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Antagonistic properties of species-groups of Trichoderma
December 1971
Vol. 57, Part 3 Trans. Br. mycol. Soc. 57 (3), 363-369 (1971) Printed in GreatBritain
ANTAGONISTIC PROPERTIES OF SPECIES-GROUPS OF TRICHODERMA III. HYPHAL INTERACTION
By C. DENNIS*
AND ].
WEBSTER t
Department of Botany, University oj Sheffield (With Plates 22 and 23) When grown in dual culture, hyphae of the majority of Trichoderma isolates coiled around hyphae of different test fungi. Penetration of hyphae by Trichoderma hyphae seldom occurred. The importance of antibiotics and extracellular enzymes during hyphal interaction is discussed.
Weindling (1932) reported coiling by hyphae of Trichoderma species around hyphae of certain other fungi. Webster & Lomas (1964) showed that Weindling's strains were not Trichoderma species, but belonged to the genus Gliocladium. Other workers, however, have reported hyphal interactions by isolates considered to be true Trichoderma species (Table I). Experiments have been carried out to test the ability of a number of isolates from the different species-groups of Trichoderma to coil around or to penetrate hyphae of other fungi, including a basidiomycete, an ascomycete, a zygomycete, an oomycete and several fungi imperfecti, The Trichoderma isolates and the test fungi were those used in studies on antibiotic production by Trichoderma isolates (Dennis & Webster, 1971 a, b). MATERIALS AND METHODS
Trichoderma isolates were grown in dual culture with each of six test fungi, on 2 % malt extract agar (15 ml per dish). Except for Fames annosus (Fr.) Cooke, both fungi were inoculated at the same time. Disks (6 mm) cut from the margins of vigorously growing cultures were placed 2-3 cm apart. Fomes annosus, because of its comparatively slow growth rate, was inoculated 2 days before the Trichoderma isolates; the inoculum disk of each Trichoderma isolate was then placed about 2 em from the edge of the F. annosus colony. The plates were incubated under a bank oflights at 2225°C. Direct inspection by light and phase-contrast microscopy was carried out before and after contact of the hyphae of the two fungi. For high
* Present address: Agricultural Research Council, Food Research Institute, Colney Lane, Norwich, NOR 7oF. t Present address: Department of Biological Sciences, Prince of Wales Road, Exeter, Devon. Vol. 56, Part 3 was issued 24 June 1971 MYC
57
364
Transactions British Mycological Society
magnifications, a cover-slip was carefully placed over the area to be examined and a small volume of water was pipetted underneath the cover-slip. Sometimes the hyphae were stained with cotton blue in lactic acid before being examined. Table
I.
Report ofinteractions between hyphae ofTrichoderma species andhyphae ofother fungi
Susceptible fungus
Gaumannomyces graminis Armillaria mellea Polyporus schweinitzii Fusarium osysporum F. solani F. roseum Rhizopus oryzae Actinomueor repens Phycomyces nitens Circinella musae Mucor uarians M. spinosus zYgorhynchus vuilleminii Syncephalastrum raeemosum Rhizoctonia solani Leniinus edodes
Type of hyphal interaction
Author
Penetration Slight c~iling and penetratIon } (only at acid pH)
SIagg & Fellows (1947)
} Coiling
Chi (1960)
Aytoun (1953)
Penetration
Durrell (1966)
Coiling
Komatsu (1968)
RESULTS
Survey ofcoiling action ofTrichoderma isolates The results ofa survey ofhyphaI coiling by eighty isolates are summarized in Table 2. No attempt was made to express the results quantitatively, because of the diversity of growth pattern of the different isolates. It also proved impossible to select a standard time at which to examine the cultures for interaction. Some Trichoderma isolates coiled around the 'host' hyphae as soon as the two colonies made contact. With other isolates, coiling did not occur until the Trichoderma hyphae had penetrated far into the 'host' fungus colony. Of the 80 Trichoderma isolates tested, only ten did not show coiling. Most isolates coiled around hyphae of all the test fungi, but some did not interact with hyphae of Fusarium oxysporum Schlecht. ex Fr., especially isolates belonging to the T. polysporum (Link ex Pers.) Rifai and T. piluliferum Webster & Rifai species groups. Some variation in coiling behaviour was observed between isolates of the same species groups, though to a lesser extent than variation in antibiotic production (Dennis & Webster, 1971 a, b). Isolates of T. harzianium Rifai which showed no apparent ability to produce antibiotics were capable of coiling around other hyphae. In addition, isolates of the T. polysporum and T. piluliferum groups, which produced non-volatile antibiotics (Dennis & Webster, 197oa), did not coil around hyphae of other fungi as severely as some isolates which showed no antibiotic activity. This was also true of antibioticproducing isolates from other species groups.
