Assessment of the entomopathogenic nematode bacteria against the termite, Microtermes mycophagus D. (Isoptera: Termitidae)

The capability of symbiotic bacteria of entomopathogenic nematodes against the termite, Microtermes mycophagus D. (Isoptera: Termitidae), was assessed. Different fractions of Pakistani isolates of entomopathogenic bacteria viz., Xenorhabdus indica strain (Pak.S.B.50), X. indica strain (Pak.S.B.56), X. stockiae strain (Pak.S.B. 65), and X. steinernematis strain (C.B.10) were assessed against M. mycophagus by direct contact method (spraying method) and sand assay in laboratory conditions. Mortality response of cell-free filtrates after 24 h at 20 °C for X. indica (Pak.S.B.50), T2 = X. indica (Pak.S.B.56), T3 = X. stockiae (Pak.S.B. 65), and T4 = X. steinernematis (C.B.10) ranged (88.3–100%) as 33 ± 9.34, 98.33 ± 6.22, 88.33 ± 7.22, and 100.00% ± 0.00, respectively. In the case of sand assay, the most effective treatment was T4, where (100%) mortality rate was recorded 24 h post application of B.S. (bacterial suspension) (4 × 104 CFU/ml) and CFF (cell-free filtrate) (100 μl/10 ml) at 20 and 25 °C.


Background
The termite, Microtermes mycophagus D. (Isoptera: Termitidae: Macrotermitinae), is a cosmopolitan pest of wood and wood products that can be distinguished by its colonial behavior. Colony members are distinctly varied morphologically, i.e., propagative (king and queen), soldiers, and workers. The head termites, the king and queen, are sexually functional but pheromonal regulation that is responsible for the caste production is only produced by the queen (Noirot and Noirot-Timothee, 1970). Wingless individuals, workers or soldiers, are usually non-reproductive males or females. Soldiers play a major role to defend the colony and represent 1/10th of the population of a colony (Bignell and Eggleton, 1998). Termites are highly devastating and cause damage to furniture, buildings, trees, and agricultural crops, such as cereals, oil crops, pulses, sugarcane, fruits, and vegetables. Estimated losses by this pest are about US$22 billion annually across the world (Govorushko, 2011). The genera viz., Microtermes, Odontotermes, and Termes, are the most prevalent termites in Pakistan (Manzoor and Naeem, 2010).
Chemicals control applications to the wood or to the soil have been determined time by time. Chemical fumigants containing methyl bromide, sulfuric fluoride, or a combination of carbon dioxide and methyl bromide is the suitable procedure of eradicating dry wood termites. Biocontrol agents are environment friendly and proficient in working but its cost effective feature is debatable. Abiotic factors such as warm and moist favored by subterranean termites, which promote epizootics, also have the potential for biological control (Verma et al., 2009). Few studies have reported the potential of entomopathogenic nematodes (EPN) to control termites. EPN exposure to termites resulted in significant response of parasitization (47% after 4 days) and 100% mortality after 12 days under lab conditions. Fujii (1975) gained 96% mortality results of C. formosanus within 7 days after treating with infective-stage Steinernema carpocapsae (Weiser) (Steinernematidae) in laboratory analysis. Mortality rate, more than 95%, was documented within 3 days by Georgis et al., (1982) for both Reticulitermes sp. and Zootermopsis sp. after laboratory exposure to S. carpocapsae; further termites were also serve as vectors for EPN that take back to their colonies. S. carpocapsae has shown high rates of infection to Nasutitermes costalis and R. flavipes obtained under laboratory conditions (Laumond et al., 1979 andTrudeau, 1989).
The present study aimed to assess the efficacy of symbiotic bacteria Xenorhabdus species as biopesticide against the termite, M. mycophagus, under laboratory conditions.

Bacterial culture (isolation of bacteria from insect hemolymph)
Entomopathogenic nematodes (EPN) were obtained from the storage unit, maintained by Prof. Dr. Shahina Fayyaz at NNRC, University of Karachi, Karachi, Pakistan. All nematodes were propagated in last instar larvae of the greater wax moth, Galleria mellonella L., using the method of Dutky (1974). Infective juveniles were collected by White traps (White, 1927), harvested, and stored in sterilized distilled water at 10-15°C for no more than 2 weeks before they were used.

