JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND Maataloustieteellinen Aikakauskirja 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 , 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 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 sclerotiorum (Lib.) de Bary (Tahvonen 1982 a, b, LahdenperA 1987, Tahvonen and Avi- Streptomyces griseoviridis Anderson et al., kainen 1987, Tahvonen and LahdenperA isolated from Finnish light coloured Sphag- 1988). In vitro tests with these fungi showed num peat, has been reported to be antagonis- their growth to be inhibited by 5. griseoviri- tic to the plant pathogens Alternaria brassici- dis (Tahvonen 1982 a). It controlled damp- cola (Schw.) Wiltshire, Botrytis cinerea Pers.: ing-off caused by A. brassicicola and R. solani Fr., Fusarium avenaceum (Fr.: Fr.) Sacc., F. on cauliflower and P. debaryanum on sugar culmorum (W.G. Sm.) Sacc., F. oxysporum beet in in vivo tests. It also reduced the mor- Schlecht. f. sp. dianthi (Prill. & Dol.) Snyd. tality of barley sprouts and foot rot caused by & Hans., Pythium debaryanum auct. non F. culmorum (Tahvonen 1982 b, Tahvonen Hess., Phomopsissclerotioides van Kesteren, and Avikainen 1987). Treatment of lettuce Rhizoctonia solani Kuhn and Sclerotinia seedlings with a spore suspension prepared

435 from the Streptomyces isolate significantly mens were then criticalpoint dried in a Bal- reduced yield losses caused by B. cinerea, but zers CPD 020 Critical point dryer using car- had no effect on those caused by R. solani. bon dioxide, coated with gold in a vacuum The antibiotic effect against R. solani in vitro, evaporator with a Jeol Fine Coat JFC-1100 was weaker than its effect against other test- Sputtering device, and examined under a scan- ed pathogens (Tahvonen 1982 a). It has been ning electron microscope (Jeol JSEM-820) at shown that the antagonistic effect is based on the Department of Electron Microscopy, Uni- antibiosis, in which aromatic heptaene poly- versity of Helsinki. enes are the active substances (Raatikainen et The hyperparasitism of S. griseoviridis on al. 1990). Many authors (Cooper & Chilton the pathogens Phomopsis sclerotioides and 1949, Johnson 1954, Rangaswami & Ethiraj Pythium ultimum, isolated from cucumber, 1962) have shown that the antibiotics pro- was studied in the same way on cucumber duced by Streptomyces spp. affect some seedlings. pathogenic fungi. Skinner (1956 b), Lock- The interaction between Mycocentrospora wood (1959) and Lockwood & Lingappa acerina Deighton, isolated from carrot and S. (1963) observed, in addition to antibiosis, a griseoviridis was examined directly on myce- lytic effect for actinomycetes when these at- lia grown on dextrose agar (PDA) in tacked the fungus mycelium directly. Tu Petri dishes. The antagonist was inoculated on (1986, 1988) described the hyperparasitism of the one-week-old mycelia of M. acerina and Streptomyces albus on Nectria inventa which the samples were prepared three days later. itself is a mycoparasite of several fungi includ- Agar blocks with mycelia were frozen in liquid ing Sclerotinia sclerotiorum, and the hyper- nitrogen at —lBO°C (Hexland CT 100), de- parasitism of S. griseus against Colletotrichum hydrated at —BO°C in vacuum in a cryonit, lindemuthianum. The present study was done coated with gold and examined under a scan- to elucidate the nature of the host-mycopara- ning electron microscope (Cambridge Instru- site interactions between Streptomyces griseo- ment S 360) at the Kemira Research Center. viridis and some plant pathogenic fungi.

