Novel biovar (biovar 6) of Pseudomonas syringae pv. actinidiae causing bacterial canker of kiwifruit (Actinidia deliciosa) in Japan
Novel biovar (biovar 6) of Pseudomonas syringae pv. actinidiae causing bacterial canker of kiwifruit (Actinidia deliciosa) in Japan
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DOI：
10.3186/jjphytopath.82.101
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发表时间：
2016-05-01
期刊：
影响因子：
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通讯作者：
Nakaune, R.
中科院分区：
文献类型：
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作者：
Sawada, H.;Kondo, K.;Nakaune, R.
2015 年 4 月，在日本长野县的美味猕猴桃“海沃德”的树干、树枝和小枝上观察到细菌性溃疡病的典型症状（例如，有红色渗出物的溃疡病、修剪切口渗出白色渗出物、树皮下褐变和变色）。从患病树枝中分离出 13 株致病细菌，并通过接种和重新分离对 A. deliciosa 具有致病性。这些菌株是革兰氏阴性、需氧的杆状体，具有一到两个极性鞭毛，并形成不透明的淡黄色圆形菌落。根据生化和生理特征，针对 ITS、hopZ3 和 hoPO1-2 进行 PCR 检测，以及使用七个管家基因（acnB、cts、gapA、gyrB、pfk、pgi 和 rpoD）的串联序列进行多位点序列分析 (MLSA)，我们将病原体鉴定为丁香假单胞菌 pv。猕猴桃 (Psa)。然而，基于使用 API 20NE 的表型表征和额外的 PCR 检测，该病原体被证明与 Psa 的四种现有生物变型（生物变型 1、2、3 和 5）不同，这表明该病原体不具有 hopH1、hopH3 或 hopZ5 基因。此外，MLSA 的结果显示它们与其他 Psa 生物变型分开聚集。此外，生物测定证实它们同时产生菜豆毒素和冠菌素，尽管尚未发现植物病原细菌产生这两种毒素。因此，基因型和表型分析的综合结果支持将这些菌株分类为 Psa 的新生物变种，建议将其命名为“生物变种 6”。除生物变种 6 外，Psa 的其他三个生物变种（生物变种 1、3 和 5）在日本也被确认为猕猴桃细菌性溃疡病病原体。尽管目前biovar 6的分布极其有限，但它可能在帮助我们了解Psa的起源和进化方面发挥重要作用。我们的结果还表明，尽管菜豆毒素和冠菌素生物合成基因已被用作分别鉴定生物变型 1 和 2 的 PCR 靶标，但可在细菌间传播的毒素相关基因不足以区分 Psa 生物变型。
Typical symptoms of bacterial canker disease (e.g., cankers with red exudates, white ooze exuding from pruning cuts, browning and discoloration beneath bark) were observed on trunks, branches and twigs of Actinidia deliciosa 'Hayward' in Nagano Prefecture, Japan, in April 2015. Thirteen strains of the causal bacterium were isolated from the diseased twigs and demonstrated by inoculation and reisolation to be pathogenic on A. deliciosa. The strains were gram-negative, aerobic rods with one to two polar flagella, and formed opaque, pale yellowish circular colonies. On the basis of biochemical and physiological characterization, PCR assays targeting ITS, hopZ3 and hoPO1-2, and a multilocus sequence analysis (MLSA) using concatenated sequences of seven housekeeping genes (acnB, cts, gapA, gyrB, pfk, pgi and rpoD), we identified the pathogen as Pseudomonas syringae pv. actinidiae (Psa). However, the pathogen proved to be different from the four existing biovars (biovars 1, 2, 3 and 5) of Psa based on phenotypic characterization using API 20NE, and additional PCR assays, which showed that the pathogen did not possess hopH1, hopH3 or hopZ5 genes. Also, the results of the MLSA revealed that they clustered separately from the other Psa biovars. In addition, bioassays confirmed that they produced both phaseolotoxin and coronatine, although no plant pathogenic bacteria have ever been identified as producing both toxins. The combined results of genotypic and phenotypic analyses therefore support the classification of these strains as a novel biovar of Psa, for which the name "biovar 6" is proposed. In addition to biovar 6, three other biovars (biovars 1, 3 and 5) of Psa were confirmed as bacterial canker pathogens of kiwifruit in Japan. Although the distribution of biovar 6 is extremely limited at present, it may play an important role in helping us to understand the origin and evolution of Psa. Our results also show that toxin-related genes, which could be transmissible among bacteria, are not adequate markers to discriminate Psa biovars, although phaseolotoxin and coronatine biosynthetic genes have been utilized as PCR targets to identify biovars 1 and 2, respectively.