BIOLOGICAL CONTROL OF PHYTOPHTHORA

BIOLOGICAL CONTROL OF PHYTOPHTHORAPARASITICA VAR.NICOTIANAE ON TOBACCO SEEDLINGS WITH NON-PATHOGENIC BINUCLEATERHIZOCTONIA FUNGID.KELLY CARTWRIGHT1*and H.W.SPURR JR.21Agricultural Research Initiatives,Inc.,10052,206W.,Harrison,72601AR,U.S.A.and2Department of Plant Pathology,North Carolina State University,Raleigh,27695NC,U.S.A.(Accepted13January1998)SummaryÐNonpathogenic binucleate Rhizoctonia fungi(BNR)controlled black shank caused by Phy-tophthora parasitica var.nicotianae on greenhouse-grown tobacco seedlings in styrofoam¯oat trays. Three BNR isolates were incorporated into a soil-less mix on colonized,pulverized,sifted rice particles; colonized whole rice grains;or on pelleted tobacco seeds coated with0.5%methyl cellulose.Five-wk-old seedlings were inoculated with zoospores of P.parasitica var.nicotianae and disease rated over 10d.The level of protection varied with method of BNR application,ranging from40to70%.Overall, control was better when BNR isolates were applied on rice inocula rather than on BNR-colonized tobacco seeds.From15%to80%of individual roots from seedlings grown in soil-less mix amended with BNR-colonized rice grains were colonized while only0±20%of roots from seedlings grown from BNR-colonized tobacco seeds were colonized.Likewise,37±100%of soil-less mix amended with BNR-colonized rice grains contained BNR's while less than3%of soil-less mix was colonized when seedlings emerged from BNR-colonized tobacco seeds.This is the®rst demonstration of biocontrol of Phy-tophthora by BNR fungi.#1998Elsevier Science Ltd.All rights reservedINTRODUCTIONBlackshank of tobacco,caused by Phytophthora parasitica Dastur var.nicotianae(Breda de Haan) Tucker,is a destructive disease of¯ue-cured tobacco(Nicotiana tabacum L.)in North Carolina and other major tobacco-producing areas through-out the United States(Lucas,1975).Losses can occur at all stages of growth and range from minor injury to complete destruction of a tobacco plant. Currently,black shank is managed through the use of healthy transplants,resistant cultivars,crop ro-tation,and the fungicide metalaxyl(Lucas,1975; Shew,1984).Nevertheless,black shank continues to pose a major problem for farmers and increasing concerns over environmental problems and meta-laxyl-resistant strains of Phytophthora support the need to®nd supplemental or alternative control methods such as biocontrol(Hunger et al.,1982; Baker,1983;Tweedy,1983;Staub and Sozzi,1984; Shew,1984;Hallberg,1987;Campbell,1989). Recently,binucleate Rhizoctonia fungi(BNR) were investigated as biocontrol agents for a number of important diseases caused by pathogenic Rhizoctonia species and damping-o of bedding plants caused by Pythium(Burpee and Goulty, 1984;Cardoso and Echandi,1987;Harris et al.,1993,1994).BNR fungi o er several advantages compared to other biocontrol agents.For example, BNR fungi typically colonize the target area rapidly and tolerate a broad range of environmental and edaphic conditions.In addition,some BNR fungi may induce systemic resistance in plants which could extend the spectrum of control to other target pathogens or lengthen the protection period (Burpee and Goulty,1984;Cardoso and Echandi, 1987).There have been few evaluations of biocontrol agents for control of P.parasitica var.nicotianae. Several investigators targeted induced resistance as a mechanism to control P.parasitica var.nicotianae (McIntyre and Miller,1978;Bonnet,1988;Bonnet et al.,1988),however no attempts have been made using BNR fungi.We evaluated BNR fungi for control of black shank on tobacco seedling trans-plants grown in¯oat trays in the greenhouse,a sys-tem commonly used to grow transplants.This system was e ective for evaluating the resistance of tobacco cultivars to black shank(Cartwright et al., 1995),and could provide a rapid and convenient system to test antagonists against P.parasitica var. nicotianae.The objectives of this research were:(1) screen BNR fungi for e cacy of black shank con-trol in the¯oat system,(2)compare di erent methods of introducing BNR fungi to the¯oat sys-tem for black shank control,and(3)determine the Soil Biol.Biochem.Vol.30,No.14,pp.1879±1884,1998#1998Elsevier Science Ltd.All rights reservedPrinted in Great Britain0038-0717/98$19.00+0.00PII:S0038-0717(98)00019-4*Author for correspondence.1879extent of colonization of BNR fungi on roots and in soil-less mix after application.Preliminary results have been reported(Cartwright and Spurr,1995).MATERIALS