Isolation and Characterization of Streptomycetes with-Plant Growth Promoting Potential from Mangrove Ecosystem.

A total of 66 actinomycetes isolates were isolated from mangroves of Andhra Pradesh, India, using various enrichment techniques and pretreatments. The samples were collected from Coringa mangrove ecosystem and pre-treated by enrichment with CaCO3, sodium dodecyl sulphate and phenol, plated on the media supplemented with cycloheximide (50 mg/ml), nystatin (25 mg/ml) and nalidixic acid (50 mg/ml). The population count of actinomycetes fluctuated from 1.9 x 10(5) to 8.0 x 10(5)/g soil. Out of the isolated 66 actinomycetes, 8 isolates possessing plant growth promoting potential were further studied and characterized by physiological and biochemical traits and identified by 16S rRNA gene sequencing as different species of Streptomycetes genera.

Genetic and functional diversity of fluorescent Pseudomonas from rhizospheric soils of wheat crop.

Wheat rhizospheric soils were collected from different part of northern and eastern Indo-Gangetic plains, which is being irrigated from water of Ganga River. Isolation of fluorescent Pseudomonas species was carried out from the soil samples collected. The percentage of isolates positive for indolic compound, P-solubilisation, siderophore production and ACC deaminase activity were 64.0, 38.6, 63.5, and 19.7, respectively. A total of 543 isolates were randomly selected for studies based on the genus specific confirmation by the Pseudomonas specific primer. Among the 543 isolates, 26 different clusters were formed from 16S rDNA-RFLP whereas 27 clusters were generated by the rpoB-RFLP with similarity percent ranging from 3 to 100%. 16S rDNA sequencing showed 9 different species of Pseudomonas whereas, rpoB sequencing showed 13 different species of Pseudomonas. Phylogenetic analysis based on 16S rDNA gene sequences generated 15 branches showing the more than 70% of boot strap value, whereas 18 branches in the rpoB based phylogenetic tree were supported by bootstrap values above 70%. Diversity indices based on rpoB were higher than the ribosomal RNA gene.

Isolation and characterization of siderophore producing antagonistic rhizobacteria against Rhizoctonia solani.

Plant protection through siderophore producing rhizobacteria (SPR) has emerged as a sustainable approach for crop health management. In present study, 220 bacteria isolated from tomato rhizosphere were screened for in vitro antagonistic activity against Rhizoctonia solani AG-4. Nine potent antagonistic strains viz., Alcaligenes sp. (MUN1, MB21, and MPF37), Enterobacter sp. (MPM1), Pseudomonas sp. (M10A and MB65), P. aeruginosa (MPF14 and MB123) and P. fluorescens (MPF47) were identified on the basis of physiological characters and 16S rDNA sequencing. These strains were able to produce hydrolytic enzymes, hydrogen cyanide, indole acetic acid, although, only few strains were able to solubilize phosphate. Two strains (MB123 and MPF47) showed significant disease reduction in glasshouse conditions were further evaluated under field conditions using three different application methods. Application of P. fluorescens (MPF47) in nursery as soil mix + seedling root treatments prior to transplantation resulted in significant disease reduction compared to control. Total chlorophyll and available iron were significantly higher in the MPF47 treated plants in contrast to infected control. In conclusion, siderophore producing bacteria MPF47 have strong biocontrol abilities and its application as soil mix + seedling root treatments provided strong shield to plant roots against R. solani and could be used for effective bio-management of pathogen.

Cold-active hydrolases producing bacteria from two different sub-glacial Himalayan lakes.

Microorganisms, native to the cold environments have successfully acclimatized their physiological, metabolic, and biological features, exhibiting uniqueness in their enzymes, proteins, and membrane structures. These cold-active enzymes have immense biotechnological potential. The diversity of culturable bacteria in two different water lakes (the sub-glacial freshwater and the brackish) of Himalayas was analyzed using SYBR green staining and cultural methods. A total of 140 bacteria were isolated and were grouped as psychrophiles, psychrotrophs, and psychrotolerant organisms, based on their optimal temperature for growth. The amplified ribosomal DNA restriction analysis using three restriction enzymes facilitated the grouping of these isolates into 96 genotypes at ≥85% polymorphism. Phylogenetic analysis using 16S rRNA gene sequences revealed that the bacterial strains from both lakes belonged to Firmicutes, Proteobacteria (α, ß, and γ) or Actinobacteria. Screening of the germplasm for the activity of different cold-active hydrolases such as protease, amylase, xylanase, and cellulase, revealed that about 16 isolates were positive, and exhibiting a wide range of stability at various temperature and pH. Our results suggest that the distinctly different ecosystems of sub-glacial freshwater and brackish water lakes have diverse groups of bacteria, which can be an excellent source of extracellular hydrolases with a wide range of thermal stability.

