Fungal recolonization of heat-treated glasshouse soils

Agro-Ecosysterns, 1 (1974) 1 3 9 - 1 5 5 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

FUNGAL RECOLONIZATION OF HEAT-TREATED GLASSHOUSE SOILS

G.J. BOLLEN

Laboratory of Phytopathology, Wageningen (The Netherlands) (Received September 26th, 1973)

ABSTRACT Bollen, G.J., 1974. Fungal recolonization of heat-treated glasshouse soils. Agro-Ecosystems, 1: 139--155. Experimental data and a literature review on the restoration of mycoflora and microbial activities after heat treatment of soil are given. Four sources for the origin of the initial flora in steamed soil were distinguished, i.e. heat-resistant survivors, air-borne infectants, colonists from the untreated subsoil, and microorganisms introduced with planting material. Among fungi, relatively high heat resistance was found for a number of ascomycetes, especially cleistothecia-forming Penicillium spp. and Aspergillus spp. High heat resistance was also recorded for some deuteromycetes with dark aleuriospores and a basidiomycete, Tephrocybe sp., which forms heavy-walled chlamydospores. Incidence of disease caused by root-infecting fungi, introduced with planting material, can be highly dependent on the biological environment. This was found for t o m a t o seedlings, which were inoculated with Didyrnella lycopersici before they were planted in pasteurized soils. In fungal colonization of steamed soils, the various components showed a marked succession as to their ability to compete for the available carbon sources. During the first weeks after steaming Trichoderma spp. became predominant only in pots, where a considerable " d e p t h of kill" had been achieved by the treatment.

In field or glasshouse, 1 g of soil contains the following number o f viable counts: 5--1 000 million bacteria; 10 thousand--10 million actinomycetes; 10 thousand--1 million fungi; and together many algae and representatives of the microfauna. In this realm of soil microbes, disinfestation of the soil by heat or chemicals means a real catastrophe. Within a few hours, ecosystems built up after long integration of the various components have been destroyed or profoundly modified. This paper deals with the restoration of the microbial population after heat treatment of the soil. A survey is given of data from the literature together with the results of studies in our laboratory at Wageningen. In dealing with the recolonization of steamed soil by the microflora and the restoration o f their physiological activities the fungal part of the microflora will be emphasized. The extent to which the biophase is altered -- to use Kreutzer's (1956) term, " t h e depth of kill" -- is dependent on the temperature and the duration

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of the treatment as well as on soil conditions, especially its humidity and nutrient content. These conditions determine the metabolic state of the biophase. High metabolism appears to be correlated with high serlsitivity of organisms to heat treatments. This relation may be either direct or indirect; the indirect relation also includes the effect of the level of metabolism on the structures in which microorganisms are present. A decrease in metabolism is associated with the formation of dormant cells, e.g. spores of bacteria and chlamydospores, sclerotia and in some cases generative and vegetative spores of fungi. These structures are generally more resistant to unfavourable conditions than those associated with normal metabolism. As a warning against generalizations it should be pointed o u t that among fungicides a few compounds, e.g. actidione, are known that are more effective on spores than on mycelium. In layers of soil where during steaming a temPerature of 100 ° C is reached, for more than 15 min, the biophase is eliminated, apart from some sporeforming bacteria. Treatment of this kind is usually given with relatively small quantities of soil (potting mixtures in containers etc.). When glasshouse softs are steamed in situ, the temperatures reached are largely dependent on the methods used. Solid systems with clay tiles buried in the soil make it possible to reach fairly deep layers (Newhall, 1955; Nederpel, 1971). According to results recently obtained in The Netherlands, this method shows great promise. At the present time, however, treatment is usually given b y blowing steam from the surface into the soil, e.g. under tarpaulin covers. In these cases the upper layers are kept at 100°C for severalhours, while in deeper layers (30--40 cm) the temperature seldom risesabove 50--60 ° C. At this depth the soil is in fact pasteurized and a considerable part of the biophase survives the treatment; physicochemical properties as well are also less radically altered than at the surface of the soft. THE SUBSTRATE A F T E R STEAMING

Steaming causes many nutrients to become available n o t only from the dead biomass, b u t also from substances liberated by the conversion of complex compounds. From the ecological point of view the carbohydrates are interesting. Unfortunately data on the effect of steaming on these c o m p o u n d s are scarce. Soluble carbohydrates increase significantly (Salonius et al., 1967). To the best of my knowledge there are no detailed qualitative studies on this fraction. Since the beginning of this century, however, many investigators have studied the effect of steaming on nitrogen c o m p o u n d s and the solubility of cations. This is due to the importance of these c o m p o u n d s for agriculture. The most marked effects are an increased solubility of salts, especially of manganese, and an increase in water-soluble nitrogen compounds, mainly NH~ -N. Some ammonia is formed during steaming, but most of it soon after;

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this persists for several weeks (Waksman and Starkey, 1923; Davies and Owen, 1953; Jager et al., 1969). A considerable increase in amino acids has been reported by Coleno et al. (1965). Thus many easily decomposable substances that serve as sources of carbon and nitrogen become available during steaming. Sterilized soil, therefore, becomes a fertile substrate for microbes. The pioneers among these are soon followed b y species belonging to the mesofauna. In a glasshouse on a sandy soil at Naaldwijk, enchytraeids (white worms) occurred in such great masses that they interfered with our experiments on the influence of steaming on cellulose decomposition. In untreated control soil they did not occur to any marked extent. ORIGIN OF THE INITIAL M I C R O F L O R A

For the origin of the recolonizing organisms four sources can be distinguished (see Fig.l).

