Isolation of larval gut bacteria of diamondback moth collected from different geographical regions
From different replicates of each geographical region, four different types of bacteria were isolated revealing thereby that in the larvae of Solan population Enterococcus casseliflavus was prevalent but the same was not observed in the larvae of diamondback moth from other geographical regions. Enterobacter hormaechei was found to be in the larvae of Solan, Keylong, Ludhiana and Hyderabad populations, however, the same was not recorded in Bengaluru population. Serratia marcescens and Proteus mirabilis were found only in Bengaluru population. E. hormaechei was the dominant bacterium which was present in majority of the population of diamondback moth collected from different geographical regions of India (Table 2).
Taxonomical position of larval gut bacteria of diamondback moth collected from different regions
The taxonomical position of all the four types of bacteria are presented in Table 3. E. casseliflavus belongs to Lactobacillales, whereas, E. hormaechei, S. marcescens and P. mirabilis belong to Enterobacterales. E. casseliflavus, E. hormaechei, S. marcescens and P. mirabilis belong to family Enterococcaceae, Enterobacteriaceae, Yersiniaceae and Morganellaceae. Majority of the bacteria belong to Enterobacteriaceae followed by Yersiniaceae, Morganellaceae and Enterococcaceae (Fig. 1).
Morphological and biochemical characteristics of larval gut bacterial isolates of diamondback moth
Morphological and biochemical characteristics of E. casseliflavus, E. hormaechei, S. marcescens and P. mirabilis were studied under laboratory conditions. Morphological characters like size, shape, colour, texture, elevation and margins of all the bacterial species were studied. The morphological features of all these are presented in Table 4 which revealed that the size of E. casseliflavus was punctiform, whereas, the size of E. hormaechei, S. marcescens and P. mirabilis was medium. The shape of E. casseliflavus was ovoid, whereas, the shape of E. hormaechei and P. mirabilis was circular. The shape of S. marcescens was filamentous. The colour of all the four bacteria was opaque. Similarly, the texture and elevation of the four bacteria were found to be dry and flat, respectively. The margin of E. casseliflavus and E. hormaechei was entire, whereas, that of S. marcescens was undulate and P. mirabilis was found to have lobate margin.
Biochemical studies on all the four bacteria were also studied. Among the four bacteria, E. casseliflavus was gram positive, whereas, E. hormaechei, S. marcescens and P. mirabilis were gram negative. Catalase test for E. hormaechei was positive, whereas, for E. casseliflavus, S. marcescens and P. mirabilis, it was negative. All the four bacteria were found to be positive for oxidase test and oxidative fermentation test. On the basis of starch hydrolysis test, E. casseliflavus and E. hormaechei were found to be negative, whereas, S. marcescens and P. mirabilis were positive. Nitrate test for E. casseliflavus and P. mirabilis was negative, whereas, E. hormaechei and S. marcescens were positive. For urease test, E. casseliflavus, E. hormaechei and P. mirabilis were positive and S. marcescens was negative (Table 4).
Molecular identification and phylogeny analysis of bacterial isolates
A total of fourteen gut bacterial isolates were obtained from the larval gut of diamondback moth collected from five different geographical regions of India and further subjected to PCR amplification of 16S rRNA with 27F and 1492R primers having 5’-3’ sequences of AGAGTTTGATCMTGGCTCAG and GGTTACCTTGTTACGACTT with annealing temperature of 63oC and amplicon sizes of 1500bp, respectively (Table 5). The PCR amplified DNA bands of these isolates were further visualized on 1 per cent agarose gel (Fig. 2).
The bacterial isolates were identified as Enterococcus casseliflavus, Enterobacter hormaechei and Enterobacter hormaechei with accession number of MK806672, MK806673 and MK806674 for Solan population; Enterobacter hormaechei, Enterobacter hormaechei and Enterobacter hormaechei with accession number of MK806675, MK806676 and MK806677 for Keylong population; Enterobacter hormaechei, Enterobacter hormaechei and Enterobacter hormaechei with accession number of MK806678, MK806679 and MK806680 for Ludhiana population; Serratia marcescens, Proteus mirabilis and Serratia marcescens with accession number of MK806681, MK806682 and MK806683 for Bengaluru population and Enterobacter hormaechei and Enterobacter hormaechei with accession number of MK806684 and MK806685, respectively for Hyderabad population (Table 6) and the per cent similarity ranged from 95 to 99 per cent.
Phylogeny analysis of all the bacterial isolates collected from five different geographical regions of India revealed that all the isolated strains are resolved in well-defined clades with their closest type species (Fig 3). The phylogenetic tree was divided into clades 1, 2, 3 and 4 belonging to Enterobacter hormaechei, Proteus mirabilis, Serratia marcescens and Enterococcus casseliflavus, respectively. Phylogenetic tree was rooted with E. casseliflavus (MK806672) from Solan population which was different from all other bacteria. Two S. marcescens (MK806681 and MK806683) isolated from Bengaluru population matched 100 per cent to each other. Proteus mirabilis (MK806682) from Bengaluru population did not show any similarity to E. casseliflavus, E. hormaechei and S. marcescens. E. hormaechei was the most common in Keylong, Solan, Ludhiana and Hyderabad populations. E. hormaechei from Keylong (MK806675) and Solan population (MK806674) were closely related to each other showing 91 per cent similarity whereas Ludhiana population (MK806680) was showing 44 per cent similarity to these two bacteria. E. hormaechei from Ludhiana population (MK806678) and Keylong population (MK806677) was showing 63 per cent similarity. E. hormaechei from Solan population (MK806673), Keylong population (MK806676) and Hyderabad population (MK806684) were slightly different from each other. E. hormaechei from Ludhiana (MK806679) and Hyderabad population (MK806685) was showing 46 per cent similarity to each other.