Optimization of media components for chitinase production by chickpea rhizosphere associated Lysinibacillus fusiformis B-CM18.

Chitinase producing strain B-CM18 was isolated from chickpea rhizosphere and identified as Lysinibacillus fusiformis B-CM18. It showed in vitro antifungal activity against a wide range of fungal plant pathogens and was found to produce several PGPR activities. Further, a multivariate response surface methodology was used to evaluate the effects of different factors on chitinolytic activity and optimizing enzyme production. A central composite design was employed to achieve the highest chitinase production at optimum values of the process variables, viz., temperature (20-45 °C), sodium chloride (2-7%), starch (0.1-1%) and yeast extract (0.1-1%), added in the minimal medium supplemented with colloidal chitin (1-10%; w:w). The fit of the model (R(2) = 0.5692) was found to be significant. The production medium to achieve the highest chitinase production (101 U ml(-1) ) was composed of the minimal medium composed of chitin (6.09%), NaCl (4.5%), starch (0.55%) and yeast extract (0.55%) with temperature (32.5 °C). The results show that the optimization strategy led to an increase in chitinase production by 56.1-fold. The molecular mass of the chitinase was estimated to be 20 kDa by anion exchange and gel filtration chromatography. Further, purified chitinase showed strong antifungal activity against test pathogens. Overall, these results may serve as a base line data for enhancing the chitinolytic potential of bacterial antagonists for bio-management of chickpea pathogens.

Epiphytic pink-pigmented methylotrophic bacteria enhance germination and seedling growth of wheat (Triticum aestivum) by producing phytohormone.

Methylotrophic bacteria were isolated from the phyllosphere of different crop plants such as sugarcane, pigeonpea, mustard, potato and radish. The methylotrophic isolates were differentiated based on growth characteristics and colony morphology on methanol supplemented ammonium mineral salts medium. Amplification of the mxaF gene helped in the identification of the methylotrophic isolates as belonging to the genus Methylobacterium. Cell-free culture filtrates of these strains enhanced seed germination of wheat (Triticum aestivum) with highest values of 98.3% observed using Methylobacterium sp. (NC4). Highest values of seedling length and vigour were recorded with Methylobacterium sp. (NC28). HPLC analysis of production by bacterial strains ranged from 1.09 to 9.89 µg ml(-1) of cytokinins in the culture filtrate. Such cytokinin producing beneficial methylotrophs can be useful in developing bio-inoculants through co-inoculation of pink-pigmented facultative methylotrophs with other compatible bacterial strains, for improving plant growth and productivity, in an environment-friendly manner.

Characterization of mycolytic enzymes of Bacillus strains and their bio-protection role against Rhizoctonia solani in tomato.

Four antagonists bacteria namely, Bacillus megaterium MB3, B. subtilis MB14, B. subtilis MB99 and B. amyloliquefaciens MB101 were able to produce chitinase, ß-1,3-glucanase and protease in different range with the presence of Rhizoctonia solani cell wall as a carbon source. Amplification of chitinase (chiA) gene of 270 bp and ß-1, 3-glucanase gene of 415 bp was given supportive evidence at molecular level of antibiosis. After in vitro screening, all antagonists were tested against R. solani under greenhouse conditions. Root treatment of Bacillus strains showed superior defense during pathogen suppression in terms of chitinase, glucanase, peroxidase, poly phenol oxidase, phenylalanine ammonia-lyase activity and total phenolic content in leaves of tomato. All these enzymes accumulated high in tomato leaves as compared to roots. Pathogenesis-related proteins and defense-related enzymes accumulation was directly correlated with plant protection and greenhouse results indicated that B. amyloliquefaciens MB101- and B. subtilis MB14-treated plants offered 69.76 and 61.51 % disease reductions, respectively, over the infected control. These results established that these organisms have the potential to act as biocontrol agents. This study could be highlighted a mutual importance of liquid formulation of antagonistic Bacillus spp. against root associated sclerotia former pathogen R. solani.

Isolation of oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein.

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10 mM of oxalic acid whereas only 15 isolates were grow at 50 mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23 %) after 60 min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.