Hyphal interaction. C. Dennis and J. Webster
365
In dual culture experiments on malt extract agar with R. solani as the test fungus no apparent difference was observed between hyphal interaction at pH 4'0 and 6'5. The amount of coiling by Trichoderma hyphae appeared to be the same at both pH levels. These observations contrast with those of Aytoun (1953) who observed coiling by Trichoderma hyphae around hyphae of Armillaria mellea and Polyporus schmeinitzii to be more intense at pH 3'4 than at pH 5'1 or pH 7'0. Table
2.
Summary of hyphal interaction experiments
No. of Rhizoctonia isolates solani
T. piluliferum T. polysporum
3 13 5 3
T. koningii
4 4
Fusarium Pyronema oxysporum domesticum
Mucor hiemalis
Pythium ultimum
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+
+ +
+ +
+ +
+ + +
+ +
+ +
+ + +
+ + +
+ + +
+ +
+
+
+ +
+ +
+ +
+ + + +
+ + + +
+ + +
+ + + +
+ + + +
+ + + +
I
T. hamatum
Fomes annosus
I
I
T. harzianum T. aureooiride
5 9 I
2
T. pseudokoningii T. longihrachiatum T. viride
4 2
17 4 I
Key:
+ denotes coiling by
Trichoderma hyphae; - denotes no coiling by Trichoderma hyphae.
Observations before contact between hyphae of the test fungi and Trichoderma hyphae Some isolates of Trichoderma, already shown to produce non-volatile antibiotics, caused inhibition of growth of the test fungus colony long before contact between the hyphae occurred, often when the two colonies were still 3-5 mm apart. The hyphae of the test fungi nearest to the Trichoderma colony showed the morphological changes reported by Dennis & Webster (1971 a). Vacuolation and coagulation of cytoplasm were especially noticeable in hyphae of F. annosus and R. solani. Bursting of Fomes and Rhizoctonia hyphae was occasionally observed to occur before contact with the Trichoderma hyphae. Rishbeth (1950) reported similar morphological changes when Trichoderma isolates were grown in dual culture with F. annosus isolates. Recently, Komatsu (1968) reported that hyphae of Lentinus edodes showed similar effects when placed in culture filtrates of Trichoderma isolates. Such observations provide supporting evidence that Trichoderma isolates produce diffusible antibiotics, which are active in advance of the hyphae. Both volatile and non-volatile antibiotics could be effective in the case of some isolates.
Transactions British Mycological Soct"ety Observations after contact between hyphae of Trichoderma isolates and oftest fungi The types of hyphal interaction observed are shown in PI. 22. Trichoderma hyphae frequently coiled around aerial hyphae, including sporangiophores, and hyphae growing on the agar surface. No coiling around subsurface hyphae was observed. The coiling could be separated into three types: (I) Numerous short branches were produced by the main hyphae, and each branch coiled tightly about the susceptible hyphae (PI. 22, figs. 1,2). (2) The main hyphae coiled about the susceptible hyphae. In this case the coils formed at a narrower angle to the' host' hypha and there were fewer coils per unit length of hypha (PI. 22, figs. 3,4), (3) The Trichoderma hyphae followed the' host' hyphae, and at intervals the Trichoderma hyphae produced a short branch which coiled around the 'host' hypha (PI. 23, fig. 5). When this occurred, the Trichoderma hyphae grew in a zig-zag or wavy manner along the surface of the 'host' hyphae. All three types of coiling were usually observed when any Trichoderma isolate was grown in dual culture with a susceptible fungus. Vacuolation, coagulation of cytoplasm and sometimes bursting of hyphae (F. annosus and R. solani) were induced by the antibiotic-producing strains, but not by other strains. Presumably, the effect of any antibiotic on the susceptible 'host' hyphae was enhanced by the intimate contact of the Trichoderma hyphae. Orynbaev (1963) studied cytochemical changes in R. solani induced by Trichoderma in dual culture experiments. He observed premature vacuolation, the cytoplasm becoming granular and finally disintegrating. Also, sclerotia were formed by young hyphae of R. solani in the dual cultures, but not in pure cultures of the same age. This induction of sclerotia was observed frequently in the present investigation. It was evident from these