Maataloustieteellinen A ikakauskirja Vol. 63: 435—441, 1991 Scanning electron microscopy of hyphal interaction between Streptomyces griseoviridis and some plant pathogenic fungi EEVA TAPIO and ARJA POHTO-LAHDENPERA Department of Plant Pathology, University of Helsinki, SF-00710 Helsinki Abstract. The interaction between Streptomycesgriseoviridis and the pathogens Alternaria brassicicola, Botrytis cinerea, Fusarium oxysporum, Mycocentrospora acerina, Rhizoctonia solani and Sclerotinia sclerotiorum was studied by SEM both on autoclaved seeds and living seedlings of turnip rape and carrot and the fungi Phomopsis sclerotioides and Pythium ulti- mum on cucumber seedlings. The samples were prepared by the standard method for exami- nation by scanning electron microscope. The hyperparasitism of S. griseoviridis was clearly shown. S. griseoviridis tightly wound around Alternaria conidia and Sclerotinia hyphae, even- tually disintegrating them. It grew along the hyphae of B. cinerea, P. sclerotioides and M. acerina, dissolving them. The hypha of F. oxysporum seemed to be slightly affected, and its conidia not at all. The hyperparasite grew only loosely on the hypha of R. solani and on the mycelium and oogonia of Pythium which seemed not to sustain much injury. Index words: Hyperparasitism, biological control, Streptomyces griseoviridis, plant pathogenic fungi Introduction Streptomyces griseoviridis Anderson et al., isolated from Finnish light coloured Sphag- num peat, has been reported to be antagonis- tic to the plant pathogens Alternaria brassici- cola (Schw.) Wiltshire, Botrytis cinerea Pers.: Fr., Fusarium avenaceum (Fr.: Fr.) Sacc., F. culmorum (W.G. Sm.) Sacc., F. oxysporum Schlecht. f. sp. dianthi (Prill. & Dol.) Snyd. & Hans., Pythium debaryanum auct. non Hess., Phomopsissclerotioides van Kesteren, Rhizoctonia solani Kuhn and Sclerotinia sclerotiorum (Lib.) de Bary (Tahvonen 1982 a, b, LahdenperA 1987, Tahvonen and Avi- kainen 1987, Tahvonen and LahdenperA 1988). In vitro tests with these fungi showed their growth to be inhibited by 5. griseoviri- dis (Tahvonen 1982 a). It controlled damp- ing-off caused by A. brassicicola and R. solani on cauliflower and P. debaryanum on sugar beet in in vivo tests. It also reduced the mor- tality of barley sprouts and foot rot caused by F. culmorum (Tahvonen 1982 b, Tahvonen and Avikainen 1987). Treatment of lettuce seedlings with a spore suspension prepared 435 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=nr-SaCmfkb8IkRui.aX0O3k8LJc0vPyyLw26sDg.fakA77jKf6jIZwB3zMa06_NktZ2GAYQ7ntrtjo-EmQAQnHppzY5AP6sE4p-fr88DFjgPxVSD8kkJmsi30LT5iiIYfojOA7crXstFJDWaNRx0y1k1kRbZ7x4xSgOjZbV0HyWhmTUt9KfzSsj44hAg4rw4Ie9QG_RndFoB04bQUo6DM4_UW1xegJN5aUibWL3Jn4AHnqRzPpD1KhQR2jigGz5SRQJbL7HFYwjm2mujijI3XhaNEjP2ZNPjWIRG6cciNzSlxGDDoalWprVogfr_bVbGLD8eJTOTUpgv_5jViOk from the Streptomyces isolate significantly reduced yield losses caused by B. cinerea, but had no effect on those caused by R. solani. The antibiotic effect against R. solani in vitro, was weaker than its effect against other test- ed pathogens (Tahvonen 1982 a). It has been shown that the antagonistic effect is based on antibiosis, in which aromatic heptaene poly- enes are the active substances (Raatikainen et al. 1990). Many authors (Cooper & Chilton 1949, Johnson 1954, Rangaswami & Ethiraj 1962) have shown that the antibiotics pro- duced by Streptomyces spp. affect some pathogenic fungi. Skinner (1956 b), Lock- wood (1959) and Lockwood & Lingappa (1963) observed, in addition to antibiosis, a lytic effect for actinomycetes when these