Pestic. Sci. 1993, 37, 417426

Biocontrol of Soil-borne Plant Pathogens : Concepts and Their Application* James W. Deacon1 & Lorraine A. Berry Institute of Cell and Molecular Biology, University of Edinburgh, Daniel Rutherford Building, King’s Buildings, Mayfield Road, Edinburgh EH9 3JH, UK

(Revised manuscript received 23 March 1993; accepted 6 April 1993)

Abstract: A few soil-borne plant pathogens have been controlled successfully by commercial formulations of biocontrol agents, but many attempts to develop biocontrol inoculants, although promising under experimental conditions, have met with difficulties in practice. The reasons for this are discussed in this review, which outlines some of the major findings on the behaviour of microbial inoculants in soil. It is emphasized that biocontrol also occurs naturally in current agricultural practice and can be exploited purposely, but it is vulnerable to disruption by agrochemicals or mismanagement. The future of biocontrol of soil- borne plant pathogens probably lies in integrated (biorational) control systems that combine the use of commercial inoculants, where appropriate, with man- agement practices that maintain and enhance the natural biocontrol systems.

1 INTRODUCTION

Garrett stated ‘There are no short cuts to biological control; that is the mistake that many of us have made in the past and that some of us, no doubt, will make again in the future’.l

There is increasing opportunity to put biocontrol into practice. The opportunity is generated by consumer demand for residue-free produce, by concern about pesticides in the environment and by the ever-increasing costs of meeting the registration requirements for new chemical products. With increasing opportunity come increasing expectations and increasing pressures for commercialization. Thus, much of the research on biocontrol of soil-borne plant pathogens in recent years has focused on the development of inoculant biocontrol agents (BCAs)-those that can be produced in culture, formulated as products and used commercially in place of chemicals. However, the limitations of this approach have become apparent in the widening gulf between promise, as evidenced in the research literature, and practice, as evidenced by the paucity of commercial

* Based on a paper presented at the meeting ‘Biological Control: Use of Living Organisms in the Management of Invertebrate Pests, Pathogens and Weeds’, organised by the SCI Pesticides Group and held at the SCI, 14/15 Belgrave Square, London SWlX 8PS, UK on 19-20 October 1992. $ To whom correspondence should be addressed.

products. There are major difficulties in applying inoculant organisms in practice, especially in field sites rather than in protected environments. Meanwhile, the opportunities for exploiting ‘know-how’ to make better use of resident soil-borne antagonists, or even inoculant organisms, have tended to be ignored.2 This impinges directly on the agrochemical industry, because ‘mild ’ chemicals and behaviour-modifying compounds could have profound effects on the biological balance in soil.

Because there have been many recent, detailed reviews of biocontrol of soil-borne plant pathogen^,^-^ our purpose here is to ask what concepts have emerged from recent work in this field and how these concepts might be applied in practice. These issues will be approached through a number of statements and propositions, relating first to the use of biocontrol inoculants and, second, to the activities of resident control agents in soil.

2 BEHAVIOUR OF INOCULANT BIOCONTROL AGENTS

2.1 define the circumstances in which it can be used

The mode of action of an inoculant BCA helps to

The modes of action against soil-borne pathogens fall into four broad categories : pre-emptive competitive exclusion; antagonism by antibiosis, interference, para-

417 Pestic. Sci. 0031-613X/93/$06.00 0 1993 SCI. Printed in Great Britain

418 J . W. Deacon, L. A . Berry

sitism or predation; induction of host resistance, and transmission of hypovirulence genes. BCAs that act locally on a pathogen-whether directly or through the host-can be used to give short-term protection at the site of application but otherwise will need to be, or become, generally distributed through the soil or on roots. Conversely, BCAs that induce systemic host resistance against a pathogen can be effective without spreading from the site of application.

2.2 Competition (pre-emptive competitive exclusion) is probably the major mechanism of pathogen control in soil

Even if competition is not the major mechanism it is at least a contributory factor when other modes of action can be dem~nstrated.~ However, it requires that the BCA is ecologically adapted to exploit the same resource as the pathogen-the same site or nutrient, at the same time.