experiments that the hyphal interaction between Trichoderma isolates and other fungi is not the same phenomenon as the hyphal interference shown by coprophilous fungi (Ikediugwu, 1969; Ikediugwu & Webster 1970a, b; Ikediugwu, Dennis & Webster, 1970). Hyphal interference occurred only on contact between hyphae, and only contacted cells were affected. Coiling by Trichoderma hyphae was also far more frequent than the hyphal interference between Coprinus heptemerus and other coprophilous fungi, and between Peniophora gigantea and F. annosus. Experiments were carried out with plastic threads ofsimilar diameter to hyphae of P. ultimum. These indicated that the coiling was not merely a contact stimulus. The Trichoderma hyphae never coiled around the threads, but generally grew over them. When Trichoderma hyphae followed the threads, they did so in a rather straight course and not in the wavy manner observed on fungal hyphae. Signs of hyphal penetration were seldom seen, and were restricted to tests with P. ultimum (PI. 23, Fig. 6). Isolates from the T. hareianum and T. viride species groups were observed to penetrate Pythium hyphae occasionally. Antibiotic production was not detected in these isolates of
Hyphal interaction. C. Dennis and J. Webster
367
T. harzianum by Dennis & Webster (1971 a, b), so that it is unlikely that antibiotics were directly responsible for promoting penetration. Amongst the six test fungi used routinely, Pythium ultimum Trow. represented the Oomycetes, a group characterized by having cellulose rather than chitin in their cell walls. Two other Oomycete species, Phytophthora cactorum Leb. & Cohn (Schroet.) and Phytophthora erythroseptica Pethyb. were also tested in dual culture. Penetration similar to that of P. ultimum was shown by two isolates of T. viride, but again infrequently. It is known that Trichoderma isolates often have strong cellulolytic activity (Iwasaki, Hayashi & Funatsu, 1964; Li, Flora & King, 1965; Selby & Maitland, 1967) and this property may enable Trichoderma hyphae to penetrate hyphae of Oomycetes. However, Trichoderma species appear to possess the necessary enzymes to attack fungal walls composed of chitin, glucan and xylan polymers, as well as cellulose (Nomura, Yasui, Kiyooka & Kobayashi, 1968; Toyama & Ogawa, 1968; Jones & Watson, 1969). Durrell (1966), using more refined techniques, showed that penetration by Trichoderma hyphae is not limited to the Oomycetes; several members of the Mucorales and also R. solani were observed to be penetrated by Trichoderma hyphae. The size of the' host' hyphae could have provided a physical barrier to penetration: if the hyphae of Trichoderma were wider than those of the host hyphae then penetration would be physically impossible. Durrell reported members of the Phycomycetes and R. solani, which have wide hyphae, to be particularly susceptible to penetration. The' host' fungus generally ceased growth soon after contact, whether or not the coiling fungus was an antibiotic-producing isolate. The Trichoderma isolates continued to grow and eventually covered the whole plate. Attempts to reisolate the test fungi from the zone of interaction between the two fungi generally failed, but very occasionally it proved possible to reisolate F. oxysporum. It has been mentioned that the hyphae of some fungi burst, resulting in the release of cytoplasm from the hyphae, when grown in dual culture with certain Trichoderma isolates. Because of this leakage of cytoplasm, the Trichoderma isolates may have obtained nutrients from the other fungi. The action of cyclic peptide antibiotics (e.g. tyrocidin, gramicidin) is characterized by a rapid leakage of low molecular weight material (including inorganic ions, amino acids, purines and pyrimidines) from bacteria (see Newton, 1965). Alamethicine is a cyclic polypeptide antibiotic produced by a Trichoderma isolate, which might induce such leakage of materials from fungal hyphae. Thus, by the action of antibiotics, by the activity of enzyme systems, or by the complementary action of both, it is feasible that Trichoderma isolates can gain nutrients from other fungi. The type of hyphaI interaction investigated would enhance the effect of such substances.
