Exopolysaccharide producing bacteria from plant microbiome for drought stress mitigation in tomato

Abstract

The study entitled “Exopolysaccharide producing bacteria from plant microbiome for drought stress mitigation in tomato” was carried out at the Department of Molecular Biology and Biotechnology and Department of Agricultural Microbiology, College of Agriculture, Vellayani, in the year 2023–2024 with the objective to isolate and characterise exopolysaccharide producing bacteria from rhizosphere and phyllosphere of tomato and to evaluate them for drought stress mitigation. The leaf samples and rhizosphere soil were collected from tomato varieties cultivated in different locations for the isolation of exopolysaccharide (EPS) producing bacteria. Isolation was carried out by serial dilution and plating on EPS selective medium amended with sucrose, from which mucoid colonies were selected, purified, and used for further studies. On quantification of EPS production of the isolates, rhizosphere bacterial isolate TRE15 (0.36 ± 0.06g) and phyllosphere isolate TPE10 (0.19 ± 0.01g) produced significantly higher EPS. The isolates were screened for their ability to withstand water stress induced by higher concentrations (0%, 10%, and 20%) of Polyethylene Glycol (PEG) 6000 and their growth was assessed by measuring OD600. Among them rhizosphere isolates TRE15 (0.38±0.02) and phyllosphere isolate TPE8 (0.08±0.00) showed better growth in 20% PEG 6000 compared to other isolates. In oil spread assay rhizosphere isolates showed better biosurfactant property compared to phyllosphere isolates, with TRE8 showing maximum oil dispersion diameter of 3.90±0.14 cm. Microtiter plate assay for determining the biofilm forming potential of the isolates revealed that TRE33 produced significant amount of biofilm (0.13±0.02), where as in phyllosphere isolates TPE16 (0.14±0.02) was the best. Most of the phyllosphere isolates showed lesser biofilm production compared to rhizosphere isolates. Based on these results six isolates each were selected from the rhizosphere and phyllosphere for further assessment of plant growth promotion traits. Rhizosphere isolates TRE 33 (32.69±0.86 mg L-1) and TRE39 (107.87±8.29 mg L-1) showed 203 significantly higher extracellular ammonia production whereas all selected phyllosphere isolates except TPE7 produced extracellular ammonia. Among rhizosphere bacteria, maximum indole acetic acid (IAA) production was observed in TRE8 (41.10±0.61 mg L-1) in medium containing tryptophan and TRE39 (107.87±8.29 mg L-1) in medium without tryptophan. Phyllosphere isolate TPE13 (67.68±8.27 mg L-1) showed higher concentration of IAA in medium containing tryptophan and TPE8 (65.82±1.84 mg L-1) produced maximum IAA in the absence of tryptophan in medium. TRE33 (187.21±4.06 mg L-1) and TPE9 (413.93±9.34 mg L-1) produced significantly higher concentration of gibberellic acid. The isolates exhibiting plant growth promotion traits were selected for further in vivo studies. Growth promotion potential of the selected isolates was analysed in tomato seeds by roll towel assay. Based on the germination percentage, seedling vigour index and survival percentage, the rhizosphere isolates TRE4, TRE8, and TRE21, and phyllosphere isolates TPE8, TPE9, and TPE13 were selected for preparation of consortium and further evaluation in tomato plant. The selected isolates were characterised morphologically, biochemically and identified by 16S rRNA gene sequencing as Preistia aryabhatti (TRE4), Enterobacter cloacae (TRE8), Bacillus pumilus (TRE21), Bacillus stercoris (TPE8), Stenotrophomonas pavanii (TPE9), and Pantoea dispersa (TPE13). Separate consortium of rhizosphere isolates (RC) and phyllosphere isolates (PC) were used for pot culture experiment in tomato. Three treatments were included viz., seed priming with RC, foliar spray with PC at 20 days after transplanting and a combination of both seed priming (RC) and foliar spray (PC), along with control. Ten days after foliar spray, drought stress was induced by withholding irrigation. The stress induced plants were analysed for cell membrane integrity, proline and malondialdehyde (MDA) concentration, and for stress related enzymes viz., super oxide dismutase (SOD) and peroxidase (PO) activity, when the relative water content in stressed control plants reached 60–65%. The results indicate that level of stress factors was less in the plants which received both seed priming with RC and foliar spray with 204 PC, showing high cell membrane integrity (39.995±0.503), reduced proline content (20.000±0.555 mg L-1), reduced MDA content (1.0872±0.002 μmol g-1 tissues), higher SOD (1.165±0.013 activity g-1), and lower PO (103.183±0.212 activity g-1min-1) activity. Expression analysis of stress inducing genes, namely SlNCED3, SlAREB, and SlERF024 in leaf samples of tomato was conducted by real-time PCR. The SlAREB showed an upregulation of 1.16 fold and 4.64 fold in control plants after 3 and 6 hours of drought induction, while in the treatment with seed priming and foliar spray, it was downregulated up to 6 hours. In the case of SlERF024 gene, it showed downregulation up to 6 hours after drought induction. SlNCED3 demonstrated that plants treated with seed priming and foliar spray was drought tolerant as they were upregulated after 6 hours of drought induction. The present study reveals that seed priming of tomato with EPS producing rhizosphere consortium of Preistia aryabhatti (TRE4), Enterobacter cloacae (TRE8), and Bacillus pumilus (TRE21) and foliar spray with 2% solution of EPS producing phyllosphere consortium of Bacillus stercoris (TPE8), Stenotrophomonas pavanii (TPE9), and Pantoea dispersa (TPE13) can induce drought stress tolerance in tomato plants. 2