PREVALENCE OF FLACHERIE DISEASE AND PATHOGENICITY OF ISOLATED PATHOGENS IN SILKWORM, BOMBYX MORI (L.) UNDER DIFFERENT ENVIRONMENTAL CONDITIONS

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1 Agric. Sci. Digest., 33 (4) : , 2013 DOI /j AGRICULTURAL RESEARCH COMMUNICATION CENTRE / indianjournals.com PREVALENCE OF FLACHERIE DISEASE AND PATHOGENICITY OF ISOLATED PATHOGENS IN SILKWORM, BOMBYX MORI (L.) UNDER DIFFERENT ENVIRONMENTAL CONDITIONS T. Selvakumar Silkworm Seed Production Centre, NSSO., Central Silk Board,K.R. Nagar , India Received: Accepted: ABSTRACT Flacherie is one of the diseases in silkworm caused by different bacteria and viruses. Flacherie diseased silkworm larvae were collected from different sericultural areas of Karnataka during different seasons and the pathogens were isolated. The data revealed that prevalence of flacherie was high in summer ( %) followed by rainy ( %) and winter ( %) seasons. It was also observed that the flacherie larvae were infected individually with bacteria ( %) or viruses ( %) and combined infections of both ( %). Streptococcus faecalis Andrewes and Horder, Staphylococcus aureus Rosenbach, Serratia marcescens Bizio and Bacillus thuringiensis Berliner were found pathogenic to silkworm. BmIFV and BmDNV1 were detected in the midgut larval samples and BmNPV was noticed in very few haemolymph samples. value was calculated for the pathogenic bacteria viz., S. faecalis, S. aureus and S. marcescens under three different environmental conditions (E 1, E 2 and E 3 ) and the value against these bacteria was , and cells/ml, respectively at E 3. could not be made for the above bacteria at E 1 and E 2 environment as there was less than 50% mortality even in high concentration. In the case of B. thuringiensis, the mortality was due to toxicity and the value was , and cells/ml at E 1, E 2 and E 3, respectively. was also calculated against all the viruses (BmIFV, BmDNV1 and BmNPV) and it was dilutions, dilutions and polyhedra/ ml (E 1 ), dilutions, dilutions and polyhedra/ml (E 2 ) and dilutions, dilutions and polyhedra/ml (E 3 ), respectively. Key words: Environmental conditions, Flacherie, Pathogenicity, Prevalence, Silkworm. INTRODUCTION The success of silkworm rearing depends upon the protection of crops from diseases and parasitoids. The major diseases affecting silkworm are flacherie, grasserie, muscardine and pebrine. The diseases in silkworm cause an estimated crop loss of 27 35% with cocoon yield loss of 11 15kg / 100 dfls (Selvakumar et al., 2002). It is reported that flacherie is caused by different species of bacteria (Nakasuji and Kodama, 1970; Samson, 1987; Selvakumar et al., 1995; Selvakumar et al., 1999), vi ruses (Shimi zu, 1975; Watanabe, 1994; Selvakumar, 2003) and their mixed infections (Matsumoto et al., 1985; Sivaprasad et al., 2000; Selvakumar et al., 2009). Under different physiologi cal stress conditions, the multiplication of bacteria and viruses enhances the development of flacherie. Adverse environmental conditions such as high temperature and humidity (Inoue, 1972; Miyajima, 1978), polluted air (Kanke et al., 1987) and starvation (Samson et al., 1981) are considered important predisposing factors for flacherie and cocoon crop loss. Vijaya Kumari et al. (2001) reported that fourth and fifth instar silkworms are known to be sensitive to temperature and with the rise in temperature the metabolic activities are accelerated while they slacken, as the temperature goes down. Temperature higher or lower than 25 C acts as a stress factor and increases the susceptibility of silkworm to viral infections (Steinhaus, 1958). If temperature and humidity are extremely high, the susceptibility of silkworm larvae increases to viral infections (Venugopala Pillai and Krishnaswamy, 1980). Kato

