The role of bacteria in the biological control of Gaeumannomyces graminis by suppressive soils

Sod Bd

Bio~firm. Vol. 8. pp 269 to 27 3 Pergamon

Press 1976 Printed m Great Britain

THE ROLE OF BACTERIA IN THE BIOLOGICAL CONTROL OF GAEUMANNOMYCES GRAMINIS BY SUPPRESSIVE SOILS R. J. COOK* and A. D.

ROWRAt

*Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington 99163. U.S.A. tDivision of Soils, C.S.I.R.O.. Glen Osmond, 5064 S.A.. Australia. (Accepted 15 No~:emher 1975) Summary-The suppression of Gaeurnannornyrrs gra~inis var. critici by certain soils or following certain soil treatments is considered to be an expression of either specific or general antagonism sensu Gerlagh (1968). Specific antagonism is effective in dilutions as high as 1 in 1,000, can be transferred from soil to soil, operates near or on wheat roots. is destroyed by 60°C moist heat for 30 min, or desiccation, is fostered bq wheat monoculture but may be lost from a soil by fallow or rotation with certain crops, especially legume hay or pasture crops. Strains of Pseudomonas ,fluorescms may be involved. General antagonism is a soil property which cannot be transferred and is resistant to 80°C moist heat for 30 min. to methyl bromide and chloropicrin, but not to autoclaving. Take-all control by organic amendments, minimum tillage, or a soil temperature of 28 C may be expressions of increased general antagonism. In much of the southern Australian wheat belt, where take-all can cause heavy crop losses, some general but rarely specific antagonism is apparently operative. Both types of antagonism are probably operative in long-term wheat growing areas of the Pacific Northwest U.S.A. where take-all is virtually nonexistent.

INTRODUCTION

There are many documented examples of soils that suppress root pathogens (Baker and Cook. 1974), but probably none has proved more fascinating for studies of natural biological control than soils suppressive to the wheat take-all fungus, &~eLlnzumzomyces yruminis (Sacc.) Arx and Olivier var. tritici Walker (= Ophiobolus graminis Sacc.). Soils can become suppressive to G. gruminis with various treatments such as the addition of organic material (Fellows and Ficke, 1934), elevation of the soil temperature up to 25”28”C (Henry, 1932), fertilization with NH:- rather than NO:-N (Smiley and Cook, 1973). or minimum tillage (Brooks and Dawson, 1968). Then there is the “take-all decline” phenomenon in which the suppression develops with 2 or 3 yr of wheat monoculture and severe take-all; this soil becomes “immune” to subsequent outbreaks of take-all if cropped exclusively thereafter to wheat or barley (Shipton, 1975). Gerlagh (1968) described this as “specific antagonism”, and distinguished it from “general antagonism” present to some degree in all soils. Vojinovic’ (1972) has confirmed the existence of these two kinds of antagonism. The recognition of a specific as distinct from general antagonism represents a significant advance in the understanding of the take-all suppressive soils. In this paper. we discuss suppressiveness in different soils or following certain treatments as expressions of one or the other of these two kinds of antagonism. We also discuss the role of certain soil bacteria in specific and general antagonism. Other reviews (Shipton, 1975) should be consulted for more details of the take-all decline phenomenon. The antagonism by Phialophora rudicicolu and related fungi probably rep269

resents still another antagonism, which is covered by Deacon (1975). Finally, our paper deals primarily with suppressiveness that affects G. graminis var tritici in its parasitic stage; the influence of soil organisms on saprophytic survival of this pathogen is reviewed by Zogg (1975). Eristrncr qf suppressive soils in Northwestern und Southern Australia

U.S.A.

