Centella asiatica harbors a rich taxonomic diversity of endophytes; however, many studies have focused on fungal endophytes, and very few have explored endophytic bacteria. In this study, twenty bacterial endophytes (Table 1) were isolated from the leaves of C. asiatica and identified as Pseudomonas sp. strain SGM1, Pseudomonas sp. strain SGM2, Pseudomonas sp. strain SGM3, Pseudomonas sp. strain SGM4, Pseudomonas sp. strain SGM5, Pseudomonas sp. strain SGM6, Pseudomonas sp. strain SGM7, Novosphingobium sp. strain SGM8, Pseudomonas sp. strain SGM9, Pseudomonas sp. strain SGM10, Chryseobacterium sp. strain SGM11, Enterobacter sp. strain SGM12, Enterobacter sp. strain SGM13, Pseudomonas sp. strain SGM14, Enterobacter sp. strain SGM15, Enterobacter sp. strain SGM16, Agrobacterium sp. strain SGM17, Pantoea sp. strain SGM18, Paraburkholderia sp. strain SGM19, and Pseudomonas sp. strain SGM20. These isolates were classified into two phyla: Bacteroidetes and Proteobacteria (Table 2).
Pseudomonas and Enterobacter were dominant species with eleven and four endophytes respectively. Some bacterial endophytic strains of Xanthomonas axonopodis, Pseudomonas fulva, Providencia vermicola, Erwinia sp., Pantoea agglomerans, Methylobacterium radiotolerans, and Bacillus gibsonii were isolated from C. asiatica leaf petioles and stems and reported in 2012 [36]. Ernawati et al. [37] identified six genera: Gordonia, Actinoplanes, Couchioplanes, Verrucosispora, Streptomyces, and Micromonospora of C. asiatica from Indonesia. Thirty-one bacterial strains classified into the genera Bacillus sp., Cohnella sp., Acinetobacter sp., Paenibacillus sp., Microbacterium sp., Achromobacter sp., Lysinibacillus sp., Pseudomonas sp., Pantoea sp., Klebsiella sp., and Delftia sp. were reported earlier from surface-disinfected C. asiatica leaves, and they showed the capability to reduce the disease occurrence and growth rate of the hemibiotrophic fungus Colletotrichum higginsianum [38].
Seemingly, the most dominant genera of bacterial endophytes from C. asiatica are Pseudomonas, Pantoea, and Bacillus. Besides Pseudomonas, Pantoea, and Bacillus, other common fungal endophytes such as Fusarium and Colletotrichum have also been identified from C. asiatica [28, 30, 31]. Martín-García et al. (2011) noted that the diversity of the endophytic community of bacterial endophytes relies on several factors such as the geographical location, plant age, species, and cultivation conditions [39]. For example, nine bacterial endophytes were isolated from aerial parts of C. asiatica harvested in Malaysia [36]. In another study, three bacterial endophytes were isolated from the leaves of subtropical forest-cultivated C. asiatica in Meghalaya, India [40]. This result is in agreement with the statement above made by Martín-García et al. (2011) and Liu et al. (2017) for further stating that growth factors such as soil pH, annual temperature, organic matter, annual rainfall, and phosphate availability could result in variation in the distribution and composition of bacterial endophytes [41]. In other studies, endophytes isolated (mostly fungi) from C. asiatica were tested in vitro for their antioxidant, antimicrobial, and plant growth-promoting activities, and these studies proved these isolates to be good candidates with pharmaceutical importance and for application as biocontrol and biofertilizer agents [42,43,44].
Phylogenetic analysis showed that strain Pseudomonas sp. strain SGM20 had a biphyletic cluster with Pseudomonas sp. strain SGM6 (Fig. 1). In addition, other Pseudomonas sp. strains under study were closely related to each other (Fig. 1). As shown in Figs. 2 and 3, a sister relation was observed between Novosphingobium sp. strain SGM8 and Novosphingobium clariflavum strain supported by a 96% bootstrap value, whereas Chryseobacterium sp. strain SGM11 is closest to Chryseobacterium bernardetii strain supported by a 92% bootstrap value. Phylogenetic analysis further revealed that Enterobacter sp. strain SGM12, Enterobacter sp. strain SGM13, Enterobacter sp. strain SGM15, Enterobacter sp. strain SGM16 (Fig. 4), and Agrobacterium sp. strain SGM17 (Fig. 5) were clustered and formed a separate lineage. Therefore, the phylogenetic positioning of Enterobacter sp. strain SGM12, Enterobacter sp. strain SGM13, Enterobacter sp. strain SGM15, Enterobacter sp. strain SGM16, and Agrobacterium sp. strain SGM17 is an indication that these species are novel members of the Enterobacter and Agrobacterium genus.
Pantoea species revealed Pantoea sp. strain SGM18 had a polyphyletic relationship with Pantoea brenneri strain and Pantoea conspicua strain (Fig. 6), while Paraburkholderia sp. strain SGM19 was closely related to Paraburkholderia caledonica strain supported by a 75% bootstrap value (Fig. 7). The isolated endophytic bacteria were identified at the genus level using sequencing of 16S rRNA. However, the evolutionary and phylogenetic relationships between bacterial endophytes from the genera, Pseudomonas, Enterobacter, and Pantoea species, and closely related endophytic strains were not resolved as polyphyletic relationship was observed from the results obtained. Therefore, for phylogenetic delineation and species description, it is suggested to identify and further phylogenetically analyze the bacterial endophytes from the three genera using the multilocus sequencing analysis (MLSA) [27, 45].
Based on the morphological analysis, unique morphological characteristics such as colony size, shape, color, and margins were observed for each isolate (data not shown). According to our results, all isolated endophytic bacteria were gram-negative, rod-shaped bacteria. The scanning electron microscopy results further showed distinct characteristics in terms of the shape and size of the pure cultures from small to long rod-shaped bacteria (Fig. 9). Although in some studies the quantity of gram negative is equal to that of the gram positive, it has been reported that gram-negative bacterial endophytes are more likely to be abundant than gram-positive bacteria [46]. Therefore, this supports the obtained results in our study.