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The fungus flora of fumigated soils
[ 441
1
Trans . Br. mycol. Soc. 51 (3 and 4).441-459 (1968) Printed in Great Britain
THE FUNGUS FLORA OF FUMIGATED SOILS By L. K. MUGHOGHO· Botany School, Uniuersity of Cambridge (With 4 Text-figures) A study was made of the extent to which Trichoderma recolonized fumigated soils with a view to biological control of root disease fungi. Treatment of Kettering soil with allyl alcohol, chloropicrin, dichloropropene-dichloropropane mixture (D-D) , methyl bromide, sorbic acid or acetylenedi carboxylic acid promoted the development of dominant populations of Trichoderma reaching about 100 % of the recolonizing fungus flora as assessed by the dilution plate method. Trichoderma was the dominant fungal recolonizer of both acid and alkaline soils treated with allyl alcohol. Analyses of Trichoderma isolates showed that several species-groups of Trichoderma, namely, T. hamatum, T. harzianum, T . koningii and T. viride, were included within these dominant populations of Trichoderma in the fumigated soils. T . harzianum was, however, the predominant species-group except in limed Kettering soil where it was replaced by T. hamatum. There was no d ifference in the general spectrum of Trichoderma species groups between naturally acid and alkaline soils treated with allyl alcohol. Fumigated soils containing dominant populations of Trichoderma were tested for their effect on the growth of two root disease fungi, Rhizoctonia solani and Armillaria mellea, which are known to be susceptible to antagonism by T richoderma. No inhibitory effect was observed; in fact, both pathogens grew better in the fumigated soils than in untreated soil. Cultural studies of the interaction between R. solani and A. mellea, respectively, and Trichoderma species-groups from fumigated soils showed that not all the species-groups of Trichoderma nor even all isolates of anyone species-group were antagonistic to the two pathogens. It is suggested that the failure of Trichoderma in fumigated soils to control R. solani and A. mellea was due to the fact that the dominant populations of Trichoderma must have been made up largely by strains of the fungus that were not effective antagonists against the two pathogens.
Biological control of root disease fungi by Trichoderma has received much attention since Weindling's (1932) discovery of Trichoderma as an antagonist of root disease fungi in culture. However, no method has as yet been found for the practical exploitation of this phenomenon. In recent years great interest has been aroused by a new approach to the problem suggested by Bliss (1951) who postulated that the high population of Trichoderma (ascribed to T. viride) following carbon disulphide fumigation of the soil was the factor primarily responsible for the killing of Armillaria mellea in infected citrus roots in Californian orchards, rather than the direct fungicidal effect of the fumigant. While plating out samples of root segments from fumigated soil, Bliss regularly isolated Trichoderma from root segments in which A. mellea was no longer viable. Fumigation of infected • Present address: Agricultural Research Council of Malawi, P.O. Box 2 15, Lilongwe. Malawi.
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root segments in sterile soil did not kill A . mellea. However, A. mellea was killed when infected root segments were buried in a pure culture of Trichoderma grown in sterilized soil. In roots in unsterile soil, A. mellea remained viable when not fumigated, but was killed wh en fumigated, Trichoderma showing rapid development in the fumigated soil. From these observations and experiments Bliss concluded that in natural soil Trichoderma is not sufficiently dominant to destroy A. mellea in infected citrus roots. But if the equilibrium of the soil microflora is disturbed by fumigation with carbon disulphide, the recolonizing fungus flora is dominated by Trichoderma which, at this higher population level, is able to kill A. mellea by antagonistic action. In a series of experiments designed to test Bliss's hypothesis Darley & Wilbur (1954) and Garrett (195 7) found that, in small pieces of infected wood, A. mellea is killed directly by fumigation with carbon disulphide. However, in massive roots, the fumigant while killing A. mellea in the outer tissues may not permeate all the root tissue and kill all the mycelium of the pathogen. Garrett postulated that under such conditions biological control may be possible due to the subsequent development of Trichoderma which may invade the roots, and spread inwards, killing the remainder of the pathogen that had been beyond the direct reach of the fumigant. Garrett (1958) also showed that the maximum antagonistic effect of Trichoderma on A. mellea, and doubtless on other fungi too, is exerted by a pure culture of Trichoderma which has a maximum inoculum potential (sensu Garrett, I956a). Nearly 100 % kill of A. mellea was obtained when woody inoculum segments were incubated in a pure culture of T. viride (from Hypocrea rufa) grown in sterilized soil. When woody inoculum segments were incubated in dilutions of the pure culture with unsterile soil, the antagonistic action of T. viride upon A. mellea was reduced. Three conclusions emerge from this brief summary of the work of Bliss and that of Garrett on the role of soil fumigation treatments for promoting biological control of root disease fungi by Trichoderma. These are: (a) the selective action of soil fumigants offers a means ofincreasing artificially the soil population of Trichoderma above its natural level, (b) such an increase may be used for the biological control of root disease fungi, (c) the population level of Trichoderma in the fumigated soil would determine the extent of biological control obtained. The testing of these conclusions provided a basis for this investigation; the objective was to study the ecology of Trichoderma in fumigated soils with a view to finding a soil fumigant that would promote a maximum dominance of Trichoderma, and to test whether this dominant population of Trichoderma in fumigated soil could control root-disease fungi. MATERIALS AND METHODS
Two soil types, Kettering soil and Botanic Garden soil, were used throughout this study. Kettering soil is a reddish, slightly acidic (pH 6-6'2) clay loam collected from under old pasture in Kettering, Northants. Botanic Garden soil is a greyish alkaline (pH 7"5), light-textured loam from the Cambridge University Botanic Garden. Organic and nitrogen contents in both soils are somewhat high, whereas available potash and phosphate
Fungus flora. L. K. Mughogho
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are high- in Botanic Garden soil and unusually low in Kettering soil. Kettering soil, which has been used in previous fumigation studies (Evans, 1955; Garrett, 1957; Saksena, 1960; Moubasher, 1963) with carbon disulphide and formalin, was used for most of the experiments. Both soils were collected when required from dumps at the Botany School Field Station. At the time of collection they were passed through a i in (6 mm) sieve and then mixed thoroughly. The procedure for soil fumigation and incubation was essentially as described by Garrett (1957). Soil was fumigated at a low moisture content of 40 % saturation to facilitate diffusion of the fumigants and to provide a well-aerated soil, which is essential for recolonization by Trichoderma (Evans, 1955). Screw-topped jars of capacity 2400 ml were used as containers, each receiving 1800 g soil. Fumigants were injected into the soil at the mid-point of the jar, after which the soil was resettled by tamping the jar on the bench; the cover was screwed on immediately afterwards and bound to the jar with' Sellotape ', Thereafter jars were left in an incubator at 25°C for 7 days for the fumigant to work. Jars were then opened and left on the laboratory bench for 7 days to allow for the fumigant to escape; they were then returned, still uncovered, to the 25° chamber for further incubation. During incubation the moisture content of the soil was maintained by regular weighing on a box balance and addition of distilled water to original weight. The population levels of Trichoderma in the fumigated and incubated soils were determined by the soil-dilution plate method. Soil dilutions were made from the well-mixed soil of each jar according to the method recommended by Johnson, Curl, Bond & Fribourg (1959) except that a dipper of vol. I ml was used in place of a pipette to transfer the various dilutions of the soil suspension from one vessel to another. Soil dilutions were plated (using the dipper) in replicates offour with 15 ml of the Smith & Dawson (1944) soil extract agar as amended by Moubasher (1963) in each plate. All plates were incubated overnight at 25° and thereafter on the laboratory bench. Plates were examined daily and each fungal colony was marked on the bottom of the plate as soon as it appeared. Where fastgrowing fungi were likely to overgrow and suppress slowly growing ones, they were removed from the plates and cultured separately for later identification. All fungal colonies were counted, identified and recorded as number of colonies of either Trichoderma or 'other fungi' per g air-dry soil. The population level of Trichoderma was calculated as a percentage of the total number of fungi recorded. ECOLOGY OF TRICHODERMA IN FUMIGATED SOILS
if various chemicals on the development if Trichoderma in Kettering soil Trichoderma has been reported as the dominant fungal recolonizer of soils treated with allyl alcohol (Overman & Burgis, 1956), chloropicrin (Smith, 1938), D-D mixture (Altson, 1950), formalin (Warcup, 1951), methyl bromide (Hodges, 1960), sorbic and acetylenedicarboxylic acids (Moje, Martin & Baines, 1957) and thiram (Richardson, 1954). The The effect
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chemicals selected for testing in the present study were, therefore, those listed above but omitting formalin and thiram. The general procedure for treating soil with these chemicals and for estimating population levels of Trichoderma in the fumigated and incubated soils has already been described. Sorbic and acetylenedicarboxylic acids, being of low volatility, were mixed well with the soil, and incubation at 25° was started immediately after the fumigation period. Soil treated with allyl alcohol was also incubated at 25° immediately after fumigation, without a period on the laboratory bench to allow for the fumigant to escape. For each chemical a wide range of dosages was tested in duplicate; nothing was added to the control (untreated) soil. Table I. Percentage population of Trichoderma in the recolonizing fungus flora of Kettering soil treated with various chemicals AcetylenediCarbon Allyl Chloro- Methyl disulD-D Sorbic carboxylic phide alcohol pircin bromide mixture acid acid
Fumigation period/days Incubation period/days Soil moisture content as % saturation Dosagev o (control) 0'08 0'1 0'2 0'3 0'4 0,6 0,8 0'9 1'5 1,6 3'3
4 35
7 21
7 28
7 28
7 21
7 21
7 21
4 0'4
41
4 1'4
4 2'4
42'4
4 1'2
42
3'1 6,8
0'0 9 6'3
0'0
1'7
100'0
77'3
100'0
14'3
80'S
100'0
100'0 100'0
9 6'9 9 6'7
96'3
9 8'8 99'4
4'2
3'5 64'3
0'0 4'2
4'1 12,8 8'3
100'0 100'0
100'0
98.8
100'0
45"8 82'1 89'2
93'3
97.8
• ml/IOOO ml space, or g/750 g soil for sorbic acid and acetylenedicarboxylic acid,
Results are given in Table I. The population level of Trichoderma in untreated soil ranged from I to 5 %. In samples of untreated soils used for the methyl bromide, D-D mixture and acetylenedicarboxylic acid tests, the population of Trichoderma recorded was zero. This does not mean that Trichoderma was totally absent; since Trichoderma appeared in the treated soils, it implies rather that its population level was so low that it could not be detected in the soil samples used for the dilution plates. Changes in the population levels of Trichoderma following treatments were as follows. All chemicals except carbon disulphide promoted dominant recolonization of Kettering soil by Trichoderma. Within the range of dosages tested, there was a minimum dosage level at which Trichoderma became dominant, i.e, its population exceeded 50 % of the total fungus population recorded on the dilution plates. Increase in dosage above the minimum required for dominance resulted in a corresponding increase in the population level of Trichoderma to almost 100 % at the higher dosages.