AND METHODS Collection and discovery of BNR fungiEighteen BNR fungal isolates from Dr Eddie Echandi's collection at North Carolina State University were screened in preliminary trials.Two of these,isolates P9023and L9125,were selected for further tests.Isolate KC94J,isolated from the roots of buckhorn plantain(Plantago lanceolata L.) collected at the edge of a cultivated®eld at the North Carolina Tobacco Research Station Farm, Oxford,NC also was selected for further tests.All isolates were subcultured on potato dextrose agar (PDA)(Difco,Detroit,MI)before being stored as mycelial plugs in sterile water at48C. Application of BNR fungi for control of black shank on¯oat-grown tobacco seedlingsTwo mycelial plugs from individual BNR isolates cultured on PDA were used to inoculate twice-auto-claved rice grains(25g rice per18ml sterile,deio-nized water)in250ml glass¯asks.Rice cultures were incubated at268C for6±8d and were shaken every2±3d to disperse the mycelium.Several methods of preparation and application of BNR fungi were used:(1)BNR-colonized rice grains were pulverized at high speed in a blender for30s fol-lowed by passage through a 1.68mm pore size sieve.One g of sieved,colonized rice particles was mixed thoroughly with1l of potting mix consisting of a uniform blend of peat humus,sphagnum peat and washed coarse sand(Carolina's Choice Tobacco Mix,Carolina Soil,Kinston,NC)using a 3.9l rotating drum for5min.Non-colonized rice particles were used as a control.Styrofoam¯oat trays(200cells;27ml per cell)were cut in half and soil-less mix(amended with BNR isolates or non-amended)was deposited in each half.Soil-less mix was pressed(dibbled)and pelletized tobacco seed (cv.K-326)were hand-seeded(1to2pelletized seed per cell).(2)Non-amended soil-less mix was placed in half trays as described.Individual BNR-colo-nized rice grains from each BNR isolate were placed on top of soil-less mix in each cell(1grain per cell)and covered with mix.The mix was then dibbled lightly and hand-seeded as described.Non-colonized rice grains were used for controls.(3) Pelletized tobacco seeds of cv.K-326were surface disinfested for2min in70%ethanol,air-dried asep-tically,and coated with methyl cellulose(Sigma Chemical,St.Louis,MO)by soaking for5min in a 0.5%solution.After drying,coated seeds were deposited into50ml glass¯asks,seeded with2 mycelial plugs from BNR isolates,and incubated at 268C as previously stated for the rice cultures.Only BNR isolates P9023and L9125were used to colo-nize methyl cellulose-coated seeds.Non-colonized, methyl cellulose-coated seeds were used for con-trols.Soil-less mix was deposited into half trays, dibbled,and colonized seed were hand seeded as described.Colonization by BNR isolates of screened rice particles,rice grains,and methyl cellulose-coated seeds(at least50seeds)was checked at the time of application by plating on Flower's medium(FM),a semi-selective medium for Rhizoctonia spp.(Flower, 1976).All rice inocula were colonized by BNR fungi and more than95%of the methyl cellulose-coated seeds were colonized.Germination of seeds was not a ected by any BNR treatment compared to the respective controls.After seeding,half trays were¯oated in water contained by black plastic in wooden frames measuring10cm deepÂ36cmÂ142cm.At time of seeding,150m g mlÀ1of a commercial20-20-20(N-P-K)fertilizer was thoroughly mixed with the¯oat water.The depth of water was maintained near the top of the wells throughout the experiments. Frames were placed on heating mats(Hummert International,St.Louis,MO)with thermostats set at308C.After3±4wk,seedlings were thinned and transplanted to50per half-tray(10seedlings in every other row of100cell half tray).To simulate commercial conditions,seedlings were hand-clipped with scissors3±4d after inoculation with P.parasi-tica var.nicotianae by excising approximately50% of leaves extending above the growing point.Soil moisture of the potting mix was H85%(w/w).Monitoring of BNR fungi on roots and in soil-less mixAdditional trays of each treatment(non-inocu-lated)consisting of the BNR isolates applied via in-dividual whole rice grains or on colonized tobacco seeds only were established at the beginning of each test and used to sample seedlings and soil-less mix to monitor colonization by the BNR isolates.Three wk after seeding,3seedlings were randomly sampled from each tray.The root mass,still very small,was excised,rinsed in sterile deionized water, and the entire root system from each seedling was