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Biological Characterization and Genetic Diversity of Indian Strains

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Biological Characterization and Genetic Diversity of Indian Strains atholog t P y & n M Singh et al., J Plant Pathol Microbiol 2018, 9:7 la ic P f r o o b DOI: 10.4172/2157-7471.1000443 l i Journal of a o n l o r g u y o J ISSN: 2157-7471 Plant Pathology & Microbiology ReviewResearch Article Article OpenOpen Access Access Biological Characterization and Genetic Diversity of Indian Strains of Ralstonia solanacearum Biovars 3 and 4 Causing Bacterial Wilt of Tomato Singh D*, Sinha S, Chaudhary G and Yadav DK Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, India Abstract Ralstonia solanacearum biovars 3 and 4 causing bacterial wilt of tomato (Solanum lycopersicum L.) is a devastating soil borne plant pathogen across the world. Eighty seven isolates of R. solanacearum were isolated from wilted tomato plants from Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Jharkhand, Orissa states of India and characterized them by traditional and molecular methods. Biovar of R. solanacearum was determined using set of carbon sources and it showed that biovar 3 of R. solanacearum were found most prominent (90.2 per cent) in all the states of India, whereas biovar 4 was found in states of Jharkhand and Himachal Pradesh. The phylotype specific multiplex PCR assigned all 87 the isolates ofR. solanacearum infecting tomato under phylotype I. To study the genetic diversity, BOX-PCR and multilocus sequence typing approaches were used. Amplification products yielded in BOX-PCR fingerprint pattern ranging from 500 bp -4 kb and found 23 DNA typing groups of 87 isolates of R. solanacearum at 50% similarity coefficient. Under multilocus sequence typing, three virulence genes viz., hrp (regulatory transcription regulation) and egl (endoglucanase precursor) and fli C genes of 18 strains of R. solanacearum belonging to different agro-climatic zones was done. Based on sequence analysis of egl gene, majority of the Indian strains of R. solanacearum were very close to each other except ORT-8, UTT-23 and JHT- 2 and there were very close to strain GMI1000. A lot of genetic variability was found in Indian isolates of R. solanacearum irrespective of place of isolation and climatic conditions. Keywords: Biovars; BOX-PCR; egl; fliC; hrpB gene; Phylotyping; Classical restriction fragment length polymorphism analysis initially Race revealed fundamental heterogeneity of pathogen strains, but large-scale DNA sequence analyses more profoundly influenced our understanding Introduction of pathogen systematics [4,14,16]. As the diversity of the strains examined increased, it became clear that the R. solanacearum species Ralstonia solanacearum [1,2], causing bacterial wilt disease is one of complex has four major subdivisions, denoted as phylotypes [8,16]. A the most devastating pathogen of tomato. R. solanacearum is distributed multiplex polymerase chain reaction (PCR) can rapidly assign strains to in wet tropical, subtropical, warm temperate regions and even in some a phylotype [8]. Each phylotype is subdivided into numbered sequevars cool temperate regions of the world and has an unusually broad host and closely related individual strains, which are usually identified by range [3,4]. Tomato (Solanum lycopersicum L.) is one of the most analyzing sequence similarity of the egl endoglucanase structural gene. important protective food crops of India and grown in 0.458 M ha area The phylotypes correspond roughly to the strains’ geographic origin: with 7.277 MT production and 15.9 tons/ha productivity. Among various Asia (phylotype I), the Americas (II), Africa (III), and Indonesia (IV). diseases, bacterial wilt is considered the world’s single most destructive Phylotype II has two clearly recognizable subclusters (IIA and IIB) plant disease, damages the tomato crop 4% to 95% [5,6] depends on the [14-16]. The phylotyping scheme proposed by Fegan and Prior [15] is seasons and cultivars, which is one of the most important disease and broadly consistent with the former phenotypic and molecular typing a major constraint on tomato production worldwide. R. solanacearum schemes and adds valuable information about the geographical origin is a genetically and physiologically diverse pathogen and has five races and in some cases the pathogenicity of strains. Wicker et al. [17] on the basis of differences in host range [7], six biovars on the ability also used coalescent genealogy reconstruction to deduce the order in to metabolize three sugar alcohols and three disaccharides carbon which phylotypes likely evolved, and along with known ecotypes they utilization and four phylotypes [8]. The phylogenetic analysis based on described eight clades superimposed on the phylotypes. In multilocus different molecular methods have shown thatR. solanacearum is a highly sequence typing, virulence and housekeeping genes are used for study heterogeneous group of bacteria probably belonging to several species genetic diversity [14]. The selection of these genes was based on their [8-10] that cannot be taxonomically