diversity in biological control

8.5

I

Review

Diversity in biological control Ralph Baker Plant Pathology and Weed Science, Colorado State University, Fort Collins, CO 80523, USA

Abstract

Keywords

Strange! All this difference should be 'Twixt Tweedledum and Tweedledee'

On the feuds between Handel and Bononcini: Miscellaneous Poems (Byrom, 1773)

There is suppressiveness to plant disease in almost all crop systems, largely attributable to the indigenous micro-organisms. Such populations are diverse and if they are eradicated by treatment, the soil is much more likely to be reinfested by a pathogen. Enhancement of this general suppressiveness may be induced by one or more specific antagonistic components of the soil biota, alone or in combination. The activity of these biological control agents can be increased in diverse microhabitats by manipulation of the nutritional, physical and biological environment of the soil. Diversity of agents with new attributes can also be brought about by mutation and/or genetic engineering.

Biocontrol; genetic manipulation; integrated control; plant diseases; soil-borne plant pathogens

Introduction

Diversity leads to good athletic events and horse races, as well as to progress in the biocontrol of plant pathogens. It is fortunate, therefore, that there are as many opinions, biocontrol agents and approaches to research on the subject as there are investigators in the discipline.

The discipline is based on diversity, whether the fasci- nation is in the disease problem to be solved, the biocontrol agent involved, or the interest of the investigator. This review therefore focuses on biocontrol viewed in the light of diversity. Authors may not have formally approached the subject by this mental pathway in the past, but it is the essential raison d'~tre underlying the bold assumption that plant diseases can be controlled by biological strategies.

Diversity in biological control agents

An antagonistae vitae lists at least 44 agents that potentially can suppress plant diseases (Cook and Baker, 1983). The authors predict that 'when the next Antagonistae Vitae is prepared, it certainly will be much longer'. Indeed, there are more reports in the recent literature of agents that potentially are useful in biocontrol (Lifshitz, Stanghellini and Baker, 1984; Utkhede, 1987: Turhan and Turhan, 1989: Klecan, Hippe and Somerville, 1990); however, one also could speculate that the major groups of biocontrol agents have been discovered and future success in their application to plant disease problems lies in strategies for enhancement (Baker and Scher, 1987; Baker, 1990a).

There is wide diversity in entities that induce suppressiveness. Cross-protection of plants from injury from virus

0261/2194/91/02/0085-10 ,~". 1991 Butterworth-Heinemann Ltd

has been provided by prior inoculation with avirulent virus (Dodds, 1990). The double-stranded RNA in Endothia parasitica (Fulbright, 1990) and Rhizoctonia solani (Cas- tanho and Butler, 1978) can be transmitted to, and induce sickness in, virulent types of the same plant pathogens. Among bacteria, Agrohacterium radiobacter, fluorescent pseudomonads, and Bacillus spp., have been most studied (Hemming, 1990). Of the 44 agents used in biocontrol listed by Cook and Baker (1983), 25 are fungi. The species contain agents from every mycological class, although Trichoderma spp. have received the most attention (Papavi- zas, 1985: Baker, 1989: Lynch, 1990). Even members of the animal kingdom, such as vampyrellid amoebae (Old and Darbyshire, 1978), destroy plant pathogens. These predators have been implicated in the decrease in inoculum density of certain fungal soil-borne pathogens (Chak- raborty, Old and Warcup, 1983), but may be more an interesting oddity than utilitarian: they require high levels of moisture for activity, and few crops that are hosts of pathogens susceptible to such predators are grown in swamps.

Curl (1988) recently reviewed the literature relating to biological control by mycophagous amoebae and other soil microfauna. Although field experiments have not yet tested their potential, many species ofmycophagous nematodes prevent initial fungai growth in soil, the rhizosphere, and in some cases, even feed on pathogens (e.g. Pythium spp.) that have invaded roots. Curl particularly calls attention to the potential of mycophagous species of Collembola and postulates a role for these insects in the reduction of inoculum density of fungal pathogens by destructive pathogens, especially in the rhizosphere.

CROP PROTECTION Vol. 10 April 1991

86 Diversity in biocontrol: R. Baker

Diversity in Infection courts

Plant pathogens typically parasitize different organs or tissues of hosts. Biological control agents are therefore necessary for protection of diverse sites. The phylioplane, including leaves, stems, flowers and fruits, may be colonized by antagonists (Windels and Lindow, 1985). Below the soil surface, fixed infection courts (e.g. hypocotyls, seeds), or developing organs (e.g. root tips) could be protected by biocontrol agents (Baker, 1977).

Diversily by increase of biomass

When organic matter is added to soil, short-term and/or long-term effects may occur. Often, these are associated with treatments involving addition of substrates that stimulate increases in biomass of micro-organisms, leading to suppression of pathogens. Strategic manipulations may be required, however, because nutrients associated with crop residues, added organic matter, or soil treatments can increase the activity of pathogens and/or deleterious microbes as well as that of antagonists. The principles and mechanisms involved in this balancing act still are not well defined, even after more than half a century since Sanford (1926) first suggested that control of potato scab by green manure was an example of biological control. In field trials, Weinhold and Bowman (1968) provided more information on the phenomenon; over 13 years, scab increased when a cover crop of green barley was incorporated into the soil, but was prevented from increasing with a soybean crop. A hypothesis was advanced that nutrition of antagonist and antibiotic production, especially by Bacillus subtilis, were factors leading to suppression of the pathogen, but direct evidence was difficult to obtain.

Another example illustrating the difficulty of assessing factors in suppressiveness of this type was reviewed in detail by Baker and Cook (1974). An avocado grove in Queensland, Australia was infested with virulent Phytoph- thora cinnamomi under climatic conditions favourable for development of disease. The grove was managed in such a way as to duplicate the environment typically associated with trees growing in a native ecosystem in which there are considerable amounts ofdeposited organic matter. No less than eight factors are listed as contributing to the observed suppressiveness of the soil, with an assumption that it is associated with complex ecological interactions inducing biological control. Again, turf and wheat diseases caused by infection by Gaeumannomvces graminis are among the easiest to control with organic amendments (Fellows and Ficke, 1934). The usual conclusion is that amendments change the quantity and quality of microbial populations in a way similar to that accomplished by long-term monoculture (Gerlagh, 1968).

Biomass, particularly that generated in composted tree bark, releases inhibitors of plant pathogens (Hoitink and Fahy, 1986). Evidently, bacterial and fungal antagonists are involved in suppression of various plant pathogens in such bark compost-amended substrates. Diseases induced by Pythium uhimum and R. solani also were reduced by

Increase in inoculum density due to active growth other than in the primary host

Relatively rapid l death of propagules

to the ill-adapted soil environment

>

t~

Long-term survival of

Extinction of inoculum

Time

Figure 1. Idealized elements of the non-transformed survival curve. (By permission from Baker, 1981)

addition ofcomposted organic household rubbish, mostly kitchen waste (Schuler et al., 1984).