Fungus flora. L. K. Mughogho
445
In general, this high level of dominance did not fall with further increases in dosage. Comparison of the efficiency of the various chemicals shows that allyl alcohol and methyl bromide promoted maximum dominance of T richoderma at lower dosages than the other chemicals. At higher dosages, however, methyl bromide was slightly less effective than allyl alcohol and chloropicrin. In allyl alcohol-treated soil, recolonization by Trichoderma was usually complete within 14 days' incubation; in soils treated with the other chemicals complete recolonization took 21-28 days. Allyl alcohol was, therefore, chosen for further experiments; chloropicrin was also used but mainly for comparative studies.
The effect of soil pH on the development of Trichoderma in fumigated soils Lily (1961) reported that in an Indian soil of pH 7-7"5, the place of Trichoderma (viride) as a dominant recolonizer after fumigation with carbon disulphide was taken by Penicillium nigricans. When the soil was acidified, Trichoderma replaced P. nigricans as the dominant recolonizer. The replaceTable 2. Percentage population of Trichoderma in allyl alcohol-treated soils at different pH values Dosages o (control)
Botanic Garden soil, pH 7'5
Acidified Botanic Garden soil, Limed Kettering pH 5,8 soil, pH 7'5
0'2
4'0 98'4
3'1 89'7
0'4 0,8 1'6
100'0 100'0 100'0
100'0 100'0 100'0
2'3
97'6 96'7
71 ' 2
49'2
• rnl/looO rnl space.
ment of Trichoderma by P. nigricans was attributed to the alkalinity of the soil. P. nigricans may be more abundant quantitatively than Trichoderma in acid soils, e.g, Evans (1954) recorded 5 % of the former and only I % of the latter in the fungus flora of Kettering soil ; nevertheless, Trichoderma was the dominant recolonizer after treatment with carbon disulphide. It appeared, therefore, that Trichoderma was a more efficient recolonizer of acid soils than P. nigricans and vice versa in alkaline soils. This hypothesis was examined using allyl alcohol, which is a more efficient fumigant than carbon disulphide for promoting dominant recolonization by Trichoderma in fumigated soils. The soils used were: (a) Botanic Garden.soil, a naturally alkaline soil of pH 7'5; (b) Botanic Garden soil acidified with sulphur to pH 5.8; (c) Kettering soil limed to pH 7"5. These soils were treated with allyl alcohol for 7 days and then incubated for 3 weeks. Thereafter population levels of Trichoderma were determined. The results in Table 2 show that, depending upon the fumigant applied, Trichoderma can be the dominant recolonizer of both acid and alkaline soils.
Transactions British Mycological Society Species-groups of Trichoderma which recolonize fumigated soils In previous investigations, the specific name T. viride was frequently applied to all Trichoderma isolates from fumigated soils, although some authors, e.g, Smith (1938), called them Trichoderma sp. or simply Trichoderma. It is known from the recent taxonomic study of the genus Trichoderma (Rifai, 1964) that most Trichoderma isolates can be assigned to one of Table 3. The spectrum of Trichoderma species-groups in Kettering soil treated with albl alcohol and chloropicrin Percentage population of Trichoderma species-groups
Dosage" Allyl 0'0 alcohol 0'2 0'4 0,8 1,6 Chloro- 0'0, picrin 0'2 0'4
No, colonies fungi permg air-dry soil 94 4 12 IOB7Bo 1631 70 lOB780 109
o-s
1·6
182 40 16121 23535
,
Total
longipolybra- pseudosporum hamatum chiatum koningii koningii 1'0 7'7 10'9 4 6'4 0'0
4'0
0 0 0 0 0 0
94'6 95'2 94'1
0 0 0
1'9 60'0 100'0 100'0 100'0
2'7
0 0 0 0 0 0
0 0 0 0 0 0
0'0 0'0 0'0
0 0 0
0 0 0
aureociride horzianum
viride
0'4 9'2 25'5 14'3 5'9 0'0
0 0 0 0 0 0
0'0 43'1 63'6 34'S 94'1 0'9
0'4
4'3 3'B o,B
0 0 0
B9'2 9 1'4 93'3
1'1 0'0 0'0
0'5 0'0
0'0 4.8 0'0
• mljrooo ml space,
Table 4. The spectrum tifTrichoderma species-groups in
Botanic Garden soil treated with albl alcohol No, colonies fungi per mg Dosage" air-dry soil 0'0 0'2 0'4 o'B 1,6
161 1355 4235 5929 10164
Percentage population of !richoderma species-groups Total 3'9 94'4 100'0 100'0 100'0
longibra- pseudopolysporum hamatum chiatum koningii 1'3 0'0 0'0 0'0 0'0
1'3 0'0 4,8 3'7 0'0
0 0 0 0 0
0 0 0 0 0
koningii
aureoviride
0'0 4'7 9'5 0' 0'0
0 0 0 0 0
harzianum viride 1'3 79'7 66'7 BB'9 95'7
0'0 14'0 19'0 7'4 4'3
• ml/IOOO ml space,
Table 5. The spectrum tifTrichoderma species-groups in albl alcohol treated-Kettering soil limed to pH 7'5 No, colonies fungi per mg Dosage. air-dry soil 0'0 0'2 0'4
o-s 1,6
241 225 1 B07 10443 12050
Percentage population of, Trichoderma species-groups Total 2'2 97'6 96'7 7 1"2 49'2
longi-bra pseudopolysporum hamatum chiatum koningii 0'0 0'0 0'0 0'0 0'0
1'1 92'B 7B'9 4 0'3 27'9
0 0 0 0 0
0 0 0 0 0
• mljrooo ml space.
koningii 0'0 2'4 5'5 9'6 1,6
aureo.. viride harzianum 0 0 0 0 0
0'0 2'4 10'0 19'2 19'7
viride 1'1 0'0 2'2 1'9 0'0
Fungus flora. L. K. Mughogho
447
eight species groups, viz. T. polysporum, T. hamatum, T. longibrachiatum, T. pseudokoningii, T. koningii, T. aureo-viride, T. harzianum and T. oiride. An analysis was, therefore, made of the species groups of Trichoderma which develop in fumigated soils, using Rifai's (1969) key for identification. The spectrum of Trichoderma species groups was studied in: (a) Kettering soil treated with allyl alcohol; (b) Kettering soil treated with chloropicrin; (c) Botanic Garden soil treated with allyl alcohol; (d) Kettering soil limed to pH 7'S and then treated with allyl alcohol. Incubation periods were 3 and 4 weeks for allyl alcohol- and chloropicrin-treated soils respectively. In estimating Trichoderma populations especial care was taken to remove all fungal colonies as soon as they appeared on the dilution plates and to culture them separately for identification. All Trichoderma isolates were identified to species-group level by following the procedure of Rifai (1964). Fresh subcultures were made on 2 % malt agar plates. These were incubated at 25° overnight, and then in front of a window to promote sporulation. Young sporulating colonies were examined 3-6 days after inoculation, to record the mode of branching of the conidiophores and the shape and disposition of the phialides. Records of spore size and character were made 2 weeks later when spores were matured. Results are presented in Tables 3 to S. The total percentage population of Trichoderma at each dosage (given in column 3) is split up into percentages of the various species groups of Trichoderma which compose it in the remaining columns of the Tables. T. hamatum, T. harrianum, T. koningii and T. viride were recorded in untreated Kettering and Botanic Garden soils. In addition, T. polysporum was present in Botanic Garden soil. Species groups not actually recorded in untreated soil but which appeared in treated soils, e.g, T. harzianum and T. koningii in Table 3, were assumed to have been present in the untreated soil. In Kettering soil treated with allyl alcohol and chloropicrin, respectively, T. viride occurred sporadically at very low population levels, and T. hamatum was absent from chloropicrin-treated soil. T. koningii and T. harrianum were recorded at all dosages of the two fmnigants; T. harzianum was predominant over all other species-groups present at any dosage. A similar spectrum of Trichoderma species-groups was found in Botanic Garden soil treated with allyl alcohol (Table 4). In limed Kettering soil treated with allyl alcohol, T. hamatum replaced T. harrianum as the predominant species-group (Table 5). These results show that more than one species-group of Trichoderma was included within the dominant populations of Trichoderma in the fumigated soils. TRIAL OF FUMIGATION TREATMENTS FOR BIOLOGICAL CONTROL OF ROOT DISEASE
The experiments to be described in this section were designed to test if fumigated soils containing dominant populations of Trichoderma, as reported in the preceding section, would reduce growth and pathogenic activity of introduced root disease fungi. Rhizoctonia solani Kuhn and Armillaria mellea (Vahl ex Fr.) Kummer about which there is evidence of 29
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susceptibility to antagonism by Trichoderma (Weindling, 1932, 1934; Aytoun, 1953), were used as test pathogens. Experiments with Rhizoctonia solani Growth of Rhizoctonia solani throughfumigated soils R. solani grows well as a saprophyte through natural unsterile soil (Blair, 1943). This ability made it a suitable test pathogen for comparing growth in untreated soil with that in fumigated soils containing dominant populations of Trichoderma. The experiment was carried out in open-ended glass tubes, 1'9 em internal diam x 25 cm long (hereafter called soil tubes), and set up as in Fig. 1. Tubes were first capped at the bottom end with lacquered moisture-proof 'Cellophane' fastened by a rubber band, and then filled with the well-mixed fumigated and incubated soil. Each tube was inoculated above the soil column with an agar inoculum disk (16 mm MOisture-proof cellophane Air space - - 4 - -
~Q"~J----Agar inoculum disk of R. solani
Glass tube
:-,;-:+-- Soi I
Fig.
I.
Growth of Rhiroctonio solani through soil in a 'soil tube'.
diam x 4 mm thick, mycelial face downwards) of R. solani cut from the margin of a colony on Garrett's (1962) synthetic medium, and the top end was then capped with Cellophane. Ten tubes were prepared from the soil in each jar. The growth of R. solani from the inoculum disk down through the soil was measured under the stereoscopic microscope with reflected light, at z-day intervals for 8 days (Fig. 2). Growth of R. solani in untreated soil was taken as 100 %, and its growth in the soils treated with allyl alcohol and chloropicrin, respectively, was calculated as percentages of its growth in untreated soil. Differences between its growth in untreated soil and in soils treated with 0·2, 0·4 and 0·8 ml dosages of allyl alcohol were not significant. Its growth through soil treated with the 1·6 ml dosage was significantly (P = 0'001) greater than
Fungus flora. L. K. Mughogho
449
that through all the other soils, treated and untreated. In chloropicrintreated soil, the growth of R. solani was less in the untreated soil than in the treated soils at all dosage levels. Differences in growth were significant at the 5 % level for 0'2 ml dosage, and at the 1 % level for 0'4, 0,8 and 1·6 ml dosages. These results thus show that this isolate of R. solani (from swede) grew better in the treated soils than in the untreated soil, although the former had been colonized by dominant populations of Trichoderma. Similar results were obtained using isolates of R. solani from tomato and potato. It is concluded, therefore, that fumigated soils containing dominant populations of Trichoderma were not inhibitory to the growth of R. solani. 200
Allyl alcohol
Chloropicri n r--
-
180 160
r--
r--
.s:
~
~ 140
e
DO
~ 120
100
-
~
r--
~I--
80 60
o Dosage
Fig.