Wheat is produced in the Northwest U.S.A. in four distinct management systems (Fig. 1). Little or no takeall occurs in two of these systems, namely, the annually-cropped area of eastern Washington/adjacent Idaho (Zone A), and the semi-arid wheat/fallow areas of the same region (Zone B). in spite of great diversities of soils. topography (including low, wet areas), management (including supplemental sprinkler irrigation), and cropping systems (continuous wheat, wheat/fallow, wheat/peas/lentils, or combinations thereof). However, severe take-all is a problem whenever wheat is grown for 2 or 3 consecutive yr in the same field in the once virgin desert which is now intensively irrigated and rotated (Zone C) and in the area west of the Cascade Mountains where wheat is grown in rotation with other crops (Zone D); but take-all decline occurs if wheat is grown in extended monoculture in these two zones. The lack of take-all under the first two management systems in dryland or supplementally-irrigated portions of the regions is probably due in part to a high water potential requirement of G. graminis var tritici (Cook et a[., 1972). On the other hand, irrigation in the dryland area has not resulted in the anticipated increase in take-all (Cook et ul., 1968), although inoculum apparently is not the limiting factor (Shipton rt al.. 1973). However, take-all develops in exper-

270

K. J. COOKand A. D. ROVIRA

Al

Annual-cropped wheat or wheat/peas/lentils; mostly subhumid; silt and clay loams; native vegetation mostly grassland. BB Dryland or sprinkler-irrigated wheat/fallow; semiarid; silt and fine sandy loams; native vegetation grassland and some sagebrush/bunchgrass. C@ Intensively irrigated wheat rotated with many other crops; arid; sands, fine sandy loams and silt loams; native vegetation sagebrush/bunchgrass. Om Wheat rotated with many crops including grasses; ornamentals. and small fruits and vegetables; humid; sandy and silt loams; acid soil; native vegetation mostly conifer forest. Fig. I. The wheat-producing areas of the Pacific Northwest, U.S.A. showing the four management systems: A-subhumid. annual cropped; B-semi-arid, wheat/Mlow; C--arid, irrigated, extensively rotated; D-humid, extensively rotated. Take-all is rare or nonexistent in the first two and occurs in the latter two but exhibits the decline phenomenon in extended wheat monoculture. imental plots in eastern Washington if the plots are fumigated first with methyl bromide, and then inoculated with G. ~~~~z~~~is var. tritici as infested oat-kernel inoculum (Cook, unpublished data) where the oat inoculum produces virtually no take-all in adjacent nonfumigated plots. In western Washington (Zone D), such oat inoculum at the same rates destroys a crop regardless of whether or not the site is fumigated. In much of Australia take-all has been a continual threat to wheat production since early settler days, with one possible improvement in that the once-common seedling blight phase of take-all has been largely replaced by the less acute “white-heads” or “hay-die” phase. This suggests that perhaps some suppressiveness now exists, but the level is obviously inadequate. The lack of antagonism to G. ~~~~~j~~~.s var. tritici in the southern Australian wheat belt probably relates to the grass/legume pasture used in rotation with wheat in that area. We have shown that any suppressiveness to take-all detectable after one wheat crop apparently is lost if the field is converted to pasture for more than 1 yr. The grass/legume pasture used in Australia has the added disadvantage in that G. yrarninis var. tritici may persist on grass hosts while antagonists die out-a double-edged effect. There is no pasture phase in the eastern Washington wheat farming system.

(a) Firici rri&. Shipton et nl. (1973) transferred a factor from a longterm wheat monoculture soil in Zone C (Quincy field, QF), that suppressed take-all.

