PJZ_57_6_2625-2636

INTRODUCTION

Soil microorganisms i.e., bacteria, fungi, and actinomycetes involved in the soil formation, improving the soil fertility via degradation and detoxification of organic materials in soil, and maintain the ecological balance (Emperor et al., 2015; Zhu et al., 2017). Vermicomposting is an ecofriendly, bio-oxidative, and non-thermophilic organic decomposition technology to recycle the organic waste into fine granular product called vermicompost via the joint action of earthworms and microorganisms (Lazcano et al., 2008; Chitrapriya et al., 2013) (Supplementary Fig. 1). Various earthworm species i.e., Metaphire californica, Eudrilus eugeniae, Eisenia andrei, Perionyx excavates, and Eisenia fetida were used for vermicomposting (Deka et al., 2011; Varma et al., 2016; Boruah et al., 2019; González-Moreno et al., 2022).

Earthworms ingest soil microbes, their population increases via passage through the intestinal track due to favorable environment, and excrete them along with nutrients in their vermicast (Huang et al., 2013; Pathma and Sakthivel, 2012). Several antibiotics and enzymes play an important role in the decomposition and degradation of organic molecules which were released by microbes in the gastrointestinal track of earthworms (Lazcano et al., 2008).

Vermicompost is not only rich in micro- and macronutrients, enzymes, plant growth hormones, plant growth regulators, and antibiotics (Ravindran et al., 2016; Shafique et al., 2021) but also improves the plant growth, plant nutrition and productivity, soil fertility, soil structure as well as growth of soil beneficial microbes (Pathma and Sakthivel, 2012; Zhu et al., 2017) (Supplementary Fig. 1). In addition, vermicompost improves soil aeration, moisture contents, pH, electrical conductivity, and water holding capacity (Shafique et al., 2021). Vermicompost suppresses plant diseases and increased microbial population like phosphate solubilizers, nitrogen fixers, Indole acetic acid producers, siderophore producers, which could significantly enhance the vegetative plant growth of ornamental plants (Shafique et al., 2021; Chauhan and Singh, 2015). Previous literature reported the presence of bacteria in vermicompost produced via L. mauritii, P. excavatus and E. eugeniae (Upadhyay et al., 2015; Selvi and Koilraj, 2015; Begum and Bora, 2018). Devi et al. (2009) and Pathma and Sakthivel (2012) illustrated the presence of Proteobacteria, Bacteroidetes Planctomycetes, Actinobacteria, and Firmicutes in vermicompost (Yasir et al., 2009a).

In view of above the objectives of the current research were to isolate, characterize, and identify vermicompost associated bacteria from vermicompost produced via E. fetida, to explore the agricultural traits and to screen the prevalence of antibiotic resistance among vermicompost associated bacteria. No research has been done before on the beneficial vermi-bacterium isolation and identification from vermicompost in Pakistan. Hopefully, this identified bacterial isolates could be used as microbial biofertilizer to enhance the seed germination and plant growth of various crops in future.

MATERIALS AND METHODS

Chemicals used

Nutrient broth (lennox), Luria Bertani (LB) broth (lennox), nutrient agar medium (sigma-aldrich), hydrogen peroxide, oxidase reagent, gram staining kit (merck), skim milk agar (neogen), Wattman No.1 disc, MacConkey agar (sigma-aldrich), mannitol salt agar (oxoid), Nessler’s reagent (sigma-aldrich), Kovac’s reagent, phenol, 0.5% Picric acid (sigma-aldrich), King’s B medium (Sigma), 2% sodium carbonate (merck), dilute Iodine, 3% KOH starch (sigma-aldrich) and bacteriological Peptone (oxoid). The antibiotics used were trimethoprim (TMP 5µg), aztreonam (ATM 30µg), kanamycin (K 30µg), erythromycin (E 15µg), sulfonamide (S 300µg), tobramycin (TOB 10µg), penicillin (P 10µg), norfloxacin (NOR 10µg), amoxicillin (AMC 30µg) and ampicillin (AMP 25µg). All these antibiotics were taken from Oxide company.

