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Antagonistic properties of species-groups of Trichoderma
[ 25 ] Trans. Br, mycol, Soc. 57 (I), 25-39 (1971) Printed in Great Britain
ANTAGONISTIC PROPERTIES OF SPECIES-GROUPS OF TRICHODERMA I. PRODUCTION OF NON-VOLATILE ANTIBIOTICS
By C. DENNIS* AND J. WEBSTERt Department ofBotany, The University, Sheffield (With Plate 3 and
I
Text-figure)
Isolates from different species-groups of Trichoderma were tested for production of non-volatile antibiotics, by an agar layer technique. Preliminary studies on the chemical nature of these antibiotics were made. Many isolates produced non-volatile antibiotics active against a range of fungi. The ability to produce such antibiotics varied between isolates of the same species-group as well as between isolates of different species groups. The susceptibility of fungi to these antibiotics varied widely; Fames annosus (Fr.) Cooke was the most susceptible and Fusarium osysporum Schlecht. ex Fr. the most resistant of the test fungi used. Gliotoxin and viridin were not produced, but other chloroform-soluble antibiotics, including trichodermin and peptide antibiotics were detected.
The toxic metabolites produced by Trichoderma spp. have been the subject of intensive study ever since Weindling (1934) reported that culture filtrates of T. lignorum (Tode) Harz were toxic to Rhiroctonia solani Kuhn and other fungi even at high dilution. Unfortunately, there has been some confusion about the nomenclature of the fungus used for antibiotic production. The taxonomic confusion was cleared up by Webster & Lomas (1964), who showed that Weindling's gliotoxin-producing isolate and the two isolates from which Brian and co-workers obtained gliotoxin and viridin (Brian & Hemming, 1945; Brian, Curtis, Hemming & McGowan, 1946) were not Trichoderma viride Pers. ex S. F. Gray but were morphologically similar to Gliocladium virens Miller, Giddens & Foster. This report supported Weindling's announcement (Weindling, 1937) that his gliotoxin-producing fungus was a Gliocladium species. Gibbs (1966) and Mughogho (1967) have reported Weindling's confirmation of this. The report of Webster & Lomas therefore casts doubt on the correct identity of some Trichoderma isolates used by workers investigating antagonism of this fungus. However, as Webster & Lomas pointed out, G. virens is not common in soil, whereas Trichoderma is quite common (especially in acid soil). Thus it seems likely that most reports on antagonism by Trichoderma actually relate to this genus.
* Present address: Agricultural Research Council, Food Research Institute, Colney Lane, Norwich, NOR 70F. t Present address: Department of Biological Sciences, Prince of Wales Road, Exeter.
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For a long time the genus Trichoderma was considered to comprise several distinct species, but there was disagreement on nomenclature and the delimitation of each species was obscure. Rifai (1964, 1969) made a taxonomic revision which provides satisfactory separation of species within the genus. Rifai stressed that his new classification is not exhaustive, and as more species of Hypocrea are grown in culture the delimitation of species may need to be redefined. Nevertheless, Rifai's work cleared up much of the previous confusion on the species concept in Trichoderma and is likely to form the basis offuture work. His key to the species-groups will be used here. It is important to note, however, that Soviet mycologists (Pidoplichko, Bilai, Shklyar and others) consider that there are only two distinct species: T. koningii Oud. and T. lignorum (Tode) Harz (Bilai, 1963) The. rough-spored T. lignorum corresponds to Persoon's T. viride. Webster & Lomas (1964) investigated the antibiotic activity of isolates of G. virens. Using the bioassay procedures of Brian & Hemming (1945) and of Brian et al. (1946), they found that all three isolates examined were active against Botrytis allii Munn and Bacillus subtilis; gliotoxin and viridin were isolated in crystalline form. No activity was shown by four isolates of Hypocrea rufa (Pers. ex Fr.) Fr. (the perfect state of T. viride) or by several other isolates of Trichoderma from wood and soil, some of which were morphologically distinct from T. viride. These results contrast with those ofJeffreys, Brian, Hemming & Lowe (1953), who showed that some Trichoderma isolates collected from acid heath soil (thus likely to be true Trichodermas) were active against B. allii. Also, Rishbeth (1950) showed that culture filtrates of ten Trichoderma strains from East Anglian forest soils all inhibited germination of conidia of Fomes annosus. Gibbs (1966) has confirmed this. Using an agar-layer technique he found that some isolates of Trichoderma species, identified according to Rifai's classification, showed antibiotic activity against Fomes annosus. Thus it appears that some true Trichoderma isolates, but not all, produce antibiotics under suitable conditions. Antibiotics reported to be produced by Trichoderma species are discussed by Dennis (1970). A sequisterpene antibiotic, trichodermin (Godtfredsen & Vangedal, 1965) has been reported to be produced by T. viride. This substance is distinct from "trichodermin-r , -2, and -3' which are reported in the Russian literature; these are crude preparations which have been used to control diseases caused by phytopathogenic fungi (Fedorinchik, 1963). Suzukacillin (Ooka et al. 1966), alamethicine (Meyer & Reusser, 1967; Reusser, 1967) and U-21963 ('Dermadine') (Pyke & Dietz, 1966; Meyer, 1966) are further antibiotics reported to have been extracted from culture filtrates of Trichoderma viride. Suzukacillin and alamethicine are peptide antibiotics, with antibacterial and antifungal properties. U -21963 is an unsaturated monobasic acid, active against Gram-negative and Grampositive bacteria and a wide range of fungi. 'Does Trichoderma produce gliotoxin and viridin?', the question which Webster & Lomas asked in 1964 still remains to be answered. In the present work an investigation of the antagonistic properties of the different Trichoderma species was made with respect to production of non-volatile
Antibiotics of Trichoderma. C. Dennis and J. Webster
27
antibiotics. The production of volatile antibiotics is described in a subsequent paper (Dennis & Webster, 1971). SURVEY OF ANTAGONISM IN TRICHODERMA SPECIES
Materials and Methods Trichoderma isolates were obtained from wood, bark, herbaceous vegetation and other substrates, or from soil samples collected from different parts of the world. Mono-ascospore isolates from different Hypocrea species were also used. Some isolates were obtained from mycologists in various countries. Material of some of the isolates has been preserved in the University of Sheffield Mycological Herbarium. Full details of the origin of the isolates are given by Dennis (1970). Bioassays of antifungal antibiotics conducted by many previous workers involved effects on spore germination of some sensitive fungus. While these methods may be fairly sensitive, antibiotic substances are possibly more important in inhibiting actively growing hyphae during intense competition for nutrients. Such competition would be most intense during the exploitation of localized areas of organic debris, which is also when antibiotic substances are most likely to be produced in concentrations sufficient to affect the growth of other soil fungi. The report of Webster & Lomas (1964) indicates that if Trichoderma strains do produce active substances, they may be produced in small quantities or may have a lower activity than either gliotoxin or viridin. For these reasons and because it provided a more convenient quantitative technique, the agarplate method devised by Heatley (1947) for use with bacteria and adapted by Gibbs (1966, 1967) for use with filamentous fungi was preferred. The antagonist was grown from an inoculum disk over the surface of a cellophane membrane laid on an agar medium, and the metabolites produced were allowed to diffuse through the cellophane into the agar. Antibiotic activity was then assessed by growing a test fungus on the medium after removal of the antagonist. Gibbs (1966, 1967) and Mughogho (1967, 1968) were unable to differentiate Trichoderma species according to their antibiotic production. In the present work, representative isolates from the different species-groups were assayed against a wider range of fungi than used by the above workers. The test fungi used (Table I) comprised a basidiomycete, an ascomycete, two phycomycetes and two fungi imperfecti. All these fungi had a reasonably high growth rate and any inhibition of growth could therefore be detected after a short incubation. Petri dishes were prepared containing 15 ml of 2 % malt extract agar. The pH was adjusted before autoclaving to two values, pH 4'0 and pH 6'5, with dilute hydrochloric acid or sodium hydroxide. A single sterile sheet of cellophane, 50 flm thick, was placed aseptically over the agar in each dish and the dishes left overnight to allow excessmoisture to evaporate. Disks 6 mm in diameter were cut with a cork-borer from the margin of a 3- to {-day-old culture of each Trichoderma isolate growing on malt extract agar, and each of the prepared plates was inoculated in a central position.
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The plates were incubated under a bank of lights at 22-25 °C for 2 days. After this time the cellophane and adhering fungus were removed. A 6-mm disk of a test fungus was placed immediately on the medium, at the central position previously occupied by the antagonist. Each antagonist was tested against each of the six test fungi in turn at the two pH levels, and the tests were conducted in triplicate. In every experiment, control plates were also set up in triplicate. A high-nutrient medium was chosen because several workers (e.g. Wright, 1952) have observed greater antibiotic production on rich media. This trend was confirmed in preliminary experiments where tapwater agar was used as an alternative to the malt medium. Table
1.
Testfungi used in the agar-plate bioassay
Fomes annosus (Fr.) Cooke Rhizoctonia solani Kuhn. Pyronema domesticum (Sow ex Fr .) Sacco Fusarium oxysporum Schlecht. ex Fr . and Pythium ultimum Trow. Mucor hiernalis Wehm.
Pathogen of pine trees. Some strains of Trichoderma shown to be antagonistic to it (Gibbs, 1966, 1967) Soil pathogen, fungus used by Weindling (1932) in his original studies of Trichoderma antagonism One of the first colonizers of burned soil. Trichoderma species observed to be antagonistic (El-Abyad, 1966; El-Abyad & Webster, 1968) Soil pathogens. Trichoderma species observed to be antagonistic against th ese fungi Soil saprophyte.
Periods of 1 day for Pyronema domesticum and Pythium ultimum and 2 days for the remaining test fungi were allowed to elapse before colony diameters of the test fungi were measured. After these incubation periods the colonies were large enough for measurement with a millimetre scale. The use of longer incubation periods would have increased the risk of loss of biologically active substances. Apparent loss of activity was observed in some experiments where measurements were made after 4- days' growth of the test fungi. It is assumed that the observed instances of severe inhibition of growth were caused by metabolites of the Trichoderma isolates which had diffused through the cellophane membrane into the agar. It could be argued that some inhibition of growth of the test fungi was caused by depletion of nutrients in the medium by previous growth of the Trichoderma isolates. However, this is unlikely because the medium employed was rich, and because incubation periods were short. Furthermore, stimulation ofgrowth of the test fungi was observed on occasions. In such cases it is possible that the Trichoderma isolates produced diffusible growth factors.