to field plots in western Washington (Zone D) by introducing 0.5% (w/w) of QF soil and rotovating to 15cm depth. In the first yr only minor disease suppression occurred but in the second year the wheat showed no take-all. In contrast, plots amended with soil from take-all conducive virgin sites from the same zone showed no disease suppression in the second crop, In the third crop, all wheat was healthy regardless of the soil amendments made 3 yr earlier, apparently because the take-all decline factor had built up in all plots due to three consecutive years of wheat. However, the results show that the QF soil acted as “starter soil” to initiate take-all decline I pr sooner than was possible in adjacent plots treated with other or no foreign soil. (b) Pot trials. The glasshouse “pot test” (Shipton et al., 1973) bioassays soils for suppressiveness to G. graminis; the test involves diluting I part of a test wheat field soil with 100 parts of a standardized soil fumigated with methyl bromide, amending this soil mixture with 0.1-0.5~~ (w/w) ground oat-kernel inoculum of G. ~~~~~~~i~~ planting to wheat, and recording infection severity on the washed seedlings after 21 days at 20°C. This method has confirmed the transferability of the decline factor and has served as a valuable technique for the detection of suppressiveness in various soils and for studies on the nature of the suppressive factors (Fig. 2). The method has also confirmed the general absence of antagonism to G. graminis var. tritici in soils of the southern Australian wheat belt; 45 soils representing various stages in the wheat-pasture fallow rotation collected in southern Australia showed little or no suppression of take-all relative to QF soil. The only Austraiian soil with suppressive activity comparable with QF soil was one from an experimeI~~1 plot which had been cropped to wheat for 57 yr; much less suppression was present in soil from the adjacent wheat fallow plots. Apparently, the organisms responsible for take-all decline are sensitive to even 1 yr of bare failow under Australian conditions. With other soils collected from five nearby plots in various phases of wheat-pasture-pasture--fallow rotation, the most suppressive soil occurred in the first yr pasture following wheat; by the second year of pasture, all suppressiveness was lost despite an increase in the general soil microflora with 2 yr of pasture. Subsequent tests with soils from other southern Australian wheat farms have confirmed that any alltagonisnl to take-ail generated by a wheat crop is lost during the grass:’ legume pasture following wheat. Although soils of the southern Australian wheat belt normally lack antagonism. one or two consecutive outbreaks of take-all in a given site apparently generates considerable antagonism. thus showing that Australian soils contain the active agents. Nature

of‘ gerwral

arid .sprc$c

suppressions

01 tdie-all

Gerlagh (1968) presumed that the suppression of take-all in warm soils (Henry, 1932) is due to the specific antagonism, but our results indicate that this antagonism is of the general type, present to some degree in all soils, as we have ~onsjstent1~ observed a marked suppression of take-all in glasshousc cxperiments if temperatures rise above 25 C. cvcll \~li~~i fumigated soils are used. Treatment of soil with 70’ C

Biological

control

of take-all

27-l

Fig. 2. Suppression of take-all by two wheat-monoculture soils (E = Quincy Field, Washington State, U.S.A.; F = Longerenong College, Horsham, Victoria, Australia) but not by three soils not recently cropped to wheat (D = virgin soil near Quincy; G = long-term wheat-pasture rotation, Turretfield Research Centre, South Australia; H = soil from long-term mixed pasture from Long Ashton. England.)

Each soil was diluted 1:I00 with a fumigated (methyl bromide) soil. A = fumigated soil planted to wheat; without added inoculum of the pathogen; B = same soil not fumigated but 0.3% (w/w) inoculum of pathogen added; C = fumigated soil amended with pathogen inoculum.

moist heat for 30min likewise did not remove the antagonism operative at 28°C. However, when the soil was autoclaved (121 “C for 2 hr), the antagonism at 28’C was lost unless contamination of the soil occurred. This is similar to the experience of Ludwig and Henry (1943). The antagonism expressed at 28°C may thus be due to spore-forming bacteria which survive 70°C but not autoclaving. The paper in this series by Smith (1976) points out that ethylene C2H,, produced in soil by bacteria is suppressive to certain aerobic organisms, particularly fungi. Circumstantial evidence suggests it may play a role in Gerlagh’s “general suppression”. CzH4 production in soil increases as the temperature is increased from 15” to 35°C (Smith and Restell, 1971). C2H, production is delayed by NO;-N but is not affected or is possibly enhanced by NH:-N. The addition of organic materials increases the C2H, producing ability of a soil. C,H, production is also best in undisturbed sites (Smith and Cook, 1974). These are the treatments that also suppress G. Evaminis (Table 1). Moreover, preliminary work indicates that C2H, at concentrations below 5 parts/IO’ in the soil atmosphere is suppressive to G. gruminis in its parasitic state (Cook. unpllblished data). Shipton (1975)has summarized the various theories on mechanisms of take-all decline (specific antagonism) as follows: (1) The fungal inoculum alone induces a highly specific microbial antagonism (Gerlagh, 1968). (2) Trophic response of the pathogen hyphae to host roots is inhibited by a bacterial flora in the rhizosphere, possibly through antibiotic action (Pope and Jackson, 1973). (3) A specific antagonistic microflora develops near infected roots or in lesions in response to pathogen mycelia which subsequently decreases the survival of

