Manoj V. Parakhia

Functional characterization of PGPR and its identification through 16 S rRNA sequencing

Plant growth promoting rhizobacteria are known to rapidly colonize the rhizosphere and suppress soil borne pathogens at the root surface. These organisms can also be beneficial to the plant by stimulating growth. Pseudomonas spp. have been studied mainly because of their widespread distribution in soil, their ability to colonize the rhizosphere of host plants and ability to produce a wide range of compounds inhibitory to number of serious plant pathogens.For performing this study, soil sample was collected from six different locations of Junagadh district.Total thirty six bacteria were isolated from the collected soil samples by using general microbiological media, among them ten prominent phosphate solubilizer were selected for studying other plant growth promoting potentials and biochemical characterization. Most efficient Plant growth promoting rhizobia was identified at their strain level by using 16 S rRNA sequencing technology. INTRODUCTION The rhizosphere, representing the thin layer of soil surroundingplant roots and the soil occupied by the roots, supports largeactive groups of bacteria (Villacieroset al., 2003) known asplant growth promoting rhizobacteria (PGPR) (Kloepperet al.,1980).Plant growth promoting rhizobacteria are known torapidly colonize the rhizosphere and suppress soil bornepathogens at the root surface (Rangajaranet al., 2003). These organisms can also be beneficial to the plant by stimulatinggrowth (Bloemberg and Lugtenberg, 2001; Moeinzadehet al.,2010). PGPR can affect plant growth by different direct and indirect mechanisms (Glick 1995; Gupta et al. 2000). Some examples of these mechanisms are (1) increased mineral nutrient solubilization and nitrogen fixation, making nutrients available for the plant; (2) repression of soil borne pathogens (by the production of hydrogen cyanide, siderophores, antibiotics, enzymes like chitinase and/or competition for nutrients); (3) improving plant stress tolerance to drought, salinity, and metal toxicity; and (4) production of phytohormones such as indole-3-acetic acid (IAA) (Gupta et al. 2000). The bacteria presenting one or more of these characteristics are known as plant growth promoting rhizobacteria. Pseudomonas spp. have been studied mainly because of their widespread distribution in soil, their ability to colonize the rhizosphere of host plants and ability to produce a wide range of compounds inhibitory to a number of serious plant pathogens (Weller 1988). The soil bacteria that aggressively colonize the root zone and promote plant growth are generally termed as Plant Growth Promoting Rhizobacteriaand primarily Pseudomonas fluorescence s is identified as an important organism with ability for plant growth promotion and effective disease management properties (Mazzolaet al., 1992). Their applicability as biocontrol agents has drawn wide attention because of the production of secondary metabolites such as siderophore, antibiotics, volatile compounds, Hydrogen Cyanide, enzymes and phyto-hormones (Nagarajkumaret al., 2004). In vitro antibiosis of P. fluorescence strains towards the rice sheath rot pathogen S. oryzaedemonstrated that this pathogen is sensitive to P. fluorescence (Sakthivel and Gnanamanickam, 1987). The biocontrol mechanism to suppress fungal pathogens by Pseudomonas spp. normally involves the production of antibiotics and P. fluorescence s has a gene cluster that produces a suite of antibiotics, including compounds such as 2,4-diacetylphloroglucinol (DAPG), phenazine, pyrrolnitrin, pyoluteorin and bio-surfactantantibiotics (Angayarkanniet al., 2005). Fluorescence t Pseudomonas is uniquely capable of synthesizing many of these antibiotics, not only to enhance its own fitness but also to help in the maintenance of soil health and bioprotection of crops from pathogens (Gaur et al., 2004). Presently, there are number of commercial isolates of Pseudomonas available in the market. MATERIALS AND METHODS ISOLATION AND PURIFICATION Collection of soil sample: The soil sample was collected for the Isolation of plant growth promoting rhizobiafrom rhizosphere of Junagadh district fields of Gujarat. Intact root system was plowed out and the rhizospheric soil samples were carefully taken in sterilized plastic bags. Samples were collected from six different points within area and mix thoroughly; likewise six different locations were selected from the Junagadh district. Isolation of bacteria: For the isolation, 1 gram of each soil samples collected from the various collection sites was dissolved in 10 ml sterile distilled water and mix well for 10 minutes. Serial dilution was made from each sample and appropriate dilution was spread on Nutrient agar plate and incubated at 37±0.1 0C for 24hrs. From the mixed population of microbes, microbial strains were isolated by single colony isolation from the nutrient agar medium. Out of total 57 microbial isolates, 36 isolates were selected for the test of phosphate solubilizing activity. Streak plate method was used for obtaining pure culture, as and when required.

Draft whole genome sequence of groundnut stem rot fungus Athelia rolfsii revealing genetic architect of its pathogenicity and virulence

Groundnut (Arachis hypogaea L.) is an important oil seed crop having major biotic constraint in production due to stem rot disease caused by fungus, Athelia rolfsii causing 25–80% loss in productivity. As chemical and biological combating strategies of this fungus are not very effective, thus genome sequencing can reveal virulence and pathogenicity related genes for better understanding of the host-parasite interaction. We report draft assembly of Athelia rolfsii genome of ~73 Mb having 8919 contigs. Annotation analysis revealed 16830 genes which are involved in fungicide resistance, virulence and pathogenicity along with putative effector and lethal genes. Secretome analysis revealed CAZY genes representing 1085 enzymatic genes, glycoside hydrolases, carbohydrate esterases, carbohydrate-binding modules, auxillary activities, glycosyl transferases and polysaccharide lyases. Repeat analysis revealed 11171 SSRs, LTR, GYPSY and COPIA elements. Comparative analysis with other existing ascomycotina genome predicted conserved domain family of WD40, CYP450, Pkinase and ABC transporter revealing insight of evolution of pathogenicity and virulence. This study would help in understanding pathogenicity and virulence at molecular level and development of new combating strategies. Such approach is imperative in endeavour of genome based solution in stem rot disease management leading to better productivity of groundnut crop in tropical region of world.

Assessment of genetic variability among Muskmelon (Cucumis melo L.) genotypes through biometrical traits and molecular markers

Fifty genotypes of muskmelon ( Cucumis melo L .) were evaluated for variability through yield attributing characters and molecular markers. Analysis of variance for yield attributing character showed significant variation for all the traits, indicating presence of sufficient variability. D 2 values distributed all the genotypes in seven clusters. Maximum genetic distance was obtained between clusters II and V, while clusters III and VII displayed the lowest degree of divergence. Total soluble sugars followed by total soluble solids and fruit yield per plant contributed the most towards divergence. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) analysis using 130 and 62 primers generated 1108 and 462 discrete markers, respectively ranging from 200-1800 bp in size. The UPGMA analysis showed that genotypes were distributed in different groups based on similarities matrix. The dendrogram obtained by combining the data of both the molecular marker revealed genetic similarities ranging between 57% to 81% with highest genetic similarity between MM-68 and MMM-61. These results suggest that RAPD and ISSR markers are useful for muskmelon genetic diversity analysis from different region of India, which will be helpful for further genetic improvement program of plant. Knowledge on the genetic diversity of muskmelon can be used to future breeding programmes to improve fruit quality.