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Survivors; heat-resistance Various microorganisms differ considerably in their heat resistance. Most root-infecting fungi and weed molds of commercial mushrooms are fairly heat-sensitive (Baker, 1962; Bollen, 1969; Wuest and Moore, 1972). On the

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other hand spore-forming bacteria and some soil fungi survive treatment even at 80 ° C for 30 min. The occurrence in soil of these fungi was studied for 54 glasshouse soils in The Netherlands. In glasshouse softs the temperature rarely exceeds 30 ° C. Therefore, we limited our search for heat-resistant fungi to mesophilic species. Suspensions of fresh soil and water I : 1 (v/v) were heated in a thermostatic waterbath for 30 min after the desired temperature had been reached. The temperature was measured with thermocouples. For analysis of the fungal flora, the heated suspension was diluted up to ten times with potato-dextrose agar containing an oxytetracyclin, Vendarcin, 50 ~g/ml. Most heat-resistant fungi were ascomycetes. Although some discomycetes with a Botrytis-like conidial stage were found, most ascomycetes were sclerotia-forming or cleistothecia-forming penicillia and aspergilli. Some of these were f o u n d after treatment of the soil at 70 ° C but n o t at 80 ° C, e.g. Eupenicillium lapidosum and Penicillium thomi; others survived 80 ° C, e.g. Eupenicillium brefeldianum (st. con. Penicillium brefeldianum ), Hamigera avellanea (st. con. Penicillium avellaneum), and Sartorya fumigata (st. con. Aspergillus fisheri). Whereas Talaromyces flavus (st. con. Penicillium vermiculatum) has frequently been isolated from soils treated at 70 ° C, it was found only rarely after treatment at 80 ° C. The same holds for Byssochlamys nivea (st. con. Paecilomyces niveus). A second group of heat-resistant soil fungi was found among deuteromyceres with dark aleuriospores, e.g. Gilmaniella humicola, Trichocladium opacum and T. piriformis; these survived the 80°C treatment. Some other species, e.g. some Humicola spp., survived treatment at 70 ° C but n o t at 80 ° C. Although basidiomycetes were rarely recorded in soil pasteurized at 70°C or more, occasionally one resistant species was found. This species, determined as Tephrocybe carbonaria by Mr H.S.C. Huijsman, attracted our attention because of the formation of heavy-walled chlamydospores both in soil and on culture media. Moreover, in vitro toadstools developed abundantly on oatmeal agar. Little is known about the heat resistance of actinomycetes. Kurzweil (1943) reported t h a t some species survived steaming for m a n y hours. As he tested survival by incubating treated softs at 45 ° C, these heat-resistant actinomycetes may have been thermophilic. For five types of soil, Broadbent et al. (1971) recorded about 1 000 viable counts of mesophilic Streptomycetes spp. per g soil when suspensions were heated at 70°C for 10 min, but few in suspensions heated at 80°C. In one glasshouse soil (loamy sand; pH water 7.1) pasteurized at 70°C for 1 h, we found two types among actinomycetes that were especially predominant when the soil had been amended with cellulose. For many of the survivors a heat shock seems to be needed to break the dormancy of their spores. In vitro this has been studied extensively for spores of Bacillus spp. (Powell and Hunter, 1955) and other spore-forming bacteria and for ascospores of Neurospora and other Sordariaceae among the fungi (cf. Sussman and Halvorson, 1966). Heat-activated germination of fungal

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Fig.2. CO 2 release f r o m p o t t i n g soil a f t e r t r e a t m e n t a t 60°C a n d a f t e r i n o c u l a t i o n o f autoclaved soil w i t h Trichoderma sp. T h e p o t t i n g soil c o n s i s t e d o f a d e c o m p o s e d S p h a g n u m p e a t t o w h i c h s o m e clay a n d 'marl were a d d e d ; p H - w a t e r 5.9; organic m a t t e r 44.0%. U n d e r sterile c o n d i t i o n s t h e samples o f soil ( 2 0 0 g fresh weight; 60 g d r y w e i g h t ) were a e r a t e d w i t h CO 2 -free air ( f l o w rate 9 0 0 1 1 0 0 m l / h ) at 21 ° C. CO 2 was a b s o r b e d in K O H a n d d e t e r m i n e d b y d i f f e r e n t i a l t i t r a t i o n w i t h HC1 w i t h p h e n o l p h t h a l e i n a n d b r o m o p h e n o l - b l u e as i n d i c a t o r s in e a c h o f t w o replicates.

spores in soil has been reported by Warcup and Baker (1963). In their softs this was especially evident for ascospores of Aspergillus fisheri. In glasshouse softs it was f o u n d for aleuriospores of Gilmaniella humicola (Bollen, 1970). Although heat-resistant structures of microorganisms can be detected in most soils, heat tolerance as such is an exception among microbes. Most of them are killed by treatment of the soil at 60 ° C for 30 min. That the biomass killed at this temperature must be substantial can be concluded from the marked increase of CO2 production -- up to nine times that of untreated soil -during the first days after treatment (Fig.2). The biomass that has been destroyed serves as food for the survivors; these thrive on it in abundance. This results in a high CO2 production. As autoclaved Saml~les of the same soil did not show such an increase in CO2 production the increase may n o t be due to physicochemical liberation. Inoculation of autoclaved soil with one of its original inhabitants revealed a pattern of respiration similar to that found after the 60 ° C treatment (Fig.2).

