The Importance of Antibiotics and Inhibiting Substances

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Chapter 14

The Importance of Antibiotics and Inhibiting Substances

DAVID PARK

Department of Botany, Queen's University Belfast, Northern Ireland

I. Introduction . . .

II. Existence in Soil

III. Properties of the Soil Toxicity IV. Origin in Soil

V. Biological Significance in Soil . . . . References

. 435 . 435 . 438 . 439 . 440 . 445 I. I N T R O D U C T I O N

This article surveys the probable effects of inhibiting substances upon the biology of micro-organisms, mainly fungi and bacteria, in soil. Antibiotics in the defined sense (Waksman, 1945) include only those substances produced by micro-organisms, an artificial distinction in this context, and the wider term is preferred here so as to include substances of probable activity in soil from all sources. Parts of this discussion deal with antagonisms, and in some of the examples there is no proof that inhibiting substances mediate the antagonistic phenomena. However, antagonisms have the same significance biologically; moreover, most investigated antagonisms are produced by inhibiting substances (Park, 1960).

II. E X I S T E N C E IN SOIL

Evidence for the presence in soil of substances inhibitory to micro- organisms is of two sorts : direct, involving specific detection by isolation, bioassay or chromatography; and indirect, from observations of the effects produced on selected micro-organisms. Various authors have demonstrated specific antibiotics to be present in soil that has been inoculated with organisms known to produce the antibiotics, and lists of references on this topic are given by Brian (1949, 1957). Most of such work has been done with sterilized soil, either additionally supplemented with energy-rich materials, or un- supplemented except for the increase in nutrients that normally occurs as a result of sterilization. In either case, such work is of little relevance to normal soil conditions, in which the over-all level of nutrients is low and the population

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large and varied. Much greater difficulty has attended the identification of antibiotics in unsterilized soil; most attempts being unsuccessful, although some workers, e.g. Gregory et al. (1952), have shown anti-microbial activity without being able to identify the antibiotic. An outstanding exception is the work of Wright (1952), who demonstrated that gliotoxin could be produced in amounts detectable by paper chromatography in unsterilized but heavily- supplemented soil. The significance of this work has since been extended by the discovery (Wright, 1956) that small local substrata, e.g. seed coats, may in unsterilized normal soil provide conditions for the production of detectable amounts of antibiotics by a fungus resident as inoculum in the soil.

Although the evidence for the presence of specific antibiotics in soil under normal conditions is thus scanty, there exists more data bearing on the existence in soil of unidentified substances inhibitory to micro-organisms.

Greig-Smith (1912) described bacteriotoxic substances which he called the agricere. From the sub-surface layers of soil, Newman and Norman (1943) extracted inhibiting substances that were held partly responsible for the differences between surface and sub-surface microbial populations. Bublitz (1954) and Winter (1955) have extracted inhibiting substances from soil litter and from raw humus, both being materials of unusually low microbial activity. More recently, lignin and chemically related substances present in soil have been demonstrated to be toxic to micro-organisms and have been implicated as a possible cause of inhibition in soil (Lingappa and Lockwood, 1960, 1962). There is thus direct evidence that there may be present in soils substances able to inhibit microbial development. There is, however, little conclusive data on the amounts of such substances in soil, and on the sort of levels that would be necessary under soil conditions to result in inhibition of microbial growth and activity. This knowledge would be necessary before one could accept these substances as being responsible for the effects recorded by indirect methods.

The indirect methods for observing inhibitory phenomena, and thus infer- ring the presence of inhibiting substances in soil, involve placing test organisms, usually fungi, in or in contact with soil and comparing their germination, growth or morphological development, with control sets. Owing to the opacity of soil the observation of microbial structures in direct contact with it is difficult, and most methods involve some carrier medium to facilitate subsequent observation of the exposed test organisms. The carrier medium may incidentally entail some separation of organism from soil. Neilson-Jones (1941) and Jackson (1958a) used a layer of agar on the surface of a layer of soil, making observations on the upper surface. Jefferys and Hemming (1953) buried in soil slabs of agar which were later removed, washed, and seeded for test. Chinn (1953) incorporated test spores in a thin layer of agar supported on a glass slide which could, after burial in soil, be lifted for observation.

Dobbs and Hinson (1953) separated fungus spores from soil during the period of exposure by using a layer of Cellophane, and Kerr (1958) used a similar method to observe the effects of soil on growing mycelium. Data provided by such methods agree together and give a picture of most soils inhibiting fungal

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14. ANTIBIOTICS AND INHIBITING SUBSTANCES 437

germination and growth, often producing morphological peculiarities in growing hyphae, and, by some methods, causing death and lysis of structures.

