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

Abstract

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.

Bacterial Wilt of Solanaceous Crops: Diagnosis, Diversity and Management

“…Standardization of PCR and real time PCR analysis could play an important role for detection R. solanacearum from the disease in the seeds planting material, soil and irrigation water, where the pathogen survives in low number ( Table 1). The bacterial pathogen causing wilt disease can be detected by using conventional PCR with detection level from 10 2 cfu/ ml (Singh et al, 2014a) to 10 5 cfu/ ml (Khakvar et al, 2008). PCR based methods for the detection of R. solanacearum are usually based on the amplification of ribosomal sequences (16S rRNA or ITS region) and other conserved genes of bacteria (Pastrick et al, 2002;Ranajn et al, 2016;Singh et al, 2014a;Weller et al, 2000).…”

“…The bacterial pathogen causing wilt disease can be detected by using conventional PCR with detection level from 10 2 cfu/ ml (Singh et al, 2014a) to 10 5 cfu/ ml (Khakvar et al, 2008). PCR based methods for the detection of R. solanacearum are usually based on the amplification of ribosomal sequences (16S rRNA or ITS region) and other conserved genes of bacteria (Pastrick et al, 2002;Ranajn et al, 2016;Singh et al, 2014a;Weller et al, 2000). Molecular based detection of R. solanacearum causing bacterial wilt of tomato, chilli, brinjal and potato has been done by using specific primers Y2 and OLII and it also distinguished R. solanacearum from other saprophytic bacteria grown on TTC medium (Singh et al, 2010(Singh et al, , 2014a.…”

“…PCR based methods for the detection of R. solanacearum are usually based on the amplification of ribosomal sequences (16S rRNA or ITS region) and other conserved genes of bacteria (Pastrick et al, 2002;Ranajn et al, 2016;Singh et al, 2014a;Weller et al, 2000). Molecular based detection of R. solanacearum causing bacterial wilt of tomato, chilli, brinjal and potato has been done by using specific primers Y2 and OLII and it also distinguished R. solanacearum from other saprophytic bacteria grown on TTC medium (Singh et al, 2010(Singh et al, , 2014a. Various modifications have been made to improve the sensitivity of a single round PCR, viz., enrichment in semi-selective SMSA broth prior to PCR (Elphinstone et al, 1996;Ranjan et al, 2016;Singh et al, 2014a) or real time PCR called as BIO-PCR (Ozakman and Schaad, 2003) as well as double round PCR as nested-PCR (Khakvar et al, 2008).…”

“…Molecular based detection of R. solanacearum causing bacterial wilt of tomato, chilli, brinjal and potato has been done by using specific primers Y2 and OLII and it also distinguished R. solanacearum from other saprophytic bacteria grown on TTC medium (Singh et al, 2010(Singh et al, , 2014a. Various modifications have been made to improve the sensitivity of a single round PCR, viz., enrichment in semi-selective SMSA broth prior to PCR (Elphinstone et al, 1996;Ranjan et al, 2016;Singh et al, 2014a) or real time PCR called as BIO-PCR (Ozakman and Schaad, 2003) as well as double round PCR as nested-PCR (Khakvar et al, 2008). Grover et al (2009) detected R. solanacearum as low as 1 cfu/ml of within 8 h including DNA isolation.…”

Biological Characterization and Genetic Diversity of Indian Strains of Ralstonia solanacearum Biovars 3 and 4 Causing Bacterial Wilt of Tomato

“…The phylogenetic analysis based on different molecular methods have shown that R. solanacearum is a highly heterogeneous group of bacteria probably belonging to several species [8][9][10] that cannot be taxonomically resolved by the race/biovar system. Genetic diversity of plant pathogenic bacteria including R. solanacearum has been reported to use different methods such as RAPD [11], PCR-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 analysis resulted in the identification of two clusters of strains denoted divisions 1 (Asiaticum) and 2 (Americanum) [8][9][10] proposed a new hierarchical classification scheme to distinguish the genetic diversity within R. solanacearum species complex.…”