Observations and discussion Severe inhibition of mycelial growth was induced by many isolates. The inhibited colonies were often usually compact and dense; this was particularly marked with Rhizoctonia solani and Fomes annosus. Microscopic examination revealed obvious morphological differences: the hyphae
Antibiotics of Trichoderma. C. Dennis and J. Webster
29
were generally more branched, and sometimes thicker than normal hyphae. Vacuolation of the hyphae occurred in R. solani and F. annosus (PI. I, figs. I, 2). When such colonies were subcultured on to fresh malt medium, apparently normal growth of the test fungi occurred. Severe inhibition of growth of surface mycelium was often accompanied by abundant aerial growth from the inoculum disk. Neither sporangial nor conidial formation appeared to be affected by the presence of the antibiotics. Evidence of a concentration gradient of antibiotic in the medium was observed in some tests: the growth rate of th e test fungi on the test plates was initially considerably lower than on control plates, but the hyphal tips eventually reached a region where growth at the normal rate of the test fungus occurred. This is an additional reason why the colonies were measured at the chosen times. When no growth, either surface or aerial, of the test fungi occurred 4 days after inoculation, no subsequent growth took place even if the inoculum disk was transferred to fresh malt extract medium. This effect was observed most frequently with F. annosus, at both pH levels. Thus antibiotics produced by some Trichoderma isolates had fungicidal as well as fungistatic effects, depending on the test fungus used. Certain isolates produced antibiotics which were fungicidal to one fungus but only fungistatic to others. F. annosus was the most susceptible of the fungi tested, followed by R. solani and P. domesticum ; F. oxysporum was the most resistant, only the most active Trichoderma isolates causing appreciable inhibition of mycelial growth, and none being fungicidal to this fungus. Other isolates of these test fungi might have reacted differently. Gibbs (1966, 1967) showed that F. annosus isolates from different parts of the world varied widely in their sensitivity to Trichoderma antibiotics, and Mughogho (1967, 1968) found a similar variation between R. solani isolates. There was considerable variation within most species-groups as well as between species-groups with regard to production of diffusible inhibitory substances by Trichoderma isolates. This is in keeping with the findings of Moubasher (1963) and Komatsu & Hashioka (1964) that strains can differ physiologically although th ey are morphologically very similar and are placed in the same species-group. The results obtained for each species group will be considered separately. T . piluliferum Webster & Rifai. The three isolates tested from this species group caused appreciable inhibition of growth of all the test fungi at both pH levels. There did not appear to be greater antagonism at the lower pH, as was reported by Gibbs (1966, 1967). T.piluliferum is isolated most frequently from acidic habitats, and Gibbs suggested that this species-group showed an ecological adaptation for antibiosis with respect to acidity. T . polysporum (Link ex Pers.) Rifai . With one exception, isolates of this group also proved to be active antibiotic producers. Again pH seemed to have little influence, whereas Gibbs reported a separation of acid-toxic and base-toxic groups. It may be significant that a rather lower nutrient medium was used by Gibbs. T. hamatum (Ben.) Bain. All isolates were acti ve against Fomes annosus
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Transactions British Mycological Society
to different degrees, but varied greatly in their effects on the other test fungi. T. koningii Oud. Some strains were highly active, whereas others showed no detectable antagonism towards most of the test fungi. Three of the 14 isolates tested caused more inhibition of most of the test fungi at pH 4'0 than at pH 6'5. In general, F. annosus was the most susceptible test fungus. Table
Production ofantifungal antibiotics by species-groups ofTrichoderma and by Gliocladium virens
2.
Isolates tested
T. piluliferum T. polysporum T. hamatum T. koningii T. harzianum T. aureoviride T. pseudo-koningii T. longibranchiatum T. viride G. virens
3 14 9 14 9 3 4 2
Antibiotic producers"
Non-antibiotic producers
3 13
6 (2) 10
(4)
s (3)
-(I) 4
3 2
2
22 19 (2) 4 5 ... Figures in parentheses are the numbers of isolates active against Fornes annosus only.
T. harzianum Rifai. Only two of the nine isolates tested caused appreciable inhibition of growth of all the test fungi. Some other isolates inhibited F. annosus. Stimulation of growth of the test fungi, particularly at the lower pH was induced by a few isolates. Mughogho (1967, 1968) reported that isolates from this particular species-group did not affect the growth of Armillaria mellea and Rhiroctonia solani. The results of this survey tend to support his findings. T. aureoviride. Rifai. Of the three isolates tested only one showed any activity and this only against F. annosus. Although this group is morphologically very similar to the T. harzianum aggregate, the growth rate of the two groups differs considerably. Generally T. harzianum isolates have a very high growth rate while T. aureoviride isolates are characterized by a very low growth rate. Possibly the incubation period of 2 days was not sufficient for antibiotics to be produced at an inhibitory concentration. T. pseudo-koningii Rifai. Isolates of this group inhibited the more susceptible of the test fungi. There was no detectable difference in the degree of inhibition at the two pH levels. T. longibrachiatum Rifai. The two isolates tested inhibited the growth of most of the test fungi. Again the pH level of the medium had no apparent influence. T. viride Pers. ex S. F. Gray. Most isolates from this species group were active against the more susceptible test fungi. Two of the twenty-two isolates tested were active against F. annosus alone, and one isolate was completely inactive. One isolate obtained from bark was more antagonistic at pH 6'5 than at pH 4'0. Mughogho (1967, 1968) reported that T. viride affected the growth of R. solani and Armillaria mellea more than other
Antibiotics of Trichoderma. C. Dennis andJ. Webster
31
species-groups. The results of the present survey do not fully support his findings; however, isolates of this group were generally active antibiotic producers, along with other green-spored Trichoderma spp. and the whitespored T. piluliferm and T. polysporum isolates. Isolates of G. virens, including some claimed to produce gliotoxin and viridin, were also tested by the agar-plate procedure at the two pH levels. Both these antibiotics are more stable at acid pH. With the exception of one isolate from the five tested, strong antibiotic activity was shown against all the test fungi. Fusarium oxysporum again proved to be the most resistant test fungus. None of the G. virens isolates were fungicidal to this organism, although some isolates were fungicidal to all the other test fungi. Activity was similar at both pH levels. The results of the survey are summarized in Table 2. PRODUCTION OF ANTIBIOTICS IN LIQ.UID CULTURE
There was no apparent difference in the antagonism shown by some of the Trichoderma isolates and by the isolates of G. virens towards the test fungi used. It was therefore decided to study in more detail antibiotic production in liquid culture by some of the most active Trichoderma isolates, and by a G. virens isolate claimed to produce gliotoxin and viridin. Two media at two pH levels were used. Weindling's medium, being chemically defined, is particularly suitable for extraction of antibiotics from culture filtrates. It is also known that gliotoxin and viridin are produced by G. virensin this medium (Weindling, 1941; Brian & Hemming, 1945; Brian et al. 1946). A malt extract medium was also used. The pH of each medium was adjusted to pH 4'0 or pH 6·5 with dilute hydrochloric acid or sodium hydroxide respectively, before sterilizing. Monax flasks (500 ml), containing 100 ml of medium, were inoculated with two 12 mm disks cut from the margin of vigorously growing cultures of G. virens, T. polysporum, T. hamatum and T. viride. The flasks were incubated in a Gallenkamp orbital incubator (150 r.p.m.) at 25°e. Samples of culture liquid were removed daily with sterile Pasteur pipettes, filtered through Whatman No. I paper to remove hyphal fragments, and placed in a well which had been cut in the centre of a 2 % malt agar plate. Three such plates were set up for each sample of culture medium. Similar control plates were prepared with uninoculated medium incubated under the same conditions as the inoculated flasks. A rz-mm disk cut from the margin of 3-day-old cultures of Rhizoctonia solani or Mucor hiemalis was then placed on each of the plates, so that it just covered the well. The plates were incubated at 25° for 24 h. R. solani and M. hiemalis were chosen as test fungi because they both had a high growth rate and had shown different degrees of sensitivity to Trichoderma antibiotics in earlier experiments (R. solani was more sensitive than M. hiemalis). R. solani was preferred to F. annosus, although the latter was more susceptible, because more accurate measurements of colony diameter could be made. The colony diameters of the test fungi were measured 24 h after inoculation.
Transactions British Mycological Society
32
T. polysporum 74
-
T. viride 1
~=~=g--Q-It>
./
~~=gJ-
J
C
-,.
\
.~ '0
50
c o .;:;
•
:0 .s: c
o ~
:i
T. polysporum 74
100
~
50
'0 c o .;:;
T. viride 1
:0 ..c
c
o T. hamatum NRRL 3199
100
G. virens ACC 211
(j;J
D
~
50
.0
c
s:
c
OL.-_-L_ _...I..-_--l._ _-L._---l
T. hamatum NRRL 3199
100
/
. . '0
=~ -t!l - lIJ - lII - . - . 0
'0-0-0
o
--.-.
G. virens ACC 211
-
o~
.2
~
~ 50
.~~
.0.