Exploration and characterization of agriculturally and industrially important haloalkaliphilic bacteria from environmental samples of hypersaline Sambhar lake, India.

Screening of bacteria from Sambhar lake, an extreme hypersaline environment of India, led to the isolation of 93 haloalkaliphilic bacteria growing optimally in media with 2-25 % salt and 6-12 pH. Based on 16S rRNA gene sequences, 93 isolates were further categorized into 32 groups, with each group representing a different taxa belonging to 3 phyla (Firmicutes, Proteobacteria and Actinobacteria). Majority of the isolates (53.12 %) showed similarity with phylum Firmicutes which was followed by Proteobacteria (40.63 %) and Actinobacteria (6.25 %). The isolates belonging to 32 representative groups were further evaluated for the production of extracellular enzymes viz. amylase, cellulase, protease and xylanase, plant growth promoting attributes and BIOLOG™ substrate usage. Among all the isolates, xylanase producing isolates were in maximum (68 %) as compared to protease (56 %), cellulase (40 %), and amylase (37 %) producing strains. Similarly, among plant growth promoting activities, ammonia producing isolates were highest (56 %) when compared to those producing ACC deaminase (53 %), IAA (50 %), hydrogen cyanide (28 %), siderophore (21 %) and solubilizing P (34 %). Isolates showing enzymatic and PGP activities could be further utilized for promoting plant growth in saline affected area.

Studies on Exo-Chitinase Production from Trichoderma asperellum UTP-16 and Its Characterization.

The growth conditions for chitinase production by Trichoderma asperellum UTP-16 in solid state fermentation was optimized using response surface methodology based on central composite design. The chitinase production was optimized, using one-factor at a time approach, with six independent variables (temperature, pH, NaCl, incubation period, nitrogen and carbon sources) and 3.31 Units per gram dry substrate (U gds(-1)) exo-chitinase yield was obtained. A 21.15% increase was recorded in chitinase activity (4.01 U gds(-1)) through surface response methodology, indicates that it is a powerful and rapid tool for optimization of physical and nutritional variables. Further, efficiency of crude enzyme was evaluated against phytopathogenic Fusarium spp. and a mycelial growth inhibition up to 3.5-6.5 mm was achieved in well diffusion assay. These results could be supplemented as basic information for the development of enzyme based formulation of T. asperellum UTP-16 and its use as a biocontrol agent.

Cyanobacteria-mediated phenylpropanoids and phytohormones in rice (Oryza sativa) enhance plant growth and stress tolerance.

Phenylpropanoids, flavonoids and plant growth regulators in rice (Oryza sativa) variety (UPR 1823) inoculated with different cyanobacterial strains namely Anabaena oryzae, Anabaena doliolum, Phormidium fragile, Calothrix geitonos, Hapalosiphon intricatus, Aulosira fertilissima, Tolypothrix tenuis, Oscillatoria acuta and Plectonema boryanum were quantified using HPLC in pot conditions after 15 and 30 days. Qualitative analysis of the induced compounds using reverse phase HPLC and further confirmation with LC-MS/MS showed consistent accumulation of phenolic acids (gallic, gentisic, caffeic, chlorogenic and ferulic acids), flavonoids (rutin and quercetin) and phytohormones (indole acetic acid and indole butyric acid) in rice leaves. Plant growth promotion (shoot, root length and biomass) was positively correlated with total protein and chlorophyll content of leaves. Enzyme activity of peroxidase and phenylalanine ammonia lyase and total phenolic content was fairly high in rice leaves inoculated with O. acuta and P. boryanum after 30 days. Differential systemic accumulation of phenylpropanoids in plant leaves led us to conclude that cyanobacterial inoculation correlates positively with plant growth promotion and stress tolerance in rice. Furthermore, the study helped in deciphering possible mechanisms underlying plant growth promotion and stress tolerance in rice following cyanobacterial inoculation and indicated the less explored avenue of cyanobacterial colonization in stress tolerance against abiotic stress.

Quantitative real-time PCR assay for rapid detection of plant and human pathogenic Macrophomina phaseolina from field and environmental samples.