Transactions British Mycological Society REFERENCES
AYTOUN, R. S. C. (1953). The genus Trichoderma: its relationship with Armillaria mellea (Vahl ex Fries) Quel, and Polyporus scluoeinitzii. Transactions of theBritishMycological Society 36, 99-114. CHI, C. C. (1960). Effects of Streptomyces and Trichoderma on Fusarium. Phytopathology 50, 631 (abstr.), DENNIS, C. & WEBSTER,]. (1971 a). Antagonistic properties of species groups of Trichoderma. I. Production of non-volatile antibiotics. Transactions of the BritishMycological Society 57, 25-39· DENNIS, C. & WEBSTER,]. (1971 b). Antagonistic properties of species groups of T richoderma. II. Production of volatile antibiotics. Transactions of the British Mycological Society 57, 4 1-48. DURRELL, L. W. (1966). Hyphal invasion by Trichoderma viride. Mycopathologia et Mycologia Applicata 35, 138-144. IKEDlUGwu, F. E. O. (1969). Antagonism between Coprinus heptemerus and other coprophilous fungi. Ph.D. Thesis. University of Sheffield. IKEDlUGwu, F. E. 0., DENNIS, C. & WEBSTER,]. (1970). Hyphal interference by Peniophora gigantea against Fomes annosus. Transactions of the British Mycological Society 54, 307-308. IKEDlUGWU, F. E. O. & WEBSTER,]. (1970a). An experimental analysis of the coprophilous fungus succession. II. Antagonism between Coprinus heptemerus and other coprophilous fungi. Transactions of the British Mycological Society 54, 181-204. IKEDlUGwu, F. E. O. & WEBSTER,]. (1970b). An experimental analysis of the coprophilous fungus succession. III. Hyphal interference in a range of coprophilous fungi. Transactions of the British Mycological Society 54, 2°5-210. IWASAKI, T., HAYASHI, K. & FUNATSU, M. (1964). Purification and characterisation of two types of cellulase from Trichoderma koningii. Joumal of Biochemistry, Tokyo 55, 2°9-212. JONES, D. & WATSON, D. (1969). Parasitism and lysis by soil fungi of Sclerotinia sclerotiorum (Lib.) de Bary, a phytopathogenic fungus. Nature, London 224,287-288. KOMATSU, M. (1968). Trichoderma viride as an antagonist of the wood-inhabiting Hymenomycetes. VIII. The antibiotic activity against the mycelial growth of Lentinus edodes (Berk.) Sing. of three genera, Trichoderma, Pachybasium, Gliocladium and other sterile forms. Report of the Tottori Mycological Institute (Japan), pp. 29-42. LI, L. M., FLORA, R. M. & KING, K. W. (1965). Individual roles of cellulase components derived from Trichoderma viride. Archives ofBiochemistry and Biophysics :III, 439-447. NEWTON, B. A. (1965). Mechanisms of antibiotic action. AnnualReview ofMicrobiology 19, 20g-24°. NOMURA, K., YASUI, T., K!YOOKA, S. & KOBAYASHI, T. (1968). Xylanases of Trichoderma viride. Some properties of enzyme reactions and a preliminary experiment of xylan hydrolysis. Journal ofFermentation Technology 46, 634-640. ORYNBAEV, S. (1963). Cytochemical changes in Rhizoctonia solani as a result of the action of metabolic products of Trichoderma. Pochvennaya i sel'skokhozyaistvennaya Mikrobioliya Akademya Nauk Uzbekskii SSR, Tashkent, 286-292. (Engl. trans.) RISHBETH,]. (1950). Observations on the biology of Fomes annosus, with particular reference to East Anglian pine plantations. I. The outbreaks of disease and ecological status of the fungus. Annals ofBotany :14, 365-385. SELBY, K. & MAITLAND, C. C. (1967). Components of Trichoderma viride cellulase. Archives of Biochemistry and Biophysics 118, 254-257. SLAGG, C. M. & FELLOWS, H. (1947). Effects of certain soil fungi and their by products on Ophiobolus graminis. Joumal ofAgricultural Research 75, 279-293. TOYAMA, N. & OGAWA, K. (1968). Purification and properties of Trichoderma viride mycolytic enzymes. Joumal ofFermentation Technology 46, 626-633. WEBSTER,]. & LOMAS, N. (1964)' Does Trichoderma viride produce gliotoxin and viridin? Transactions ofthe British Mycological Society 47, 535-540. WEINDLING, R. (1932). Trichoderma lignorum as a parasite of other soil fungi. Phytopathology 22,837-845·
Trans. Br. my col. Soc.
Vol. 57.
Plate
22
3
(Facing p. 3G8)
Trans. Br. mycol. Soc.
Vol. 57. Plate 23
6
Hypha! interaction. C. Dennis andJ. Webster EXPLANATION OF PLATES 22 AND 23 PLATE 22
Figs. I, 2. Trichoderma polysporum coiling tightly around Rhizoctonia solani. x 1200. Figs. 3, 4. T. polysporum (same isolate) coiling at a narrower angle to hyphae of R. solani. x
1200.
PLATE 23
Fig. 5. Hypha of Trichoderma viride (arrowed) following a hypha of Pythium ultimum (stained with cotton blue in lactic acid). x 600. Fig. 6. Hyphae of P. ultimum (arrowed), with T. viride (same isolate) growing intracellularly. x 600.
