植物病原短小杆菌的分子检测和糖甜菜叶斑病菌的研究

This thesis gives a detailed account on the identification of the first reported pathovar of the genus of Curtobacterium. Bacterial leaf spot diseases of sugar beet (Beta vulgaris var. saccharifera) was found in 1995. The disease has not been reported before. The pathogen is Gram-positive, small irregular rods, and possess lateral flagellation. The cells become shorter to coccoid form in old culture. They are obligatedly aerobic, catalase positive. The optimum temperature for growth is 24~27℃, the highest is 37℃. Acid is produced from many carbohydrates. Several kinds of organic acids are assimilated. Urease and oxydase is not produced. Generally gelatin is not hydrolyzed. NH3 can not be produced from peptone, and H2S can be released from cysteine, cystine and Na2S2O3·5H2O. Nitric acid is not deoxidized. Indole is not produced. Casein, starch, esculin and Tween 80 can be hydrolyzed. The cell wall peptidoglycan is based upon ornithine (type B2β). The predominating menaquinone is MK-9. Polar lipid contains several glycosyldiacylglycerols. The colonies of malabar spinach isolates are yellow, and those of sugar beet strains produce light yellow pigment. The DNA base compositions are 69.952 and 67.456 mol % G+C (Tm) separately. The identification results show that the characteristics of the pathogen is consistent with that of Curtobacterium flaccumfaciens. According to the pathogenicity, it is considered to be a new pathovar of this species. Name suggested is Curtobacterium flaccumfaciens pv. beticola pv. nov. .We got the proper primer to differentiate the bacteria of genus Curtobacterium from other bacteria by RAPD method. And then, by molecular hybridization and PCR, we found the special probe of genus Curtobacterium. The classification of the new pathovar and Plant Pathogenic Coryneform Bacteria is discussed according to the results of phonetic characteristic numerical analysis and RAPD analysis in this thesis. The classification of Curtobacterium is relatively clear, but Clavibacter is still a heterogenetic genus.Curtobacterium flaccumfacies pv. flaccumfacies (Cff), the agent of of Phaseolus spp., is on the A1 list of quarantine organism for China. Curtobacterium flaccumfacies pv. batae (Cfb) is the causal agent of silvering disease of red beet. In this assay, A Polymerase chain reaction (PCR) was developed that specifically detected Cff. Generic PCR products from the intergenic transcribed spacer (ITS) region of 16S-23S ribosomal DNA of Cff and other related bacteria were cloned and sequenced. Based on a multiple sequences alignment among these obtained sequences and other-nonredundant highly homologous sequences from database, two Cff-specific PCR primers were designed, A1（5'-AGTGCTGGTCGCCCATCAT-3'）and A2（5'-CACCAGACCAACCCGAAGGT-3'）. These two oligonucleotides primed the specific amplification of a 380-bp DNA product from genomic DNA sample of Cff strain. Amplification was not observed with other 20 tested bacteria, including five strains of other Curtobacterium flaccumfacies pvs., two strains of Clavibacter species, eight model strains of other different genus and five strains of saprophytic bacteria. The similar PCR product also was amplified from Rathayibacter. tritic, which has different host with Cff. Two Cfb-specific PCR primers were designed, B1（5'-GGCCTCGTGTTGTCCCTTATC-3'）/B2 （5'-GTCACCAATCAACAACCCGAG-3'）. These two oligonucleotides primed the specific amplification of a 387bp DNA product from genomic DNA sample of Cfb strain. Amplification was not observed with other 21 tested bacteria, including 5 strains of other Curtobacterium flaccumfacies pvs., 3 strains of Clavibacter species, 8 type strains of other different genus and 5 strains of saprophytic bacteria. The PCR protocol provides a rapid, reliable, and economical tool for routine detection and identification of Cff and Cfb.

用表型特征测定，蛋白质电泳图谱分析，化学成分测定和遗传物质分析等方法鉴定糖甜菜叶斑病菌，确定该病菌在短小杆菌属下的分类地位并为其命名。同时，利用分子杂交和PCR的椒ㄕ页鲋膊《绦「司淖ɑ苑肿蛹觳馓秸耄⒘槊簟⒓虮恪⒖焖佟⒕返奶锛洳≈昙觳馐侄危『Φ目焖僬锒虾头乐翁峁┛煽康囊谰荨