Detection of Ralstonia solanacearum from Asymptomatic Tomato Plants, Irrigation Water, and Soil Through Non-selective Enrichment Medium with hrp Gene-Based Bio-PCR

• Published in: Current microbiology 2014-08, Vol.69 (2), p.127-134
• Main Authors: Singh, Dinesh, Sinha, Shweta, Yadav, D. K, Chaudhary, Garima
• Format: Article
• Language: English
• Subjects: agroclimatology; Bacteria; bacterial wilt; Bacteriological Techniques - methods; Biomedical and Life Sciences; Biotechnology; Cultivars; Culture Media - chemistry; Deoxyribonucleic acid; DNA; DNA Primers - genetics; DNA, Bacterial - genetics; Environmental conditions; Flowers & plants; Genes; hypersensitive response; India; Irrigation; Irrigation water; Life Sciences; Lycopersicon esculentum; Lycopersicon esculentum - microbiology; Microbiology; monitoring; nucleotide sequences; Pathogens; polymerase chain reaction; Polymerase Chain Reaction - methods; Ralstonia solanacearum; Ralstonia solanacearum - isolation & purification; Seedlings; Sensitivity and Specificity; Soil Microbiology; Soil properties; soil water; Soils; Tomatoes; virulence; virulent strains; Water; Water Microbiology; Xanthomonas oryzae; Xanthomonas oryzae pv. oryzae
• ISSN: 0343-8651; 1432-0991
• DOI: 10.1007/s00284-014-0566-z

Bacterial wilt of tomato caused by Ralstonia solanacearum (Smith) Yabuuchi et al. (Microbiol Immunol 39:897–904, 1995) is a serious disease, which causes losses up to 60 % depending on environmental conditions, soil property, and cultivars. In present investigation, nucleotide sequences of virulence, hypersensitive response and pathogenicity (hrp) gene were used to design a pair of primer (Hrp_rs 2F: 5′-AGAGGTCGACGCGATACAGT-3′ and Hrp_rs 2R: 5′-CATGAGCAAGGACGAAGTCA-3′) for amplification of bacterial genome. The genomic DNA of 27 isolates of R. solanacearum race 1 biovar 3 & 4 was amplified at 323 bp. The specificity of primer was tested on 13 strains of R. solanacearum with other group of bacteria such as Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, and X. citri subsp. citri. Primer amplified DNA fragment of R. solanacearum at 323 bp. The sensitivity of the primer was 200 cfu/ml and improved further detection level by using non-specific enrichment medium casamino acids-pepton-glucose broth followed by PCR (BIO-PCR). Out of 130 samples of asymptomatic tomato plants, irrigation water, and soil collected from bacterial wilt infested field in different agro-climatic regions of India, R. solanacearum was detected from 86.9, 88.5, and 90.9 per cents samples using BIO-PCR, respectively. The primer was found specific for detecting viable and virulent strains of R. solanacearum and useful for the diagnosis of R. solanacearum in tomato seedlings and monitoring of pathogen in irrigation water and soil.