The mechanisms of biological control of plant diseases

Sari Biol. Bmchem. Vol. 5, pp. 721-728.

Pergamon

Press 1973 Printed m Great Britain

THE MECHANISTS OF BIOLOGICAL OF PLANT DISEASES J. E. Department

of Plant Pathology,

CONTROL

MITCHELL

University

of Wisconsin.

(Acc
Madison.

Wisconsin

53706

1973)

Summary-Examples of biologicalcontrolofplant diseases are analyzed to identify the mechanisms by which such control is effected. The evidence supporting the hypothesis that metabolites produced by one organism may inhibit another is presented. The fact that the formation of such a toxic material is an adaptive phenomenon in response to the presence of the mhibited organism is of considerable significance. Failure to compete successfully for suitable substrates is a form of biological control, that probably ranks second in importance. Lysis appears not to be a primary mechanism of microbial antagonism in the soil. Biological control of soil-borne pathogens is an important factor in disease control in nature. Its exploitation requires sustained and imaginative effort.

INTRODUCTION

MOST NONCHEMICAL procedures to controf plant disease involve biological activity in some form or another. Most cases of so-called biological control found in plant pathological literature involve manipulations of the environment that alter biological activity in the soil to the detriment of the pathogen or the development of the disease. The objectives of this review are to examine what has been discovered about the mechanisms of biological control of plant diseases in the past 10 or more years and to see whether this work has made evident any means by which we can increase the effectiveness of cultural procedures. We are concerned with understanding mechanisms and not with practicalities. We need to ask questions such as those that follow. What is known about the mechanisms of any particular example of a phenomenon of biological control? What is not known about the interaction between microorganisms that results in control? What needs to be known if we are to understand what is actually effecting control of any specific disease? While studies apart from the soil have contributed to our understanding of the microbial interactions of importance to this discussion, they have left many questions unanswered. The problem must be considered in the context of the soil environment. Even more specifically it is the interaction of microorganisms with one another and with the environment in the soil at the root surface that we must investigate if we are to understand the mechanisms of so-tailed biological control of soil-borne plant pathogens.

INTERACTIONS

Ecologists identify some eight different types of interactions between organisms; four involve effects deleterious to one or both of the interactants. These are competition, amensalism, parasitism and predation. The latter two appear quite clear-cut in our current state of ignorance, and seem to be of limited importance in the control of soil-borne plant pathogens. Our lack of knowledge is so great, however, that there really is little basis for 721

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concluding anything about the importance of these phenomena in the natural soil environment. Huber, Anderson and Finley (1966) reported many cases of mycoparasitism in his soil immersion plates. The work done with mycoparasitism by Boosalis (1964) and Butler (1957), among others, indicates that it is as much affected by host and environmel~t variability as is parasitism of higher plants. Attempts to control nematodes with mycophagous fungi has not yet achieved any appreciable success. The occurrence of bacteriophages has long been known, but their function in soil in relation to bacterial population is not known. The possibility that the Bdellor;ihrio function in biological control has been suggested by Alexander (1971) and their presence on plant tissue indicates their wide andgeneral distribution. About all we can say about the importance of parasitism and predation is that they do occur, they are immensely interesting, but that much more needs to be known if their potential for disease control is to be known. Competition and amensalism are difficult to separate experimentally and, indeed, they both seem to be involved in many of the principal situations which have been studied. Amensalism includes situations where metabolites produced by organism A inhibit organism B while A is not affected. Competition, on the other hand, can affect both due to a lack of adequate supply of energy or other factor to satisfy the needs of both. Toxic rnetnholites A rather typical example of the state of our knowledge with respect to the ecological effects of toxic materials on soil microorganisms is found in one of the early studies of the effects of antibiotics in soil by Siminoff and Gottlieb (195 1). When Bacillus suhtiEis was added to a soil previously infested with a strain of Streplomyces gviseus, known to be a source of streptomycin, multiplication of the former was severely limited as compared with that in soil not so infested. This was patently biological control. Since the strain of S. griseus used was known to have the ability to produce streptomycin one might conclude that this was the mechanism by which the inhibition was achieved. When a strain of S. ~~is~~.~ known not to have the capacity to produce streptomycin was used, the results were the same, namely, no growth of B. subtilis. This, too, was biological control. But, why? The important point is that toxic phenomena were obviously not involved, or at least were not essential. Numerous attempts at control of plant diseases have directly or indirectly, intentionally or inadvertently, involved antibiotic producing organisms. One notable and relatively recent case is the interesting experiment reported by Weinhold, Oswald, Bowman, Bishop and Wright (1964) and by Weinhold and Bowman (1968) with potato scab in California that spanned some 12 yr. When soybeans were used as a cover crop between successive plantings ofpotatoes, scab did not increase over the years and yields remained good. When, on the other hand, barley or pigeon pea, or no cover crop was used between successive potato plantings, a striking increase in scab occurred after the 3rd4th yr of continuous potato plantings. It is significant to note that the use of soybean after the increase had occurred did not result in a reduction in scab. The treatment prevented a build-up of scab, but did not repress existing populations. A microbial analysis of the soil showed that the predominant antagonist of S. scabies present in soybean plots was a bacterium of the B. s~b~ilis type and that it produced, in culture, an antibiotic similar to subtilin. However, it was found that the population of this bacteria had increased as much in the barley amended soil as it had where soybeans were used. Of seemingly importance was the production in soybean extracts of 2-3 times as much antibiotic as in extracts of barley tissue. Circumstantial evidence indicated that

