World J Microbiol Biotechnol (2015) 31:675–679 DOI 10.1007/s11274-015-1821-6 SHORT COMMUNICATION A method of multiplex PCR for detection of field released Beauveria bassiana, a fungal entomopathogen applied for pest management in jute (Corchorus olitorius) Chinmay Biswas • Piyali Dey • B. S. Gotyal • Subrata Satpathy Received: 9 December 2014 / Accepted: 7 February 2015 / Published online: 14 February 2015 Ó Springer Science+Business Media Dordrecht 2015 Abstract The fungal entomopathogen Beauveria bassi- jewellery and home decorations (Kundu 1956). Jute is of ana is a promising biocontrol agent for many pests. Some two kinds namely, tossa jute (Corchorus olitorius) and B. bassiana strains have been found effective against jute white jute (C. capsularis). Jute is severely affected by pests. To monitor the survival of field released B. bassiana various insect-pests such stem weevil (Apion corchori), a rapid and efficient detection technique is essential. jute semilooper (Anomis sabulifera), Bihar hairy caterpillar Conventional methods such as plating method or direct (Spilosoma obliqua) etc. (Ramasubramanian et al. 2010). culture method which are based on cultivation on selective Earlier reports suggest that jute pests particularly Bihar media followed by microscopy are time consuming and not hairy caterpillar and semilooper can be controlled by so sensitive. PCR based methods are rapid, sensitive and Beauveria bassiana (Pandit and Som 1988). Apart from reliable. A single primer PCR may fail to amplify some of causing ecological hazards complete reliance on chemical the strains. However, multiplex PCR increases the possi- pesticides becomes a burden on resource poor jute farmers bility of detection as it uses multiple primers. Therefore, in of the Indian subcontinent. Therefore, efforts are on to the present investigation a multiplex PCR protocol was substitute some use of pesticides by low cost biocontrol developed by multiplexing three primers SCA 14, SCA 15 agents. B. bassiana (Balsamo) Vuillemin (Ascomycota: and SCB 9 to detect field released B. bassiana strains from Hypocreales) is an important entomopathogenic fungus. soil as well as foliage of jute field. Using our multiplex Since long it is known to cause white muscardine disease in PCR protocol all the five B. bassiana strains could be de- silkworm (Bombyx mori). Spores of B. bassiana germinate tected from soil and three strains viz., ITCC 6063, ITCC and grow directly through the cuticle to the inner body of 4563 and ITCC 4796 could be detected even from the crop their host, proliferate throughout the insect’s body and foliage after 45 days of spray. eventually kill it. It has been found useful to control many crop pests such as stem borer (Chilo partellus) in maize Keywords Multiplex PCR Á Beauveria bassiana Á Jute Á and sorghum (Maniania 1993; Reddy et al. 2009), leaf SCAR marker Á Endophyte roller (Sylepta derogata) in cotton (Ramesh et al. 1999); beetle (Leptinotarsa decemlineata) in potato (Wraight and Jute is an important fibre crop only second to cotton which Ramos 2002), aphids (Aphis spp.) as well as mites (Am- is mainly grown in the South East Asian countries like blyomma maculatum and A. americanum) in wheat (Hat- India, Bangladesh, Nepal, China, Indonesia, Thailand, ting et al. 2004; Kirkland et al. 2004) etc. Presently many Myanmar and few South American countries. It is used in commercial formulations of B. bassiana are available in making sacks, ropes, bags, carpets, shoes, geo-textiles, the market and its use is increasing. However, as it is a live product and is introduced into a particular ecosystem from outside its survival is always challenged by the native C. Biswas (&) Á P. Dey Á B. S. Gotyal Á S. Satpathy microbial populations. Therefore, monitoring of field re- Central Research Institute for Jute and Allied Fibres (CRIJAF), leased B. bassiana is essential and it requires an efficient Barrackpore, Kolkata 700120, West Bengal, India e-mail: [email protected]; and robust detection technique. Like other fungi, B. [email protected] bassiana is conventionally detected by plating or culturing 123 676 World J Microbiol Biotechnol (2015) 31:675–679 on selective media. But these cultivation methods are time K were added and kept at 37 °C for half an hour and 0.6 consuming and not so sensitive (Biswas et al. 2012). volume of ice cold isopropanol was added. The precipitate However, PCR based methods are rapid, sensitive and re- was again centrifuged at 80009g and the pellet was washed liable in microbial diagnostics (Yamamoto 2002; Baric and with 70 % ethanol and dried at room temperature. Then the Dalla-Via 2004). The technique is recently being applied in DNA pellet was dissolved in TE buffer (10 mmol-1 Tris– detection of B. bassiana (Castrillo et al. 2003; Quesada- HCl, 1.0 mmol-1 EDTA) and stored at -20 °C. Moraga et al. 2006; Ownley et al. 2008). But, a single Genomic DNA was extracted from the young leaves of primer PCR may fail to amplify some of the strains. The both B. bassiana treated as well as untreated jute (C. oli- probability of detection in PCR can be increased by using torius) plant samples collected from the field. 