:c::: c 2
4
6
Time (days)
8
4
6
8
Time (days)
Text-fig. I. Antibiotic activity of culture filtrates from Trichoderma spp, and G" oirens grown in malt extract and Weindling's medium at pH 4"0 and pH 6"5 for 10 days. 0-0, Malt extract medium pH 4"0; 0-0, malt extract medium pH 6"5; . - . , Weindlings's medium pH 4"0; . - . , Weindling's medium pH 6·5"
10
Antibiotics of Trichoderma. C. Dennis andJ. Webster
33
The amount of antibiotic present, as represented by the degree of inhibition of growth of R. solani and M. hiemalis, varied with time and in some cases with pH and the chemical nature of the medium (Text-fig. I). The results confirmed that R. solani was more sensitive than M. hiemalis. The Trichoderma isolates produced antibiotics at higher or similar concentrations in malt extract medium compared with the synthetic medium. The initial pH of the medium had no apparent effect on antibiotic activity of Trichoderma isolates growing in malt medium. Antibiotic activity of the T. viride isolate was, however, influenced by pH when grown in Weindling's medium. This difference might explain our inability to separate isolates into acid-toxic and base-toxic groups, as was done by Gibbs (1966, 1967) using a medium oflow nutrient concentration. Both the chemical nature and pH of the medium had a profound influence on the antibiotic activity of the culture liquid of G. virens. In contrast to the Trichoderma isolates, the production of antibiotics in malt extract medium was much less than in Weindling's medium at both pH levels. The strain of G. virens used was that reported by Brian & Hemming (1945) to produce gliotoxin. These workers confirmed the findings of Weindling (1941), that gliotoxin was more stable at acid pH and that a glucose salts medium (e.g. Weindling's medium) was excellent for the production of gliotoxin. Ammonium ions were superior to nitrate or to peptone as a nitrogen source. The relatively small amount of inorganic nitrogen present in malt extract medium, and possibly also its low sulphur content, may have accounted for the marked difference in antibiotic production by G. virens on malt extract and on Weindling's medium observed in the present study. These experiments indicate that Trichoderma species produce antibiotics different from those produced by G. virens (gliotoxin and viridin). However, the possibility that the latter are produced by Trichoderma isolates in low concentrations cannot be ruled out. EXTRACTION AND IDENTIFICATION OF ANTIBIOTICS FROM CULTURE FILTRATES
A number of Trichoderma isolates from the species groups found most active by the cellophane technique were grown in liquid culture, on Weindling's medium. Isolates of G. virens were also included in this experiment. Isolates of the T. piluliferum species group made little or no growth, and the culture filtrates were inactive in assays against R. solani. Culture filtrates from this species group were not studied further. The cultures were set up and grown by the methods described above and adjusted initially to pH 4'0. After 5 days the contents of each flask were filtered under vacuum through four layers of Whatman No. I paper to remove the mycelium. A sample of the culture filtrate was assayed for activity by the bioassay procedure described above. If the culture filtrate possessed high activity against R. solani (IOO %inhibition) and M. hiernalis. (75 % inhibition) it then underwent the following procedure. Culture filtrates from replicate flasks were pooled, to give 500 ml of filtrate. This was concentrated to about 100 ml on a rotary vacuum evaporator at 3
MYC
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40°. The concentrated culture filtrates were then extracted with chloroform (A.R.). Two successive extractions, each with 50 ml chloroform, were carried out by shaking the chloroform with aqueous fraction in a separating funnel. An emulsion formed which was dispersed by centrifugation at 3000 rev/min for about 5 min. After centrifugation the two layers were carefully poured into a separating funnel. The heavier chloroform fraction was allowed to separate out and run off from the aqueous layer. A white precipitate formed between the chloroform and aqueous layers, which will be referred to later. The pooled extracts were dried in a rotary vacuum evaporator at room temperature.
Table 3. Production ofantibiotics by isolates
ofTrichoderma and Gliocladium
Chloroform extract
Ethanol extract I
I
Isolate
T. polysporum 74 4982 9 8 T.hamatum NRRL 3153 NRRL 3199 T. koningii SHD-M 3029 99 T. harzianum 4 T. pseudo-koningii A/19 6fl T. longibrachiatum WBC4576 T. viride I
4 SHD-M 26II 17 G. virens 2II 213 94 1
Biological activity Gliotoxin
Viridin
Trichodermin
Biological Peptide activity antibiotic
+ + + +
+ + + +
+ +
+ +
+ +
+ + +
+ + +
+ + +
+
+
+
+
+
+
+ + + +
+ + + +
+ + + +
NT NT NT
NT
+ + + +
+
+
+ + + + + + + + + +, Presence of activity and antibiotic.
NT NT
-, Absence of activity and antibiotic. NT, Not tested.
Uninoculated medium, incubated under similar conditions, was also extracted with chloroform. Virtually no chloroform-soluble substances were found, indicating that the substances extracted from the culture filtrates were fungal metabolites. The dried chloroform extracts from culture filtrates of Trichoderma isolates were without exception resinous, whereas those from the Gliocladium virens culture filtrates were in the form of a powder. The dried extracts were tested for antibiotic activity by placing a small amount of the extract in the centre of a 2 % malt
Antibiotics of Trichoderma. C. Dennis andJ. Webster
35
extract agar plate and observing the growth of a test fungus (usually R. solani) from an inoculum disk placed I em from the extract. Growth rates of the test fungus towards and away from the point of application of the sample were compared. Morphological differences between the hyphae nearest to the extract and those furthest from the extract were also recorded. Each dried active extract was redissolved in a minimal amount of chloroform. Roughly equal quantities of each extract were spotted on to thin-layer silica gel plates with r-mm capillary tubing. Pure samples of gliotoxin and viridin were used as markers. The plates were run in chloroform: acetone (95: 5, vIv) for 2-3 h and developed by spraying with silver nitrate reagent or phloroglucinol-hydrochloric acid reagent. The former was used for the detection of gliotoxin, which gives a brownish black spot. Phloroglucinol-hydrochloric acid gives an orange-red colour with viridin. Amounts as low as 0'5 p,g of each antibiotic were detectable. Chloroform extracts of the culture filtrates of all the Trichoderma and Gliocladium uirens isolates tested showed antibiotic activity (Table 3). No quantitative data were recorded, but the activity varied between isolates (see PI. 3, fig. 3). The inhibited Rhizoctonia hyphae showed increased branching and vacuolation, as observed in the experiments with the cellophane-agar plate technique. It was not possible to detect either gliotoxin or viridin in any of the chloroform extracts from any of the Trichoderma isolates tested. Both antibiotics were detected in chloroform extracts of culture filtrates from the Gliocladium uirens isolates. Each G. uirens isolate produced both gliotoxin and viridin, and separation into gliotoxin-producers and viridin-producers was not possible. From the chromatograms it appeared that more gliotoxin than viridin was produced by the isolates tested. Khasanov ( I962) reported the antibiotic activity of chloroform extracts of Trichoderma culture filtrates to be due to the presence of gliotoxin. No chemical tests appear to have been conducted, although Khasanov (1962) reported that extracts contained an antibiotic with a similar R F , determined by bioassay, to a sample of gliotoxin in different solvent systems. Thus, it appeared that chloroform-soluble antibiotics other than gliotoxin or viridin were produced by Trichoderma isolates. Trichodermin, and antibiotic U-2I,963 ('Dermadine') are chloroform-soluble antibiotics reported to be produced by Trichoderma isolates (see introduction). Examination of cultures used for the production of these antibiotics showed them to be true Trichoderma species. The trichodermin-producing isolate is morphologically similar to Rifai's T. viride aggregate (isolate T. uiride 17), while the isolate used for the production of U -2I,963, reported to be T. uiride (Pyke & Dietz, 1966; Meyer, 1966), is morphologically similar to the T. hamatum aggregate (isolate T. hamatum NRRL 3I53)· Both U-21,963 and trichodermin possessantifungal and antibacterial properties, the latter being highly antifungal. Pure samples of trichodermin obtained from ICI Pharmaceuticals Division, Macclesfield, and from Leo Pharmaceuticals, Denmark, were chromatographed on thin-layer plates along with the chloroform extracts 3- 2
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Transactions British Mycological Society
of culture filtrates from Trichoderma isolates. An ascending system with chloroform: acetone (95: 5) and ethyl acetate: cyclohexane (50: 50) as solvents was employed. Trichodermin was detected by spraying with concentrated sulphuric acid and heating the plates to 100° for 10-15 min. It was possible to detect 1-2 /lg of the pure antibiotic. Trichodermin was detectable only in extracts of one isolate (T. polysporum 74), other than the isolate used previously for its production (T. viride 17). Other isolates tested from the T. polysporum group apparently did not produce this antibiotic (Table 3). No tests were carried out for the production of U-21,963. It was not possible to obtain a sample of this antibiotic, owing to its instability.