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antibiotic production was the mechanism involved, but it was not possible to demonstrate the presence of antibiotic activity in aqueous extracts of tissue decomposing in soil. The authors concluded that a “major obstacle to gaining a more complete understanding of the mechanism by which green manures influence the build-up of potato scab is our incomplete knowledge concerning the behavior of S. scabies in soil”. A study of the population dynamics of the pathogen in the soil would be difficult, but essential to the understanding of this phenomenon. Good circumstantial evidence linking antibiotic production to disease control has been provided by Marx (1969a, 1969b) and by Marx and Davey (1969) when they demonstrated protection of shortleaf pine (Pinus rchinuta Mill.) against Ph~tophtlzoru cinna~~lonli by ectotrophic mycorrhizae. This protection is apparently partly mechanical and partly chemical. The fungal component of the mycorrhizae has been shown to produce an antibiotic in culture as have the axenic mycorrhizae. The difficulties of observing events occurring at the surface of the mycorrhizae in the soil have precluded the demonstration of the antibiotic production in natural soil. While there is little reason to doubt the protective action of the antibiotic in the mycorrhizae, the knowledge of its persistence and activity in soil would seem to be essential to fully understand this relationship. The work of Ricard and Laird (1970) with Scytulidium sp. and Pork carbonica is a beautiful example of demonstrated biological control through the agency of a toxic metabolite. Field experiments have shown that Scytalidiurn sp. could be introduced artificially into, and would become established in, parts of Douglas fir [P.srudot.suga menziesii (Mirb.) France] poles that P. carbonicu had previously colonized. No live P. carbonica could be subsequently isolated. In uitro experiments showed that as mycelium of the two organisms grew together, a narrow inhibition zone formed and then Scytalidiurn sp. produced a yellow pigment that was toxic or accompanied by a material toxic to P. carhonicu and the latter was killed and overgrown. A similar situation was noted by Tveit and Wood (1955) who studied the mechanism of interaction of Chaetonziurn ylobosum and Fu.suriurn nivale in controlling seedling blight of oats. In culture there was no appreciable inhibition zone as the mycelium of the two organisms approached. However, strains of C. globosurn which were effective in pot tests continued to grow after mycelial contact was made and completely penetrated the colony of the pathogen while the latter ceased to grow. Neither Tveit and Wood (195.5) nor later Chang and Kommedahl (1968) were able to demonstiate antibiotic production in agar culture by Chuetomium, but the former found cell free culture extracts to be fungitoxic and the latter observed abnormal growth of hyphal tips of F. nivule as the two organisms came into contact suggesting the possibility of a nondiffusable or an induced toxic material. It would appear that an adaptive reaction may occur upon contact between the two organisms or that special conditions are required for toxin production. The suggestions by Tveit and Wood (1955) of a toxic phenomenom seems more likely than the space preemption per se suggested by the latter workers. Control of corn seedling blight by coating seeds with a strain of B. suhtilis, known to induce the formation of zones of inhibition with F. resewn f. sp. cerealis (Chang and Kommedahl, 1968), was assumed to be due to antibiotic production, but no evidence of inhibitor production on corn kernels was presented. Unfortunately, the experiment was not done using a strain of B. subtilis known not to produce a zone of inhibition. What a challenge there is to find out what initiates the production of the toxic material if the organisms come together. Park (1956) demonstrated the importanct: of the environment in an adaptive phenomena for the control of a plant pathogen in soil. Fusuriurn rosem conidia were lysed rapidly

