Repetitive Sequence-derived PCR Profiling Using the BOX-A1R Primer for Rapid Identification of the Plant Pathogen Clavibacter Michiganensis Subspecies Sepedonicus

Repetitive sequence-derived PCR using the BOX-A1R primer was used to generate genomic fingerprints of Clavibacter michiganensis subspecies sepedonicus, the causal agent of bacterial ring rot disease of potato. A total of 35 C. michiganensis subsp. sepedonicus strains were selected for study in order to represent the widest possible historical, morphological and geographical diversity of the organism. Comparison was made with genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. insidiosus, C. michiganensis subsp. tessellarius, C. michiganensis subsp. nebraskensis as well as other related Gram positive plant pathogens. The resultant genomic fingerprints and subsequent cluster analysis show C. michiganensis subsp. sepedonicus to form a remarkably homogeneous group with approximately 84% similarity between all of the strains tested. There was no evidence to suggest that fingerprints varied with historic, morphological or geographic diversity. In addition, C. michiganensis subsp. sepedonicus isolated from asymptomatic sugar beet had the same fingerprint as those which were isolated as potato pathogens. This group was easily distinguished from the clusters formed by the other subspecies of C. michiganensis and Gram positive plant pathogens. The potential for this technique to be used as a relatively rapid method to replace the time consuming and sometimes inconclusive eggplant bioassay test is discussed.     

Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

API 50CH and API ZYM systems were used to characterize fifty-three strains of Clavibacter michiganensis subsp. sepedonicus from different geographic locations and several reference strains of the same and different species, including other potato pathogens. Clavibacter michiganensis subsp. sepedonicus strains displayed a high level of homogeneity, both in carbohydrate utilization and in enzymatic activity. Using API 50CH and API ZYM it was possible to differentiate C. michiganensis subsp. sepedonicus strains from the remaining taxa analysed in this study, which included representative strains of the other subspecies of C. michiganensis as well as other bacterial pathogens affecting potatoes. Therefore, these systems could be used as an effective method to characterize C. michiganensis subsp. sepedonicus. Such a procedure would constitute an alternative system to the conventional nutritional and physiological identification tests currently included in the official methods employed in the European Union to detect and identify this bacterium. The results obtained with the API systems agreed with the current taxonomic classification of C. michiganensis, clearly separating sepedonicus from the other subspecies belonging to this species.     

广东省马铃薯块茎软腐病病原菌鉴定

为明确广东省惠州市马铃薯块茎软腐病的病原菌,采用常规病组织分离法获得4株菌株,通过生理生化特征和分子生物学特性对其进行鉴定,并测定了该病原菌对20个马铃薯品种的致病力。结果显示,该病原菌为革兰氏阳性菌,在LB平板上菌落有透明和不透明2种形态;除葡萄糖发酵阳性、对链霉素和青霉素具有抗性等特征不同外,BP-hd-1、BP-hd-2、BP-hd-3和BP-hd-4菌株与短小芽胞杆菌Bacillus pumilus菌株的其它生理生化特征均相同。系统进化分析结果表明,4株菌株的16S rDNA序列分别与短小芽胞杆菌AI-Khrj18(KY123871)、ML270(KC692158)、NCTC10337(LT906438)和ARD21(KX023236)的相似性为100.0%;4株菌株的gyrB基因序列均与短小芽胞杆菌AUEC29菌株(HM585095)的gyrB基因相似性最高,为99.7%～99.8%。生理生化特征及分子生物学鉴定结果表明,引起广东省马铃薯块茎软腐病的病原菌为短小芽胞杆菌。致病力测定结果显示,接种菌株BP-1后20个马铃薯品种的发病率均为100.0%,表明该病原菌对马铃薯有强致病力。     

Relationship Between Production of the Phytotoxin Prehelminthosporol and Virulence in Isolates of the Plant Pathogenic Fungus Bipolaris sorokiniana

A gas chromatographic method was developed to quantify the phytotoxin prehelminthosporol, which is a sesquiterpene metabolite of the plant pathogen Bipolaris sorokiniana. The toxin was extracted from mycelium or culture filtrates, pre-cleaned using solid phase extraction, and analyzed by gas chromatography as a trimethylsilyl-derivative. The detection limit of the method was 5ngl^–1 (signal to noise ratio 4:1) which corresponds to ca. 15ng prehelminthosporol per mg dry weight of mycelium or 15ng prehelminthosporol per ml culture filtrate. The total amount of prehelminthosporol (mycelium plus culture filtrate) increased with cultivation time when examined in six isolates of B. sorokiniana after 6, 9, 12 and 15 days of incubation. The screening experiment of 17 isolates for prehelminthosporol production after 8 days of incubation revealed significant differences in the toxin production between the isolates. The isolates with low toxin production had lower virulence towards barley roots compared to those with higher production of the toxin. However, the virulence did not increase with prehelminthosporol level among the high producing isolates. Prehelminthosporol was also analyzed in a number of related Bipolaris and Drechslera species. In addition to B. sorokiniana, three out of six Bipolaris species (B. setariae, B. zeicola, B. victoriae) produced prehelminthosporol, which indicates that ability to produce prehelminthosporol is conserved among closely-related Bipolaris species.     