Glassware and types of equipment used

Steam sterilizer (autoclave), Laminar flow (ESO Prod Model; EQU/03-EHC; Serial # 2000-0052), 37ºC incubator (MMM group Medcenter Enrich tungsten GmbH), Analytical balance (SARTORIUS GMBM GOTTINGEN, Germany), sterile distilled water, sterile bottles, sterile dissecting pins, dissecting box, dissecting board, gloves, 37ºC shaker (Irmeco GmbH, Germany), digital weighing machine (Jeweler Precision Balance Model: DH-V600A), 70% Ethanol, 500 ml beakers, 250 ml conical flasks, test tubes, petri plates, L shaped glass rod, microscope, bacteriological wire loop, micropipette, glass slides, glycerol, coverslips, spirit lamp and toothpicks.

Isolation, enumeration, and purification of vermi-bacteria

Vermicompost associated bacteria (VAB) were isolated from the vermicompost collected from Vermi-tech Unit, Department of Zoology, The University of Azad Jammu and Kashmir, Muzaffarabad. Vermicompost was prepared using cow dung, raw vegetables and fruits, coconut peel, wheat straw and rice straw, egg shells and waste papers. Eisenia fetida was used for the vermicomposting (Shafique et al., 2021). Hundred mg vermicompost was added in 200 ml of distilled water, mixture was shaken for 20 min, and kept at room temperature for two days. After incubation, serial dilution method was used as illustrated by Somasegaran and Hoben (2012) for the isolation of vermicompost associated bacteria. Nutrient broth medium was used for bacterial culturing. After incubation, serial dilution 10-2, 10-3, 10-4, and 10-5 were made and again incubated for 24 h at room temperature to pick the single colony of VAB. After incubation, 10 µl sample was spread on nutrient agar medium and incubated at 37°C for overnight. Next day, several colonies were observed in the case of 10-2, 10-3, 10-4, and 10-5 diluted samples. From these plates, twenty bacterial isolates were taken in nutrient broth medium and incubated for 24 h at 37oC. After incubation, 14 VAB isolates (VAB-1 to VAB-14) were purified after sub-culturing and then kept at -20 oC in the form of 60% glycerol.

Morphological and biochemical characteristics of VAB

For the identification of VAB different techniques and methods i.e. microscopic (Gram staining), cultural techniques (nutrient agar medium, MacConkey agar), biochemical tests (Plant growth promoting traits like production of Indole acetic acid, siderophore, ammonia, hydrogen cyanide, potassium hydroxide), hydrolytic enzymes production like catalase, oxidase, amylase, protease, and lipase, and hemolytic test were applied (Okon et al., 1977; Dawwam et al., 2013; Kumar et al., 2015). The different colors, size, elevations, textures, shapes, and edges of vermicompost associated bacteria were recorded.

Molecular characterization

Genomic DNA was extracted from bacterial isolates using chloroform: Isoamyl alcohol method with slight modifications. The isolates were grown in Luria broth medium for 24 h at 37 oC. After incubation, medium was centrifuged at 10,000 rpm for 5 min to harvest cells in pellet form. The pellet was suspended in lysis buffer-1 (Tris EDTA and SDS; pH 4.0) and centrifuged at 10,000 rpm for 10 min. After centrifugation, 500 µL of chloroform: isoamyl alcohol (24:1) was added, mixed, and centrifuged at 10,000 rpm for 10 min. To the collected supernatant 1/10th volume of sodium acetate and 2.5 volumes of chilled 100% absolute ethanol were added, incubated at -20 oC for overnight, Next day, samples were centrifuged for 10 min at 10,000 rpm, and pellet was washed with 70% ethanol. After centrifugation, pellet was dried for 3 h at room temperature (25±2 oC) and DNA was suspended in distilled water (20 µl). For the identification of bacterial diversity, 16S rRNA primers; universal bacterial primer (27F; 5′-AGAGTTTGATCCTGGCTCAG-3′) was taken to amplify 1500 bps sequence using following PCR conditions (initial denaturation 95 °C for 10 min; cyclic denaturation at 95 °C for 30 sec; annealing at 50°C for 1 min; cyclic extension 72°C for l min 30 sec; and final extension 72°C for 7 min; 30 cycles) (de Lillo et al., 2006). After PCR analysis all PCR products were dispatch to Macrogen, Korea for sequence analysis. Obtained nucleotide sequences were further preceded for homology through BLAST at National Center for Biotechnology Information (NCBI) platform. The evolutionary history was concluded through Tamura-Nei model and neighbour-joining method (Tamura et al., 2007). This analysis contained 32 nucleotide sequences. There were 1573 positions in the final dataset. Evolutionary studies were accompanied in MEGA X (Kumar et al., 2018). After homology prediction and phylogenetic analysis, amplified sequences were submitted to Genbank, NCBI for obtaining accession numbers.