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when placed in soil in which Bacillus macerans had been growing in the presence of F. roseum. The development of the lysogenic agent occurred only in the presence of F. roseum, was less extensive in sand than in soil, and only a delayed and partial lysis occurred in solid or liquid culture media. The observations of activity through an agar film suggested the production of a toxic metabolite. Naturally

occurring

biological control

The possibility is intriguing that adaptive phenomena are involved in natural biological control of plant pathogens either at the organism or population level. There are examples of a decline in the intensity of disease caused by three important plant pathogens in certain sites after the latter had been present for a number of years. Menzies (1959) reported that soils long used for potato production became resistant to S. scabies. The production of a transferable, heat labile factor was stimulated by the addition of alfalfa meal to the soil. Menzies (personal communication) reported the frustration of futile attempts to detect the presence of a population of antagonists in resistant soil that would set it apart from the susceptible soil. The experiment of Weinhold et al. (1964) referred to earlier is another example of an apparently adaptive response in soil that resulted in a natural decline in severity of potato scab in soil after nine successive crops of potatoes. While the most striking reduction came in the barley plots where the greatest disease had occurred, it was evident in all plots except soybean where little disease occurred. The second, and less well characterized, example of adaptive resistance of soil to a pathogen was reported by Burke (1965) with bean root rot. In soils that had become resistant the chlamydospores formed were ofa less resistant and consequently less persistent form and, as a result, there was less disease in such fields. Virgin soils, on the other hand, supported production of normal persistent chlamydospores that initiated severe infection of subsequent bean crops. The critical factor was thermolabile, but the effect could not be transferred. The third example of the occurrence of an adaptive phenomenom resulting in the reduction in disease severity, and the one about which most is known, is that of “Take All Decline” known since Fellows and Ficke (1934) and Glynne (1935) reported its occurrence. Here again as the crop is planted over a period of time, the severity increases to a maximum and then declines to an equilibrium level of lower severity. The mechanism of this decline has been extensively studied by Gerlagh (1968) Lester and Shipton (1967) and Shipton (1972) and others. There is a specific antagonism to virulent 0. graminis involving a heat stable toxic material and a microorganism destroyed by heating soil to 50°C for 30 min. The antibiotic effect could only be demonstrated by inhibition of runner hyphae on wheat seedlings growing in sand and not in vitro. The antagonism is active both in the saprophytic and in the parasitic phase of the disease. Evidence that take-all decline may result when a virus infection of the mycelium of 0. graminis causes a reduction in the virulence of the pathogen has been reported recently by Lemaire and co-workers (1970). The important point of these examples of apparent biological control is the fact that an adaptive mechanism is involved. This is indicated by the fact that.a response to the presence of the pathogen by a component of the microbial population of the soil results in an interaction deleterious to the development or activity of the pathogen population. The unsuccessful searches for responsible microbial components in all cases have involved traditional isolation methods and have not looked for microbial parasites such as phages or Bdellovibrio type of agents. Menzies (personal communication) has suggested that some

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new approach is needed. It will be unfortunate indeed if these clear-cut cases of natural biological control are not studied until the mechanisms are understood and capitalized on. Competition While amensalism and the production of toxic metabolites is one kind of population interaction that may be found to explain certain instances of biological control of plant pathogens, a strictly competitive type of interaction in which nutrient depletion is a critical feature is also a possibility. Thus, according to Snyder, Schroth and Christov (1959) and Maurer and Baker (1965) and others, bean root rot is reduced when organic materials with a high C/N ratio are added to increase the competition for nitrogen present. Here populations of F. solani f. phaseoli remain high, but the disease low. The control of Fusurium stem rot of carnations by B. subtilis, recently reported by Aldrich and Baker (1970) may also be accomplished by several other organisms, according to Baker (personal communication). This makes it look like competition, but, as he points out, phytoalexins are certainly a possibility. It is a beautiful system to work with and we can expect some answers from the work. Lysis In other cases of apparent competition for specific materials as a limiting factor, the mechanism is not as well defined. In most cases reported, lysis is closely associated with the control phenomena. There are so many things that result in lysis that it is difficult to know what is cause and what is effect. Lysis could be autolysis following energy source deprivation due to competition, or to a toxic effect of a material excreted into the environment, or it could be due to lytic enzymes excreted into the environment by other organisms. The distinction between heterolysis and autolysis, when populations of two organisms are growing together is as difficult as distinguishing growth inhibition due to competition from that due to toxic action. Mitchell and Alexander (1962) controlled F. sohi f. phaseoli by adding chitin to the soil. They demonstrated that the production of chitinase and laminarinase by streptomycetes was stimulated by the treatments. They were unable to rule out toxic metabolites and concluded that these or exoenzymes could be involved. So whether lysis is a direct mechanism or a scavenging action is a moot question. Environmental

factors

It is difficult to talk of mechanisms separately from activating effects of environment. Soil environmental factors control the incidence of these biological mechanisms. In an elegant demonstration of the effect of soil water potential on pathogen activity, Cook and Flentje (1967) showed that at high water potentials, bacterial activity increased in the vicinity of the pea seedlings and that the increases lysis of germ tubes took place with an ultimate reduction in the number of infections. While it was not possible to determine whether competition, heterolysis or autolysis (from starvation or toxic action) was responsible, the modification of the physical environment of the soil resulted in a clear-cut case of biological control. Griffiths and Siddiqi (1958) reported a case where temperature and soil moisture combined to regulate the mechanism by which the activity of Fusarium culmorum was controlled. When the soil moisture was high, the success in isolation of F. culmorum was correlated with the temperature. When soil moisture was at 40 per cent of moisture holding capacity, F. culmorum was dominant and could be isolated only when the temperature was below

