来自桑树的3株青枯劳尔氏菌分离株的亚分类研究
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摘要
青枯劳尔氏菌(Ralstonia solanacearum)是导致桑青枯病的病原菌。从广东省罗岗、英德蚕区桑园的感病桑树取样,采用TZC固体培养基分离纯化得到的3株青枯劳尔氏菌菌株G11-41、G12-9、G12-50的形态特征均有差异:G11-41菌株菌落小,菌落中央深红色,菌体形态为接近球形的短杆状;G12-9菌落不规则,菌落中央呈淡红色,菌体形态为短杆状;G12-50菌落呈不规则圆形,菌体形态呈长杆状。将3个分离菌株的菌液淋灌根部接种感染桑树植株,其致病性也存在明显差异:接种G11-41菌株的植株发病率为11%,死亡率为0;接种G12-9与G12-50菌株的植株发病率和死亡率均为100%。根据Hayward分类标准进行生化型分类,3个分离株的6种碳源利用试验均呈现阳性反应,属于生化型Ⅲ。基于菌株的16S r DNA序列和内切葡聚糖酶基因(egl)序列构建的系统进化树将青枯劳尔氏菌分类为4个种群型,G11-41、G12-9、G12-50分离株同归属于种群型Ⅰ,其中:16S r DNA序列进化树上,G11-41与G12-50分离株的亲缘关系最近,分布在同一个进化小枝上;egl基因序列进化树上,强毒力分离株G12-9与G12-50分布在同一个进化小枝上,与弱毒力分离株G11-41有一定的进化距离。初步认为:在青枯劳尔氏菌的亚分类中,依据16S r DNA序列构建的系统进化树可能会更好地显示各分离株的地理来源;依据egl基因序列构建的系统进化树则可能会更好地显示出各分离株的致病性。
Ralstonia solanacearum is the pathogen causing mulberry bacterial wilt. Solid TZC medium was used to isolate and purify R. solanacearum samples collected from infected mulberry trees in mulberry field of Luogang and Yingde sericultural regions of Guangdong Province. The obtained 3 R. solanacearum isolates,i.e. G11-41,G12-9 and G12-50,had different morphological characteristics. The colony of G11-41 was small and center of the colony was in deep red color.Its bacteria were in near-spherical short-rod shape. The colony of G12-9 was irregular and center of the colony was in pale red color. Its bacteria were in short-rod shape. The colony of G12-50 was in irregular round form. Its bacteria were in long-rod shape. After bacterial solutions of the 3 isolates were sprinkled onto mulberry roots for disease inoculation,the 3 isolates showed obvious difference in pathogenicity. Mulberry trees inoculated with G11-41 had a morbidity rate of 11% and a mortality rate of 0,while those inoculated with G12-9 and G12-50 had both morbidity and mortality rates of 100%. According to Hayward criteria of classification,the 3 isolates had positive reactions in all 6carbon utilization tests,demonstrating that they belong to biochemical type Ⅲ. Phylogenetic trees constructed based on16 S r DNA and endoglucanase gene(egl) sequences displayed that R. solanacearum can be classified into 4 phylotypes.G11-41,G12-9 and G12-50 isolates all belong to phylotypeⅠ. In phylogenetic tree of 16 S r DNA sequences,G11-41 and G12-50 have the closest genetic relationship and are located on the same evolutionary clade. In phylogenetic tree of egl gene sequences,strong virulence isolates G12-9 and G12-50 are located on the same evolutionary clade which have a certain evolutionary distance with the weak virulence isolate G11-41. It is preliminarily concluded that sub-classification of R.solanacearum based on phylogenetic tree of 16 S r DNA sequences can better reflect the geographic origin of various isolates,while that based on phylogenetic tree of egl gene sequences can better reflect the pathogenicity of various isolates.
Ralstonia solanacearum is the pathogen causing mulberry bacterial wilt. Solid TZC medium was used to isolate and purify R. solanacearum samples collected from infected mulberry trees in mulberry field of Luogang and Yingde sericultural regions of Guangdong Province. The obtained 3 R. solanacearum isolates,i.e. G11-41,G12-9 and G12-50,had different morphological characteristics. The colony of G11-41 was small and center of the colony was in deep red color.Its bacteria were in near-spherical short-rod shape. The colony of G12-9 was irregular and center of the colony was in pale red color. Its bacteria were in short-rod shape. The colony of G12-50 was in irregular round form. Its bacteria were in long-rod shape. After bacterial solutions of the 3 isolates were sprinkled onto mulberry roots for disease inoculation,the 3 isolates showed obvious difference in pathogenicity. Mulberry trees inoculated with G11-41 had a morbidity rate of 11% and a mortality rate of 0,while those inoculated with G12-9 and G12-50 had both morbidity and mortality rates of 100%. According to Hayward criteria of classification,the 3 isolates had positive reactions in all 6carbon utilization tests,demonstrating that they belong to biochemical type Ⅲ. Phylogenetic trees constructed based on16 S r DNA and endoglucanase gene(egl) sequences displayed that R. solanacearum can be classified into 4 phylotypes.G11-41,G12-9 and G12-50 isolates all belong to phylotypeⅠ. In phylogenetic tree of 16 S r DNA sequences,G11-41 and G12-50 have the closest genetic relationship and are located on the same evolutionary clade. In phylogenetic tree of egl gene sequences,strong virulence isolates G12-9 and G12-50 are located on the same evolutionary clade which have a certain evolutionary distance with the weak virulence isolate G11-41. It is preliminarily concluded that sub-classification of R.solanacearum based on phylogenetic tree of 16 S r DNA sequences can better reflect the geographic origin of various isolates,while that based on phylogenetic tree of egl gene sequences can better reflect the pathogenicity of various isolates.