Competition is difficult to prove as a biocontrol mechanism and often relies on 'negative' evidence-the elimination of all other possibilities. Perhaps the clearest example is found above-ground in the control of ice- nucleation active bacteria (ice+) on leaves by mutants (ice-) that lack the surface protein that has ice-nucleation activity.' In this case, a direct antagonistic effect in biocontrol can be excluded, because the ice- and ice+ strains can be isogenic except for a mutation at a single gene locus, and antibiotic-producing organisms are naturally resistant to their own metabolites, so the near- isogenic forms would also be resistant. Moreover, the ice+ strains grow as saprophytes on the leaf surface, so any induction of host resistance against them by ice- forms can be discounted.

Competition for carbon nutrients during pre-pen- etration growth of vascular wilt fusaria (Fusarium oxysporum Schlecht) in the rhizosphere is thought to be a major mechanism of biocontrol by non-pathogenic strains of F. o x y ~ p o r u m . ~ Competition for the resources provided by naturally senescing cereal and grass root cortices seems to be a major mechanism of biocontrol of take-all by the weakly parasitic fungi that naturally exploit these tissues, including Phialophora graminicola (Deacon) Walker and Idriella bolleyi (Sprague) von Arx.", l1 These fungi are closely related ecologically, and in one case taxonomically, to the pathogen, as in the case of ice-nucleation control. Also, the pathogen needs nutrients to support its pre-penetration growth (a common feature of soil-borne plant pathogens) and the control does not operate if the pathogen has adequate nutrients in its inoculum food base.12 Nevertheless, it is impossible to exclude completely a contributory role of induced host resistance in take-all control, especially when this induced resistance is strictly localized to the vicinity of the control agent.13~14

Competition for iron is suggested to play a role in biocontrol of several pathogens by fluorescent pseudo- r n o n a d ~ , ~ ~ . ~ ~ but is considered unimportant in other cases.17 Iron competition is mediated by production of siderophores with extremely high affinity for the ferric ion, coupled with the possession of specific siderophore- receptor proteins in the cell membrane. It is amenable to precise experimental analysis because mutants of pseudo- monads deficient in these properties are easily generated, and iron availability in soil can be controlled with chelators or adjustment of pH. Production of sidero- phores has been detected in the rhizosphere, and 'reporter gene' systems can be used to assess both the concentration of iron in the rhizosphere and the in-situ expression of iron-regulated genes.IB

2.3 Production of antibiotics and other bioactive metabolites can contribute to biocontrol in soil''

The evidence for this is of three major types: mutational, in which antibiotic-deficient or over-producing strains are compared with wild-type strains for biocontrol efficiency ; correlative, in which sensitivity of pathogens in vitro to the metabolites of BCAs correlates with control in vivo, and direct detection of metabolite production in the root zone of inoculated plants. Thus, recent evidence indicates a role of the antibiotic pyrrolnitrin from Pseudomonas cepacia Burkholder in control of Rhizoctonia solani Kiihn on sugar beet," a role of phenazine- I -carboxylic acid from Pseudomonas fluorescens (Trevisan) Migula, strain 2-79, in take-all

and roles of pyoluteorin and 2,4-diacetyl- phloroglucinol from strain CHAO of P. fluorescens in control of Thieluviopsis basicola (Berk & Br.) Ferris on tobacco.22 The degree of control can depend on the amount of antibiotic produced, and this in turn relates to the degree of establishment of the BCA in the root zone-typically higher on seedling roots and in pasteurized rooting media than on older plants and in natural soil. The development of antibiotic- overproducing mutants may provide a means of over- coming this limitation, because the degree of expression of antibiotic genes in the rhizosphere can be only a few per cent of that in laboratory culture.23 On the other hand, at least some of the antibiotics can be phytotoxic, leading to growth depression of inoculated plants.24

Apart from 'conventional' antibiotics, hydrogen cy- anide from P. juorescens has been implicated in biocontrol of several diseases.25 Glucose oxidase from the fungus Taluromycesflavus (Klocker) Stolk & Samson is implicated in control of Verticillium dahliae Kleb. on aubergines, because the enzyme cleaves root-derived (or other sources of) glucose to generate hydrogen peroxide that is toxic to the pathogen.26 The widely used commercial biocontrol strain of Agrobacterium radio- bacter Conn (K84) is thought to control crown gall by producing a diffusible nucleotide analogue, agrocin 84,

