Molecular characterization of plant growth-promoting vermi-bacteria associated with Eisenia fetida gastrointestinal tract

Earthworms are highly productive invertebrates and play a vital role in organic farming and improving soil structure and function. The gastrointestinal tract of earthworms possessed agricultural important bacteria. So, the current research aimed was to examine, screen, and identify the plant growth promoting bacteria existing in the digestive tract of Eisenia fetida called plant growth promoting vermi-bacteria. The plant growth promoting traits such as siderophore, phytohormone, and hydrolytic enzymes production, and phosphate solubiliation were assessed. Eleven vermi-bacteria i.e. Bacillus mycoides, B. aryabhattai, B. megaterium, Staphylococcus hominis, B. subtilis, B. spizizenii, B. licheniformis, B. mojavensis, B. toyonensis, B. anthracis, B. cereus, B. thuringiensis, and B. paranthracis were isolated and identified based on microscopic studies, biochemical tests, ribotyping, and agricultural traits. All vermi-bacteria are Gram-positive rods except Staphylococcus hominis and produce different compounds such as siderophore, indole acetic acid, catalase, oxidase, proteases, amylases, and lipases. All vermi-bacteria also act as phosphate solubilizers. Therefore, all isolated vermi-bacteria could be used as potential microbial biofertilizers to enhance crops production in Pakistan.


Introduction
Earthworms play a vital role in soil productivity, nutrient recycling, soil structure, and agriculture [1][2][3]. Thus, earthworms may be observed as a biological indicator of soil fertility and health [4,5]. Guts of earthworms are suitable habitats for bacteria, and fungi, and proved that microbial numbers in the gut are much more compared to soil in which earthworms were living [6][7][8]. The gut environment is anoxic, with 6.9 pH having 50% water contents, enriched in total carbon, nitrogen, and organic carbon [9,10]. Medina-Sauza et al. [11] showed that the growth of beneficial microbes in soil belongs to various families such as Actinobacteria, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Ethical statement
All experiments conducted during research work have been specifically designed to avoid any distress, suffering, and unnecessary pain to the experimental animals. All procedures were performed following international regulations referred to as Wet op de dierproeven (Article 9) of Dutch Law. , sterile dissecting pins, Sterile distilled water, dissecting box, gloves, dissecting board, sterile bottles, 70% ethanol, 500 ml beakers, micropipette, 250 ml conical flasks, test tubes, bacteriological wire loop, Petri plates, glycerol, glass rod, glass slides, coverslips, spirit lamp, microscope, and toothpicks.

Sampling and dissection of E. fetida
To isolate vermi-bacteria, 4-5 mature clitellate E. fetida were collected and taken to the vermitechnology laboratory, Zoology Department, University of AJ&K (UAJ&K), Muzaffarabad. Mature earthworms were rinsed with sterile distilled water, cleaned externally with 75% ethanol in a sterile Petri plate, and dried with tissue papers. With its anterior end pointing forward, they were placed around the second, third, and fourth fingers of the left hand. Sterilized pair of dissecting scissors were used with their fine sharp tip introduced into the ventral region at the clitellum, with the help of scissors body wall was slightly raised and the cut was gently made along the length of the worm. Earthworm was held down on a board, with the help of sterile dissecting pins, and the body wall was stretched to expose the internal organs. With sterilized forceps, the gut and nephridia were then freed from surrounding blood vessels and separated into foregut, midgut, and hindgut. After washing the parts of the gut with sterile distilled water, they were suspended in another sterilized bottle containing distilled water (10 ml). The homogenized mixture was used further for bacteria isolation.

