ISOLATION AND MASS PROPAGATION OF ENTOMOPATHOGENIC FUNGI FOR BIOCONTROL ASSAYS* P.Mythili1, S.Gomathinayagam2, C.

Balachandran3,DhinakarRaj4 and Lalitha John5

Tamil Nadu Veterinary and Animal Sciences University

ABSTRACT Bioassays were conducted using the Entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against insects and ticks of veterinary importance. Isolation of theses fungi was done from dead larvae of Musca domestica and Boophilus ticks. Surface and mass culturing of both the fungi were done in Potato Dextrose Agar and Potato Dextrose Broth respectively. Cultural characters of the fungi were studied. Surface culture of fungus Beauveria bassiana yielded white or lightly coloured colonies, whereas M. anisopliae produced herbage green or olivaceous green colonies. Mass culturing of fungi using other easily available field sources was discussed. Key Words: Beauveria bassiana; Metarhizium anisopliae; Surface culture; Mass culture. Entomopathogenic fungi have played a uniquely important role in the history of microbial control of insects. Historical evidence indicated that entomopathogenic fungi were the first to be recognized as disease causing microorganisms in insects. Agostino Bassi de Lodi wrote about a disease in silkworm caused by a fungus, which was later, identified as Beauveria bassiana. (Ainsworth, 1956). Elie Metschnikoff began with study of disease of a grain beetle Anisoplia austriaca that resulted in the discovery of the fungus Metarhizium anisopliae (Ainsworth, 1956). Beauveria bassiana, commonly known as white muscardine fungus attacks a wide range of immature and adult insects. Metarhizium anisopliae a green muscardine fungus is reported to infect 200 species of insects and

arthropods. Both of these entomopathogenic fungi are soil borne and widely distributed. In this study, Isolation of theses fungi from insect cadavers was done and mass propagation of theses fungi was carried out to obtain sufficient quantity of the fungal propagules (mixture of conidia and blastospores) to be used in the bioassay against ticks and insects of veterinary importance. The cultural characteristics of these fungi were also studied. MATERIALS AND METHODS

Isolation of fungi
External sporulation of the fungi was induced by placing the cadavers of larvae of Musca domestica and Boophilus microplus ticks collected

* Part of M.V.Sc thesis submitted to Tamil Nadu Veterinary Animal Sciences University 1. Veterinary Assistant Surgeon, 2. Professor, Central University Laboratory, Madhavaram,3. Registrar, TANUVAS, 4. Professor, Department of Animal Biotechnology, MVC, 5. Professor and Head, (retd.), Department of Veterinary Parasitology,MVC

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Isolation and mass propagation of .... from wild by placing them in a BOD incubator with 70% humidity. After sporulation had occurred, primary cultures of these fungi were done by scraping the surface of cadavers by needle and inoculated into agar slants and petri dishes for further propagation. In the absence of sporulation, the cadavers were homogenized using micro-pestle and the homogenate was inoculated into agar medium. Isolation from cadavers was done to obtain pure culture of the fungi. Potato Dextrose Broth was prepared using readymade broth powder supplied by Hi Media laboratory as per the standard recommendation One hundred ml of Potato Dextrose Broth (PDB) was dispensed in to sterile 250 ml Erlenmeyer flasks. It was allowed to cool. Conidia or blastospore suspension was harvested either by direct scraping from primary culture or surface culture as above or by washing off the surface culture with sterile distilled water. It was inoculated into the PDB substrate. The flasks were loosely plugged with cotton and incubated at 25o C in BOD incubator for 7-10 days.

Surface culture of fungi Surface cultures were accomplished in Potato Dextrose Agar medium within test tube slants and glass petriplates. Potato dextrose agar was prepared from readymade powder supplied by Hi Media laboratory following their recommendation Preparation of Potato Dextrose Agar: Potato Dextrose Agar Powder (Hi media) 41 g Distilled water Boiled and sterilized by autoclaving. Agar surface was inoculated under sterile conditions with suspensions of conidia or blastospores by direct scraping from the surface of the primary culture using a sterile platinum loop. The petriplates were sealed with Para film to reduce dehydration. Slants were loosely plugged with cotton and wrapped with aluminum foil. Glass petriplates and slants were incubated at 25oC in BOD incubator for 7-10 days. 1000 ml

RESULTS AND DISCUSSION Sporulation of the green muscardine fungus was evident after 5 days from the dead Boophilus ticks whereas no evidence of sporulation of white muscardine fungus was noticed from the cadavers of larvae of Musca domestica. This could be due to the lack of conidiation resulting from poorly growing hyphal bodies or internal production of resting spores instead of conidia as reported by Lacey (1997). This could also be due to lower humidity provided in the environment. Goettel and Inglis (1997) also induced external sporulation by placing insect cadavers in higher relative humidity (97-100%). However, the growth of both the fungi was evident from cultures initiated from the homogenates of the cadavers.