Biocontrol of soil-borne plant pathogens 419

that specifically affects Agrobacterium tumefaciens Conn because only this pathogen can transport the inhibitor across its cell mernb~ane .~~*~* However, agrocin 84 has yet to be detected at biocontrol sites on roots, and transfer of the agrocin-encoding plasmid to other Agrobacterium strains does not always make these as effective as K84 in biocontr01.~~ Pre-emptive competitive exclusion from wound sites, or from other rhizosphere resources, may be an additional mechanism. This could also apply in many other cases where fungitoxic metabolites have been implicated in bio~ontrol.~

2.4 Contact or post-contact interference can contribute to biocontrol in soil

Among commercial BCAs this phenomenon is best illustrated by Phlebia gigantea ( = Peniophora gigantea (Fr.) Massee), used for biocontrol of Heterobasidion annosum (Fr.) Bref. in pine plantations in Europe and North America.30 Like many basidiomycetes, Phlebia antagonizes other fungi on contact by a phenomenon termed hyphal interferen~e.~~ The ffeterobasidion hypha shows membrane dysfunction, followed by cytoplasmic vacuolation and coagulation, in the specific region (cell) of contact with Phlebia. But Phlebia does not parasitize the other hypha, nor apparently gain directly from release of its nutrients; instead, hyphal interference seems to be a mechanism for disrupting other fungi that are competitors for the same underlying substratein this case, the freshly cut surfaces of pine stumps.

An almost identical mode of behaviour is shown by Gliocladium ruseum Bain and closely related species (G. atrum Gilman & Abbott, G. catenulatum Gilman & Abbott) when they contact hyphae of several plant pathogens on agar,32 although some pathogens are inhibited at a distance by G. roseum and yet others are reported to be parasitized by it.33 This potential overlap between contact interference, production of diffusible inhibitors and mycoparasitism (Section 2.5, below) is found in several other biocontrol agents, such as Gliocladium virens Millar, Giddens & Foster and Trichoderma spp.34-38 In some cases, as with G. roseum, the mode of action may differ for different pathogens or different strains of the antagonist.

2.5 Several biocontrol agents can parasitize other fungi, destroying them and thereby gaining nutrients

Perhaps the best evidence for this-at least in terms of nutrient transfer in laboratory conditions-oncerns the six mycoparasitic Pythium species : P. oligandrum Drechs., P. acanthicum Drechs., P. periplocum Drechs., P. nunn Lifshitz, Stanghellini & Baker, P. mycoparasiticum Deacon, Laing & Berry and P. acanthophoron Sideri~.~'~' With the possible exception of P. nunn,43-45 these fungi do not produce diffusible inhibitors in culture, nor substantial amounts of wall-

31

lytic enzymes, and thus differ from other reported mycoparasites such as T r i c h o d e r m ~ ~ ~ and G. v i r e n ~ . ~ ~ Nevertheless, it can be debated to what degree the mycoparasitic Pythium spp. rely on mycoparasitism per se in soil; they could, alternatively, be successful competitors for crop residues or secondary invaders of diseased plant 46

Other mycoparasites that have been tested as inoculant biocontrol agents include Talaromyces flavus (which sporulated on microsclerotia of Verticillium dahliae on plant roots, but not on the roots themsel~es),~' Verticillium biguttatum Gams (which seems specifically to attack Rhizoctonia spp. and a few other sclerotial fungi),48 and Sporidesmium sclerotivorum Uecker4' and Coniothyrium minitans Campbellso which invade the sclerotia of Sclerotinia spp. and Sclerotium cepivorum Berk. in soil. Again, there is potential overlap in modes of action. S. sclerotivorum is suggested as destroying sclerotia by growing in their intercellular spaces, utilizing the small amounts of nutrients there and creating nutrient stress ; the sclerotial cells respond by producing 8-glucanase, the synthesis of which is otherwise repressed by glucose, and the pathogen auto-degrades its sclerotial cell walls.'l T. Jlavus is suggested as utilizing sclerotial nutrients also, but destroying the pathogen cells by generating hydrogen peroxide, via its glucose oxidase enzyme.