Air-borne infectants The role of this group of pioneers is in direct proportion to the " d e p t h of kill" obtained by the heat treatment. The group constitutes by far the most important source of recolonizing microflora after treatment of soils at 90 ° C and higher. By exposing agar plates to aerial contamination I measured the density of the "spore rain" landing on the surface of a freshly steamed glass-

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house soft. Immediately after the plot treated last had cooled, 36 380 viable counts of fungi, bacteria, and actinomycetes per m 2 were detected within lh. The composition of the initial microflora present in the superficial layers of the soil, however, differs from that of germs which have landed on the soil even when the soil has been steamed at high temperatures. It can, therefore, be concluded that freshly sterilized soil does n o t offer an adequate substrate for every microorganism. Katznelson's work (1940) reveals that inability to grow in steamed soils may be rather the exception than the rule among soil-borne microorganisms. Katznelson used autoclaved soil as a medium for pure cultures of 13 saprophytes. Among these were bacteria, actinomycetes and fungi. Only A z o t o b a c t e r chro(~coccum failed to multiply in this medium. He suggested that this might be explained by a lack of suitable nutrients or by the production of toxic materials during autoclaving. In a study on the fate of germs which were introduced into steamed soil, Rapilly and Coleno (1967) distinguished two patterns of behaviour. Botrytis cinerea and Pseudomonas phaseolicola failed to develop when introduced into freshly steamed soil. Once the nitrifying bacteria had reappeared and the activity of the ammonifying microflora had decreased, B. cinerea was able to develop at the surface of the soft. This was about 3 weeks after the treatment. Organisms showing the second pattern of behaviour established themselves only if they were introduced within a short period after steaming. Their inability to develop when introduced after this period was thought to be due to sensitivity to antagonism exerted by the initial microflora. The organisms were able to play a part in the ammonification. A detailed study on Erwinia varotovora revealed that this species was unable to develop when introduced into the soil sooner than 6 h or later than 48 h after sterilization. The authors attributed the lag period of at least 6 h after steaming to the liberation of volatile toxic substances from the soil. Untreated subsoil as a source o f colonists

A third group of invaders originates from the untreated subsoil. Because of the temperature gradient in the soil during steaming the heat resistance of the microbial population present directly after treatment decreases with depth. In this way fast growing and fairly heat-resistant fungi have a chance to become the first invaders from subsoil into sterilized layers. However, Warcup (1951) found that recolonizing fungi occurred most frequently near the surface of the soil. He suggested that fungal colonization from the untreated subsoil into the steamed soil was of minor importance. Unlike the upward spread, a rapid horizontal growth of many fungi into the treated plots from untreated soil at the sides of the experimental plots was found. Because this t o o k place especially in surface soil, Warcup indicates that some of this recolonization may even have been aerial. Using Evans' recolonization tubes,

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Lily (1965) found fairly rapid growth from untreated soil into steam-sterilized soil in the following sequence: PeniciUium nigricans, Aspergillus niger,

Paecilomyces varioti, Aspergillus flavus, Trichoderma viride, Aspergillus sydowi, Fusarium sp. and Curvularia lunata. However, bacteria had colonized the sterile soil before the fungi. These findings are not directly pertinent to the colonization of glasshouse soils in practice since in Lily's experiments the steamed soil was directly in contact with untreated soft. After steaming in situ this transition is gradual, viz. via partially sterilized layers with more or less heat-resistant organisms which may act as a barrier against rapidly growing pioneers. Growers of crops that root deeply are usually confronted with a type of colonization specific for a number of pathogenic fungi whose occurrence is restricted to roots and which maintain themselves for a certain time in the debris of previously invaded root tissue, i.e. the root-inhabiting fungi sensu Garrett. When these roots are present in the subsoil below the layers treated during steaming, the root-inhabiting fungi survive in the root-debris either as sapropl~ytes or as resting structures. At a given time after a susceptible crop has been planted in these soils, the roots reach the infested subsoil. After infecting the roots the fungu s spreads upwards via the r o o t system, in this way colonizing the steamed soil, using roots as colonization roads (Fig.l). An example of this can be found in the reinfestation by Phialophora cinerescens of soil planted with carnations. From 6 months to 1 year after steaming, the first s y m p t o m s of wilting caused by this pathogen often appear.

