Detection Of Three Phytopathogenic Bacteria By Real-Time Quantitative Assays

Curtobacterium flaccumfaciens pv. beticola (Cfb) are the causative agent of sugar beet(Beta vulgaris var. saccharifera) leaf spot disease. According to the 16S-23S rRNA ITSsequence from Cfb(accession number AY191513), Primers CfbF (5'-TCAGTGCTGGTCGCCCATTAG-3') and CfbR (5'-AACAACGTGC ACCAGCCAGCA-3') were designed.Results of screening the primers CfbF/CfbR with three strains of Curtobacteriumflaccumfaciens pv. beticola and the other 29 strains of bacteria by PCR showed the primersto be specific and the size of production was 191 bp. In real-time PCR the target DNA wasdetected and melting curve analysis showed each PCR product exhibited a characteristicpeak at its melting temperature (Tm) maximum and there were nontarget PCR products. Inorder to create standard curve real-time PCR amplifications were performed using theSYRB Green I fluorescent dye with a 10-fold dilution series of target DNA from 0.001-10ng. The regression equation is y=-0.2741x+7.51 and the determination coefficient R²=0.99.The strain Tc7(Pseudomonas spp) was isolated from sugar beet(Beta vulgaris var. sacc-harifera) too. 16S rRNA gene was amplified from the strain Tc7 using universal primers(16sF:5'-ATTGAACGCTGGCGGCAGGCCT-3' and 16sR: 5'-TCCCCTACGGTTACCTTGTTA-3')and sequenced. Alignment of the 16S rRNA gene showed the sequence washighly homologous to that of Pseudomonas argentinensis, Pseudomonas flavescens,Pseudomonas fulva and so on and there existed no specific loci. Used Eubacteria universalprimers(U1: 5'-GTGGATCACCTCCTTC-3' and U2: 5'-TTCGCTCGCCCTAC-3') toamplify the16-23S rRNA ITS region of the strain Tc7 and get a DNA fragment of 420 bp(accession number EF205021). Based on this sequence primers PF (5'-AGGCGCTCAAGCAAATCATAGA-3') and PR(5'-TT GTTCCCAA TCACGTAGG GTG-3') were designed,with which a 238-bp PCR product was obtained from the strain Tc7, whereas no PCRproducts were observed with the other 29 strains.The real-time PCR result showed theamplification curve of the strain Tc7 was smooth and the main peak was obvious,indicating that unspecific amplification had not occurred. To create a standard curve, 10-fold serial dilutions of target DNA (ranging from 0.00084 to 84 ng) were used for the experiment. Additionally, each experiment also included a negative controls (ddH₂O).There were two replicates for each sample, The amplification curves of every two replicateswere almost superposable and the Ct values were nearly equal. The regression equation isy=-3.33x+20.42 and the determination coefficient R²=0.99. This assay could be used todetect the strain Tc7 from sugar beet.Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops.A fluorogenic (SYBR Green I) PCR assay was developed to detect Ralstonia solanacearumstrain with Rsol_fliC primers reported by J. Schonfeld et al. A standard curve was createdtoo. The regression equation is y=-3.74x+44.15 and the determination coefficient R²=0.99.