2.6 Some biocontrol agents act by inducing systemic host resistance to pathogens

There are many experimental examples in which in- oculation of plants with weak pathogens or non- pathogens leads to induced systemic plant resistance against subsequent challenge by pathogen^.^^^^^ The mechanisms remain largely unknown but, typically, the induced resistance operates against a wide range of pathogens and can persist for three to six weeks; then a booster treatment is required. Induced resistance is thought to be the principal means by which non- pathogenic strains of Fusarium oxysporum protect sweet potato cuttings from attack by pathogenic strain^.'^ The technique is used commercially in Japan as a pre-plant treatment of the bases of sweet potato cuttings. It requires sufficient inoculum to cause localized rotting of the stem base, and the induced resistance lasts for only a limited time, but it is sufficient to give season-long control of vascular wilt because the plants are most susceptible when young and then naturally develop resistance to the pathogen. Induced systemic resistance has also been demonstrated when carnation roots were inoculated with a Pseudumonas strain and the plant stems were subsequently challenged with a vascular wilt f ~ s a r i u m . ~ ~ Treatment of the roots with lipopoly- saccharide from bacterial cultures also induced the phen~menon.~~ This raises the prospect that a stable biological product rather than a living biological agent

EPS 3 1

420 J. W. Deacon, L. A. Berry

might be developed commercially as an inducer of resistance.

Perhaps also relevant is the role of manganese in plant resistance, reviewed recently.57 The availability of manganese to plants is strongly influenced by its state of oxidation in soil. This in turn can be influenced by rhizosphere micro-organisms, which thus might act indirectly to promote resistance through plant nutrition.

2.7 The hypovirulent phenotype can be transferred to pathogens by anastomosis, leading to disease control

The classic example of practical exploitation of this phenomenon, reviewed re~ently,~' is in the control of Cryphonectria parasitica (Murrill) Barr, the chestnut blight pathogen, in Europe. Hypovirulent strains of this pathogen contain virus-like double-stranded RNA, which can be transmitted to virulent strains during hyphal fusions, rendering them hypovirulent. Attempts to exploit the same phenomenon in soil-borne pathogens are still at an early stage and are complicated by conflicting evidence on the role of dsRNA in fungal v i r u l e n ~ e . ~ ~ ~ ~ ~ Hypovirulent strains of R. solani have been shown to control virulent strains on seedlings in experimental conditions, but whether by transmission of hypovirulence or by other mechanisms is still unclear.

2.8 Biocontrol is often multifactorial, with a combination of mechanisms even in single control agents

Evidence for this has already been seen in many examples above. For instance, the production of hydrogen cyanide and of the antibiotics pyoluteorin and 2,4-diacetyl- phloroglucinol are jointly implicated in the suppression of diseases by strain CHAO of P. f l ~ o r e s c e n s . ~ ~ We have argued el~ewhere~.~' that access to nutrient resources at, or near, the site where the pathogen is active will underpin all other mechanisms, because the production of toxic metabolites, or attempted invasion to induce host resistance, is nutrient-dependent. Also video- microscopy of interactions between single hyphae of pathogens and antagonistic fungi on agar films indicates that the maximum distance over which antibiotics diffuse from single hyphae to affect others is often 300pm or less, but was up to 600 pm when Trichoderma harzianum Rifai grew from a nutrient-rich agar block and inhibited Pythium aphanidermatum (Edson) fit^.^',^^ It follows that, in order to antagonize by diffusible antibiotics, a control agent must have access to nutrients close to the pathogen.

2.9 In general, biocontrol agents are most effective when (a) applied to the sites where they will act, (b) supplied with 'starter' nutrients and (c) introduced into non-competitive environments

All these factors relate to the need of biocontrol agents

to establish high populations in order to be effective. It is widely recognized that Trichoderma spp. and Gliocladium virens need to be supplied with a food base when added to soil, to support their population increase and antibiotic production in competition with resident soil micro-organisms. To achieve this, wheat bran or other such nutrients are usually added to prills composed of inert carrier materials, and a further advantage can be gained by 'activating' the BCA before it is added to soiLB2 Seed-derived nutrients can also support population increase of inoculant BCAs such as bacteria,63 Penicillium oxalicum ThornB4 and Idriella b ~ l l e y i . ~ ~ In cases where the inoculant control agent may not exploit the seed nutrients or compete with resident microbes rapidly, the inoculant can be favoured by activation during seed priming treatments or by incorporation of nutrients into seed coatings.66 The widespread success of Agrobacterium radiobacter and Phlebia gigantea as commercial bio- control agents is similarly related to the fact that they are applied to sites (bare-rooted transplants and freshly cut pine stumps, respectively) where they have immediate and preferential access to nutrients for their initial establishment.

