Jiang H H, Meng Q X, Hanson L E, Hao J J
Department of Plant Pathology, Michigan State University, East Lansing, MI 48824.
Plant Dis. 2012 Jun;96(6):904. doi: 10.1094/PDIS-02-12-0132-PDN.
Potato (Solanum tuberosum L.) common scab can be caused by multiple Streptomyces spp., with S. scabies as a predominant species (2,3). However, according to our survey in August 2007, many symptomatic potato tubers did not have S. scabies in Michigan. To identify the pathogen, potato tubers with scab symptoms were collected from two fields in Michigan, and Streptomyces spp. were isolated using Streptomyces selective medium (STR) (2). Pure cultures of the isolates were obtained by transferring single colonies and incubation at 28°C on STR. Three isolates, designated HER21, HER24, and HER26, were identified as Streptomyces stelliscabiei based on morphological and physiological characterization (1). Bacterial cultures were prepared in liquid yeast malt extract at 28°C on an incubator shaker at 150 rpm. Genomic DNA was extracted from the cultures and used as a template for PCR with species-specific primers for Streptomyces spp. (4). The isolates gave a positive PCR reaction with primers Stel3 and T2st2 for S. stelliscabiei, but negative for any other Streptomyces spp. reported as pathogenic to potato. The 16S rRNA genes were amplified using primers previously reported (4) and amplicons were sequenced and submitted to GenBank (Accession Nos. HM018115, HM018116, and HM018117 for the three isolates, respectively). BLAST analysis of these sequences against the NCBI GenBank database determined these sequences to have 99 to 100% sequence identity with S. stelliscabiei sequences such as Accession No. FJ546728 (4). These isolates were all confirmed by PCR, using the same conditions described above, to have txtAB, nec1, and tomA genes (4), which are associated with pathogenicity of scab-causing Streptomyces spp. To complete Koch's postulates, cell suspensions of the isolates were mixed in vermiculate media (3) at a final concentration of 10 colony-forming units/ml, which were used as inocula. Potato (cv Snowden) tubers were incubated in sterilized potting mix in a growth chamber at 25°C until the seed germinated. Each potato seedling was transferred to a new pot in the greenhouse. Two weeks later, the potting mix was infested with the bacterial spore suspensions of either HER21, HER24, or HER26, with five pots per isolate. Potting mix with only media or media with S. scabies isolate 49173 were used as negative and positive controls, respectively. Three months later, potato tubers were harvested and evaluated for scab symptoms (3). The experiment was done twice. Potato tubers inoculated with either S. stelliscabiei or S. scabies exhibited superficial, raised, or pitted scabby symptoms, and no symptoms were observed on tubers grown in noninfested potting mix. Disease index values from the combined trials averaged 0, 37.8, 26.5, 11.1, and 30.5% for negative control and isolates HER21, HER24, HER26, and 49173, respectively. The pathogen was reisolated from the lesions and confirmed identical to the original isolate by DNA sequences. To our knowledge, this is the first report of S. stelliscabiei causing potato common scab in Michigan (4). References: (1) K. Bouchek-Mechiche et al. Int. J. Syst. Evol. Microbiol. 50:91, 2000. (2) Conn et al. Plant Dis. 82:631, 1998. (3) Hao et al. Plant Dis. 93:1329, 2009. (4) L. A. Wanner. Am. J. Potato Res. 86:247, 2009.
马铃薯（Solanum tuberosum L.）的普通疮痂病可由多种链霉菌引起，其中疮痂链霉菌是主要病原菌（2,3）。然而，根据我们2007年8月的调查，密歇根州许多有症状的马铃薯块茎中并未检测到疮痂链霉菌。为了鉴定病原菌，从密歇根州的两块田地中采集了有疮痂症状的马铃薯块茎，并使用链霉菌选择性培养基（STR）分离链霉菌（2）。通过转移单菌落并在28°C的STR培养基上培养，获得了分离菌株的纯培养物。根据形态和生理特征，将三个分离菌株HER21、HER24和HER26鉴定为星状链霉菌（1）。在28°C的液体酵母麦芽提取物中，于摇床培养箱中以150 rpm的转速制备细菌培养物。从培养物中提取基因组DNA，并用作链霉菌属特异性引物PCR的模板（4）。这些分离菌株与星状链霉菌的引物Stel3和T2st2发生阳性PCR反应，但对其他报道的对马铃薯致病的链霉菌均为阴性。使用先前报道的引物扩增16S rRNA基因（4），对扩增子进行测序并提交至GenBank（这三个分离菌株的登录号分别为HM018115、HM018116和HM018117）。将这些序列与NCBI GenBank数据库进行BLAST分析，确定这些序列与登录号为FJ546728的星状链霉菌序列具有99%至100%的序列同一性（4）。使用上述相同条件的PCR方法，确认这些分离菌株均具有txtAB、nec1和tomA基因（4），这些基因与引起疮痂病的链霉菌的致病性相关。为了完成柯赫氏法则，将分离菌株的细胞悬浮液以终浓度10个菌落形成单位/毫升混入蛭石培养基（3）中，用作接种物。将马铃薯（品种Snowden）块茎在25°C的生长室中于无菌盆栽混合物中培养，直至种子发芽。将每株马铃薯幼苗转移至温室中的新花盆中。两周后，用HER21、HER24或HER26的细菌孢子悬浮液侵染盆栽混合物，每个分离菌株处理五盆。仅含培养基的盆栽混合物或含疮痂链霉菌分离菌株49173的培养基分别用作阴性和阳性对照。三个月后，收获马铃薯块茎并评估疮痂症状（3）。该实验重复进行了两次。接种星状链霉菌或疮痂链霉菌的马铃薯块茎表现出表面凸起、凹陷或有麻点的疮痂症状，而在未侵染的盆栽混合物中生长的块茎未观察到症状。综合试验的病情指数值，阴性对照、分离菌株HER21、HER24、HER26和49173分别平均为0、37.8%、26.5%、11.1%和30.5%。从病斑中重新分离出病原菌，并通过DNA序列确认与原始分离菌株相同。据我们所知，这是星状链霉菌在密歇根州引起马铃薯普通疮痂病的首次报道（4）。参考文献：（1）K. Bouchek-Mechiche等人，《国际系统与进化微生物学杂志》50:91，2000年。（2）Conn等人，《植物病害》82:631，1998年。（3）Hao等人，《植物病害》93:1329，2009年。（4）L. A. Wanner，《美国马铃薯研究杂志》86:247，2009年。