引文
[1]HAYWARD A C.Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum[J].Annu Rev Phytopathol,1991,29:65-87
[2]MANSFIELD J,GENIN S,MAGORI S,et al.Top 10 plant pathogenic bacteria in molecular plant pathology[J].Mol Plant Pathol,2012,13(6):614-629
[3]BUDDENHAGEN I,KELMAN A.Biological and physiological aspects of bacterial wilt caused by Pseudomonas solanacearum[J].Phytopathology,1964(2):203-230
[4]华静月,张长岭,何礼远.我国植物青枯菌的生化型和其他生理差异[J].植物保护学报,1984,11(1):43-50
[5]HAYWARD A C.Bacteriophage sensitivity and biochemical group in Xanthomonas malvacearum[J].J Gen Microbiol,1964,35:287-298
[6]GILLINGS M,FAHY P,DAVIES C.Restriction analysis of an amplified polygalacturonase gene fragment differentiates strains of the phytopathogenic bacterium Pseudomonas solanacearum[J].Lett Appl Microbiol,1993,17(1):44-48
[7]TAGHAVI M,HAYWARD C,SLY L I,et al.Analysis of the phylogenetic relationships of strains of Burkholderia solanacearum,Pseudomonas syzygii,and the blood disease bacterium of banana based on 16S r DNA gene sequences[J].Int J Syst Bacteriol,1996,46(1):10-15
[8]VANEECHOUTTE M,KAMPFER P,DE BAERE T,et al.Wautersia gen.nov.,a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species,and proposal of Ralstonia[Pseudomonas]syzygii(Roberts et al.1990)comb.nov.[J].Int J Syst Evol Microbiol,2004,54(2):317-327
[9]FEGAN M,PRIOR P.How complex is the Ralstonia solanacearum species complex?[M]∥Allen C,Prior P,Hayward A C.Bacterial wilt disease and the Ralstonia solanacearum species complex.Bethesda:APS Press,2005:229-461
[10]王艳,常润磊,周旭东.青枯病快速检测研究概况[J].桉树科技,2012,29(2):53-58
[11]王胜坤,王军,徐大平.植物青枯菌检测方法研究进展[J].南京林业大学学报,2007,27(2):118-122
[12]罗国庆,唐翠明,王振江.桑树细菌性青枯病的研究概况[J].蚕业科学,2011,37(6):1093-1097
[13]HAYWOWD A C.Characteristics of Pseudomonas solanacearum[J].J Appl Bacteriol,2010,27(2):265-277
[14]PITKETHLEY N.Host range and biotypes of Pseudomonas solanacearum in the northernterritory[J].Australas Plant Path,1981,10(3):46-47
[15]曾宪铭,董春.广东农作物青枯病菌的生化型[J].华南农业大学学报,1995,16(1):50-53
[16]吕志强,王汉荣,周勤.浙江省桑树青枯病菌生理小种及生化型的测定[J].浙江农业学报,2007,9(4):306-309
[2]MANSFIELD J,GENIN S,MAGORI S,et al.Top 10 plant pathogenic bacteria in molecular plant pathology[J].Mol Plant Pathol,2012,13(6):614-629
[3]BUDDENHAGEN I,KELMAN A.Biological and physiological aspects of bacterial wilt caused by Pseudomonas solanacearum[J].Phytopathology,1964(2):203-230
[4]华静月,张长岭,何礼远.我国植物青枯菌的生化型和其他生理差异[J].植物保护学报,1984,11(1):43-50
[5]HAYWARD A C.Bacteriophage sensitivity and biochemical group in Xanthomonas malvacearum[J].J Gen Microbiol,1964,35:287-298
[6]GILLINGS M,FAHY P,DAVIES C.Restriction analysis of an amplified polygalacturonase gene fragment differentiates strains of the phytopathogenic bacterium Pseudomonas solanacearum[J].Lett Appl Microbiol,1993,17(1):44-48
[7]TAGHAVI M,HAYWARD C,SLY L I,et al.Analysis of the phylogenetic relationships of strains of Burkholderia solanacearum,Pseudomonas syzygii,and the blood disease bacterium of banana based on 16S r DNA gene sequences[J].Int J Syst Bacteriol,1996,46(1):10-15
[8]VANEECHOUTTE M,KAMPFER P,DE BAERE T,et al.Wautersia gen.nov.,a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species,and proposal of Ralstonia[Pseudomonas]syzygii(Roberts et al.1990)comb.nov.[J].Int J Syst Evol Microbiol,2004,54(2):317-327
[9]FEGAN M,PRIOR P.How complex is the Ralstonia solanacearum species complex?[M]∥Allen C,Prior P,Hayward A C.Bacterial wilt disease and the Ralstonia solanacearum species complex.Bethesda:APS Press,2005:229-461
[10]王艳,常润磊,周旭东.青枯病快速检测研究概况[J].桉树科技,2012,29(2):53-58
[11]王胜坤,王军,徐大平.植物青枯菌检测方法研究进展[J].南京林业大学学报,2007,27(2):118-122
[12]罗国庆,唐翠明,王振江.桑树细菌性青枯病的研究概况[J].蚕业科学,2011,37(6):1093-1097
[13]HAYWOWD A C.Characteristics of Pseudomonas solanacearum[J].J Appl Bacteriol,2010,27(2):265-277
[14]PITKETHLEY N.Host range and biotypes of Pseudomonas solanacearum in the northernterritory[J].Australas Plant Path,1981,10(3):46-47
[15]曾宪铭,董春.广东农作物青枯病菌的生化型[J].华南农业大学学报,1995,16(1):50-53
[16]吕志强,王汉荣,周勤.浙江省桑树青枯病菌生理小种及生化型的测定[J].浙江农业学报,2007,9(4):306-309