbearing different letters are significantly different from each other a

FARTH and FOXY were not pathogenic to any of thevegetable crops tested when washed spores or myceliawere used as inoculum in the greenhouse pot trials(Table 3).

There are many problems in the field application ofsoil-applied mycoherbicides. Initially, more than a tonof inoculum plus medium was applied per hectare(Walker, 1981) and more recently inoculum pelletingand other technologies have been developed (Boyette etal., 1993; Amsellem et al., 2000). It seems that, unlikethe control of nonparasitic weeds, the direct applica-tion of mycoherbicides to transplants might be an effi-cient and simple way to effect initial control. The datapresented (Fig. 8) indicate that control is far betterwhen augmented with a soil drench of the organisms.One might assume on theoretical grounds that sporesor mycelia may not sufficiently penetrate into the soil,but elegant experiments by Alabouvette et al. (1993)indicate that a Fusarium sp. used as a biocontrol agentagainst F. oxysporum f. sp. lycopersici, a crop-rootpathogen, was highly effective when applied as adrench.

Eventually, it may be possible to use seed dressingsof mycoherbicides to control parasitic weeds. However,if mycoherbicides are to be used as seed dressings, the

TH and FOXY growing along the tomato root rhizoplane. Arrowsar young tomato roots 6 cm from the edge of the initial inoculation,the crop.

ing soil drenches with Fusarium spp., 2 and 3 weeks after a predipd. (B) Dry weight per live tubercle. Treatments within each panelhe P 5 0.05 level using Student’s t test.

ARg neto

antount t

A

A

B

B

283MYCOHERBICIDAL PATHOGENS OF BROOMRAPE

seed cannot be treated with an incompatible fungicide.Selection for fungicide-resistant strains of the myco-herbicidal organisms may have to be performed. Theuse of seed-applied biocontrol agents against fungalpathogens of the crop may also be contraindicated, assuch agents could also be antagonistic to the mycoher-bicide used to control broomrape. We have recentlyshown that the virulence of these strains can be en-hanced transgenically, using exogenous genes (B. Co-hen, Z. Amsellem, R. Maor, A. Sharon and J. Gressel,unpublished).

Reservations have been expressed about the use of F.oxysporum strains as biochemical agents due to thepossibility that they might acquire genetic traitsthrough mutation, parasexuality, or other ways of hor-izontal gene transfer to become pathogenic to crops.The molecular data (Table 2) support a divergent ori-gin of the broomrape-pathogenic strains from othercrop-pathogenic F. oxysporum formae specialis. Recentinvestigations on gene genealogies in the F. oxysporumspecies complex led to the conclusion that even some ofthe wilt- and rot-causing pathogenic forms of this fun-gus could be nonmonophyletic in their origins. Ampli-fied fragment length polymorphism-based phyloge-netic analysis for 89 strains including nonpathogenicisolates used as commercialized biocontrol agents indi-cated strongly that these biocontrol strains are notlikely to convert to pathogenic strains (Baayen et al.,2000).

There are still many experiments to be performedbefore agricultural use of FARTH and FOXY can be

TABLE 3

Pathogenicity of Fusarium spp. Pathogenic to Orobancheand Crop Plant Species

FARTHa FOXYaF. oxysporum f. sp.

orthoceras

Pathogenicity to Orobanche species

O. aegyptiaca Pb P NPO. cernua P P NPO. ramosa P P NPO. cumana NPc NPc Pc

Pathogenicity to crop plant speciesd

Carrot NP NPCelery NP NPChickpea NP NPMelon NP NPPepper NP NPTomato NP NPSunflower NP NPc NPc

Vetch NP NP NP

a FARTH, Fusarium arthrosporioides E4a; FOXY, Fusarium oxys-porum E1d.

b P, pathogenic; NP, nonpathogenic.c Data from Sauerborn’s group (J. Sauerborn, University of Hohen-

heim, Stuttgart, Germany, personal communication).d See Materials and Methods for details of cultivars tested.

envisaged. These include soil residue analyses and fur-ther pathogenicity testing on crops cultivated in thesame geographical region to determine incompatibili-ties. Both DNA analyses and host range studies indi-cate that the F. oxysporum isolated in these studies isclosely related to but different from the F. oxysporum f.sp. orthoceras isolated by Bedi (1994).

Basic studies of the defenses of broomrape may leadto synergies with chemical synergists to suppress de-fenses and/or facilitating microorganisms (Gressel etal., 1997). Additionally, it is possible to envisage theuse of transgenic hypervirulent pathogens of weedsbearing failsafes to prevent their spread and introgres-sion into crop pathogens (Gressel, 2001). Still, it ispromising to see that native pathogens can be foundthat can reduce broomrape growth to an extent thatthe resulting balance may allow crop production with-out further procedures or with small amounts of her-bicides. Preferably this technology will be integratedwith other technologies, as it would be unwelcome tosee Orobanche rapidly evolve strains resistant to itspathogens.

ACKNOWLEDGMENTS

This research has been supported by Grant HNE-0158-G-00-3060-00 of the Trilateral USA–Egypt–Israel Program of the U.S.Agency for International Development (A.I.D.), by a DFG TrilateralGrant, and by the Gilbert de Botton Chair of Plant Sciences to J.G.Useful discussions with Professor Yaakov Katan are greatly appre-ciated.

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