Instinct tells us that diversity, related to an increase in the proportion of ambient beneficial microbes, is at the heart of these biocontrol systems; the apple always lies potentially in the blossom (Lowell). Indeed, in theory development, at least three mechanisms have been related to the induction of suppressiveness by manipulation of biomass: (i) effects on survival; (ii) competition for elements essential for successful infection by a pathogen, and (iii) saprophytic competition for substrate.

Effects on s u r v i v a l

Quantitative analyses of the effects of organic amendments on survival of soil-borne plant pathogens have been developed (Baker, 1981). The thalli of these pathogens typically increase through parasitism in their host. When reintroduced into soil by cultivation or other means of debris incorporation, their inoculum density may occasionally increase through saprophytism on dead substrates, commensalism in the rhizosphere of a plant, or parasitism of an alternate host (Park, 1965). In most cases, however, there is a rapid increase in death rate soon after inoculum is introduced into soil (Figure 1), as illustrated by n umerous examples from the literature (Baker, 198 ! ). The rapid increase in death rate apparently is caused by propagules that are ill suited to survive the vicissitudes encountered in the antagonistic soil environment. The increase in antagonistic biomass due to introduction ot" organic substrate serves only to hasten the process and the truc test of such treatments to limit survival hinges on its influence on the death rate of resistant propagules. There are few examples, however, where organic substrates influence this type of survival. The classic system was described by Clark and involved the digestion of sclerotia of Phymatotrichum omnivorum when manure was added to soil (Clark, 1942). Any amendment, as long as it is organic,


results in the death of these resistant structures; however, the strategy of hastening eradication of long-term survival units by elevating biomass may depend on the susceptibil- ity of a specific pathogen to such treatment and may not be effective for application to every soil-borne pathogen.

Competition for e lements

The examples treated above involve relatively non-specific interactions induced by organic substrates introduced to elevate biomass. However, it is possible to manage a biocontrol system so that selective enhancement of a portion of the biomass results in environmental c6nditions that suppress pathogens. The strategy simply involves immobilization of one or more nutritional factors essential for successful penetration by a soil-borne pathogen. These essential factors may be nitrogen (N), simple carbon (C) or iron (Fe) in an available form for those pathogens requir- ing an exogenous source of such nutrients (Baker, 1971; Paulitz, 1990). For example, the small chlamydospores produced by forma specialis of Fusarium oxysporum did not contain enough Fe for efficient germination and penetration, whereas the larger propagules of Fusarium solani f. sp. solani were not affected by Fe deficiency (Elad and Baker, 1985; Simeoni, Lindsay and Baker, 1987).

Immobilization of N may be accomplished by addition of organic substrates with a wide C/N ratio. The increased microbial biomass utilizes the C. In a matter of hours, inorganic N in the soil and/or organic N exudates from the host are metabolized, resulting in N levels insufficient for successful penetration by the pathogen (Maurer and Baker, 1965). Obviously, such a biocontrol system can only be applied to legumes that can rely on fixation for a source of N.

Ifcomplex C is added to soil, a biomass is activated that is capable of metabolizing these substrates. Such management of diversity can result in biocontrol. When chitin and lignin were added to soil, reduction in severity of Fusarium root rot of bean was achieved (Maurer and Baker, 1964). Later, Benson and Baker (1970) followed the utilization of simple C, typical of the exudates from a host, in the rhizosphere of an analogue system. When chitin and lignin were added to soil, these simple C compounds were utilized much more rapidly than in an unamended system. The hypothesis was advanced that the elevated biomass resulting from metabolism of complex C also could utilize any simple C introduced into the rhizosphere by the host. In this case, management of diversity in saprophytic colonization resulted in simple C competition deleterious to a pathogen.

Because biocontrol related to competition for Fe has usually been associated with only one component to the biomass, this subject is treated later in this review.

Saprophyt ic compet i t ion

Saprophytic colonization of substrates is necessary in the life cycle of some pathogens for survival and/or growth into the infection court of a host. For example, Fusarmm avenaceum, pathogenic on lentils, added to raw soil in stems

Diversity in biocontrol: R. Baker 87

of the host or oat grains, had much less inoculum potential than in soil previously treated with aerated steam at 55°C for 30 min (Lin and Cook, 1979). Fast-growing members of the biomass such as Trichoderma viride. Mucor hiemalis and Mucor plumbeus colonized the residue occupied by F. avenaceum and reduced its activity.

Competition by the total biomass or diverse portions of it, therefore, can be a mechanism of biocontrol. There probably are more examples, not yet fully explored, that are associated with profound decreases in inoculum potential of a pathogen within a substrate when later invasion of components of the biomass occurs. More attention, however, has been given to those that efficiently survive in organic matter by prior colonization ofa substrate. Bruehl (1987) reviewed principles involved in such relationships; indeed, Bruehl's Law is an application of the old statute that 'possession is nine-tenths of the law'. Once a substrate is occupied by these fortified pathogens, subtle or indirect manipulations of the biomass may have little effect or the outcomes may be complex. Consider Gaeurnannomyces graminis: the addition of N supports saprophytic activity of the pathogen, increasing inoculum potential in the short term (Garrett, 1976); in the absence of a host, however, the biomass further depletes the substrate, and potential diminishes.

The strategy most often advanced by researchers for making sure that beneficial microbes occupy substrates is akin to the 'sledgehammer approach'. A pathogen is eradicated from its substrate by heat or fumigation and an added biomass re-established to prevent reinfestation of substrates released by this process. Although there have been instances where a small amount of raw soil not containing the pathogen was added and biocontrol was achieved (Scher and Baker, 1979; Yuen, Schroth and McCain, 1985; Alabouvette, Couteaudier and Lemanceau, 1986), attention usually is centred on selective reinfestation by a single biocontrol agent. For example, Trichoderma spp. was reintroduced into fumigated or heat-treated soils to provide biocontrol of a number of soil-borne pathogens that can rapidly reinvade soils (Strashnov et al., 1985) that are not 'biologically buffered' (Kreutzer, 1965).

These examples of 'garbage biological control' often involve multiple beneficial components that may be associated with physical and chemical modifications as well as biological ones. Experimental approaches are therefore more on a trial and error basis than are those based on theory. Some researchers have preferred to abstract components of biocontrol systems with identification of associated mechanisms and enhancement strategies (Baker, 1990a).