(Accepted for publication 25 September 1970)
Vol. 57, Part 3 Trans. Br. mycol. Soc. 57 (3), 363-369 (1971) Printed in GreatBritain
ANTAGONISTIC PROPERTIES OF SPECIES-GROUPS OF TRICHODERMA III. HYPHAL INTERACTION
By C. DENNIS*
AND ].
WEBSTER t
Department of Botany, University oj Sheffield (With Plates 22 and 23) When grown in dual culture, hyphae of the majority of Trichoderma isolates coiled around hyphae of different test fungi. Penetration of hyphae by Trichoderma hyphae seldom occurred. The importance of antibiotics and extracellular enzymes during hyphal interaction is discussed.
Weindling (1932) reported coiling by hyphae of Trichoderma species around hyphae of certain other fungi. Webster & Lomas (1964) showed that Weindling's strains were not Trichoderma species, but belonged to the genus Gliocladium. Other workers, however, have reported hyphal interactions by isolates considered to be true Trichoderma species (Table I). Experiments have been carried out to test the ability of a number of isolates from the different species-groups of Trichoderma to coil around or to penetrate hyphae of other fungi, including a basidiomycete, an ascomycete, a zygomycete, an oomycete and several fungi imperfecti, The Trichoderma isolates and the test fungi were those used in studies on antibiotic production by Trichoderma isolates (Dennis & Webster, 1971 a, b). MATERIALS AND METHODS
Trichoderma isolates were grown in dual culture with each of six test fungi, on 2 % malt extract agar (15 ml per dish). Except for Fames annosus (Fr.) Cooke, both fungi were inoculated at the same time. Disks (6 mm) cut from the margins of vigorously growing cultures were placed 2-3 cm apart. Fomes annosus, because of its comparatively slow growth rate, was inoculated 2 days before the Trichoderma isolates; the inoculum disk of each Trichoderma isolate was then placed about 2 em from the edge of the F. annosus colony. The plates were incubated under a bank oflights at 2225°C. Direct inspection by light and phase-contrast microscopy was carried out before and after contact of the hyphae of the two fungi. For high
* Present address: Agricultural Research Council, Food Research Institute, Colney Lane, Norwich, NOR 7oF. t Present address: Department of Biological Sciences, Prince of Wales Road, Exeter, Devon. Vol. 56, Part 3 was issued 24 June 1971 MYC
57
364
Transactions British Mycological Society
magnifications, a cover-slip was carefully placed over the area to be examined and a small volume of water was pipetted underneath the cover-slip. Sometimes the hyphae were stained with cotton blue in lactic acid before being examined. Table
I.
Report ofinteractions between hyphae ofTrichoderma species andhyphae ofother fungi
Susceptible fungus
Gaumannomyces graminis Armillaria mellea Polyporus schweinitzii Fusarium osysporum F. solani F. roseum Rhizopus oryzae Actinomueor repens Phycomyces nitens Circinella musae Mucor uarians M. spinosus zYgorhynchus vuilleminii Syncephalastrum raeemosum Rhizoctonia solani Leniinus edodes
Type of hyphal interaction
Author
Penetration Slight c~iling and penetratIon } (only at acid pH)
SIagg & Fellows (1947)
} Coiling
Chi (1960)
Aytoun (1953)
Penetration
Durrell (1966)
Coiling
Komatsu (1968)
RESULTS
Survey ofcoiling action ofTrichoderma isolates The results ofa survey ofhyphaI coiling by eighty isolates are summarized in Table 2. No attempt was made to express the results quantitatively, because of the diversity of growth pattern of the different isolates. It also proved impossible to select a standard time at which to examine the cultures for interaction. Some Trichoderma isolates coiled around the 'host' hyphae as soon as the two colonies made contact. With other isolates, coiling did not occur until the Trichoderma hyphae had penetrated far into the 'host' fungus colony. Of the 80 Trichoderma isolates tested, only ten did not show coiling. Most isolates coiled around hyphae of all the test fungi, but some did not interact with hyphae of Fusarium oxysporum Schlecht. ex Fr., especially isolates belonging to the T. polysporum (Link ex Pers.) Rifai and T. piluliferum Webster & Rifai species groups. Some variation in coiling behaviour was observed between isolates of the same species groups, though to a lesser extent than variation in antibiotic production (Dennis & Webster, 1971 a, b). Isolates of T. harzianium Rifai which showed no apparent ability to produce antibiotics were capable of coiling around other hyphae. In addition, isolates of the T. polysporum and T. piluliferum groups, which produced non-volatile antibiotics (Dennis & Webster, 197oa), did not coil around hyphae of other fungi as severely as some isolates which showed no antibiotic activity. This was also true of antibioticproducing isolates from other species groups.