Materials and methods Results

The interaction of Streptomyces griseoviri- The antagonist, S. griseoviridis, was easily dis isolated from light coloured peat and some distinguished from its hosts by its fine (0.5 pm plant pathogenic fungi was studied on au- in diameter) sporulating hyphae in contrast to toclaved seeds and living seedlings of turnip the coarse hyphae of the host fungi. The rape and carrot. The seeds were placed on wa- hyperparasite grew epiphytically on the ter agar in Petri dishes and inoculated with hyphae of the plant pathogenic fungi. pathogens, Alternaria brassicicola, isolated The conidia of A. brassicicola were heavi- from cauliflower seeds, Botrytis cinerea and ly colonized by Streptomyces and almost to- Sclerotinia sclerotiorum, isolated from carrots tally destroyed (Fig. 1). Its hyphae were tightly or Fusarium oxysporum and Rhizoctonia coiled around the Sclerotinia hyphae, dissolv- solani, isolated from turnip rape. These were ing and disintegrating them (Figs. 2 and 3). inoculated three days later with Streptomyces The hyperparasite also grew on the hyphae griseoviridis. The dishes were incubated in of Botrytis cinerea causing their lysis and de- room temperature. After three days the sam- struction (Fig. 4). At the early state of para- ples were fixed in a mixture of 2 °7o glutardal- sitism, single strands of S. griseoviridis hypha dehyde and 1 % formaldehyde in 0.1 M phos- growing closely pressed to the host hypha phate buffer, pFI 7.3, rinsed in buffer and de- seemed to secrete cell wall-dissolving enzymes hydrated in a graded ethanol series. The speci- (Fig. 5). The hyphae of the hyperparasite

436 seems also to grow inside the host hypha of Most of the mycelia and oogonia of Pythi- B. cinerea (Fig. 4). um were not visibly affected at the moment The sporulating hyphae of S. griseoviridis of fixing the preparation, although the hyper- grew along and coiled around the hyphae of parasite seems to have penetrated its mycelial Fusarium oxysporum slightly dissolving them, wall (Figs. 10, 11). A portion of the host but the conidia did not seem to be affected hyphae abundantly covered with the hyper- (Fig. 6). parasite showed some disintegration (Fig. 11). The hyphae of Phomopsis sclerotioides were collapsed in the presence of S. griseoviri- Discussion dis, but the sclerotia did not seem to be affected (Fig. 7). The first observations made by Tahvonen The mycelia of Mycocentrospora acerina (1982 a) about the antagonism of Strep- were dissolvedand flattened by S. griseoviri- tomyces spp. against many plant pathogenic dis in a similar manner to those of P. sclero- fungi in vitro demonstrated their antibiotic tioides (Fig. 8). The hyphae of the hyperpara- and growth inhibiting effect. The hyper- site grew loosely on the hypha of Rhizocto- parasitism of one of these antagonists was nia solani at the early stages of infection. Later clearly shown in the present study. The lytic on, Streptomyces hyphae seemed to be close- activity of actinomycetes has been earlier ly associated with the collapsed host hyphae shown by Lockwood (1959). According to Tu (Fig. 9). (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. griseoviridis are 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- served that the germination ofFusarium sola- tion of appressoria-like structures and S. ni f. pisi conidia was not affected by Strep- griseus produced appressorium-like swellings tomyces isolates, although the mycelia of F. on the hyphal surface of Colletotrichum lin- oxysporum f. pisi and F. solani f. pisi were demuthianum. Appressoria were not observed lysed and disappeared. in our studies at S. griseoviridis. The internal In our studies the parasitized hyphae of P. parasitism of A. brassicicola, S. sclerotiorum, sclerotioides and M. acerina were collapsed, B. cinerea and P. sclerotioides was evidenced dissolved and flattened without disruption of by the fact that hyperparasitic hyphae were the cell walls. S. griseoviridis only slightly frequently found inside partly fractured host parasitized on R. solani, at least at the begin- hyphae, similar to the findings at Tu (1986, ning of the infection. According to Tahvo- 1988) who found parasitism of hyphae of N. nen (1982 a) and Tahvonen and LahdenperA inventa and C. lindemuthianum by Strep- (1988) its growth inhibiting effect in vivo tests tomyces albus. S. griseoviridis seemed also to was weaker on R. solani than on the other grow inside the collapsed Rhizoctonia- hypha. pathogens studied. Tahvonen (1982 a) ob- There is some doubt as to whether the cells served a strong antibiotic effect of S. griseo- were penetrated before or after death. The viridis against P. ultimum, as did Knauss hyperparasitized hyphae of F. oxysporum (1976) for two other Streptomyces species. In were abnormal, but not clearly disintegrated. our study oospores of P. ultimum were not Skinner (1956 a) found that Streptomyces al- parasitized by S. griseoviridis. Sneh et al. bidoflavus limited early growth of Fusarium (1977) and Sutherland and Lockwood (1984) culmorumby antibioticaction and that it also found a zoospore-producing actinomycete, directly attacked preformed fungus mycelium. Actinoplanes missouriensis Couch, frequent- He also observed that the actinomycete lysed ly infecting oospores of Pythium sp. and some the contents of its host hyphae (Skinner other primarily on flooded soil. S. 1956 b). The conidia of F. oxysporum seemed griseoviridis seems, however, to be able to not to be affected at least in three days these penetrate the hyphae of Pythium sp. and dis- were hyperparasitized. Lockwood (1959) ob- integrate them.

