THE EFFECT OF VARIOUS TREATMENTS OF CULTURE MEDIUM UPON THE GROWTH OF CERTAIN FUNGAL ANTAGONISTS Leila-Riitta Erviö Department of Plant Pathology, University of Helsinki Received December 18, 1964 Many soil fungi have been found to be antagonists of Sclerotinia trifoliorum Erikss., S. sclerotiorum (Lib.) Bref. and Botrytis cinerea Pers. (1, 2,9, 10). Since external conditions have an influence on these antagonists (3, 4,5) and thus indirectly affect theirability to suppress the occurrence of harmful parasitic fungi of cultivated plants, this study dealt with the effect of various treatments of culture medium upon the growth of such soil fungi, whose antagonism is based on their capacity to destroy the sclerotia of their hosts. Materials and methods The following fungal antagonists, which have been found to be antagonists of 5. trifoliorum, S. sclerotiorum and B. cinerea, were used in these studies: Acrostalagmus roseus Banier, isolate I (cf. 10). Isolated from a sclerotium of S. trifoliorum in 1954. A. roseus, isolate 11. Isolated from a sclerotium of 5. trifoliorum in 1963. Coniothyrium minitans, Campb., isolate I. Isolated from foreign red clover seed in 1962. C. minitans, isolate 11. Isolated from a sclerotium of S. trifoliorum in 1962. Fusarium avenaceum (Fries) Sacc. Isolated from carnation at the Department of Plant Pathology, Tikkurila, in 1955. Gliocladium roseum (Link) Thom. (cf. 7). Isolated from a sclerotium of 5 trifoliorum in 1955. Gliocladium sp. Isolated from a sclerotium of S. trifoliorum in 1955. Mucor hiemalis Wehm. (cf. 7). Isolated from a sclerotium of Claviceps purpurea in 1959. 85 M. spinosus Tiegh. Isolated from Henneberg agar medium treated with pentachloronitrobenzene in 1962. Rhizopus nigricans Ehrenb. Isolated from dead clover in 1962. Sporotrichum carnis Brooks & Hansf. Isolated from a sclerotium of S. trifo- liormn in 1962. Trichoderma viride (Tode) Hartz (cf. 7). Isolated from a sclerotium of S. trifoliorum in 1955. Trichothecium roseum Link. Isolated from mycelia of S. trifoliorum in potato stem in 1963. The fungi were cultured on a basic medium consisting of Henneberg agar (10 g peptone, 2 g NH 4 H 2P0 4 , 2 g KN0 3 , 0.5 g MgS04 , 0.1 g CaCl 2 , 100 g glucose, 20 g agar and 1000 ml distilled water). In the different trials the following additions were made to the basic medium: Potassium was added at a rate of 200 g/m2 in the form of 50 % potassium chloride (KCI) or as potassium sulphate (K 4SO 4). In the tests with pentachloronitrobenzene (PCNB), Brassicol dust at rates of 50 and 100 g/m2 was applied to the solidified surface of the agar in the Petri dishes. The pH of the Henneberg agar normally 6.7 was made more acidic b} r adding 10 % hydrogen chloride (HCI) or more basic by adding 10 % potassium hydroxide (KOH). In addition to the above treatments, the effect of oxygen deficiency on the growth of the fungi was studied by sealing the Petri dishes with modelling clay after inoculation of the fungi. The control dishes were left unsealed. The fungi were transferred with an inoculating needle to the centre of the agar medium in the Petri dish, and the size of the colony was measured from time to time. In determining the effect of PCNB, the vigour of the fungal growth was estimated visually on a scale of o—3.0—3. All the trials were carried out at room tempera- ture in the laboratory. The control organism used was 5. trifoliorum which had been isolated in the autumn of 1960 from a first-year clover ley at Viik. Results Effect of potassium. The addition of potassium either as chloride or as sulphate had no appreciable effect on the growth of the fungi (Fig. 1). The differences in colony size of the same isolate on the differently treated plates were either very small or completely lacking. Effect of pentachloronitrobenzene (PCNB). The fungi in these tests were cultivated on Henneberg agar which had been treated with Brassicol dust, and their growth was compared with that of fungi on untreated plates. It was found that the different isolates responded differently to PCNB. All of them grew more poorly on the treated than on the control medium on which their growth 86 was rated as »3» on the scale. In comparison with that the fungi grew on the PCNB- treated medium as seen below: S. trifoliorum O Gliocladium sp. 2 A. roseus I 1 M. hiemalis I A. roseus II 2 M. spinosus 2 C. minitans I 1 Ä. nigricans O C. minitans II 1 S. ca»-H!s O The species 5. trifoliorum, R. nigricans and S. carnis were most sensitive to PCNB since their development ceased completely. In contrast, such parasites of S. trifo- liorum as A. roseus, Gliocladium sp. and M. spinosus were considerably more resistant to the chemical. The tolerance of A. roseus isolate II to PCNB was further studied by culturing it for 4 weeks on Henneberg agar to which Brassicol dust had been added at rates of 50 and 100 g/m2 . According to the results shown below, the fungus grew relatively well at both consentrations. Rate of PCNB Diameter of colony, cm 0 9.4 50 g/m2 