Subbiah Sundaram

Growth promotion and induction of systemic resistance in rice cultivar Co-47 (Oryza sativa L.) by Methylobacterium spp.

Pink-pigmented facultatively methylotrophic bacteria (PPFMs), persistent colonizers of plant leaf surfaces, belong to the genus Methrlobacterium and are mostly transmitted through seeds. Plant growth-promoting activity of methylotrophic bacteria and their effects on disease suppression were evaluated on rice under greenhouse conditions. Rice seeds were inoculated with Methylobacterium sp. strain PPFM-Os-07 and seed germination was evaluated in terms of morphometric measurements, seedling growth, rate of germination (R(subscript G)), and seedling vigour index (SVI). Another experiment was carried out to study the induction of pathogenesis-related proteins (PR-proteins) in rice plants that were inoculated with methylotrophic bacteria by seed imbibition or foliar spray. In the third experiment, sixty-day-old rice plants grown in pots were challenge inoculated with Rhizoctonia solani strain TNAU-01. Methylobacterium inoculation promoted seed germination and plant growth. Increased plant height, number of tillers, plant biomass, and grain yield were observed. The average yield increases for seed imbibition and phyllosphere spray were, respectively, 22.1% and 24.3% greater than control. The bacteria also significantly reduced the sheath blight incidence when applied as either bacterial culture through seed imbibition and or phyllosphere spray. The percent disease reduction recorded for seed imbibition alone and for combined applications of seed imbibition and phyllosphere spray were 17.8% and 23.5%. Rice plants sprayed with PPFM-Os-07 strain showed increased presence of PR-proteins and phenolic contents on day 1 after application. Maximum phenylalanine ammonia lyase (PAL) and peroxidase activity on day 4 and β-1,3-glucanase and chitinase activity on day 5 were recorded. The results suggest that Methylobacterium inoculation may alter rice susceptibility to R. solani. This work emphasizes the importance of evaluating induced systemic resistance while studying plant-associated growth promoting bacteria.

Pink-pigmented facultative methylotrophic bacteria accelerate germination, growth and yield of sugarcane clone Co86032 (Saccharum officinarum L.)

The existence of Methylobacterium as a symbiont with sugarcane and its influence on crop growth at various stages was examined. Pink-pigmented facultative methylotrophic bacteria (PPFMs) strains isolated from different parts of the sugarcane clone Co86032 showed growth on methanol, and were further confirmed based on the mxaF gene encoding the α-subunit of the methanol dehydrogenase by polymerase chain reaction amplification using specific primers. True seeds inoculated with PPFMs had a higher germination percent and rate of germination than the control. A combined treatment of seed imbibition, soil application and phyllosphere spray increased specific leaf area, plant height, number of internodes, and cane yield. Immunological determination of cytokinin in young and mature leaves significantly increased when the epiphytic population on the leaf surface increased. Trends in sugar qualities in the form of Pol (sucrose) % in cane, Brix % in cane, and commercial cane sugar were similar to that of cane yield. These effects might be mediated by the production or synthesis of plant hormones.

Mucilaginibacter gossypii sp. nov. and Mucilaginibacter gossypiicola sp. nov., plant-growth-promoting bacteria isolated from cotton rhizosphere soils.

Two isolates from rhizosphere soil of cotton, designated Gh-67(T) and Gh-48(T), which produced large amounts of extracellular polysaccharide and possessed plant-growth-promoting traits, were characterized phenotypically and genotypically. The strains were Gram-negative and cells were non-motile rods that grew optimally at 28°C and grew between pH 4 and 7. 16S rRNA gene sequence analysis of strains Gh-67(T) and Gh-48(T) placed them in the genus Mucilaginibacter, with pairwise sequence similarity between them and type strains from related genera ranging from 93.9 to 98.2 %. The major fatty acids were iso-C₁₅:₀, C₁₆:₀ and summed feature 3 (C₁₆:₁ω7c and/or iso-C₁₅:₀ 2-OH). The strains contained MK-7 as the major isoprenoid quinone. The DNA G+C contents of strains Gh-67(T) and Gh-48(T) were 46.7 and 44.2 mol%, respectively. The low DNA-DNA hybridization value (18 %) and a number of phenotypic differences between strains Gh-48(T) and Gh-67(T) indicated that they represent two separate species. Results of phenotypic, phylogenetic and genotypic analysis revealed that the strains were separated from the species of Mucilaginibacter described to date. Therefore, strains Gh-67(T) and Gh-48(T) represent novel species of Mucilaginibacter, for which we propose the names Mucilaginibacter gossypii sp. nov. (type strain Gh-67(T) =NCIMB 14470(T) =KCTC 22380(T)) and Mucilaginibacter gossypiicola sp. nov. (type strain Gh-48(T) =NCIMB 14471(T) =KCTC 22379(T)).

Flavobacterium glycines sp. nov., a facultative methylotroph isolated from the rhizosphere of soybean.