Antibiogram analysis

The emergence of multi-drug resistant among microbial biofertilizers is serious concern in developing countries now a days due to anthropogenic activities. Antibiotic resistant biofertilizers carry antibiotic resistant genes (ARGs) which aggravate negative impact not only on environment as well as on wildlife and public health (Mahdi et al., 2022). So, in the current study, various antibiotics like kanamycin (30µg), norfloxacin (10µg), erythromycin (15µg), amoxicillin (30µg), ampicillin (25µg), trimethoprim (5µg), penicillin (10µg), tobramycin (10µg), sulfonamide (300µg) and aztreonam (30µg) were used to display the sensitivity/resistivity of vermibacteria using agar disc diffusion method (Bauer et al., 1959; Brown and Kothari, 1975). Nutrient agar (Oxoid: CMOO3) and nutrient broth media (Oxoid: CM1) were used for bacterial growth. The bacteria were added to a nutrient broth medium for the growth and incubated for 24 h on a rotary shaker at 37°C. The incubated culture was mixed in a freshly prepared nutrient agar medium (NAM) at 45°C. The mixture was poured into sterilized Petri dishes, solidified in laminar flow at room temperature. After solidification, antibiotic discs were placed on the surface and then incubated for 24 h at 37ºC. According to Seeley et al. (2001), the growth of bacteria was determined in 24-48 h, and the diameter of the inhibition zone in mm was also measured with the help of ruler (Hammer et al., 1999).

Statistical analysis

Each experiment was repeated in triplicates and Mean ± Standard Deviation from absolute data was calculated: (http://easycalculation.com/statistics/standard-deviation. php).

RESULTS

Morphological features

The isolated bacterial isolates were characterized by morphological features, microscopic studies, and biochemical characterizations (Table I). Results revealed that different colonies were appeared on the various agar culture media plates i.e., MacConkey agar and nutrient agar medium. The colonies of vermicompost associated bacteria were found with different colors, size, elevations, texture, shape, and edge, respectively. The colonies of VAB showed yellow, opaque, white, cream, and cherry in color; the surface and shape of colonies were recorded as spherical, rough, oval, smooth, circular, and oval. On the other hand, colony elevations (convex, flat, umbonate), margins (entire, irregular, filiform), and wet texture were recorded. Gram staining indicated that VAB-1, VAB-3, VAB-4, VAB-10, and VAB-12 were observed as Gram-negative rod bacteria while VAB-2, VAB-5, VAB-6, VAB-7, VAB-8, VAB-9, VAB-11, and VAB-14 are rod shaped Gram-positive bacteria.