Diversity in mechanisms

The importance of detailed studies on basic mechanisms as an essential step in enhancing success in the development and application of biocontrol was highlighted in a keynote address by R. R. Schmidt (Schmidt, 1990) in a recent symposium. Knowledge of mechanisms involves studies of the parts of a phenomenon, taken collectively, and how


88 Divers i ty in b iocontro l : R. Bake r

Interact ion of agent in host I . Induced resistance 2. I nh ib i to rs Icompet i t ion 3. Hypov i ru lence

Host

Pathogen ~

Biological control agent

Antagot~ism ex te r io r to host 1. Ant ib ios is 2. Competit ion 3. Exploi tat ion

a. predat ion b. hyperparas i t ism

Figure 2. Some pathways of the mechanisms involved in biological control of plant pathogens. (By permission from Baker, 1985)

they are related to produce an effect (Baker, 1990a). A diagrammatic illustration of the pathways involved in mechanisms is given in Figure2.

In biological control systems operating in localities exterior to the subterranean organs of a plant, antibiosis was a logical candidate mechanism. Such an association was not easily demonstrated (Baker, 1985); indeed, until the pioneering research of Wright (reviewed by Brian, 1957), little was known about how constraints on production and inhibitory factors of antibiotics in soil limited potential for biocontrol. Wright established that antibiotics can be produced by micro-organisms in specific sites where nutrients are sufficient and at locations in which compounds are not inactivated by adsorption (see review by Weller and Thomashow, 1990). These sites are diverse. An ectomycorrhizal fungus, Leucopaxillus cerealL~" var. piceina, produced diatretyne nitrile on shortleaf pine roots and effectively provided protection against Phytophthora cinnamomi (Marx, 1972). Gliovirin, produced by Gliocla- dium virens, suppressed seed and seedling damping-off induced by P. ultimum (Howell and Stipanovic, 1983). The same pathogen was suppressed in the rhizosphere by an inhibitory factor produced by T. harzianum growing on the rhizoplane (Baker, 1989). Phenazine-l-carboxylate, produced by Pseudomonads' fluorescens and P. aureofaciens, was detected in the rhizosphere of wheat (Thomashow and Wcller, 1988: Wellcr and Thomashow, 1990) and may be a thctor in the induction of takc-all decline.

As reviewed previously, examples of biocontrol systems where competition for nitrogen and/or carbon is involved arc known. For those soil-borne pathogens producing small propagules, like chlamydospores, that require an exogenous source of Fe (Simconi et al., 1987), siderophores produced by fluorescent pseudomonads reduced the avail- ability of this element and suppressed Fusarium wilt diseases (Bakcr, Elad and Sneh, 1986). The competitive saprophytic ability of Pythium nunn apparently inhibits the activity and sporulation of another primary colonizer of plant tissue such as P. ultimum (Paulitz and Baker, 1988a.b).

Exploitation involves predation, usually by members of the Animalia (sec above), and hyperparasitism. The latter mechanism is fascinating and has been thc impetus for

artistic studies involving illustrations (Figure 3) of various stages of parasitism on a microlevel (Lifshitz et al., 1985).

Bdellovibrio hacteriovorus attaches to cells of other bacteria, revolves rapidly with no rotation of the attachment tip, drills a hole in the cell wall and digests the interior contents (Starr and Baigent, 1966). Simultaneous infestation of soybean with the parasite and the pathogen Pseudomonas srringae pv. glycmea (9 : 1 ratio) reduced the necrotic lesions and systemic toxaemia that usually are induced by the latter micro-organism (Scherff, 1973).

Most attention, however, has been centred on mycoparasites. As might be expected, these exhibit a wide variety of parasitic phenomena and have interactions with their hosts that often are similar to those of higher plant pathogens (Baker, 1987). There are necrotrophs that destroy their hosts. Biotrophs are (balanced) mycoparasites that accrue nutrients but do not necessarily kill unless they predispose the host to invasion by secondary micro-organisms that have metabolites resulting in lysis. Some produce toxins that induce necrosis. Others establish an intimate nutritional relationship with their hosts, with or without the production of specialized haustorium-like appendages. Most frequently, the necrotrophs have relatively sophisticated prepenetration stages in which they coil around the hyphae of their hosts, form appressium-like structures, and release enzymes appropriate for degrading cell walls. After penetration, the contents of the cells are digested.

Some cxamples of biocontrol induced by mycoparasites have becn reported. Sporidesmium sclerotivorum grows through soil and may decay sclerotia of Sclerotinia minor

• ~1~,

Figure3. Scanning electron micrographs of Pythium nunn - fungi interactions, a, Hyphae of Phytophthora parasi t ica penetrated by P. nunn with advanced degradation of the host's cell wall x 2000. b-d, Hyphae of Phytophthora c innamomi parasitized by Pythium nunn. Arrows indicate appressoria and infection pegs of the mycoparasite at sites of interaction, b x 580. c x 950: d x 225. (By permission from Lifshitz etal . , 1984)

CROP PROTECTION Vol. 10 Apri l 1991


(Adams and Ayers, 1981). Mycoparasitism by Coniothy- rium minitans was an important factor in survival of Sclerotinia spp. in the field and even parasitized sclerotia formed inside the pith cavities of stems (Tribe, 1957; Huang, 1976). Soil suppressiveness to R. solani after monoculture of radish was correlated with increased population densities of T. harzianum due to mycoparasitism (Liu and Baker. 1980). However, in Hawaii, suppressiveness to the same pathogen generated in monoculture was associated with increased bacterial population densities (Chern and Ko, 1989), which again illustrates diverse mechanisms of biocontrol in similar crop systems.

It is possible for plants to be 'immunized' by introduction ofa biocontrol agent that switches on a natural system in response to stress. Such resistance involves recognition between host and an agent that initiates plant reactions, either generally or specifically inhibitory to later challenge inoculation with a virulent pathogen.

The inherent complexity of induction, in which interactions among pathogen, biocontrol agent(s), host and environment occur, again illustrates the diverse mechanisms associated with the phenomenon. Sequeira (1990) covers these mechanisms in detail, including highly specific hypersensitive gene-for-gene reactions, induction ofphyto- alexin biosynthesis, changes in enzymes involving ligni- fication, protease inhibition, chitin degradation, and synthesis of isoflavonoids, and extensions involved in cell-wall strengthening. The agents inducing resistance may be avirulent types of the same closely related races or differentformae speciales of the pathogen. In other cases, a wide variety of micro-organisms may enhance resistance to a specific pathogen (Baker, Hanchey and Dottarar, 1975). There is even an inter-kingdom interaction between spider mites and Verticillium wilt of cotton (Karban, Adamchak and Schnathorst, 1987).

