Phyllosphere-associated Methylobacterium: a potential biostimulant for ginger (Zingiber officinale Rosc.) cultivation.

Methanol, a by-product associated with plant metabolism, is a substrate for pink pigmented facultative methylotrophs (PPFMs) of phyllosphere. The symbiotic interaction of PPFMs has many desirable effects on plant growth and disease resistance. The present study investigated the potential of native PPFMs for mitigating biotic stress and plant growth promotion in ginger. PPFMs were isolated from ginger phyllosphere by leaf imprint technique and screened against major fungal phytopathogens of ginger viz. Macrophomina phaseolina, Sclerotium rolfsii, Pythium myriotylum, Colletotrichum gloeosporioides and Fusarium oxysporum. Among the 60 PPFMs, IISRGPPFM13 was selected for its highly inhibitory activity against the target pathogens. The isolate was useful for mineral solubility, production of IAA, siderophores and hydrolytic enzymes like cellulase, pectinase, lipase, amylase and chitinase. On in planta experiments revealed that IISRGPPFM13 considerably increased plant growth parameters when the bacterium was applied as soil drenching cum foliar spraying. Methanol utilization potential of the isolate was confirmed by mxaF gene analysis where the sequence showing 95.51% identity towards Methylobacterium platani and M. iners. Further, 16S rRNA gene sequence showing 98.73% identity with M. komagatae 002-079 T (AB252201). This is the first report of its kind that a genus of Methylobacterium with biostimulant potential isolated from the phyllosphere of ginger.

Evaluation of pink-pigmented facultative methylotrophic bacteria for phosphate solubilization.

Thirteen pink-pigmented facultative methylotrophic (PPFM) strains isolated from Adyar and Cooum rivers in Chennai and forest soil samples in Tamil Nadu, India, along with Methylobacterium extorquens, M. organophilum, M. gregans, and M. komagatae were screened for phosphate solubilization in plates. P-solubilization index of the PPFMs grown on NBRIP-BPB plates for 7 days ranged from 1.1 to 2.7. The growth of PPFMs in tricalcium phosphate amended media was found directly proportional to the glucose concentration. Higher phosphate solubilization was observed in four strains MSF 32 (415 mg l(-l)), MDW 80 (301 mg l(-l)), M. komagatae (279 mg l(-l)), and MSF 34 (202 mg l(-l)), after 7 days of incubation. A drop in the media pH from 6.6 to 3.4 was associated with an increase in titratable acidity. Acid phosphatase activity was more pronounced in the culture filtrate than alkaline phosphatase activity. Adherence of phosphate to densely grown bacterial surface was observed under scanning electron microscope after 7-day-old cultures. Biochemical characterization and screening for methanol dehydrogenase gene (mxaF) confirmed the strains as methylotrophs. The mxaF gene sequence from MSF 32 clustered towards M. lusitanum sp. with 99% similarity. This study forms the first detailed report on phosphate solubilization by the PPFMs.

Cellulase production by pink pigmented facultative methylotrophic strains (PPFMs).

Pink pigmented facultative methylotrophs (PPFM) isolated from water samples of Cooum and Adyar rivers in Chennai and soil samples of forests located in various districts of Tamil Nadu, India were screened for cellulase production using carboxymethylcellulose agar (CMC agar) medium. The strains showed wide variations in the production of clearing zones around the colonies on CMC agar medium flooded with Congo red. CMCase and filter paper assays were used to quantitatively measure the cellulase activity of 13 PPFM strains. Among the strains, Methylobacterium gregans, MNW 60, MHW 109, MSF 34, and MSF 40 showed cellulolytic activity ranging from 0.73 to 1.16 U mL(-1) with wide temperature (35-65°C) and pH (5 to 8) tolerance. SDS-PAGE analysis of the crude enzyme of PPFM strain MNW 60 exhibited several protein bands, and zymogram analysis revealed two dimeric cellulase bands with molecular mass of ~92 and 42 kDa. Scanning electron microscopic studies revealed significant morphological differences between the cells grown in normal and CMC amended medium. The strain MNW 60 was identified as Methylobacterium sp. based on biochemical, physiological, and morphological analyses, and the methylotrophic nature was authenticated by the presence of mxaF gene, encoding methanol dehydrogenase as a key indicator enzyme of methylotrophs, with 99% similarity to Methylobacterium lusitanum. With the 16S ribosomal RNA sequence showing 97% similarity to M. lusitanum strain MP2, this can be proposed as a novel taxon of the genus Methylobacterium. The study forms the first detailed report on the extracellular cellulase production by pink pigmented Methylobacterium sp., and it is expected that this might be the basis for further studies on cellulase production by PPFMs to explore the molecular mechanism, strain improvement, and large-scale cellulase production for its application.