Peptide antibiotics Polypeptide antibiotics have been reported to be produced by Trichoderma isolates (see introduction). Trichoderma viride is again the name which has been applied to the isolates. It was not possible to obtain the culture used for the production of suzukacillin, but from the published photomicrograph (Ooka et at. 1966) it appears to be a true Trichoderma species. Isolate NRRL 3 I 99, used for the production of alamethicine (Meyer & Reusser, 1967), was examined and placed in the T. hamatum aggregate of Rifai. Both antibiotics contain glutamic acid or glutamine, proline, glycine, alanine, valine and leucine. In addition, alamethicine is reported to contain z-methyl-alanine (a-amino-isobutyric acid). Isoleucine, alloisoleucine and an unidentified ninhydrin-positive substance are present in hydrolysates of suzukacillin. Alloisoleucine is thought to have derived from isoleucine during hydrolysis of the peptide. Both peptides were active against a range of fungi and bacteria. The production of such antibiotics by isolates from the different species groups of Trichoderma was investigated. The cultural conditions were those described above, Weindling's medium being used. After the concentrated culture filtrates were extracted with chloroform, attempts were made to extract polypeptide antibiotics from the aqueous fraction. It was stated earlier that after extraction with chloroform, precipitates formed between the chloroform and aqueous layers. Following extraction with chloroform the precipitate was separated from the aqueous fraction. Acetone (100 ml) was added to the precipitate and the acetone-soluble fraction of the precipitate was separated by filtration through Whatman No. I paper. The acetone fraction was dried in a rotary vacuum evaporator, the dry material being redissolved in a minimal amount of ethanol. Further purification of the peptide fraction was not carried out. Although this method did not necessarily ensure complete extraction of peptide antibiotics from the culture liquid, it proved convenient when several isolates were tested. After concentration of the culture filtrates from isolates T. polysporum 74, T. polysporum 9 and T. viride 17, a white precipitate formed on standing. This precipitate was removed by centrifugation at 3000 rev/min for about 5 min. The collected precipitate was then treated in the same way as
Antibiotics of Trichoderma. C. Dennis and J. Webster
37
the precipitates formed after chloroform extraction. Antibiotic acti vity of the dried alcohol extracts was tested by a similar technique to that used for the chloroform extracts. Ethanolic solutions of pure samples of alamethicine, suzukacillin and extracts and precipitates from several isolates of Trichoderma were chromatogrammed on thin-layer plates in two solvent systems - ethanol: water (70: 30) and n-butanol: acetic acid: water (60 : 20: 20). Initially two methods were used to develop the chromatograms. The polypeptide was halogenated with iodine and then detected by spraying with a I % starch solution to yield a blue colour (Waldi, 1965). The second method (Rydon & Smith, 1952) used chlorine for the halogenation. The peptide was then detected by spraying with 1 % potassium iodide-starch solution. The dried plates were exposed to the halogen in a closed vessel for 5-10 min, and left to stand in a fume cupboard for about 30 min to remove excess halogen before being sprayed with the appropriate solution. The first method was normally employed, because of the more convenient production of iodine vapour. Antibiotic activity was observed in all dried ethanol extracts tested. Increased branching and vacuolation of affected hyphae were the only morphological changes visible. Alamethicine and suzukacillin were observed to have similar RF values in both solvent systems. The ethanol solutions prepared from all the isolates investigated contained a substance detectable by halogenation with RF values in both solvents similar to that of alamethicine and suzukacillin. Only a single spot could be detected in chromatograms run with ethanol: water. The n-butanol-acetic acid-water solvent, however, separated another substance with a higher R F value than the two pure peptide antibiotics. The intensity of this second spot varied between isolates and was never as great as that of the spot with an R F value very similar to that of alamethicine and suzukacillin. Ethanol extracts of precipitates from concentrated culture filtrates behaved similarly to extracts from the pr ecipitates after chloroform extraction. Hydrolysis of the two pure peptide antibiotics and of the dried ethanol extracts was carried out by dissolving in 6 N hydrochloric acid and hydrolysing at 100° for 24 h in an ampoule sealed under vacuum. The dried hydrolysates were dissolved in a minimal amount of water. One-way descending chromatograms of the hydrolysates were run in n-butanol: acetic acid: water (120: 30: 50) and phenol: 0-880 ammonia (200 : I) on Whatman No. I paper. Amino acids were detected by dipping in ninhydrin-pyridine reagent. Marker amino acids were run in the two solvent systems. The following amino acids were detected in hydrolysates from alamethicine and suzukacillin: glutamic acid, alanine, glycine, valine, leucine and proline. During hydrolysis, glutamine would be converted to glutamic acid . Meyer & Reusser (1967) reported glutamine to be present in alamethicine. With the solvent systems used, no separation of leucine and its isomer could be made. If the chromatograms were left at room temperature for more than 24 h after treatment with the ninhydrin reagent, an additional spot could be detected on the chromatograms run in n-butanol:acetic acid :water. From its RF value (Smith, 1960) it was possible that this spot could be a-amino-isobutyric acid, reported to
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Transactions British Mycological Society
be present in alamethicine (Meyer & Reusser, 1967). The spot was also detected in hydrolysates of both alamethicine and suzukacillin. Hydrolysates of extracts from a range of Trichoderma isolates (Table 3) contained the same six amino acids as alamethicine and suzukacillin, along with the unknown spot. No other amino acids were detected in any of the hydrolysates. These chromatographic results confirmed the earlier indications that the ethanol extracts contained a peptide similar to alamethicine and suzukacillin. It is notable that amino acids such as phenylalanine, ornithine, tyrosine, tryptophan, lysine, aspartic acid and sulphur-containing amino acids such as cysteine and fi-methyl-Ianthionine, which are commonly present in other polypeptide antibiotics (e.g. tyrocidine, gramicidin, subtilin, cinnamycin, bacillomycin, licheniform and bacitracin), appear not to be present in the polypeptide antibiotics of Trichoderma species. REFERENCES
BlLAI, V.1. (1963). Antibiotic-producing microscopic fungi, pp. 115-121. Amsterdam: Elsevier Publishing Co. BRIAN, P. W., CURTIS, P.J., HEMMING, H. G. & MCGOWAN,]. C. (1946). The production of viridin by pigment-forming strains of Trichoderma viride. Annals of AppliedBiology 33, 190-200. BRIAN, P. W. & HEMMING, H. G. (1945). Gliotoxin, a fungistatic metabolic product of Trichoderma viride. Annalsof AppliedBiology 32, 214-220. DENNIS, C. (1970). The antagonistic properties of the different species groups of Trichoderma. Ph.D. thesis, University of Sheffield. DENNIS, C. & WEBSTER,]. (1971). Antagonistic properties of species-groups of Trichoderma. II. Production of volatile antibiotics. These Transactions. EL-ABYAD, M. S. H. (1966). Ecology of some pyrophilous discomycetes. Ph.D. thesis, University of Sheffield. EL-ABYAD, M. S. H. & WEBSTER,]. (1968). Studies of pyrophilous discomycetes. II. Competition. These Transactions 5:1, 369-375. FEDORINCHIK, N. S. (1963). Some results and aims of research at the Microbiological Control Laboratory of the All-Union Plant Protection Institute. Trudy Vsescryuznogo Instituta Zashchity Rastenii, pp. 138-161. (Eng. trans.) GIBBS,]. N. (1966). Fomes annosus: host-parasite relationships. Ph.D. thesis, University of Cambridge. GIBBS, ]. N. (1967). The role of host vigour in the susceptibility of pines to Fomes annosus. Annalsof Botany 3:1, 803-815. GODTFREDSEN, W. O. & VANGEDAL, S. (1965)' Trichodermin, a new sesquiterpene antibiotic. Acta chemica scandinavica 19, 1088-1102. HEATLEY, N. G. (1947). A simple plate method for multiple tests of the anti-bacterial activity of many bacteria against other bacterial strains. Journal of General Microbiology I, 168-170. JEFFREYS, E. G., BRIAN, P. W., HEMMING, H. G. & LOWE, D. (1953). Antibiotic production by the micro-fungi of acid heath soils. Journal of General Microbiology 9, 3 14-341. KHASANOV, O. K. (1962). The antibiotic properties of fungi of the genus Trichoderma Pers. found in the swamp soil of Uzbekistan. Uzbekskii Biologicheskii Zhurnal, Tashkent 6, 62-67 (Eng. trans.) KOMATSU, M. & HAsHIOKA, Y. (1964). Trichoderma viride, as an antagonist of the woodinhabiting hymenomycetes. IV. Physiological properties of the different forms of Trichoderma derived from the different Hypocrea species and soil. Report of the Tottori Mycological Institute 4, 6- 10. MEYER, C. E. (1966). U-21, 963 - a new antibiotic. II. Isolation and characterization. AppliedMicrobiology 14, 511-512.
Trans. Br. mycol. Soc.
Vol. 57·
Plate 3
2
A
B
c
) D
/ E
F
(Facing p. 39)
3
Antibiotics of Trichoderma. C. Dennis and J. Webster
39
MEYER, C. E. & REUSSER, F. (1967). A polypeptide anti-bacterial agent isolated from Trichoderma viride. Experientia 23, 85-86. MOUBASHER, A. M. (1963). Effect of duramycin on some isolates of Trichoderma viride. Nature, London 200, 492. MUGHOGHO, L. K. (1967). The fungus flora of fumigated soils. Ph.D. thesis, University of Cambridge. MUGHOGHO, L. K. (1968). The fungus flora of fumigated soils. These Transactions 51, 44 1-459. OOKA, T., SHIMOJlMA, Y., AKIMOTO, T., TAKEDA, I., SENOH, S. & ABE,]. (1966). A new antibacterial peptide 'SuzukaciIlin.' Agricultural and Biological Chemistry 30, 700-7 02. PYKE, T. R. & DIETZ, A. (1966). U-21, 963, a new antibiotic 1. Discovery and biological activity. Applied Microbiology 14, 5°6-510. REUSSER, F. (1967). Biosynthesis of antibiotic U-22, 324, a cyclic polypeptide. Journal if' Biological Chemistry 242, 243-247. RIFAI, M. (1964). A reinvestigation of the taxonomy of the genus Trichoderma Pel's. M.Sc. thesis, University of Sheffield. RIFAI, M. (1969). A Revision of the genus Trichoderma. Mycological Papers 116. RISHBETH,]. (1950). Observations on the biology of Fomes annosus, with particular reference to East Anglian pine plantations. 1. The outbreaks of disease and evological status of the fungus. Annals rif Botany 14, 365-385. RYDON, H. N. & SMITH, P. W. G. (1952). A new method for the detection of peptide and similar compounds on paper chromatograms. Nature, London IGg, 922-923. SMITH, 1. (1960). Chromatographic and electrophoretic techniques. Vol. 1. Chromatography. London: Heinemann. WALDI, D. (1965). Spray reagents for thin layer chromatography. In Thin-layer chromatography - A laboratory handbook (ed. E. Stahl), PP.483-502. New York: Academic Press. WEBSTER,]. & LOMAS, N. (1964). Does Trichoderma viride produce gliotoxin and viridin? These Transactions 47, 535-540. WEINDLING, R. (1932) . Trichoderma lignorum as a parasite of other soil fungi. Phytopathology 22, 837-845. WEINDLING, R. (1934). Studies on a lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizocionia solani and other soil fungi. Phytopathology 24, 1153- 1179. WEINDLING, R. (1937). The isolation of toxin substances from the culture filtrates of Trichoderma and Gliocladium. Phytopathology 27, 1175-1177. WEINDLING, R. (1941). Experimental consideration of the mold toxins of Gliocladium and Trichoderma. Phytopathology 31, 991-1003. WRIGHT,]. M. (1952). Production of gliotoxin in unsterilized soil. Nature, London 170, 673-674· EXPLANATION OF PLATE
3
Figs. I, 2. Morphology of normal (Fig. I) and affected (Fig. 2) hyphae of Rhizotonia solani. Note greater branching and vacuolation of affected hyphae. x 600. Fig. 3. Inhibition of growth of R. solani caused by dried chloroform extracts of culture filtrates from Trichoderma isolates and an isolate of G. oirens. A, T.po!Jsporum 74; B, T. polysporum 9; C, T.koningii SHD-M 3029; D, T.longibrachiatumWBC4576; E, T. viride 17; F, G. virens ACC 211.