Fungi introduced with planting material This group of colonists differs from the previous ones in that they are introduced into soil by man, although unintentionally. Among them pathogens may be present even on roots of healthy plant material, for a number of these fungi, e.g. Cylindrocarpon destructans, can live both in the r o o t tissue and epiphytically on the surface of the roots. Their infection and subsequent development in the r o o t system is largely dependent on the biological environment. To a certain extent the presence of an antagonistic microflora can prevent this. This is illustrated by an experiment in which pasteurization was used to reduce the antagonism potential. Lots of soil treated at various temperatures were planted with t o m a t o seedlings whose roots had previously been dipped in a spore suspension of Didymella lycopersici. We found that as the temperature of soil treatment increased -- thus with decreasing antagonism potential -- the incidence of wilting rose (Table I). S U C C E S S I O N IN F U N G A L C O L O N I Z A T I O N

Long before mycologists began studying the recolonization of disinfested soils, growers were familiar with phenomena associated with a succession in the fungal recolonization. They had realized that steaming of their glasshouse

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TABLE I Effect of soil t r e a t m e n t on wilting of t o m a t o seedlings inoculated with Didymella lyco-

persici before planting No treatment

Wilted plants* (%) at 2 weeks after planting

28

T r e a t m e n t (° C, 30 min) 50

55

60

70

80

autoclaved

24

21

39

35

49

94

Soil: potting mixture, organic m a t t e r 58.9%, pH 5.9. *n = 100 seedlings/treatment. Inoculation by dipping the r o o t system in a spore suspension (3.106 spores/ml). The plants were k e p t at 20 ° C.

soils had been successful if the colourful "steam fungi" appeared in abundance some days after the treatment. The most c o m m o n of these are the rose Monilia sitophila and M. crassa, the red Pyronema species, and the brown Peziza ostracoderma. Sometimes the soil becomes green through Trichoderma spp. On steamed potting soils in warm (> 20 ° C) glasshouses, the yellow toadstools of Lepiota lutea may be found. The growers also know that these phenomena are only temporary; no sooner do the fungi appear than they vanish. The steam fungi are generally characterized by fast growth on a substrate containing easily decomposable nutrients and by a lack of tolerance to antibiotics produced by other fungi. With a few exceptions, e.g. Peziza ostracoderma, their fructifications disappear as soon as other colonizing fungi develop. In colonizing the steamed soil, the various components show marked succession in their ability to compete for the available substrates. This succession can be studied by analyzing the microflora and also by measuring the biological activities in the successive stages of the nitrogen and carbon cycles. Among the most important inorganic sources of nitrogen there is presumptive evidence of fungal preference for NH~. Whereas nitrates are excellent sources of. nitrogen for most fungi, an inability to utilize them is c o m m o n in s0me groups, e.g. the higher basidiomycetes. A large number of fungi fail to utilize nitrit~ at all (Cochrane, 1958). As mentioned earlier, many studies have been devoted to the occurrence of these compounds in soil after steaming. Build-up of ammonia is followed by a relatively slow increase in nitrite and subsequently nitrate. This is due to the sequence in the microbial colonization of the steamed soil. Among the survivors and pioneers many ammonifiers are found; these form ammonia out of organic nitrogen compounds e.g. amino acids liberated by the heat treatment (Coleno et al., 1965). Nitrifying bacteria, however, reappear in the soil relatively late. The same sequence holds for sterilization with chemicals (cf. Kreutzer, 1965).

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Because most fungi can utilize NH~ as well as nitrate, the effect of nitrogen source on succession in fungal colonization is often considered to be of minor importance: The inability of Botrytis cinerea to develop when introduced into the soil during the "ammonia phase" and the ability to grow on the soil when nitrification has been restored (Rapilly and Coleno, 1967), however, suggest that nitrogen sources may play a role -- either directly or indirectly -in the succession of fungal colonization. Since Garrett (1963) discussed the succession in fungal colonization of dead plant tissue in soil, we have been aware of the key-role of carbon sources. These substrates in particular "are more or less ephemeral, and each passes in turn through the three phases: colonization, exploitation, and exhaustion". Firstly, fast-growing species appear that utilize sugars and carbon sources simpler than cellulose. These fungi are followed by those that metabolize cellulose, accompanied by species that thrive on sugars liberated during cellulose decomposition. Finally, the fungi that break down lignin and other complex compounds have a chance. Our study on the fungal recolonization of one glasshouse soil clearly illustrates this succession. Using the soil-dilution plate method, we found that sugar-consuming yeasts increasedto a peak 3 days after steaming, later the cellulose-degrading Chaetomium spp., and finally Botryotrichum piluliferum (Fig.3). This fungus is able to utilize lignified plant material (Haider and Domsch, 1969).

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Fig.3. Colonization of steamed glasshouse soft by fungi with differing substrate preferences. Clay soil; pH-KC1 7.0; organic m a t t e r 10.0%. During the period in which the colonization was studied soil moisture was a b o u t 50% o f field capacity. The data represent averages o f numbers of colonies on three soil dilution plates (cellulose agar and soil extract agar provided with 30 m g / m l Terramycin).

