M. Madhaiyan

Regulation of ethylene levels in canola (Brassica campestris) by 1-aminocyclopropane-1-carboxylate deaminase-containing Methylobacterium fujisawaense

We report the presence of ACC deaminase in Methylobacterium fujisawaense and its lowering of ethylene levels and promotion of root elongation in canola seedlings under gnotobiotic conditions. To test a part of the previous model proposed for ACC deaminase producing bacteria with Methylobacterium, ACC levels and various enzyme activities were monitored in canola. Lower amounts of ACC were present in the tissues of seeds treated with M. fujisawaense strains than in control seeds treated with MgSO4. Though the increased activities of ACC synthase in the tissue extracts of the treated seedlings might be due to bacterial indole-3-acetic acid, the amount of ACC was reduced due to bacterial ACC deaminase activity. The activities of ACC oxidase, the enzyme catalyzing conversion of ACC to ethylene remained lower in M. fujisawaense treated seedlings. This consequently lowered the ethylene in plants and prevented ethylene inhibition of root elongation. Our results collectively suggest that Methylobacterium commonly found in soils, as well as on the surfaces of leaves, seeds, and in the rhizosphere of a wide variety of plants could be better exploited to promote plant growth.

Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing Methylobacterium oryzae and interactions with auxins and ACC regulation of ethylene in canola (Brassica campestris)

The possible interaction of the plant hormones auxin and ethylene and the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing bacteria on ethylene production in canola (Brassica campestris) in the presence of inhibitory concentrations of growth regulators were investigated. The effects of auxin (indole-3-acetic acid and 2,4-dichlorophenoxy acetic acid), auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid, ethylene precursor 1-aminocyclopropane-1-carboxylate and ethylene synthesis inhibitor l-α-(2-aminoethoxyvinyl)glycine hydrochloride on root elongation were concentration dependent. Exogenous addition of growth regulators influences the enzyme activities of ethylene production and we have presented here evidences that support the hypothesis that inhibitory effects of auxin on root elongation are independent of ethylene. Additionally, we have proved that inoculation of ACC deaminase containing Methylobacteriumoryzae sequester ACC exuded from roots and hydrolyze them lowering the concentration of ACC in root exudates. However, the inhibitory actions of exogenous additions of auxins could not be ameliorated by bacterial inoculation that reduces ethylene concentration in canola seedlings.

Synergistic effects of inoculating arbuscular mycorrhizal fungi and Methylobacterium oryzae strains on growth and nutrient uptake of red pepper (Capsicum annuum L.)

A greenhouse experiment was conducted to examine the effects of inoculation with two Methylobacterium oryzae strains (CBMB20 and CBMB110) and a consortium of three arbuscular mycorrhizal (AM) fungi on the growth of red pepper (Capsicum annum L.). Inoculation of red pepper plants with the M. oryzae strains resulted in a significant increase in root length and root fresh weight compared to untreated control plants. The combined inoculation of M. oryzae strains and AM fungi significantly increased various plant growth parameters and chlorophyll content compared to uninoculated controls. Mycorrhizal colonisation and the number of AM fungal spores were higher in co-inoculation treatments. In addition, the combined inoculation of M. oryzae strains and AM fungi resulted in significantly higher nitrogen (N) accumulation in the roots and shoots of red pepper plants compared to uninoculated controls. The combined inoculation of M. oryzae strain CBMB110 and AM fungi increased the phosphorus (P) content by 23.3% compared to untreated controls. The micronutrient content of the red pepper plants also increased in most of the inoculation treatments. A perfect mutualism among CBMB100-AMF was found which was attributed to the improved macro- and micronutrient uptake along with higher chlorophyll content in red pepper. Further research on in-depth understanding of the co-operative microbial interactions will facilitate the successful application of Methylobacterium-AM fungi products in biotechnology.