However, the results of methods involving carrier media must be evaluated with some caution since, first, the carriers may necessitate diffusion, possibly differential, of substances between soil and test organisms; second, the carriers may provide nutrients to one or other of the test organisms or soil microbial population; and, third, even inert additions to soil, e.g. clean glass (Dobbs and Hinson, 1953), may result in behaviour not found in normal soil.

Lingappa and Lockwood (1961) have critically analysed the drawbacks of such carrier medium methods of testing for soil toxins, and demonstrate convincingly that it is possible for agar or Cellophane to act as enrichment media and to allow the activity of sections of the normal soil microbia that are able, within the period of the test, to produce toxic substances as a direct result of the presence of the carrier medium. While this evidence does not disprove the existence in soils of inhibiting substances, it indicates that methods of this type are inappropriate for the detection of the normal presence in soil of such substances. The methods may, however, be used to indicate the presence in soils of organisms able to produce inhibiting substances under

appropriate conditions.

Methods not involving carrier media have been used. Park (1955) added spores directly to the soil surface and later obtained some of them for subsequent observation by means of glass slides pressed onto the inoculated surface. Data obtained by such direct contact methods give general agree- ment with those from carrier media methods. Lingappa and Lockwood (1961) also confirm by direct contact methods that the failure of fungal spores to germinate in natural soils is a fact and not an artefact of carrier media methods, but they suggest that the surface of the spore, or materials exuded from it, may act as a nutrient supplement simulating antagonistic organisms in the environment to produce inhibiting substances in consequence of the spores' presence in the soil. Convincing evidence for this mechanism has more recently been presented by these authors (Lingappa and Lockwood, 1964).

These authors were unable to demonstrate fungitoxicity in any extracts from the toxic soil. Some of these findings may be related to those of Jackson (1959), whose unsterilized extracts from soil showed toxicity, but if subjected to any of a variety of sterilization treatments showed no toxicity. Jackson's interpretation was that sterilization removed toxicity, but Lingappa and Lockwood's data could be used to argue that the sterilized extracts had remained non-toxic, while the unsterilized extracts had developed toxicity on testing.

There is then no conclusive evidence that normal mature soils contain effective concentrations of inhibiting substances. Much of the evidence suggests that such substances do exist and are effective, and this is supported by the very low level of activity of micro-organisms in the normal mature soils, but none of this evidence is sufficiently critical to be regarded as adequate proof. What is known is that inhibiting substances may locally exist in effective concentration in special situations, and that in the soil more generally

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inhibiting substances may at least develop quickly under suitable conditions of stimulation.*

III. P R O P E R T I E S OF THE SOIL T O X I C I T Y

The properties of specific antibiotics have been investigated in some detail in connection with possible therapeutic value, and in relation to soil biology they have been reviewed by Brian (1949, 1957). There is a small number of antibiotics that could, under specified conditions of pH, etc., remain active in soil for effective periods, but most are likely to be adsorbed by soil colloids or otherwise inactivated fairly quickly in soil. Antibiotics of this sort, therefore, may require a specific range of conditions under which to be effective.

The more general soil fungistasis on the other hand appears to be effective under a wide range of conditions, and it is of most interest to examine the recorded general properties of this toxicity.

The toxicity of soil for fungi can be counteracted by the proximity of living roots (Kerr, 1956; Jackson, 1957) and by the addition to soil of energy sources for microbes (Neilson-Jones, 1941; Dobbs and Hinson, 1953), the more readily available energy sources having the greatest effect (Jackson, 1960). These facts have led some workers to suppose that the inhibition is caused by nutrient deficiency or competition for nutrients, but Park (1956, 1960) and Lingappa and Lockwood (1961) have shown that the arguments are inadequate.

The inhibitory effect disappears when the soil is sterilized by heat or by exposure to chemical disinfectants (Neilson-Jones, 1941 ; Dobbs and Hinson, 1953; Park, 1955; Jackson, 1960; Hack and Williams, 1960), and it has been argued from this that the inhibiting substances are thermolabile and sensitive to chemicals. This conclusion is not necessarily true. As already stated, Jackson (1959) showed that all tested forms of sterilization prevented the demonstration of inhibition in normally toxic extracts, and the lack of toxicity may be a function of the state of sterility rather than an effect of the mechanism of sterilization. Partial sterilization may similarly remove or reduce the inhibition (Martin and Aldrich, 1952; Stover et al, 1953). A possible ex- planation for this situation comes in part from the statement of Dobbs et al.