2.
N
'V
6
66':-
Q)
..,g
Growth of Rhizoctonia solani (swede isolate) through Kettering soil treated with allyl alcohol and chloropicrin respectively.
Seedling testfor the activity of Rhizoctonia solani in fumigated soils Kettering soil was treated with allyl alcohol and then incubated for 3 weeks, R. solani inoculum was prepared by growing the swede isolate for 3 weeks at 22'5° in 500 ml flasks containing 250 g sand, 12'5 g maize-meal and 50 ml water autoclaved for 1 hat 15lbJin2 (103'4 kNJm 2 ) . Cultures were homogenized by passing through a 3 mm sieve; 12 g of this inoculum was mixed with 228 g fumigated soil and put in a crystallizing dish (9'4 em diam x 5 em depth), which was then covered with a glass lid. Six dishes were prepared from soil at each dosage level. Inoculated soils were incubated at 22'5° for 7 days to enable R. solani to establish itself throughout the soil. Thereafter, twenty germinated swede seeds were sown equidistantly in each dish. Dishes were kept at 22'5° until seedlings emerged, when lids were removed and the dishes kept in a glasshouse (mean temp. 20' 7 ± 2'4°). The soil moisture content optimum for infection by R. solani is about the same as that for soil fumigation, i.e. 40-50 % saturation (Blair, 1943). This moisture level was maintained by weighing the dishes daily and adding distilled water to original weight. To prevent drying of the soil surface, the dishes were kept on metal trays containing a shallow layer of water and covered by a polyglaze box.
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Fourteen days after sowing, numbers of seedlings killed before and after emergence were recorded. Seedlings which failed to emerge were assumed to have been killed by R. solani, because in preliminary tests all pregerminated seeds emerged in uninoculated soil. Seedlings still standing were removed, washed carefully and examined for lesions caused by R. solani. Table 6 shows no significant difference in infection of seedlings by R. solani between the untreated and treated soils. These results thus again show that fumigated soils containing dominant populations of Trichoderma were not inhibitory to the growth of R. solani. Table 6, Infection of swede seedlings by Rhizoctonia solani in Kettering soil treated with allyl alcohol
Dosage'" 0
o-z 0'40·8 1"6
Percentage population of Trichoderma 2,6 61"5 89'1 100 100
Seedlings emerged out of 120 88 7485 70 39
Seedlings Total seedlings killed or killed or infected after infected out of 120 emergence 81 61 67 63 35
113 107 102 113 116
Percentage infection 94'2 89'2 85'0 94'2 94'7
• mlj rooo ml space,
Antagonism of Trichoderma species groups to Rhizoctonia solani. All Trichoderma isolates except T. viride isolate no. 73 (from Hypocrea rufa) were obtained from Kettering soil treated with allyl alcohol or chloropicrin. Experiment 1. Growth of R. solani isolates through cultures of Trichoderma species-groups on sterilized soil. The set-up of this experiment was similar to that shown in Fig. I. Kettering soil at a moisture content of 45 % saturation was sterilized in lots of 250 gin 50 ml conical flasks for I h at I5lb/in2 (103' 4 kN1m2 ) . Each soilseries was then inoculated with a Trichoderma species group and incubated at 22'5 0 for 21 days. Thereafter the replicate cultures of each series were well mixed and filled into sterilized soil tubes; ten tubes were prepared from cultures of each species group of Trichoderma. Each tube was inoculated above the soil column with an agar inoculum disk of R. solani grown on Garrett's (1962) medium, A control set of tubes was prepared from soil which had been sterilized and incubated without inoculation with Trichoderma. The growth of R. solani was measured every 2 days for 8 days. Results in Fig. 3 show that growth of R. solani isolates was better through uninoculated sterilized soil than through the same soil colonized by T. harzianum (14), T. koningii (17) and T. viride (I and 73) respectively. Soil colonized by T. hamatum (8) actually promoted growth of R. solani, The poor growth of R. solani through soil sterilized and then inoculated with Trichoderma isolates can be attributed partly to the depletion of nutrients released by autoclaving; superimposed upon this nutrientdepletion effect, however, must have been further differences due to varied antagonistic effects produced by the different species groups of Trichoderma.
451
Fungus flora. L. K. Mughogho
Experiment 2. Antibiotic activity of Trichoderma species groups against R. solani. A simple 'Cellophane plate' technique designed by Heatley (1947) for use with bacteria and adapted by Gibbs (1966) for use with fungi, was used for testing the antibiotic activity of Trichoderma species groups against R. solani (swede isolate). Sheets of 'Cellophane' (grade
% gr ow t h 20
40
60
80
100
120
Contro l
T. hamatum (8)
T harzianum (14)
T. koningi (17)
T. v;r ide (1)
~
Sw ed e and
If f Wll l
Swede isolate
l---J tomato iso late s
~ To mato iso lat e T v;ride (73)
Fig. 3. The effect of various species groups of Trichoderma on the growth of the swede and tomato isolates of Rhizoctonia solani.
300 PT) cut to fit a Petri dish were washed in boiling distilled water to remove plasticizers, sterilized in distilled water and then spread singly on 15 ml agar (Kettering soil extract, 11; agar, 20 g, adjusted to pH 6'2) in a Petri dish. The' Cellophane' was inoculated in the centre with a 4 mm diam agar inoculum disk ofa Trichoderma species-group cut from the margin of a colony on 2 % malt agar. After 2 days' incubation at 22'5°, the , Cellophane' together with the Trichoderma colony growing on it was stripped off and the plates were inoculated (below the site previously occupied by the Trichoderma inoculum) with a 4 mm disk of R. solani cut marginally from a colony on Garrett's (1962) medium. The growth of R. solani was measured daily for 2 days (Table 7),
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452
Three of the five isolates of T. hamatum showed strong antibiotic activity against R. solani. No antibiotic activity was shown by most of the T. harzianum isolates. Most of the T. koningii isolates were antagonistic to R. solani. All four T. viride isolates showed strong antibiotic activity. The mean antibiotic effects of the different isolates for each Trichoderma species-group are shown in Fig. 4. The highest and lowest levels of antibiotic activity against R. solani were shown by T. viride and T. harrianum, respectively. Table 7. Antibiotic activity of Trichoderma species-groups against Rhizoctonia solani (swede isolate) Trichoderma sp, and isolate no, Nil (control) T, hamatum
T. harzianum
T. koningii
T. viride
* t
7 8 9 24 25 II
12 13 14 15 36 37 42 48 19 21 3° 33 1 2 5 73
Mean * radial growth of Trichoderma sp, (mm/2 days)
Mean * radial growth of R. solani (mm/2 days) 14'03±0'24
28'58 28'00 28'4 1 21"30 23'15 23'75 17'75 24'5° 21'75 24'4 1 22'3 2 17'75 19'5° 18'75 3°'75 22'9° 25'00 26'90 26'25 26'5° 23'58 27'00
4' 00±0'02 13'35±o'15 4' 16±o'02 ° 12'91 ±O'IO 15"83±0'20 15'38±O'40 13'42±o'31 3'16±o'38 15"58±0'24 3'08±o'15 11'25± 0'1 7 15"70± 0'20 3'33±0'11 5'33±0'03 5'58±O'17 8'20±O'10 11'50±O'28 o'75±o'12 ° 0 3'40±o'02
%
inhibition'[ of the growth of R. solani 7 1'49 7°'35 100'00
77"48 78'°5 76'27 62'22 60'23 4 1'55 93'94 100'00 100'00 75'77
Mean of six replicates. Percentage inhibition is only given where it is statistically significant,
These results show dearly, as in the previous experiment, the range of variation in the antagonism of Trichoderma species-groups to R. solani. They also suggest the existence of a number of strains, differing in their antagonism to a given root disease fungus, within each Trichoderma species group. The relation of this to the failure of dominant populations of Trichoderma to inhibit the growth of R. solani will be discussed later.
Experiments with Armillaria mellea Rhizomorph production by Armillaria mellea from woody inoculum segments incubated in fumigated soils. The following experiment was designed to study the effect of dominant populations of Trichoderma in fumigated soils on the production of rhizomorphs by A. mellea. Woody inoculum segments of A. mellea were prepared
Fungus flora. L. K. Mughogho
453
by the method of Garrett (1956b). Shoots (1'75 em diam) of pollarded willows (Salix spp.) were sawn serially into 2'5 cm segments. Eight segments in a 500 ml conical flask were covered with a maize-meal/sand culture medium (150 g sand; 5 g maize-meal; 30 ml water). After autoclaving for I h at 221b/in2 (151'7 kN/m2) , flasks were inoculated with agar disks of A. mellea cut from a colony on 3 % malt agar, and incubated at 22'5° for 13-16 weeks before use. Mean radial growth of R. so/ani in mm/2 days o 10 20 I
I
Control
I
T. hamatum
I
T. harzianum
I
T. koningi
T. viride
0
Fig. 4, Mean antibiotic effects of isolates of Trichoderma species groups against Rhiroctoniasolani (swede isolate).
Table 8. Production of rhizomorphs by Armillaria mellea from woody inoculum segments buried in chloropicrin-treated Kettering soil Mean* percentage population of
Dosage]
Trichoderma 5'1 63'0 100'0 100'0 100'0
No.of'segments (out of 40) which produced rhizomorphs 28 38
f
Total
39
40
t
\
Mean per segment
245 1,052 1,016 944 855
40
* Mean of two replicates.
.
Dry wt. (in mg) of rhizomorphs
8'75 27'68 25'40 24'21 21'38
ml/rooo ml space.
Kettering and Botanic Garden soils were treated with chloropicrin and allyl alcohol, respectively. After incubation for 4 weeks, the well mixed soils were put in 2 lb jam jars, with A. mellea inoculum segments buried in them. Each jar received 660 g soil and ten segments arranged in two layers of five, and the moisture content of the soil was then raised from 40 to 50 % saturation, optimal for rhizomorph production (Garrett, 1956h). Jars were finally capped with moisture-proof 'Cellophane' and incubated at 22'5° for 13 weeks; numbers of segments which produced
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Transactions British Mycological Society
rhizomorphs at each dosage level were then recorded. Rhizomorphs were then cut from the segments, washed, dried at 80° for 48 h and weighed. Table 8 shows that 70 and 90-100 % of the segments in the untreated soil and in the fumigated soils, respectively, produced rhizomorphs; the more important difference, however, is that in yield of rhizomorphs, as shown by the total dry weight of rhizomorphs produced in each series. All the segments had been selected for uniformity in fresh weight, because of the relationship between inoculum weight and extent of rhizomorph production (Garrett, 1956b). Inoculum segments incubated in the fumigated soils gave higher yields of rhizomorphs than segments in the untreated soil. The differences were highly significant (P < 0'001). Similar results were obtained from segments buried in Botanic Garden soil treated with allyl alcohol (Table 9). The conclusion to be drawn from these results is that Table 9. Production of rhizomorphs by Armillaria mellea from woody inoculum segments buried in allyl alcohol-treated Botanic Garden soil
Dosagc'"
Percentage population of Trichoderma
No, ofsegments (out of 10) which produced rhizomorphs
0 0'2 0'4 0·8 1,6
3'9 98'4 100 100 100
10 9 9 9 8
.