The influence of cultivation methods on fungi developing after soil steaming is illustrated by the following example. A fairly c o m m o n practice in cultivation of cucumbers is to grow the plants on straw that has been buried in soil and to which fertilizer containing nitrogen has been added. Fig.4 shows the effect of this amendment on the occurrence of Botryotrichum and Dorato-

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log number of colon,es I gromr~e of sod I dw)

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myces spp. (cf. De Waard, 1968). These fungi are able to utilize cellulose and lignin (Haider and Domsch, 1969). Many microbiological activities are quickly restored. To a certain extent this is shown by the considerable increase in respiration after heat treatment of the soil (Fig.2). Within 2 weeks after steaming, Coleno et al. (1965) found that anaerobic cellulolysis, amylolysis and ammonification even exceeded the level obtained for soil before steaming. For a long time after sterilization, however, the composition of the microflora -- at least the fungal population differs qualitatively and quantitatively from that of the flora in the untreated soil. Data on the recolonization o f fungi in steamed soil .are a matter of controversy, as was indicated by Warcup (1957) and Welvaert (1962). Some authors have found an increase in fungi many times the level of that in untreated soil. Most of the studies, however, have indicated slow restoration of the fungal population. Working with dilution plates, Coleno et al. (1965) obtained about 7% of the viable counts recorded for the unsteamed soil 28 days after steaming. 83% of the fungal flora then consisted of Mortierella spp. In an extensive study on the recolonization of a steamed forest nursery soil in the field, Warcup (1951) found that even 18 months after treatment the fungal flora had n o t been restored either quantitatively or qualitatively. Later on he realized that the method of analysis influenced this conclusion. Since some basidiomycetes fruited in the second year after treatment, these fungi must have been present as mycelium. However, they were n o t recorded at all on soil plates or on soil-dilution plates (J.H. Warcup, personal communication). From 7 to 10 weeks after steaming, the flora consisted mainly o f species of Mortierella, Other dominant species were Coniothyrium spp. and Phoma eupyrena. In p o t experiments, an analysis of the fungal flora carried out 3 months after steaming revealed significantly more viable counts in

149

treated than in untreated soils (Martin, 1950). Whereas more than 20 species were identified in the untreated series only one to five species were found in the treated soils. On soil plates, Welvaert (1962) obtained a b o u t half the number of colonies from annually steamed glasshouse soils, as from those which had never been steamed. Furthermore, when working under laboratory conditions he found a rapid fungal colonization, mainly Penicillium spp., of steamed soil in p o t experiments, b u t a slow restoration of the population in glasshouse soils under field conditions. He thinks that this difference might be an explanation for the controversy in the literature data on the rate of recolonization. Herzog (1938) related the rate of fungal colonization to the acidity of the soils steamed. On soil-dilution plates, she found an increase in viable counts of fungi in acid soils b u t a decrease in most of the neutral and alkaline soils. The succession of the various groups of fungi with regard to their ability to utilize various substrates follows a fairly clear line, but qualitative studies on fungal recolonization reviewed by Warcup (1957), Welvaert (1962), and Kreutzer (1965) snow that the species composition within the substrate groups may differ greatly in various soils. Martin (1950) reported considerable differences even between replicates of the same treatment. It seems to be oependent largely on the species that happens to arrive first in the soil in which the treatment has created a biological vacuum. R E S T O R A T I O N OF ANTAGONISM TO ROOT-1NI~ECTING FUNGI

Many ecologists among the plant pathologists are more interested in the antagonism to pathogens than in any other microbial activity of the saprophytes. The marked decrease in this antagonism as a result of steaming is regarded as an undesirable side-effect (Baker, 1967). In his review on soil disinfestation, Newhall {1955) mentioned a number of cases where pathogenic fungi spread much faster in steamed than in unsteamed soils. More damage to crops in steamed than in unsteamed soil was reported for Rhizoctonia solani on pepper (Baker, 1947), Didymella lycopersici on t o m a t o (Williams et al., 1953), and Cylindrocarpon destructans on cyclamen (Scholten, 1956). The effects of heat treatment of soil on Didymella stem rot of previously inoculated t o m a t o seedlings as shown in Table I are consistent with these data. On the other hand, rapid rebuilding of the bacterial flora (Katznelson and Richardson, 1943; Tam and Clark, 1943; Coleno et al., 1965), in some cases accompanied by an abundant increase of fungi, suggests a quick restoration of antagonistic activities. I tested this for the antagonism to Phy tophthora cryptogea. Samples of a potting mixture were treated at various temperatures and subsequently stored in the greenhouse both under sterile conditions and exposed to air-borne infections. After one week, samples were placed in Petri-dishes and the antagonism to P. cryptogea exerted by the microflora was determined. This was done by placing an inverted disc of agar with mycelium onto a cellophane membrane covering the soil, which had previously been smoothed with a thin layer of water agar. Fig.5 shows the mycelial i

150

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Fig. 5. Mycelial growth of Phytophthora eryptogeaon potting soil immediately and 7 days after the soil has been steamed. T h e g r o w t h was m e a s u r e d in each o f five replicates; a t each m e a n value s t a n d a r d deviat i o n is indicated. The soil was incubated at approx. 20° C. Soil moisture was 44% of field

capacity; pF 1.9. For further data of soil see legend of Fig.2. growth on the various soil samples. The results of the experiment revealed that in autoclaved soil within 1 week the antagonism had been restored almost completely. In the absence of air-borne infectants, the heat-resistant microfloraespecially the microorganisms surviving the 80 ° C treatment -- had almost completed the restoration of the antagonism. The inhibition of mycelial growth on autoclaved soil stored for 7 days under sterile conditions, as compared to t h a t on autoclaved soil inoculated immediately after the treatment, may be due to bacterial antagonism. It was f o u n d t h a t a few bacteria had survived autoclaving. Besides bacteria in the 80 ° C-treated soil, an unidentified Cladosporium-like fungus forming heavy walled chlamydospores survived the treatment. In the autoclaved soil exposed to the air, Trichoderma was found. One should be careful using these results to generalize about the restoration of antagonism. Antagonism is n o t only specific for the microflora exerting it, but also highly specific for the organism towards which it is exerted. With regard to the microflora of a potting soft, Phytophthora cryptogea was very sensitive to fungal antagonism, and Fusarium solani and Rhizoctonia solani to bacterial antagonism (Bollen, 1971). Among the bacteria both Pseudomonas fluorescens and Bacillus cereus (Katznelson, 1940) and B. subtilis (Olsen and Baker, 1968) were effective against R. solani.