Hyphal interaction. C. Dennis and J. Webster
365
In dual culture experiments on malt extract agar with R. solani as the test fungus no apparent difference was observed between hyphal interaction at pH 4'0 and 6'5. The amount of coiling by Trichoderma hyphae appeared to be the same at both pH levels. These observations contrast with those of Aytoun (1953) who observed coiling by Trichoderma hyphae around hyphae of Armillaria mellea and Polyporus schmeinitzii to be more intense at pH 3'4 than at pH 5'1 or pH 7'0. Table
2.
Summary of hyphal interaction experiments
No. of Rhizoctonia isolates solani
T. piluliferum T. polysporum
3 13 5 3
T. koningii
4 4
Fusarium Pyronema oxysporum domesticum
Mucor hiemalis
Pythium ultimum
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+
+ +
+ +
+ +
+ + +
+ +
+ +
+ + +
+ + +
+ + +
+ +
+
+
+ +
+ +
+ +
+ + + +
+ + + +
+ + +
+ + + +
+ + + +
+ + + +
I
T. hamatum
Fomes annosus
I
I
T. harzianum T. aureooiride
5 9 I
2
T. pseudokoningii T. longihrachiatum T. viride
4 2
17 4 I
Key:
+ denotes coiling by
Trichoderma hyphae; - denotes no coiling by Trichoderma hyphae.
Observations before contact between hyphae of the test fungi and Trichoderma hyphae Some isolates of Trichoderma, already shown to produce non-volatile antibiotics, caused inhibition of growth of the test fungus colony long before contact between the hyphae occurred, often when the two colonies were still 3-5 mm apart. The hyphae of the test fungi nearest to the Trichoderma colony showed the morphological changes reported by Dennis & Webster (1971 a). Vacuolation and coagulation of cytoplasm were especially noticeable in hyphae of F. annosus and R. solani. Bursting of Fomes and Rhizoctonia hyphae was occasionally observed to occur before contact with the Trichoderma hyphae. Rishbeth (1950) reported similar morphological changes when Trichoderma isolates were grown in dual culture with F. annosus isolates. Recently, Komatsu (1968) reported that hyphae of Lentinus edodes showed similar effects when placed in culture filtrates of Trichoderma isolates. Such observations provide supporting evidence that Trichoderma isolates produce diffusible antibiotics, which are active in advance of the hyphae. Both volatile and non-volatile antibiotics could be effective in the case of some isolates.
Transactions British Mycological Soct"ety Observations after contact between hyphae of Trichoderma isolates and oftest fungi The types of hyphal interaction observed are shown in PI. 22. Trichoderma hyphae frequently coiled around aerial hyphae, including sporangiophores, and hyphae growing on the agar surface. No coiling around subsurface hyphae was observed. The coiling could be separated into three types: (I) Numerous short branches were produced by the main hyphae, and each branch coiled tightly about the susceptible hyphae (PI. 22, figs. 1,2). (2) The main hyphae coiled about the susceptible hyphae. In this case the coils formed at a narrower angle to the' host' hypha and there were fewer coils per unit length of hypha (PI. 22, figs. 3,4), (3) The Trichoderma hyphae followed the' host' hyphae, and at intervals the Trichoderma hyphae produced a short branch which coiled around the 'host' hypha (PI. 23, fig. 5). When this occurred, the Trichoderma hyphae grew in a zig-zag or wavy manner along the surface of the 'host' hyphae. All three types of coiling were usually observed when any Trichoderma isolate was grown in dual culture with a susceptible fungus. Vacuolation, coagulation of cytoplasm and sometimes bursting of hyphae (F. annosus and R. solani) were induced by the antibiotic-producing strains, but not by other strains. Presumably, the effect of any antibiotic on the susceptible 'host' hyphae was enhanced by the intimate contact of the Trichoderma hyphae. Orynbaev (1963) studied cytochemical changes in R. solani induced by Trichoderma in dual culture experiments. He observed premature vacuolation, the cytoplasm becoming granular and finally disintegrating. Also, sclerotia were formed by young hyphae of R. solani in the dual cultures, but not in pure cultures of the same age. This induction of sclerotia was observed frequently in the present investigation. It was evident from these