(1960) that the inhibiting substance in soil is volatile or of limited stability, particularly where oxygen is available. Dobbs (1960) has stated that small particles of soil lose their inhibiting property when exposed to air for short periods. It is of interest in this connection that Bilai (1956) has raised the possibility of the existence of volatile antibiotics. Such instability could explain the effect of sterilization, if the normal state were one in which the toxins present were continually diminishing owing to their instability, their level in soil being maintained by a balanced production by some of the organisms in the soil; given these conditions, sterilization, even without

* The considerations discussed in this section are reviewed in detail by Lockwood, J. L. (1964). This article appeared too late for it to be appraised in this chapter, but should be regarded as an authoritative account of this aspect of the topic.

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14. ANTIBIOTICS AND INHIBITING SUBSTANCES 4 3 9

assuming any direct destruction of inhibiting substances, could result in their disappearance without the means of replacement. This hypothetical relationship could help to explain an important anomalous property of the inhibiting factor in soil, namely that attempts to isolate it (Dobbs et al, 1960; Jackson, 1959; Lingappa and Lockwood, 1961) or even to demonstrate its presence in extracts or filtrates (e.g. Bamberg, 1930; Tveit and Wood, 1955; Barton, 1961) have frequently failed when the extracts have been cell- free : this would be expected with a substance of limited stability. While rapid lability and continual replacement of the inhibiting substance in soil is hypothetical, it may be noted that the operation in pure culture of an autogenic inhibiting system with these properties has been demonstrated (Park,

1961).

Summarizing, mainly from the works of Neilson-Jones (1941), Dobbs and Hinson (1953), Park (1955), Jackson (1958a, 1959) and Dobbs et al. (1960), a list of the more important properties of the general inhibitory factor of soil can be made. It is of biological origin, is volatile or unstable in air or water at normal temperatures, or is produced best under microaerobic conditions, is not a very large molecule, and is water-soluble and filterable through paper.

It is lost after Sterimat filtration or on sterilization by ultra-violet rays, by heat, or by chemicals. The toxicity is temporarily overcome by energy rich materials, and is less marked in acid soils. Under some test conditions the substance is fungistatic, not fungicidal, but under others can cause lysis of cells.

IV. O R I G I N IN SOIL

Substances in soil known to be inhibitory to micro-organisms may have their origin in a number of different sources. It is to be expected that higher plants produce inhibiting substances, since they have the capacity chemically to exclude entry of many potentially parasitic micro-organisms. Several investigations have confirmed that green plant parts contain inhibiting substances and that some of these may remain active on their removal from the plant (Kavanagh, 1947; Hughes, 1952; Topps and Wain, 1957; Nickell, 1959). Roots may also exude into their environment inhibiting substances that may accumulate in soil and there remain effective (Desai, 1946; Stiven,

1952; Naumova, 1953; Buxton, 1957a, b; Woods, 1960). Toxins may be liberated into the soil by decomposition of higher plant parts, or may even be produced there during decomposition (Winter and Willeke, 1952; Winter, 1955; Borner, 1960). A soil animal has been cited as the cause of one specific fungal antagonism in soil (Timonin, 1961a, b). Here an hemipterous insect living on banana roots in soil proved able to reduce the local soil population of Fusarium oxysporum f. cubense, the cause of the serious Panama disease of the banana plant.

Probably most of the toxicity of soil for micro-organisms comes from the micro-organisms themselves. The emphasis on searches for specific antibiotics from soil micro-organisms reflects this relationship. Moreover, the

fact that the toxicity disappears on sterilizing the soil and can be re-introduced

16+S.B.

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by re-inoculating with a small amount of normal soil (Neilson-Jones, 1941;

Park, 1955), strongly suggests its microbial origin. The work described in the previous section shows that the inhibiting factors in different soils have general properties in common, indicating a general phenomenon of a widespread nature, a conclusion reached by several workers prominent in this field (e.g.

Dobbs and Hinson, 1953; Jefferys and Hemming, 1953; Jackson, 1958b).

Moreover, the level of fungitoxicity at a site commonly varies with variations in the level of its general microbial activity (Jackson, 1958a, b; Dobbs et al, 1960). In addition, just as the soil toxicity can be restored to sterilized soil by a small inoculum of normal soil, so may it be by inoculation with individual micro-organisms (Katznelson, 1942; Lockwood, 1959), and in this respect any of a number of species may be effective (Park, 1957a, b; Weltzien, 1959).