Isolation and enumeration of vermi-bacteria
Somasegaran and Hoben's [20] used the serial dilution method for the isolation of bacteria. A homogenized mixture (1 ml) was poured into another test tube to make 10 −1 dilution. Similarly, other dilutions 10 −2 , 10 −3 , 10 −4 , 10 −5, and 10 −6 were prepared accordingly. After making dilutions, the mixture was spread on a nutrient agar medium and placed for 24 h at 37˚C. The diverse bacterial colonies developed on the media were estimated and expressed as colony-forming units (CFU). The concentration of bacteria in the original sample was calculated as:

Bacterial purification
From the well-separated dilution plates, a total of eleven bacterial strains were picked, grown in a nutrient broth medium, and placed for 24 h at 37˚C. The next day, overnight culture was streaked on freshly prepared nutrient agar plates and incubated at 37˚C for 24 h. These plates were labeled as U1, U2, U3, U4, U5, U6, B1, B2, B3, B4, and B6, respectively. After sub-culturing, these eleven vermi-bacterial isolates were picked and stored in 60% glycerol for future work.

Genomic DNA extraction
From vermi-bacterial isolates, genomic DNA extraction was carried out using the method of Sambrook et al. [36], with slight modifications. All vermi-bacterial isolates were grown in a Luria broth medium and incubated for 24 h at 37˚C. After incubation, centrifugation was carried out at 10,000 rpm for 5 min to harvest cells. The pellet was suspended in lysis buffer-1 (Tris EDTA and SDS; pH 4.0) and then centrifuged for 10 min at 10,000 rpm. After centrifugation, 500 μL of chloroform: isoamyl alcohol (24: 1) was added, mixed, and centrifuged at 10,000 rpm for 10 min. In the collected supernatant 2.5 volumes of chilled 100% absolute ethanol and 1/10 th volume of sodium acetate was added and incubated at -20˚C overnight. The next day, samples were centrifuged for 10 min at 10,000 rpm, and the pellet was washed with 70% ethanol. After centrifugation, pellets were dried for 3 h, and DNA was dissolved in distilled water (20 μl).

Amplification and sequencing of 16S rDNA
For the identification of vermi-bacterial isolates, full-length 16S rRNA primers (341F 5'-CCTACGGGNGGCWGCAG-3'; 806R 5'-GGACTACNNGGGTATCTAAT-3') were taken to amplify the V3-V4 region (Approx. 470 bps) using following PCR conditions (initial denaturation 95˚C for 2 min; Cyclic denaturation at 95˚C for 20 sec; Annealing at 50˚C for 30 sec; Cyclic extension 72˚C for l min 30 sec; and Final extension 72˚C for 5 min; 35 cycles). After PCR analysis, all PCR products were sent to Macrogen, Korea for sequence analysis. The obtained nucleotide sequences further proceeded for homology through BLAST at National Center for Biotechnology Information (NCBI) platform.

Phylogenetic analysis
The phylogenetic relationship was determined using the Maximum Likelihood method and Tamura-Nei model [37]. This analysis involved 32 nucleotide sequences. There were a total of 1573 positions in the final dataset. Evolutionary analyses were conducted in MEGA X [38]. After BLAST and phylogenetic analysis, all amplified sequences were submitted to NCBI, Genbank for the provision of accession numbers.

Morphological and biochemical characterization of vermi-bacteria
Several well-separated colonies were observed in the case of 10 −3 , 10 −4 , and 10 −5 diluted samples. These plates were selected for the isolation of pure vermi-bacterial isolate. Eleven bacterial strains were isolated from these dilutions such as 2 isolates from 10 −3 , 3 isolates from 10 −4 , and 6 isolates from 10 −5 , respectively. These vermi-bacterial isolates were further grown on the nutrient agar, mannitol salt agar, and MacConkey agar, respectively. Out of 11 isolates, U1, U2, and U3 form yellow colonies with yellow zones indicating their ability to ferment mannitol whereas U4, U5, U6, B1, B2, B3, B4, and B6 showed no growth in mannitol salt agar. Out of 11 isolates U2, U4, U5, B3, and B6 produced pink colonies indicating their ability to ferment lactose. Whereas U1, U3, U6, B1, B2, and B4 form yellow zones indicating that cannot ferment the lactose (Fig 1). The color of colonies was creamy, yellow, and white, showing entire and undulate margins, the elevation of colonies was also varied from raised flat, and convex, and colonies' forms were circular, punctiform, and irregular, respectively. Most colonies were translucent and opaque. The shape of vermi-bacterial isolates was also varied from singly rods coccobacilli, and branching rods. All isolated vermi-bacteria were Gram-positive rods (Fig 2;  Table 1).