2 254 AGRICULTURAL SCIENCE DIGEST et al. (1989) reported that exposure of fifth instar larvae to high temperature causes decline in their survival rate. In the present study, the observations on the prevalence of flacherie, different pathogens causing flacherie disease and the pathogenicity of different pathogens causing flacherie under different environmental conditions have been discussed and out come of the study can be utilized for the development of flacherie under different environmental and nutritional conditions which in turn will help in prevention of flacherie in silkworm. MATERIALS AND METHODS Collection of flacherie diseased silkworm larvae: Silkworm larvae exhibiting typical symptoms of flacherie were collected from 120 farmers crops covering 10 farmers in each sericultural areas viz., Kunigal (multivoltine silkworm seed area), Anekal (bivoltine silkworm seed area), Ramanagar and Chamarajanagar (commercial sericultural areas) during three well defined seasons viz., summer, rainy and winter. The number of crops infected with bacterial flacherie, viral flacherie and their mixed infections were also recorded. Isolation of pathogens: The collected flacherie diseased silkworm larvae were sterilized and their midgut and haemolymph were utilized for the isolation of different bacteria. Midgut homogenate were also subjected to agar gel immunodiffusion test for the detection of BmIFV and BmDNV1 using antiserum of BmIFV and BmDNV1. The haemolymph of typical flacherie diseased silkworm larvae were also observed for any other pathogens like BmNPV, N. bombycis or any fungus. Identification of pathogens causing flacherie disease: The different isolated bacteria at cells/ml were inoculated to second instar bivoltine silkworm larvae and reared at normal room temperature (25 ± 1ºC and 75 ± 5% relative humidity). The 10% midgut homogenate (10g midgut tissue+ 90 ml steri le water) of silkworm infected with BmIFV and BmDNV1 and purified polyhedra of BmNPV (identified viruses) were serially diluted and inoculated to second instar bivoltine silkworm larvae. All the inoculated larval batches of both bacteria and viruses were reared for 15 days and the cause of mortality due to inoculated pathogens were confirmed as per Koch s postulates. The only pathogenic bacteria causing mortality to silkworm viz., Streptococcus sp. (15-17%), Staphylococcus sp. (15-16%), Serratia sp. (12-14%) and Bacillus sp. (100%) isolated as above was subjected to identification upto species level by their colony, morphological, selective biochemical and other culture characteristics as mentioned in Bergeys manual of Systematic Bacteriology (Brenner, 1984; Schleifer, 1986; Sneath, 1986). Agar gel immuno diffusion test was conducted to detect viruses in the midgut homogenate. of bacteria / viruses in silkworm under different environmental conditions: Different sets of silkworm larvae were inoculated by per os method with specific inoculum of specific dosage ( to cells/ml) of actively multiplying young culture of different bacteria such as S. faecalis, S. aureus, S. marcescens and old cultures of B. thuringiensis, viruses viz., BmNPV ( to polyhedras/ml), BmIFV and BmDNV1 ( to dilutions) immediately after 1 st moult. Normal control group was maintained separately without inoculum. Each treatment / control had three replications of 100 larvae. The larvae were reared for 15 days and mortality due to the respective pathogens was recorded. The values for different bacteria & viruses and IC 50 & IC 10 values for viruses were calculated under different environmental conditions viz., E1, E2 and E3 (E1: 25 ± 1 C and 75 ± 5 % RH; E2: 28 ± 1 C and 60 ± 5 % RH; E3: Fluctuating temperature and humidity of 25 & 28 ± 1 C and 75 & 60 ± 5 % RH for 12 h in each day) following Probit analysis method (Finney, 1971). The required environmental conditions were maintained using thermostatic controlled heater and humidistat controlled humidifier. RESULTS AND DISCUSSION The data presented in Table 1 revealed that the prevalence of both bacterial and viral flacherie was more during summer ( %) followed by rainy ( %) and winter ( %) seasons. The crops were also infected individually with bacteria ( %) / viruses ( %) or with combined infections of both bacteria and viruses ( %) (Table 1). A total of 66 isolates of bacteria were isolated and all the bacterial isolates were classified upto

causal agents in different sericultural areas during

5 genus level as Streptococcus sp., Staphylococcus sp.