resolved by the race/biovar system. use in an MLST scheme of other bacterial species and the availability of Genetic diversity of plant pathogenic bacteria including R. solanacearum some sequence data from the virulence-related egl, fli C and hrpB genes has been reported to use different methods such as RAPD [11], PCR- in databases. The virulence-related genes are implicated directly (egl) or RFLP of hrp gene [12], Rep-PCR [13] and multilocus sequence typing (MLST) sequencing [14]. The assessment of the genetic diversity of R. solanacearum employing restriction arrangement length polymorphism *Corresponding author: Dinesh Singh, Division of Plant Pathology, Indian analysis resulted in the identification of two clusters of strains denoted Agricultural Research Institute, New Delhi-110012, India, Fax: 91+ 11- 25840772; divisions 1 (Asiaticum) and 2 (Americanum) [8-10] proposed a new E-mail: [email protected] hierarchical classification scheme to distinguish the genetic diversity Received June 29, 2018; Accepted July 27, 2018; Published July 31, 2018 within R. solanacearum species complex. This classification is based Citation: Singh D, Sinha S, Chaudhary G, Yadav DK (2018) Biological on sequence analysis of the internal transcribed spacer (ITS) region, Characterization and Genetic Diversity of Indian Strains of Ralstonia solanacearum the endoglucanase (egl) gene and hrpB gene, that subdivides R. Biovars 3 and 4 Causing Bacterial Wilt of Tomato. J Plant Pathol Microbiol 9: 443. solanacearum into four phylotypes, defined as “a monophyletic cluster doi: 10.4172/2157-7471.1000443 of strains revealed by phylogenetic analysis of sequence data” [15]. A Copyright: © 2018 Singh D, et al. This is an open-access article distributed under variety of genetic techniques have been used to investigate the diversity the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and and interrelatedness within the R. solanacearum species complex [4]. source are credited. J Plant Pathol Microbiol, an open access journal ISSN: 2157-7471 Volume 9 • Issue 7 • 1000443 Citation: Singh D, Sinha S, Chaudhary G, Yadav DK (2018) Biological Characterization and Genetic Diversity of Indian Strains of Ralstonia solanacearum Biovars 3 and 4 Causing Bacterial Wilt of Tomato. J Plant Pathol Microbiol 9: 443. doi: 10.4172/2157-7471.1000443 Page 2 of 12 indirectly (hrpB, fliC) in disease-causing process. The egl gene encodes bromide (CTAB) method [19]. The total genomic DNA was dissolved and endoglucanases that likely acts at the front line of host invasion in 1X TE buffer (10 mM × Tris-hydrochloric acid and 0.5 mM sodium by partially degrading host cell-wall, hrpB encodes an araC (1-β-D- EDTA, pH 8) and stored at -20°C. The estimation of quantity and arabinofuranosyl cystosine) type transcriptional regulatory protein quality of DNA was done by Nanodrop and gel electrophoresis. that governs multiple virulence pathways. Flagellin encoded by the fliC Molecular characterization and Phylotyping of Ralstonia gene, is the essential subunit of the flagellar filament that is needed for solanacearum invasion virulence and. However, flagellin is not a major elicitor of host defense in R. solanacearum. All 87 isolates of R. solanacearum were PCR amplified at 288 bp using a set of primers corresponding to 16S rDNA (OLI1 and Y2) Although bacterial wilt of tomato is known to be prevalent in India, as described by Seal et al. [20], using universal primers. Phylotype the genetic diversity of R. solanacearum strains affecting tomato crop affiliation of these isolates of R. solanacearum was determined as in this country is not known particularly north and eastern parts of described [8,16]. Multiplex PCR was carried in 25 µl volume of master India. In this study, a collection of R. solanacearum strains originating mix, containing PCR buffer (5X), MgCl (25 mM), dNTPs (10 mM), from tomato plants and different states of India under different agro- 2 Primers: Nmult:21:1F, Nmult:21:2R, Nmult:22:InF, Nmult:22:InR, climatic regions was characterized by biochemical and genotypic Nmult:23:AF, Nmult:23:AR, OLI1, Y2 (10 pmol) (Table 1), Taq methods assigned to biovar, DNA typing, phylotype and analyze polymerase 1 U/µl and molecular grade H2O. The following cycling multilocus sequence typing using virulence genes. program was used in the Master cycler Gradient: Initial denaturation : Materials and Methods 96°C for 5 min, 30 cycle s of 94°C for 15sec, annealing 59°C for 30 sec, extension 72°C for 30 sec followed by final extension at 72°C for 10 Bacterial strains, media and growth conditions min. A 10 µl aliquot of each primer amplified product was subjected to electrophoresis on 1.5% agarose gel, stained with ethidium bromide A total of 87 strains of R. solanacearum were isolated from bacterial and visualized as a phylotype 1, which produced 2 bands, 144 bp wilted tomato plants from Uttarakhand, Himachal Pradesh, Jammu and 288 bp. Amplification of 16S rRNA primer was determined by and Kashmir, Jharkhand, Orissa and West Bengal states of India on casamino acid peptone glucose (CPG) agar medium and TZC medium producing specific bands at 288 bp.
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