Characterization and evaluation of Pseudomonas spp. for abiotic stress tolerance

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Department of Plant Pathology, College of Horticulture, Vellanikkara
Pseudomonas spp. are one among the most extensively used biocontrol agent in plant disease management to control foliar, soil borne or seed borne pathogens. However, prevalence of abiotic stresses such as drought, high temperature, salinity and acidity may affect the field performance due to poor survival under adverse conditions. To date, very little effort has been taken to tap microbial diversity from stressed ecosystems of Kerala. Thus, a study was undertaken to isolate native strains of Pseudomonas spp. having inherent stress tolerance. Purposive soil sampling survey was conducted during the period February- April 2018 and a total of 26 representative soil samples were collected from four districts viz., Ernakulam, Palakkad, Thrissur and Alappuzha. It was revealed that various locations of Palakkad and Thrissur district experienced high temperature (40-46oC) and low moisture of 1.1- 3.9 per cent. Samples procured from Alappuzha and Ernakulam districts were extremely acidic (pH 3.4- 4.5) andsaline (EC 4.1-6.68 dSm-1). The results of soil analysis confirmed that the collected soil samples were abiotically stressed and there could be chances of obtaining stress tolerant native isolates of Pseudomonas spp. For isolation of Pseudomonas spp., soil samples weresubjected to serial dilution and plating technique in King’s B agar and Pseudomonas agar base, among which King’s B agar yielded optimum number of Pseudomonas colonies at 105 dilution.Maximum population of Pseudomonas spp. was recorded in samples procured from Moncombu (1.3-7.8x106cfu g-1), whereas, minimum population was observed in soil samples collected from Vyttila (3.14- 6.01 x 104cfu g-1). A total of 15 isolateswere purified and designated with sample codes based on the place of collection. Among the nine fluorescent isolates, M7 and P7 were identified as Pseudomonas aeruginosa, an opportunistic human pathogen based on its growth at 42oC and on King’s A agar medium and thus, were eliminated. Isolates of Pseudomonas spp. were subjected to in vitro screening for abiotic stress tolerance and compared with reference culture of KAU. The isolates P2, M4 and T5 were selected as temperature tolerant (50oC), M4 and V4 as salt tolerant (1.5 M NaCl), P4 and T4 as xxxii drought tolerant (30% PEG) and M4 and M5 as acid tolerant (pH 3.5) strains. Subsequently, a total of seven abioticstress tolerant isolates viz., M4, M5, P2, P4, V4, T4 and T5 were evaluated further for their in vitro antagonistic potential against five major soil borne fungal pathogens using Bangle method.The four isolatesviz., P2, P4, M4 and M5 were selected as the potential antagonistic strains since they outperformed reference culture of KAU and showed higher and consistent biocontrol activity with per cent growth inhibition ranging from 62.21 to 91.00 per cent against Phytophthora capsici, Pythium aphanidermatum, Sclerotium rolfsii, Rhizoctonia solani and Fusarium oxysporum. Further studies revealed M4, M5 as better producers of ACC deaminase enzyme and M4, P4 as better producers of exopolysaccharide. Highest cellulase and β-1, 3 glucanase production was recorded by the isolates P2 and M4 respectively. A preliminary screening of the isolates were carried out for testing the bioefficacy on cowpea seeds against Rhizoctonia solani using roll towel method. The results revealed that the isolate M4 was highly efficient, reference culture as efficient and the other isolates P2, P4 and M5 as moderately efficient.All the four isolates were further evaluated for their in vivo activity through a pot culture experiment using cowpea- R. solani system. Eventhough, M4 outperformed all other isolates with highest seed germination, biometric characters and yield as well as with lowest per cent disease incidence, other three isolates were also found superior or equally efficient as reference culture of KAU. The best performing isolates with promising traits of stress tolerance, antagonism and plant growth promotion viz., P2, P4, M4 and M5 were identified based on cultural, morphological, biochemical and molecular characterization. The isolate P2 was identified as Pseudomonas putida, P4 as P. fluorescens and M4 and M5 as P. aeruginosa. Thus, the current investigation has thrown light on the prevalence of abiotic stress tolerant strains of Pseudomonas spp. in stressed ecosystems which would significantly help the farming community to overcome such drastic climate changes. However, the study should further be complemented with large scale multilocational trials to prove their efficacy under field conditions.