Cross protection among viruses first was discovered by McKinney (1929). As pointed out by Dodds (1990), cross protection usually is associated with strong specificity, indicating that specific but subtle mechanisms underlie the phenomenon; however, it is possible to find strains of a single virus that can cross protect against unrelated viruses. The mechanisms hypothesized to account for cross protection are reviewed by Sherwood (1987), but diversity of interactions and research in different studies has induced confusion over probable mechanisms.

One of the few biocontrol agents currently on the market is strain K84 of Agrobacterium radiobacter, which is effective against the related pathogen A. tumefaciens, inducer of crown gall. When K84 is introduced into wounds, it produces an antibiotic (agrocin) inhibitory to the pathogen, but also competes for infection sites (reviewed by Farrand, 1990). Competition for infection sites also appears to be involved in protection of cotton hypocotyls from penetration by R. solani by 'hypovirulent' isolates of the same fungus (Sneh, Ichielevich-Auster and Stomer, 1989).

Scientists despaired of ever containing - much less suppressing - the devastating chestnut blight induced by Endothia parasitica. However, an unexpected and unique mechanism for biocontrol of this disease appeared that is

competently reviewed by Griffin (1986) and Fulbright (1990). Eight years after its first introduction into New York City in 1904, the disease already had spread in alarming proportions in the American chestnut. In the 1930s the disease spread to the European chestnut; however, by the 1950s, sprouts originating from trees killed by blight had 'healing cankers'. The phenomenon was labelled hypovirulence because a transmissible cytoplasmic genetic factor was responsible for the reduced aggressiveness of E. parasitica. The genetic factor is thought to be a number of double-stranded RNA (dsRNA) molecules that vary in size and frequently are multigenomic among isolates. Hypovirulent strains carrying the appropriate dsRNA can be inoculated into the bark surrounding the margin of an expanding canker. Subsequently, wound tissue forms and restricts canker development. The primary factor limiting dissemination of hypovirulent biocontrol agents in nature may be incompatibility (i.e. inability to anastomose) among isolates. Even so, there is some evidence of spread of such agents in nature from isolates released over a decade ago.

Weakly pathogenic isolates of R. solani contained plasmids that were responsible for a slow growth and low virulence upon inoculation to Japanese radish seedlings (Hashiba, 1987). These are examples of linear mitochondrial DNAs and have been found in at least eight other microbial eukaryotes. Their function is unknown, but the existence of inverted repeats at both ends may be inter- preted to mean that they act as transposons. If the linear DNAs control the genetic determinants of pathogenicity, they may be useful in the biological control of some fungi.

Enhancement of diversity through combinations of applied biological control agents

Obviously, two or more biocontrol agents might be better than one in suppression of a disease. Besides additive or synergistic effects, it could be possible to use multiple agents that would become active (when others become inactive) as environmental conditions change, or become active at various time intervals as host organs develop and concomitantly initiate different microniches and/or infection courts. This, indeed, involves not only multivariant approaches, but also an intimate knowledge of mechanisms. Little has been accomplished in this direction, therefore, and incompatibility between agents was noticed in one case (Hubbard, Harman and Hadar, 1983).

The beneficial use of combinations is being explored, however. For example, combinations of fluorescent pseudomonad isolates 2-79 and 13-79 were superior in control of take-all than either isolate alone (Weiler, 1988). Combinations of fluorescent pseudomonads and avirulent species of Fusarium were superior in control of Fusarium wilt of cucumber to either one alone (Park, Paulitz and Baker, 1988b). This may be an example of the principle of suum cuique (to each his own): i.e. in this example, two agents with different mechanisms (competition for Fe by pseudomonads and induced resistance initiated by the avirulent fungus) can be compatible and provide additive



control. Again, the principle ofsuo loco (in its proper place) may be illustrated by the combination of T. harzianum protecting against infection by P. ultimum in the rhizosphere and P. nunn reducing inoculum density of the same pathogen in the soil mass (Paulitz, Ahmad and Baker, 1990). Furthermore, a mixed population of Collembola altered the quantitative, and perhaps qualitative, nature of the rhizosphere microflora of cotton seedlings, resulting in a significant decrease in viable inoculum density ofF. o.~v- .~porum (Curl, 1988).

Theory development related to biological control of take-all of wheat is progressing to the point that at least two different agents may be involved, depending on soil reaction. Fluorescent pseudomonads flourish under alkaline conditions and were implicated in take-all decline (Weller, 1988). Alternatively, in soils of lower pH or in those acidified by treatment with ammonium sulphate, increased activity of Tricho&,rma spp. was also implicated (Simon and Sivasithamparam, 1989). Again, suppressiveness in soil to R. solani spp. by monoculture of radish developed more rapidly in acid than in alkaline soil (Chet and Baker, 1980; Liu and Baker, 1980). Successful biological control demands diversity of agents in diverse habitats.

Creating diversity by genetic manipulation

So far, the extent of diverse activity among biocontrol agents has been limited by their inherent characteristics in concrete systems. The challenge in the new age of biotechnology is to confer new or enhanced attributes into the microbes found in nature. Obviously, permanent installa- tion requires tinkering with genomes.

Great advances were made by geneticists in the past by use of conventional breeding techniques with higher plants and animals. Although biocontrol agents obey the ancient axiom to be fruitful and multiply, they seldom do this in traditional ways by sexual reproduction. The microbial geneticist therefore must devise clever techniques for manipulation: these were reviewed by Papavizas (1987) for fungi. An overview of potentials for application of geneti- cally engineered bacteria has been given by Lindow, Panopoulos and McFarland (1989). More specific reviews treating genetic manipulation of bacteria in biocontroi are also available (Handelsman and Parke, 1989; Baker, 1990b).

For fungi without functional or easily induced sexual stages, three methods are usually employed for genetic manipulation: (i) conventional mutagenesis; (ii) protoplast fusion, and (iii) transformation.

Conventional mutagenesis

Howell and Stipanovic (1983) induced mutation in G. virens by use of ultraviolet (u.v.) light and developed strains with enhanced ability to produce gliovirin. Treatment of cotton seeds with the mutant provided control superior to that given by the parent strain. U.v. light also was employed for mutation of Trichoderma spp. by Papavizas and coworkers in 1982 (reviewed by Papavizas, 1987).

Mutation to benomyl resistance improved the efficiency of these antagonists in biocontrol of R. solani, P. ultimum, Sclerotium cepivorum and S. rolfsii. In this case, two previously undetected antibiotic metabolites were produced by some of the mutants in the fermentation medium. The strain producing the highest amounts of antibiotic was the most effective biological control agent against S. cepivorum.