Biochemical characterization

Results revealed that all vermicompost associated bacteria were shown catalase and oxidase positive results. In case of proteolytic assay, VAB-1, VAB-4, VAB-5, VAB-6, VAB-8, VAB-9, VAB-10, VAB-11, VAB-12, and VAB-14 were shown positive hydrolysis of casein in the range of 2.0±0.0 mm to 8.0±0.0 mm, while VAB-2, VAB-3, VAB-7 showed negative results. Similarly, positive lipolytic activity was shown by all tested VAB isolates. All VAB isolates showed amylolytic activity except VAB-3. VAB-1, VAB3, VAB-4, and VAB-9 showed urease and citrate positive results (Table I). All VAB showed the mannitol fermentation when streaked on the mannitol salt agar medium except VAB-1, VAB-2, and VAB-3 (Table I). Hemolytic test results revealed that all isolated VABs are non-pathogenic in nature.

Plant growth promoting traits

Results revealed that all VAB isolates were involved in the production of siderophore and Indole acetic acid (except VAB-6), and act as phosphate solubilizes (Table II). Ammonia production test revealed that VAB-1, VAB-6, VAB-7, VAB-8, VAB-9, VAB-10, VAB-12, and VAB-14 were positive for this test. Yellow color was observed in the medium, denoting the production of ammonia by these isolates. All VAB involved in the HCN production except VAB-6 (Table II). In case of KOH test, VAB-3, VAB-4, and VAB-6 showed negative results.

Molecular identification

The 16S rDNA genes from isolated bacteria were amplified using 27F bacterial universal primer and the size of amplified genes was recorded in the range of 1100 bps to 1769 bps. The partial nucleotide sequences were proceeded for further BLAST analysis at National Center for Biotechnology Information (NCBI) and results reveal that no significant similarities of VAB-8, VAB-9, and VAB-11 were found (Table III). On the other hand, VAB-1 indicated 91% similarity with Achromobacter insolitus (AB642187.1), Achromobacter denitrificans (KY475779.1), Achromobacter anxifer (MN197585.1), Achromobacter ruhlandii (KY087962.1), Achromobacter agilis (MK791141.1), and Achromobacter xylosoxidans (MK587674.1); VAB-2 showed 99.55% homology with Bacillus cereus (JX971533.1) and 99.46% with

Table II. Plant growth promoting traits of vermicompost associated bacteria.

Agricultural traits → Bacterial strains ID↓	Siderophore	Phosphate	Potassium hydroxide	Indole acetic acid	Hydrogen cyanide	Ammonia
Achromobacter ruhlandii (VAB-1)	+	+	+	+	+	+
Bacillus wiedmannii (VAB-2)	+	+	+	+	+	-
Pseudomonas sp. (VAB-3)	+	+	-	+	+	-
Achromobacter xylosoxidans (VAB-4)	+	+	-	+	+	-
Oceanobacillus oncorhynchi (VAB-5)	+	+	+	+	+	-
Bacillus mycoides (VAB-6)	+	+	-	-	-	+
Serratia nematodiphila (VAB-7)	+	+	+	+	+	+
Unknown (VAB-8)	+	+	+	+	+	+
Unknown (VAB-9)	+	+	+	+	+	+
Serratia marcescens (VAB-10)	+	+	+	+	+	+
Unknown (VAB-11)	+	+	+	+	+	-
Serratia marcescens (VAB-12)	+	+	+	+	+	+
Paenibacillus dendritiformis (VAB-14)	+	+	+	+	+	+

Table III. BLAST outcomes of vermicompost associated bacteria.