Physiological enhancement of early growth of rice seedlings (Oryza sativa L.) by production of phytohormone of N2-fixing methylotrophic isolates

Three plant-growth promoting, N2-fixing methylotrophic strains isolated from rice cultivars (Oryza sativa L.), viz, Methylobacterium sp. CBMB20, Enterobacter sp. CBMB30, Burkholderia sp. CBMB40, were selected, and their activities in promoting the early growth of rice were studied. Seeds treated with the methylotrophic strains improved seed germination, seedling vigor index (SVI), and biomass of rice seedlings. The methylotrophic population in the treated seedlings increased in the vegetative stages when compared to seeding stages. Treated seedlings showed a higher accumulation of plant hormones viz trans-zeatin riboside, isopentenyladenosine, and indole-3-acetic acid than untreated seedlings. Plant hormones were detected immunologically using the phytodetek kit. Conformational evidence suggested that cytokinins were produced by the epiphytic bacteria colonizing the plants rather than by the plants themselves. In addition, the inoculated early stage rice seedlings also exhibited a wide range of acetylene reduction activity. The results suggest the potential use of these bacteria to stimulate germination, SVI, and biomass production, which is mediated by production of plant hormone accumulation and nitrogen fixation.

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.

Colonization pattern of plant root and leaf surfaces visualized by use of green-fluorescent-marked strain of Methylobacterium suomiense and its persistence in rhizosphere

The localization of bacterial cell, pattern of colonization, and survival of Methylobacterium suomiense CBMB120 in the rhizosphere of rice and tomato plants were followed by confocal laser scanning, scanning electron microscopy, and selective plating. M. suomiense CBMB120 was tagged with green fluorescent protein (gfp), and inoculation was carried out through seed source. The results clearly showed that the gfp marker is stably inherited and is expressed in planta allowing for easy visualization of M. suomiense CBMB120. The colonization differed in rice and tomato—intercellular colonization of surface-sterilized root sections was visible in tomato but not in rice. In both rice and tomato, the cells were visible in the substomatal chambers of leaves. Furthermore, the strain was able to compete with the indigenous microorganisms and persist in the rhizosphere of tomato and rice, assessed through dilution plating on selective media. The detailed ultra-structural study on the rhizosphere colonization by Methylobacterium put forth conclusively that M. suomiense CBMB120 colonize the roots and leaf surfaces of the plants studied and is transmitted to the aerial plant parts from the seed source.

Cultivation-dependent characterization of rhizobacterial communities from field grown Chinese cabbage Brassica campestris ssp pekinensis and screening of traits for potential plant growth promotion

The composition of the bacterial community associated with plant roots is influenced by a variety of plant, environmental factors and also management practices. Our study aimed at detecting the root associated bacterial communities of Chinese cabbage under different fertilization regimes using cultivation dependent methods. The cultivable population was studied using plate count assay, fatty acid methyl ester (FAME) analysis and carbon substrate utilization␣(SU)using BIOLOG™ plates. Taxonomical identification of the isolates by FAME resulted in about 83% identification and they represented 9 and 14 different known bacterial genera from the rhizosphere and root interior respectively from Proteobacteria (α, β, and γ), firmicutes (actinobacteria and the Bacillus groups) and Bacteroidetes. Pseudomonas and Bacillus were associated with the plants grown under all the fertilized conditions and actinobacteria could be observed only in rhizosphere of plants grown on unfertilized plots. FAME and BIOLOG profiles of the rhizosphere and endophytic isolates could separate them with reference to fertilization. Principal component analysis (PCA) on the BIOLOG SU revealed that the isolates were metabolically dissimilar. The diversity, as revealed by the diversity indices was greater among the isolates obtained from unfertilized samples than that of fertilized ones. The isolates analyzed for different traits related to plant growth promotion revealed differences between rhizosphere and endophytic isolates and also with reference to the treatments. The highest percentage of phosphate solubilizing bacteria (PSB) and 1-aminocyclopropane-1-carboxylic acid (ACC) utilizers was recorded in chemical fertilizer treated samples, followed by the organic fertilizer treated. The results from this study indicate that fertilizers have an effect on the root associated bacterial communities of Chinese cabbage and also on their physiological characteristics related to plant growth promotion.