Among the desirable attributes of biocontrol agents effective against soil-borne pathogens is the ability to colonize plant rhizospheres (rhizosphere competence). Broadcast or even band application of agents is impractical because of cost constraints, except in certain agricultural industries such as container-grown high-income plant products. Subsequent rhizosphere colonization following seed application is therefore a desirable attribute of agents, but (in the limited studies available) appears to be charac- teristic of certain bacteria only (Weller, 1988). The reason why these are rhizosphere competent is a fascinating unknown (Lam, 1990); however, species and strains of incompetent fungi in the genus Trichoderma have been mutated to rhizosphere competence (reviewed by Baker, 1989). Mutation to benomyl resistance with N-methyl-N'- nitro-N-nitrosoguanidine was accompanied in most cases by colonization of rhizospheres from seed treatments up to 106c.f.u.g ~ soil 8cm from the seed, whereas parents never were found below 2cm. The association of this phenomenon with benomyl resistance has not been explained; however, rhizosphere competence was always associated with cellulase production but not, in all cases, with benomyl resistance. The hypothesis was advanced that the mutants colonized the cellulose-rich mucigel of the root more efficiently than their parents. By use of these mutants, significant increases in plant growth (Baker, 1988) and protection of roots against P. ultimum in comparison to wild types was noticed (Baker, 1989).

Protoplast fusion

Protoplast fusion is really a reckless, random shot at genetic improvement, but 'the gambling person lurks at the bottom of every heart' (Balzac). Peberdy (1980) summed up the situation well: there is a complex pattern of chromosome segregation that defies traditional criteria used in the recovery of segregants. Even when fusion is successful, the progeny may be unstable. Even so, Seh and Kenerley (1988) fused protoplasts and regenerated three Gliocladium spp. Seed treatment with two fusants of T. harzianum or parent strains (Harman, Taylor and Stasz, 1989) or their parents, were as effective in increasing plant stands of various crops as was treatment with thiram. Moreover, stands were significantly improved when seeds were treated with progeny strains in comparison with those treated with either parent strain when combined with solid-matrix priming.

Transformat ion

Transformation-induced mutations in fungi certainly a r e

more specifically identifiable than those in protoplast



fusion; however, identification and localities of genes instrumental in biocontrol are necessary before genetic improvement can be rationally attempted. As reviewed above, mechanisms were identified in a number of systems, but the determinants may be multistage components of complex biochemical pathways. For improved aggressiveness in mycoparasitism, for example, genetic manipulation may be a monumental task, although speculators may ask whether the addition of improved genes for chitinase production (see Lindow et al., 1989) could enhance fungai cell-wall penetration by such an endowed transformant. So far, however, trends in biotechnology are associated with cloning and mapping of plasmids and transfer of specific well-studied genes. The majority of strains ofG. virens and Talaromycesflavus contained one or more piasmids in the mitochondrial DNA fraction, and cloning and mapping are under way to determine relationships among the plasmids (Mischke, 1988a,b; Mischke and Papavizas, 1987). Henson, Blake and Pilgeram (1988) transformed G. graminis var. graminis (Ggg) and var. tritici (Ggt) by use of pBT6 (the plasmid-encoding fungicide- resistant fl-tubulin) to achieve benomyl resistance. Ggg is not virulent on wheat but can induce resistance to Ggt (Wong and Southwell, 1980). The possibility exists, therefore, of combining biological and chemical control with a fungicide-resistant biocontrol agent, although benomyl is not particularly effective in control of Ggt.

Preoccupation with the genetic basis of some of the less-complicated mechanisms (e.g. antibiosis, competition for Fe with siderophores versus induced resistance, hyperparasitism) associated with biocontrol by bacteria as a necessary first step toward creating diversified enhancement has been a limiting factor in research. Nevertheless, progress is being made in retarding the transfer of agrocin 84 (produced by A. radiohacter K84) resistance to A. tumt:/'aciens by generating a stable mutant in which transfer genes have been deleted (Farrand, 1990). In P, fluorescens strain Hv37a, genes for biosynthesis of oomycin A, an antibiotic effective against P. uhimum, were cloned and regulation for increased production biosynthesis in an enhanced strain was demonstrated (Gutterson, Howie and Suslow, 1990). A further potential step might be to engineer plants to produce antibiotic secondary metabolites such as oomycin, although energy tradeoffand current nutrition and environmental concerns could make this approach less attractive (Smedegaard-Petersen and Tol- strup, 1985).

As Loper (1990) pointed out, the immense contribution of molecular genetics to the study ofsiderophores and their influence on biological control has been in basic research. Because of this, logical hypotheses for strain improvement of fluorescent pseudomonads are now possible. Certain of these microbes possess the capacity to take up and utilize Fe from siderophores produced by others, but they may not themselves secrete siderophores (Marugg et al., 1985; Bakker, Weisbeck and Schippers, 1988). The ability of a fluorescent pseudomonad to utilize the siderophores of another is dependent upon the possession o f an outer membrane reception protein for that pseudomonad's ferric siderophore (Magazin, Moores and Leong, 1986). Thus,

the most beneficial strains used in biocontrol would produce siderophores that are not used by deleterious strains, but could use Fe-transport systems produced by deleterious strains (Buyer, Wright and Leong, 1986). In addition, there is wide genotypic diversity among beneficial siderophore-producing organisms so that strain selection and/or genetic engineering could provide enhanced biocontrol potential. For example, a more efficient biocontrol agent, Pseudomonas putida strain N IR, had higher rhizosphere competence and produced siderophores at higher available Fe levels than an inferior strain (A 12) of the same species (Park, Paulitz and Baker, 1988a).

The fruits of diversity Diverse management techniques of modern agriculture have ensured excess production of food and fibre in the United States. A considerable increment of the surplus resulted from application of chemical pesticides (Baker and Dunn, 1990). It is apparent that the levels of such applications cannot continue in the future because of public concern over contamination ofthe environment and of foodstuffs. A logical alternative is to diversify pest management strategies to include integration of biological control measures for control of mites, nematodes, plant pathogens and weeds (Tauber and Baker, 1988).

Even in the face of this challenge to develop control strategies conducive to a healthy environment, research support for biocontrol has been inconsiderable. The trends in funding for Federal R & D, 1978-88 (Office of Legisla- tive and Public Affairs, National Science Foundation), indicate a 67% decrease in support of agricultural science (Baker and Dunn, 1990). Currently there is little evidence that these trends will change. Further, the diverse approaches to molecular studies in biocontrol initiated by private enterprise are being curtailed.

Perhaps a reason for inattention to support for biological control lies in lack of success in developing products. Only a few are being marketed today. The pessimism reflected by the title of a discussion session at the 1971 annual meeting of the American Phytopathological Society, Biological Control... Mission Impossible?, is also apparent today. Optimists believe that the problems can be solved by strategies employing integration and/or enhancement procedures (Baker, 1990a). Consumers may have to accept lower-quality agricultural products; however, one advantage of biocontrol agents is that some induce increased growth responses (Baker, 1988, 1989). This feature could increase marketability.