Two particular problems have been found in the control of seedling disease by seed-applied BCAs, perhaps explaining why this seemingly simple goal has proved so difficult in practice. First, pathogens such as Pythium spp. can respond remarkably quickly to seed- derived volatile compounds or other seed diffusates, being triggered to germinate within 1-13 h and colonizing and rotting the seeds a few hours later; BCAs should thus respond equally q~ickly.~' Second, even when pre-emergence damping off and seed rot have been controlled effectively by seed-applied BCAs, the plants can die subsequently from attack on the hypocotyl or stem base if this remains relatively free from colonization by the BCA. Seed-applied BCAs can almost invariably be expected to move downwards into the rhizosphere more than upwards towards the soil surface, unless the seed coat is carried upwards by epigeal germination, as in soybean.6s

2.10 Biocontrol agents are seldom effective against high pathogen pressure

There are two separate issues here, although they may not always be easy to disentangle. First, a biocontrol agent may have some effect against a high pathogen population, but the effect may be insufficient to give worthwhile, economic control. This seems to be true of many attempts to control sclerotial pathogens (e.g. Sclerotinia spp.) when mycoparasites such as Coniothyrium minitans are added to A large proportion of the sclerotia may be destroyed, but those remaining can be sufficient to cause economic losses. Nevertheless, a degree of control can carry over to subsequent crops. Second, the mechanism of biocontrol

Biocontrol of soil-borne plant pathogens 42 1

may depend on the population balance of pathogen and control agent, or on the food base available to the pathogen. For example, successful control of crown gall by strain K84 of A. radiobacter depends on the population of A . radiobacter being at least equal to that of the pathogen, though the reason for this is unclear. Phialophora graminicola, and other weak parasites such as Zdriella bolleyi, can control take-all only when their populations greatly exceed that of the pathogen71 and, in particular, when the pathogen must infect from a critically low food base.12 Typically, this occurs after a break in a sequence of cereals, when the crop residues that contain the surviving pathogen inoculum are progressively fragmented and decompo~ed.~' Then the pathogen may need to exploit the naturally senescing root cortex of the succeeding cereal crop in order to rebuild its food base for infection, and the weak parasites competitively exclude it from this resource.

In practice, it may be necessary to weaken a pathogen or otherwise reduce its inoculum before an inoculant BCA can have effect. The weakening effect is a long- established principle, reviewed For example, sub-lethal concentrations of soil fumigants can serve to eliminate pathogens such as Armillaria mellea (Vahl. ex. Fr.) Kummer from woody host residues, by promoting invasion by resident Trichoderma spp. which oust the pathogen.74 Solar heating beneath plastic mulches is also effective for many field crops.75 Most of the commercial soil fumigants such as methyl bromide pose serious environmental and health hazards. Less dangerous compounds could be used in conjunction with biocontrol agents, a recent example being the experimental use of furfuraldehyde for control of Sclerotium rolfsii S ~ C C . ~ ' These and the other examples in this section illustrate that biocontrol agents often need to be used differently from fungicides : to prevent the build-up of damaging pathogen populations or, in conjunction with other management strategies, to reduce existing pathogen populations.

2.11 Biocontrol agents can seldom be expected to colonize thoroughly and to persist in soil at levels effective for season-long control

Micro-organisms typically have phases of activity, corresponding to windows of opportunity when nutrients and physicochemical conditions are optimal, interspersed with phases of stasis or decline when conditions are not optimal. Even the rhizosphere is a highly structured env i r~nmen t~~ consisting of a succession of niches as the root tip extends through soil and the root progressively ages behind the tip. For example, Pseudomonas spp. are particularly well adapted to exploit the relatively rich source of nutrients near the root tip so these bacteria achieve high populations on inoculated seedlings. But they compete relatively poorly with other rhizosphere micro-organisms on older root regions, and then would

not be expected to produce antibiotics sufficient for maximum biocontrol. Strain 2-79 of P. JEuorescens can give excellent early control of take-all in wheat crops but the control breaks down by mid-season when the inoculant population has declined. But its numbers rise again in root regions that become infected by the take-all fungus, presumably because the pseudomonad can exploit the locally abundant nutrients from disease lesions. 78

2.12 Biocontrol agents can be crop and soil-specific

Crop specificity and even cultivar specificity is common in plant growth-promoting rhizoba~teria.~~ The weakly parasitic fungi that control take-all are specialized colonizers of roots of graminaceous plants,so although some of them grow poorly on oats." Phlebia gigantea is a specialized colonizer of pines.'* On the other hand, strain K84 of Agrobacterium radiobacter is effective for crown gall control on a range of plants, not just almonds from which it was first isolated.

