2016 RELS ISSN: Res. Environ. Life Sci. 9(8) (2016)

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1 2016 RELS ISSN: Res. Environ. Life Sci. 9(8) (2016) Efficacy of antagonistic soil bacteria in management of subterranean termites (Isoptera) Y.S. Rakshiya, M.K. Verma and S.S. Sindhu* Department of Microbiology, CCS Haryana Agricultural University, Hisar, India * (Received: September 28, 2015; Revised received: June 01, 2016; Accepted: June 07, 2016) Abstract: Subterranean termites are important pest of the Indian subcontinent and cause extensive damage to major agricultural crops and forest plantation trees. Due to long persistence of synthetic insecticides in soil, entry of residual toxic chemicals in food chain and other environmental concerns, attempts are being made to characterize microorganisms for biological control of termites. In this study, 220 bacterial isolates were obtained from the nest soil collected from termite mounds. Sixty three bacterial isolates along with eight reference strains were found to kill the termites under in vitro conditions at 2 days of observation and killing frequency of different bacterial isolates varied from 5 to 90 percent. Nine isolates i.e., KPM35, KBM79, PBM106, PPM115, PPM126, PPM147, PPM195, PPM203, PPM204 were found to cause more than 60% killing of termites in Petri plates even on 2 nd days of observation. Six bacterial isolates i.e., PPM119, PPM123, PPM167, PPM194, PPM199 and PPM203 caused even 100 percent killing at 5 days of observation. At 10 days of incubation, forty eight bacterial isolates caused 90 to 100 per cent killing of termites. To explore the mechanism of termite killing, all the 71 bacterial isolates were tested for expression of proteolytic, lipolytic and chitinolytic activities. Only 20 bacterial isolates expressed proteolytic activity on modified casein agar medium and proteolytic activity varied from 1.24 to 2.64 among different bacterial isolates. Lipolytic activity was expressed by 46 bacterial isolates on tributyrin supplemented medium plates and fourteen isolates showed very high lipolytic activity. The range of lipolytic activity varied from 1.16 to Only 21 bacterial isolates expressed chitinolytic activity on plates containing colloidal chitin and it varied from 1.15 to 2.96 in different bacterial isolates. Nine bacterial isolates i.e., KPM15, KPM30, KPM31, KPM32, KPM35, PPM94, PPM100, PPM119 and WPS73 showed all the three enzyme activities. Some bacterial isolates i.e., NBM8, KPM72, PPM147, PPM162, PPM167, PPM203 and PPM204, which showed high termite killing ability, did not show any of the three enzyme activities. Thus, termite killing was not correlated with any of these enzyme activities and probably, some other compound such as toxin, hydrocyanic acid or siderophore along with these enzymes may be involved in killing of termites. Key words: Termites, Biological control, Bacteria, Proteolytic, Lipolytic, Chitinolytic activities Introduction Termites are soft-bodied, polymorphic, cellulose-eating social insects living in large communities and over 2650 known species of termites have been described worldwide (Wood, 1988; Kambhampati and Eggleton, 2000). The individuals are differentiated morphologically into distinct forms, i.e. reproductives (the king and queen), workers and soldiers. The workers are most numerous individuals in a colony and perform the work of nest building and repair, foraging and feeding. Termites play an important role in the tropical ecosystems by decomposing dead wood and other plant material rich in cellulose (Wood and Sands, 1978; Wood and Johnson, 1986; Abe, 1995; Bajya et al., 2015). Termites also provide a possible input of nitrogen through symbiont fixation (Wood and Sands, 1978; Collins, 1984) and also contribute to carbon-flux (Jones, 1990). They also release a high amount of methane in the atmosphere worldwide. Due to the feeding function, the worker caste causes the wide spread destruction resulting into major economic losses in tropical and subtropical areas. A number of subterranean termite species destroy valuable agricultural/ornamental crops, live forest trees and even wooden structures, causing significant losses annually throughout the world. About 300 species have been reported to cause significant damage to agricultural