Biochemical characterization
The results of the KOH test showed that all isolates were Gram-positive bacteria, and their cell wall was not affected by 3% KOH and do not form stringy, sticky, and viscous material within the first 30 sec (Table 1). All vermi-bacterial isolates form a cherry red ring at the top of the medium denoting the indole production action (Fig 3). All vermi-bacteria did not show yellow color which means that all vermi-bacteria cannot produce ammonia (Table 1). All vermi-bacterial isolates were not able to produce HCN as the color of the filter paper did not change ( Table 1). All vermi-bacterial isolates were catalase-positive. Similarly, all vermi-bacterial isolates were oxidase-positive except U2 and B3 as purple color spots appeared within 5-10 secs on the filter paper after adding the oxidase reagent (Table 1; Fig 3). All vermi-bacterial isolates showed amylase and lipase production ( Table 1; Fig 3). All vermi-bacterial showed proteolytic activity in the range of 10.0 ± 0.0 mm to 15.0 ± 0.0 mm except B1 and B2 (Fig 3). The maximum clear zone for lipolytic activity was recorded in the range of 15.0 ± 0.0 mm to 20.0 ± 0.0 mm (Fig 3). All bacterial isolates were screened for siderophore production on CAS agar plates and results revealed that all isolates showed siderophore production. All vermi-bacterial isolates produced maximum siderophore except U3, B3, and B6. The activity diameter was recorded in the range of 12.0 ± 0.0 mm to 33.0 ± 0.0 mm. The zone of activity indicated the amount of siderophore excreted by the bacterial isolates (Fig 3). Results revealed that all bacterial isolates were phosphate solubilizers. The clear zone around the colonies indicated positive phosphate solubilization activity. The clear zones were recorded to have a range of 12.0 ± 0.0 mm to 33.0 ± 0.0 mm (Fig 3).