Surface culture of fungi Surface culture of fungus Beauveria bassiana yielded white or lightly coloured colonies after 10 days of incubation Conidiophores were single globose and conidia were small, round to oval measuring 5 mm (Fig.1). Surface culture of Metarhizium anisopliae produced dark herbage green or yellowish green,

Mass Cultivation of fungi Nutritive substrate Potato Dextrose Broth was used for large scale production of propagules. 272

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Mythili et al., olivaceous colonies after 7 days of incubation (Fig.2). Colonies were closely packed, highly branched with cylindrical conidiophores. Conidial chains were round, columnar and conidia were cylindrical to oval measuring 7.5 mm. Many authors have used Sabourauds Dextrose Agar (SDA) medium for the culture of fungi but in this study both the fungi grew very well in PDA medium. Goettel and Inglis (1997) also used Potato Dextrose Agar (PDA) for culture of entomopathogenic fungi. The morphological characters of both fungi are akin to the description of Humber (1997).He observed the colonies of B. bassiana as densely clustered conidiogenous cells, which had denticulate apical extension bearing one conidium. Conidia were long and ovoid measuring 3.5 mm. He also described colonies of Metarhizium as broadly branched interwined conidiogenous cells which formed chains or cylindrical colonies with conidia being ovoid and light green measuring < 9mm. It was seen in this study that M. anisopliae had a faster growth compared to B. bassiana under same incubation conditions. This is in contrast to the findings of Humber (1997), who reported M. anisopliae would develop relatively slow in slant cultures. This might be due to strain variation of fungus used in the study.

Mass cultivation of fungi Colonies of B. bassiana in liquid medium appeared as white fluffy mycelial mat on the surface of liquid medium .This is in agreement to reports of Kwon-chung and Bennet (1992), who described the colonies of B. bassiana as white or lightly coloured and fluffy to powdery in liquid medium. M. anisopliae formed olivegreen mycelial mat on surface of liquid medium This is akin to the observations of Bridge et al. (1993). They described the colonies of M. anisopliae as yellowish green or

Fig.1 White colored colonies of Beauveria bassiana

Fig.2 Green color colonies of Metarhizium anisopliae

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Isolation and mass propagation of .... olivaceous green, sometimes even as pink buff colonies. of Metarhizium anisopliae and Metarhizium flavoviridae. Journal of General Microbiology,139: 1163-1169 Goettel, M.S.and Inglis, G.D.(1997) Fungi: Hyphomycetes. In: A manual of Techniques in insect Pathology. L.A.Lacey(ed). Academic Press, New York. pp : 213-249 Humber,R.A.(1997) Fungi: Identification .In: A manual of Techniques in insect Pathology. Academic Press, New York. pp :153-167 Kaaya, G.P(1989) Glossina morsitans morsitans: Mortalities caused in adults by experimental infection with entomopathogenic fungi. Acta tropica, 46: 107-114 Kwon-chung, K.J and Bennet, M.D.J.E.(1992) Common laboratory saprophytes. (AppendixA) in: Medical Mycology. Lea and Febiger Publications. Philadelphia and London. Pp.797-815. Lacey,L.A.(1997) A manual of Techniques in insect Pathology. Academic Press, New York. pp :187-210

Mass cultivation of the fungi was done in other media like Sabourauds dextrose agar (Kaaya, 1989) and Potato Carrot Agar( Bridge, et al.1993). This could be achieved in autoclaved rice, minced carrot and half broken sorghum grains. Thus use of cost of effective media could pave way for mass cultivation of fungi under field conditions and their use in the biological control of insects of veterinary importance. REFERENCES Ainsworth,G.C.(1956). Agostino Bassi, 1773-1856, Nature.,177: 255-257 Booth, C. (1971). Introduction to General Methods In: Methods in Microbiology, Academic Press, London. pp: 2-45. Bridge,P.D.,Williams,M.A.J., Prior,C and Paterson,R.R.M.(1993). Morphological, biochemical and molecular characteristics

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