crops and have been recorded as pests (Su and Scheffrahn, 1998). Species of Microtermes and Odontotermes have been found to damage different crops like sugarcane, wheat, barley, maize, vegetables, garden crops, valuable ornamental crops and even forest trees (Lai et al., 1983; Tamashiro et al., 1987). The estimated damage to wood and wood products caused by subterranean termites (family; Rhinotermitidae) annually in the United States is reported to exceed $750 million to $1 billion (Su and Scheffrahn, 1990). In India, they cause 15-25% yield loss in various crops leading to a net loss of 1478 million rupees. Therefore, the challenge of controlling these destructive pests is enormous in most developing countries (Raina et al., 2001). For the control of termites in agriculture, synthetic insecticides such as chlorpyrifos, bifenthrin, imidacloprid, endosulfan and lindane are currently being used (Su et al., 1999: Mahapatro and Kumar, 2015). Seed treatment with chlorpyrifos, lindane or thiomethoxam resulted in lesser termite damage in cotton, maize, rice, sorghum and sugarcane crop, and found effective against three species of termites Trinernitermes trinervius, Odontotermes smeathmani and Amitermea evuncifer (Bhanot et al., 1991; Bhanot and Singal, 2007). However, the indiscriminate use of pesticides to control the pests have increased the resistance in insects/termites and has resulted in contamination of ground water and soil along with destruction of non-target entomofauna including natural predators, parasitoids and pollinators. Thus, excessive use of these toxicants has generated several problems including safety risks for humans and domestic animals because of their long persistence in soil and due to the entry of residual toxic chemicals in food chain. Therefore, there is an urgent need for development of environment-friendly, microbe-based insecticides which act differently from known pesticide chemicals (Ruiu et al., 2013; Ratna Kumari et al., 2014). Several microbes including bacteria, viruses, fungi, protozoa and nematodes have been identified as potential pathogens of termites which could be developed as biological control agents against termites (Grace, 1997; Sindhu et al., 2011; Lacey et al., 2015; Ruiu, 2015). The moist and warm microenvironment preferred by subterranean termites supports the growth of epizootics and thus enhances the Research in Environment and Life Sciences 949 August, 2016

2 potential for biological control (Verma et al., 2009; Sindhu et al., 2011). Khan et al. (1992) reported that mortality of M. championi, H. indicola and Coptotermes heimi (Rhinotermitidae) by the pathogenic Pseudomonas aeruginosa (Schroeter) ranged from per cent seven days post-inoculation to per cent 25 days post-inoculation in the laboratory. Connick et al. (2001) reported that Serratia marcescens isolate T8 was highly virulent to the C. formosanus and termite mortality was 24 per cent by 2 days and 99 per cent after 19 days of the experiment. Other bacterial isolates involved in mortality of termites included Acinetobacter calcoaceticus, Aeromonas caviae, Alcaligenes latus, Arthrobacter sp., Bacillus sp., Chromobacterium sp., Corynebacterium urealyticum, Enterobacter gergoviae, Micrococcus, Neisseria, Pseudomonas and Rhizobium radiobacter (Osbrink et al., 2005; Kanchana Devi et al., 2007, 2009; Singh, 2007). Termites are also susceptible to infection by the bacterium Serratia marcescens, Bacillus thuringiensis and Pseudomonas aeruginosa (Khan et al., 1985). Regarding control of termites using microorganisms, very little work has been done in Northern plains of India. In this study, antagonistic bacterial isolates were obtained from the nest soil collected from termite mounds, which killed the termites under in vitro conditions. These termite killing bacterial isolates were screened for different enzyme activities to explore the mechanism of termite killing. Materials and Methods Collection of samples: Soil samples from termite nest (mound) were collected from eight different termite colonies located at different sites in the farm area and campus of CCS Haryana Agricultural University, Hisar. The surface soil was removed and mounds were dug (up to mm) with the help of spade. Nest soil samples were taken from different areas of the particular