Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, Gent B-9000, Belgium.
BMC Microbiol. 2013 Jun 5;13:126. doi: 10.1186/1471-2180-13-126.
Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato. Cmm is present nearly in all European countries. During the last three years several local outbreaks were detected in Belgium. The lack of a convenient high-resolution strain-typing method has hampered the study of the routes of transmission of Cmm and epidemiology in tomato cultivation. In this study the genetic relatedness among a worldwide collection of Cmm strains and their relatives was approached by gyrB and dnaA gene sequencing. Further, we developed and applied a multilocus variable number of tandem repeats analysis (MLVA) scheme to discriminate among Cmm strains.
A phylogenetic analysis of gyrB and dnaA gene sequences of 56 Cmm strains demonstrated that Belgian Cmm strains from recent outbreaks of 2010-2012 form a genetically uniform group within the Cmm clade, and Cmm is phylogenetically distinct from other Clavibacter subspecies and from non-pathogenic Clavibacter-like strains. MLVA conducted with eight minisatellite loci detected 25 haplotypes within Cmm. All strains from Belgian outbreaks, isolated between 2010 and 2012, together with two French strains from 2010 seem to form one monomorphic group. Regardless of the isolation year, location or tomato cultivar, Belgian strains from recent outbreaks belonged to the same haplotype. On the contrary, strains from diverse geographical locations or isolated over longer periods of time formed mostly singletons.
We hypothesise that the introduction might have originated from one lot of seeds or contaminated tomato seedlings that was the source of the outbreak in 2010 and that these Cmm strains persisted and induced infection in 2011 and 2012. Our results demonstrate that MLVA is a promising typing technique for a local surveillance and outbreaks investigation in epidemiological studies of Cmm.
密歇根棒杆菌亚种。密歇根州（Cmm）引起番茄细菌性萎蔫和溃疡。Cmm 几乎存在于所有欧洲国家。在过去的三年中，比利时检测到几起局部暴发。缺乏一种方便的高分辨率菌株分型方法，阻碍了 Cmm 的传播途径和番茄种植中的流行病学研究。在这项研究中，通过 gyB 和 dnaA 基因测序研究了全球范围内 Cmm 菌株及其亲缘菌株的遗传相关性。此外，我们开发并应用了一种多位点可变串联重复分析（MLVA）方案来区分 Cmm 菌株。
对 56 株 Cmm 菌株的 gyB 和 dnaA 基因序列进行的系统发育分析表明，2010-2012 年最近爆发的比利时 Cmm 菌株在 Cmm 进化枝内形成了一个遗传上均匀的群体，Cmm 在系统发育上与其他棒杆菌亚种和非致病性棒杆菌样菌株明显不同。用 8 个微卫星位点进行的 MLVA 检测到 Cmm 内 25 个单倍型。2010 年至 2012 年间分离的所有比利时暴发菌株，以及 2010 年来自法国的两个菌株，似乎形成了一个单态群。无论分离年份、地点或番茄品种如何，来自最近暴发的比利时菌株都属于同一单倍型。相反，来自不同地理位置或分离时间较长的菌株大多为单倍型。
我们假设这种引入可能起源于一批种子或受污染的番茄幼苗，这些幼苗是 2010 年暴发的源头，这些 Cmm 菌株在 2011 年和 2012 年持续存在并引发感染。我们的研究结果表明，MLVA 是一种有前途的分型技术，可用于 Cmm 的局部监测和暴发调查，以及流行病学研究。