Stover (1954) has argued that a high level of toxicity developing in flooded soils has a non-biological origin. His evidence does not support this conclusion, however, and Hollis (1948), Mitchell and Alexander (1960) and New- combe (1960), investigating closely similar situations, all conclude that the increased toxicity in the flood-fallowed soil has a biological origin.

There are several possible origins for substances in soil inhibitory to micro- organisms, and in different soils different sets of factors may operate; but while specific substances may be produced by particular organisms, and may be effective under some conditions, the evidence suggests that there is a common inhibitory phenomenon that is a function of the previous general microbial activity in the soil, and which may be produced by several or many species inhabiting the soil.

V. B I O L O G I C A L S I G N I F I C A N C E IN SOIL

In a settled and mature soil there is little microbial activity and the soil has a predominating population of inactive propagules and a residual background inhibition (Park, 1960). When decomposable material is added to such a soil, a succession of events begins in which antagonisms and inhibitions play a large part. The background inhibition is diluted in the immediate vicinity of the amendment, and spores in contact with it are thereby allowed to germinate (Park, 1956). Of the four properties of a successful competitive saprophyte listed by Garrett (1956), the three likely to be of greatest advan- tage in the initial colonization phase are rapid germination, rapid growth and enzyme production, and antibiotic production. The first two will enable an organism to obtain a large proportion of a newly presented substratum:

antibiotic production may, in this relatively simple situation, with very few organisms present, help the producer to exclude other micro-organisms for a time. It was in respect of just such a situation that Wright (1956) demonstrated antibiotic production in non-sterile soil by a colonizing fungus resident in that soil. Regarding Garrett's fourth criterion, that of tolerance for antibiotic substances, it should be pointed out that species occupying this niche of early colonization may have a relatively low tolerance of inhibiting

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14. ANTIBIOTICS AND INHIBITING SUBSTANCES 441 substances, and thus be dependent on obtaining early entry to a substratum.

Barton (1960,1961) has shown that it is sensitivity to antagonism that restricts Pythium mamillatum to the role of pioneer colonizer. It is the subsequent colonizers of a substratum that need to be tolerant of those inhibiting substances produced by the organisms previously and contemporaneously active.

These later colonizers must, of course, also be capable of utilizing the continually diminishing and decreasingly available energy sources. However, as argued by Park (1960), with these later organisms the first three of the above mentioned criteria might be of smaller importance than the fourth, which, with an increasing level of inhibiting substances in the substratum, will become of greater significance. At a relatively late stage in the colonization process, with fewer and more refractory materials available, and microbial activity less intense, the level of inhibiting substances in the substratum may decrease, depending on their degree of lability. This could allow late colonizers to be successful, although possessing a low tolerance for inhibiting substances and therefore a low apparent competitive saprophytic ability in some tests.

Thus Rhizoctonia solani and Armillaria mellea both appear as early colonizers, in fact as parasites of still living plants, and may also be saprophytically active in soil in the later stages of decomposition (Blair, 1943; Rao, 1959;

Garrett, 1960). Garrett (1960) has discussed how this type of biphasic development may be regarded as exhibiting two forms of escape from the intense antagonism characteristic of the middle stages of utilization of substrata.

Following its period of activity in a substratum in soil, or subsequent to its addition to soil from outside, a micro-organism may or may not survive there. One of the factors controlling this is the level and type of inhibiting substances in the soil, and the tolerance of the micro-organism to them.

These will vary with the soil, with the organism, and with the local conditions.

Waksman and Woodruff (1940) showed that some micro-organisms not already in the soil die out when added to it. Katznelson (1940a, b) presented data supporting this conclusion, but by altering the local soil conditions he was able to get survival and even increase of the added organisms. Kubiena and Renn (1935) found that exposure of soil to aerial laboratory contami- nation had no effect on the soil population, and they stated the general principle that a soil determines its own microbial population. However, Park (1958) records the survival in soil of an added non-indigenous soil fungus. The differences here may depend on whether the species has previously had the opportunity of reaching that particular soil, and of being tested against the local inhibiting substances.

Hawker (1957) lists four attributes contributing to survival in an habitat and, with respect to soil, two of these are relevant to inhibiting substances.