Microbe coding	Amplified length	Percent identity (%)	Scientific name	Sequence id	Accession length
VAB-1	1170	91%	Achromobacter insolitus	AB642187.1	1295
		91%	Achromobacter denitrificans	KY475779.1	1339
		91%	Achromobacter anxifer	MN197585.1	1420
		91%	Achromobacter ruhlandii	KY087962.1	1448
		91%	Achromobacter agilis	MK791141.1	1398
		91%	Achromobacter xylosoxidans	MK587674.1	1426
VAB-2	1171	99.55	Bacillus cereus	JX971533.1	1374
		99.46	Bacillus subtilis	MG593998.1	1188
		99.46	Bacillus tropicus	ON878141.1	1351
		99.46	Bacillus thuringiensis	ON974260.1	1423
		99.46	Bacillus paranthracis	ON939702.1	1419
		99.46	Bacillus pacificus	ON885910.1	1417
		99.46	Bacillus wiedmannii	ON340605.1	1370
		99.46	Bacillus paramycoides	ON000574.1	1448
VAB-3	1679	94	Bacillus firmus	LC385949.1	710
		89	Escherichia coli	MH793567.1	1371
		93	Stenotrophomonas maltophilia	MK493713.1	643
		93	Pseudomonas sp.	MT793102.1	1498
VAB-4	1224	96.82	Achromobacter insolitus	MN044760.1	1300
		96.52	Achromobacter denitrificans	MT275805.1	1374
		96.52	Achromobacter ruhlandii	KY087962.1	1448
		96.56	Achromobacter xylosoxidans	MK587674.1	1426
		96.09	Achromobacter aegrifaciens	NR_117707.1	1483
			Table continues on next page............
Microbe coding	Amplified length	Percent identity (%)	Scientific name	Sequence id	Accession length
VAB-5	1100	78.95	Bacillus pumilus	KM222185.1	1448
		78	Bacillus subtilis	OM760707.1	1454
		83	Bacillus cereus	FJ404785.1	539
		77	Oceanobacillus oncorhynchi	JX280491.1	1485
		75	Pediococcus pentosaceus	ON545700.1	1102
VAB-6	1100	81	Bacillus anthracis	KP813652.1	1221
		81	Bacillus toyonensis	LN995802.1	1111
		81	Bacillus thuringiensis	HM032789.1	948
		81	Bacillus cereus	MF726967.1	1267
		80	Bacillus proteolyticus	MK418822.1	1028
		80	Bacillus albus	MN658863.1	1235
		81	Bacillus manliponensis	MW519873.1	1083
		81	Bacillus pseudomycoides	MT573519.1	1040
		82	Bacillus mycoides	KU687331.1	1257
VAB-7	1100	87.03	Serratia marcescens	MF462933.1	1479
		87	Staphylococcus aureus	MH603394.1	1161
		87	Serratia surfactantfaciens	NR_169468.1	1495
		87	Enterobacter cloacae	KJ850207.1	1434
		87	Raoultella planticola	MZ203710.1	1363
		87	Serratia nematodiphila	KM099143.1	1392
		87	Klebsiella sp.	MZ203699.1	1363
VAB-8	1100	No significant similarity found
VAB-9	1100	No significant similarity found
VAB-10	1100	83.16	Serratia marcescens	MT386168.1	1385
		83.16	Pseudomonas sp. strain jx-18	KY780232.1	1454
		83.16	Serratia nematodiphila	ON651725.1	1261
VAB-11		No significant similarity found
VAB-12	1591	94	Serratia marcescens	KM099142.1	1411
VAB-12	1591	92	Serratia nematodiphila	MN691576.1	1251
VAB-14	1100	89	Paenibacillus dendritiformis	MG592704.1	1414
		89	Paenibacillus popilliae	KC107790.1	1380
		89	Paenibacillus thiaminolyticus	EU330645.1	1474

