Identification Of Pathogenic Bacteria Of Four New Flower Bacterial Diseases In Guangzhou

Flower industry is developing rapidly in China, especially in the past ten years, the planting area has expanded year by year, and the number of flower varieties is increasing each year. With the development of the flower industry, some new pests occurred subsequently, especially the disease of flower occurred seriously. Guangzhou is an important area for production and sale of flowers, the flower diseases cause greater impact to flower industry every year. But the kinds of flower diseases are not known clear. Therefore, it is an important role to understand the pathogens of flower diseases for control of flower diseases. The purpose of this study was to determinate the pathogens of flower diseases based on the pathogenicity test, physiological and biochemical characteristics test and molecular identification, the results are shown in the following:1, Identification of the pathogen of leaf spot disease of Philodendron congo. Pathogenicity of isolate JZMLR from the diseased tissue was determined according to Koch’s postulate. The pathogenic strain JZMLR was identified based on analysis of 16 S rRNA gene, RNA polymerase beta subunit encoding gene, and physiological and biochemical characteristics test. The result showed that, the near full-length sequence of 16 S rRNA gene was amplified from strain JZMLR(GenBank Accession No. KU758911) with primers 27 F and 1492 R, obtaining a PCR product of 1401 bp. A BLAST search in GenBank revealed the highest similarity(99%) to sequences of Dickeya dadantii, D. solani, Erwinia chrysanthemi. By construction of phylogenic tree between the pathogenic strain JZMLR and the related strains from Gen Bank, the pathogenic strain JZMLR and other strains were not in the same clade. Then, amplification of rpoB gene of strain JZMLR was conducted with primers CM7 and CM13, obtaining a PCR product of 1035 bp. A BLAST search in GenBank revealed the highest similarity(98%) to sequences of D. dadantii. Then, according to difference of Dickeya dadantii and the related bacteria in physiological and biochemical characteristics referring to Bergey’s Manual of Systematic Bacteriology(2005), 19 physiological and biochemical characteristics were selected to determine the pathogen, the results indicated that, similarity of physiological and biochemical characteristics between pathogenic strains JZMLR and D. dadantii was 68.42%. Therefore, pathogen of leaf spot of Philodendron congo was preliminarily identified as a Dickeya dadantii.2, Identification of the pathogen of leaf spot disease of Polyscias guifoylei. Pathogenicity of isolate FLT from the diseased tissue was determined according to Koch’s postulate. The pathogenic strain FLT was identified based on analysis of 16 S rRNA gene and physiological and biochemical characteristics test. The result showed that, the near full-length sequence of 16 S rRNA gene was amplified from strain FLT with primers 27 F and 1492 R, obtaining a PCR product of 1415 bp. A BLAST search in Gen Bank revealed the highest similarity(99%-100%) to sequences of Xanthomonas campestris, Xanthomonas axonopodis, Xanthomonas citri. By construction of phylogenic tree between the pathogenic strain FLT and the related strains from GenBank, the pathogenic strain FLT and other strains were in a group. Then, according to difference of pathogenic strain in physiological and biochemical characteristics referring to Bergey’s Manual of Systematic Bacteriology(2005), 18 physiological and biochemical characteristics were selected to determine the pathogen, the results indicated that, similarity of physiological and biochemical characteristics between pathogenic strains FLT and X. axonopodis was 55.56%. Therefore, preliminary, pathogenic strains of leaf spot of Polyscias guifoylei was preliminarily identified as a Xanthomonas axonopodis. 3, Identification of the pathogen of leaf spot disease of Dracaena reflexa. Pathogenicity of isolate BHZ from the diseased tissue was determined according to Koch’s postulate. The pathogenic strain BHZ was identified based on analysis of 16 S rRNA gene, gry B and physiological and biochemical characteristics test. The result showed that, the near full-length sequence of 16 S rRNA gene was amplified from strain BHZ with primers 27 F and 1492 R, obtaining a PCR product of 1402 bp. A BLAST search in GenBank revealed the highest similarity(99%) to sequences of Bacillus aryabhattai, B. megaterium, Bacterium JCSHGTR2A02-2 Bacterium JCSHGTTSA01-13 Bacterium enrichment By construction of phylogenic tree between the pathogenic strain BHZ and the related strains from GenBank, the pathogenic strain BHZ and others were in the same group. Then, amplification of gryB gene of strain BHZ was conducted with primers gryb1 f and gryb2 f, obtaining a PCR product of 500 bp. A BLAST search in GenBank revealed the highest similarity(99%) to sequences of B. megaterium, P. syringae. By construction of phylogenic tree between the pathogenic strain BHZ and the related strains from GenBank, the pathogenic strain BHZ and Bacillus megaterium strain Q3 were in the same clade, and the bootstrap value is 74%. Then, according to difference of pathogenic strain in physiological and biochemical characteristics referring to Bergey’s Manual of Systematic Bacteriology(2005), 26 physiological and biochemical characteristics were selected to determine the pathogenic strain, the results indicated that, similarity of physiological and biochemical characteristics between pathogenic strains BHZ and B. megaterium was 92.3%. Therefore, pathogen of leaf spot of Dracaena reflexa was preliminarily identified as a Bacillus megaterium.4, Identification of the pathogen of leaf blight of Ficus elastica. Pathogenicity of isolate YDXJR from the diseased tissue was determined according to Koch’s postulate. The pathogenic strain YDXJR was identified based on analysis of 16 S rRNA gene, RNA polymerase beta subunit encoding gene, and physiological and biochemical characteristics test. The result showed that, the near full-length sequence of 16 S rRNA gene was amplified from strain YDXJR with primers 27 F and 1492 R, obtaining a PCR product of 1393 bp. A BLAST search in GenBank revealed the highest similarity(99%) to sequences of Pseudomonas monteilii, Pseudomonas putida, Marinobacter arcticus and Pseudomonas plecoglossicida. By construction of phylogenic tree between the pathogenic strain YDXJR and the related strains from GenBank, the pathogenic strain YDXJR and other strains were not in the same clade. Then, amplification of rpo B gene of strain YDXJR was conducted with primers CM7 and CM13, obtaining a PCR product of 1031 bp. A BLAST search in Gen Bank revealed the highest similarity(99%) to sequences of Pseudomonas putida, Pseudomonas monteilii, Pseudomonas oleovorans strain, Pseudomonas plecoglossicida. By construction of phylogenic tree between the pathogenic strain YDXJR and the related strains from GenBank, the pathogenic strain JZMLR and Pseudomonas plecoglossicida were in the same clade, and the bootstrap value is 39%. Then, according to difference of Pseudomonas plecoglossicida and the related bacteria in physiological and biochemical characteristics referring to Bergey’s Manual of Systematic Bacteriology(2005), 10 physiological and biochemical characteristics were selected to determine the pathogenic strain, the results indicated that, similarity of physiological and biochemical characteristics between pathogenic strains YDXJR and Pseudomonas plecoglossicida was 70%. Therefore, pathogen of brown spot of Ficus elastica was preliminarily identified as a Pseudomonas plecoglossicida.