First Report of Bacterial Soft Rot Disease on Pak Choi (Brassica rapa subsp. chinensis) caused by Pectobacterium brasiliense in the United States.
First Report of Bacterial Soft Rot Disease on Pak Choi (Brassica rapa subsp. chinensis) caused by Pectobacterium brasiliense in the United States.
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DOI：
10.1094/pdis-08-20-1854-pdn
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发表时间：
2021-03-17
期刊：
影响因子：
4.5
通讯作者：
Arif M
中科院分区：
文献类型：
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作者：
Klair D;Boluk G;Silva J;Arizala ED;Dobhal S;Arif M
白菜（Brassica rapa subsp. chinensis）是原产于中国的重要蔬菜作物，以高含水量和低热值而闻名，含有高质量的蛋白质、碳水化合物、纤维、维生素、矿物质和次生植物代谢产物（Acikgoz 2016）。一个白菜田（8,000平方米）（英尺）于二零二零年五月到访夏威夷奥伯斯岛。约10%的植物被感染，并表现出软腐病、湿病、浸渍感染茎和坏死叶的特征症状，导致怀疑是果胶杆菌属中最具破坏性的细菌病原体之一（Arizala和Arif 2019; Arizala等人2020; Boluk等人2020; Li等人2019）。从田间收集四株感染的植物，用0.6%次氯酸钠溶液对茎进行表面灭菌30秒，然后在蒸馏水中连续冲洗三次。无菌浸渍茎，在结晶紫果胶酸盐培养基（CVP）上划线（Hélias等人，2011），并在26±2 ℃下孵育48 h。观察到果胶溶解细菌病原体的特殊形态特征，在CVP上形成凹坑（Meng等人，2017）。通过在葡萄糖蛋白胨琼脂（DPA，不含氯化四唑; Norman和Alvarez 1989）上重新破碎单个菌落来进行细菌菌落的纯化。使用DNeasy血液和组织试剂盒（Qiagen，日耳曼敦，MA）从细菌培养物中分离DNA。通过使用Pec对管家基因dnaA（染色体复制起始蛋白）的测序区域进行4个菌株（PL 243至PL 246）的分子鉴定。dnaA-F1/R1引物组（Dobhal等人，2020）。使用ExoSAP-IT（Affyestry，Santa Clara，CA）酶促清洁扩增的PCR产物，并在Genewiz机构（拉霍亚，CA）使用正向和反向引物进行测序。使用Geneious比对dnaA基因序列，并手动编辑以去除错误。用NCBI BLASTn工具分析共有序列，并以登录号MT 899920至MT 899923保藏在NCBI GenBank中。NCBI BLASTn报告表明，所有序列具有99%至100%的同一性，查询覆盖巴西果胶杆菌登录号MN 544627至MN 544629。使用Geneious，用代表不同果胶杆菌属的dnaA序列进行系统发育分析，并且所有菌株都分组在巴西假单胞菌的进化枝内（Arizala等人，2020）。对在含有商业盆栽混合物的盆栽中生长并保持在受控温室（温度26 - 30 ℃;相对湿度50 - 58%）中的小白菜进行致病性测定，重复三次。用100 μl PL 243（1.3× 108 CFU/ml）、PL 244（1.2× 108 CFU/ml）、PL 245（1.2 × 108 CFU/ml）和PL 246（1.1× 108 CFU/ml）的细菌悬浮液人工接种三周龄植物茎;对照植物用100 μl蒸馏水接种。接种后两天，所有四种供试菌株均出现与田间观察到的症状相似的软腐病和萎蔫症状。从接种的植物中成功地重新分离细菌;分离DNA，扩增，测序dnaA区域，并分析与原始菌株的100%同源性，以满足Koch假设。根据分子特征，再分离物与原始菌株相同。据我们所知，这是巴西拟青霉在美国小白菜上的首次报道。最近的报告表明，该病原体可能对十字花科作物构成潜在威胁，因此强调需要在全州范围内对果胶分解菌进行调查，并实施更好的预防措施。
Pak choi (Brassica rapa subsp. chinensis) is an important vegetable crop native to China, known for high water content and low caloric value, containing high quality of protein, carbohydrates, fiber, vitamins, minerals, and secondary plant metabolites (Acikgoz 2016). A pak choi field (8,000 sq. ft.) on Oahu, Hawaii, was visited in May 2020. About 10% plants were infected and showed characteristic symptoms of soft rot, wet lesions, macerated infected stem, and necrotic leaves, leading to the suspecting of one of the most devastating bacterial pathogens within genus Pectobacterium (Arizala and Arif 2019; Arizala et al. 2020; Boluk et al. 2020; Li et al. 2019). Four infected plants were collected from the field, and stems were surface sterilized with 0.6% sodium hypochlorite solution for 30 s, followed by three consecutive rinses in distilled water. The stems were aseptically macerated, streaked on crystal violet pectate medium (CVP)(Hélias et al. 2011), and incubated for 48 h at 26±2 C. The peculiar morphological characteristic of pectolytic bacterial pathogen, forming pits on CVP, was observed (Meng et al. 2017). Purification of bacterial colonies were done by restreaking of a single colony on dextrose peptone agar (DPA, without tetrazolium chloride; Norman and Alvarez 1989). DNA was isolated from bacterial cultures using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA). Molecular identification of four strains (PL243 to PL246) was performed by the sequencing region of the housekeeping gene dnaA (chromosomal replication initiation protein) using Pec. dnaA-F1/R1 primer set (Dobhal et al. 2020). The amplified PCR product was enzymatically cleaned using ExoSAP-IT (Affymetrix, Santa Clara, CA) and sent for sequencing at the Genewiz facility (La Jolla, CA) using both forward and reverse primers. The dnaA gene sequences were aligned using Geneious and manually edited to remove the errors. The consensus sequences were analyzed with the NCBI BLASTn tool and were deposited in NCBI GenBank under the accession numbers MT899920 to MT899923. The NCBI BLASTn report indicated that all the sequences shared 99 to 100% identity and query cover with Pectobacterium brasiliense accession numbers MN544627 to MN544629. A phylogenetic analysis, using Geneious, was performed with the dnaA sequences representing different Pectobacterium spp., and all strains grouped within the clade of P. brasiliense (Arizala et al. 2020). A pathogenicity assay was carried out in three replications on pak choi grown in pots containing commercial pot mixture and maintained in the controlled greenhouse (temperature 26 to 30 C; relative humidity 50 to 58%). Threeweek-old plant stems were artificially inoculated with 100 μl of bacterial suspension of PL243 (1.3× 108 CFU/ml), PL244 (1.2× 108 CFU/ml), PL 245 (1.2× 108 CFU/ml), and PL246 (1.1× 108CFU/ml); control plants were inoculated with 100 μl of distilled water. Two days after inoculation, soft rot and wilting symptoms similar to the ones observed in the field developed for all four strains tested. Bacteria were successfully reisolated from the inoculated plants; DNA was isolated, amplified, sequenced for the dnaA region, and analyzed for 100% homology with original strains, to fulfill Koch, s postulates. Based on the molecular characteristics reisolates were identical to the original strains. To the best of our knowledge, this is the first report of P. brasiliense on pak choi in the United States. Recent reports indicated that the pathogen could potentially pose a threat to cruciferous crops, therefore highlighting a need to conduct a state-wide survey for pectinolytic bacteria and to implement better …