Molecular Detection Of Plant Pathogenic Coryneform Bacteria

Plant pathogenic coryneform bacteria are important pathogens which cause many plant disease including 8 quarantine pathogens in our country. It is important to detect pathogens rapidly and specifically for plant quarantine and disease control.Universal PCR primers Ll(5'- AGTCGTAACAAGGTAGCCGT-3') and L2 ( 5'-GTGCCAAGGCATCCACC-3') that target the Intergenic Transcribed Spacers (ITS) region between 16S and 23 S rDNA genes were utilized in this assay. A similar fragment with 500bp was amplified from each of 13 tested strains (except Curbacterium. plantarum ), by PCR protocol using primer pair LI/L2. The PCR products of 14 strains were recovered, cloned and sequenced. Based on multiple sequence alignments among these sequences and other-nonredundant highly homologous sequences from the database, 9 primer pairs of SCAR markers (A1/A2, B1/B2, C1/C2, D1/D2, E1/E2, F1/F2, G1/G2, H1/H2, 11/12), were designed and synthesized. Primers were used to amplified the ITS genes from 22 strains genomic DNA in order to study their specificity.Primer pairs of B1/B2, E1/E2 and 11/12 amplified of a unique 387bp, 390bp and 350bp fragment respectively from genomic DNA samples strain of Cur. f. pv. betae, Cur. luteum and Cl.fangii respectively, product was not obtained from other 21 related strains. These primer pairs are specific and can be used for the rapid detection of their specific bacteria according to the PCR product.Using primer pairs F1/F2, an amplified fragment was obtained from each of the genomic DNA of Cur. albidum and Cur. pusillum, but molecular weight of these two products were obviously different. No product was obtained from for other 20 strains. Primer pairs F1/F2 can also be used for specific detection of Cur. pusillum according to thepresence and pattern of PCR products.Primer pairs A1/A2 can amplified the genomic DNA from both Cur. f. pv. flaccumfaciens and Rathayibacter.. tritici; H1/H2 can amplified genomic DNA from both Arthrobacter ilicis and R.. tritici, but no product was amplified from Cur. f. pv. flaccumfaciens DNA. By using these two primer pairs and two times of PCR protocols, Cur. f. pv. flaccumfaciens and R.. tritici can be differentiated.Primer pairs C1/C2, D1/D2 and G1/G2 amplified different fragments from several genomic DNA samples. Further study should be done to improved PCR conditions or search for other specific primers for the detection of Cur. f. pv. basellae, Cur. f. pv. beticila and Cur. plantarum.