Dry wt. (in mg) of rhizomorphs Total 14 1'1 66'0 182'4 334'5 4 63'2
~ean
per segment 14'10 7'33 20'27 37'17 57'90
• ml/rooo ml space.
fumigated soils containing dominant populations of Trichoderma were more favourable than untreated soils for growth of A. mellea. The favourable effect of fumigation due to release of nutrients evidently outweighed any unfavourable effect due to the populations of Trichoderma.
Replacement of Armillaria mellea in woody inoculum segments by Trichoderma species groups. In order to investigate whether Trichoderma species-groups from fumigated soils were antagonistic to A. mellea, a 'replacement' method was developed. In this method, inoculum segments of A. mellea were incubated on pure cultures of Trichoderma species-groups grown on a nutrient-poor medium. After incubation for about 3 months, the segments were removed and the viability of A. mellea determined by plating on a basidiomyceteselective medium (Russell, 1956). The merit of this method was that it represented what would be expected to happen to Armillaria-infected roots in soil following fumigation. The roots would be invaded by the recolonizing species-groups of Trichoderma; if the latter were antagonistic to A. mellea, they would kill and replace it in the roots. Eight inoculum segments of A. mellea were put aseptically into I 1conical flasks containing 5-day-old cultures of Trichoderma species groups on 250 ml soil extract agar (Kettering soil extract, I 1; agar, 20 g, adjusted to pH 6'2) and incubated at 22'5°. The control flasks had been left
Fungus flora. L. K. Mughogho
455
uninoculated prior to adding the inoculum segments. After incubation for 3 months, the viability of A. mellea in the segments was determined as follows. The segments were washed, dipped in 70 % alcohol for 3 min and then surface-sterilized in o- 1 % mercuric chloride for 5 min, followed by thorough washing in sterile distilled water. Thereafter the segments were split down the middle; four small chips were taken aseptically from different positions in the innermost tissues and plated on 15 ml 3 % malt agar containing o· 1 ml 1 % o-phenyl phenol; 0'5 ml 1 % streptomycin was added to suppress bacterial development. Table 10. Viability of Armillaria mellea in woody inoculum segments incubated in pure cultures of Trichoderma species-groups grown on soil extract agar
Trichoderma species-group
Nil (control) T. hamatum (8) T. harzianum (14) T. koningii (I 7) T. oiride (I) T. oiride (73)
No. of segments (out of 32) with living A. mellea 32 30
o
° °5
Percentage viability of A. mellea as determined by cultures from wood chips 100
9°·6
° °o 15.6
Where A. mellea was viable, it grew out from the chips and produced rhizomorphs in the agar. Where A. mellea failed to grow out, it was assumed it had been killed by Trichoderma. As shown in Table 10, A. mellea was viable in all the segments incubated in the uninoculated flasks. In the inoculated flasks, T. harzianum (14), T. koningii (I 7) and T. viride (I) were most effective in killing and replacing A. mellea; T. viride (73) was less effective, and T. hamatum (8) was ineffective. These results are further evidence that different species-groups of Trichoderma, and even different isolates of the same species-group, may vary in their antagonism to a given root disease fungus. DISCUSSION
The conclusion from Bliss's (1951) hypothesis, that dominant populations of Trichoderma (viride) in fumigated soils may be used for the biological control of root disease fungi, may now be examined in the light of results from this investigation. Garrett (1958) modified Bliss's hypothesis by a qualification that the extent of biological control obtained would depend on the inoculum potential of Trichoderma (viride) that developed in the fumigated soil. The inoculum potential of a pathogen, or in the present case, of a root disease antagonist (sensu Garrett, 1956a, 1958) has been resolved by Baker (1965) into four components: (I) inoculum density or intensity (mass or units ofinoculum per unit ofsoil), (2) available nutrients (3) environmental factors, (4) genetic capacity of the organism. The failure of dominant populations of Trichoderma species-groups in fumigated soils to limit the growth of Rhizoctonia solani and Armillaria mellea can thus be attributed to anyone or to any combination of these four factors.
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It is unlikely that ( 1) was limiting, because the inoculum densities of Trichoderma species groups, as assessed by the dilution plate method, were certainly much higher in fumigated soil than in untreated soil. With respect to (2), i.e. available nutrients, it is known that soil fumigation increases the nutrient status of the soil, largely by the killing of microorganisms and the consequent release of nutrients bound in microbial protoplasm (Kreutzer, 1965; Jenkinson, 1966). These nutrients were Table 11. Antagonism of different isolates of Trichoderma species-groups against Rhizoctonia solani and Armillaria mellea
Isolate no.
T. hamatum 7 8 9 24 25 T . harzianum II
12 13 14 15 36 37 42 48 T. koningii 17 19 21 30 33 T . viride I
2
5 73
, Cellophane plate ' technique. R. solani, Swede strain A NA A A NA NA NA NA A NA A NA NA A
,
Tomato strain
A . mellea. Replacement test
NA
NA
NA
A A
SA
A
A
A
A
A
A
A
A
A
SA
'Soil culture.' R. solani
Swede strain
,
A A A NA A A A A
A, Antagonistic; SA , slightly antagonistic; NA, not antagonistic.
available for colonization by Trichoderma species-groups. The fact that both R. solani and A. mellea grew better through fumigated soils than through untreated soil, even though the former had been colonized by T richoderma, suggests that residual nutrients in the fumigated and incubated soils were still higher than in untreated soil. In the case of A. mellea, the woody inoculum segments served as additional substrates for colonization by Trichoderma, which was observed to grow profusely on the segments incubated in the fumigated soils. However, the level of nutrients available, and the ability of Trichoderma species groups to use them, could have been affected by environmental factors (3) such as the presence of bacteria,
Fungus flora. L. K. Mughogho
457
actinomycetes and other micro-organisms which might also have developed in the fumigated soils. This analysis leads on to (4), the genetic capacity of Trichoderma speciesgroups to antagonize root disease fungi. It has been shown in this investigation that more than one species-group of Trichoderma was included within the dominant populations of Trichoderma in fumigated soils, and that not all of them were antagonistic to R. solani or A. mellea (Table 1I). It is suggested, therefore, that the failure of dominant populations of Trichoderma in fumigated soils to limit the growth of R. solani and A. mellea was due to the fact that these dominant populations must have been made up largely by strains of Trichoderma species-groups that were not effective antagonists against the two pathogens. This explanation makes it possible to interpret the results of Smith (1953), who found that the percentage damping-off of seedlings caused by Pythium ultimum introduced into formalin-treated soil was highest in those treated soils with the maximum dominance of Trichoderma (viride). It is interesting to note that isolates of T. viride, which occurred sporadically and at low population levels in fumigated soils, were on the whole more effective antagonists against R. solani and A. mellea than were isolates of T. harrianum, which was the predominant species group in the fumigated soils. The success in biological control of A. mellea obtained by Bliss (1951) and Garrett (1957) may have been due to their choice of carbon disulphide as a fumigant, if we admit the possibility (not tested in this investigation) that this particular fumigant may selectively stimulate the development of antagonistic strains of T. viride (sensu Rifai, 1969). The general conclusion emerging from this investigation is that indirect, i.e. biological, control of root disease fungi through soil fumigation must so far have depended on the chance development of strains of Trichoderma species-groups antagonistic to the particular root disease fungus. It seems, therefore, that practical success must await the discovery of a fumigant that will stimulate the development of a Trichoderma population consisting predominantly of strongly antagonistic strains. A first step in this direction would be to find a fumigant promoting particularly the development of T. viride (Rifai, 1969), since this investigation has shown that antagonistic strains seem to be more common in this than in the other newly delimited species of Trichoderma. This work forms part of a thesis for the Ph.D degree of the University of Cambridge. It was carried out during the tenure of a Beit Fellowship and theJohn Stothert Bye Fellowship of Magdalene College, Cambridge. I am most grateful to my supervisor, Dr S. D. Garrett, F.R.S., for much valuable advice throughout this investigation and in the preparation of the manuscript. REFERENCES
ALTSON, R. A. (1950). Diseases of the root system. Rep. Rubb. Res. Inst. Malaya, 1945-48,
PP·96- 190.
AYTOUN, R. S. C. (1953). The genus Trichoderma: its relationship with Armillaria
mellea (Vahl ex Fries) Que!. and Polyporus schweinitzii. Trans. Proc, bot. Soc. Edinb. 36, 99- 1 1 4.
Transactions British Mycological Society BAKER, R. (1965). The dynamics of inoculum. Ecology of soil-borne plant pathogens (ed. by K. F. Baker and W. C. Snyder), pp, 395-403. University of California Press. BLAIR, I. D. (1943). Behaviour of the fungus Rhizoctonia solani Kuhn in the soil. Ann. appl. Biol. 30, 118-127. BLISS, E. D. (1951). The destruction of Armillaria mellea in citrus soils. Phytopathology 4I, 665-683. DARLEY, E. F. & WILBUR, W. D. (1954). Some relationships of carbon disulfide and Trichoderma viride in the control of Armillaria mellea (Abstr.). Phytopathology 44, 485. EVANS, E. (1954). The effect of soil sterilizing agents on fungal ecology. Ph.D. Thesis, University of Cambridge. EVANS, E. (1955). Survival and recolonization by fungi in soil treated with formalin or carbon disulphide. Trans. Br. mvcol, Soc. 38, 335-346. GARRETT, S. D. (1956a). Biology of root-infecting fungi. Cambridge University Press. GARRETT, S. D. (1956b). Rhizomorph behaviour in Armillaria mellea (Vahl) Quel. II. Logistics of infection. Ann. Bot. N.S. 20, 189-209. GARRETT, S. D. (1957). Effect of a soil microflora selected by carbon disulphide fumigation on survival of Armillaria mellea in woody host tissues. Can. ]. Microbiol. 3, 135- 149. GARRETT, S. D. (1958). Inoculum potential as a factor limiting lethal action by Trichoderma viride Fr. on Armillaria mellea (Fr.) Quel. Trans. Br, mycol. Soc. 4I, 158-164. GARRETT, S. D. (1962). Decomposition of cellulose in soil by Rhizoctonia solani Kuhn. Trans. Br. mycol. Soc. 45, 115-120. GARRETT, S. D. (1965). Toward biological control of soil-borne plant pathogens. Ecology of soil-borne plant pathogens (ed. by K. F. Baker and W. C. Snyder), pp. 4-16. University of California Press. GIBBS, J. N. (1966). Fomes annosus : host-parasite relationships. Ph.D. Thesis, University of Cambridge. HEATLEY, N. G. (1947). A simple plate method for multiple tests of the anti-bacterial activity ofmany bacteria against other bacterial strains. ]. gen. Microbial. I, 168-170. HODGES, C. S. (1960). Studies of black root rot of pine seedlings. Diss. Abstr. 20, 24672468. JENKINSON, D. S. (1966). Studies on the decomposition of plant material in the soil. II. Partial sterilization of the soil and the soil biomass. ]. Soil Sci. I7, 280-302. JOHNSON, L. F., CURL, E. A., BOND,J. H. & FRIBOURG, H. A. (1959). Methods for studying soil microflora-plant relationships. Minneapolis: Burgess Publishing Co. KREUTZER, W. A. (1965). The infestation of treated soil. Ecology of soil-borne plant pathogens (ed. by K. F. Baker and W. C. Snyder), pp 495-508. University of California Press. LILY, K. (1961). Ecological studies of soil fungi. Ph.D. Thesis, University of Saugar, India. Cited by Garrett (1965). MOJE, W., MARTIN, J. P. & BAINES, R. C. (1957). Structural effects of some organic compounds on soil organisms and citrus seedlings grown in an old citrus soil. ]. agric. Fd Chem. 5, 32-36. MOUBASHER, A. H. (1963). Selective effects of fumigation with carbon disulphide on the soil fungus flora. Trans. Br. mycol. Soc. 46, 338-344. OVERMAN, A.J. & BURGIS, D. S. (1956). Allyl alcohol as a soil fungicide. Phytopathology 46, 53 2-535. RICHARDSON, L. T. (1954). The persistence of thiram in soil and its relationship to the microbiological balance and damping-off control. Can. ]. Bot. 32, 335-346. RIFAI, M. A. (1964). A reinvestigation of the taxonomy of the genus Trichoderma Pers. M.Sc. Thesis, University of Sheffield. RIFAI, M. A. (1969). Mycol. Pap. II6 (in the Press). RUSSELL, P. (1956). A selective medium for the isolation of Basidiomycetes. Nature, Lond. II7, 1038-1039. SAKSENA, S. B. (1960). Effect of carbon disulphide fumigation on Trichoderma viride and other soil fungi. Trans. Br. mycol. Soc. 43, I I I-I 16. SMITH, H. C. (1953). Biology of Pythium species in soil. Ph.D. Thesis, University of Cambridge. SMITH, N. R. (1938). The partial sterilization of soil by chloropicrin. Proc. Soil Sc. Soc. Am. 3, p. 188.