(Table 1) based on their cultural and morphological

, Staphylococcus sp., Serratia sp. and Bacillus sp.

species level as Streptococcus faecali s,

thuringiensis by following specific biochemical and

Mi crococcus sp. was not pathogenic to silkworm.

samples by agarose gel immuno diffusion test.

fluctuating temperature and relative humidity (E 3 )

aureus and S. marcescens were 5.57 10 7, 3.

could not be worked out for the above bacteria as there

of bacteria at E1 and E2 environment even upto 15 days

This indicates that the environmental factors influence

The results of studies on the i nfluence of

pathogens are influenced by the fluctuating temperature

The influence of optimum temperature and humidity (E1)

3 Vol. 33, No. 4, TABLE 1: Prevalence of flacherie and their causal agents in different sericultural areas during different seasons. bacterial isolates genus level as Streptococcus sp., Staphylococcus sp., Serratia sp., Bacillus sp. and Micrococcus sp. (Table 1) based on their cultural and morphological characteristics. Among them, Streptococcus sp., Staphylococcus sp., Serratia sp. and Bacillus sp. were found pathogenic to silkworm and identified upto species level as Streptococcus faecali s, Staphylococcus aureus, Serratia marcescens and Bacillus thuringiensis by following specific biochemical and other characteristics mentioned in Bergey s Manual. Mi crococcus sp. was not pathogenic to silkworm. BmIFV and BmDNV1 were detected in the midgut larval samples by agarose gel immuno diffusion test. In few samples, the BmNPV was also observed in the haemolymph. The values calculated for the mortality of silkworm at fluctuating temperature and relative humidity (E 3 ) for the three pathogenic bacteria viz., S. faecalis, S. aureus and S. marcescens were , and cells/ml, respectively (Table 2). could not be worked out for the above bacteria as there was less than 50 % mortality even in high concentration of bacteria at E1 and E2 environment even upto 15 days of rearing. This indicates that the environmental factors influence the pathogenicity and cause of mortality. The results of studies on the i nfluence of environmental factors on the individual pathogens viz., S. faecalis, S. aureus and S. marcescens indicate that the infectivity of these pathogens are influenced by the fluctuating temperature and relative humidity (E3) conditions. The influence of optimum temperature and humidity (E1) and constant high temperature of 28 C and low humidity (E2) conditions are low. However, the influence of high temperature and low humidity (E2) is comparatively high. But in the case of B. thuringiensis, these environmental factors have little impact on the mortality as the mortality itself is due to toxicity and not due to infection by the bacteria. The values at different environmental conditions for this bacteria was (E1), (E2) and cells/ml (E3) (Table 2). It is also observed that the virus, BmIFV caused mortality in silkworm under E1 conditions, at dilution s of 10% stock inoculum while at E2 conditions at and E3