Finally, even research results in the discipline reflect diversity! It is not uncommon to report repeated experiments in terms of proportions of trials where biocontrol was significant. Nevertheless, 'they fail and they alone, who have not striven' (Aldrich) and, we might add, 'in diverse pathways'.

References

Adam, P. B. and Ayers, W. A. (1981) Sporidesmium sclerotivorum" distribution and function in natural biological control of sclerotial fungi. Phytopatholog.v 71, 90-93



Alabouvette, C., Couteaudier, Y. and Lemanceau, P. (1986) Nature of intrageneric competition between pathogenic and non-pathogenic Fusarium in a wilt-suppressive soil. In: Iron, Siderophores, and Plant Diseases, pp. 165-178 (ed. by T. R. Swinburne). New York: Plenum Press

Baker, K. F. and Cook R., J. (1974) Biological Control of Pathogert~. San Francisco: W. H. Greeman & Co. 433 pp

Baker, R. (1971) Ecology of the fungus, Fusarium" competition, in: Fusarium: Diseases. Biology, and Taxonomy, pp. 245-249 (ed. by P. E. Nelson, T. A. Toussoun and R. J. Cook) University Park: Penn. State Univ. Press

Baker, R. (1977) Inoculum potential. In: Plant Pathology. an Advanced Treatise. Vol. H. How Disease Develops in Populations. pp. 137-157 (ed. by J. G. Horsfall and E. Cowling). New York: Academic Press

Baker, R. (1981) Biological control: eradication of plant pathogens by adding organic amendments to soil. In: Handbook of Pest Management in Agriculture, Vol. Ii, pp. 317 327 (ed. by D. Pimentel). Boca Raton: CRC Press, Inc

Baker, R. (1985) Biological control of plant pathogens: definitions. In: Biological Control in Agricultural IPM Systems, pp. 24-39 (ed. by M. A. Hoy and D. ('. Herzog). Orlando: Academic Press, Inc

Baker, R. (1987) Mycoparasitism: ecology and physiology. Can. J. Plant Pathol. 9, 370-379

Baker, R. (1988) Trichoderma spp. as plant growth stimulants. C'RC Crit. Rev. 7.97 106

Baker, R. (1989) Some perspectives on the application of molecular approaches to biocontrol problems. In: Biotechnology Of Fungi for Improving Plant Growth, pp. 220-233 (ed. by M. Whipps and R. D. Lumsden). Cambridge: Cambridge Univ. Press

Baker, R. ([990a) An overview of current and future strategies and models for biological control. In: Biological Control o]'Soil-borne Plant Pathogens, pp. 375-388 (ed by D. Hornby). Slough: CABI.

Baker, R. (1990b) Molecular biology in control of fungal pathogens. In: ttandbook ~! Applied Mycology. Vol. I. Soil and Plants. pp. 259-271. New York: Marcel Dekker, Inc., in press

Baker, R. and Dunn, P. R. (1990) Preface. in: New Directions in Biological Control. pp. xix-xxii (ed. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss, Inc

Baker, R. and Scher, F. M. (1987) Enhancing the activity of biological control agents. In: Innovative Approaches to Plant Disease Control, pp. I -17 (ed. by 1. Chet). New York: John Wiley & Sons

Baker, R., Elad, Y. and Sneh, B. (1986) Physical, biological, and host factors in iron competition in soils. In: Iron. Siderophores, and Plant Disea.~es, pp. 77 -84 (ed. by T. R. Swinburne). New York: Plenum Press

Baker, R., Hanchey, P. and Dottarar, S. D. (1975) Protection of carnation against Fusarium stem rot by fungi. Phytopathology 68, 1495-.1501

Bakker, P. A. H. M., Weisbeck, P. J. and Schippers, B. (1988) Siderophore production by plant growth-promoting Pseudomonas spp. J. Plant Nutr. I I, 925 933

Benson, D. M. and Baker, R. (1970) Rhizosphere competition in model soil systems. Phvtopathology 60, 1058- 1061

Brian, P. W. (1957) The ecological significance of antibiotic production. In: MicrobialEcology, pp. 168-188 (ed. by R. E. O. Williams and C. C. Spicer). London: Cambridge University Press

Bruehl, (;. W. (1987) Soilborne Plant Pathogens. New York: Macmillan. 368 pp.

Buyer, J. S., Wright, J. M. and Leong, J. (1986) Structure of pseudobactin A214, a siderophore from a bean-deleterious Pseudomonas. Biochemistry 25. 5492. 5499

Castanho, B. and Butler, E. E. (1978) Rhizoctonia decline: a degenera- tive disease of Rhizoctonia solani. Phytopathology 68, 1505-1510

Chakraborty, S., Old, K. M. and Warcup, J. H. (1983) Amoebae from a take-all suppressive soil which feed on Gaeumannomyces graminis trith'i and other soil fungi. Soil Biol. Bioehem. 15, 17.-24

Chern, L. L. and Ko, W. H. (1989) Characteristics of inhibition of suppressive soil created by monoculture with radish in the presence of Rhizocmma s~hlni. J. Phvtopathol. 126. 237 245

Chet, I. and Baker, R. (19801 Induction of suppressiveness of Rhi:oc- Ionia sohmi in soil. Phytopathology 70, 994.-998

Clark, R. E. (1942) Experiments Toward~ the Control ~['Take-all Disease ~!f Wheat and the Phymatotrichum Root Rot ~['Cotton. US Deparmwnt ~/ Agricuhure Technh'al Bulletin 835.27 pp

Cook, R. J. and Baker, K. F. (1983) The Nature and Practwe ~fBiologieal ('ontrol o[" Plant Pathogens. St. Paul: American Phytopathological Society. 539 pp

Curl, E. A. (1988) The role of soil microfauna in plant-disease suppression. CRC Crit. Rev. Plant Sci. 7. 175-196

Dodds, J. A. (1990) Approaches to studying viral cross protection. In: New Directions in Biological Control, pp. 213 221 (ed. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss. Inc

Elad, Y. and Baker, R. (1985) The role of con]petition for iron and carbon in suppression ofchlamydospore germination of Fusarium spp. by P~'eudomonas spp. Pto'topatholog.l' 75, 1053 1059

Farrand, S. K. (1990) Agrohacterium radiohacter strain K84: a model biocontrol system. In: New Directions m Biological Control pp. 679-691 (cd. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss, Inc

Fellows, H. and Ficke, C. H. (1934) Wheat take-all. Kansa.~ Agr. Exp. Sta. Annu. Rep., 1932-34. 95 96

Fulbright, D. W. (1990) Molecular basis for hypovirulence and its ecological relationships. In: New Direction~' in Biological Control. pp. 693 702 led. by R R. Baker and P. ['. Dunn) New York: Alan R. 1.iss. Inc