Fungus flora. L. K. Mughogho
459
N. R . & DAWSON, U . T. ( [944) . The bacteriostatic action of rose bengal in media used for plate coun ts of soil fungi. Soil Sci. 58, 467-47 r , WARCUP, J. H. ( [95 [) . Effect of partial sterilization by steam or formalin on the fungus flora of an old forest nursery soil. Trans. Br. mycol. Soc. 34, 5 [9-532. WE[NDL[NG, R. ([932). Trichoderma lignorum as a parasite of other soil fungi. Phytopathology 22, 837-845. WE[NDLING, R. ( r934 ). Studies on a lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other soil fungi. Phytopathology 24, 1[53-1 179. SM[TH,
(Accepted for publication
22
June 1967)
1
Trans . Br. mycol. Soc. 51 (3 and 4).441-459 (1968) Printed in Great Britain
THE FUNGUS FLORA OF FUMIGATED SOILS By L. K. MUGHOGHO· Botany School, Uniuersity of Cambridge (With 4 Text-figures) A study was made of the extent to which Trichoderma recolonized fumigated soils with a view to biological control of root disease fungi. Treatment of Kettering soil with allyl alcohol, chloropicrin, dichloropropene-dichloropropane mixture (D-D) , methyl bromide, sorbic acid or acetylenedi carboxylic acid promoted the development of dominant populations of Trichoderma reaching about 100 % of the recolonizing fungus flora as assessed by the dilution plate method. Trichoderma was the dominant fungal recolonizer of both acid and alkaline soils treated with allyl alcohol. Analyses of Trichoderma isolates showed that several species-groups of Trichoderma, namely, T. hamatum, T. harzianum, T . koningii and T. viride, were included within these dominant populations of Trichoderma in the fumigated soils. T . harzianum was, however, the predominant species-group except in limed Kettering soil where it was replaced by T. hamatum. There was no d ifference in the general spectrum of Trichoderma species groups between naturally acid and alkaline soils treated with allyl alcohol. Fumigated soils containing dominant populations of Trichoderma were tested for their effect on the growth of two root disease fungi, Rhizoctonia solani and Armillaria mellea, which are known to be susceptible to antagonism by T richoderma. No inhibitory effect was observed; in fact, both pathogens grew better in the fumigated soils than in untreated soil. Cultural studies of the interaction between R. solani and A. mellea, respectively, and Trichoderma species-groups from fumigated soils showed that not all the species-groups of Trichoderma nor even all isolates of anyone species-group were antagonistic to the two pathogens. It is suggested that the failure of Trichoderma in fumigated soils to control R. solani and A. mellea was due to the fact that the dominant populations of Trichoderma must have been made up largely by strains of the fungus that were not effective antagonists against the two pathogens.
Biological control of root disease fungi by Trichoderma has received much attention since Weindling's (1932) discovery of Trichoderma as an antagonist of root disease fungi in culture. However, no method has as yet been found for the practical exploitation of this phenomenon. In recent years great interest has been aroused by a new approach to the problem suggested by Bliss (1951) who postulated that the high population of Trichoderma (ascribed to T. viride) following carbon disulphide fumigation of the soil was the factor primarily responsible for the killing of Armillaria mellea in infected citrus roots in Californian orchards, rather than the direct fungicidal effect of the fumigant. While plating out samples of root segments from fumigated soil, Bliss regularly isolated Trichoderma from root segments in which A. mellea was no longer viable. Fumigation of infected • Present address: Agricultural Research Council of Malawi, P.O. Box 2 15, Lilongwe. Malawi.
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Transactions British Mycological Society
root segments in sterile soil did not kill A . mellea. However, A. mellea was killed when infected root segments were buried in a pure culture of Trichoderma grown in sterilized soil. In roots in unsterile soil, A. mellea remained viable when not fumigated, but was killed wh en fumigated, Trichoderma showing rapid development in the fumigated soil. From these observations and experiments Bliss concluded that in natural soil Trichoderma is not sufficiently dominant to destroy A. mellea in infected citrus roots. But if the equilibrium of the soil microflora is disturbed by fumigation with carbon disulphide, the recolonizing fungus flora is dominated by Trichoderma which, at this higher population level, is able to kill A. mellea by antagonistic action. In a series of experiments designed to test Bliss's hypothesis Darley & Wilbur (1954) and Garrett (195 7) found that, in small pieces of infected wood, A. mellea is killed directly by fumigation with carbon disulphide. However, in massive roots, the fumigant while killing A. mellea in the outer tissues may not permeate all the root tissue and kill all the mycelium of the pathogen. Garrett postulated that under such conditions biological control may be possible due to the subsequent development of Trichoderma which may invade the roots, and spread inwards, killing the remainder of the pathogen that had been beyond the direct reach of the fumigant. Garrett (1958) also showed that the maximum antagonistic effect of Trichoderma on A. mellea, and doubtless on other fungi too, is exerted by a pure culture of Trichoderma which has a maximum inoculum potential (sensu Garrett, I956a). Nearly 100 % kill of A. mellea was obtained when woody inoculum segments were incubated in a pure culture of T. viride (from Hypocrea rufa) grown in sterilized soil. When woody inoculum segments were incubated in dilutions of the pure culture with unsterile soil, the antagonistic action of T. viride upon A. mellea was reduced. Three conclusions emerge from this brief summary of the work of Bliss and that of Garrett on the role of soil fumigation treatments for promoting biological control of root disease fungi by Trichoderma. These are: (a) the selective action of soil fumigants offers a means ofincreasing artificially the soil population of Trichoderma above its natural level, (b) such an increase may be used for the biological control of root disease fungi, (c) the population level of Trichoderma in the fumigated soil would determine the extent of biological control obtained. The testing of these conclusions provided a basis for this investigation; the objective was to study the ecology of Trichoderma in fumigated soils with a view to finding a soil fumigant that would promote a maximum dominance of Trichoderma, and to test whether this dominant population of Trichoderma in fumigated soil could control root-disease fungi. MATERIALS AND METHODS
Two soil types, Kettering soil and Botanic Garden soil, were used throughout this study. Kettering soil is a reddish, slightly acidic (pH 6-6'2) clay loam collected from under old pasture in Kettering, Northants. Botanic Garden soil is a greyish alkaline (pH 7"5), light-textured loam from the Cambridge University Botanic Garden. Organic and nitrogen contents in both soils are somewhat high, whereas available potash and phosphate
Fungus flora. L. K. Mughogho
443
are high- in Botanic Garden soil and unusually low in Kettering soil. Kettering soil, which has been used in previous fumigation studies (Evans, 1955; Garrett, 1957; Saksena, 1960; Moubasher, 1963) with carbon disulphide and formalin, was used for most of the experiments. Both soils were collected when required from dumps at the Botany School Field Station. At the time of collection they were passed through a i in (6 mm) sieve and then mixed thoroughly. The procedure for soil fumigation and incubation was essentially as described by Garrett (1957). Soil was fumigated at a low moisture content of 40 % saturation to facilitate diffusion of the fumigants and to provide a well-aerated soil, which is essential for recolonization by Trichoderma (Evans, 1955). Screw-topped jars of capacity 2400 ml were used as containers, each receiving 1800 g soil. Fumigants were injected into the soil at the mid-point of the jar, after which the soil was resettled by tamping the jar on the bench; the cover was screwed on immediately afterwards and bound to the jar with' Sellotape ', Thereafter jars were left in an incubator at 25°C for 7 days for the fumigant to work. Jars were then opened and left on the laboratory bench for 7 days to allow for the fumigant to escape; they were then returned, still uncovered, to the 25° chamber for further incubation. During incubation the moisture content of the soil was maintained by regular weighing on a box balance and addition of distilled water to original weight. The population levels of Trichoderma in the fumigated and incubated soils were determined by the soil-dilution plate method. Soil dilutions were made from the well-mixed soil of each jar according to the method recommended by Johnson, Curl, Bond & Fribourg (1959) except that a dipper of vol. I ml was used in place of a pipette to transfer the various dilutions of the soil suspension from one vessel to another. Soil dilutions were plated (using the dipper) in replicates offour with 15 ml of the Smith & Dawson (1944) soil extract agar as amended by Moubasher (1963) in each plate. All plates were incubated overnight at 25° and thereafter on the laboratory bench. Plates were examined daily and each fungal colony was marked on the bottom of the plate as soon as it appeared. Where fastgrowing fungi were likely to overgrow and suppress slowly growing ones, they were removed from the plates and cultured separately for later identification. All fungal colonies were counted, identified and recorded as number of colonies of either Trichoderma or 'other fungi' per g air-dry soil. The population level of Trichoderma was calculated as a percentage of the total number of fungi recorded. ECOLOGY OF TRICHODERMA IN FUMIGATED SOILS
if various chemicals on the development if Trichoderma in Kettering soil Trichoderma has been reported as the dominant fungal recolonizer of soils treated with allyl alcohol (Overman & Burgis, 1956), chloropicrin (Smith, 1938), D-D mixture (Altson, 1950), formalin (Warcup, 1951), methyl bromide (Hodges, 1960), sorbic and acetylenedicarboxylic acids (Moje, Martin & Baines, 1957) and thiram (Richardson, 1954). The The effect
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chemicals selected for testing in the present study were, therefore, those listed above but omitting formalin and thiram. The general procedure for treating soil with these chemicals and for estimating population levels of Trichoderma in the fumigated and incubated soils has already been described. Sorbic and acetylenedicarboxylic acids, being of low volatility, were mixed well with the soil, and incubation at 25° was started immediately after the fumigation period. Soil treated with allyl alcohol was also incubated at 25° immediately after fumigation, without a period on the laboratory bench to allow for the fumigant to escape. For each chemical a wide range of dosages was tested in duplicate; nothing was added to the control (untreated) soil. Table I. Percentage population of Trichoderma in the recolonizing fungus flora of Kettering soil treated with various chemicals AcetylenediCarbon Allyl Chloro- Methyl disulD-D Sorbic carboxylic phide alcohol pircin bromide mixture acid acid
Fumigation period/days Incubation period/days Soil moisture content as % saturation Dosagev o (control) 0'08 0'1 0'2 0'3 0'4 0,6 0,8 0'9 1'5 1,6 3'3
4 35
7 21
7 28
7 28
7 21
7 21
7 21
4 0'4
41
4 1'4
4 2'4
42'4
4 1'2
42
3'1 6,8
0'0 9 6'3
0'0
1'7
100'0
77'3
100'0
14'3
80'S
100'0
100'0 100'0
9 6'9 9 6'7
96'3
9 8'8 99'4
4'2
3'5 64'3
0'0 4'2
4'1 12,8 8'3
100'0 100'0
100'0
98.8
100'0
45"8 82'1 89'2
93'3
97.8
• ml/IOOO ml space, or g/750 g soil for sorbic acid and acetylenedicarboxylic acid,
Results are given in Table I. The population level of Trichoderma in untreated soil ranged from I to 5 %. In samples of untreated soils used for the methyl bromide, D-D mixture and acetylenedicarboxylic acid tests, the population of Trichoderma recorded was zero. This does not mean that Trichoderma was totally absent; since Trichoderma appeared in the treated soils, it implies rather that its population level was so low that it could not be detected in the soil samples used for the dilution plates. Changes in the population levels of Trichoderma following treatments were as follows. All chemicals except carbon disulphide promoted dominant recolonization of Kettering soil by Trichoderma. Within the range of dosages tested, there was a minimum dosage level at which Trichoderma became dominant, i.e, its population exceeded 50 % of the total fungus population recorded on the dilution plates. Increase in dosage above the minimum required for dominance resulted in a corresponding increase in the population level of Trichoderma to almost 100 % at the higher dosages.