A real-time qPCR assay was developed to detect and quantify Macrophomina phaseolina abundance in rhizosphere soil and plant tissue. Both TaqMan and SYBR green techniques were targeted on ~ 1 kb sequence characterized amplified region (SCAR) of M. phaseolina and two sets of specific primers were designed for SYBR green (MpSyK) and TaqMan (MpTqK) assays. No cross-hybridization and no fluorescent signal exceeding the baseline threshold was observed in TaqMan and SYBR green assays, respectively. The minimum detection limit or sensitivity of TaqMan assay was 30 fg/µL of M. phaseolina DNA and limit of quantification of M. phaseolina viable population was estimated as 0.66 × 10(5) CFU/g soil(-1) equivalent to 10 pg/µL of target DNA. This is the first report which demonstrated real-time qPCR assays with greater specificity and sensitivity to detect M. phaseolina population in soil and plant materials.

Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic soil.

The molecular diversity of aerobic endospore-forming bacteria, typically Bacillus and its derived genera, has been investigated in various environments. However, there have been few investigations concerning Bacillus in acidic soils. In this study, the genotypic diversity and phylogenetic relationships among plant growth-promoting (PGP) bacilli isolated from the rice rhizosphere growing in acidic soils of Kerala (pH varying from 6.3 to 6.8) were investigated. For assessing their biocontrol potential and PGP attributes, 115 isolates were randomly selected and 49 isolates that were positive for multiple traits were selected. Metabolic characterization of representative strains, using the Biolog GP2 (Gram Positive) MicroPlate(TM) , revealed a large versatility with respect to carbohydrate utilization. Amplified ribosomal DNA restriction analysis revealed 13 clusters at 65% similarity level, which consisted of 1-21 strains. 16S rDNA partial sequencing assigned all the isolates, except for one, to the Bacillus genus, with close relatedness to Bacillus humi, B. megaterium, B. drentensis, B. pocheonensis, B. aestuarii, B. arbutinivorans, B. niacini, and Brevibacterium casei. The Bacillus species with different metabolic capabilities, PGP abilities, and genetic diversity found in this study are likely to have ecological relevance.

Genetic diversity of Macrophomina phaseolina isolates from certain agro-climatic regions of India by using RAPD markers.

Genetic diversity analysis of Macrophomina phaseolina isolates obtained from different host range and diverse geographical locations in India was carried out using RAPD fingerprinting. Of the thirteen 10-mer random primers used, primer OPB-08 gave the maximum polymorphism and the UPGMA clustering could separate 50 isolates in to ten groups at more than 65% similarity level. The ten clusters correlated well with the geographical locations with exceptions for isolates obtained from Eastern and Western Ghats. There was a segregation of isolates from these two geographical locations in to two clusters thus, distributing 10 genotypes in to eight geographical locations. All the isolates M. phaseolina irrespective of their host and geographical origin, exhibited two representative monomorphic bands at 250 bp and 1 kb, presence of these bands suggests that isolates might have evolved from a common ancestor but due to geographical isolation fallowed by natural selection and genetic drift might have segregated in to subpopulations. Genetic similarity in the pathogenic population reflects the dispersal of single lineage in all locations in India.

A comparative analysis of distribution and conservation of microsatellites in the transcripts of sequenced Fusarium species and development of genic-SSR markers for polymorphism analysis.

We used an in silico approach to survey and compare microsatellites in transcript sequences of four sequenced members of genus Fusarium. G + C content of transcripts was found to be positively correlated with the frequency of SSRs. Our analysis revealed that, in all the four transcript sequences studied, the occurrence, relative abundance and density of microsatellites varied and was not influenced by transcript sizes. No correlation between relative abundance and transcript sizes was observed. The relative abundance and density of microsatellites were highest in the transcripts of Fusarium solani when compared with F. graminearum, F. verticillioides and F. oxysporum. The maximum frequency of SSRs among all four sequence sets was of trinucleotide repeats (67.8%), whereas the dinucleotide repeat represents <1%. Among all classes of repeats, 36.5% motifs were found conserved within Fusarium species. In order to study polymorphism within Fusarium isolates, 11 polymorphic genic-SSR markers were developed. Of the 11 markers, 5 were from F. oxysporum and remaining 6 belongs to F. solani. SSR markers from F. oxysporum were found to be more polymorphic (38%) as compared to F. solani (26%). Eleven polymorphic markers obtained in this study clearly demonstrate the utility of newly developed SSR markers in establishing genetic relationships among different isolates of Fusarium.

Horizontal and vertical movement of Pseudomonas fluorescens toward exudate of Macrophomina phaseolina in soil: influence of motility and soil properties.

