Scanning electron microscopy of hyphal interaction between Streptomyces griseoviridis and some plant pathogenic fungi

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 ultimum on cucumber seedlings. The samples were prepared by the standard method for examination by scanning electron microscope. The hyperparasitism of S. griseoviridis was clearly shown. S. griseoviridis tightly wound around Alternaria conidia and Sclerotinia hyphae, eventually 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

. In vitro tests with these fungi showed their growth to be inhibited by 5. griseoviridis (Tahvonen 1982 a). It controlled damping-off caused by A. brassicicola and R. solani on cauliflower and P. debaryanum on sugar beet in in vivo tests. It also reduced the mortality of barley sprouts and foot rot caused by F. culmorum (Tahvonen 1982 b, Tahvonen andAvikainen 1987). Treatment of lettuce seedlings with a spore suspension prepared 435 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND 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 tested pathogens (Tahvonen 1982 a). It has been shown that the antagonistic effect is based on antibiosis, in which aromatic heptaene polyenes are the active substances (Raatikainen et al. 1990). Many authors (Cooper & Chilton 1949, Johnson 1954, Rangaswami & Ethiraj 1962 have shown that the antibiotics produced by Streptomyces spp. affect some pathogenic fungi. b), Lockwood (1959 and Lockwood & Lingappa (1963) observed, in addition to antibiosis, a lytic effect for actinomycetes when these attacked the fungus mycelium directly. Tu (1986,1988) described the hyperparasitism of Streptomyces albus on Nectria inventa which itself is a mycoparasite of several fungi including Sclerotinia sclerotiorum, and the hyperparasitism of S. griseus against Colletotrichum lindemuthianum. The present study was done to elucidate the nature of the host-mycoparasite interactions between Streptomyces griseoviridis and some plant pathogenic fungi.

Materials and methods
The interaction of Streptomyces griseoviridis isolated from light coloured peat and some plant pathogenic fungi was studied on autoclaved seeds and living seedlings of turnip rape and carrot. The seeds were placed on water 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 samples were fixed in a mixture of 2°7o glutardaldehyde and 1 % formaldehyde in 0.1 M phosphate buffer, pFI 7.3, rinsed in buffer and dehydrated in a graded ethanol series. The speci-mens were then criticalpoint dried in a Balzers CPD 020 Critical point dryer using carbon dioxide, coated with gold in a vacuum evaporator with a Jeol Fine Coat JFC-1100 Sputtering device, and examined under a scanning electron microscope (Jeol JSEM-820) at the Department of Electron Microscopy, University 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 mycelia 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), dehydrated at -BO°C in vacuum in a cryonit, coated with gold and examined under a scanning electron microscope (Cambridge Instrument 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 heavily colonized by Streptomyces and almost totally destroyed (Fig. 1). Its hyphae were tightly coiled around the Sclerotinia hyphae, dissolving and disintegrating them (Figs. 2 and 3).
The hyperparasite also grew on the hyphae of Botrytis cinerea causing their lysis and destruction ( Fig. 4). At the early state of parasitism, 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 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. griseoviridis, but the sclerotia did not seem to be affected (Fig. 7).
The mycelia of Mycocentrospora acerina were dissolved and flattened by S. griseoviridis in a similar manner to those of P. sclerotioides (Fig. 8). The hyphae of the hyperparasite grew loosely on the hypha of Rhizoctonia solani at the early stages of infection. Later on, Streptomyces hyphae seemed to be closely associated with the collapsed host hyphae (Fig. 9).
Most of the mycelia and oogonia of Pythium were not visibly affected at the moment of fixing the preparation, although the hyperparasite seems to have penetrated its mycelial wall (Figs. 10,11). A portion of the host hyphae abundantly covered with the hyperparasite showed some disintegration (Fig. 11).

Discussion
The first observations made by Tahvonen (1982 a) about the antagonism of Streptomyces spp. against many plant pathogenic fungi in vitro demonstrated their antibiotic and growth inhibiting effect. The hyperparasitism 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 (1986Tu ( , 1988, Streptomyces albus could act as           In figure 5 the scale bar is 1 tun, in all other figures it is 10 am. a surface parasite with or without the formation of appressoria-like structures and S. griseus produced appressorium-like swellings on the hyphal surface of Colletotrichum lindemuthianum. 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 Streptomyces 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.  found that Streptomyces albidoflavus limited early growth of Fusarium culmorum by antibiotic action 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 of Fusarium solani f. pisi conidia was not affected by Streptomyces isolates, although the mycelia of F. oxysporum f. pisi and F. solani f. pisi were lysed and disappeared.