Evaluation of newly isolated probiotic bacteria from various plant roots harboring growth promoting traits for the cultivation of organic tomato

Abstract

In recent year the overuse of chemical fertilizer increases day by day and their effects on the environment and human health have been reported by many studies. Due to the adverse effect of chemical fertilizer scientist should think of a new and alternative way for sustainable agriculture. The plant probiotic bacteria (Endophytic bacteria) occur inside the plants and has a diverse effect on plant growth promotion, which can solve this problem by providing sustainable agriculture. This study was carried out by 20 isolated endophytic bacteria from rice, tomato and cauliflower roots and also evaluated their role as plant growth promoters. The isolates were named as P1 – P10 form rice roots, TR1 – TR5 form tomato roots and CR1– CR5 form cauliflower roots. All the isolates except P7 were continued for next studies. On the basis of KOH string test the 15.79% isolates were gram positive and the rest of 84.21% isolates were gram negative endophytes. The biochemical tests reveal that among the isolated strains 84.21% were positive for MIU and Urease test, 73.68% were positive for CIU test, 52.63% were positive for Oxidase test, 47.37% were positive for Voges-Proskauer (VP), 15.79% were positive for Methyl Red (MR) test. All endophytic bacterial isolates were positive for Catalase and TSI test. In carbohydrate utilization test 47.37%, 78.95%, 94.77% and 100% were positive for Lactose, Maltose, Sucrose and Dextrose, respectively. For extracellular hydrolytic enzyme assay all the isolates showed positive results. Among the 20 endophytic isolate 18 isolates were molecularly characterized using 16s rRNA gene sequencing and were identified 38.89% as Enterobacter sp., 22.22% as Klebsiella sp., 16.67% as Pantoae sp., 11.11% as Bacillus sp., 5.56% as Pseudomonas sp., and 5.56% as Burkholderia sp. These identified strains have been submitted to gene bank in National Centre for Biotechnology Information (NCBI). Three Phylogenetic tree has also been described for all the 18 strains with maximum likelihood method and proper node and branch lengths has been maintained to identify all of these strains. The 19 isolated bacteria were checked for their plant growth promoting (PGP) ability and the 100% of the isolates were shown positive results for phosphate solubilization which was done on Pikovskayas Agar medium. The ability of nitrogen fixation of the isolates were checked both in N- free (Azotobacter agar medium) and N- containing (Yeast extract mannitol agar) medium. In both the medium all the isolates were able to fix atmospheric nitrogen and showed positive results. In N- containing (Yeast extract mannitol agar) medium the isolates P4, P5, P8, P9, CR1 and CR2 were shown the maximum nitrogen fixation ability. For indole acetic acid (IAA) production 100% of the strains were able to produced IAA and the maximum amount were produced by P2 (3.514μg/ ml), P5 (4.038μg/ ml), TR1 (6.093μg/ ml), TR3 (7.002μg/ ml), TR4 (3.65μg/ ml) and TR5 (6.729μg/ ml). The ability of isolates to utilize ACC as a sole source of N was assessed on Dworkin and Foster (DF) minimal medium and the isolates TR3, CR4 and CR5 gave the maximum absorbance at 600nm. The growth promoting ability of all the isolates were checked on tomato plant individually and also in the consortium. The plate assay for germination effect of isolated strains were carried out on tomato seeds and the highest significant amount of germination percentage, root length, shoot length, seedling length, vigour index was found in CR4 (100%), P9 (7.19 cm), CR3 (6.57 cm), P9 (13.23 cm) and CR4 (1262.67 cm), respectively. For pot (field) application of 40 days recorded data, the highest significant root length, shoot length, fresh weight and dry weight were recorded in TR2 (7.03 cm), TR1 (18.77 cm), P9 (2.50 gm/ plant) and TR5 (0.48 gm/ plan), respectively. The application of these endophytes as consortia in pot field experiment also showed the significant development of root length, shoot length and seedling length and the results was significant in soil+bacterial treated tomato plant compared to soil+compost+bacteria treated plant. The highest significant root length was recorded with group 2 (P6+P7+P8+P9+P10) treated plant that is 13.60 cm and the highest significant shoot length was recorded with group 3 (TR1+TR2+TR3+TR4+TR5) treated plant that is 20.17 cm. The significant seedling length was recorded with group 1 (P1+P2+P3+P4+P5) treated plant that is 32.67 cm. All this result strongly suggests that the endophytic bacteria characterized in this study could be successfully used to promote plant growth.