(Accepted for publication 25 September 1970)
ANTAGONISTIC PROPERTIES OF SPECIES-GROUPS OF TRICHODERMA I. PRODUCTION OF NON-VOLATILE ANTIBIOTICS
By C. DENNIS* AND J. WEBSTERt Department ofBotany, The University, Sheffield (With Plate 3 and
I
Text-figure)
Isolates from different species-groups of Trichoderma were tested for production of non-volatile antibiotics, by an agar layer technique. Preliminary studies on the chemical nature of these antibiotics were made. Many isolates produced non-volatile antibiotics active against a range of fungi. The ability to produce such antibiotics varied between isolates of the same species-group as well as between isolates of different species groups. The susceptibility of fungi to these antibiotics varied widely; Fames annosus (Fr.) Cooke was the most susceptible and Fusarium osysporum Schlecht. ex Fr. the most resistant of the test fungi used. Gliotoxin and viridin were not produced, but other chloroform-soluble antibiotics, including trichodermin and peptide antibiotics were detected.
The toxic metabolites produced by Trichoderma spp. have been the subject of intensive study ever since Weindling (1934) reported that culture filtrates of T. lignorum (Tode) Harz were toxic to Rhiroctonia solani Kuhn and other fungi even at high dilution. Unfortunately, there has been some confusion about the nomenclature of the fungus used for antibiotic production. The taxonomic confusion was cleared up by Webster & Lomas (1964), who showed that Weindling's gliotoxin-producing isolate and the two isolates from which Brian and co-workers obtained gliotoxin and viridin (Brian & Hemming, 1945; Brian, Curtis, Hemming & McGowan, 1946) were not Trichoderma viride Pers. ex S. F. Gray but were morphologically similar to Gliocladium virens Miller, Giddens & Foster. This report supported Weindling's announcement (Weindling, 1937) that his gliotoxin-producing fungus was a Gliocladium species. Gibbs (1966) and Mughogho (1967) have reported Weindling's confirmation of this. The report of Webster & Lomas therefore casts doubt on the correct identity of some Trichoderma isolates used by workers investigating antagonism of this fungus. However, as Webster & Lomas pointed out, G. virens is not common in soil, whereas Trichoderma is quite common (especially in acid soil). Thus it seems likely that most reports on antagonism by Trichoderma actually relate to this genus.
* Present address: Agricultural Research Council, Food Research Institute, Colney Lane, Norwich, NOR 70F. t Present address: Department of Biological Sciences, Prince of Wales Road, Exeter.
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Transactions British Mycological Society
For a long time the genus Trichoderma was considered to comprise several distinct species, but there was disagreement on nomenclature and the delimitation of each species was obscure. Rifai (1964, 1969) made a taxonomic revision which provides satisfactory separation of species within the genus. Rifai stressed that his new classification is not exhaustive, and as more species of Hypocrea are grown in culture the delimitation of species may need to be redefined. Nevertheless, Rifai's work cleared up much of the previous confusion on the species concept in Trichoderma and is likely to form the basis offuture work. His key to the species-groups will be used here. It is important to note, however, that Soviet mycologists (Pidoplichko, Bilai, Shklyar and others) consider that there are only two distinct species: T. koningii Oud. and T. lignorum (Tode) Harz (Bilai, 1963) The. rough-spored T. lignorum corresponds to Persoon's T. viride. Webster & Lomas (1964) investigated the antibiotic activity of isolates of G. virens. Using the bioassay procedures of Brian & Hemming (1945) and of Brian et al. (1946), they found that all three isolates examined were active against Botrytis allii Munn and Bacillus subtilis; gliotoxin and viridin were isolated in crystalline form. No activity was shown by four isolates of Hypocrea rufa (Pers. ex Fr.) Fr. (the perfect state of T. viride) or by several other isolates of Trichoderma from wood and soil, some of which were morphologically distinct from T. viride. These results contrast with those ofJeffreys, Brian, Hemming & Lowe (1953), who showed that some Trichoderma isolates collected from acid heath soil (thus likely to be true Trichodermas) were active against B. allii. Also, Rishbeth (1950) showed that culture filtrates of ten Trichoderma strains from East Anglian forest soils all inhibited germination of conidia of Fomes annosus. Gibbs (1966) has confirmed this. Using an agar-layer technique he found that some isolates of Trichoderma species, identified according to Rifai's classification, showed antibiotic activity against Fomes annosus. Thus it appears that some true Trichoderma isolates, but not all, produce antibiotics under suitable conditions. Antibiotics reported to be produced by Trichoderma species are discussed by Dennis (1970). A sequisterpene antibiotic, trichodermin (Godtfredsen & Vangedal, 1965) has been reported to be produced by T. viride. This substance is distinct from "trichodermin-r , -2, and -3' which are reported in the Russian literature; these are crude preparations which have been used to control diseases caused by phytopathogenic fungi (Fedorinchik, 1963). Suzukacillin (Ooka et al. 1966), alamethicine (Meyer & Reusser, 1967; Reusser, 1967) and U-21963 ('Dermadine') (Pyke & Dietz, 1966; Meyer, 1966) are further antibiotics reported to have been extracted from culture filtrates of Trichoderma viride. Suzukacillin and alamethicine are peptide antibiotics, with antibacterial and antifungal properties. U -21963 is an unsaturated monobasic acid, active against Gram-negative and Grampositive bacteria and a wide range of fungi. 'Does Trichoderma produce gliotoxin and viridin?', the question which Webster & Lomas asked in 1964 still remains to be answered. In the present work an investigation of the antagonistic properties of the different Trichoderma species was made with respect to production of non-volatile
Antibiotics of Trichoderma. C. Dennis and J. Webster
27
antibiotics. The production of volatile antibiotics is described in a subsequent paper (Dennis & Webster, 1971). SURVEY OF ANTAGONISM IN TRICHODERMA SPECIES
Materials and Methods Trichoderma isolates were obtained from wood, bark, herbaceous vegetation and other substrates, or from soil samples collected from different parts of the world. Mono-ascospore isolates from different Hypocrea species were also used. Some isolates were obtained from mycologists in various countries. Material of some of the isolates has been preserved in the University of Sheffield Mycological Herbarium. Full details of the origin of the isolates are given by Dennis (1970). Bioassays of antifungal antibiotics conducted by many previous workers involved effects on spore germination of some sensitive fungus. While these methods may be fairly sensitive, antibiotic substances are possibly more important in inhibiting actively growing hyphae during intense competition for nutrients. Such competition would be most intense during the exploitation of localized areas of organic debris, which is also when antibiotic substances are most likely to be produced in concentrations sufficient to affect the growth of other soil fungi. The report of Webster & Lomas (1964) indicates that if Trichoderma strains do produce active substances, they may be produced in small quantities or may have a lower activity than either gliotoxin or viridin. For these reasons and because it provided a more convenient quantitative technique, the agarplate method devised by Heatley (1947) for use with bacteria and adapted by Gibbs (1966, 1967) for use with filamentous fungi was preferred. The antagonist was grown from an inoculum disk over the surface of a cellophane membrane laid on an agar medium, and the metabolites produced were allowed to diffuse through the cellophane into the agar. Antibiotic activity was then assessed by growing a test fungus on the medium after removal of the antagonist. Gibbs (1966, 1967) and Mughogho (1967, 1968) were unable to differentiate Trichoderma species according to their antibiotic production. In the present work, representative isolates from the different species-groups were assayed against a wider range of fungi than used by the above workers. The test fungi used (Table I) comprised a basidiomycete, an ascomycete, two phycomycetes and two fungi imperfecti. All these fungi had a reasonably high growth rate and any inhibition of growth could therefore be detected after a short incubation. Petri dishes were prepared containing 15 ml of 2 % malt extract agar. The pH was adjusted before autoclaving to two values, pH 4'0 and pH 6'5, with dilute hydrochloric acid or sodium hydroxide. A single sterile sheet of cellophane, 50 flm thick, was placed aseptically over the agar in each dish and the dishes left overnight to allow excessmoisture to evaporate. Disks 6 mm in diameter were cut with a cork-borer from the margin of a 3- to {-day-old culture of each Trichoderma isolate growing on malt extract agar, and each of the prepared plates was inoculated in a central position.
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The plates were incubated under a bank of lights at 22-25 °C for 2 days. After this time the cellophane and adhering fungus were removed. A 6-mm disk of a test fungus was placed immediately on the medium, at the central position previously occupied by the antagonist. Each antagonist was tested against each of the six test fungi in turn at the two pH levels, and the tests were conducted in triplicate. In every experiment, control plates were also set up in triplicate. A high-nutrient medium was chosen because several workers (e.g. Wright, 1952) have observed greater antibiotic production on rich media. This trend was confirmed in preliminary experiments where tapwater agar was used as an alternative to the malt medium. Table
1.
Testfungi used in the agar-plate bioassay
Fomes annosus (Fr.) Cooke Rhizoctonia solani Kuhn. Pyronema domesticum (Sow ex Fr .) Sacco Fusarium oxysporum Schlecht. ex Fr . and Pythium ultimum Trow. Mucor hiernalis Wehm.
Pathogen of pine trees. Some strains of Trichoderma shown to be antagonistic to it (Gibbs, 1966, 1967) Soil pathogen, fungus used by Weindling (1932) in his original studies of Trichoderma antagonism One of the first colonizers of burned soil. Trichoderma species observed to be antagonistic (El-Abyad, 1966; El-Abyad & Webster, 1968) Soil pathogens. Trichoderma species observed to be antagonistic against th ese fungi Soil saprophyte.