宁夏回族自治区马铃薯镰刀菌根腐病病原菌的分离鉴定与致病性测定

为明确宁夏回族自治区马铃薯镰刀菌根腐病的病原菌种类,2015—2018年从宁夏回族自治区固原市、吴忠市、中卫市和石嘴山市4个市12个区县采集马铃薯病样,采用组织分离法对病样进行分离纯化,利用形态学鉴定方法和基于rDNA-ITS与EF-1α序列分析的分子鉴定方法对分离物进行鉴定,并按照柯赫氏法则对其致病性进行测定。结果显示,经组织分离法共获得真菌菌株347株,其中230株镰刀菌属真菌菌株引起马铃薯镰刀菌根腐病,这些镰刀菌分属于5个种,分别为尖孢镰刀菌Fusarium oxysporum、茄病镰刀菌F. solani、锐顶镰刀菌F. acuminatum、木贼镰刀菌F. equiseti、接骨木镰刀菌F. sambucinum,这5种镰刀菌分别为67、29、50、39和45株,分离频率分别为29.13%、12.61%、21.73%、16.95%和19.56%;这5种镰刀菌的致病性有差异,其中接骨木镰刀菌和茄病镰刀菌的发病率分别为91.81%和99.99%,病情指数分别为69.56和82.23,均高于其它3种镰刀菌,是引起宁夏回族自治区马铃薯镰刀菌根腐病的优势病原菌。     

宁夏马铃薯镰刀菌根腐病病原菌的ISSR-PCR标记体系建立及其遗传多样性分析

为明确宁夏马铃薯镰刀菌根腐病病原菌种内的遗传差异与亲缘关系,利用ISSR-PCR技术对影响PCR扩增效果的因素和ISSR引物进行优化和筛选,建立适合马铃薯镰刀菌根腐病病原菌的ISSR-PCR反应体系,并通过ISSR分子标记技术对30株地理来源不同的镰刀菌进行遗传多样性分析。结果显示:马铃薯镰刀菌根腐病病原菌的ISSR-PCR扩增最适引物为807,20μL反应体系中2×Easy Taq PCR Super Mix为8μL、引物浓度为0.6μmol/L、DNA模板浓度为56 ng/μL,循环次数为36次,退火温度为54℃。在选用的18条ISSR引物中有13条对30株镰刀菌扩增出84条条带,大小多分布在250～2 000 bp之间,其中多态性条带为82条,多态性比例平均为98.3%。30株镰刀菌的遗传相似系数在0.349～0.976之间,在0.478水平上可划分为2个类群,其中类群I包括尖孢镰刀菌Fusarium oxysporum、木贼镰刀菌F. equiseti、茄病镰刀菌F. solani和锐顶镰刀菌F. acuminatum;类群II全部为接骨木镰刀菌F. sambucinum;在0...     

云南小麦条锈菌群体对18个抗条锈近等基因系的毒性分析

为监测云南省小麦条锈菌群体毒性及小麦抗条锈基因的有效性动态,2016年采用18个抗条锈近等基因系鉴别寄主对云南省9个州市的136个小麦条锈菌株进行毒性分析,并按八进制法对小种进行命名。结果表明,云南省小麦条锈菌群体毒性丰富,共鉴定出64个小种类型,其中居于前2位的小种是550273和550073,出现频率分别为28.68%和11.76%,是本年度优势小种;其它小种出现频率均在4.41%以下,为次要小种。条锈菌群体对Yr5、Yr10、Yr15、Yr32四个抗条锈基因的毒力频率均为0,对Yr24、Yr Tr1、Yr8、Yr17四个抗条锈基因的毒力频率在0.74%~11.76%之间,表明这8个基因是云南省当前有效的抗条锈基因;对Yr27的毒力频率为52.94%,对Yr1、Yr6、Yr7、Yr9、Yr43、Yr44、Yr SP、Yr Exp2、Yr Tye九个抗条锈基因的毒力频率为77.94%~91.91%,表明这10个抗条锈基因的抗性已减缓或丧失,说明这些基因在云南省已失效。     

小麦条锈病菌新菌系G22-9(CYR34)和G22-14流行趋势预测

为预测小麦条锈病菌新菌系G22-9(CYR34)和G22-14的未来流行趋势,科学指导小麦抗病育种及条锈病治理,采用常规鉴定法于2009—2016年对甘肃省2 330份小麦条锈病菌标样进行了生理小种鉴定及消长动态监测,并对G22-9(CYR34)和G22-14的致病性及寄生适合度进行了测定。结果发现:新菌系G22-9(CYR34)和G22-14在甘肃省的出现频率加快,已从2009年的0分别上升到2016年的34.85%和6.43%,其中G22-9(CYR34)已发展为甘肃省第1位流行生理小种。与主流生理小种CYR32和CYR33相比较,G22-9(CYR34)和G22-14具有毒性谱宽、致病力强、寄生适合度高、哺育品种多等特点。建议在甘肃省特别是小麦条锈病常发易变区的陇南麦区,针对新菌系的出现,及时调整小麦品种布局及育种目标,以持续控制条锈病的发生流行。