7.0 100 » 5.9 Effect of pH. It is generally known that fungi can exist over a wide range of pH values (6, 8). In the present tests, S. trifoliorum, A. roseus I, Gliocladium sp., T. roseum and T. viride were cultivated on Henneberg agar whose pH varied by 1-unit intervals from 2 to 9. It was found that all the fungi grew when the pH was between 4 and 9. The pH optimum of 5. trifoliorum was 4—6 while Gliocladium sp. and A. roseus favoured a more alkaline substrate. T. viride was the only fungus which grew at pH 2. In this highly acidic medium, however, the appearance of the colony Fig. 1. The effect of potassium on the growth of some fungi. The trial period was 14 days. 87 differed from normal: it was slimy and pale-coloured, and no conidia were formed. At pH B—98—9 the normal green colour of this fungus changed to yellow; abundant formation of conidia was not inhibited. Effect of oxygen deficiency. A deficiency in the oxygen supply slowed the development of S. trifoliorum, A. roseus I and 11, M. hiemalis and T. viride. Toward the end of the 4-week trial period their growdh stopped completely. In such dishes no conidia were formed, nor did S. trifoliorum produce sclerotia. However, even four months after the end of the trial, all the fungi in the sealed dishes were still viable. Conclusions The addition of potassium chloride and potassium sulphate at a rate of 200 g/m2 to the culture medium had no effect on the growth of Sclerotinia trifoliorum, Acrostalagmus roseus I and 11, Coniothyrium minitans, Gliocladium sp., Mucor hiemalis, M. spinosus, Sporotrichum carnis and Trichothecium roseum. Treatment of the culture medium with PCNB suppressed to some extent the development of A. roseus I and 11, Gliocladium sp., C. minitans I and 11, Mucor hiemalis and M. spinosus. The fungal species S. trifoliorum, Rhizopus nigricans and Sporotrichum carnis did not grow at all on PCNB-treated medium. S. trifoliorum, A. roseus I, Gliocladium sp., T. viride and T. roseum were able to grow in the pH range of 4—9. When the pH of the medium was 2, only T. viride grew. Oxygen deficiency suppressed the growth of S. trifoliorum, and the growth and the conidia formation of A. roseus I and 11, M. hiemalis and T. viride. REFERENCES (1) Erviö, L-R. Certain parasites of fungal sclerotia. Maatal.tiet. aikak. 1965, painossa. (2) —» Halkilahti, A-M. & Pohjakallio, O. 1964. The survival in the soil of sclerotia of two Sclerotinia species and their ability to form mycelia. Repr. from Advancing Frontiers of Plant Sci. 8: 121-134. (3) Garret, S. 1). 1956. Biology of root infecting fungi. 292 p. Cambridge. (4) Halkilahti, A-M 1962. The survival of sclerotia of Sclerotinia trifoliorum Erikss. on the soil and the occurence of clover rot in various years. Maatal.tiet. aikak. 34; 154 161. (5) — »— 1964. The significance of soil microorganisms as a limiting factor in infection of clover by Sclerotinia trifoliorum Erikss. at different times of the year. Ibid. 36: 120 134. (6) Jensen, H. L. 1931. The fungus flora of the soil. Soil Sci. 31: 123 158. (7) Karhuvaara, L. 1960. On the parasites of the sclerotia of some fungi. Acta Agr. Scand. 10; 127 -134. (8) Kaufman, D. D. & Williams, L, E. 1964. Effect of mineral fertilization and soil reaction on soil fungi. Phytopath. 54: 134 139. (9) Makkonen R. & Pohjakallio, O. 1960. On the parasites attacking the sclerotia of some fungi pathogenic to higher plants and on the resistance of these sclerotia to their parasites. Acta Agr. Scand. 10: 105 126. (9) Pohjakallio, O. 1960. Untersuchungen iiber Antagonisten der Erreger von Pflanzenkrankheiten. Verhandl. IV Intern. Pflanzenschutz. Kongresses Hamburg 1957 Bd. 2:1541 1543. (10) —» Salonen, A., Ruokola, A-L. & Ikäheimo, K. 1956. On a mucous mold fungus Acrosta- lagmus roseus Bainier, as antagonist to some plant pathogens. Acta Agr. Scand. 3: 53 60. 88 SELOSTUS: KASVUALUSTAN ERILAISTEN KÄSITTELYJEN VAIKUTUKSESTA ERÄIDEN TUHOSIE- NIEN ANTAGONISTIEN KASVUUN Leila-Riitta Erviö Yliopiston kasvipatologian laitos, Helsinki Tutkittaessa kasvualustan erilaisten käsittelyjen vaikutusta eräiden maasienien kasvuun, todet- tiin, että kaliumkloridin (KCI) ja kaliumsulfaatin (K2 SO 4 ) 200 g/m2 vastaava lisäys ravintoalustaan ei vaikuttanut Sclerotinia trifoliorumin, Acrostalagmus roseus I:n ja II:n, Coniothyrium minitansin, Glio- cladium sp:n, Mucor hiemalisen, M. spinosusen, Sporotrichum carnisen ja Trichothecium roseum in kas- vuun. Ravintoalustan käsittely PCNBIIä heikensi jonkin verran A. roseus I;n ja II:n, Gliocladium sp;n, C. minitans I:n ja II:n, Mucor hiemalisen sekä M. spinosusen kasvua. Sensijaan S. trifoliorum, Rhizopus nigricans ja Sporotrichum carnis eivät kasvaneet olleenkaan PCNBdlä käsitellyllä ravinto- alustalla. Sienien S. trifoliorum, A. roseus I, Gliocladium sp., T. viride ja T. roseum todettim menestyvän pH- rajoissa 4—9. Ravintoalustan pH-arvon ollessa 2 kasvoi vain T. viride. Hapenpuute hidasti sienien S. trifoliorum, A. roseus I ja 11, M. hiemalis ja T. viride kasvua ja kuromanmuodostusta.