The role of motility and cell surface hydrophobicity in transport and dispersal of Pseudomonas fluorescens strains LAM1-hydrophilic, LAM2-hydrophobic and LAM(NM) (non-motile mutant of LAM2) under different soil conditions was studied. Maximum adhesion was recorded for LAM2 in clay loam (70%), followed by sandy loam (68%) and sandy soil (40%). Vertical migration of P fluorescens isolates in soils was recorded at 5 and 25 cm flow of wafer or M. phaseolina exudate. In all the treatments, LAM1 exhibited maximum migration followed, by LAM2 and LAM(NM). The rate of migration of such isolates was lowered in water irrigated soils compared to those irrigated with M. phaseolina exudate. In sandy soil, cells of LAM1 migrated up to 13 cm in comparison to LAM2 (11 cm) and LAN(NM) (9 cm) at 5 cm flow of fungal exudate. Population of LAM1, LAM2 and LAM(NM) was 5.7, 5.68 and 5.61 log cfu g(-1) soil at 1 cm depth, but it decreased to 2.56, 2.21 and 1.99 log cfu during migration up to 11 cm in sandy soil at 5 cm flow of fungal exudate. Greater motility was observed in sandy soil irrigated with water or fungal exudate, followed by sandy loam and clay loam. In general, filtration coefficient (lambda) of P. fluorescens was higher in soils irrigated with 5 cm of water or exudate than with 25 cm of irrigation. The horizontal movement of P. fluorescens strains in sandy soil adjusted at different psi m showed marked reduction with decrease in psi m. The non-motile LAN(NM) did not show chemotactic response and migrated up to a maximum of 3 mm in saturated soils (0 kPa). After 96 h, LAM1 and LAM2 migrated upto 35 and 29 mm respectively in sandy soil. Motile isolates had significantly greater colonization of M. phaseolina sclerotia over the non-motile mutant.

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique.

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.

Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens against Fusarium oxysporum f. sp. ciceri in chickpea.

Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic acid, DL-norvaline, indole-3-carbinol and lichenan) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea. A marked increase in shoot and root length was observed in P. fluorescens treated plants. The isolates of P. fluorescens systemically induced resistance against Fusarium wilt of chickpea caused by Fusarium. oxysporum f.sp. ciceri (FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to control. Varied degree of protection against Fusarium wilt was recorded with chemical inducers. The reduction in disease was more pronounced when chemical inducers were applied with P. fluorescens. Among chemical inducers, SA showed the highest protection of chickpea seedlings against wilting. Fifty two- to 64% reduction of wilting was observed in soil treated with isolate Pf4-92 along with chemical inducers. A significant (P = 0.05; r = -0.946) negative correlation was observed in concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration of 2000 microg ml(-1) mycelial growth was completely arrested. Exogenously supplied SA also stimulated systemic resistance against wilt and reduced the disease severity by 23% and 43% in the plants treated with 40 and 80 microg ml(-1) of SA through root application. All the isolates of P. fluorescens produced SA in synthetic medium and in root tissues. HPLC analysis indicated that Pf4-92 produced comparatively more SA than the other isolates. 1700 to 2000 nanog SA g(-1) fresh root was detected from the application site of root after one day of bacterization whereas, the amount of SA at distant site ranged between 400-500 nanog. After three days of bacterization the SA level decreased and was found more or less equal at both the detection sites.

A comparative in silico analysis on frequency and distribution of microsatellites in coding regions of three formae speciales of Fusarium oxysporum and development of EST-SSR markers for polymorphism studies.

Fusarium oxysporum is a ubiquitous species complex of soil-borne plant pathogens comprising of many different formae speciales, each characterized by a high degree of host specificity. In the present investigation, we surveyed microsatellites in the available express sequence tags and transcript sequences of three formae speciales of F. oxysporum viz. melonis (Fom), cucumerium (Foc), and lycopersici (Fol). The relative abundance and density of microsatellites were higher in Fom when compared with Foc and Fol. Thirty microsatellite primers were designed, ten from each forma specialis, for genetic characterization of F. oxysporum isolates belonging to five formae speciales. Of the 30 primers, only 14 showed amplification. A total of 28 alleles were amplified by 14 primers with an average of two alleles per marker. Eight markers showed 100% polymorphism. The markers were found to be more polymorphic (47%) in Fol as compared to Fom and Foc; however, polymorphic information content was the maximum (0.899) in FocSSR-3. Nine polymorphic markers obtained in this study clearly demonstrate the utility of newly developed markers in establishing genetic relationships among different isolates of F. oxysporum.