4 256 AGRICULTURAL SCIENCE DIGEST TABLE 2: values against different bacteria and viruses in silkworm B. mori at different environmental conditions. Probit values values against different bacteria and viruses under different environmental conditions Bacteria Viruses S. faecalis S. aureus S. marcescens B. thuringiensis BmNPV BmIFV BmDNV1 E1: 25 C with 75 ± 5% RH 0.00* 0.00* 0.00* Upper fiducial limits Lower fiducial limits Regression Equation Y= 2.54 Y= 1.75 Y = 8.29 Y = x x x x E2: 28 C with 60 ± 5% RH 0.00* 0.00* 0.00* Upper fiducial limits Lower fiducial limits Regression Equation Y= 2.73 Y= 2.14 Y = 7.11 Y = x x x x T3: 25 C & 28 C with 75 & 60 ± 5 % RH Upper fiducial limits Lower fiducial limits Regression Equation Y= Y= Y= 0.71 Y= 2.95 Y= 2.04 Y = 8.90 Y = x x x x x x x *: could not be worked out as there was less than 50 % mortality even in high concentration of bacteria at E1 & E2 environment even upto 15 days of rearing. conditions at dilution s of stock inoculum. Apart from, infectivity concentrations (IC 50,) were also calculated for the viruses. The IC 50 value for BmIFV at E1, E2 and E3 conditions were , and dilutions of stock inoculum, respectively (Table 3). It indicates the requirement of lower inoculum dosage to cause infection at fluctuating temperature and humidity (E3) and high temperature (28 C) and low humidity of 60 ± 5 % (E2) compared to optimum condition (E1). Similar were the observations to obtain IC 30 values at E1, E2 and E3 treatment conditions. The inoculums dosage required at E1, E2 & E3 conditions to obtain IC 30 values were , & and , & dilutions of stock inoculum, respectively (Table 3). Similarly BmDNV1 caused mortality at dilution of 10 % stock inoculum under E1 conditions while under E2 and E3 conditions, its requirement was and dilutions, respectively (Table 2). The dosage required for infectivity differed under different environmental conditions. Under E1 conditions, the IC 50 dose was while IC 30 was and , respectively. Under E2 and E3 conditions, IC 50 & IC 10 doses were , & (T2) and , & (E3) dilutions, respectively (Table 3). But in the case of BmNPV, the mortality in silkworm was , and polyhedra/ml in E1, E2 and E3 conditions, respectively (Table 2). LC 30 and LC 10 against BmNPV was also recorded and it was , & polyhedra/ml and , & polyhedra/ml under all the T1, E2 and E3 conditions (Table 3) was also comparatively higher at constant high temperature and low humidity (E2) than at optimum temperature and humidity (E1). However, it is different with B. thuringiensis, since the cause of mor tali ty was toxicity only. The impact of environmental factors viz., temperature and humidity (E1, E2 and E3) on the infection of BmIFV and BmDNV1 though observed, is not as significant as on the bacterial pathogens. However, at fluctuating temperature and humidity (E3), the pathogen dose for causing IC 50 infection values were lower than the values of optimum temperature and humidity (E1) indicating higher susceptibility under fluctuating temperature and humidity.

5 TABLE 3: Probit values of different viruses in silkworm under different environmental conditions. T2 T3 T1 T2 T2 T1 T2 T3 T1 Vol. 33, No. 4, 2013 E1: 25 ± 1 C and 75 ± 5 % RH; E2: 28 ± 1 C and 60 ± 5 % RH; E3: 25 & 28 ± 1 C and 75 & 60 ± 5 % for 12 h in each day IC= Infective concentration; LC= Lethal Concentration 257 Si mi lar observati o n of l ow pathogenicity of S. faecalis (Selvakumar et al., 1995; Nataraj u et al., 1999), S. aureus (Nataraju et al., 1999) and S. marcescens (Sevakumar, 2003) at optimum environmental conditi ons and i ncreased pathogeni ci ty of Streptococcus sp., Staphylococcus sp. and S. marcescens under fluctuating temperature and humidity (Sivaprasad et al., 2000) have also been reported. The viruses at their infective concentrations did not cause mortality during the observat i on peri o d of 15 days post inoculation. These observations are true with all the bacterial pathogens viz., S. faecalis, S. aur eus and S. mar cescens ex cept f or B. thuri ngiensis and the viral pathogens viz., BmIFV and BmDNV1. It may be possible as reported by several workers that high temperature may weaken the larvae resulting in their high susceptibility (Miyajima, 1978;). Vijaya Kumari et al. (2001) reported that minimum incidence of flacherie ( %) was obser ved under t he optimum temperature of 25 C and humidity of 70 ± 5 % whereas hi ghest i nci dence of flacherie was about & % under high temperature of 30 C & 80 ± 5 % RH and 35 C & 50 ± 5 % RH, respectively. Inoue (1977) also reported that the multiplication of flacherie virus was less at 21 C and high at 31 C in the silkworm, B. mori. Chishti and Schaf (1990) reported that silkworm reared under controlled envi ronmental conditions developed low level of mortality ( %) due to nuclear polyhedrosis, compared to rearing under un-controlled temperature and humidity which result in high level of mortality ( %).