One has significance in a positive way ("ability to alter the environment in a direction unfavourable to other organisms") and has been discussed already;

the other ("escape from an unsuitable habitat") has significance in a negative way and can be considered further. Garrett (1950, 1956) has discussed how some micro-organisms have during evolution exchanged tolerance to the

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inhibiting influence of antagonism for tolerance to that of host resistance, concurrent with the parasitic existence. The more highly specialized parasites have escaped further along this route, and, in general, are found to be less tolerant of soil antagonism, having a lower competitive saprophytic ability (e.g. Anwar, 1949; Butler, 1953a, b). Thus, there appears to be an antithesis between the two tolerances; this may be partly explained by the fact that organisms with demanding requirements can, in the presence of organisms with more simply satisfied requirements, survive only if the latter are inhibited or prevented from competing successfully. Parasites generally have more demanding requirements.

Just as some organisms evade exposure to antagonism by the evolution of tolerance to host resistance, others evade it by developing a tolerance to extremes of physical and chemical conditions, and occupying the early stages in the formation of mature soil (Brown, 1958).

Inhibiting substances may influence the direction of evolution in the reverse direction to that just described, namely towards an increased tolerance. There are records (Wiltshire, 1932; Christensen and Davies, 1940; Miller, 1946;

Brown and Wood, 1953; Bistis, 1959) of saltants or mutants appearing in inhibited colonies, and possessing increased tolerance to the inhibiting influence. Goodlow et al. (1950) and Goodlow et al. (1952) describe sequences of autogenic evolution directionally determined by inhibitory substances.

In addition to influencing the direction of evolution, inhibiting substances of microbial origin may influence the rate of evolution by increasing the inci- dence of saltation in inhibited cultures (Isaac and Abraham, 1959).

In addition to their influence on the evolution of individual species, inhibiting substances help to determine the evolution of community structure.

Nissen (1954) added antibiotics to soils and subsequently isolated soil fungi able to utilize these inhibitory substances as sole carbon source. Just as inhibiting substances of a green plant may determine the local population of its pathogens (Turner, 1960), so Newman and Norman (1943) found that the development of distinctive microbial populations in surface and sub-surface soils, while influenced partly by the substrata presented, was largely determined by the antagonisms developing. These two workers also illustrated in soil a phenomenon of general ecological significance, namely the edge effect (Elton, 1958), by showing that the surface soil with its bigger variety of niches, bore a bigger variety of species; this effect was also shown by England and Rice (1957). The edge effect depends partly upon the fact that an antagonism shown by one species for another is not absolute but depends on the local environment. In part the principle put forward by Brown (1922), that an organism is less susceptible to inhibiting substances when its energy for growth is great, is applicable here, and the effect of inhibiting substances may be greater at the limit of range of an organism (Hawker, 1957). Thus, micro-organisms may be eliminated from the soil matrix but still survive in infected organic material in the soil (Stover, 1953; Venkata Ram, 1953;

Macer, 1961). Apparently anomalous results are of the sort where, in the more homogeneous conditions of culture, one organism is inhibitory to another

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14. ANTIBIOTICS AND INHIBITING SUBSTANCES 443 organism and eventually eliminates it ; whereas eradication or even inhibition is not exhibited in the soil with its variety of habitats (Sanford and Broadfoot, 1931 ; Slagg and Fellows, 1947 ; Isaac, 1953 ; Wood and Tveit, 1955) and may be related to the edge effect and the greater chances of escape from antagonism in a more varied habitat. Not only can nutritional status of the habitat exert an effect on antagonistic relationships, but so may temperature also. Griffiths and Siddiqi (1961) investigated a soil antagonism shown above 10°C but not below. This example gives further illustration to the general principle that the more varied the habitat the greater the opportunities for escape or evasion of inhibition. In this context relatively small variations may result in markedly different outcomes, as is illustrated by the work of Keynan et al. (1961),who demonstrated that cellulose presented to a soil as Cellophane film was colonized by a particular soil fungus, but if presented as filter paper was colonized by a particular soil bacterium. On analysis, the situation was found to be caused by an antagonism between the two organisms. It is clear then that caution must be exercised in respect of any claim that one organism is antagonistic to a second organism, unless specific conditions are stated. Such a claim cannot be accepted in an absolute sense otherwise the recipient would have become extinct.