References

Cooper, W.E. & Chilton, S.J.P. 1949. Antibiosis of Ac- Lockwood, J.L. & Linoappa, B.T. 1963. Fungitoxicity of tinomycete strains to Pythium arrhenomanes, P. ul- sterilized soil inoculated with soil microflora. Phyto- timum and Rhizoctonia solani. Phytopathology pathology 53, 917—920. 39, 5. Raatikainen, 0., Tahvonen, R., Tuomisto, J. and Ro- Johnson, L.F. 1954. Antibiosis in relation to Pythium senqvist, H. 1990. In vitro production of heptaene root rot of sugarcane and corn. Phytopathology 44, polyene by suppressive Streptomyces sp. isolated from 69—73. Sphagnum peat. In sth Europea Congress on Biotech- Knauss, J.F. 1976. In vitro antagonistic activity of sever- nology, Copenhagen, July B—l3, 1990. Abstract al Streptomyces spp. against species of Pythium and Book, ed, by Christiansen, C., Mucsk, L. and Vil- Phytophtora Plant Disease Reporter 60, 846—850. ladsen, J. LahdenperA, M-L. 1987. Streptomyces -sadesienivalmiste Ranoaswami, G. & Ethiraj, S. 1962. Antibiotic produc- ja sen kaytto Fusarium oxysporum f.sp. dianthi tion by Streptomyces sp. in unamended soil. -sienen aiheuttaman neilikanlakastumistaudin torjun- Phytopathology 52, 989—992. nassa. Licentiate-thesis. Department of Plant Pathol- Skinner, F.A. 1956 a. Inhibition of the growth of fungi ogy, University of Helsinki. by Streptomyces spp. in relation to nutrient condi- Lockwood, J.L. 1959. Streptomyces spp as a cause of tions. Journal of General Microbiology 14, 381 —392. natural fungitoxicityin soils. Phytopathology 49, 327 Skinner, F.A. 1956. The effect of adding clays to mixed —331. cultures of Streptomyces albidoflavus and Fusarium

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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- aiheuttavat mm. kurkun tyvi- ja mustajuuriraataa, levi- kittu pyyhkaisyelektronimikroskoopilla (SEM) sadesienen, tettiin kurkun siementaimiin. Kolmen vuorokauden ku- Streptomyces griseoviridis, ja useiden kasvitautia aiheut- luttua ne kasiteltiin S. gmeovincfe-suspensiolla. Sen jal- tavien sienten vuorovaikutusta. 5. griseoviridis eristettiin keen ne inkuboitiin kolme vuorokautta huoneenlammdssa 1980-luvun alussa vaaleasta rahkaturpeesta ja silla todet- ennenkuin niista valmistettiin tavanomaisia menetelmia tiin olevan antagonistisia eli kasvitauteja ehkaisevia omi- kayttaen SEM-preparaatit elektronimikroskopointia var- naisuuksia. Se erittaaantibioottisia aromaattisia heptee- ten. nipolyeeneja. Tassa tutkimuksessa selvitettiin pyslyyko Kuvauksissa oli nahtavissa, etta sadesienirihmat loisi- se myds loisimaan kasvipatogeeneilla sienilla. Alternaria vat useiden sienten rihmoilla. S. griseovirtdis kietoutui tiu- brassicicola (kaalikasvien taimipoltesieni), Botrytis cine- kasti Alternaria -kuromien ja Sderotinia -rihmojen ym- rea (harmaahome), Fusarium oxysporum (lakastumistau- parille hajoittaen ilmeisesti niita. Sadesieni kasvoi B. ci- tisieni), Mycocentrospora acerina (porkkananmustama- nerea, P. sclerotioides ja M. acerina rihmoilla liuottaen tasieni), Rhizoctonia solani (mm. seittirupi- ja taimipol- niita. 5. griseoviridis vioitti vain vahan F. oxysporum tesieni) ja Sderotinia sderoliorum (pahkahome) -sienilla -sienirihmaa eika lainkaan sen kuromia. Se kasvoi loy- tartutettiin rypsin japorkkanan siemenia ja siementaimia. hasti R. so/am-rihmalla jaPythium sienen rihmalla ja mu- Phomopsis sclerotioides jaPythium ultimum -sienet, jotka naitiolla, jotka eivat paljoakaan vioittuneet.

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