placed on FM to check for the presence of BNR fungi.Four and5wk after seeding,3seedlings were randomly sampled from each tray and the entire root system from each seedling was rinsed3times in sterile,deionized water,blotted dry,cut into 1cm sections,and bined sections were thoroughly stirred and10root sections were randomly selected,blotted dry,and placed on FM. At the4and5wk sampling dates,adjacent soil-less mix from each sampled cell(containing seedling) was collected with the seedlings.Additional soil-less mix was removed from the root system by shaking. The soil-less mix from all three sampled seedlingsD.Kelly Cartwright and H.W.Spurr Jr. 1880and cells were combined and thoroughly mixed. Plastic tubing(4.8mm[inside dia]Â0.8mm[wall thickness]Â6.4mm[outside dia])(A.Daigger, Wheeling,IL)was surface-disinfested in70%etha-nol,air dried,and cut into5-cm sections.Each sec-tion of tubing was used to lightly tap the soil-less mix contained in a sterile,plastic Petri dish,®lling the tubing with the mix.The tubing was then asep-tically cut into3-mm sections(pellets)and placed (mix side down)on FM(15pellets per plate).Two replicate plates were used for each treatment.All plates were placed at268C and monitored for3±5d.The presence of BNR fungi was con®rmed visually by the characteristic growth and mor-phology of mycelium on FM and by comparison to the respective controls.Preparation of and inoculation with P.parasitica var. nicotianaeIsolate RMT6of P.parasitica var.nicotianae, obtained from Dr H.David Shew,North Carolina State University,was used in all tests.Cultures were grown on oatmeal agar(Difco,Detroit,MI) for about3wk at268C,mycelial mats were care-fully removed,wetted with sterile water,and incu-bated for an additional7±10d at268C.Mats werethen rinsed in sterile water several times,resus-pended in sterile water,chilled at48C for30min, and warmed to room temperature for30min to release zoospores.Zoospore numbers were quanti-®ed with a hemacytometer and suspensions diluted to5000zoospores mlÀ1for inoculum.After4±5wk, seedlings were inoculated by pipetting1ml of the zoospore suspension to the base of each seedling in an individual cell.Disease assessment and experimental design Disease development was monitored on seedlings 7and10d after inoculation.Disease incidence(%) was recorded and a disease severity scale was used to evaluate disease development on individual seed-lings in which0=no visible symptoms;1=¯ecks, small lesions,wilting;2=moderate lesions,26±50%of stem with lesion,severe wilting;3=large lesions extending up and down the stem covering 51±75%of stem;and4=death of plant.Disease incidence data are presented from the10d readings. Each row of seedlings in a half-tray was con-sidered a replicate(10seedlings per replicate,5 replicates per treatment)and the trays were arranged randomly within the framed wells(4trays per well).Each experiment was repeated,and data were pooled based on similar variances and ana-lyzed with PROC MEANS and PROC GLM with PC SAS(SAS Institute,Cary,NC).Treatment means were separated with the Waller-Duncan k-ratio test.Based on replicates,data from soil colo-nization tests were pooled,analyzed,and means separated as previously stated.Means only are pre-sented for the root colonization assays.RESULTSApplication of BNR fungi for control of black shank on¯oat-grown tobacco seedlingsEach of the three BNR isolates decreased disease incidence compared with the infested controls when BNR isolates were applied on screened,colonized rice particles incorporated into the soil-less mix. Isolates L9125,P9023and KC94J decreased black shank incidence(P=0.05)to35,42and43%,re-spectively(from60%incidence observed on seed-lings in the infested controls)(Fig.1).There was a similar trend when BNR isolates were introduced via individual,colonized rice grains in each cell of the trays,although overall disease incidence was less.An average of31%incidence occurred on seedlings in the infested control in these tests. However,incidence was limited to10,18or24%in treatments where isolate P9023,L9125or KC94J were used,respectively(Fig.1).When isolate P9023 or L9125was used to colonize tobacco seeds before seeding,the level of control was similar to that observed with rice inocula with isolate L9125(16% incidence)compared to the infested control(39% incidence).However,signi®cantly higher disease incidence was observed on seedlings in treatments where BNR isolate P9023was applied to the seeds (60%incidence).No disease occurred