151

Because of the antagonism of Trichoderma spp. to many root-infecting pathogens -- either b y antibiosis or by hyperparasitism -- their occurrence in steamed soils is of particular interest. The colonization of disinfested softs by Trichoderma is concisely reviewed b y Warcup (1957). He pointed o u t that after treatment with chemicals like formalin, carbon disulphide, and chloropicrin, this fungus is c o m m o n l y the dominant species, b u t that data on dominance in steamed soils are inconsistent. He cites Evans (1955), who found that if the soil Was loosely packed Trichoderma was the pioneer among the fungi, b u t not if the soil was closely packed. Evans suggested that this might explain w h y Trichoderma does n o t always become dominant in steamed soils. An additional explanation m a y be found in the effect of the " d e p t h of kill" of the treatment on subsequent microbial colonization of the soft. In a sandy glasshouse soil at Naaldwijk, plots were steamed for at least 1 h at 70, 85 and 100 ° C to a depth of 50 cm. 14 days after steaming analyses of soil from these plots revealed exceptionally poor colonization b y fungi; Trichoderma was predominant only in soil steamed at 100°C (Table II). Evidently in its turn this well-known antagonist has its own antagonists among the microflora that survive milder heat treatments. The literature on survival in soil after steaming justify the inference that there is considerable difference in the severity of these treatments, i.e. in the " d e p t h of kill", achieved b y them. This may be a second explanation as to why in steamed soils Trichoderma has sometimes, b u t n o t always, been found to be dominant.

TABLE II Frequency of Trichoderma spp. in glasshouse soil 14 days after the soil had been steamed Soil

Depth (cm below surface)

N u m b e r of washed particles plated out*

N u m b e r of fungal isolates Total n u m b e r

Untreated

Trichoderma spp.

0-- 5 . 30--35

100 200

78 139

5 3

Steamed at 85°C for 1 h

0--- 5 30--35

200 200

9 4

0 0

Steamed at 100 ° C for 1 h

0-- 5 30--35

200 200

57 34

55** 33

For the analysis five samples of each t r e a t m e n t were mixed together in one composite sample. Soil: loamy, organic matter 6.9%, pH 7.1. Soil temperature varied f r o m 20 to 26°C (15 cm below surface). *After t h o r o u g h washing of the soil, particles of organic matter were plated o u t on carboxyme thylcellulose-agar. **84 of the 88 isolates of Trichoderma obtained f r o m steamed soil were identified as T. pseudo-koning£

152 Because of its high degree of resistance to fumigants (cf. Warcup, 1957), the origin of Trichoderma in chemically disinfested softs is mainly ascribed to Survival. Data on heat resistance are controversial. Survival after soil steaming (Ludwig and Henry, 1943; Welvaert, 1962), as well as lack of heat resistance (Bollen, 1969) have been reported. As suggested earlier, its occurrence in freshly steamed soil may be due rather to a rapid spread after re-invasion than to survival. Whatever the cause, a dominance of this fungus can contribute greatly to the restoration of antagonism towards a number of pathogens. However, the antagonism in treated soil, even when the availability of more substrate causes it to reach a level higher than in untreated soil, will be delicate because of the relatively few species exerting it. Alteration in the conditions will result in a disturbance in the "equilibrium" within the population, rather than in a well-balanced pluriformal microflora. For this reason, treatments like Baker's soil pasteurization, allowing the survival of a considerable part of the saprophytic microflora, are preferable to treatments that eradicate it. CONTROLLED RECOLONIZATION The question is, whether it will prove possible to manage the recolonization of steamed soils in such a way that rapid rebuilding of a stable antagonism can be achieved (cf. Kreutzer, 1965). Although many cases are known where the inoculation of planting material with antagonists has shown promise for the control of soil-borne pathogens, e.g. the use of a strain of Bacillus subtilis for Fusarium-wilt in carnations (Aldrich and Baker, 1970), relatively few examples are available of treated soils having been protected from infestation by pathogens through a previous inoculation with their antagonists. In 1957, in the famed U.C.-manual 23, under the title "Controlled recolonization: a future step,,, Ferguson presented his positive results on controlling dampingoff in pepper seedlings caused by Rhizoctonia. In spite of some successful attempts (e.g. Novagrudsky, 1935; Olsen and Baker, 1968; Wensley, 1969; Broadbent et al., 1971), in 1973 this is still a matter for the future for practical application to glasshouse soils. Reasons for this may be: (1) In most experiments only one antagonist is used -- a selected strain of Bacillus subtilis, Trichoderma sp. or Penicillium sp. As mentioned above, this creates an unstable situation, so that the results are inconsistent. More stability in antagonism can be expected from inoculation with a group of antago° nists which are suited to each other, in other words a compatible combination. (2) For the key-role of the substrate in colonization, the inoculants must be provided with an adequate substrate. When this is a general substrate that can be used by many microbes, competition will cause the effect of inoculation to be short-lived. This is typical of many inoculations with antagonists.