Fungus flora. L. K. Mughogho
445
In general, this high level of dominance did not fall with further increases in dosage. Comparison of the efficiency of the various chemicals shows that allyl alcohol and methyl bromide promoted maximum dominance of T richoderma at lower dosages than the other chemicals. At higher dosages, however, methyl bromide was slightly less effective than allyl alcohol and chloropicrin. In allyl alcohol-treated soil, recolonization by Trichoderma was usually complete within 14 days' incubation; in soils treated with the other chemicals complete recolonization took 21-28 days. Allyl alcohol was, therefore, chosen for further experiments; chloropicrin was also used but mainly for comparative studies.
The effect of soil pH on the development of Trichoderma in fumigated soils Lily (1961) reported that in an Indian soil of pH 7-7"5, the place of Trichoderma (viride) as a dominant recolonizer after fumigation with carbon disulphide was taken by Penicillium nigricans. When the soil was acidified, Trichoderma replaced P. nigricans as the dominant recolonizer. The replaceTable 2. Percentage population of Trichoderma in allyl alcohol-treated soils at different pH values Dosages o (control)
Botanic Garden soil, pH 7'5
Acidified Botanic Garden soil, Limed Kettering pH 5,8 soil, pH 7'5
0'2
4'0 98'4
3'1 89'7
0'4 0,8 1'6
100'0 100'0 100'0
100'0 100'0 100'0
2'3
97'6 96'7
71 ' 2
49'2
• rnl/looO rnl space.
ment of Trichoderma by P. nigricans was attributed to the alkalinity of the soil. P. nigricans may be more abundant quantitatively than Trichoderma in acid soils, e.g, Evans (1954) recorded 5 % of the former and only I % of the latter in the fungus flora of Kettering soil ; nevertheless, Trichoderma was the dominant recolonizer after treatment with carbon disulphide. It appeared, therefore, that Trichoderma was a more efficient recolonizer of acid soils than P. nigricans and vice versa in alkaline soils. This hypothesis was examined using allyl alcohol, which is a more efficient fumigant than carbon disulphide for promoting dominant recolonization by Trichoderma in fumigated soils. The soils used were: (a) Botanic Garden.soil, a naturally alkaline soil of pH 7'5; (b) Botanic Garden soil acidified with sulphur to pH 5.8; (c) Kettering soil limed to pH 7"5. These soils were treated with allyl alcohol for 7 days and then incubated for 3 weeks. Thereafter population levels of Trichoderma were determined. The results in Table 2 show that, depending upon the fumigant applied, Trichoderma can be the dominant recolonizer of both acid and alkaline soils.
Transactions British Mycological Society Species-groups of Trichoderma which recolonize fumigated soils In previous investigations, the specific name T. viride was frequently applied to all Trichoderma isolates from fumigated soils, although some authors, e.g, Smith (1938), called them Trichoderma sp. or simply Trichoderma. It is known from the recent taxonomic study of the genus Trichoderma (Rifai, 1964) that most Trichoderma isolates can be assigned to one of Table 3. The spectrum of Trichoderma species-groups in Kettering soil treated with albl alcohol and chloropicrin Percentage population of Trichoderma species-groups
Dosage" Allyl 0'0 alcohol 0'2 0'4 0,8 1,6 Chloro- 0'0, picrin 0'2 0'4
No, colonies fungi permg air-dry soil 94 4 12 IOB7Bo 1631 70 lOB780 109
o-s
1·6
182 40 16121 23535
,
Total
longipolybra- pseudosporum hamatum chiatum koningii koningii 1'0 7'7 10'9 4 6'4 0'0
4'0
0 0 0 0 0 0
94'6 95'2 94'1
0 0 0
1'9 60'0 100'0 100'0 100'0
2'7
0 0 0 0 0 0
0 0 0 0 0 0
0'0 0'0 0'0
0 0 0
0 0 0
aureociride horzianum
viride
0'4 9'2 25'5 14'3 5'9 0'0
0 0 0 0 0 0
0'0 43'1 63'6 34'S 94'1 0'9
0'4
4'3 3'B o,B
0 0 0
B9'2 9 1'4 93'3
1'1 0'0 0'0
0'5 0'0
0'0 4.8 0'0
• mljrooo ml space,
Table 4. The spectrum tifTrichoderma species-groups in
Botanic Garden soil treated with albl alcohol No, colonies fungi per mg Dosage" air-dry soil 0'0 0'2 0'4 o'B 1,6
161 1355 4235 5929 10164
Percentage population of !richoderma species-groups Total 3'9 94'4 100'0 100'0 100'0
longibra- pseudopolysporum hamatum chiatum koningii 1'3 0'0 0'0 0'0 0'0
1'3 0'0 4,8 3'7 0'0
0 0 0 0 0
0 0 0 0 0
koningii
aureoviride
0'0 4'7 9'5 0' 0'0
0 0 0 0 0
harzianum viride 1'3 79'7 66'7 BB'9 95'7
0'0 14'0 19'0 7'4 4'3
• ml/IOOO ml space,
Table 5. The spectrum tifTrichoderma species-groups in albl alcohol treated-Kettering soil limed to pH 7'5 No, colonies fungi per mg Dosage. air-dry soil 0'0 0'2 0'4
o-s 1,6
241 225 1 B07 10443 12050
Percentage population of, Trichoderma species-groups Total 2'2 97'6 96'7 7 1"2 49'2
longi-bra pseudopolysporum hamatum chiatum koningii 0'0 0'0 0'0 0'0 0'0
1'1 92'B 7B'9 4 0'3 27'9
0 0 0 0 0
0 0 0 0 0
• mljrooo ml space.
koningii 0'0 2'4 5'5 9'6 1,6
aureo.. viride harzianum 0 0 0 0 0
0'0 2'4 10'0 19'2 19'7
viride 1'1 0'0 2'2 1'9 0'0
Fungus flora. L. K. Mughogho
447
eight species groups, viz. T. polysporum, T. hamatum, T. longibrachiatum, T. pseudokoningii, T. koningii, T. aureo-viride, T. harzianum and T. oiride. An analysis was, therefore, made of the species groups of Trichoderma which develop in fumigated soils, using Rifai's (1969) key for identification. The spectrum of Trichoderma species groups was studied in: (a) Kettering soil treated with allyl alcohol; (b) Kettering soil treated with chloropicrin; (c) Botanic Garden soil treated with allyl alcohol; (d) Kettering soil limed to pH 7'S and then treated with allyl alcohol. Incubation periods were 3 and 4 weeks for allyl alcohol- and chloropicrin-treated soils respectively. In estimating Trichoderma populations especial care was taken to remove all fungal colonies as soon as they appeared on the dilution plates and to culture them separately for identification. All Trichoderma isolates were identified to species-group level by following the procedure of Rifai (1964). Fresh subcultures were made on 2 % malt agar plates. These were incubated at 25° overnight, and then in front of a window to promote sporulation. Young sporulating colonies were examined 3-6 days after inoculation, to record the mode of branching of the conidiophores and the shape and disposition of the phialides. Records of spore size and character were made 2 weeks later when spores were matured. Results are presented in Tables 3 to S. The total percentage population of Trichoderma at each dosage (given in column 3) is split up into percentages of the various species groups of Trichoderma which compose it in the remaining columns of the Tables. T. hamatum, T. harrianum, T. koningii and T. viride were recorded in untreated Kettering and Botanic Garden soils. In addition, T. polysporum was present in Botanic Garden soil. Species groups not actually recorded in untreated soil but which appeared in treated soils, e.g, T. harzianum and T. koningii in Table 3, were assumed to have been present in the untreated soil. In Kettering soil treated with allyl alcohol and chloropicrin, respectively, T. viride occurred sporadically at very low population levels, and T. hamatum was absent from chloropicrin-treated soil. T. koningii and T. harrianum were recorded at all dosages of the two fmnigants; T. harzianum was predominant over all other species-groups present at any dosage. A similar spectrum of Trichoderma species-groups was found in Botanic Garden soil treated with allyl alcohol (Table 4). In limed Kettering soil treated with allyl alcohol, T. hamatum replaced T. harrianum as the predominant species-group (Table 5). These results show that more than one species-group of Trichoderma was included within the dominant populations of Trichoderma in the fumigated soils. TRIAL OF FUMIGATION TREATMENTS FOR BIOLOGICAL CONTROL OF ROOT DISEASE
The experiments to be described in this section were designed to test if fumigated soils containing dominant populations of Trichoderma, as reported in the preceding section, would reduce growth and pathogenic activity of introduced root disease fungi. Rhizoctonia solani Kuhn and Armillaria mellea (Vahl ex Fr.) Kummer about which there is evidence of 29
MyC.51
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Transactions British Mycological Society
susceptibility to antagonism by Trichoderma (Weindling, 1932, 1934; Aytoun, 1953), were used as test pathogens. Experiments with Rhizoctonia solani Growth of Rhizoctonia solani throughfumigated soils R. solani grows well as a saprophyte through natural unsterile soil (Blair, 1943). This ability made it a suitable test pathogen for comparing growth in untreated soil with that in fumigated soils containing dominant populations of Trichoderma. The experiment was carried out in open-ended glass tubes, 1'9 em internal diam x 25 cm long (hereafter called soil tubes), and set up as in Fig. 1. Tubes were first capped at the bottom end with lacquered moisture-proof 'Cellophane' fastened by a rubber band, and then filled with the well-mixed fumigated and incubated soil. Each tube was inoculated above the soil column with an agar inoculum disk (16 mm MOisture-proof cellophane Air space - - 4 - -
~Q"~J----Agar inoculum disk of R. solani
Glass tube
:-,;-:+-- Soi I
Fig.