the pathogen and its infectivity by inhibition of hyphal branching (Vojinovic’, 1972). (4) The occurrence of take-all causes a change in the soil microflora that subsequently limits further disease by modifications of the nutritional environment of the root perhaps by shifts in the ratio of NOT/NH:-N (Brown et al., 1973). All theories involve a role for antagonistic microorganisms. The possible role of a mycovirus (Lapierre et al., 1970) in take-all decline is now generally discounted (Rawlinson et ai., 1973: Baker and Cook. 1974). The pot bioassay of Shipton rt al. (1973) demonstrates that the transfer of specific antagonism depends upon a specific microflora that develops when wheat is infected with G. gruntinis but which does not develop with other plants nor when wheat grows without the pathogen. In fact, we have shown that the non-suppressive soils from legume/grass pasture have a more abundant and varied microtlora than soil which has had continuous wheat for many years. This specific antagonism is eliminated by 6O’C moist heat in contrast with the general, non-transferable antagonism which survives this heat treatment (Gerlagh, 1968; Shipton et al., 1973). Both forms of antagonism are destroyed by heating to 121°C indicating that biological factors are responsible. There is substantial evidence that the sites of specific and general antagonism differ, the former operating in the rhizoplane and the latter more in the bulk soil extending into the rhizosphere. The results of Pope and Jackson (1973) in which they transferred the “take-ail decline factor” by dipping the roots of wheat in a suspension of suppressive soil and planting into a conducive soil, and those of Shipton (1969) and Vojinovic’ (1972) who found that initial attack of roots by G. graminis was not prevented but rather,