at- tacked the fungus mycelium directly. Tu (1986, 1988) described the hyperparasitism of Streptomyces albus on Nectria inventa which itself is a mycoparasite of several fungi includ- ing Sclerotinia sclerotiorum, and the hyper- parasitism of S. griseus against Colletotrichum lindemuthianum. The present study was done to elucidate the nature of the host-mycopara- site interactions between Streptomyces griseo- viridis and some plant pathogenic fungi. Materials and methods The interaction of Streptomyces griseoviri- dis isolated from light coloured peat and some plant pathogenic fungi was studied on au- toclaved seeds and living seedlings of turnip rape and carrot. The seeds were placed on wa- ter agar in Petri dishes and inoculated with pathogens, Alternaria brassicicola, isolated from cauliflower seeds, Botrytis cinerea and Sclerotinia sclerotiorum, isolated from carrots or Fusarium oxysporum and Rhizoctonia solani, isolated from turnip rape. These were inoculated three days later with Streptomyces griseoviridis. The dishes were incubated in room temperature. After three days the sam- ples were fixed in a mixture of 2 °7o glutardal- dehyde and 1 % formaldehyde in 0.1 M phos- phate buffer, pFI 7.3, rinsed in buffer and de- hydrated in a graded ethanol series. The speci- mens were then criticalpoint dried in a Bal- zers CPD 020 Critical point dryer using car- bon dioxide, coated with gold in a vacuum evaporator with a Jeol Fine Coat JFC-1100 Sputtering device, and examined under a scan- ning electron microscope (Jeol JSEM-820) at the Department of Electron Microscopy, Uni- versity of Helsinki. The hyperparasitism of S. griseoviridis on the pathogens Phomopsis sclerotioides and Pythium ultimum, isolated from cucumber, was studied in the same way on cucumber seedlings. The interaction between Mycocentrospora acerina Deighton, isolated from carrot and S. griseoviridis was examined directly on myce- lia grown on potato dextrose agar (PDA) in Petri dishes. The antagonist was inoculated on the one-week-old mycelia of M. acerina and the samples were prepared three days later. Agar blocks with mycelia were frozen in liquid nitrogen at —lBO°C (Hexland CT 100), de- hydrated at —BO°C in vacuum in a cryonit, coated with gold and examined under a scan- ning electron microscope (Cambridge Instru- ment S 360) at the Kemira Research Center. Results The antagonist, S. griseoviridis, was easily distinguished from its hosts by its fine (0.5 pm in diameter) sporulating hyphae in contrast to the coarse hyphae of the host fungi. The hyperparasite grew epiphytically on the hyphae of the plant pathogenic fungi. The conidia of A. brassicicola were heavi- ly colonized by Streptomyces and almost to- tally destroyed (Fig. 1). Its hyphae were tightly coiled around the Sclerotinia hyphae, dissolv- ing and disintegrating them (Figs. 2 and 3). The hyperparasite also grew on the hyphae of Botrytis cinerea causing their lysis and de- struction (Fig. 4). At the early state of para- sitism, single strands of S. griseoviridis hypha growing closely pressed to the host hypha seemed to secrete cell wall-dissolving enzymes (Fig. 5). The hyphae of the hyperparasite 436 seems also to grow inside the host hypha of B. cinerea (Fig. 4). The sporulating hyphae of S. griseoviridis grew along and coiled around the hyphae of Fusarium oxysporum slightly dissolving them, but the conidia did not seem to be affected (Fig. 6). The hyphae of Phomopsis sclerotioides were collapsed in the presence of S. griseoviri- dis, but the sclerotia did not seem to be affected (Fig. 7). The mycelia of Mycocentrospora acerina were dissolvedand flattened by S. griseoviri- dis in a similar manner to those of P. sclero- tioides (Fig. 8). The hyphae of the hyperpara- site grew loosely on the hypha of Rhizocto- nia solani at the early stages of infection. Later on, Streptomyces hyphae seemed to be close- ly associated with the collapsed host hyphae (Fig. 9). Most of the mycelia and oogonia of Pythi- um were not visibly affected at the moment of fixing the preparation, although the hyper- parasite seems to have penetrated its mycelial wall (Figs. 10, 11). A portion of the host hyphae abundantly covered with the hyper- parasite showed some disintegration (Fig. 11). Discussion The first observations made by Tahvonen (1982 a) about the antagonism of Strep- tomyces spp. against many plant pathogenic fungi in vitro demonstrated their antibiotic and growth inhibiting effect. The hyper- parasitism of one of these antagonists was clearly shown in the present study. The lytic activity of actinomycetes has been earlier shown by Lockwood (1959). According to Tu (1986, 1988), Streptomyces albus could act as Fig. I. Thin hyphae (width 0,5 pm) of Streptomyces griseoviridis are covering and gradually dissolving the hyphae and especially the conidia (arrow) of Alternaria brassicicola. Fig. 2. The sporulating Streptomyces hyphae are coiling around the hyphae of Sclerotinia sclerotiorum disintegrat- ing them (arrow). Fig. 3. The hyperparasite is dissolving the hyphae of Sclerotinia sclerotiorum. Fig. 4. Thin hyphae and spores of S. griseoviridisare growing on the broad hyphae of Botrytis cinerea. The hyper- parasite are growing both epiphytically and internally (arrow) on the pathogen, which is disintegrated and dissolved. Fig. 5. Eroded wall of hyphae of Bolrylis cinerea adjacent to hyphae of S. griseoviridis. Fig. 6. The speculating hyphae of 5. griseoviridis only slightly affecting the hyphae of Fusarium oxysporum (arrow). The conidia do not seem to be affected. Fig. 7. Streptomyces hyphae degrading the hyphae of Phomopsis sclerotioides, but seem not to affect the sclerotia of the pathogen. Fig. 8. S. griseoviridis hyphae are growing from the right towards Mycocentrospora acerina hyphae, which are col- lapsing and disintegrating. Fig. 9. Streptomyces hyphae are growing on the hyphae of Rhizoctonia solani (arrow), which are only slightly af- fected. The hyperparasite seems, however, be associated with sunken area of some collapsed host hyphae (double arrow). Fig. 10. The hyphae and oogonia of Pythium are not much affected, although the Streptomyces hyphae seem to be able to penetrate (arrow) its hyphal wall. Fig. 11. The hyphae of Pythium sp. are penetrating the cucumber root cells (arrow), although the Streptomyceshyp- hae are abundant. In some portions of the preparation, the Pythium hypha are clearly disintegrated (double arrow). In figure 5 the scale bar is 1 tun, in all other figures it is 10 am. 437 438 439 a surface parasite with or without the forma- tion of appressoria-like structures and S. griseus produced appressorium-like swellings on the hyphal surface of Colletotrichum lin- demuthianum. Appressoria were not observed in our studies at S. griseoviridis. The internal parasitism of A. brassicicola, S. sclerotiorum, B. cinerea and P. sclerotioides was evidenced by the fact that hyperparasitic hyphae were frequently found inside partly fractured host hyphae, similar to the findings at Tu (1986, 1988) who found parasitism of hyphae of N. inventa and C. lindemuthianum by Strep- tomyces albus. S. griseoviridis