I.
Growth of Rhiroctonio solani through soil in a 'soil tube'.
diam x 4 mm thick, mycelial face downwards) of R. solani cut from the margin of a colony on Garrett's (1962) synthetic medium, and the top end was then capped with Cellophane. Ten tubes were prepared from the soil in each jar. The growth of R. solani from the inoculum disk down through the soil was measured under the stereoscopic microscope with reflected light, at z-day intervals for 8 days (Fig. 2). Growth of R. solani in untreated soil was taken as 100 %, and its growth in the soils treated with allyl alcohol and chloropicrin, respectively, was calculated as percentages of its growth in untreated soil. Differences between its growth in untreated soil and in soils treated with 0·2, 0·4 and 0·8 ml dosages of allyl alcohol were not significant. Its growth through soil treated with the 1·6 ml dosage was significantly (P = 0'001) greater than
Fungus flora. L. K. Mughogho
449
that through all the other soils, treated and untreated. In chloropicrintreated soil, the growth of R. solani was less in the untreated soil than in the treated soils at all dosage levels. Differences in growth were significant at the 5 % level for 0'2 ml dosage, and at the 1 % level for 0'4, 0,8 and 1·6 ml dosages. These results thus show that this isolate of R. solani (from swede) grew better in the treated soils than in the untreated soil, although the former had been colonized by dominant populations of Trichoderma. Similar results were obtained using isolates of R. solani from tomato and potato. It is concluded, therefore, that fumigated soils containing dominant populations of Trichoderma were not inhibitory to the growth of R. solani. 200
Allyl alcohol
Chloropicri n r--
-
180 160
r--
r--
.s:
~
~ 140
e
DO
~ 120
100
-
~
r--
~I--
80 60
o Dosage
Fig.
2.
N
'V
6
66':-
Q)
..,g
Growth of Rhizoctonia solani (swede isolate) through Kettering soil treated with allyl alcohol and chloropicrin respectively.
Seedling testfor the activity of Rhizoctonia solani in fumigated soils Kettering soil was treated with allyl alcohol and then incubated for 3 weeks, R. solani inoculum was prepared by growing the swede isolate for 3 weeks at 22'5° in 500 ml flasks containing 250 g sand, 12'5 g maize-meal and 50 ml water autoclaved for 1 hat 15lbJin2 (103'4 kNJm 2 ) . Cultures were homogenized by passing through a 3 mm sieve; 12 g of this inoculum was mixed with 228 g fumigated soil and put in a crystallizing dish (9'4 em diam x 5 em depth), which was then covered with a glass lid. Six dishes were prepared from soil at each dosage level. Inoculated soils were incubated at 22'5° for 7 days to enable R. solani to establish itself throughout the soil. Thereafter, twenty germinated swede seeds were sown equidistantly in each dish. Dishes were kept at 22'5° until seedlings emerged, when lids were removed and the dishes kept in a glasshouse (mean temp. 20' 7 ± 2'4°). The soil moisture content optimum for infection by R. solani is about the same as that for soil fumigation, i.e. 40-50 % saturation (Blair, 1943). This moisture level was maintained by weighing the dishes daily and adding distilled water to original weight. To prevent drying of the soil surface, the dishes were kept on metal trays containing a shallow layer of water and covered by a polyglaze box.
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Transactions British' Mycological Society
Fourteen days after sowing, numbers of seedlings killed before and after emergence were recorded. Seedlings which failed to emerge were assumed to have been killed by R. solani, because in preliminary tests all pregerminated seeds emerged in uninoculated soil. Seedlings still standing were removed, washed carefully and examined for lesions caused by R. solani. Table 6 shows no significant difference in infection of seedlings by R. solani between the untreated and treated soils. These results thus again show that fumigated soils containing dominant populations of Trichoderma were not inhibitory to the growth of R. solani. Table 6, Infection of swede seedlings by Rhizoctonia solani in Kettering soil treated with allyl alcohol
Dosage'" 0
o-z 0'40·8 1"6
Percentage population of Trichoderma 2,6 61"5 89'1 100 100
Seedlings emerged out of 120 88 7485 70 39
Seedlings Total seedlings killed or killed or infected after infected out of 120 emergence 81 61 67 63 35
113 107 102 113 116
Percentage infection 94'2 89'2 85'0 94'2 94'7
• mlj rooo ml space,
Antagonism of Trichoderma species groups to Rhizoctonia solani. All Trichoderma isolates except T. viride isolate no. 73 (from Hypocrea rufa) were obtained from Kettering soil treated with allyl alcohol or chloropicrin. Experiment 1. Growth of R. solani isolates through cultures of Trichoderma species-groups on sterilized soil. The set-up of this experiment was similar to that shown in Fig. I. Kettering soil at a moisture content of 45 % saturation was sterilized in lots of 250 gin 50 ml conical flasks for I h at I5lb/in2 (103' 4 kN1m2 ) . Each soilseries was then inoculated with a Trichoderma species group and incubated at 22'5 0 for 21 days. Thereafter the replicate cultures of each series were well mixed and filled into sterilized soil tubes; ten tubes were prepared from cultures of each species group of Trichoderma. Each tube was inoculated above the soil column with an agar inoculum disk of R. solani grown on Garrett's (1962) medium, A control set of tubes was prepared from soil which had been sterilized and incubated without inoculation with Trichoderma. The growth of R. solani was measured every 2 days for 8 days. Results in Fig. 3 show that growth of R. solani isolates was better through uninoculated sterilized soil than through the same soil colonized by T. harzianum (14), T. koningii (17) and T. viride (I and 73) respectively. Soil colonized by T. hamatum (8) actually promoted growth of R. solani, The poor growth of R. solani through soil sterilized and then inoculated with Trichoderma isolates can be attributed partly to the depletion of nutrients released by autoclaving; superimposed upon this nutrientdepletion effect, however, must have been further differences due to varied antagonistic effects produced by the different species groups of Trichoderma.
451
Fungus flora. L. K. Mughogho
Experiment 2. Antibiotic activity of Trichoderma species groups against R. solani. A simple 'Cellophane plate' technique designed by Heatley (1947) for use with bacteria and adapted by Gibbs (1966) for use with fungi, was used for testing the antibiotic activity of Trichoderma species groups against R. solani (swede isolate). Sheets of 'Cellophane' (grade
% gr ow t h 20
40
60
80
100
120
Contro l
T. hamatum (8)
T harzianum (14)
T. koningi (17)
T. v;r ide (1)
~
Sw ed e and
If f Wll l
Swede isolate
l---J tomato iso late s
~ To mato iso lat e T v;ride (73)
Fig. 3. The effect of various species groups of Trichoderma on the growth of the swede and tomato isolates of Rhizoctonia solani.
300 PT) cut to fit a Petri dish were washed in boiling distilled water to remove plasticizers, sterilized in distilled water and then spread singly on 15 ml agar (Kettering soil extract, 11; agar, 20 g, adjusted to pH 6'2) in a Petri dish. The' Cellophane' was inoculated in the centre with a 4 mm diam agar inoculum disk ofa Trichoderma species-group cut from the margin of a colony on 2 % malt agar. After 2 days' incubation at 22'5°, the , Cellophane' together with the Trichoderma colony growing on it was stripped off and the plates were inoculated (below the site previously occupied by the Trichoderma inoculum) with a 4 mm disk of R. solani cut marginally from a colony on Garrett's (1962) medium. The growth of R. solani was measured daily for 2 days (Table 7),
Transactions British Mycological Society
452
Three of the five isolates of T. hamatum showed strong antibiotic activity against R. solani. No antibiotic activity was shown by most of the T. harzianum isolates. Most of the T. koningii isolates were antagonistic to R. solani. All four T. viride isolates showed strong antibiotic activity. The mean antibiotic effects of the different isolates for each Trichoderma species-group are shown in Fig. 4. The highest and lowest levels of antibiotic activity against R. solani were shown by T. viride and T. harrianum, respectively. Table 7. Antibiotic activity of Trichoderma species-groups against Rhizoctonia solani (swede isolate) Trichoderma sp, and isolate no, Nil (control) T, hamatum
T. harzianum
T. koningii
T. viride
* t
7 8 9 24 25 II
12 13 14 15 36 37 42 48 19 21 3° 33 1 2 5 73
Mean * radial growth of Trichoderma sp, (mm/2 days)
Mean * radial growth of R. solani (mm/2 days) 14'03±0'24
28'58 28'00 28'4 1 21"30 23'15 23'75 17'75 24'5° 21'75 24'4 1 22'3 2 17'75 19'5° 18'75 3°'75 22'9° 25'00 26'90 26'25 26'5° 23'58 27'00
4' 00±0'02 13'35±o'15 4' 16±o'02 ° 12'91 ±O'IO 15"83±0'20 15'38±O'40 13'42±o'31 3'16±o'38 15"58±0'24 3'08±o'15 11'25± 0'1 7 15"70± 0'20 3'33±0'11 5'33±0'03 5'58±O'17 8'20±O'10 11'50±O'28 o'75±o'12 ° 0 3'40±o'02
%
inhibition'[ of the growth of R. solani 7 1'49 7°'35 100'00
77"48 78'°5 76'27 62'22 60'23 4 1'55 93'94 100'00 100'00 75'77
Mean of six replicates. Percentage inhibition is only given where it is statistically significant,
These results show dearly, as in the previous experiment, the range of variation in the antagonism of Trichoderma species-groups to R. solani. They also suggest the existence of a number of strains, differing in their antagonism to a given root disease fungus, within each Trichoderma species group. The relation of this to the failure of dominant populations of Trichoderma to inhibit the growth of R. solani will be discussed later.