Bacillus subtilis (MG593998.1), Bacillus tropicus (ON878141.1), Bacillus thuringiensis (ON974260.1), Bacillus paranthracis (ON939702.1), Bacillus pacificus (ON885910.1), Bacillus wiedmannii (ON340605.1), Bacillus paramycoides (ON000574.1); VAB-3 showed 94% resemblance with Bacillus firmus (LC385949.1), 89% with Escherichia coli (MH793567.1), 93% Stenotrophomonas maltophilia (MK493713.1), 93% with Pseudomonas sp. (MT793102.1); VAB-4 showed 96.82% similarity with Achromobacter insolitus (MN044760.1), 96.52% with Achromobacter denitrificans (MT275805.1), 96.52% with Achromobacter ruhlandii (KY087962.1), 96.56% with Achromobacter xylosoxidans (MK587674.1), 96.09% with Achromobacter aegrifaciens (NR_117707.1); VAB-5 indicated 78.95% similarity with Bacillus pumilus (KM222185.1), 78% with Bacillus subtilis (OM760707.1), 83% with Bacillus cereus (FJ404785.1), 77% with Oceanobacillus oncorhynchi (JX280491.1), 75% with Pediococcus pentosaceus (ON545700.1); VAB-6 showed 81% homology with Bacillus anthracis (KP813652.1), Bacillus toyonensis (LN995802.1), Bacillus thuringiensis (HM032789.1), Bacillus cereus (MF726967.1), Bacillus manliponensis (MW519873.1), Bacillus pseudomycoides (MT573519.1), 80% with Bacillus proteolyticus (MK418822.1), Bacillus albus (MN658863.1), 82% with Bacillus mycoides (KU687331.1); VAB-7 displayed 87.03% resemblance with Serratia marcescens (MF462933.1), 87% with Staphylococcus aureus (MH603394.1), Serratia surfactantfaciens (NR_169468.1), Enterobacter cloacae (KJ850207.1), Raoultella planticola (MZ203710.1), Serratia nematodiphila (KM099143.1), Klebsiella sp. (MZ203699.1); VAB-10 indicated 83.16% similarity with Serratia marcescens (MT386168.1), Pseudomonas sp. strain jx-18 (KY780232.1), Serratia nematodiphila (ON651725.1); VAB-12 showed 94% resemblance with Serratia marcescens (KM099142.1) and 92% with Serratia nematodiphila (MN691576.1); and VAB-14 indicated 89% homology with Paenibacillus dendritiformis (MG592704.1), Paenibacillus popilliae (KC107790.1), and Paenibacillus thiaminolyticus (EU330645.1). The results of phylogenetic tree using the NJ method were recorded as VAB-1 indicated relationship with Achromobacter ruhlandii (81%), VAB-2 with Bacillus wiedmannii (81%), VAB-3 with Pseudomonas sp. (82%); VAB-4 with Achromobacter xylosoxidans (96%), VAB-5 with Oceanobacillus oncorhynchi (74%), VAB-6 with Bacillus mycoides (87%), VAB-7 with Serratia nematodiphila (74%), VAB-10 with Serratia marcescens (95%), VAB-12 with Serratia marcescens (79%), and VAB-14 with Paenibacillus dendritiformis (75%).

Antibiogram analysis

Erythromycin and norfloxacin showed the maximum inhibition of tested vermicompost associated bacteria (Table IV). Erythromycin showed the maximum inhibition of Achromobacter ruhlandii (18.0±0.0 mm), Pseudomonas sp. (14.0±0.0 mm), Oceanobacillus oncorhynchi (14.0±0.0 mm), Bacillus mycoides (12.0±0.0 mm), unknown (18.0±0.0 mm), unknown (18.0±0.0 mm), Serratia marcescens (20.0±0.0 mm), unknown (16.0±0.0 mm), Serratia marcescens (20.0±0.0 mm). Similarly, norfloxacin indicated the maximum inhibition of Achromobacter ruhlandii (14.0±0.0 mm), Achromobacter xylosoxidans (13.0±0.0 mm), Bacillus mycoides (11.0±0.0), Unknown (15.0±0.0 mm), unknown (14.0±0.0 mm), Serratia marcescens (13.0±0.0 mm), unknown (17.0±0.0 mm), Serratia marcescens (11.0±0.0 mm) Paenibacillus dendritiformis (17.0±0.0 mm). Trimethoprim, kanamycin, sulfonamide showed the maximum inhibition of Oceanobacillus oncorhynchi and VAB-11 with 17.0±0.0 mm, 15.0±0.0 mm, 11.0±0.0 mm, 19.0±0.0 mm, 14.0±0.0 mm, 12.0±0.0 mm. On the other hand, all tested VAB isolates displayed resistant towards trimethoprim, aztreonam, ampicillin, and amoxocillin (Table IV). Tobramycin and penicillin showed lowest and moderate inhibition of tested vermicompost associated bacterial isolates.