Soil type can strongly influence the growth of biocontrol agents by either direct effects-for example, Trichoderma spp. are typically acidophilics3-or indirect effects operating through competition by resident soil microbes. Transport in percolating water is known to be important for spread of inoculant biocontrol agents in soil or on root^.^^,^^ It can be facilitated by irrigation, but in any case is influenced by, among other things, soil pore distribution (e.g. sands versus clays) and divalent cation availability.86 Metabolite production can be influenced specifically by soil properties, an example being the ability of P. Juorescens to antagonize pathogens in montmorillonite but not illite clay soils, because only the former seem to make iron readily available to the control agent.17ss7 Soil type also significantly affects the success of inoculant pseudo- monads in take-all control.ss

2.13 Mixtures of biocontrol agents may be more effective than single agents

Mixtures of biocontrol agents might be particularly effective when they have different modes of action or different ecological attributes so that they function at different times or locations. Mutual compatibility of fluorescent pseudomonads and non-pathogenic Fusarium oxysporum enables these agents to act additively or synergistically in vascular wilt Even for single agents it may be necessary to include more than one strain in a formulation. This point has been demonstrated for ice- mutants of Erwinia, which were more effective in controlling their relevant parent strains than in controlling other wild-type strains on potato leaves.eo It seems that Erwinia strains have different patterns of resource utilization and the mutants retain these patterns, so mixtures of ice- strains may be

422 J. W. Deacon, L. A . Berry

necessary for effective control of mixed wild-type populations. Similarly, mixtures of fluorescent pseudo- monads can be more effective than single strains in disease control on roots.91

2.14 Biocontrol agents can be unstable in culture and possibly change after application to soil

Early work on plant growth-promoting rhizobacteria was plagued by problems of instability during culture maintenance, leading to inconsistent results in practice." Even one or two routine sub-cultures on laboratory media can alter the growth-promoting or growth- reducing effects of p~eudomonads.~~ A related problem might occur in Agrobacterium, because the original biocontrol strain (K84) presumably was motile but some routinely maintained subcultures of it are now non- motile (Deacon, J. W., unpublished). This might even be true of strain K84 in some commercial biocontrol products.

Stability after application to soil has received little study, but there could be selection for mildly deleterious activity in the competitive environment of the rhizosphere.' There is some evidence that strains of Zdriella bolleyi from continuous cereal cropping sequences are more damaging to barley seedlings than are strains from rotation-cropped cereals, but this deleterious activity is lost during laboratory

3 MANIPULATION AND MANAGEMENT OF RESIDENT BIOCONTROL AGENTS

Many of the points above do not pose insurmountable problems for the development of biocontrol inoculants, but they have focused attention increasingly on the need to use biocontrol agents in conjunction with other disease-control measures. These measures include cul- tivation and fertilizer practices, use of soil-amendments, use of partially resistant cultivars and even the use of compatible chemical treatments in what has become known as ' biorational' disease control. Similarly, at- tention has re-focused on the natural, resident biocontrol agents in cropping systems and on ways in which their activities can be enhanced.

Below we list a number of points that are relevant in these respects, with special emphasis on recent work.

3.1 Natural biological control of soil-borne plant pathogens occurs by both general and specific mechanisms

Soil has a natural biological buffering effect, tending to limit population increase by pathogens and inoculant biocontrol agents alike. This ' general antagoni~m"~ or general soil suppressivene~s~~ is non-specific : it operates against most, if not all, pathogens. It involves the

activities of many resident soil organisms, and can be enhanced by addition of composted materials such as composted hardwood bark which may have particular value when used as a component of soil-less plant growth media.96

Specific suppressiveness operates in addition to general soil suppressiveness but against only certain types of pathogemS5 Specific suppressiveness has been described, inter uliu, for fusarium take-all," P y t h i ~ m , ~ ~ ~ Thielaviopsis ba~icola,'~ cereal cyst nematode," and R. solani.loO In all such cases the particular pathogen causes significantly less disease in suppressive soils than in other (conducive) soils; the effect is lost when the soil is treated with biocides, indicating the involvement of micro- organisms, and typically the effect can be serially transmitted to conducive soils (indicating the involve- ment of an agent capable of multiplying), provided that the microflora of the conducive soil is first simplified by partial sterilization.