mound and the composite samples were prepared by mixing the soil. Isolation of bacteria from termite nest soil: Ten grams of each composite soil sample, was dissolved in 90 ml water blank and further serial dilutions were made in 9.0 ml sterilized water blank. Diluted suspension (0.1 ml) of 10-3 to 10-5 dilutions of each sample was plated on pre-labelled nutrient agar, soil extract agar and King s B medium plates. The plates were incubated at 28±2 C in BOD incubator for 3-4 days and total counts of bacteria were determined as colony forming units (CFU) per gram of soil. Based on variation in colony morphology and pigment production, 220 bacterial isolates were streaked on respective medium plates. Purified bacterial isolates were transferred on Luria Bertani (LB) agar slants. Eight reference bacterial strains were procured from the Department of Microbiology, CCS Haryana Agricultural University, Hisar. Bacterial strains were maintained by periodic transfer on LB agar slants (Sambrook et al., 1989). Liquid cultures of all the isolates were preserved in 50% glycerol at -20 C. Collection of termite for pathogenic interaction with bacterial isolates: Termites belonging to genus Odontotermes were collected routinely from different sites to study antagonistic interactions of bacterial strains with the termites. Soil from termite colony along with live termites was collected in plastic bucket traps. The termites were maintained on water soaked filter papers in plastic containers ( cm size) at 25±2 0 C and ~100% relative humidity. Termites were identified using the key of soldier identification (Scheffrahn and Su, 1994). Fifty Research in Environment and Life Sciences 950 termites (soldiers and workers class) were picked up manually and subsequently transferred to the Petri plates containing filter paper discs impregnated with bacterial growth suspension. Preparation of bacterial culture: Selected bacterial isolates were grown in LB broth by transferring the pure culture aseptically into the sterilized liquid broth in 150 ml Erlenmeyer flasks containing 50 ml LB broth medium. The inoculated flasks were incubated for 2 days at 28±2 0 C on shaker with 200 rotations per minute. The bacterial population in the selected cultures varied from to colony forming units (CFU) per ml of the medium broth as determined by plating on nutrient agar medium. These cultures were used to test the termite killing activity and for preparation of bacterial cell free supernatant. Sterilization of filter paper, nest soil and saw dust: Two types of filter papers (180 mm, 90 mm diameter) were placed in large size Petri plate ( mm size) and small size petri plate ( mm size) and moistened with water. The Petri plates were wrapped with aluminium foil. Soil collected from termite s nest was grounded with pestle mortar, put into bucket (2 litre capacity) and it was covered with aluminium foil. Similarly, saw dust was kept in 2 litre flask and covered with aluminium foil. Filter papers, nest soil and saw dust were sterilized in the autoclave at 15 psi pressure for 20 minutes. Antagonistic interactions of whole cell broth of bacterial isolates on local soil termites: Log phase growing bacterial growth suspension (2.0 ml), containing 2.4 x 10 9 to 7.6 x c.f.u./ ml, was added on sterilized filter paper discs in Petri plates. Worker and soldier castes of termites (fifty termites) belonging to genus Odontotermes, were added manually in each Petri plate. The double layer of filter paper was used as a bed for termites. After moistening the Whatman paper with 2 ml of 48 hour-old bacterial culture suspension, Petri dishes were covered and incubated at 25±2 0 C with 100% relative humidity. Termites were allowed to walk over the bacterial-inoculated filter paper. Observations for killing of termites were recorded at 2, 5 and 10 days. The termites were considered dead if they did not move and did not respond detectably to tapping of the Petri dish. Dead termites were removed from the Petri plates and placed in separate sterilized Petri plates. This enabled the calculation of total mortality (i.e. the number of dead termites over a period of 2, 5 and 10 days) from exposure to different bacterial cultures. A Petri dish with termites and filter paper moistened with sterile LB medium broth served as the