An aerobic, yellow-pigmented, facultatively methylotrophic, Gram-staining-negative, non-spore-forming bacterium, designated strain Gm-149(T), was isolated from the rhizosphere of cultivated soybean in India. Cells were motile by gliding. The predominant cellular fatty acids were iso-C(15 : 0), summed feature 3 (comprising iso-C(15 : 0) 2-OH and/or C(16 : 1) ω 7c), C(16 : 0) 3-OH and anteiso-C(15 : 0), and the major isoprenoid quinone was MK-6. The G+C content of the genomic DNA of strain Gm-149(T) was 35.6 mol%. Comparative 16S rRNA gene sequence analysis showed that strain Gm-149(T) formed a distinct phyletic line within the genus Flavobacterium. Based on levels of pairwise 16S rRNA gene sequence similarity, strain Gm-149(T) was related most closely to the type strain of Flavobacterium daejeonense (97.1 %), but the level of DNA-DNA relatedness between these two strains was about 11.2 %. On the basis of phenotypic and genotypic data, strain Gm-149(T) is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium glycines sp. nov. is proposed. The type strain is Gm-149(T) (=ICMP 17618(T)=NBRC 105008(T)).

Genetic diversity and phylogeny of pink-pigmented facultative methylotrophic bacteria isolated from the phyllosphere of tropical crop plants

Genetic diversity of pink-pigmented facultative methylotrophic bacteria belonging to the genus, Methylobacterium, was assessed using 16S ribosomal RNA (rRNA) gene sequencing, amplified ribosomal DNA restriction analysis (ARDRA), and differential carbon-substrate utilization profile in the phyllosphere of cotton, maize, sunflower, soybean, and mentha plants. Methylobacterium populi, Methylobacterium thiocyanatum, Methylobacterium suomiense, M. aminovorans, and Methylobacterium fujisawaense were identified to colonize the phyllosphere of these crop plants. Among these, M. populi found to be the dominating species followed by M. aminovorans. The diversity indices like Shannon index of diversity, Pielou index of evenness, and Margalef index of richness calculated based on combined data of ARDRA, 16S rRNA gene sequence, and differential carbon-substrate utilization revealed that sunflower leaves showed richest methylobacterial diversity, followed by soybean, while cotton and mentha leaves recorded the lowest diversity. The outcome of this study indicates that diversity of Methylobacterium in phyllosphere depends upon the host plant species colonized.

Leifsonia soli sp. nov., a yellow-pigmented actinobacterium isolated from teak rhizosphere soil.

Two yellow-pigmented, Gram-stain-positive, aerobic, motile, short rod-shaped bacteria were isolated from natural teak tree rhizosphere soil and their taxonomic positions were determined by using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strains TG-S248(T) and TG-S240 formed a distinct phyletic line within the genus Leifsonia. 16S rRNA gene sequence analysis of strain TG-S248(T) with sequences from Leifsonia shinshuensis DB 102(T), L. poae VKM Ac-1401(T), L. naganoensis DB 103(T), L. aquatica DSM 20146(T) and L. xyli subsp. cynodontis JCM 9733(T) revealed pairwise similarities ranging from 98.7 to 99.1 %. The major fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0). The G+C content of the DNA of the type strain was 69.4 mol%. DNA-DNA hybridization experiments revealed low levels of DNA-DNA relatedness (32 % or less) between strain TG-S248(T) and its closest relatives. Based on differences in phenotypic and genotypic characteristics, strain TG-S248(T) (=LMG 24767(T) =JCM 15679(T)) is designated the type strain of a novel species of the genus Leifsonia, for which the name Leifsonia soli sp. nov. is proposed.

Control of Wilt and Rot Pathogens of Tomato by Antagonistic Pink Pigmented Facultative Methylotrophic Delftia lacustris and Bacillus spp.

The studies on the biocontrol potential of pink pigmented facultative methylotrophic (PPFM) bacteria other than the genus Methylobacterium are scarce. In the present study, we report three facultative methylotrophic isolates; PPO-1, PPT-1, and PPB-1, respectively, identified as Delftia lacustris, Bacillus subtilis, and Bacillus cereus by 16S rRNA gene sequence analysis. Hemolytic activity was tested to investigate the potential pathogenicity of isolates to plants and humans, the results indicates that the isolates PPO-1, PPT-1, and PPB-1 are not pathogenic strains. Under in vitro conditions, D. lacustris PPO-1, B. subtilis PPT-1, and B. cereus PPB-1 showed direct antagonistic effect by inhibiting the mycelial growth of fungal pathogens; Fusarium oxysporum f. sp. lycopersici (2.15, 2.05, and 1.95 cm), Sclerotium rolfsii (2.14, 2.04, and 1.94 cm), Pythium ultimum (2.12, 2.02, and 1.92 cm), and Rhizoctonia solani (2.18, 2.08, and 1.98 cm) and also produced volatile inhibitory compounds. Under plant growth chamber condition methylotrophic bacterial isolates; D. lacustris PPO-1, B. subtilis PPT-1, and B. cereus PPB-1 significantly reduced the disease incidence of tomato. Under greenhouse condition, D. lacustris PPO-1, B. subtilis PPT-1, and B. cereus PPB-1 inoculated tomato plants, when challenged with F. oxysporum f. sp. lycopersici, S. rolfsii, P. ultimum, and R. solani, increased the pathogenesis related proteins (β-1,3-glucanase and chitinase) and defense enzymes (phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and catalase) on day 5 after inoculation. In the current study, we first report the facultative methylotrophy in pink pigmented D. lacustris, B. subtilis, and B. cereus and their antagonistic potential against fungal pathogens. Direct antagonistic and ISR effects of these isolates against fungal pathogens of tomato evidenced their possible use as a biocontrol agent.