Isolation of bacteria from insect hemolymph
To isolate bacteria from hemolymph, G. mellonella larvae were inoculated by EPN (Table 1) 100 IJs in a Petri dish lined with moistened filter paper. After 48 h the dead larvae were surface sterilized by 75% ethanol for 15 min; then, the cadavers were passed through the flame for further sterilization. Cadavers were dissected with sterilized scissors at the second foot, a loop full of hemolymph streaked onto NBTA agar medium (Akhurst, 1980). The streaked plates were incubated in the dark at 28°C for 48 h for the development of primary colonies. For further purification, single colonies of bacteria were sub cultured on new plates of agar medium. Then, single colony was transferred to the nutrient broth (0.81 broth + 61 ml water) and kept it for incubation on shaking bath for 2 days at 150 rpm ND 28°C. The bacterial suspension was used for bioassay.

Biochemical analysis of bacterial isolates
The pure cultures of different species of Xenorhabdus were subjected to biochemical test through API 20E test kit of Biomerieux Ltd., USA.

Spray method
Heavy infested branches of different trees were selected and kept in a plastic shopper after cutting with a hammer or cutter and brought in to the laboratory. Plastic containers about the size of 8 × 6 in. lined with wax at the edges were used for the experiment. Six-inch pieces of tree branches carrying approximately 50 termite individuals were placed in each container and sprayed with 20 ml of each treatment separately. Each container was sealed with a parafilm and each set of experiment incubated at different temperatures 20, 25, and 30°C. Different EPB formulations were examined viz., B.S. (bacterial suspension) (4 × 10 4 CFU/ml), CFF (cell-free filtrate) (100 μl/10 ml), B.R. (bacterial residue) (100 μl/10 ml) of T 1 (Xenorhabdus indica (Pak.S.B.50)), T 2 (X. indica (Pak.S.B.56)), T 3 (X. stockiae (Pak.S.B. 65)), and T 4 (X. steinernematis (C.B.10)). This combination was replicated 3 times with control treatment, which was sprayed only with water. Mortality rate was assessed each after 24, 48, and 72 h.

Data analysis
Data are expressed as means, standard deviation and the significance of mean differences was determined with Duncan's multiple range test (SAS Institute, Cary, NC).

Effect of different application methods for controlling
Microtermes species Spray method All bacterial isolates were found to be significantly effective against termites by spray method. Different fractions of bacterial formulations showed significant differences of mortality rate (P < 0.001). Bacterial suspension and cell-free filtrates of all treatments (bacterial isolates) had the potential to control termites at 20 and 25°C even after 24 h, whereas the bacterial residue of all the bacterial isolates had least potential for controlling termites. Due to direct contact of formulations with termites, effective results were obtained within 24 h in most of the cases. Mortality response of cell-free filtrates after 24 h at 20°C in Xenorhabdus indica (Pak.S.B.50), T 2 (X. indica (Pak.S.B.56)), T 3 (X. stockiae (Pak.S.B. 65)), and T 4 (X. steinernematis (C.B.10)) ranged between 88.3 and 100% as 88. 33 ± 9.34, 98.33 ± 6.22, 88.33 ± 7.22, and 100.00% ± 0.00, respectively. No mortality response was found in the control treatment (Table 3).
Different bacterial species have the capability to control termites. The effects of Bacillus thuringiensis subspecies was examined under laboratory conditions against Nasutitermes ehrhardti (Castilhos-Fortes et al. 2002). They observed that B. thuringiensis subspecies kurstaki produced 80% mortality of termite species. B. thuringiensis proteins having insecticidal properties are highly specific as gut toxins and it has shown a superior safety in reference to the effectiveness for non-target organism (Sarwar, 2015).
Pseudomonas sp., P.maltophilia, Bacillus strains, and Paenibacillus sp., are reported to produce chitinase (Suyal et al., 2015;Verma et al., 2016a;Yadav et al., 2016a). It was also reported that ten bacterial strains along with two control strains have been evaluated as biocontrol against termites. Different bacterial strains having termite-killing ability showed > 80% mortality after 5 days of incubation (Dua, 2014). Four bacterial strains caused 100% killing at 10 days of observation. The cell-free culture filtrate of these cultures showed that the antagonistic substance was extracellular having protein properties. Bacterial strains of Bacillus subtilis KBM79 and Pseudomonas synxantha KPM35 possessed proteolytic, chitinolytic, and lipolytic enzyme activities and caused 100% killing of termites (Yadav et al., 2016b). In previous studies, X. nematophila has been proved as a potential candidate of biocontrol agent against termites (Hiranwrongwera et al., 2007).

Conclusion
The present results proved that different bacterial fractions of Xenorhabdus species were found effective against termites in certain adequate conditions and can be a successful candidate for integration in termites' controlling strategy.