R. J.

‘72

Coon

and A. D. Rovt~n

and of the pathogyn accotprtnied by Igsis of the hyphae and protectIon of the roots. (f, Pseudomonads are common inhabitants of the rhizospherc and rhizoplane, especially with wheat (Martin. t 971 f. (g) Dilution plate counts using a seiective medium Survives methyl bromide Eliminated by methly bromide (Simon and Ridge, 1974) have shown that Ruorcsccnt and chloropicrin and chloropicrin pseudomonads arc 100&1000-fold more numerous on increased by warm SoiIs (>25’C) Qperares in cooler 50i15 lesioned thnn on healthy roots (Rovira and Coak. ilO0 - 25%) 1975). Not transfercable Transferrable (h) Ridge (1976) has shown that over 7%:, of fiuorProbably operater :n bulk soil Operates in rhizoplane and cscent pseudomonads isolated from soil were antarhizosphere gonistic on agar to I;. ~~~~~~77~/7~scompared with some ?Oy:, of bacteria isolated on non-sefective media and 7”;; of ~ff~~~~~,~,~ spp. (i) In a field trial (Warcup. 1976; Rovira, 1976) G. <~ran?inisrecolonized soif and colonized the roots of the ectotrophic growth of the fungus along the root was reduced, both support the hypothesis that specific wheat growing in soil fumigated with methyl bromide or c~lloropi~ril~. hut in titc rncth\l hrcmidc phttr;. ~~nt~lgonisrnoperates in the rhizoplane. take-all was severe while tvheat iri the chloropicrin We grew wheat in axenic culture in sterilized sand in 3 x 20cm test tubes and inoculated these with plots showed no above-ground qmptoms and grain agar plugs of G. graminis, together with dilutions from yields were high. The explanation offered is that for 4 months following chloropicrin, the populations of bulk soil, the rhizosphere and the rhizoplane of wheat fluorescent pseudomo~ds were from 500 to lK!O-fold grown in fumigated soil inoculated with G. ~~~~~?~~~~js greater in the chtoropicrin-fumigated plots than in the and the suppressive QF soil; we found the antagonissoil treated with methyl bromide, and that the high tic factors in highest concentrations in the rhizoplane. ~pulatioIls of these ~~n~~gonistic bacteria suppressed Our hypothesis that fluorescent ps~~ldon~on~~dsare on the roots. responsible for specific suppression is based upon the c. ~~~?~~~j~~~~~ Although the evidence strongly favors the Iluorcsfol~o\~~n~obser~~~~~ions: cent pseudomonads as being responsible for the speci(al In one trial over 100 isolates of bacteria and actinomycetes from soil, and from diseased and pro- fic suppression of take-all, our results indicate that tected wheat roots were screened for their ability to it is necessary for specific strains to be present in suppress take-all in the pot bioassay; only eight ctd- order to obtain sil~~ression of the disease under turcs gave suppression equal to, or better than the either natural field conditions or in the pot bioassay. suppressive QF soil and these were all Psrudon~orzus This conclusion is based upon our findings that, spp. (seven were fluorescent), despite the fact that we although many pseudomonads inhibited G. yr~~~ir~is did not use any selective procedure to obtain the iso- on agar media or protected wheat under axenic conlates. Among the isolates were many Buciltt~s spp., ditions in sterile sand, few isolates protected wheat in the more competitive soil and rhizosphere environStwptmr;ccs spp., Gram-positive and Gram-negative rods. but apart from some protection given bv two ment of fumigated soil used in the pot bioassay. This paper sLIrnrn~~r~~es our views on the nature isolates of ~ir~~p~or~?~e~~, none was protective in the of both the general and specific sqqmssion of G. p-apot bioassay. (b) The specific antagoll~sm is eliminated by 6WC mid in the field and. ~~ithough at this stage of our moist best for 3Omin which kills pseudomonads but knowledge we have to say that each type of suppression develops under quite different conditions, we not sporing bacilli nor many actinomycetes. (c)The suppressive QF soil contains 104 fluorescent hope that the two mechanisms are not mutually er;clusive. Further study is required to define more ps~Lldomollads~g compared with 10/g in nearby conducive Quincy virgin soil, but the counts of fungi. precisely the processes involved and by such resolutotal bacteria, aerobic sporing bacteria and actinomytion of the problem we hope that it will be possible cetes wcrc similar in the two soils. to develop agronomic techniques to exploit the two (df VojinoviC (1972) observed that when wheat was control rnec)~al~isrnsand so reduce the losses in cereal protected against G. yraminis, lysis of the hyphae on grain production caused by G. orclrltiizis. the wheat root occurred in association with a prolific growth of bacteria and that, upon isolation, these bacteria were “asporogenous, Gram-negative+ small rodshaped bacteria”,--- a description which fits the pseudomonads. REFERENCES (e) Rovira and Campbell (1975) observed under BAKER K. F. nnd COOK R. J. (1974) scanning electron microscopy a massive development Plartr Palhog~ns. Freeman. San Franc~sco. of small, non-sporing rods on the roots of wheat in BIXKXSD. f-i. and DAWSO~: M. G. (196X) Intiuence of dirthe vicinity of lesions caused by G, gr~~ninis~ and colect-drilling of winter wheat on incidence of take-all and onization of the hyphae by small rod-shaped bacteria eyespot. Ann. qpl. Biol. 61. 57 64. followed by lysis of the hyphae in a suppressive situa- BROWN M. E., HOKWY D. md PEARSON V. (1973) Mition. They also found that under axenic culture condicrobial populations Rand nitrogen in soil growing contions. a culture of Ps~~~i[~f~zo~?~~~~ ,~~~~~sc~ffsfollowed secutive cereal crops infected with take-all. J. Soil Sci. 24. 296. 3IO. ;I similar pattern of colonization of the infected root Table