experiments that the hyphal interaction between Trichoderma isolates and other fungi is not the same phenomenon as the hyphal interference shown by coprophilous fungi (Ikediugwu, 1969; Ikediugwu & Webster 1970a, b; Ikediugwu, Dennis & Webster, 1970). Hyphal interference occurred only on contact between hyphae, and only contacted cells were affected. Coiling by Trichoderma hyphae was also far more frequent than the hyphal interference between Coprinus heptemerus and other coprophilous fungi, and between Peniophora gigantea and F. annosus. Experiments were carried out with plastic threads ofsimilar diameter to hyphae of P. ultimum. These indicated that the coiling was not merely a contact stimulus. The Trichoderma hyphae never coiled around the threads, but generally grew over them. When Trichoderma hyphae followed the threads, they did so in a rather straight course and not in the wavy manner observed on fungal hyphae. Signs of hyphal penetration were seldom seen, and were restricted to tests with P. ultimum (PI. 23, Fig. 6). Isolates from the T. hareianum and T. viride species groups were observed to penetrate Pythium hyphae occasionally. Antibiotic production was not detected in these isolates of
Hyphal interaction. C. Dennis and J. Webster
367
T. harzianum by Dennis & Webster (1971 a, b), so that it is unlikely that antibiotics were directly responsible for promoting penetration. Amongst the six test fungi used routinely, Pythium ultimum Trow. represented the Oomycetes, a group characterized by having cellulose rather than chitin in their cell walls. Two other Oomycete species, Phytophthora cactorum Leb. & Cohn (Schroet.) and Phytophthora erythroseptica Pethyb. were also tested in dual culture. Penetration similar to that of P. ultimum was shown by two isolates of T. viride, but again infrequently. It is known that Trichoderma isolates often have strong cellulolytic activity (Iwasaki, Hayashi & Funatsu, 1964; Li, Flora & King, 1965; Selby & Maitland, 1967) and this property may enable Trichoderma hyphae to penetrate hyphae of Oomycetes. However, Trichoderma species appear to possess the necessary enzymes to attack fungal walls composed of chitin, glucan and xylan polymers, as well as cellulose (Nomura, Yasui, Kiyooka & Kobayashi, 1968; Toyama & Ogawa, 1968; Jones & Watson, 1969). Durrell (1966), using more refined techniques, showed that penetration by Trichoderma hyphae is not limited to the Oomycetes; several members of the Mucorales and also R. solani were observed to be penetrated by Trichoderma hyphae. The size of the' host' hyphae could have provided a physical barrier to penetration: if the hyphae of Trichoderma were wider than those of the host hyphae then penetration would be physically impossible. Durrell reported members of the Phycomycetes and R. solani, which have wide hyphae, to be particularly susceptible to penetration. The' host' fungus generally ceased growth soon after contact, whether or not the coiling fungus was an antibiotic-producing isolate. The Trichoderma isolates continued to grow and eventually covered the whole plate. Attempts to reisolate the test fungi from the zone of interaction between the two fungi generally failed, but very occasionally it proved possible to reisolate F. oxysporum. It has been mentioned that the hyphae of some fungi burst, resulting in the release of cytoplasm from the hyphae, when grown in dual culture with certain Trichoderma isolates. Because of this leakage of cytoplasm, the Trichoderma isolates may have obtained nutrients from the other fungi. The action of cyclic peptide antibiotics (e.g. tyrocidin, gramicidin) is characterized by a rapid leakage of low molecular weight material (including inorganic ions, amino acids, purines and pyrimidines) from bacteria (see Newton, 1965). Alamethicine is a cyclic polypeptide antibiotic produced by a Trichoderma isolate, which might induce such leakage of materials from fungal hyphae. Thus, by the action of antibiotics, by the activity of enzyme systems, or by the complementary action of both, it is feasible that Trichoderma isolates can gain nutrients from other fungi. The type of hyphaI interaction investigated would enhance the effect of such substances.
