ANTAGONISTIC EFFECT AGAINST BEE PATHOGENS BY GUT BACTERIA IN CAVITY NESTING HONEYBEE Apis cerana

Abstract

In an apiary, honeybees have had problems with pests and diseases leading to economic loss in apiculture. For example, American foulbrood (AFB) disease was caused by Paenibacillus larvae. Nosema disease was caused by Nosema ceranae and N. apis. Sacbrood disease was caused by sacbrood bee virus (SBV) and chalkbrood disease was caused by Ascosphaera apis. In this research, infection of P. larvae, N. ceranae, N. apis, SBV, and A. apis were investigated in Apis cerana. Fifty colonies were collected from ten apiaries in Samut-songkhram and Chumphon provinces in the central and the south of Thailand, respectively. Total DNA was extracted from larvae, pupae, and adults and amplified by multiplex PCR. In each reaction, it comprised of two pairs of primers (16S rRNA of P. larvae and cytochrome b of A. cerana), three pairs of primers (16S rRNA of N. ceranae, of N. apis, and RpS5 of A. mellifera), two pairs of primers (pol of SBV and 28S rRNA of A. mellifera), and two pairs of primers (5.8S rRNA of A. apis and RpS5 of A. mellifera). The results showed that 80%, 94%, 96%, and 80% of samples were not infected by P. larvae, N. ceranae, N. apis, and A. apis, respectively. The phylogenetic lineage depending on the partial sequences of 16S rRNA of P. larvae, N. ceranae, N. apis, and 5.8S rRNA of A. apis exhibited six, two, single, and four distinct clusters, respectively. Bacteria in healthy bee’s gut such as Bifidobacterium sp., Lactobacillus sp. were identified by fermentation test. Bacillus sp., Lactobacillus sp., and lactic acid bacteria were identified by the analyzed sequence of 16S rRNA. Then, the antagonistic effect of selected gut bacteria which were Bacillus sp., Pantoea sp., Azotobacter sp., Klebsiella sp., and Lactobacillus sp. against P. larvae, strain 01 and 02 was determined by agar well diffusion assay. It revealed that Azotobacter sp. showed the highest relative inhibitory zone effect on strain 02 with 56.68c±3.17% in diameter. The supernatant of targeted bacteria showing the antibacterial activities against strain 01 and 02 at 37 ºC were from Bacillus sp., Lactobacillus sp., Klebsiella sp., Azotobacter sp., and at 90 ºC were from Lactobacillus sp., Klebsiella sp., Azotobacter sp., and Bacillus sp. The supernatant treated at 121 ºC, with proteinase K and RNase A did not show the inhibitory effect. Furthermore, the supernatant from Pantoea sp. at pH 5, Bacillus sp. at pH 3, Klebsiella sp. at pH 3, Lactobacillus sp. at pH 8 and 10, and Klebsiella sp. at pH 10 showed the highest inhibitory effect against strain 01 and 02. The statistical analysis of mean difference was significant at the 0.05 level. The inhibitory activity of supernatant at pH 8-10 was reduced, comparing to supernatant at pH 5 which was the condition near the value of pH in bee stomach. Supernatant treated at 90 ºC showed the higher inhibitory effect against strain 01 and 02, comparing to supernatant at 37 ºC. In conclusion, gut bacteria which could inhibit P. larvae growth could be obtained from healthy bee.