Garrett, S. D. (1976) Influence of nitrogen on cellulolysis rate and saprophytic survival in soil of some cereal fi~ot-rot fungi. Soil Biol Biochem. 8. 229-234

Gerlagh, M. (1968) Introduction of Ophioholus graminis into new poldcrs and its decline. Neth. J. Plant Pathol. 74 (Suppl. 2), I--97

Griffin, G. J. (1986) Chestnut blight and its control. Hort. Rev. 8. 291 336

Gutterson, N., Howie, W. and Suslow, T. (1990) Enhancing etficiencies of biocontrol agents by use of biotechnology In: :Vew Directums in BtohJgical Control. pp. 749-765 (ed. by R. R. Baker and P. E. Dunn). Ncw York: Alan R. Liss, Inc

Handelsman, J. and Parke, J. L. (1989) Mechanisms in biocontrol of soilborne plant pathogens. In: Plant Microbe Interactions: Molecular and Genetic Perspective.~. Vol. 3, pp. 27 61. New York: McGraw-Hill Pub. ('o

Harman, G. E., Taylor, A. G. and Stasz, T. E. (1989) Combining effective strains of Trichoderma har:ianum and solid matrix priming to provide improved biological seed treatment systems. Plant Dis. 73, 631 -637

Ilashiba, T. (1987) An improved system for biological control ot damping-off by using plasmids in fungi. In: Innovative Approaches to Plant Disease Control. pp. 337 351 (ed. by I. Chet). New York: John Wiley & Sons

itemming, B. C. (1990) Bacteria as antagonists in biological control of plant pathogens. In: New Directions in Biological Control. pp. 223-242 (cd. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss. Inc

I len.~n, J. M., Blake, N. K. and Pilgeram, A. L. (1988) Transformation of Gaeumannomyces gramhd~" to benomyl resistance. Curr. Goner. 14. 113--117

Hoitink, H. A. J. and Fahy, P. C. (1986) Basis for the control of soilborne plant pathogens with composts. Annu. Rev. Phytopathol. 24. 93-114

Ilowell, C. R. and Stipanovic, R. D. (1983) Glioviren, a new antibiotic from Gliocladium virens and its role in the biological control of pvthium uhimum. Can. J. Microbiol. 29, 321 324

|luang, 14. C. (1976) Importance of Coniothyrium minitans in survival of sclerotia of Sch'rotinia selerotiorum in wilted sunflower. ('an. J. Bot. 55, 289 295

Hubbard, J. P., Harman, G. E. and Hadar, Y. (1983) Effect of soilborne Pseudomonas spp. on the biological control agent, Trichoderma hamatum, on pea seeds. Phytopatholog.v 73, 655--659

Karban, R., Adamchak, R. and Schnathorst, W. C. (1987) Induced resistance and interspecific competition between spider mites and a ',ascular wilt fungus. Science. N. Y.. 235, 678 679



Klecan, A. L., Hippe, S. and Somerville, S. C. (1990) Reduced growth of Erysiphe graminis f.sp. hordei induced by Tilletiopsis pallescens. Phyto- pathology 80, 325 -331

Kreutzer, W. A. (1965) The reinfestation of treated soil. In: Ecology of Soil-borne Plant Pathogens, pp. 495-508 (ed. by K. F. Baker and W. C. Snyder). Berkeley: Univ. Calif. Press

Lam, S. T. (1990) Microbial attributes associated with root colonization, in: New Directions in Biological Control, pp. 767-778 (ed. by. R. R. Baker and P. E. Dunn). New York: Alan R. Liss, Inc

Lifshitz, R., Stanghellini, M. and Baker, R. (1984) A new species of Pythium isolated from soil in Colorado. Mycotaxon 20, 373-379

Lifshitz, R., Dupler, M., Elad, Y. and Baker, R. (1985) Hyphal interactions between a mycoparasite Pythium nunn and several soil fungi. ('an. J. Microbiol. 30, 1482-1487

Lin, J. A. and Cook, R. J. (1979) Suppression of Fusariurn roseum "Avenaceum" by soil microorganisms. Phytopathology 69, 384-388

Lindow, S. E., Panopoulos, N. J. and MeFarland, B. L. (1989) Genetic engineering of bacteria from managed and natural habitats. Science. N.Y. 244. 1300-1306

Liu, S. and Baker, R. (1980) Mechanism of biological control in soil suppressive to Rhicoctonia solani. Phytopathology 70, 404--412

Loper, J. E. (1990) Molecular and biochemical bases for activities of biological control agents: the role of siderophores. In: New Directions in Biological Control, pp. 735-747 (ed. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss, inc

Lynch, J. M. (1990) Fungi as antagonists. In: New Directions in Biological Control, pp. 243-253 (ed. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss, Inc

McKinney, H. H. (1929) Mosaic diseases in the Canary Islands, West Africa and Gibraltar. J. Agric. Res. 39, 557-578

Magazin, M., Moores, J. C. and Loong, J. (1986) Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain. J. Biol. Chem. 261,795-799

Marugg, J. D., Voo Spanje, M., Hockstra, W. P. M., Schippers, B. and Weisbeck, P. J. (1985) Isolation and analysis of genes involved in siderophore-biosynthesis in the plant growth stimulating Pseudomonas putida strain WCS 538. J. Bacteriol. 164, 563-570

Marx, D. H. (1972) Ectomycorrhizae as biological deterrents to pathogenic root infections. Annu. Rev. Phytopathol. I10, 429-454

Maurer, C. L. and Baker, R. (1964) Ecology of plant pathogens in soil. I. Influence of chitin and lignin amendments on development of bean root rot. Phytopathology 54, 1425-1426

Maurer, C. L. and Baker, R. (1965) Ecology of plant pathogens in soil. II. Influence of glucose, cellulose and inorganic nitrogen amendments on development of bean root rot. Ph.vtopathology 55, 69-72

Mischke, S. (1988a) The nature of mitochondrial plasmids in Gliocla- dium virens, a biocontrol fungus. Phytopathology 78, 591 (abstract)

Mischke, S. (1988b) The presence of plasmids in the biocontrol fungus Gliocladium. Phytopathology 78, 863 (abstract)

Mischke, S. and Papavizas, G. C. (1987) Mitochondrial DNA of the biocontrol agent Talaromycesflavus. Phytopathology 77, 1765 (abstract)

Old, K. M. and Darbyshire, J. F. (1978) Soil fungi as food for giant soil amoebae. Soil Biol. Biochem. 10, 93-100

Papavizas, G. C. (1985) Trichoderma and Gliocladium: biology, ecology, and potential for biocontrol. Annu. Rev. Phytopathol. 78° 190-194