Periods of 1 day for Pyronema domesticum and Pythium ultimum and 2 days for the remaining test fungi were allowed to elapse before colony diameters of the test fungi were measured. After these incubation periods the colonies were large enough for measurement with a millimetre scale. The use of longer incubation periods would have increased the risk of loss of biologically active substances. Apparent loss of activity was observed in some experiments where measurements were made after 4- days' growth of the test fungi. It is assumed that the observed instances of severe inhibition of growth were caused by metabolites of the Trichoderma isolates which had diffused through the cellophane membrane into the agar. It could be argued that some inhibition of growth of the test fungi was caused by depletion of nutrients in the medium by previous growth of the Trichoderma isolates. However, this is unlikely because the medium employed was rich, and because incubation periods were short. Furthermore, stimulation ofgrowth of the test fungi was observed on occasions. In such cases it is possible that the Trichoderma isolates produced diffusible growth factors.
Observations and discussion Severe inhibition of mycelial growth was induced by many isolates. The inhibited colonies were often usually compact and dense; this was particularly marked with Rhizoctonia solani and Fomes annosus. Microscopic examination revealed obvious morphological differences: the hyphae
Antibiotics of Trichoderma. C. Dennis and J. Webster
29
were generally more branched, and sometimes thicker than normal hyphae. Vacuolation of the hyphae occurred in R. solani and F. annosus (PI. I, figs. I, 2). When such colonies were subcultured on to fresh malt medium, apparently normal growth of the test fungi occurred. Severe inhibition of growth of surface mycelium was often accompanied by abundant aerial growth from the inoculum disk. Neither sporangial nor conidial formation appeared to be affected by the presence of the antibiotics. Evidence of a concentration gradient of antibiotic in the medium was observed in some tests: the growth rate of th e test fungi on the test plates was initially considerably lower than on control plates, but the hyphal tips eventually reached a region where growth at the normal rate of the test fungus occurred. This is an additional reason why the colonies were measured at the chosen times. When no growth, either surface or aerial, of the test fungi occurred 4 days after inoculation, no subsequent growth took place even if the inoculum disk was transferred to fresh malt extract medium. This effect was observed most frequently with F. annosus, at both pH levels. Thus antibiotics produced by some Trichoderma isolates had fungicidal as well as fungistatic effects, depending on the test fungus used. Certain isolates produced antibiotics which were fungicidal to one fungus but only fungistatic to others. F. annosus was the most susceptible of the fungi tested, followed by R. solani and P. domesticum ; F. oxysporum was the most resistant, only the most active Trichoderma isolates causing appreciable inhibition of mycelial growth, and none being fungicidal to this fungus. Other isolates of these test fungi might have reacted differently. Gibbs (1966, 1967) showed that F. annosus isolates from different parts of the world varied widely in their sensitivity to Trichoderma antibiotics, and Mughogho (1967, 1968) found a similar variation between R. solani isolates. There was considerable variation within most species-groups as well as between species-groups with regard to production of diffusible inhibitory substances by Trichoderma isolates. This is in keeping with the findings of Moubasher (1963) and Komatsu & Hashioka (1964) that strains can differ physiologically although th ey are morphologically very similar and are placed in the same species-group. The results obtained for each species group will be considered separately. T . piluliferum Webster & Rifai. The three isolates tested from this species group caused appreciable inhibition of growth of all the test fungi at both pH levels. There did not appear to be greater antagonism at the lower pH, as was reported by Gibbs (1966, 1967). T.piluliferum is isolated most frequently from acidic habitats, and Gibbs suggested that this species-group showed an ecological adaptation for antibiosis with respect to acidity. T . polysporum (Link ex Pers.) Rifai . With one exception, isolates of this group also proved to be active antibiotic producers. Again pH seemed to have little influence, whereas Gibbs reported a separation of acid-toxic and base-toxic groups. It may be significant that a rather lower nutrient medium was used by Gibbs. T. hamatum (Ben.) Bain. All isolates were acti ve against Fomes annosus
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Transactions British Mycological Society
to different degrees, but varied greatly in their effects on the other test fungi. T. koningii Oud. Some strains were highly active, whereas others showed no detectable antagonism towards most of the test fungi. Three of the 14 isolates tested caused more inhibition of most of the test fungi at pH 4'0 than at pH 6'5. In general, F. annosus was the most susceptible test fungus. Table
Production ofantifungal antibiotics by species-groups ofTrichoderma and by Gliocladium virens
2.
Isolates tested
T. piluliferum T. polysporum T. hamatum T. koningii T. harzianum T. aureoviride T. pseudo-koningii T. longibranchiatum T. viride G. virens
3 14 9 14 9 3 4 2
Antibiotic producers"
Non-antibiotic producers
3 13
6 (2) 10
(4)
s (3)
-(I) 4
3 2
2
22 19 (2) 4 5 ... Figures in parentheses are the numbers of isolates active against Fornes annosus only.
T. harzianum Rifai. Only two of the nine isolates tested caused appreciable inhibition of growth of all the test fungi. Some other isolates inhibited F. annosus. Stimulation of growth of the test fungi, particularly at the lower pH was induced by a few isolates. Mughogho (1967, 1968) reported that isolates from this particular species-group did not affect the growth of Armillaria mellea and Rhiroctonia solani. The results of this survey tend to support his findings. T. aureoviride. Rifai. Of the three isolates tested only one showed any activity and this only against F. annosus. Although this group is morphologically very similar to the T. harzianum aggregate, the growth rate of the two groups differs considerably. Generally T. harzianum isolates have a very high growth rate while T. aureoviride isolates are characterized by a very low growth rate. Possibly the incubation period of 2 days was not sufficient for antibiotics to be produced at an inhibitory concentration. T. pseudo-koningii Rifai. Isolates of this group inhibited the more susceptible of the test fungi. There was no detectable difference in the degree of inhibition at the two pH levels. T. longibrachiatum Rifai. The two isolates tested inhibited the growth of most of the test fungi. Again the pH level of the medium had no apparent influence. T. viride Pers. ex S. F. Gray. Most isolates from this species group were active against the more susceptible test fungi. Two of the twenty-two isolates tested were active against F. annosus alone, and one isolate was completely inactive. One isolate obtained from bark was more antagonistic at pH 6'5 than at pH 4'0. Mughogho (1967, 1968) reported that T. viride affected the growth of R. solani and Armillaria mellea more than other
Antibiotics of Trichoderma. C. Dennis andJ. Webster
31
species-groups. The results of the present survey do not fully support his findings; however, isolates of this group were generally active antibiotic producers, along with other green-spored Trichoderma spp. and the whitespored T. piluliferm and T. polysporum isolates. Isolates of G. virens, including some claimed to produce gliotoxin and viridin, were also tested by the agar-plate procedure at the two pH levels. Both these antibiotics are more stable at acid pH. With the exception of one isolate from the five tested, strong antibiotic activity was shown against all the test fungi. Fusarium oxysporum again proved to be the most resistant test fungus. None of the G. virens isolates were fungicidal to this organism, although some isolates were fungicidal to all the other test fungi. Activity was similar at both pH levels. The results of the survey are summarized in Table 2. PRODUCTION OF ANTIBIOTICS IN LIQ.UID CULTURE
There was no apparent difference in the antagonism shown by some of the Trichoderma isolates and by the isolates of G. virens towards the test fungi used. It was therefore decided to study in more detail antibiotic production in liquid culture by some of the most active Trichoderma isolates, and by a G. virens isolate claimed to produce gliotoxin and viridin. Two media at two pH levels were used. Weindling's medium, being chemically defined, is particularly suitable for extraction of antibiotics from culture filtrates. It is also known that gliotoxin and viridin are produced by G. virensin this medium (Weindling, 1941; Brian & Hemming, 1945; Brian et al. 1946). A malt extract medium was also used. The pH of each medium was adjusted to pH 4'0 or pH 6·5 with dilute hydrochloric acid or sodium hydroxide respectively, before sterilizing. Monax flasks (500 ml), containing 100 ml of medium, were inoculated with two 12 mm disks cut from the margin of vigorously growing cultures of G. virens, T. polysporum, T. hamatum and T. viride. The flasks were incubated in a Gallenkamp orbital incubator (150 r.p.m.) at 25°e. Samples of culture liquid were removed daily with sterile Pasteur pipettes, filtered through Whatman No. I paper to remove hyphal fragments, and placed in a well which had been cut in the centre of a 2 % malt agar plate. Three such plates were set up for each sample of culture medium. Similar control plates were prepared with uninoculated medium incubated under the same conditions as the inoculated flasks. A rz-mm disk cut from the margin of 3-day-old cultures of Rhizoctonia solani or Mucor hiemalis was then placed on each of the plates, so that it just covered the well. The plates were incubated at 25° for 24 h. R. solani and M. hiemalis were chosen as test fungi because they both had a high growth rate and had shown different degrees of sensitivity to Trichoderma antibiotics in earlier experiments (R. solani was more sensitive than M. hiemalis). R. solani was preferred to F. annosus, although the latter was more susceptible, because more accurate measurements of colony diameter could be made. The colony diameters of the test fungi were measured 24 h after inoculation.
Transactions British Mycological Society
32
T. polysporum 74
-
T. viride 1
~=~=g--Q-It>
./
~~=gJ-
J
C
-,.