DISCUSSION

Vermicompost associated bacteria

Based on the morphological, microscopic, biochemical tests, and molecular characterization, 10 VAB strains were identified viz. Achromobacter ruhlandii, Bacillus wiedmannii, Pseudomonas sp., Achromobacter xylosoxidans, Oceanobacillus oncorhynchi, Bacillus mycoides, Serratia nematodiphila, Serratia marcescens, and Paenibacillus dendritiformis. The current results agreed with previous literature (Upadhyay et al., 2015; Selvi and Koilraj, 2015; Begum and Bora, 2018). Khyade (2018) and Vaz-Moreira et al. (2008) also demonstrated the presence of B. cereus, B. pumilus, B. macrolides, B. licheniformis, Bacillus benzoevorans, B. megaterium, and B. subtilis from vermicompost. Several bacteria i.e. Pseudomonas, Klebsiella, Bacillus, Serratia, Azospirillum, Acetobacter, Burkholderia, and Azotobacter, have been recorded as plant growth-promoting bacteria (PGPB) while Pseudomonas and Bacillus spp. have been identified as the predominant communities. Similar results were found by Kang et al. (2014), and our findings agreed with them. Andleeb et al. (2022) isolated and identified eleven vermibacteria from the gut of E. fetida such as B. megaterium, Bacillus mycoides, Staphylococcus hominis, B. aryabhattai, B. subtilis, B. licheniformis, B. spizizenii, B. mojavensis, B. cereus, B. toyonensis, B. thuringiensis, B. anthracis, and B. paranthracis. which enhanced the quality of vermicompost during vermicomposting?

Vermicompost associated bacteria possessed agricultural traits

Vermibacteria associated with the gut of E. fetida possessed agricultural traits and their impact was done on ornamental plants. These plant growth promoting vermibacteria (PGPVB) not only used for the develop of plant growth but also enhance the plant nutrition value either directly or indirectly mechanisms (Andleeb et al. 2022). The direct assistance of plant growth is carried out by various ways such as supplementation of essential nutrients such as phosphate, nitrogen, potassium, zinc, and iron (Divjot et al., 2020; Fasciglione et al., 2015). Similarly, PGPB also enhanced the plant growth by the production of phytohormones (Al-Kahtani et al., 2020). On the other side, the indirect plant growth promotion occurs through the prevention of deleterious effects of pathogens and pest on plants by releasing compounds or defense enzymes/proteins by PGPBs (Simon et al., 2019; Frampton et al., 2012). Several reports have also been known for the imperative role of PGPRs in mitigating salt stress in different crop plants tomato, groundnut, wheat, rice and red pepper (Upadhyay and Singh, 2015; Bal et al., 2013; Shukla et al., 2012). The current results showed that vermicompost associated bacterial strains i.e., Achromobacter ruhlandii, Bacillus wiedmannii, Pseudomonas sp., Achromobacter xylosoxidans, Oceanobacillus oncorhynchi, Bacillus mycoides, Serratia nematodiphila, Serratia marcescens, and Paenibacillus dendritiformis have ability to produce siderophores, hydrolytic enzymes, hydrogen cyanide, ammonia, and IAA, and solubilize phosphates and our findings agreed with the outcomes of Mahdi et al. (2020) and Ahemad and Kibret (2014). Achromobacter spp. are endophytic bacteria, showed plant growth promoting properties like production of IAA, HCN, and ammonia, and have phosphate solubilizing activity. Oceanobacillus oncorhynchi is gram-positive rod shaped endophytic bacteria, and also possessed agricultural traits. Our outcomes agreed with previous reports indicated that Oceanobacillus oncorhynchi and Achromobacter spp could be used as PGPB (Mapelli et al., 2013; Orhan, 2016; Jha and Kumar, 2009).