control. Screening of bacterial isolates for enzymatic activities: Selected bacterial isolates were screened for lipolytic, proteolytic and chitinolytic activities by plate assay method. (i) Lipolytic activity: Qualitative detection of lipase production was done by growing bacterial isolates on TDYA medium (supplemented with 1.0% tributyrin) plates (Kasana et al., 2002). All the selected bacterial isolates were grown overnight on LB medium slants to log phase conditions. The growth from each culture was harvested in 2.0 ml sterile water. The growth suspension (10.0 µl) of each culture was spotted on tributyrin agar medium plate. The plates were incubated at 28±2 0 C for 2-3 days. Bacterial isolates showing clearance zone (CZ) around colonies (CS, colony size) indicated the production of lipase. Efficiency of all the isolates was calculated based on the ratio of diameter of clearance zone to the colony size. August, 2016

3 (ii) Proteolytic activity: Qualitative detection of protease production production of protease. Efficiency of all the strains was calculated was done by growing bacterial isolates on casein agar medium based on the ratio of diameter of clearance zone to the colony size. plates (Lawrence and Sanderson, 1969). The bacterial growth (iii) Chitinolytic activity: Chitin agar plate was used for the screening from each of the isolate was harvested in 2.0 ml sterile water. The of chitinolytic microorganisms and the plates were observed for a growth suspension (10.0µl) of each culture was spotted on casein clearing zone surrounding the colony of microorganism (Cody, 1989). agar medium plate. The plates were incubated at 28±2 0 C for 2 Calcoflour white incorporated medium is more sensitive for plate days. After 48 hours of incubation, the plates were flooded with 1% clearing assays and chitinolytic microorganisms were directly observed mercuric chloride solution. Colonies showing clearance zone (CZ) by the formation of distinct clearing zone within 4-5 days of incubation around the growth of colonies (CS, colony size) indicated the on the substantially darker background of the medium (Vaidya et al., Table-1: Total count of bacteria in termite nest soil on different media 2003). The bacterial growth from each of the selected isolate was harvested in 2.0 ml sterile water. The growth suspension (10.0 µl) of Bacterial colonies (x 10 6 /g soil) each culture was spotted on minimal medium supplemented with Sample no. Nutrient agar Soil extract King s B colloidal chitin and chitin binding fluorescent calcoflour white M2R dye medium agar medium medium (NAM) (SEM) (KBM) (0.001%, w/v). The plates were incubated for 28±2 0 C for 4-5 days. After incubation, chitinase producing strains were selected on the I II basis of clearance zone formed when seen under UV light. The III diameter of the colony size and clearance zone of each bacterial IV isolate was measured (mm). Efficiency of all the strains was calculated V based on the ratio of diameter of clearance zone to the colony size. VI Results and Discussion VII VIII Isolation of bacteria from termite nest soil: The serial dilutions of the eight soil samples collected from different termite nests were The values represents colony forming units (CFU) per gram of the soil plated on prelabelled nutrient agar, soil extract agar and King s B Table-2: Termite killing efficiency of different bacterial cultures at 2 days of observation Killing frequency (%) Table-4: Termite killing efficiency of different bacterial cultures at 10 days of observation Killing frequency (%) Bacterial isolates High level ( %) KPM35, KBM79, PBM106, PPM115, PPM126, PPM147, PPM195, PPM203, PPM204 Intermediate level NBM4, KPM32, NBM65, KPM72, KPM80, KPM85, KAM87, PBM95, PBM97, PBM103, PAM107, PBM108, PPM110, ( %) PPM119, PPM123, KBM139, PPM140, PBM144, PPM145, PPM158, PPM159, PPM162, PPM166, PPM168, PBM183, PPM194, PPM199, PPM205 Low level ( %) NBM8, KPM39, KPM46, KBM77, PPM94, PPM100, PPM129, PPM134, PPM136, PPM151, PPM161, PPM167, PPM172, PPM173, PPM180, NSY3, CBS16, WPS90, NNY60, SB153 < 10.0% killing KPM15, KPM20, PPM43, KPM86, NNY19, WPS73 No killing KPM16, KPM18, KPM23, KPM30, KPM31, KPM57, PPM142, WPS3 Table-3: Termite killing efficiency of different bacterial cultures at 5 days of observation Killing frequency (%) Bacterial