on seedlings in the non-infestedcontrols.Fig.1.Biocontrol of black shank on tobacco seedlings grown in greenhouse¯oat trays with BNR fungi.Disease incidence(%)assessed after10d.Values within each ap-plication method are signi®cantly di erent(P=0.05)if followed by a di erent letter according to the Waller Duncan k-ratio test.CSRP=BNR isolates applied uni-formly to soil-less mix on colonized,screened rice par-ticles;CWRG=BNR isolates applied to soil-less mix on colonized,whole rice grains;CPTS=BNR isolates applied on colonized,pelletized tobacco seeds. Control+=Infested control.Bars represent standarderror of the mean.Biological control of phytophthora1881Monitoring of BNR fungi on roots and in soil-less mixAll BNR isolates applied on a single colonized rice grain colonized the soil-less mix and root sys-tems of the seedlings.Three,4,and 5wk after seed-ing,isolates P9023,L9125,and KC94J were present in the roots-rhizosphere of all the sampled seedlings (data not shown).Isolates P9023and L9125colo-nized more of the soil-less mix than isolate KC94J (Fig.2).After 4or 5wk,isolate P9023colonized 100%of the soil-less mix per sampled cell and iso-late L9125colonized 98or 100%,compared to 43or 37%colonization for isolate KC94J,respectively.Individual roots of sampled seedlings also were colonized well using the rice grain application method.Forty percent of the roots were colonized by isolate P9023,15%by isolate L9125and 25%by isolate KC94J,respectively,at 4wk after seed-ing.Five wk after seeding,80%of the roots were colonized by isolate P9023,45%by isolate L9125and 20%by isolate KC94J,respectively (Fig.2).No BNR fungi were detected in the control treat-ments.Very little colonization by the BNR isolates occurred in the soil-less mix or on roots of seedlings grown from BNR-colonized tobacco seeds (Fig.3).Fig.2.Colonization of roots and soil-less mix by BNR fungi applied to soil-less mix on colonized,whole rice grains for control of black shank on tobacco seedlings grown in greenhouse ¯oat trays.Assays performed 4or 5wk after seeding.Values representing percent soil-less mix samples colonized by BNR isolates are signi®cantly di erent (P =0.05)if followed by a di erent letter according to theWaller Duncan k -ratio test.=root colonization;q =soilcolonization.Fig.3.Colonization of roots and soil-less mix by BNR fungi applied on colonized,pelletized tobacco seeds coated with 0.5%methyl cellulose for control of black shank on tobacco seedlings grown in greenhouse ¯oat trays.Assays performed 4or 5wk after seeding.Values representing percent soil-less mix samples colonized by BNR isolates are signi®cantly di erent (P =0.05)if followed by a di erentletter according to the Waller Duncan k -ratio test.=root colonization;q =soil colonization.Three wk after seeding,both isolate P9023and iso-late L9125were present in67%of the sampled seedling roots when the entire root mass was plated on a selective medium.After4wk,however,only 1.7%of the volume of soil-less mix per cell was colonized by isolate P9023and isolate L9125,and only3%of the soil-less mix was colonized by iso-late P9023after5wk(Fig.3).With isolate P9023, only20%and5%of individual roots of sampled seedlings were colonized after4or5wk,respect-ively.DISCUSSIONIn these tests,three non-pathogenic BNR isolates signi®cantly decreased the incidence of black shank on¯oat-grown tobacco seedlings.The use of the ¯oat system was bene®cial in a number of ways. The test area was small and manageable,a large number of plants could be tested,the seedlings used were at a very susceptible stage of growth,environ-mental conditions were precisely controlled,and precise amounts of inoculum were easy to apply uniformly to each seedling.In addition,monitoring of the BNR isolates in the target area was practical with the use of the plastic tubing and the FM med-ium.Applying the BNR isolates using di erent methods was important to test the e cacy of black shank control and to observe colonization of the soil-less mix and seedling roots.Distributing the isolates uniformly on small,colonized rice particles or incorporating the isolates via a single,colonized rice grain resulted in the highest control using iso-late P9023.Isolate P9023also colonized the roots and surrounding mix well when applied on a single, colonized rice grain.However,when this isolate was grown on pelletized tobacco seeds,colonization was minimal on the roots and in the soil-less mix and black shank was not controlled.Disease incidence was