Molecular characterization
After morphological and biochemical characterization, genomic DNA was extracted, 16S rRNA was amplified, and sequenced. The range of amplified PCR products was 419 bps-443 bps and the length of obtained accessions (419 bps-1517 bps) is shown in
Morphologically, colonies indicated the variations in the margin, elevation, color, form, opacity, and shape, respectively. Gram staining technique indicated that all isolated bacterial species were coccobacilli and filamentous rods which was consistent with the results of Karsten and Drake, [41] who revealed the presence of cocci, rod-shaped bacteria, and filamentous microbes in Lumbricus terrestris L. and Octolasion cyaneum through scanning electron microscopy. Bacterial strains can be identified by growing them on selective culture media [42]. In current study, vermi-bacteria were grown on MacConkey agar and results revealed that B. mojavensis, B. toyonensis, B. anthracis, S. hominis, and B. licheniformis produced pink colonies indicating their ability to ferment lactose whereas B. subtilis, B. paranthracis, B. mycoides (B1, U3, and B4), B. megaterium are non-lactose fermenters. It was also observed that B. subtilis, B. mojavensis, B. mycoides have ability to ferment mannitol whereas B. toyonensis, B. thruiginesis, B. paranthracis, B. mycoides (B2 and B4), B. megaterium, S. hominis, and B. licheniformis showed no growth on mannitol salt agar. Our finding agreed with the outcomes of Silawat et al. [43]. They isolated and identified nine bacteria from the soil and compost. They showed that P. aeruginosa, A. calcoacet, P. pseudomalli, P. pickettii, P. cepacia have ability to ferment mannitol whereas P. putida, P. shutzeri, and P. pickettii fermented lactose.
Plant growth promoting bacteria (PGPB) play an essential role in the metabolism and growth of plants. Variety of PGPB such as Bacillus, Arthobacter, Enterobacter, Azotobacter, Serratia, and Rhizobium are being used worldwide to enhance the crop production [44]. Ammonia production is a significant trait of PGPR's and act as metabolic inhibitors towards phytopathogens [45,46]. In the current research all vermi-bacteria have ability to produce ammonia except S. hominis and our findings are agreed with Kumar et al. [45] and could be used as antimicrobial agents. Our study revealed that all vermi-bacterial isolates are involved in IAA production except B. mycoides and B. megaterium suggesting the ability to suppress plant diseases, promote plant growth and development, and seed germination. Our results agreed with Khare and Arora, [47], who reported that bacterial indole acetic acid has a role in the suppression of rot diseases in various plants and IAA regulates several fundamental cellular processes including cell divisions, elongation, and differentiation. Our results are agreed with the previous literature [48][49][50]. They illustrated that IAA is produced by PGPB and helps in plant-microbe interactions.
Present work revealed that all vermi-bacterial isolates didn't produce HCN. Our findings are thus contrary to those of Nadège et al. [51] who isolated and identified nine PGPR i.e. five Bacillus species (B. pantothenticus, B. circulans, B. thuringiensis, B. polymyxa and B. anthracis) three Pseudomonas species (P. cichorii, P. putida, and P. syringae) and Serratia marcescens from the rhizospheric region of maize and all of this PGPR were capable of HCN production. The synthesis and production of HCN is varied from species to species and our findings agreed with the outcomes of Rijavec and Lapanje, [52]. Results revealed that all vermi-bacterial isolates produce siderophores and are phosphate solubilizers. Siderophores act as a biocontrol agent [53]. According to Indiragandhi et al. [54], siderophores form a stable complex with the trace elements (Fe, Ca, Zn, and Cu, etc.) which help in plant growth promotion. Similarly, phosphate solubilizing bacteria have a great impact on agriculture and are considered promising natural microbial biofertilizers [55]. Bacillus megaterium has been commercialized as BioPhos by AgriLife (India) [56]. Bhattacharyya and Jha, [57] also reported some phosphate solubilizing bacteria like Beijerinckia, Erwinia, Azotobacter, Bacillus, Flavobacterium, Microbacterium, Burkholderia, Enterobacter, Rhizobium, Serratia, and Pseudomonas, respectively.
In the current study, production of hydrolytic enzymes by PGPVB were screened and results revealed that all vermi-bacterial isolates were involved in the production of catalase, amylases, lipases, proteases, and oxidases that act as biocontrol agents to be used in the fields of medicine, environment and agriculture [58,59]. These vermi-bacteria could be used in the plant disease management system. Our findings agreed with the outcomes of Parashar et al. [60] who demonstrated that PGPB also produces antifungal agents. Protease and amylase-producing microbes such as Pythium spp and Phytophthora were not only played a major role in the plant growth promotion, decomposition of organic matter, and nutrient mineralization however also act as biocontrol agents [18]. Bacterial strains containing catalase action which showed resistance to environmental, chemical, and mechanical stress [45]. Our results revealed that all isolated vermi-bacteria were catalase-positive, and our results are parallel to the work of Silawat et al. [43] who reported that P. aeruginosa, P. malli, Achromobacter group, P. putida, P. shutzeri, A. calcoacet, P. pseudomalli, P. pickettii, P. cepacia isolated from compost were catalase positive. It was observed that vermi-bacterial isolates were oxidase-positive except Bacillus mycoides (B1, B4 U3), Bacillus/Priestia megaterium (B2) and Staphylococcus hominis while our findings are contrary to the outcomes of Kaur and Brar, [61] who showed that B. subtilis is oxidase-negative. In our study, all bacteria were amylase positive except Staphylococcus hominis, parallel to the work of Geetha et al. [44] who confirmed that rhizospheric bacteria were able to produce amylases. The current research reveals that all isolated and identified plant growth promoting vermi-bacterial isolates showed the agricultural traits (siderophore, phytohormones, ammonia, and hydrolytic enzymes production, and also act as phosphate solubilizers). The current outcomes are consistent with the findings of previously reported data [56,[62][63][64][65].

Conclusion
It was concluded that, earthworm gut is favorable host for the isolation of plant growth promoting bacteria which could not only be used as a microbial biofertilizers to enhance the crop production in Pakistan but also used in sustainable disease management system. Vermi-bacteria play an important role in certain soil processes such as growth hormone production, phosphorous solubilizers, nitrogen fixation, and control of microbial pathogens.