isolates High level NBM8, KPM32, KPM35, KPM39, NBM65, KPM72, KBM77, KBM79, KPM85, KPM86, KAM87, PPM94, ( %) PBM95, PBM97, PBM103, PBM106, PAM107, PBM108, PPM110, PPM115, PPM119, PPM123, PPM126, PPM134, PPM136, KBM139, PBM144, PPM147, PPM158, PPM161, PPM162, PPM166, PPM167, PPM168, PBM183, PPM194, PPM195, PPM199, PPM203, PPM204, PPM205 Intermediate level NBM4, KPM16, KPM20, KPM23, KPM30, KPM46, KPM80, PPM100, PPM129, PPM140, PPM145, PPM159, ( %) PPM172, PPM173, PPM180, NSY3, WPS90, NNY60, SB153 Low level ( %) KPM15, KPM18, KPM31, PPM151, CBS16, WPS3, WPS73 < 10.0% killing PPM43, NNY19 No killing KPM57, PPM142 Bacterial isolates High level NBM4, NBM8, KPM16, KPM35, KPM39, NBM65, KPM72, KBM77, KBM79, KPM85, KPM86, KAM87, PPM94, ( %) PBM95, PBM97, PBM103, PBM106, PAM107, PBM108, PPM110, PPM115, PPM119, PPM123, PPM126, PPM134, KBM139, PPM140, PBM144, PPM145, PPM147, PPM151, PPM158, PPM161, PPM162, PPM166, PPM167, PPM168, PPM173, PBM183, PPM194, PPM195, PPM199, PPM203, PPM204, PPM205, SB153, WPS3, NSY3 Intermediate level KPM18, KPM20, KPM23, KPM30, KPM31, KPM32, KPM46, KPM80, PPM100, PPM129, PPM136, PPM159, ( %) PPM180, WPS90, CBS16, NNY19, NNY60, WPS73 Low level ( %) KPM15, PPM172 < 40.0% killing PPM43, KPM57, PPM142 Research in Environment and Life Sciences 951 August, 2016

4 medium plates. The population of bacteria in termite nest soil varied from 1.2 x 10 6 (in sample V) to 90.0 x 10 6 (in sample IV) cfu/g soil on nutrient agar medium plates (Table 1). On soil extract agar medium, the bacterial population varied from 28.3 x 10 6 (in sample VI) to x 10 6 (in sample II) cfu/g soil. The population of bacteria on King s B medium plates varied between 2.6 x 10 6 (in sample VII) to x 10 6 (in sample III) cfu/g soil. The colonies of bacteria from different media plates were selected on the basis of colony morphology, gum production, colony characteristics and pigment production. Typical pigment producing bacteria (yellow, blue, creamish, red or green colour) and Bacillus colonies were observed on all the three media plates. A total of 220 purified bacterial isolates were selected. Screening of bacteria for pathogenic interactions with termites: Two hundred and twenty bacterial isolates along with eight reference bacterial strains were tested for pathogenic interactions with termites in Petri plates. Numbers of the dead termites were counted manually at 2, 5 and 10 days. Sixty three bacterial isolates caused killing of termites and killing frequency of different bacterial isolates varied from 5 to 90 percent at 2 days of observation (Table 2). Nine isolates i.e., KPM35, KBM79, PBM106, PPM115, PPM126, PPM147, PPM195, PPM203, PPM204 were found to cause more than 60% killing of termites in Petri plates even on 2 nd days of observation. Twenty eight isolates showed % killing of termites. At 2 days of observation, eight bacterial cultures did not kill the termites. Six bacterial isolates i.e., PPM119, PPM123, PPM167, PPM194, PPM199 and PPM203 caused even 100 percent killing at 5 days of observation (Table 3). Twenty six isolates were found to kill more than 80.0% of the termites. Nineteen isolates showed % killing of termites whereas seven bacterial isolates caused only % of the termites. No killing of termites was observed with two bacterial isolates KPM57 and PPM 142. At 10 days of incubation, forty eight bacterial isolates caused 90 to 100 per cent killing of termites (Table 4). Nineteen isolates were found to kill 50 to 90 per cent of termites. Even in control plates, 35 per cent killing of termites was observed at 10 th day of incubation, where filter paper soaked with only broth of LB medium was used. In some bacterial treatments, the termites survived even up to 20 days of incubation just like that of control plates. Screening of antagonistic bacterial isolates for proteolytic, chitinolytic and lipolytic activity: Seventy one bacterial isolates obtained from different termite nests and having variation in the termite killing ability at 5 days of observation were screened for expression of proteolytic, lipolytic and chitinolytic activities (Table 5). Growth of different bacterial cultures was spotted on the modified casein agar medium, tributyrin agar medium and minimal medium containing colloidal chitin for determination of proteolytic, lipolytic and