seemed also to grow inside the collapsed Rhizoctonia- hypha. There is some doubt as to whether the cells were penetrated before or after death. The hyperparasitized hyphae of F. oxysporum were abnormal, but not clearly disintegrated. Skinner (1956 a) found that Streptomyces al- bidoflavus limited early growth of Fusarium culmorumby antibioticaction and that it also directly attacked preformed fungus mycelium. He also observed that the actinomycete lysed the contents of its host hyphae (Skinner 1956 b). The conidia of F. oxysporum seemed not to be affected at least in three days these were hyperparasitized. Lockwood (1959) ob- served that the germination ofFusarium sola- ni f. pisi conidia was not affected by Strep- tomyces isolates, although the mycelia of F. oxysporum f. pisi and F. solani f. pisi were lysed and disappeared. In our studies the parasitized hyphae of P. sclerotioides and M. acerina were collapsed, dissolved and flattened without disruption of the cell walls. S. griseoviridis only slightly parasitized on R. solani, at least at the begin- ning of the infection. According to Tahvo- nen (1982 a) and Tahvonen and LahdenperA (1988) its growth inhibiting effect in vivo tests was weaker on R. solani than on the other pathogens studied. Tahvonen (1982 a) ob- served a strong antibiotic effect of S. griseo- viridis against P. ultimum, as did Knauss (1976) for two otherStreptomyces species. In our study oospores of P. ultimum were not parasitized by S. griseoviridis. Sneh et al. (1977) and Sutherland and Lockwood (1984) found a zoospore-producing actinomycete, Actinoplanes missouriensis Couch, frequent- ly infecting oospores of Pythium sp. and some other Oomycetes primarily on flooded soil. S. griseoviridis seems, however, to be able to penetrate the hyphae of Pythium sp. and dis- integrate them. References Cooper, W.E. & Chilton, S.J.P. 1949. Antibiosis of Ac- tinomycete strains to Pythium arrhenomanes, P. ul- timum and Rhizoctonia solani. Phytopathology 39, 5. Johnson, L.F. 1954. Antibiosis in relation to Pythium root rot of sugarcane and corn. Phytopathology44, 69—73. Knauss, J.F. 1976. In vitro antagonistic activity of sever- al Streptomyces spp. against species of Pythium and Phytophtora Plant Disease Reporter 60, 846—850. LahdenperA, M-L. 1987. Streptomyces -sadesienivalmiste ja sen kaytto Fusarium oxysporum f.sp. dianthi -sienen aiheuttaman neilikanlakastumistaudin torjun- nassa. Licentiate-thesis. Department of Plant Pathol- ogy, University of Helsinki. Lockwood, J.L. 1959. Streptomyces spp as a cause of natural fungitoxicity in soils. Phytopathology 49, 327 —331. Lockwood, J.L. & Linoappa, B.T. 1963. Fungitoxicity of sterilized soil inoculated with soil microflora. Phyto- pathology 53, 917—920. Raatikainen, 0., Tahvonen, R., Tuomisto, J. and Ro- senqvist, H. 1990. In vitro production of heptaene polyene by suppressive Streptomyces sp. isolated from Sphagnum peat. In sth Europea Congress on Biotech- nology, Copenhagen, July B—l3, 1990. Abstract Book, ed, by Christiansen, C., Mucsk, L. and Vil- ladsen, J. Ranoaswami, G. & Ethiraj, S. 1962. Antibiotic produc- tion by Streptomyces sp. in unamended soil. Phytopathology 52, 989—992. Skinner, F.A. 1956 a. Inhibition of the growth of fungi by Streptomyces spp. in relation to nutrient condi- tions. Journal of General Microbiology 14, 381 —392. Skinner, F.A. 1956. The effect of adding clays to mixed cultures of Streptomyces albidoflavus and Fusarium 440 culmorum. Journal of General Microbiology 14, 393 —405. Sneh, 8., Humble, S.J. & Lockwood, J.L. 