\
.~ '0
50
c o .;:;
•
:0 .s: c
o ~
:i
T. polysporum 74
100
~
50
'0 c o .;:;
T. viride 1
:0 ..c
c
o T. hamatum NRRL 3199
100
G. virens ACC 211
(j;J
D
~
50
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s:
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OL.-_-L_ _...I..-_--l._ _-L._---l
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. . '0
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8
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Text-fig. I. Antibiotic activity of culture filtrates from Trichoderma spp, and G" oirens grown in malt extract and Weindling's medium at pH 4"0 and pH 6"5 for 10 days. 0-0, Malt extract medium pH 4"0; 0-0, malt extract medium pH 6"5; . - . , Weindlings's medium pH 4"0; . - . , Weindling's medium pH 6·5"
10
Antibiotics of Trichoderma. C. Dennis andJ. Webster
33
The amount of antibiotic present, as represented by the degree of inhibition of growth of R. solani and M. hiemalis, varied with time and in some cases with pH and the chemical nature of the medium (Text-fig. I). The results confirmed that R. solani was more sensitive than M. hiemalis. The Trichoderma isolates produced antibiotics at higher or similar concentrations in malt extract medium compared with the synthetic medium. The initial pH of the medium had no apparent effect on antibiotic activity of Trichoderma isolates growing in malt medium. Antibiotic activity of the T. viride isolate was, however, influenced by pH when grown in Weindling's medium. This difference might explain our inability to separate isolates into acid-toxic and base-toxic groups, as was done by Gibbs (1966, 1967) using a medium oflow nutrient concentration. Both the chemical nature and pH of the medium had a profound influence on the antibiotic activity of the culture liquid of G. virens. In contrast to the Trichoderma isolates, the production of antibiotics in malt extract medium was much less than in Weindling's medium at both pH levels. The strain of G. virens used was that reported by Brian & Hemming (1945) to produce gliotoxin. These workers confirmed the findings of Weindling (1941), that gliotoxin was more stable at acid pH and that a glucose salts medium (e.g. Weindling's medium) was excellent for the production of gliotoxin. Ammonium ions were superior to nitrate or to peptone as a nitrogen source. The relatively small amount of inorganic nitrogen present in malt extract medium, and possibly also its low sulphur content, may have accounted for the marked difference in antibiotic production by G. virens on malt extract and on Weindling's medium observed in the present study. These experiments indicate that Trichoderma species produce antibiotics different from those produced by G. virens (gliotoxin and viridin). However, the possibility that the latter are produced by Trichoderma isolates in low concentrations cannot be ruled out. EXTRACTION AND IDENTIFICATION OF ANTIBIOTICS FROM CULTURE FILTRATES
A number of Trichoderma isolates from the species groups found most active by the cellophane technique were grown in liquid culture, on Weindling's medium. Isolates of G. virens were also included in this experiment. Isolates of the T. piluliferum species group made little or no growth, and the culture filtrates were inactive in assays against R. solani. Culture filtrates from this species group were not studied further. The cultures were set up and grown by the methods described above and adjusted initially to pH 4'0. After 5 days the contents of each flask were filtered under vacuum through four layers of Whatman No. I paper to remove the mycelium. A sample of the culture filtrate was assayed for activity by the bioassay procedure described above. If the culture filtrate possessed high activity against R. solani (IOO %inhibition) and M. hiernalis. (75 % inhibition) it then underwent the following procedure. Culture filtrates from replicate flasks were pooled, to give 500 ml of filtrate. This was concentrated to about 100 ml on a rotary vacuum evaporator at 3
MYC
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40°. The concentrated culture filtrates were then extracted with chloroform (A.R.). Two successive extractions, each with 50 ml chloroform, were carried out by shaking the chloroform with aqueous fraction in a separating funnel. An emulsion formed which was dispersed by centrifugation at 3000 rev/min for about 5 min. After centrifugation the two layers were carefully poured into a separating funnel. The heavier chloroform fraction was allowed to separate out and run off from the aqueous layer. A white precipitate formed between the chloroform and aqueous layers, which will be referred to later. The pooled extracts were dried in a rotary vacuum evaporator at room temperature.
Table 3. Production ofantibiotics by isolates
ofTrichoderma and Gliocladium
Chloroform extract
Ethanol extract I
I
Isolate
T. polysporum 74 4982 9 8 T.hamatum NRRL 3153 NRRL 3199 T. koningii SHD-M 3029 99 T. harzianum 4 T. pseudo-koningii A/19 6fl T. longibrachiatum WBC4576 T. viride I
4 SHD-M 26II 17 G. virens 2II 213 94 1
Biological activity Gliotoxin
Viridin
Trichodermin
Biological Peptide activity antibiotic
+ + + +
+ + + +
+ +
+ +
+ +
+ + +
+ + +
+ + +
+
+
+
+
+
+
+ + + +
+ + + +
+ + + +
NT NT NT
NT
+ + + +
+
+
+ + + + + + + + + +, Presence of activity and antibiotic.
NT NT
-, Absence of activity and antibiotic. NT, Not tested.
Uninoculated medium, incubated under similar conditions, was also extracted with chloroform. Virtually no chloroform-soluble substances were found, indicating that the substances extracted from the culture filtrates were fungal metabolites. The dried chloroform extracts from culture filtrates of Trichoderma isolates were without exception resinous, whereas those from the Gliocladium virens culture filtrates were in the form of a powder. The dried extracts were tested for antibiotic activity by placing a small amount of the extract in the centre of a 2 % malt
Antibiotics of Trichoderma. C. Dennis andJ. Webster
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extract agar plate and observing the growth of a test fungus (usually R. solani) from an inoculum disk placed I em from the extract. Growth rates of the test fungus towards and away from the point of application of the sample were compared. Morphological differences between the hyphae nearest to the extract and those furthest from the extract were also recorded. Each dried active extract was redissolved in a minimal amount of chloroform. Roughly equal quantities of each extract were spotted on to thin-layer silica gel plates with r-mm capillary tubing. Pure samples of gliotoxin and viridin were used as markers. The plates were run in chloroform: acetone (95: 5, vIv) for 2-3 h and developed by spraying with silver nitrate reagent or phloroglucinol-hydrochloric acid reagent. The former was used for the detection of gliotoxin, which gives a brownish black spot. Phloroglucinol-hydrochloric acid gives an orange-red colour with viridin. Amounts as low as 0'5 p,g of each antibiotic were detectable. Chloroform extracts of the culture filtrates of all the Trichoderma and Gliocladium uirens isolates tested showed antibiotic activity (Table 3). No quantitative data were recorded, but the activity varied between isolates (see PI. 3, fig. 3). The inhibited Rhizoctonia hyphae showed increased branching and vacuolation, as observed in the experiments with the cellophane-agar plate technique. It was not possible to detect either gliotoxin or viridin in any of the chloroform extracts from any of the Trichoderma isolates tested. Both antibiotics were detected in chloroform extracts of culture filtrates from the Gliocladium uirens isolates. Each G. uirens isolate produced both gliotoxin and viridin, and separation into gliotoxin-producers and viridin-producers was not possible. From the chromatograms it appeared that more gliotoxin than viridin was produced by the isolates tested. Khasanov ( I962) reported the antibiotic activity of chloroform extracts of Trichoderma culture filtrates to be due to the presence of gliotoxin. No chemical tests appear to have been conducted, although Khasanov (1962) reported that extracts contained an antibiotic with a similar R F , determined by bioassay, to a sample of gliotoxin in different solvent systems. Thus, it appeared that chloroform-soluble antibiotics other than gliotoxin or viridin were produced by Trichoderma isolates. Trichodermin, and antibiotic U-2I,963 ('Dermadine') are chloroform-soluble antibiotics reported to be produced by Trichoderma isolates (see introduction). Examination of cultures used for the production of these antibiotics showed them to be true Trichoderma species. The trichodermin-producing isolate is morphologically similar to Rifai's T. viride aggregate (isolate T. uiride 17), while the isolate used for the production of U -2I,963, reported to be T. uiride (Pyke & Dietz, 1966; Meyer, 1966), is morphologically similar to the T. hamatum aggregate (isolate T. hamatum NRRL 3I53)· Both U-21,963 and trichodermin possessantifungal and antibacterial properties, the latter being highly antifungal. Pure samples of trichodermin obtained from ICI Pharmaceuticals Division, Macclesfield, and from Leo Pharmaceuticals, Denmark, were chromatographed on thin-layer plates along with the chloroform extracts 3- 2
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of culture filtrates from Trichoderma isolates. An ascending system with chloroform: acetone (95: 5) and ethyl acetate: cyclohexane (50: 50) as solvents was employed. Trichodermin was detected by spraying with concentrated sulphuric acid and heating the plates to 100° for 10-15 min. It was possible to detect 1-2 /lg of the pure antibiotic. Trichodermin was detectable only in extracts of one isolate (T. polysporum 74), other than the isolate used previously for its production (T. viride 17). Other isolates tested from the T. polysporum group apparently did not produce this antibiotic (Table 3). No tests were carried out for the production of U-21,963. It was not possible to obtain a sample of this antibiotic, owing to its instability.