chitinolytic activities, respectively. The diameter of zone formation on respective medium plates was scored for estimation of the expression level of enzyme activities. Proteolytic activity on casein agar plates varied from 1.24 to 2.64 among different bacterial isolates and five bacterial isolates i.e., KAM85, PBM103, PAM107, KPM31 and CBS16 showed high proteolytic activity (Table 5). Only 20 bacterial cultures expressed proteolytic activity on modified casein agar medium. Lipolytic activity was expressed by 46 bacterial isolates on tributyrin supplemented Research in Environment and Life Sciences 952 Table-5: Screening of bacterial isolates for proteolytic, chitinolytic and lipolytic activity Bacterial Percent killing Proteolytic Lipolytic Chitinolytic of termites at activity activity activity Isolate 2 days 5 days (CZ/CS) (CZ/CS) (CZ/CS) NBM NBM KPM KPM KPM KPM KPM KPM KPM KPM KPM KPM PPM KPM KPM NBM KPM KBM KBM KPM KAM KPM KAM PPM PBM PBM PPM PBM PBM PAM PBM PPM PPM PPM PPM PPM PPM PPM PPM KBM PPM PPM PBM PPM PPM PPM PPM PPM PPM PPM PPM PPM PPM PPM PPM PPM PBM PPM PPM PPM PPM PPM PPM WPS SB WPS NSY CBS NNY NNY WPS Proteolytic, lipolytic and chitinolytic production efficiency was calculated on the basis of diameter of clearance zone (CZ) to diameter of colony size (CS). - : indicates the absence of enzyme activity August, 2016

5 medium plates and fourteen isolates i.e., KPM16, KPM32, KPM35, KAM85, KPM86, KAM87, PBM108, PPM110, PPM151, PBM183, WPS90, SB153, CBS16 and NNY60 showed very high lipolytic activity. The range of lipolytic activity varied from 1.24 to On the other hand, only 21 bacterial isolates expressed chitinolytic activity on plates containing colloidal chitin and five bacterial isolates i.e., KPM15, KPM30, KPM35, PPM100 and PPM161 showed high chitinolytic activity. Chitinolytic activity varied from 1.15 to 2.96 in different bacterial isolates. Nine isolates showed all the three enzyme activities, 16 isolates showed two enzyme activities and 27 isolates showed only one enzyme activity. Global crop yields are reduced by 20 to 40% annually due to plant pests and diseases (Ratna Kumari et al., 2014). For the control of pests in agriculture, farmers have mostly relied on the intensive application of synthetic pesticides and the global pesticide market is presently growing at a rate of 3.6% per year (BCC Research, 2010). The excessive use of pesticides has generated several problems including insecticide resistance, outbreaks of secondary pests, and safety risks for humans and domestic animals because of the entry of residual toxic chemicals in food chain. These environmental and health concerns, emphasized the need for environmental friendly method of pest management using biological control agents (Ratna Kumari et al., 2014; Ruiu, 2015). Identification of microbes associated with common termite species could lead to optimization of a biological control strategy involving endemic natural pathogens or opportunistic microorganisms that could weaken/destroy the colony (Ferron, 1978; Mahapatro and Kumar, 2015). Our interest was to isolate and characterize bacteria from nest soil and to determine the pathogenic interactions of these bacterial isolates with local Odentotermes termites under laboratory conditions. The population of bacteria in the different termite nest soil varied depending on the location of soil sample and the medium used for plating. Bacterial population varied from 1.2 x 10 6 (in sample V) to x 10 6 cfu/g soil (in sample III), when plated on nutrient agar and King s B medium plates (Table 1). On soil extract agar medium plates, the bacterial population varied from 28.3 x 10 6 (in sample VI) to x 10 6 (in sample II). Variation in pigment production (yellow, blue, creamish, red or green colour), colony morphology, gum production and colony characteristics were observed on all the three media plates. Similar variations in the population of different bacterial species were reported by other workers during isolation of bacteria from different rhizosphere or soil samples on different media. Gupta et al. (1998) isolated rhizobacteria from the rhizotic zones of green gram using 7 selective and 4 non-selective media. A total of 121 bacteria were isolated of which Gram negative bacteria accounted for 65%. The dominant genera were