6 258 AGRICULTURAL SCIENCE DIGEST REFERENCES Brenner, D. J. (1984) In: Bergeys manual of Systematic Bacteriology [J. G. Holt, ed] William s & Wilkins, Baltimore, 1: Chisti, M. Z. and Sehaf, K. A. (1990) Indian J. Seric., 29: Finney, D. J. (1971) Probit analysis. Third Edition. S. Chand & Company Ltd. New Delhi, pp Inoue, H. (1972) Appl. Entomol. Zool., 7: Inoue, H. (1977) J. Seric. Sci. Jpn., 46: Kanke, E., Iwasita, Y. and Iwasaki, K. (1987) Bull. Coll. Agric. Utsunomiya Uni., Jpn., 13: Kato, M. O., NAGAYASU, N., Hara, W. and Watanabe, A. (1989) Proceedings of VI th International Congress of Sabro, 11: Kobayashi, M., Inagaki, S. and Kawase, S. (1981) J. Invertebr. Pathol., 38: Matsumoto, T., Yafeng Zhu and KAZuhiko K. (1985) J. Seric. Sci. Jpn., 54: Miyajima, S. (1978) Res. Bull. Aichi. Ken. Agri. Res. Centr. Series D. Seric. Jpn., 9: Nakasuji, Y. and Kodama, R. (1970) J. Seric. Sci. Jpn., 39: 187. Nataraju, B., Sivaprasad, V. and Datta, R. K. (1999) Indian J. Seric., 38: Ssmson, M. V. (1987) Ph. D Thesis, University of Mysore, Mysore. pp. 46: Ssmson, M. V., Nataraju, B., Baig, M. and Krishnaswamy, S. (1981) Indian J. Seric., 20: Savanurmath, C. J., Basavarajappa, S., Hinchigeri, S. B., Ingalhalli, S. S., Singh, K. K. and Sanakal, R. D. (1992) National Conference on Mulberry Sericulture Research, CSR & TI., Mysore, Dec Schleifer, K. H. (1986) In: Bergeys manual of Systematic Bacteriology [J. G. ed] Holt, William s and Wilkins, Baltimore, pp. Selvakumar, T. (2003) Ph. D. Thesis, University of Mysore, Mysore, India. Selvakumar, T., Nataraju, B. and Utsumi, S. (1995) Current Technology Seminar on Silkworm Disease Management, Silkworm Rearing Technology and Mulberry Pathology, CSR & TI., Berhampore, West Bengal, India, Oct , p. 6. Selvakumar, T., Nataraju, B. and Datta, R. K. (1999) Indian J. Seric., 38: Selvakumar, T., Nataraju, B., Balavenkatasubbaiah, M., Sivaprasad, V., Baig, M., Virendra Kumar, Sharma, S.D., Thiagarajan, V. and Datta, R. K. (2002) In: Advances in Indian sericulture research (Edt. by Dandin S. B. & Gupta V. P.). pp Selvakumar, T., Prabha, B.S., Nataraju, B., Balavenkatasubbaiah, M., Thiagarajan, V. and Datta, R. K. (2009) Bull. Ind. Acad. Seri., 13: Shinmizu, T. (1975) J. Seric. Sci. Jpn., 44: 45. Sivaprasad, V., Selvakumar, T., Nataraju, B. and Datta, R. K. (2000) Sericologia, 40 (Suppli.), 122. Sneath, P. H. A. (1986) Bergeys manual of Systematic Bacteriology (Eds.) [J. G.ed] Holt, William s and Wilkins, Baltimore, 2: Steinhaus, E. A. (1958) Proc. Tenth. Int. Congr. Entomol., 4: Venugopala Pillai, S. and Krishnaswamy, S. (1980) Proceedings of Sericultural Symposium and Seminar, TNAU, Vijaya Kumari, K. M., Balavenkatasubbaiah, M., Rajan, R. K., Himantharaj, M. T., Natraju, B. and Rekha, M. (2001) Int. J. Ind. Entomol., 3: Watanabe, H. (1964) J. Seric. Sci. Jpn., 33: Watanabe, H. (1994) Indian J. Seric., 33:

K.P. Kiran Kumar and S. Sankar Naik ISSN

K.P. Kiran Kumar and S. Sankar Naik ISSN International Journal of Integrative sciences, Innovation and Technology (A Peer Review E-3 Journal of Science Innovation Technology) Section A Basic Sciences; Section B Applied and Technological Sciences;