16S rRNA SEQUENCE ANALYSIS OF CAUSAL ORGANISM OF FLACHERIE IN MUGA SILKWORM (ANTHERAEA ASSAMA WW.) Merrylina Marak 1 , Pranab Dutta 2 and L. C. Dutta 3 1&3 Department of Sericulture, Assam Agricultural University, Jorhat-785 013, India. 2 Department of Plant Pathology, Assam Agricultural University, Jorhat-785 013, India. e-mail : marakmerrylina19@gmail.com (Accepted 17 June 2016) ABSTRACT : Muga silkworm is a polyphagous, semi-domestic and multivoltine sericigenous insect. It completes half of its lifecycle outdoors so it responses to many biotic and abiotic environmental factors, which makes it susceptible to various diseases. Amongst these Flacherie, a bacterial disease is a prime one causing around 14-40% crop loss. Bacteria causing flacherie in muga silkworm includes genus Bacillus, Pseudomonas etc. A pathogenic bacterium was isolated from diseased silkworm cadavers. The bacterium was identified on the basis of its physiological and biochemical properties and the results of sequence analysis of its 16S rRNA gene. The bacterium was identified as Bacillus subtilis. Key words : Muga silkworm, Flacherie disease, Pathogenic bacterium, Bacillus subtilis. INTRODUCTION Muga silkworm, Antheraea assama Westwood (Saturniidae : Lepidoptera) is an important bioresource of India and mainly confined to the Brahmaputra valley of Assam and foothills of East Garo hills of Meghalaya. This confinement of the silkworm to this region is maybe due to salubrious climatic regiments prevailing here because of which it was awarded geographical indication (GI) registration in 2007. The silk spun by the insect is called “Muga” in Assamese for golden yellow/amber colour of the cocoon. India in the global silk market is a noted player for being the second largest producer of silk after China. It is renowned worldwide for the muga silk in particular. Outdoor rearing in natural conditions is indispensable for the muga silkworm; therefore it is subjected to a number of diseases leading to crop loss (Choudhury, 1981; Thangavelu et al, 1988; Das et al, 2005). Among all the silkworm diseases, flacherie of muga silkworm is an ill-defined disease because of lack of precise knowledge; perhaps because of the complexity of symptoms and causal agents involved. Flacherie is an infectious bacterial disease though some authors like to categorize it as a viral disease (Choudhury, 1981). The disease starts with the larvae becoming weak, lethargic and stop to feed. As the disease progresses, the larvae shows retarded growth, with body swelling develops diarrhoea vomiting its gut juices. They excrete semi-solid excreta, which is sticky with chain of faecal beads being observed sometimes (Chakravorty et al, 2007). Bacteria of the genus Bacillus (CMER&TI & AAU Project Progress Report, 2006-07), Pseudomonas (Dutta et al, 2007), Streptococcus, Aeromonas (Unni et al, 2011) have been associated with the disease. Therefore, the present investigation was carried out to identify the causal organism of the disease and confirm it through 16S rRNA sequence analysis. MATERIALS AND METHODS Source of inoculum Samples were collected from different locations i.e. farms in and around Jorhat district. The pathogenic bacteria were isolated from freshly infected diseased samples. Purification of the isolated culture was done through streak plate technique. Isolation of associated organism from diseased sample Isolation of the disease causative agent of flacherie disease was done from freshly collected disease samples showing typical symptoms of flacherie disease of muga silkworm. The infected larvae were washed thoroughly in sterile water. The infected tissues along with adjacent small unaffected tissue were cut into small pieces (2–5 mm squares) and by using flame-sterilized forceps, sections were transferred to sterile petridishes containing 0.1% mercuric chloride (HgCl 2 ) solution used for surface sterilization of the tissues for a period of 30–60 sec. Sections were again washed in sterile water to remove traces of HgCl 2 The sterilized pieces were aseptically transferred to slants containing PDA medium,@ 1 section per slant and incubated at room temperatures (25–27°C). J. Exp. Zool. India Vol. 19, No. 2, pp. 683-687, 2016 www.connectjournals.com/jez ISSN 0972-0030