Screening Of Bacterial Colonies On The Basis Of Salt & Antibiotic Tolerance Properties

Introduction

In this study, we conducted a thorough screening of 285 bacterial colonies from various Tea gardens in the Terai, Dooars, and Darjeeling regions. The screening was based on their salt and antibiotic tolerance properties. Bacterial isolates demonstrating good tolerance to these abiotic stresses were further subjected to screening based on total nitrogen (N) content using the Kjeldahl Process for N estimation. Additionally, their ability to fix nitrogen (N) was assessed using ARA and Glutamine Synthetase assays, followed by molecular characterization to determine their identity and molecular phylogeny.

Results and Findings

After analyzing the 16SrRNA and accessioning the data to the NCBI database, we discovered that most of the finally screened bacteria exhibited striking similarities to various free-living nitrogen-fixing bacteria. Among the 23 characterized bacteria, the dominant species were Burkholderia, Stenotrophomonas, Herbaspirillum, Methylobacter, Acinetobacter, Bacillus, and Azospirillum.

From the six most promising isolates in terms of total N content, ARA, and Glutamine Synthetase assay, we identified the following bacteria: DS-1-20 (Stenotrophomonas sp Accession No. KY636360), DS-2-10 (Herbaspirillum sp Accession No. KX587468), DJ-1-22 (Burkholderia sp Accession No. KY 859855), DJ-1-3 (Burkholderia sp Accession No. KY 636359), TS-3-15 (Stenotrophomonas sp Accession No. KY631488), and AS-1-4 (Stenotrophomonas sp Accession No. KY636361). These six isolated screened bacteria were selected for further study to explore their potential as biofertilizers or bioaccelerants in Tea fields.

Discussion

The genus Burkholderia comprises 19 species, including soil and root-associated bacteria, as well as plant and human pathogens. Similarly, the aerobic, rod-shaped, endospore-producing genus Bacillus is taxonomically diverse, with at least 10 phylogenetic groups identified. Bacillus cereus AR156, a member of this genus, has been found to exhibit plant growth-promoting activities. Bacillus cepacia is also known for promoting maize growth and enhancing crop yields while suppressing soil-borne plant pathogens and degrading various pesticides. The genus Bacillus has been predominantly observed in the rhizoplane and the zone adjacent to the roots of tea bushes.

The genus Stenotrophomonas, comprising about eight species, includes Stenotrophomonas maltophilia, which functions as a plant growth promoter. Stenotrophomonas maltophilia is a ubiquitous, aerobic, non-fermentative, gram-negative bacillus closely related to Pseudomonas species. It exhibits some pathogenic effects and shows tolerance to certain antibiotics, as observed in previous studies. Azospirillum, belonging to the α-subclass of Proteobacteria, is also well-known as a plant growth-promoting rhizobacteria, isolated from the root zone of many grasses and cereals worldwide.

Herbaspirillum was initially thought to be a new Azospirillum species due to its cell shape, growth behavior, and habitat within grass roots. However, RNA-RNA hybridization experiments revealed no relationship with Azospirillum species or Aquaspirillum itersonii. Some Herbaspirillum species, such as Herbaspirillum seropedicae, Herbaspirillum frisingense, and Herbaspirillum lusitanum, have been reported to fix nitrogen (N). An endophytic Herbaspirillum sp isolated from tea plants has shown potential in promoting tea-plant rooting and budding due to its ability to produce indole-3-acetic acid (IAA), ammonia, and siderophores.

Members of the genus Acinetobacter are widely distributed in nature and have been isolated from the rhizosphere of various plants. Acinetobacter indicus, for instance, was first described in soil samples collected from hexachlorocyclohexane dump sites in India. Some isolates belonging to the genus Acinetobacter have exhibited plant growth-promoting properties in vitro cultures. However, no work has been done in tea field soil related to their potential as biofertilizers or PGPR.

Methanotrophs are methane-oxidizing bacteria, and certain types, such as Type I genus Methylococcus and Type II methanotrophs, have shown the capability of N fixation.

The genus Ralstonia was established to accommodate species previously known as Alcaligenes eutrophus, Pseudomonas solanacearum, and Pseudomonas pickettii. Ralstonia eutropha and Ralstonia brasilensis have been isolated from sludge, soil, and wastewater, with Ralstonia taiwanensis identified as a promising symbiont N fixer in Mimosa.

Conclusion

Our comprehensive screening of bacterial colonies from various Tea gardens in the Terai, Dooars, and Darjeeling regions has led to the identification of six promising isolates with excellent nitrogen-fixing potential and growth-promoting properties. These isolates, specifically DS-1-20, DS-2-10, DJ-1-22, DJ-1-3, TS-3-15, and AS-1-4, hold great promise as biofertilizers or bioaccelerants in Tea fields. Their application could significantly benefit organic and conventional Tea gardens, especially in North Bengal. Further research and experimentation will be crucial to fully realize the potential of these bacterial isolates for sustainable agricultural practices in the tea industry.

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