Experiments with Armillaria mellea Rhizomorph production by Armillaria mellea from woody inoculum segments incubated in fumigated soils. The following experiment was designed to study the effect of dominant populations of Trichoderma in fumigated soils on the production of rhizomorphs by A. mellea. Woody inoculum segments of A. mellea were prepared
Fungus flora. L. K. Mughogho
453
by the method of Garrett (1956b). Shoots (1'75 em diam) of pollarded willows (Salix spp.) were sawn serially into 2'5 cm segments. Eight segments in a 500 ml conical flask were covered with a maize-meal/sand culture medium (150 g sand; 5 g maize-meal; 30 ml water). After autoclaving for I h at 221b/in2 (151'7 kN/m2) , flasks were inoculated with agar disks of A. mellea cut from a colony on 3 % malt agar, and incubated at 22'5° for 13-16 weeks before use. Mean radial growth of R. so/ani in mm/2 days o 10 20 I
I
Control
I
T. hamatum
I
T. harzianum
I
T. koningi
T. viride
0
Fig. 4, Mean antibiotic effects of isolates of Trichoderma species groups against Rhiroctoniasolani (swede isolate).
Table 8. Production of rhizomorphs by Armillaria mellea from woody inoculum segments buried in chloropicrin-treated Kettering soil Mean* percentage population of
Dosage]
Trichoderma 5'1 63'0 100'0 100'0 100'0
No.of'segments (out of 40) which produced rhizomorphs 28 38
f
Total
39
40
t
\
Mean per segment
245 1,052 1,016 944 855
40
* Mean of two replicates.
.
Dry wt. (in mg) of rhizomorphs
8'75 27'68 25'40 24'21 21'38
ml/rooo ml space.
Kettering and Botanic Garden soils were treated with chloropicrin and allyl alcohol, respectively. After incubation for 4 weeks, the well mixed soils were put in 2 lb jam jars, with A. mellea inoculum segments buried in them. Each jar received 660 g soil and ten segments arranged in two layers of five, and the moisture content of the soil was then raised from 40 to 50 % saturation, optimal for rhizomorph production (Garrett, 1956h). Jars were finally capped with moisture-proof 'Cellophane' and incubated at 22'5° for 13 weeks; numbers of segments which produced
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Transactions British Mycological Society
rhizomorphs at each dosage level were then recorded. Rhizomorphs were then cut from the segments, washed, dried at 80° for 48 h and weighed. Table 8 shows that 70 and 90-100 % of the segments in the untreated soil and in the fumigated soils, respectively, produced rhizomorphs; the more important difference, however, is that in yield of rhizomorphs, as shown by the total dry weight of rhizomorphs produced in each series. All the segments had been selected for uniformity in fresh weight, because of the relationship between inoculum weight and extent of rhizomorph production (Garrett, 1956b). Inoculum segments incubated in the fumigated soils gave higher yields of rhizomorphs than segments in the untreated soil. The differences were highly significant (P < 0'001). Similar results were obtained from segments buried in Botanic Garden soil treated with allyl alcohol (Table 9). The conclusion to be drawn from these results is that Table 9. Production of rhizomorphs by Armillaria mellea from woody inoculum segments buried in allyl alcohol-treated Botanic Garden soil
Dosagc'"
Percentage population of Trichoderma
No, ofsegments (out of 10) which produced rhizomorphs
0 0'2 0'4 0·8 1,6
3'9 98'4 100 100 100
10 9 9 9 8
.
Dry wt. (in mg) of rhizomorphs Total 14 1'1 66'0 182'4 334'5 4 63'2
~ean
per segment 14'10 7'33 20'27 37'17 57'90
• ml/rooo ml space.
fumigated soils containing dominant populations of Trichoderma were more favourable than untreated soils for growth of A. mellea. The favourable effect of fumigation due to release of nutrients evidently outweighed any unfavourable effect due to the populations of Trichoderma.
Replacement of Armillaria mellea in woody inoculum segments by Trichoderma species groups. In order to investigate whether Trichoderma species-groups from fumigated soils were antagonistic to A. mellea, a 'replacement' method was developed. In this method, inoculum segments of A. mellea were incubated on pure cultures of Trichoderma species-groups grown on a nutrient-poor medium. After incubation for about 3 months, the segments were removed and the viability of A. mellea determined by plating on a basidiomyceteselective medium (Russell, 1956). The merit of this method was that it represented what would be expected to happen to Armillaria-infected roots in soil following fumigation. The roots would be invaded by the recolonizing species-groups of Trichoderma; if the latter were antagonistic to A. mellea, they would kill and replace it in the roots. Eight inoculum segments of A. mellea were put aseptically into I 1conical flasks containing 5-day-old cultures of Trichoderma species groups on 250 ml soil extract agar (Kettering soil extract, I 1; agar, 20 g, adjusted to pH 6'2) and incubated at 22'5°. The control flasks had been left
Fungus flora. L. K. Mughogho
455
uninoculated prior to adding the inoculum segments. After incubation for 3 months, the viability of A. mellea in the segments was determined as follows. The segments were washed, dipped in 70 % alcohol for 3 min and then surface-sterilized in o- 1 % mercuric chloride for 5 min, followed by thorough washing in sterile distilled water. Thereafter the segments were split down the middle; four small chips were taken aseptically from different positions in the innermost tissues and plated on 15 ml 3 % malt agar containing o· 1 ml 1 % o-phenyl phenol; 0'5 ml 1 % streptomycin was added to suppress bacterial development. Table 10. Viability of Armillaria mellea in woody inoculum segments incubated in pure cultures of Trichoderma species-groups grown on soil extract agar
Trichoderma species-group
Nil (control) T. hamatum (8) T. harzianum (14) T. koningii (I 7) T. oiride (I) T. oiride (73)
No. of segments (out of 32) with living A. mellea 32 30
o
° °5
Percentage viability of A. mellea as determined by cultures from wood chips 100
9°·6
° °o 15.6
Where A. mellea was viable, it grew out from the chips and produced rhizomorphs in the agar. Where A. mellea failed to grow out, it was assumed it had been killed by Trichoderma. As shown in Table 10, A. mellea was viable in all the segments incubated in the uninoculated flasks. In the inoculated flasks, T. harzianum (14), T. koningii (I 7) and T. viride (I) were most effective in killing and replacing A. mellea; T. viride (73) was less effective, and T. hamatum (8) was ineffective. These results are further evidence that different species-groups of Trichoderma, and even different isolates of the same species-group, may vary in their antagonism to a given root disease fungus. DISCUSSION
The conclusion from Bliss's (1951) hypothesis, that dominant populations of Trichoderma (viride) in fumigated soils may be used for the biological control of root disease fungi, may now be examined in the light of results from this investigation. Garrett (1958) modified Bliss's hypothesis by a qualification that the extent of biological control obtained would depend on the inoculum potential of Trichoderma (viride) that developed in the fumigated soil. The inoculum potential of a pathogen, or in the present case, of a root disease antagonist (sensu Garrett, 1956a, 1958) has been resolved by Baker (1965) into four components: (I) inoculum density or intensity (mass or units ofinoculum per unit ofsoil), (2) available nutrients (3) environmental factors, (4) genetic capacity of the organism. The failure of dominant populations of Trichoderma species-groups in fumigated soils to limit the growth of Rhizoctonia solani and Armillaria mellea can thus be attributed to anyone or to any combination of these four factors.
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Transactions British Mycological Society
It is unlikely that ( 1) was limiting, because the inoculum densities of Trichoderma species groups, as assessed by the dilution plate method, were certainly much higher in fumigated soil than in untreated soil. With respect to (2), i.e. available nutrients, it is known that soil fumigation increases the nutrient status of the soil, largely by the killing of microorganisms and the consequent release of nutrients bound in microbial protoplasm (Kreutzer, 1965; Jenkinson, 1966). These nutrients were Table 11. Antagonism of different isolates of Trichoderma species-groups against Rhizoctonia solani and Armillaria mellea
Isolate no.
T. hamatum 7 8 9 24 25 T . harzianum II
12 13 14 15 36 37 42 48 T. koningii 17 19 21 30 33 T . viride I
2
5 73
, Cellophane plate ' technique. R. solani, Swede strain A NA A A NA NA NA NA A NA A NA NA A
,
Tomato strain
A . mellea. Replacement test
NA
NA
NA
A A
SA
A
A
A
A
A
A
A
A
A
SA
'Soil culture.' R. solani
Swede strain
,
A A A NA A A A A
A, Antagonistic; SA , slightly antagonistic; NA, not antagonistic.
available for colonization by Trichoderma species-groups. The fact that both R. solani and A. mellea grew better through fumigated soils than through untreated soil, even though the former had been colonized by T richoderma, suggests that residual nutrients in the fumigated and incubated soils were still higher than in untreated soil. In the case of A. mellea, the woody inoculum segments served as additional substrates for colonization by Trichoderma, which was observed to grow profusely on the segments incubated in the fumigated soils. However, the level of nutrients available, and the ability of Trichoderma species groups to use them, could have been affected by environmental factors (3) such as the presence of bacteria,
Fungus flora. L. K. Mughogho
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actinomycetes and other micro-organisms which might also have developed in the fumigated soils. This analysis leads on to (4), the genetic capacity of Trichoderma speciesgroups to antagonize root disease fungi. It has been shown in this investigation that more than one species-group of Trichoderma was included within the dominant populations of Trichoderma in fumigated soils, and that not all of them were antagonistic to R. solani or A. mellea (Table 1I). It is suggested, therefore, that the failure of dominant populations of Trichoderma in fumigated soils to limit the growth of R. solani and A. mellea was due to the fact that these dominant populations must have been made up largely by strains of Trichoderma species-groups that were not effective antagonists against the two pathogens. This explanation makes it possible to interpret the results of Smith (1953), who found that the percentage damping-off of seedlings caused by Pythium ultimum introduced into formalin-treated soil was highest in those treated soils with the maximum dominance of Trichoderma (viride). It is interesting to note that isolates of T. viride, which occurred sporadically and at low population levels in fumigated soils, were on the whole more effective antagonists against R. solani and A. mellea than were isolates of T. harrianum, which was the predominant species group in the fumigated soils. The success in biological control of A. mellea obtained by Bliss (1951) and Garrett (1957) may have been due to their choice of carbon disulphide as a fumigant, if we admit the possibility (not tested in this investigation) that this particular fumigant may selectively stimulate the development of antagonistic strains of T. viride (sensu Rifai, 1969). The general conclusion emerging from this investigation is that indirect, i.e. biological, control of root disease fungi through soil fumigation must so far have depended on the chance development of strains of Trichoderma species-groups antagonistic to the particular root disease fungus. It seems, therefore, that practical success must await the discovery of a fumigant that will stimulate the development of a Trichoderma population consisting predominantly of strongly antagonistic strains. A first step in this direction would be to find a fumigant promoting particularly the development of T. viride (Rifai, 1969), since this investigation has shown that antagonistic strains seem to be more common in this than in the other newly delimited species of Trichoderma. This work forms part of a thesis for the Ph.D degree of the University of Cambridge. It was carried out during the tenure of a Beit Fellowship and theJohn Stothert Bye Fellowship of Magdalene College, Cambridge. I am most grateful to my supervisor, Dr S. D. Garrett, F.R.S., for much valuable advice throughout this investigation and in the preparation of the manuscript. REFERENCES
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(Accepted for publication
22
June 1967)