300 mg tis- multiple primers in a single reaction, commonly known as sue was taken from each sample and was surface sterilized multiplex PCR (Loncaric et al. 2008). As many strains of by treating with 0.5 % sodium hypochlorite for 2 min and B. bassiana are used in pest management, multiplex PCR then with 70 % ethyl alcohol for 2 min. Then it was would be more useful for their detection. Therefore, in the thoroughly washed with sterile distilled water. DNA was present investigation a multiplex PCR protocol was de- extracted by using cetyl trimethyl ammonium bromide veloped to detect field released B. bassiana strains from (CTAB) method (Biswas et al. 2012). The sample was soil as well as foliage. crushed properly by adding Polyvinylpyrrolidone (PVP) Beauveria bassiana isolates viz., ITCC 6063, ITCC which helps to dissolve the mucilage present in jute plant. 4512, ITCC 4925, ITCC 4796 and ITCC 4644 used in the The tissues were ground in CTAB and transferred to 500 ll experiment were collected from Indian Type Culture Col- 4 M NaCl which was kept at 60 °C for 1 h with occasional lection (ITCC), Division of Plant Pathology, Indian Agri- stirring. Then equal volume of dichloromethane was added cultural Research Institute, New Delhi. All the fungal and centrifuged at 14,0009g for 15 min. The supernatant cultures were maintained at 25 °C on potato dextrose agar was carefully taken and 5 ll RNAse (5 mg/ml) was added (PDA). The leading C. olitorius variety JRO 524 was used and kept at 37 °C for half an hour. After that equal volume for the field experiments. Seeds were collected from Crop of dichloromethane was added followed by centrifugation Improvement Division, Central Research Institute for Jute at 10,0009g for 15 min. The supernatant was carefully and Allied Fibres, Barrackpore, India. taken out and was precipitated with 0.6 volume of ice cold The conidia of B. bassiana were harvested by scraping isopropanol. The precipitate was again centrifuged at the surface of fungal culture with a sterile camel hair brush 14,0009g and the pellet was washed with 70 % ethanol into a 100 ml glass beaker containing 50 ml sterile distilled and dried at room temperature. Then the DNA pellet was water. The conidial suspension was prepared by mixing the dissolved in TE buffer (10 mM Tris, 1.0 mM EDTA) and solution with a magnetic stirrer for 5 min. The concentra- stored at -20 °C. tion of conidia was adjusted to the desired concentration of For isolation of DNA from B. bassiana, monoconodial 1 9 108 conidia/ml using haemocytometer and a light cultures of all the seven strains were grown in potato microscope (409 magnifications). The conidial concen- dextrose agar (PDA) broth (pH 5.5) for 7 days at tration of B. bassiana @19 108 conidia/ml was chosen 25 ± 1 °C. The mycelia were filtered through Whatman following Biswas et al. (2012). For pest management in No. 1 filter paper. An amount of 500 mg mycelia was jute conidial suspension of the five B. bassiana isolates ground in liquid nitrogen and transferred to DNA extrac- were sprayed in the field where the crop was sown in tion buffer (100 mM Tris, 1.4 M NaCl, 20.0 mM EDTA, 3 9 2.5 m plots with four replications for each isolate in 4 % CTAB (Murray and Thompson 1980) and incubated at randomized block design along with untreated check. The 60 °C for 1 h with occasional stirring. Equal volume of crop was grown with recommended package of practices dichloromethane was added followed by centrifugation at without using any plant protection chemical. 14,0009g for 15 min. The supernatant was carefully taken Chemical lysis buffer was prepared by using CTAB out and was precipitated with 0.6 volume of ice cold iso- method (Robe et al. 2003) with major modifications. 400 mg propanol. The precipitate was again centrifuged at soil sample was grinded with liquid nitrogen and 1 ml of 14,0009g and the pellet was washed with 70 % ethanol preheated (65 °C) chemical lysis buffer was added in the and dried at room temperature. Then the DNA pellet was sample. Extraction lysis buffer was modified by adding dissolved in TE buffer (10 mM Tris, 1.0 mM EDTA) and 10 % CTAB, 40 mM Tris, 1.4 M NaCl, 20.0 mM EDTA, stored at -20 °C. The DNA yield obtained by modified 0.2 % b-marcaptoethanol and 3 % w/v PVP (Sigma). CTAB method from different soil samples varied from 370 Sample was then incubated at 60 °C for 1 h.