higher in treatments containing isolate P9023 on pelletized tobacco seeds than in the infested con-trols.Colonization of the tobacco seeds,rather than the rice grains,may have predisposed seedlings to infection by P.parasitica var.nicotianae.This pre-disposition and the lack of a suitable food base to support the growth of isolate P9023may have resulted in poor colonization and no protection for the seedlings.Some BNR isolates are themselves pathogenic(Martin et al.,1983;Burpee and Goulty, 1984),although no damping-o has been observed with the isolates tested on tobacco seedlings.Based on the high colonization in the target area and sub-sequent black shank control by isolate P9023when applied on rice,colonization of the target area seems critical in disease control.Other researchers have shown that protection of plants with avirulent Rhizoctonia solani Kuhn isolates from pathogenic R.solani isolates was the result of root colonization and competition for attachment sites.Other workers suggest that systemic,induced resistance is the mechanism involved(Cardoso and Echandi, 1987;Sneh et al.,1989).Root colonization by avirulent R.solani,however,only protected plants against R.solani and was ine ective for pathogens such as Pythium and Fusarium spp.(Sneh et al., 1989).Control with isolate L9125and isolate P9023was similar when applied to the target system on rice inocula.However,consistently less control was observed with isolate KC94J than with the other two isolates.Colonization was also less with isolate KC94J on the roots and in the soil-less mix.Like isolate P9023,isolate L9125e ectively controlled black shank when applied on rice inocula and also colonized the seedling roots and soil-less mix. However,this isolate also controlled black shank when applied on colonized,pelletized tobacco seeds, even though colonization of the root zone and sur-rounding soil-less mix was very limited.Because of the control observed here,the subsequent lack of colonization,growth and colonization in the target area may not be as critical with this isolate. Colonization and growth on the tobacco seeds and the young seedlings upon emergence may induce a systemic defense mechanism within the plant,giving protection to the seedlings.Many biocontrol antag-onists exhibit di erent mechanisms or combinations of antagonistic mechanisms depending on nutrient availability,target pathogen,host plant and existing micro¯ora(Campbell,1989;Lumsden and Locke, 1989;Sneh et al.,1989).A major constraint to the practical use of biocon-trol agents is the narrow scope of target pathogens on which they are e ective(Campbell,1989).In most cases,antagonists are suitable only for a single pathogen or group of pathogens.For example, BNR fungi have typically been used for control of R.solani(Burpee and Goulty,1984;Cardoso and Echandi,1987;Sneh et al.,1989).The ability of these BNR isolates to control an unrelated patho-gen,i.e.P.parasitica var.nicotianae,in this study expands the potential target range of these fungi for biocontrol.BNR fungi are promising as biocontrol agents due to their ubiquitous occurrence,non-pathogenic nature,aggressive colonization of roots and soil,and tolerance of a wide range of environ-mental conditions(Martin et al.,1983;Burpee and Goulty,1984;Cardoso and Echandi,1987).These data indicate the potential of BNR fungi used in diverse systems for biocontrol.AcknowledgementsÐWe thank Dr Eddie Echandi,Dr Jean Ristaino and Dr David Shew for providing cultures of BNR fungi and P.parasitica var.nicotianae.We also thank Pete Elliot and Buddy Quick for technical assist-ance.Biological control of phytophthora1883REFERENCESBaker K.F.(1983)The future of biological and cultural control of plant disease.In Challenging Problems in Plant Health,eds T.Kommedahl and P.H.Williams, pp.422±430.The American Phytopathological Society, St.Paul.Bonnet P.(1988)Culture®ltrates of three Phytophthora spp.:puri®cation and biological activity of di erent fractions.Agronomie8,347±350.Bonnet P.,Poupet A.,Abad P.,Venard P.and Cardin L.(1988)Occurrence of foliar necrosis,b.-proteins and resistance in non-compatible Phytophthora-tobacco in-teractions.Agronomie8,829±837.Burpee L.L.and Goulty L.G.(1984)Suppression of brown patch disease of creeping bentgrass by isolates of nonpathogenic Rhizoctonia spp.Phytopathology74, 692±694.Campbell R.(1989)Biological Control of Microbial Plant Pathogens.Cambridge University Press,Cambridge. 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