Papavizas, G. C. (1987) Genetic manipulation to improve the effec- tiveness of biocontrol fungi for plant disease control. In: Innovative Approaches to Plant Disease Control, pp. 193-212 (ed. by I. Chet). New York: John Wiley & Sons

Park, C. S., Paulitz, T. and Baker, R. (1988a) Attributes associated with increased biocontrol activity of fluorescent pseudomonads. Korean J. Plant Pathol. 4, 218-225

Park, C. S., Paulitz, T. C. and Baker, R. (1988b) Biocontrol of Fusarium wilt of cucumber resulting from interactions between Pseudomonas putida and nonpathogenic isolates of Fusarium o.,cysporum. Phytopath- ology 78, 190.- 194

Park, D. (1965) Survival of microorganisms in soil. !n: Ecology of Soil-borne Plant Pathogens, pp. 82-97 (ed. by K. F. B-~tker and W. C. Snyder). Berkeley: Univ. Calif. Press

Paulitz, T. C. (1990) Biochemical and ecological aspects of competition in biological control. In: New Directions in Biological Control." Alterna- tives for Suppressing Agricultural Pests and Diseases, pp. 713- 724 (ed. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss

Paulitz, T. C. and Baker, R. (1988a) Interactions between Pythiurn nunn and Pythium ultimum on bean leaves. Can. J. Microbiol. 34, 947-951

Paulitz, T. C. and Baker, R. (1988b) The formation of secondary sporangia by Pythium ultimum: The influence of organic amendments and Pythium nunn. Soil Biol. Biochem. 20, 151 - 156

Paulitz, T. C., Ahmad, J. S. and Baker, R. (1990) Integration of Pythium nunn and Trichoderma harzianum isolate T-95 for the biological control of Pythium damping-off of cucumber. Plant Soil 121,243-250

Peberdy, J. F. (1980) Protoplast fusion - a tool for genetic manipulation and breeding in industrial microorganisms. En=yme Microbial Tech. 2. 23-29

Sanford, G. B. (1926) Some factors affecting the pathogenicity of Actinomyces scabes. Phytopathology 16, 535-547

Scher, F. M. and Baker, R. (1979) Mechanisms of biological control in Fusarium-suppressive soil. Phytopathology 70, 412 417

Scherfl', R. H. (1973) Control of bacterial blight of soybean by Bdellovibrio bacteriovorus. Ph)'topathology 63, 400-402

Schmidt, R. R. (1990) Investigations of mechanisms: The key to successful use of biotechnology. In: New Directions in Biological Control. pp. 1-22 (ed. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss, Inc

Schuler, C., Biala, J., Brune, C., Gottshall, R., Ahlers, S. and Vogtmann, H. (1989) Suppression of root rot of peas, beans, and beet roots caused by Pvthium ultimurn and Rhizoctonia solani through the amendment of growing media with composted organic household waste. J. Phyto- pathol. 127, 227--238

Seh, M. L. and Kenerley, C. M. (1988) Protoplast formation and regeneration of three Gliocladium species. J. Microbiol. Methods 8, 121-130

Sequeira, L. (1990) Induced resistance: physiology and biochemistry. In: New Directions in Biological Control, pp. 1-22 (ed. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss, Inc

Sherwood, J. L. (1987) Mechanisms of cross protection between plant virus strains. In: Plant Resistance to Viruses, pp. 136-150 (ed. by D. Evered and S. Harnett). Chichester: John Wiley

Simeoni, W. L., Lindsay, W. I,. and Baker, R. (1981) Critical iron level associated with biological control of Fusarium wilt. Phytopathoh>gy 77, 1057 1061

Simon, A. and Sivasithamparam, K. (1989) Pathogen-suppression: a case study in biological suppression of Gaeumannomyces graminis var. tritici in soil. Soil Biol. Biochem. 21,331-337

Sneh, B., lchielevieh-Auster, M. and Stomer, 1. (1989) Comparative anatomy of colonization of cotton hypocotyls and roots by virulent and hypovirulent isolates of Rhi:octonia solani. Can. J. Bot. 67, 2142-2149

Smedegaard-Petersen,V. and Tolstrup, K. (1985) The limiting effect of disease resistance on yield. Annu. Rev. Phytopothol. 23, 475-490

Starr, M. P. and Baigent, N. L. (1966) Parasitic interaction of Bdeflo- vihrio hocteriovorus with other bacteria. J. Bacteriol. 91, 2006-2017

Strashnov, Y., Elnd, Y., Sivan, A. and Cbet, i. (1985) Integrated control of Rhi'_octonia solani Kiihn by methyl bromide and Trichoderma harcianum Rifai Agar. Plant Pathol. 34, 146--151

Tauber, M. J. and Baker, R. (1988) Every other alternative but biological control. Bio-Science 38. 660

Thomashow, L. S. and Welter, D. M. (1988) Role of a phenazine antibiotic from Pseudomonas/tuorescens in biological control of G. graminis var tritici. J. Bacteriol. 170, 3499-3508

Tribe, H. T. (1957) On the parasitism of Sclerotinia triJolium by Coniothmyrium minitans. Transl Br. M.vcol. Soc. 40. 489-499



Turhan, G. and Turhan, K. (1989) Suppression of damping-off on pepper caused by pvthium ultimum Trou and Rhizoctonia solani Kfihn by some new antagonists in comparison with Trichoderma harzianum Rifai. J. Phvtopathol. 126, 175-182

Utkede, R. S. (1987) Chemical and biological control of crown and apple rot of apple caused by Ph.vtophthora cactorum. Can. J. Plant Pathol. 9. 295 300

Weinlmld, A. R. and Bowman, T. (1968) Selective inhibition of the potato scab pathogen by antagonistic bacteria and substrate influence on antibiotic production. Plant Soil 28, 12-24

Weller, D. M. (1988) Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu Rev. Phytopathol. 26. 379 -407

Weller, D. M. and Thoma~ow, L. S. (1990) Antibiotics: Evidence for their production and sites where they are produced. In: New Directions

m Biological Control. pp. 1-22 (ed. by R. R. Baker and P. E. Dunn). New York: Alan R. Liss, lnc

Windels, C. E. and Lindow, S. E., Eds. (1985) Biological Control t~/the Phylloplane. St. Paul: American Phytopathological Society. 169 pp

Wong, P. T. W. and Seuthwell, R. J. (1980) Field control of take-all of wheat by avirulent fungi. Ann. Appl. Biol. 94, 41-49

Yuen, G. Y., Schroth, M. N. and MeCain, A. H. (1985) Reduction of Fusarium wilt of carnations with suppressive soils and antagonistic bacteria. Plant Dis. 69, 1071-1075

Received 28 November 1990 Accepted 29 November 199~)


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