Pseudomonas, Bacillus, Enterobacter, Proteus and Klebsiella. Cottelan et al. (1999) isolated 1131 bacteria from bulk soil and the rhizosphere of soybean. On the basis of fatty acid methyl ester (FAME) identification, 60 isolates representing 15 different species from eight different genera were selected. The most common genera were Pseudomonas, Burkholderia, Bacillus and Alcaligenes. Baig et al. (2002) isolated 105 bacteria from rhizosphere and rhizoplane of groundnut. Out of these, 67% were from the rhizosphere and 33% were from the rhizoplane. Pseudomonas was the most predominant (42%) followed by Bacillus (28%) and Enterobacter (21%). Biocontrol of subterranean termites by microbial pathogens may be facilitated by the warm, humid environment of the colony, their sharing of food (trophollaxis), their intimate contact with nest mates (e.g., allogrooming) and transporting of infected cadavers (Grace, 1994; Sindhu et al., 2011). However, effective biocontrol of termites by means of a single pathogen is unlikely because termites have evolved a complex social structure, formidable immune response and adaptive behaviour toward infected individuals (Logan et al., 1990). In this study, two hundred and twenty bacterial isolates along with eight reference strains were tested for pathogenic interactions with termites in Petri plates. Sixty three bacterial isolates were found to cause killing of termites in Petri plates on 2 nd days of observation (Table 2) and the killing frequency of different bacterial isolates varied from 5 to 90 percent. Ten isolates i.e., KPM35, KBM79, PBM106, PPM115, PPM126, PPM147, PBM185, PPM195, PPM203, PPM204 caused more than 60 per cent killing of termites in Petri plates. Six bacterial isolates i.e., PPM119, PPM123, PPM167, PPM194, PPM199 and PPM203 caused even 100 percent killing of termites at 5 days of observation (Table 3) and twenty six isolates were found to kill more than 80.0 per cent of the termites. At 10 days of incubation, 67.6 per cent bacterial isolates caused 90 to 100 per cent killing of termites (Table 4). Nineteen isolates were found to kill 50 to 90 per cent of termites. Generally, the insect pathogens have developed multiplicity of strategies to infect and kill the insect host, including production of insecticidal toxins, enzymes and metabolites with broader insecticidal spectrum (Ruiu, 2015; Sugio et al., 2015). The termite infection could have originated through direct contact with high bacterial cell concentration or ingestion of the bacterium from the filter paper in these studies. Other workers have also reported killing of termites by infection with bacterial pathogens. Exposure of laboratory colonies of the subterranean species, Reticulitermes flavipes (Kollar) and R. hesperus Banks (Rhinotermitidae) to a mixture of soluble endotoxin, spores and inclusion bodies of Bacillus thuringiensis (Berliner) resulted in greater than 95% mortality after six days (Smythe and Coppel, 1965). Workers of Microcerotermes championi (Snyder) (Termitidae) and Heterotermes indicola (Wasmann) (Rhinotermitidae) suffered 100% mortality within 13 days of exposure to two local strains of B. thuringiensis in laboratory tests (Khan et al., 1977a). Khan et al. (1977b) found that laboratory colonies of M. championi, H. indicola and B. beesoni exposed to suspensions of the spore-forming bacterium Serratia marcescens Bizio succumbed completely 7-13 days following infection. Employing a commercial preparation of Bt (Thuricide-HP concentrate), Khan et al. (1978, 1985) showed that H. indicola, M. championi and Bifiditermes beesoni (Gardner) (Kalotermitidae), were found highly susceptible to infection, exhibiting 100% mortality within six days of exposure. Most of the insecticidal activity of Bacillus thuringiensis is associated with the proteinaceous toxins located in the parasporal crystals and these are referred to as ä-endotoxins. The activated Cry1 proteins (protoxins) causes pore formation, membrane transport disruption and cell lysis leading to insect death (Schnepf et al., 1998). Serratia marcescens caused septicemia and killed the termite Coptotermes formosanus due to the production of proteolytic enzymes (Lysenko and Kucera, 1971; Osbrink et al., 2005). Research in Environment and Life Sciences 953 August, 2016