Exposure of an organism to inhibiting substances to which it is sensitive may in certain circumstances assist in its survival. Dobbs and Hinson (1953) suggest that in the absence of colonizable substrata any inhibition of spores is favourable to their survival, in the same way that in the absence of the host, obligate parasites like Plasmodiophora brassicae survive longest under conditions least favourable for germination (Bremer, 1924). The principle appears to apply best to ecologically obligate parasitic fungi of low competitive saprophytic ability, in which a temporary reduction of soil inhibition, allowing a consequent increase in general microbial activity with a subsequent intensification of inhibiting influences, tends to reduce the numbers of the pathogen (Garrett, 1938), and some methods of biological control depend upon such an increase in general microbial activity (Millard and Taylor, 1927; King et al, 1934; Stover et al, 1953; Baker, 1957). Similarly, the rhizosphere effect, which involves greater activity of soil micro-organisms in the region of the root, may protect against parasites of low competitive saprophytic ability owing to the increase in antagonism (Lochhead et a/., 1940; Timonin, 1940, 1941; Agnihothrudu, 1955). In this context it can be pointed out that the rhizosphere effect itself is a result of the root's influence in counteracting the general soil fungistasis in its vicinity (Kerr, 1956;

Jackson, 1957). This leads in the first place to a population quantitatively different from that of the soil, and then secondarily to the qualitative differences.

The principle applied above to the reduction in numbers of soil micro- organisms of low competitive saprophytic ability may also apply to less specialized parasites. Newcombe (I960) found that high levels of carbon dioxide inhibited chlamydospore formation in Fusarium oxysporum f. cubense, but allowed conidial germination to take place. This was not inimical to

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survival in pure cultures, but only in the mixed culture conditions of soil when there was no colonizable material present. Chinn and Ledingham (1961) consider a similar situation in which they differentiate between "antagonism"

and "germination lysis," terms they apply to antagonism in the inactive and active phases respectively. In their experiments, stimulation followed by the consequent intensified antagonism resulted in a decrease of Helminthosporium sativum conidia in soil.

Garrett (1955) has pointed out the difficulties in deliberately applying the foregoing principles to methods of biological control. It has for long (e.g.

Daines, 1937) been accepted that there are difficulties in the way of successfully inoculating soil with an antagonist to a pathogen. This difficulty is itself a function of the general inhibitory influence of the soil. Even where the antagonist is a normal member of the soil population or even of the host root itself (Kerr, 1961), there exists the difficulty of holding its population at levels higher than that normally obtaining. The reasons for these difficulties are readily understood in the light of what has been written.

Any successful method of biological control of a soil-borne pathogen must depend on the correct adjustment of the environment so that the appropriate antagonistic relationships are brought into play. Some naturally- resistant plant varieties secrete individual inhibiting substances from their roots which result in a biologically protective microbial population (Timonin, 1940, 1941; Buxton, 1957a). In some hosts an infection of the roots with a mild pathogen may result in a synergistic secretion of inhibiting substances (Perry, 1959) that protects against infection by a more serious pathogen (Bega, 1954; Buxton and Perry, 1959). Similarly, mycorrhizal roots are said (Harley, 1959) to be less susceptible to infection than non-mycorrhizal roots of the same plant. It might be easier to establish artificially and maintain such mild infections than to maintain an antagonist in soil or in the rhizosphere.

Most of the few effective and practicable methods of artificial biological control (Garrett, 1946) depend upon alteration of the soil environment by the addition of fairly large amounts of decomposable organic matter, but it is usually difficult to maintain any prolonged change in the composition of the soil population by this method since the added material fairly quickly disappears (Humfeld and Smith, 1932; Dawson et a/., 1945; Dunleavy, 1955), and also the relative abundance of the organisms in soil tends by antagonistic balance to be buffered as already described, and may even be controlled by balanced chains of parasites and hyperparasites such as that described by Klement and Kirâly (1957). A possible method for biological control, by which a concentration of effective antagonists may practicably be built up on local organic material in soil about the suscept, depends upon the facts demonstrated by Wright (1956), in which the seed coat can allow favourable conditions for the production of substances inhibitory to pathogens. The practicability of the method had previously been suggested by Simmonds (1947) and by Slykhuis (1947).

Attempts to alter deliberately the natural balance of micro-organisms in soil present the same sort of dangers that face workers concerned with the

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14. ANTIBIOTICS AND INHIBITING SUBSTANCES 445

control of higher plant and animal populations, whereby a treatment may set off a chain of events which finally results in the opposite of the desired effect (Odum, 1954). Gibson et al (1961) have described how a method of control of Rhizoctonia solani causing damping-off of tree seedlings subsequently gave an increase in Pythium spp. in the soil with an eventual increase in damping-off. A study of the often complex interactions in soil and a better understanding of the nature and effects of inhibiting influences in soil might enable the effects of treatments to be explained, if not forecast, with more accuracy than is at present possible.

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