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3. E. MITCHELL

10°C. Above this it was completely suppressed by Trichodertm uiridr. The root is also a significant factor in the environmental control of regulatory mechanisms. Seedling blight of rye was controlled by Griffiths and Siddiqi (1958) when the soil was simultaneously infested with a bacterium that had been isolated from roots and had been selected because of ,the inhibition zone it induced on agar plates seeded with F. culmorum. Subsequent tests showed this bacterium to be rare in the soil but abundant on root surfaces. Soil atwmltnents There is a fascinating paper by Hawn and Lebeau (1962) reporting a relationship between bacterial wilt reaction of alfalfa varieties and the inhibitory effect of extracts of dried 2-yr-old roots. The inhibitory factors were not present in sterile roots but resulted from microbial action on root tissue. The inhibitory activity of the extracts was proportional to the known resistance of the variety to wilt. This is suggestive of the phytoalexin phenomenom. The extensive work on the effect of soil amendments on the control of cotton root rot caused by P. u~~~?z~~~~~~~?~ demonstrated that a rapid decrease in sclerotial population coincides with the period of enhanced development of soil microorganisms. There is evidence of increased lysis of P. otnnicorutw mycelium produced as the sclerotia begin to germinate. The implication has been that this is due to the lytic action of enzymes produced by other microorganisms. There is no reason it could not be autolysis resulting from toxic action or simply a lack of nutrients. It is tempting to associate this with the relatively recent work of Menzies and Gilbert (1967), and Gilbert, Menzies and Griebel (1969), and of Gilbert and Griebel (1969) on the action of volatile compounds in stimulating the growth of propagules of various microorganisms. If the sclerotia of P. otnniuorutn react as do those of S. ro@ii one would expect to see the sclerotia germinate, expend their energy in fruitless growth, and die due to nutrient exhaustion. Lysis would be a sequel to the actual control mechanism not the mechanism itself. Finally, one must consider the toxicity of products resulting from the decomposition of organic amendments in the soil. These have been implicated in the reduction in activity of P. cinnammi in avocado by the work of Zentmyer (1963) and of Gilpatrick (1969). Alfalfa meal apparently gives rise first to NH, that may be phytotoxic as well as fungitoxic. Evidence for the action of other fungitoxic materials, such as the saponins, which would be released by microbial action is presented. Lewis and Papavizas (1971) have suggested that the substantial reduction in pea root rot caused by ~~~uno~~~~~.~ eutdws following incorporation of residues of several crucifers was due to sulfur-containing compounds released by microbial decomposition of plant tissue. The correlation between actual toxicant concentration in the soil and the inhibitory effect noted is still in the realm of extrapolation. Nevertheless the fact that a reduction of disease can be achieved during the period of active biological decomposition of such residues seems to be without reasonabie doubt. Increasing interest in volatile metabolites formed by microorganisms in soils (e.g. Hutchinson, 1971; Moore-tandecker, and Stotzky, 1972; and Romine and Baker, 1972) has focused attention on the fact that products of normal biological activity may function in an important way in controlling growth and development of soil-borne microorganisms and possibly of plant roots as well. CONCLUSIONS

What can we conclude from all of this‘? We knew at the start that biological control of plant pathogens is a complex situation that is difficult to unravel. We know that while a great many people have spent a great deal of time working on one or another part of the problem, there are a few cases where we have a clear picture of the mechanisms

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involved. Considerable progress has been made in understanding one of the cases of natural biological control, namely the take-all disease, but we are yet far even from this goal. Progress has been made because we have defined several areas of importance. We need to know more about a wider range of organisms and biological entities in the soil than the traditional forms. We need to know the function of phytoalexins at the root surface. We need to know about the effect of changes in the soil environment on the contribution of the root to the rhizosphere and to the pathogens. We are aware of the importance of the specificity of the substrate as far as antibiotic production is concerned and in many cases as far as growth of specific organisms is concerned, but we need to know the population dynamics of the organisms involved. I am optimistic that we have in hand several of the keys on which future progress depends. Much of the work that has been done so far has been pragmatic by necessity and for this reason superficial. Real progress can be expected only if a critical, sustained attack on the problem is organized and properly supported. We can contemplate what might be done if a group including a plant,pathologist, a plant physiologist, a biochemist, a soil microbiologist and a soil physicist got together and waded into this problem. It would not provide a quick solution, but 10 years from now we would understand many more of the mechanisms and would have made progress in applying this knowledge.

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