Potential plant growth promoting traits and bioacidulation of rock phosphate by thiosulfate oxidizing bacteria isolated from crop plants

Thiosulfate oxidizing bacteria isolated from crop plants were tested for their traits related to plant growth promotion and their ability to solubilize Morocco rock phosphate (RP) through oxidation of thiosulfate to sulfuric acid. All the tested strains grew in Nfb medium (except Dyella ginsengisoli) and possessed β‐1,3 glucanase activity (except Burkholderia kururiensis). Of the fourteen tested strains, 2 were found positive for siderophore production, 3 each for phytohormones (IAA), and salicylic acid production. Based on qualitative and quantitative assays, 5 strains were found to efficiently solubilize tri‐calcium phosphate in Pikovskayas medium. Nine strains exhibited ACC (1‐aminocyclopropane‐1‐carboxylate) deaminase activity. In gnotobiotic experiments, Pandoraea sputorum ATSB28 which possessed the lowest ACC deaminase (0.44 nM of α‐Keto butyrate formed min–1 mg of protein–1) activity increased the primary root length of canola by 166%. Inoculation of Pandoraea sp. strain ATSB30 in mixture containing RP and thiosulfate significantly enhanced the water extractable‐P (1147 μg P g RP–1) and bicarbonate extractable‐P (1144 μg P g RP–1) on day 45. Glucose amendment resulted in increased RP solubilization as compared to glucose unamended treatments. Thiosulfate oxidizing bacteria tested in this study possessed at least one or more plant growth promoting traits apart from thiosulfate oxidation and solubilized the RP. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Characterization of plant-growth promoting diazotrophic bacteria isolated from field grown Chinese cabbage under different fertilization conditions

Diazotrophic bacteria isolated from the rhizosphere of Chinese cabbage were assessed for other plant growth promoting characteristics viz., production of IAA, ethylene, ACC deaminase, phosphate solubilization, and gnotobiotic root elongation. Their effect on inoculation to Chinese cabbage was also observed under growth chamber conditions. A total of 19 strains that showed higher nitrogenase activity identified by 16S rRNA gene sequence analysis were found to be the members of the genera Pseudomonas and Agrobacterium belonging to α- and γ-Proteobacteria groups. These strains were also efficient in producing IAA and ACC deaminase though they produced low levels of ethylene and no phosphate solubilization. In addition, inoculation of selected diazotrophic bacterial strains significantly increased seedling length, dry weight, and total nitrogen when compared to uninoculated control. The colonization of crop plants by diazotrophic bacteria can be affected by many biotic and abiotic factors, and further studies are oriented towards investigating the factors that could influence the establishment of a selected bacterial community.

Methylophilus rhizosphaerae sp. nov., a restricted facultative methylotroph isolated from rice rhizosphere soil.

Three facultative methylotrophic bacterial strains, designated CBMB127(T), CBMB145 and CBMB147, were isolated from the rhizosphere soil of rice and characterized. The strains produced indole-3-acetic acid and siderophores, had 1-aminocyclopropane-1-carboxylate deaminase activity and sulfur oxidation property and also methanol dehydrogenase. Phylogenetic analysis based on the 16S rRNA and methanol dehydrogenase (mxaF) gene sequences showed that Methylophilus methylotrophus was their close relative. The results of the phenotypic, phylogenetic and genotypic analyses showed that strains CBMB127(T) and CBMB145, with 99.4 % 16S rRNA gene sequence similarity and 99 % DNA-DNA hybridization, could be distinguished from recognized species of Methylophilus. Therefore strain CBMB127(T) and CBMB145 are considered to represent a novel species of Methylophilus, for which the name Methylophilus rhizosphaerae sp. nov. is proposed, with CBMB127(T) (=KACC 13099(T)=NCCB 100233(T)) as the type strain. Strain CBMB147 represents a novel strain of the species Methylophilus methylotrophus.