All this raises the possibility that the responsible organisms could be identified, cultured and added to conducive soils to render these suppressive. Candidate control agents have, indeed, been identified in many cases (references above). But their use in practice has been limited by at least three factors. (a) Some of the suppressive agents cannot be cultured readily for production of inoculum (e.g. the fungus Nematophthora gynophila Kerry & Crump, for cyst nematode sup- pression). (b) The suppressive agents probably do not act alone but as components of mixtures of antagonists specifically adapted to particular soils. (c) The sup- pressive soils sometimes have underlying physico- chemical properties that favour the development and activity of the suppressive micro-organisms. Montmor- illonite clays have been associated with fusarium-wilt- suppressive soil in Central Arnerica'O' and Thielaviopsis- suppressive soil in S~itzerland;'~ high chloride content, among other things, has been associated with Pythium- suppressive soils in Calif~rnia;~' high soil moisture content and adequate pore size distribution, suitable for the motility of fungal zoospores, are necessary for suppression of cereal cyst nematode.sB

Attempts to mimic the natural suppressiveness of soils by addition of specific control agents may be successful, especially in soil-less systems or in soil subjected to prior biocidal treatment. However, for most field soils it may be more appropriate to manipulate the soil or site conditions so as to favour the activities of resident or introduced control agents.

3.2 Natural biocontrol agents with specific modes of action are common in soil. Their detection depends on the development of appropriate methods, and new agents remain to be discovered

These points are well illustrated by recent work on mycoparasites. These can be detected by pre-incubating

Biocontrol of soil-borne plant pathogens 423

soil with mycelia or sclerotia of pathogens, so that the mycoparasite population is enriched. Mycoparasites can then be isolated by placing the soil or retrieved mycelia/sclerotia on agar precolonized by an appropriate fungal host. Verticillium biguttatum was discovered in this way, and its soil population can be quantified by dilution plating on colonies of R. ~olani.~' The role of Trichoderma in suppression of R. solani was similarly established by burial of hyphal mats."' A recently described mycoparasite, Pythium mycoparasiticum,lo2 was detected by plating soil on agar precolonized by Phialophora. For a newly discovered species it was surprisingly common, being found in 19 of a total 51 soils from arable cropping sites throughout Britain.lo3 As an extension of this methodology, agar plates pre- colonized by different pathogenic fungi were found to differ in efficiency of detecting mycoparasites from ~ 0 i l . l ~ ~ For a total of 28 soils, Fusarium culmorum was the best detector fungus for Pythium oligandrum (detected in 18 of the soils) whereas R. solani failed to detect it in soil. Botrytis cinerea Fr. was the best detector for a Papulaspora sp. (in 19 of the soils) whereas F. culmorum did not detect it at all. Rhizoctonia solani was best for Trichoderma spp. (22 soils) and several fungi could be used to detect Gliocladium roseum (20-27 soils).

Such simple techniques could be powerful tools: (a) for identifying the resident antagonists of specific pathogens when these are used on precolonized agar plates; (b) for monitoring population changes of the resident antagonists when soils are treated in various ways, and (c) for identifying soils that are conducive to specific mycoparasites (on the basis that these soils already support their populations) so that the populations can be supplemented, if necessary, by inoculation.

Non-culturable or other, lesser-known mycoparasites can be detected by burial of pathogen structures in soil. For example, oospores of Aphanomyces, Pythium and Phytophthora spp.-the typical form in which these pathogens survive-an be heavily colonized and de- stroyed in soil by Hyphochytrium catenoides Karling, Humicola fuscoatra Traaen, Catenaria anguillulae Sorokin and Microdochium fusarioides H a r ~ - i s . ~ ' ~ - ~ ~ ~ The significance of these organisms as natural biocontrol agents is unknown, and must be balanced against their parasitic effects on spores of mycorrhizal fungi.ll'~ '11 But they might be manipulated to advantage by simple measures such as management of soil water content.lo7

3.3 Natural biocontrol is vulnerable to mismanagement

Take-all of bent turf (Agrostis spp.) occurs only under specific conditions, i.e. when existing turf is heavily limed to correct for excessive acidity, caused by neglect of turf pH during the persistent use of ammonium fertilizers, and when new turf is established following soil fu- migation. In all other circumstances the disease is

32

naturally suppressed by resident biocontrol agents, including the fungus Phialophora graminicola which is abundant on grass roots.l12 So this disease is a direct result of turf mismanagement or the destruction of natural biocontrol agents when soils are fumigated to destroy pathogens and weeds. The recent introduction of 'sand carpet' techniques for establishing golf greens also creates opportunities for the take-all pathogen to infect from air-borne ascospores, in the absence of resident antagonists. Fluorescent pseudomonads (watered onto the seedbed) and Idriella bolleyi (cited as a 'fungal BCA' and applied as fermenter biomass dried onto sand grains-Deacon, J. W., unpublished results) helped to delay the onset of take-all patch disease in a recent field trial.l13 But none of these control agents was wholly effective because the seedbed conditions, including high pH, were highly favourable to the pathogen.