Transactions British Mycological Society REFERENCES
AYTOUN, R. S. C. (1953). The genus Trichoderma: its relationship with Armillaria mellea (Vahl ex Fries) Quel, and Polyporus scluoeinitzii. Transactions of theBritishMycological Society 36, 99-114. CHI, C. C. (1960). Effects of Streptomyces and Trichoderma on Fusarium. Phytopathology 50, 631 (abstr.), DENNIS, C. & WEBSTER,]. (1971 a). Antagonistic properties of species groups of Trichoderma. I. Production of non-volatile antibiotics. Transactions of the BritishMycological Society 57, 25-39· DENNIS, C. & WEBSTER,]. (1971 b). Antagonistic properties of species groups of T richoderma. II. Production of volatile antibiotics. Transactions of the British Mycological Society 57, 4 1-48. DURRELL, L. W. (1966). Hyphal invasion by Trichoderma viride. Mycopathologia et Mycologia Applicata 35, 138-144. IKEDlUGwu, F. E. O. (1969). Antagonism between Coprinus heptemerus and other coprophilous fungi. Ph.D. Thesis. University of Sheffield. IKEDlUGwu, F. E. 0., DENNIS, C. & WEBSTER,]. (1970). Hyphal interference by Peniophora gigantea against Fomes annosus. Transactions of the British Mycological Society 54, 307-308. IKEDlUGWU, F. E. O. & WEBSTER,]. (1970a). An experimental analysis of the coprophilous fungus succession. II. Antagonism between Coprinus heptemerus and other coprophilous fungi. Transactions of the British Mycological Society 54, 181-204. IKEDlUGwu, F. E. O. & WEBSTER,]. (1970b). An experimental analysis of the coprophilous fungus succession. III. Hyphal interference in a range of coprophilous fungi. Transactions of the British Mycological Society 54, 2°5-210. IWASAKI, T., HAYASHI, K. & FUNATSU, M. (1964). Purification and characterisation of two types of cellulase from Trichoderma koningii. Joumal of Biochemistry, Tokyo 55, 2°9-212. JONES, D. & WATSON, D. (1969). Parasitism and lysis by soil fungi of Sclerotinia sclerotiorum (Lib.) de Bary, a phytopathogenic fungus. Nature, London 224,287-288. KOMATSU, M. (1968). Trichoderma viride as an antagonist of the wood-inhabiting Hymenomycetes. VIII. The antibiotic activity against the mycelial growth of Lentinus edodes (Berk.) Sing. of three genera, Trichoderma, Pachybasium, Gliocladium and other sterile forms. Report of the Tottori Mycological Institute (Japan), pp. 29-42. LI, L. M., FLORA, R. M. & KING, K. W. (1965). Individual roles of cellulase components derived from Trichoderma viride. Archives ofBiochemistry and Biophysics :III, 439-447. NEWTON, B. A. (1965). Mechanisms of antibiotic action. AnnualReview ofMicrobiology 19, 20g-24°. NOMURA, K., YASUI, T., K!YOOKA, S. & KOBAYASHI, T. (1968). Xylanases of Trichoderma viride. Some properties of enzyme reactions and a preliminary experiment of xylan hydrolysis. Journal ofFermentation Technology 46, 634-640. ORYNBAEV, S. (1963). Cytochemical changes in Rhizoctonia solani as a result of the action of metabolic products of Trichoderma. Pochvennaya i sel'skokhozyaistvennaya Mikrobioliya Akademya Nauk Uzbekskii SSR, Tashkent, 286-292. (Engl. trans.) RISHBETH,]. (1950). Observations on the biology of Fomes annosus, with particular reference to East Anglian pine plantations. I. The outbreaks of disease and ecological status of the fungus. Annals ofBotany :14, 365-385. SELBY, K. & MAITLAND, C. C. (1967). Components of Trichoderma viride cellulase. Archives of Biochemistry and Biophysics 118, 254-257. SLAGG, C. M. & FELLOWS, H. (1947). Effects of certain soil fungi and their by products on Ophiobolus graminis. Joumal ofAgricultural Research 75, 279-293. TOYAMA, N. & OGAWA, K. (1968). Purification and properties of Trichoderma viride mycolytic enzymes. Joumal ofFermentation Technology 46, 626-633. WEBSTER,]. & LOMAS, N. (1964)' Does Trichoderma viride produce gliotoxin and viridin? Transactions ofthe British Mycological Society 47, 535-540. WEINDLING, R. (1932). Trichoderma lignorum as a parasite of other soil fungi. Phytopathology 22,837-845·
Trans. Br. my col. Soc.
Vol. 57.
Plate
22
3
(Facing p. 3G8)
Trans. Br. mycol. Soc.
Vol. 57. Plate 23
6
Hypha! interaction. C. Dennis andJ. Webster EXPLANATION OF PLATES 22 AND 23 PLATE 22
Figs. I, 2. Trichoderma polysporum coiling tightly around Rhizoctonia solani. x 1200. Figs. 3, 4. T. polysporum (same isolate) coiling at a narrower angle to hyphae of R. solani. x
1200.
PLATE 23
Fig. 5. Hypha of Trichoderma viride (arrowed) following a hypha of Pythium ultimum (stained with cotton blue in lactic acid). x 600. Fig. 6. Hyphae of P. ultimum (arrowed), with T. viride (same isolate) growing intracellularly. x 600.
(Accepted for publication 25 September 1970)