Peptide antibiotics Polypeptide antibiotics have been reported to be produced by Trichoderma isolates (see introduction). Trichoderma viride is again the name which has been applied to the isolates. It was not possible to obtain the culture used for the production of suzukacillin, but from the published photomicrograph (Ooka et at. 1966) it appears to be a true Trichoderma species. Isolate NRRL 3 I 99, used for the production of alamethicine (Meyer & Reusser, 1967), was examined and placed in the T. hamatum aggregate of Rifai. Both antibiotics contain glutamic acid or glutamine, proline, glycine, alanine, valine and leucine. In addition, alamethicine is reported to contain z-methyl-alanine (a-amino-isobutyric acid). Isoleucine, alloisoleucine and an unidentified ninhydrin-positive substance are present in hydrolysates of suzukacillin. Alloisoleucine is thought to have derived from isoleucine during hydrolysis of the peptide. Both peptides were active against a range of fungi and bacteria. The production of such antibiotics by isolates from the different species groups of Trichoderma was investigated. The cultural conditions were those described above, Weindling's medium being used. After the concentrated culture filtrates were extracted with chloroform, attempts were made to extract polypeptide antibiotics from the aqueous fraction. It was stated earlier that after extraction with chloroform, precipitates formed between the chloroform and aqueous layers. Following extraction with chloroform the precipitate was separated from the aqueous fraction. Acetone (100 ml) was added to the precipitate and the acetone-soluble fraction of the precipitate was separated by filtration through Whatman No. I paper. The acetone fraction was dried in a rotary vacuum evaporator, the dry material being redissolved in a minimal amount of ethanol. Further purification of the peptide fraction was not carried out. Although this method did not necessarily ensure complete extraction of peptide antibiotics from the culture liquid, it proved convenient when several isolates were tested. After concentration of the culture filtrates from isolates T. polysporum 74, T. polysporum 9 and T. viride 17, a white precipitate formed on standing. This precipitate was removed by centrifugation at 3000 rev/min for about 5 min. The collected precipitate was then treated in the same way as
Antibiotics of Trichoderma. C. Dennis and J. Webster
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the precipitates formed after chloroform extraction. Antibiotic acti vity of the dried alcohol extracts was tested by a similar technique to that used for the chloroform extracts. Ethanolic solutions of pure samples of alamethicine, suzukacillin and extracts and precipitates from several isolates of Trichoderma were chromatogrammed on thin-layer plates in two solvent systems - ethanol: water (70: 30) and n-butanol: acetic acid: water (60 : 20: 20). Initially two methods were used to develop the chromatograms. The polypeptide was halogenated with iodine and then detected by spraying with a I % starch solution to yield a blue colour (Waldi, 1965). The second method (Rydon & Smith, 1952) used chlorine for the halogenation. The peptide was then detected by spraying with 1 % potassium iodide-starch solution. The dried plates were exposed to the halogen in a closed vessel for 5-10 min, and left to stand in a fume cupboard for about 30 min to remove excess halogen before being sprayed with the appropriate solution. The first method was normally employed, because of the more convenient production of iodine vapour. Antibiotic activity was observed in all dried ethanol extracts tested. Increased branching and vacuolation of affected hyphae were the only morphological changes visible. Alamethicine and suzukacillin were observed to have similar RF values in both solvent systems. The ethanol solutions prepared from all the isolates investigated contained a substance detectable by halogenation with RF values in both solvents similar to that of alamethicine and suzukacillin. Only a single spot could be detected in chromatograms run with ethanol: water. The n-butanol-acetic acid-water solvent, however, separated another substance with a higher R F value than the two pure peptide antibiotics. The intensity of this second spot varied between isolates and was never as great as that of the spot with an R F value very similar to that of alamethicine and suzukacillin. Ethanol extracts of precipitates from concentrated culture filtrates behaved similarly to extracts from the pr ecipitates after chloroform extraction. Hydrolysis of the two pure peptide antibiotics and of the dried ethanol extracts was carried out by dissolving in 6 N hydrochloric acid and hydrolysing at 100° for 24 h in an ampoule sealed under vacuum. The dried hydrolysates were dissolved in a minimal amount of water. One-way descending chromatograms of the hydrolysates were run in n-butanol: acetic acid: water (120: 30: 50) and phenol: 0-880 ammonia (200 : I) on Whatman No. I paper. Amino acids were detected by dipping in ninhydrin-pyridine reagent. Marker amino acids were run in the two solvent systems. The following amino acids were detected in hydrolysates from alamethicine and suzukacillin: glutamic acid, alanine, glycine, valine, leucine and proline. During hydrolysis, glutamine would be converted to glutamic acid . Meyer & Reusser (1967) reported glutamine to be present in alamethicine. With the solvent systems used, no separation of leucine and its isomer could be made. If the chromatograms were left at room temperature for more than 24 h after treatment with the ninhydrin reagent, an additional spot could be detected on the chromatograms run in n-butanol:acetic acid :water. From its RF value (Smith, 1960) it was possible that this spot could be a-amino-isobutyric acid, reported to
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be present in alamethicine (Meyer & Reusser, 1967). The spot was also detected in hydrolysates of both alamethicine and suzukacillin. Hydrolysates of extracts from a range of Trichoderma isolates (Table 3) contained the same six amino acids as alamethicine and suzukacillin, along with the unknown spot. No other amino acids were detected in any of the hydrolysates. These chromatographic results confirmed the earlier indications that the ethanol extracts contained a peptide similar to alamethicine and suzukacillin. It is notable that amino acids such as phenylalanine, ornithine, tyrosine, tryptophan, lysine, aspartic acid and sulphur-containing amino acids such as cysteine and fi-methyl-Ianthionine, which are commonly present in other polypeptide antibiotics (e.g. tyrocidine, gramicidin, subtilin, cinnamycin, bacillomycin, licheniform and bacitracin), appear not to be present in the polypeptide antibiotics of Trichoderma species. REFERENCES
BlLAI, V.1. (1963). Antibiotic-producing microscopic fungi, pp. 115-121. Amsterdam: Elsevier Publishing Co. BRIAN, P. W., CURTIS, P.J., HEMMING, H. G. & MCGOWAN,]. C. (1946). The production of viridin by pigment-forming strains of Trichoderma viride. Annals of AppliedBiology 33, 190-200. BRIAN, P. W. & HEMMING, H. G. (1945). Gliotoxin, a fungistatic metabolic product of Trichoderma viride. Annalsof AppliedBiology 32, 214-220. DENNIS, C. (1970). The antagonistic properties of the different species groups of Trichoderma. Ph.D. thesis, University of Sheffield. DENNIS, C. & WEBSTER,]. (1971). Antagonistic properties of species-groups of Trichoderma. II. Production of volatile antibiotics. These Transactions. EL-ABYAD, M. S. H. (1966). Ecology of some pyrophilous discomycetes. Ph.D. thesis, University of Sheffield. EL-ABYAD, M. S. H. & WEBSTER,]. (1968). Studies of pyrophilous discomycetes. II. Competition. These Transactions 5:1, 369-375. FEDORINCHIK, N. S. (1963). Some results and aims of research at the Microbiological Control Laboratory of the All-Union Plant Protection Institute. Trudy Vsescryuznogo Instituta Zashchity Rastenii, pp. 138-161. (Eng. trans.) GIBBS,]. N. (1966). Fomes annosus: host-parasite relationships. Ph.D. thesis, University of Cambridge. GIBBS, ]. N. (1967). The role of host vigour in the susceptibility of pines to Fomes annosus. Annalsof Botany 3:1, 803-815. GODTFREDSEN, W. O. & VANGEDAL, S. (1965)' Trichodermin, a new sesquiterpene antibiotic. Acta chemica scandinavica 19, 1088-1102. HEATLEY, N. G. (1947). A simple plate method for multiple tests of the anti-bacterial activity of many bacteria against other bacterial strains. Journal of General Microbiology I, 168-170. JEFFREYS, E. G., BRIAN, P. W., HEMMING, H. G. & LOWE, D. (1953). Antibiotic production by the micro-fungi of acid heath soils. Journal of General Microbiology 9, 3 14-341. KHASANOV, O. K. (1962). The antibiotic properties of fungi of the genus Trichoderma Pers. found in the swamp soil of Uzbekistan. Uzbekskii Biologicheskii Zhurnal, Tashkent 6, 62-67 (Eng. trans.) KOMATSU, M. & HAsHIOKA, Y. (1964). Trichoderma viride, as an antagonist of the woodinhabiting hymenomycetes. IV. Physiological properties of the different forms of Trichoderma derived from the different Hypocrea species and soil. Report of the Tottori Mycological Institute 4, 6- 10. MEYER, C. E. (1966). U-21, 963 - a new antibiotic. II. Isolation and characterization. AppliedMicrobiology 14, 511-512.
Trans. Br. mycol. Soc.
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Plate 3
2
A
B
c
) D
/ E
F
(Facing p. 39)
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MEYER, C. E. & REUSSER, F. (1967). A polypeptide anti-bacterial agent isolated from Trichoderma viride. Experientia 23, 85-86. MOUBASHER, A. M. (1963). Effect of duramycin on some isolates of Trichoderma viride. Nature, London 200, 492. MUGHOGHO, L. K. (1967). The fungus flora of fumigated soils. Ph.D. thesis, University of Cambridge. MUGHOGHO, L. K. (1968). The fungus flora of fumigated soils. These Transactions 51, 44 1-459. OOKA, T., SHIMOJlMA, Y., AKIMOTO, T., TAKEDA, I., SENOH, S. & ABE,]. (1966). A new antibacterial peptide 'SuzukaciIlin.' Agricultural and Biological Chemistry 30, 700-7 02. PYKE, T. R. & DIETZ, A. (1966). U-21, 963, a new antibiotic 1. Discovery and biological activity. Applied Microbiology 14, 5°6-510. REUSSER, F. (1967). Biosynthesis of antibiotic U-22, 324, a cyclic polypeptide. Journal if' Biological Chemistry 242, 243-247. RIFAI, M. (1964). A reinvestigation of the taxonomy of the genus Trichoderma Pel's. M.Sc. thesis, University of Sheffield. RIFAI, M. (1969). A Revision of the genus Trichoderma. Mycological Papers 116. RISHBETH,]. (1950). Observations on the biology of Fomes annosus, with particular reference to East Anglian pine plantations. 1. The outbreaks of disease and evological status of the fungus. Annals rif Botany 14, 365-385. RYDON, H. N. & SMITH, P. W. G. (1952). A new method for the detection of peptide and similar compounds on paper chromatograms. Nature, London IGg, 922-923. SMITH, 1. (1960). Chromatographic and electrophoretic techniques. Vol. 1. Chromatography. London: Heinemann. WALDI, D. (1965). Spray reagents for thin layer chromatography. In Thin-layer chromatography - A laboratory handbook (ed. E. Stahl), PP.483-502. New York: Academic Press. WEBSTER,]. & LOMAS, N. (1964). Does Trichoderma viride produce gliotoxin and viridin? These Transactions 47, 535-540. WEINDLING, R. (1932) . Trichoderma lignorum as a parasite of other soil fungi. Phytopathology 22, 837-845. WEINDLING, R. (1934). Studies on a lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizocionia solani and other soil fungi. Phytopathology 24, 1153- 1179. WEINDLING, R. (1937). The isolation of toxin substances from the culture filtrates of Trichoderma and Gliocladium. Phytopathology 27, 1175-1177. WEINDLING, R. (1941). Experimental consideration of the mold toxins of Gliocladium and Trichoderma. Phytopathology 31, 991-1003. WRIGHT,]. M. (1952). Production of gliotoxin in unsterilized soil. Nature, London 170, 673-674· EXPLANATION OF PLATE
3
Figs. I, 2. Morphology of normal (Fig. I) and affected (Fig. 2) hyphae of Rhizotonia solani. Note greater branching and vacuolation of affected hyphae. x 600. Fig. 3. Inhibition of growth of R. solani caused by dried chloroform extracts of culture filtrates from Trichoderma isolates and an isolate of G. oirens. A, T.po!Jsporum 74; B, T. polysporum 9; C, T.koningii SHD-M 3029; D, T.longibrachiatumWBC4576; E, T. viride 17; F, G. virens ACC 211.
(Accepted for publication 25 September 1970)