6 Antagonistic bacterial isolates were screened for the production of different enzymes, i.e., protease, lipase and chitinase on specific media. Nine bacterial isolates i.e., KPM15, KPM30, KPM31, KPM32, KPM35, PPM94, PPM100, PPM119 and WPS73 showed all the three enzyme activities and 16 isolates showed two enzyme activities (Table 5). Proteolytic activity on casein agar plates varied from 1.24 to 2.64 among different bacterial isolates and five bacterial isolates i.e., KAM85, PBM103, PAM107, KPM31 and CBS16 showed high proteolytic activity. Similar variations in proteolytic activity have also been observed in bacterial isolates obtained from the effluent (Mehta et al., 2004). Out of 120 bacterial isolates screened, only 29 isolates were found to be proteolytic. Two out of four strains of Planococcus sp. and one out of four strains of Micrococcus showed good proteolytic activity. Majority of the bacterial isolates (64.76%) showed lipolytic activity on tributyrin supplemented medium plates and 14 isolates showed very high lipolytic activity. The range of lipolytic activity varied from 1.16 to Microsporidians present in the body cavity and proventiculus of Microcerotermes championi, were found to attack fat body tissues and caused death of termites indicating the role of lipolytic activity in termite killing (Jafri et al., 1976). Sharma et al. (2002) optimized the cultural conditions for production of extracellular lipase from Bacillus sp. RSJ1 and its secretion started as soon as the organism entered the logarithmic phase with the maximum release in the late exponential phase. Only 21 bacterial isolates expressed chitinolytic activity on plates containing colloidal chitin and five bacterial isolates i.e., KPM15, KPM30, KPM35, PPM100 and PPM161 showed appreciable chitinolytic activity. Chitinolytic activity varied from 1.15 to 2.96 in different bacterial isolates. Chitinase production has also been reported in Serratia marcescens, Pseudomonas sp., Bacillus sp., P. stutzeri, Paenibacillus sp. and Pseudomonas maltophila (Chet et al., 1990; Lim et al., 1991; Sindhu and Dadarwal, 2001). Gohel et al. (2004) screened Pseudomonas sp., Pantoeadispersa and Enterobacter ammrenus for chitinase and protease activity. Kamensky et al. (2003) isolated Serratia plymuthica IC14 from soil around melon roots that possessed chitinolytic and proteolytic activities, produced antibiotic pyrrolnitrin and siderophores. Simultaneous production of lipase and protease has been reported in strains of Pseudomonas and Acinetobacter (Guillou et al., 1995; Cordenons et al., 1996). Bacterial strain KPM31 showed all the three enzyme activities but it showed very little termite killing ability at 5 days of observation. On the other hand, some of the bacterial isolates i.e., NBM8 (85%), KPM72 (95%) PPM147 (95%), PPM162 (90%), PPM167 (100%), PPM203 (100%) and PPM204 (85%), which showed high termite killing ability (as given in brackets) did not produce any of the three enzyme activities. Thus, no correlation of termite killing was observed with any of these enzyme activities, indicating that some other compound such as toxin/hcn/siderophore along with these enzymes could be responsible for killing of termites in these bacterial isolates. Screening of 10 most effective termite killing bacterial cultures for the production of HCN on the King s B medium supplemented with glycine showed that bacterial strain KPM35 showed significant HCN production whereas, other two strains PPM195 and PPM106 also showed HCN production (data not shown). Previous studies showed that ingestion of high doses of Serratia marcescens cause infection in many insect species (Poinar Research in Environment and Life Sciences 954 et al., 1979; O Callaghan et al., 1996). Osbrink et al. (2005) reported that Serratia marcescens Bizio caused septicemia in C. formosanus and contained proteolytic enzyme, indicating its possible role as biological control agent against Formosan subterranean termites, Coptotermes formosanus Shiraki. Forced exposure bioassays demonstrated that the T8 strain of S. marcescens killed 100% of C. formosanus by day 19. In C. formosanus cadavers infected with Serratia marcescens, the abdomens rapidly lost their integrity and degraded into a wet, amorphous mass. 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