1977. Para- sitism of oospores of Phytophthoramegasperma var. sojae, P. cactorum, Pythium sp., and Aphanomyces euleiches in soil by Oomycetes, Chytridiomycetes, Hyphomycetes, Actinomycetes, and Bacteria. Phyto- pathology 67, 622—628. Sutherland, E.D. & Lockwood, J.L. 1984. Hyper- parasitism of oospores of somePeronosporales by Ac- tinoplanes missouriensis and Humicola fuscoatra and other Actinomycetes and fungi. Canadian Journal of Plant Pathology 6, 139—145. Tahvonen, R. 1982 a. The suppressiveness of Finnish light coloured Sphagnumpeat. Journal of the Scien- tific Agricultural Society of Finland 54, 345—356. Tahvonen, R. 1982 b. Preliminary experiments into the use of Streptomyces spp. isolated from peat in the biological control of soil and seedborne diseases in peat culture. Journal of the Scientific Agricultural So- ciety of Finland 54, 357—369. Tahvonen, R. & Avikainen, H. 1987. The biological con- trol of seedborne Alternaria brassicicola of crucifer- ous plants with a powdery preparation of Strep- tomyces sp. Journal of Agricultural Science in Fin- land 59, 199—208. Tahvonen, R. & Lahdenpera, M-L. 1988. Biological control of Botrytis cinerea and Rhizoctonia solani in lettuce by Streptomyces sp. Annales AgriculturaeFen- niae, 27, 107—116. Tu, J.C. 1986, Hyperparasitism of Streptomyces albus on a destructive mycoparasite Nectria inventa Jour- nal of Phytopathology 117, 71 —76. Tu, J.C. 1988. Antibiosis of Streptomyces griseus against Colletotrichum Undemuthianum. Journal of Phyto- pathology 121, 97—102. Ms received April 15, 1991 SELOSTUS Pyyhkaisyelektronimikroskooppitutkimukset Streptomyces griseoviridis-sadesienen ja eraiden kasvitauteja aiheuttavien sienten vuorovaikutuksesta Eeva Tapio ja Arja Pohto-Lahdenpera Kasvipatologian lailos, Helsingin yliopislo, 00710 Helsinki Helsingin yliopiston kasvipatologian laitoksella on tut- kittu pyyhkaisyelektronimikroskoopilla (SEM) sadesienen, Streptomyces griseoviridis, ja useiden kasvitautia aiheut- tavien sienten vuorovaikutusta. 5. griseoviridis eristettiin 1980-luvun alussa vaaleasta rahkaturpeesta ja silla todet- tiin olevan antagonistisia eli kasvitauteja ehkaisevia omi- naisuuksia. Se erittaaantibioottisia aromaattisia heptee- nipolyeeneja. Tassa tutkimuksessa selvitettiin pyslyyko se myds loisimaan kasvipatogeeneilla sienilla. Alternaria brassicicola (kaalikasvien taimipoltesieni), Botrytis cine- rea (harmaahome), Fusarium oxysporum (lakastumistau- tisieni), Mycocentrospora acerina (porkkananmustama- tasieni), Rhizoctonia solani (mm. seittirupi- ja taimipol- tesieni) ja Sderotinia sderoliorum (pahkahome) -sienilla tartutettiin rypsin japorkkanan siemenia ja siementaimia. Phomopsis sclerotioides jaPythium ultimum -sienet, jotka aiheuttavat mm. kurkun tyvi- ja mustajuuriraataa, levi- tettiin kurkun siementaimiin. Kolmen vuorokauden ku- luttua ne kasiteltiin S. gmeovincfe-suspensiolla. Sen jal- keen ne inkuboitiin kolme vuorokautta huoneenlammdssa ennenkuin niista valmistettiin tavanomaisia menetelmia kayttaen SEM-preparaatit elektronimikroskopointia var- ten. Kuvauksissa oli nahtavissa, etta sadesienirihmat loisi- vat useiden sienten rihmoilla. S. griseovirtdis kietoutui tiu- kasti Alternaria -kuromien ja Sderotinia -rihmojen ym- parille hajoittaen ilmeisesti niita. Sadesieni kasvoi B. ci- nerea, P. sclerotioides ja M. acerina rihmoilla liuottaen niita. 5. griseoviridis vioitti vain vahan F. oxysporum -sienirihmaa eika lainkaan sen kuromia. Se kasvoi loy- hasti R. so/am-rihmalla jaPythium sienen rihmalla ja mu- naitiolla, jotka eivat paljoakaan vioittuneet. 441