153

So far these are suitable only for protection against diseases during the early stages of a crop, e.g. seedlings, cuttings etc. Once we know not only the substrate preferences, but also the compatible combinations of antagonists, then the ingredients to be added after disinfestation will be ready to supply prolonged protection against root-infecting fungi. ACKNOWLEDGEMENTS

I am greatly indebted to Mrs E. Helmet van Maanen, Ph.D., for help in preparing the English text, to Dr J.H. Warcup from the Waite Institute at Glen Osmond, South Australia, for critical comments on the manuscript, and to Mrs B. van der Pol-Luiten and Mr G.A.J.M. Willemse for their meticulous performance of part of the experiments. REFERENCES Aldrich, J. and Baker, R., 1970. Biological control of F u s a r i u m r o s e u m f. sp. d i a n t h i by B a c i l l u s subtilis. Plant Dis. Reptr., 5 4 : 4 4 6 - - 4 4 8 Baker, K.F., 1947. Seed transmission of Rhizoctonia solani in relation to control of seedling damping-off. Phytopathology, 37 : 912--9 24 Baker, K.F., 1962. Principles of heat treatment of soil and planting material. J. Aust. Inst. agric. Sci., 28:118--126 Baker, K.F., 1967. Control of soil-borne plant pathogens with aerated steam. Proc. Greenhouse Grow. Inst. Pullman, Wash., pp. 3--18 Bollen, G.J., 1969. The selective effect of heat treatment on the microflora of a greenhouse soil. Neth. J. Plant Patho]., 7~: 157--163 Bollen, G.J., 1970. Effect of pasteurization on spore germination of some saprophytic fungi from soil. Acta bot. neerl., 19:117--118 (Abstract) Bollen, G.J., 1971. Antagonism after pasteurization of greenhouse soil. Acta bot. neerl., 20:258 (Abstract) Broadbent, P., Baker, K.F. and Waterworth, Y., 1971. Bacteria and actinomycetes antagonistic to fungal root pathogens in Australian soils. Aust. J. biol. Sci., 24:925--944 Cochrane, V.W., 1958. Physiology of Fungi. Wiley, New York, N.Y., 524 pp. Coleno, A., Rapilly, F., Pionnat, J.C. and Aug,, G., 1965. Incidence des variations de la microflore d'un sol d~sinfect~ sur la gen~se de certaines maladies des plantes. I. Variations quantitatives et qualitatives de la microflore d ' u n sol trait~ ~ la vapeur. Ann. Epiphyt., 1 6 : 2 6 7 - - 2 7 8 Davies, J.N. and Owen, O., 1953. Soil sterilization. II. Ammonia and nitrate production in a glasshouse soil steam-sterilized in situ. J. Sci. Fd Agric., 4 : 2 4 8 - - 2 5 7 De Waard, M.A., 1968. De herkolonisatie van kasgrond door microorganismen na volledige grondontsmetting door stomen in de praktijk. Neth. J. Plant Pathol., 7 4 : 5 4 (Abstract) Evans, E., 1955. Survival and recolonization by fungi in soil treated with formalin or carbon disulphide. Trans. Brit. mycol. Soc., 3 8 : 3 3 5 - - 3 4 6 Ferguson, J., 1957. Beneficial soil microorganisms. In: K.F. Baker (Editor), The U.C.System for Producing Healthy Container-Grown Plants. California Agric. Exp. Stn Manual, 23: pp. 237--254 Garrett, S.D., 1963. Soil Fungi and Soil Fertility. Pergamon Press, Oxford, 165 pp. Haider, K. and Domsch, K.H., 1969. Abbau und Umsetzung von lignifiziertem Pflanzenmaterial dureh mikroskopische Bodenpilze. Arch. Mikrohiol., 6 4 : 3 3 8 - - 3 4 8