I. Characterization

~~il~~o~js~s

of genemi

in soils suppressive

and specific

to G. ~~~~~~~~s

Biological

control

Cooti R. J.. HUH~K D.. POW~LSOY R. L. and BKLIFHL G. W. (1968) Occurrence of take-all in wheat in the Pacific Northwest. PI. Dis. Rqm 52. 71&718. COOK R. _I.. PAPEUDI(.K R. 1. and GRIFFIN D. M. (1972) Growth of two root-rot fungi as affected by osmotic and matric water potentials. Proc. Soil Sci. Sot. A/II. 36. 7X-82. DI.AC‘O\I J. W. (1976) Biological control of the take-all fungus. G. qvo~~~ni,\. hq P. rtrdicrdu and similar funsi. so;/. Bio/. BiOChOJl. 8. 275-283. FI.LLO~S H. and FI~KI C. H. (1934) Wheat take-ail. Karlx~s Aqric,. t’u/x Sm. ;I~lri. R~J. 1932. 34. 9.5 96. GEKLACH M. (196X) Introduction of 0. qrcminis into new polders and its decline. .vct/i. J. PI. Ptrrh 74. (suppl. 2) I ~91. HENRY A. W. (1932) Influence of soil temperature and soil sterilization on the reaction of wheat seedlings to 0. qrtr,,lir~i.s &cc. Carl. J. Rd. 7. 198~203. LAPI~RRI: H.. LEMAIR~ J. M.. J~VAX B. and MOLIV G. (1970) Mix en evidence de particules niales associees a une perte de pathogenicltc chez le Pietin-echaudage des ccrcales. 0. yrarniuis &cc. c‘. I’. lochs. SPmc. ilc&. SC,,.. Puris 271. 1833 1836. L~owrc; R. A. and H~NRV A. W. (1943) Studies on the microbiology of recontaminated sterlhzed soil in relation to its infestation with 0. grrrn~irlic Sacc. Carl. J. Rcxc. C. 21. 343 350. MAR~IU J. K. (1971) Influence of plant species and plant age on the rhiLosphere microflora. -I~,sr. J. hid. Sci. 24. 1143~1150. POPI; A. M. S. and JACKKIN R. M. (1973) Etfects of wheat& field soil on inocula of G. grcrmiuis Sacc. Ars and Olivier var. /ritici J. Walker in relation to take-all decline. Soil Hiol. BiOchC,fU.5. 88 I 890. RAWLINSON C. J.. HORXB~ D.. PI.ARSON V. and CARI’~\ rt R J. M. (1973) Virus-like particles in the take-all fungus. G. qrar~i~f.s. A~rl. crppl. Bid. 74. 197-209.

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RIIXX E. H. (1976) Studies on soil fumigation--II. Effects on bacteria. SOI/ Biol. BIo~IzL’II~. 8. 249 153. RO~IRA A. D. (1976) Studies on soil fumigation-I. Effects on ammonium. nitrate and phosphate in soil and on the growth. nutrition and yield of wheat. Soil Biol. Bio(./1m. 8. 24 I~_247. ROVIRA A. D. and CAIMI’B~LI R. (1975) A scanning electron microscope study of the interactions between microorgmisms and G. q~a,ll,~r.s. (Syn. 0. qrtrr~i~is) on the roots of wheat. !!lic~rohiol Ecol. 2. SIIIUO~ P. J. (lY69) Take-all Decline. Ph.D. thesis. Universit4 of Reading. England. SIIII>~O\ P J. (lY75) Take-all decline durmg cereal monoculture. III Rioloy~~ ml Comd of’ Soil-BOIW P ht P~thogem (G. W. Bruehl. Ed.) The Am. Phytopath. Sot.. St. Paul. Minnesota. pp. 137 134. SHtI’.roN P. J.. C‘OOK R. J. and SITTO~ J. W. (1973) Occurrence and transfer of a biological factor in soil that suppresses take-all of wheat in eastern Washington. Phytopddo