Key role of Vermicompost associated bacteria as PGPB

The current study reveals that vermicompost associated bacteria possessed osphate solubilizing capability and phosphorous solubilizing microbes play an important role directly and indirectly in biological, physical, and chemical soil properties, and agreed with previous studies (Guo et al., 2015; Alori et al., 2017). The presence of phosphate solubilizing bacterium in soils as well as vermicompost may be considered a positive indicator of utilizing the microbes as biofertilizers for crop production and beneficial for sustainable agriculture. In addition, phosphorous solubilization, VAB involved in the IAA and siderophore production which promotes the seed germination, enhance root development, biosynthesis of various metabolites, initiate florescence, enhances root surface area and root length, resistance to biotic and abiotic stresses, and nodule formation. Bacterial isolates also able to produce ammonia which indicates that VAB supply nitrogen to their plants and not only capable to promote shoot and root elongation as well as biomass of the plant, and our findings are consistent with the outcomes of Etesami and Beattie (2018), Mahanty et al. (2017), and Dutta et al. (2015). The nitrogen fixation ability of associative bacteria provides the essential nitrogen content during the growth phase of the plant (Jha and Kumar, 2009). It was observed that Bacillus species such as B. licheniformis, B. cereus, and B. subtilis involved in the enhancement of root colonization, phytohormone production, enhanced the plant growth and possessed biocontrol properties due to presence of agricultural traits. Our findings agreed with previous (Radhakrishnan and Lee, 2016; Allard-Massicotte et al., 2016; Rijavec and Lapanje, 2016; Islam et al., 2014; Beauregard et al., 2013).

VABs are source of spread of antibiotic resistant genes

The major concern on the development of multi-drug resistant microbes is increasing day by day due to extensive use of antibiotics in livestock. These antibiotics alter the gut microbiota of the animals that started to attain antibiotic resistance and raises the transfer of antibiotic resistant genes to rhizosphere bacteria through horizontal gene transfer (Tariq et al., 2022). These bacteria might be involved in the spread of resistome to human pathogens or can infect humans through contaminated vegetables and fruits (Checcucci et al., 2020; Zeng et al., 2018). According to Gonzalez et al. (2017), some PGPB involved in the human infections on exposure of contaminated soil, water and farm products. So, in the current study, screening of antibiotic susceptibility tests was necessary to know the status of VABs that could be used as microbial biofertilizers. Results revealed that all vermibacterial isolates are not multidrug resistant bacteria and their presence in the fields reduced the possibility of transfer of resistome in foodchain.

Current study reveals that vermicompost associated bacteria i.e., Achromobacter spp., Bacillus spp., Pseudomonas sp., Oceanobacillus oncorhynchi, Serratia spp., and Paenibacillus dendritiformis could be used as microbial biofertilizers to enhance crop production, involved in vermicomposting, improves the vermicompost quality and soil health, and used as a biocontrol agent due to presence of agricultural traits.

Declarations

Acknowledgements

Authors are thankful to Transfer Technology Funds (TDF), HEC for providing funds (TDF-02-006) for current research work and also thankful to Department of Biochemistry, Islamia University Bahawalpur, Bahawalpur for the identification of vermibacterial isolates.

Funding

Whole research work is done under HEC funded project No. TDF-02-006 titled: “Establishment of the vermi-tech unit at Azad Jammu and Kashmir, Muzaffarabad for vermi product development”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Ethical statement and approval

All experiments conducted during the research were carefully designed to prevent distress, suffering, and unnecessary pain to the experimental animals. All procedures were carried out in accordance with international regulations, specifically following Article 9 of the Dutch Law on animal experimentation (Wet op de dierproeven). The current study was approved by Institutional Review Committee of Office of Research, Innovation, and Commercialization (ORIC), The University of Azad Jammu and Kashmir, Muzaffarabad vide No. 246/ORIC/2022; Dated: 3-10-2022.

Availability of data and materials

All the data generated during this study are included in this article.

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20230525060540

Statement of conflict of interest

The authors have declared no conflict of interest.

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