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Herzog, G., 1938. f.Jber den Einfluss der Dampfung auf die biologischen und chemischen Eigenschaften der Gartenerden. Bodenk. Pfl. Ern$/hrung, 1 2 : 3 3 9 - - 3 8 4 Jager, G., Van der Boon, :J. and Rauw, G.J.G., 1969. The influence of so}l steaming on some properties of the soil and on the growth and heading of winter glasshouse lettuce, I. Changes in chemical and physical properties. Neth. J. agric. Sci., 1 7 : 1 4 3 - - 1 5 2 Katznelson, H., 1940. Survival of microorganisms inoculated into sterilized soil. Soil. Sci., 49:211--217 Katznelson, H. and Richardson, L.T., 1943. The microflora of the rhizosphere of tomato plants in relation to soil sterilization. Can. J. Res., 2 1 : 2 4 9 - - 2 5 5 Kreutzer, W..A., 1965. The reinfestation of treated soil. In: K.F. Baker and W.C. Snyder (Editors), Ecology of Soil-Borne Plant Pathogens. Univ. of California Press, Berkeley and Los Angeles, Calif., pp. 495--508 Kurzweil, H., 1943. t~ber das Verhalten yon nativen und Kulturerdsporen im str0menden und gespaunten Wasserdampf. Ein Beitrag zur Frage der Eignung dieser Sporen als Testobjekte bei der Dampfsterilisation. Z. Hyg. Infekt Krankh., 1 2 4 : 1 - - 7 0 Lily, K., 1965. Ecological studies on soil fungi, I. Recolonization of steam-sterilized soil by different microorganisms. J. Indian Bot. Soc., 4 4 : 2 7 6 - - 2 8 9 Ludwig, R.A. and Henry, A.W., 1943. Studies on the microbiology of recontaminated sterilized soil in relation to its infestation with Ophiobolus graminis, Sacc. Can. J. Res., C 2 1 : 3 4 3 - - 3 5 0 Martin, J.P., 1950. Effects of fumigation and other soil treatments in the greenhouse on the fungus population of old citrus soil. Soil Sci., 6 9 : 1 0 7 - - 1 2 2 Nederpel, L., 1971. 70 ° C-stomen biedt nieuwe mogelijkheden. Groenten en Fruit, p.319 Newhall, A.G., 1955. Disinfestation of soil by heat, flooding and fumigation. Bot. Rev., 21:189--250 Novagrudsky, D., 1935. Use of microorganisms in control of fungal diseases of cultivated plants. Bull. Acad. Sci. U.S.S.R. Biol. Ser 1, pp. 277--293 (Abstract in Rev. appl. Mycol., 16 (1937): 204) Olsen, C.M. and Baker, K.F., 1968. Selective heat treatment of soil, and its effect on the inhibition of Rhizoctonia solani by Bacillus subtilis, Phytopathology., 5 8 : 7 9 - - 8 7 Powell, J.F. and Hunter, J.R., 1955. Spore germination in the genus Bacillus: the modification of germination requirements as a result of preheating, J. gen. Microbiol., 13: 59--67 Rapilly, F. and Coleno, A., 1967. Incidence des variations de la microflore d ' u n sol d~sinfect4 sur la gen~se de certaines maladies des plantes, II. ~tude du comportement des germes introduits dans un sol d~sinfect~ ~ la vapeur. Ann. ~Epiphyt., 1 8 : 4 9 5 - - 5 0 7 Salonius, P.D., Robinson, J.B. and Chase, F.E., 1967. A comparison of autoclaved and gamma-irradiated soils as media for microbial colonization experiments. Plant and Soil, 27:239--248 Scholten, G., 1956. Wortelrot by cyclamen, Vakbl. Bloemisterij, 1 1 : 4 4 3 Sussman, A.S. and Halvorson, H.O., 1966. Spores: their Dormancy and Germination. Harper and Row 7 New York, N.Y., 354 pp. Tam, R.K. and Clark, H.E., 1943. Effect of chloropicrin and other soil disinfectants on the nitrogen nutrition of the pineapple plant. Soil Sci., 5 6 : 2 4 5 - - 2 6 1 Waksman, S.A. and Starkey, R.L., 1923. Partial sterilization of soil, microbiological activities and soil fertility I--III. Soil Sci., 16: 137--158., 247--268, and 343--357 Warcup, J.H., 1951. Effect of partial sterilization by steam or formalin on the fungus flora of an old forest nursery soil. Trans. Brit. mycol. Soc., 3 4 : 5 1 9 - - 5 3 2 Warcup, J.H., 1957. Chemical and biological aspects of soil sterilization. Soils Fert., 20: 1--5 Warcup, J.H. and Baker, K.F., 1963. Occurrence of dormant ascospores in soil. Nature, 4874:1317--1318

155 Welvaert, W., 1962. Bijdrage tot de studie van de bestrijding van fytopathogene bodemschimmels door grondontsmetting. Meded. Landb. Hoogesch. Opzoek Stns Gent, 27: 1631--1789 Wensley, R.N., 1969. Experiments in biological and chemical control of Fusarium wilt (F. oxysporum f. melonis (Leach and Currence) Snyd. and H a n s . ) o f muskmelon. Can. J. Microbiol., 1 5 : 9 1 7 - - 9 2 3 Williams, P.H., Sheard, E. and Read, W.H., 1953. Didymella stem rot of tomato. J. hort. Sci., 2 8 : 2 7 8 - - 2 9 4 Wuest, P.J. and Moore, R.K., 1972. Additional data on the thermal sensitivity of selected fungi associated with Agaricus bisporus. Phytopathology, 6 2 : 1 4 7 0 - - 1 4 7 2 DISCUSSION

Question (Lebbink, G.): Is there a relation between the recovery of nitrification and the re-establishment of some fungi (ammonium toxicity). Does the well-known inhibitive action of steamed soil on added nit-riflers also inhibit the re-establishment of fungi? Answer: Rapilly and Coleno (1967) showed thatBotrytis cinerea was able to establish itself when the nitrification had started. Their results do n o t preclud e that other processes going on at the same time m a y n o t also have played a role in the ability of the fungus to grow on steamed soft. Regarding to the second question, I would like to emphasize that among fungi the behaviour of Botrytis in this respect is probably the exception rather than the rule. Many fungi grow well on steamed soil. The rate at which fungi are sometimes inhibited on steamed soil depends on the type of the soil. I do n o t know whether in these cases the inhibitive principle m a y be the same for fungi and for nitrifying bacteria.