Resident biocontrol agents can be adversely affected by fungicides in current cropping practice. For example, single applications of metalaxyl plus mancozeb to field soils at the standard recommended rate for control of cavity spot of carrots (Pythium uiolae Chester & Hickman) reduced the detectable resident population of the mycoparasite Pythium oligandrum more than 10-fold, and there was little or no recovery for at least two years.l14 In experimental conditions both the pathogen and its potential biocontrol agent were sensitive to metalaxyl-a problem that might be overcome by introduction of metalaxyl-insensitive P. oligandrum-but the control agent was much more sensitive to mancozeb than was the pathogen. This research raised doubts that the inclusion of mancozeb in the commercial fungicide formulation has any significant effect in control of P. violae, but may have serious adverse effect on resident P. oligandrum ,

3.4 Resident biocontrol agents may need to be augmented for effective disease control

One of the major advantages of exploiting resident biocontrol agents rather than inoculant BCAs is that the residents are already distributed in the soil profile whereas this can be difficult or expensive to achieve with inoculant organisms. However, the residents may not be present at sufficient levels for effective disease control at specific vulnerable sites on the plant or at specific vulnerable stages of crop growth. In these circumstances the population might be enhanced with an inoculant formulation, knowing that the site conditions are favourable for the control agent. Among many examples, Phlebia gigantea needs to be added to freshly cut pine stumps because the natural invasion of stump surfaces by this fungus is too low and unpredictable to guarantee control of Heterobasidion;30 similarly, Agrobacterium radiobacter is applied to seeds or bare-rooted transplants because the resident antagonist population may be insufficient to give complete protection. This strategy has

E P S 3 7

424 J . W. Deacon, L. A . Berry

been explored for Idriella bolleyi in take-all control. Seed-applied inoculum can significantly enhance the population near the crown of the plant, even though I . bolfeyi is almost always present on cereals in field sites. The high degree of localized establishment is expected to create a cordon sanitaire to protect the crown (and thus newly emerging adventitious roots) from early take-all infection7'

Different approaches have been necessary to exploit the mycoparasite Verticillium biguttatum for control of Rhizoctonia in potato cropping. The control agent does not grow below 13"C, whereas the pathogen can grow at nearly O"C, so potato crops are vulnerable to early infection by the pathogen, before the biocontrol agent can act from either resident or introduced inocula. In this case the proposed control strategies involve a com- bination of treatments, including low fungicide dosage to suppress the pathogen without adversely affecting V. biguttatum ; seed-tuber disinfestation by inoculation of V. biguttatum during storage, and cultivation and crop management to delay the onset of pathogen attack.4s

4 CONCLUDING REMARKS

Perhaps the greatest contribution that biocontrollers can make to current agricultural practice is to remind growers, plant breeders and agrochemical companies constantly that (a) biocontrol operates naturally in cropping systems but is vulnerable to disruption, (b) natural biocontrol systems are complex and multi- factorial, thus needing to be conserved in this state, and (c) understanding of these natural biocontrol systems, which is already highly advanced, can suggest simple, robust strategies for their enhancement. In plant breeding the trend of recent years has been away from dependence on single 'major' gene (vertical) resistance, towards niultigenic, (durable, horizontal) resistance in many crop species-a trend born of necessity because single major gene resistance proved unstable. The agrochemical in- dustry already faces the same problem, in the de- velopment of insensitivity of pathogens to fungicides with single-site modes of action. We have yet to see the full acceptance of a logical alternative strategy : the development and deployment of crop cultivars, fungicides and cropping practices in a way that is intended to exploit both natural and inoculant biocontrol agents; not alone, but in concert.

ACKNOWLEDGEMENT

L. A. Berry was supported by a Post-Doctoral Research Assistantship from the Agricultural and Food Research Council, which we gratefully acknowledge.

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