黑腿病和软腐病是马铃薯(Solanumtuberosum)中的有害疾病。还有Dickeyaspp.(Czajkowski等人。2015).福建省霞浦县周边马铃薯产区马铃薯黑腿的发生较为严重,中国,2021年(6公顷)和2022年(7公顷),发病率约为5%,达到近23%。收集三株患病植物以分离病原体。分离来自每个取样植物的单个菌落并在新鲜板上划线。用16SrRNA基因的引物对27F/1492R(泳道1991)对来自不同植物的三个菌落的DNA进行PCR扩增。因为序列是相同的,我们选择菌株M2-3进行进一步分析。菌株M2-3为革兰氏阴性,CVP上的果胶,在37°C和5%NaCl下生长。该细菌的磷酸酶活性呈阳性,红霉素敏感性,吲哚生产,明胶液化,马莱语利用,和酸的生产,Melibose,棉子糖,和阿拉伯糖。细菌对蔗糖呈阴性,α-甲基葡糖苷,山梨醇,海藻糖,乳糖,和柠檬酸钠(Fujimoto等人。2018;),虽然蔗糖和乳糖没有提供预期的结果,所有物种都有例外。对菌株M2-3的基因组进行测序并以登录号CP077422保藏在NCBI数据库中。平均核苷酸同一性(ANI)分析显示,M2-3与其他达达尼D.dadantii菌株成簇,并且与达达尼D.dadantii菌株DSM18020(CP023467)具有98.39%的同一性。管家基因(recA,dnaX,acnA,gapA,icd,mdh,mtlD和pgi)用先前设计的引物对进行扩增(Fujimoto等人。2018年;马等人。2007)和测序。通过连接8个基因序列并使用PhyloSuite版本1.2.1构建最大似然系统发育树,进行了多位点序列分析(MLSA)。2020年)和智商树版本1.6.8(Nguyen等人。2015)软件。菌株M2-3与dadantiiDickeya聚集在一起。对于致病性测试,每种处理三株植物,总共九株,被使用。在10mMPBS缓冲液中制备细菌悬浮液(1×10^8CFU/mL)。10μL的M2-3,Dadantii型菌株18020(阳性对照),将缓冲液(阴性对照)注入靠近基部的植物茎中。2天后在接种部位出现水渍,并且它们逐渐变黑和腐烂。叶子变成黄色和枯萎,接种后5天内叶柄基部腐烂,完成科赫假设。根据平均核苷酸同一性和管家基因序列分析,菌株M2-3被鉴定为dadantii。以前的研究报道了几种在中国引起马铃薯黑腿的病原体,包括嗜酸性疟原虫,P.胡萝卜素,P.巴西,P.parmentieri,P.北极星,和P.punjabense(李平等人2020年;王等人。2021)。据我们所知,本研究是福建省首次报道由dadantii引起的马铃薯黑腿病,中国。这一发现表明,该病原体可能对福建省马铃薯生产构成威胁。
Blackleg and soft rot are harmful diseases in potato (Solanum tuberosum) caused by Pectobacterium spp. and Dickeya spp. (Czajkowski et al. 2015). The occurrence of potato blackleg was serious in potato-producing areas around Xiapu County in Fujian Province, China, in 2021 (6 ha) and 2022 (7 ha), with an incidence of approximately 5%, which reached nearly 23%. Three diseased plants were collected to isolate the pathogen. Single colonies from each sampled plant were isolated and streaked onto fresh plates. DNA from three colonies from different plants was PCR amplified with primer pair 27F/1492R (Lane 1991) for the 16S rRNA gene. Since the sequences were identical, we selected strain M2-3 for further analysis. The strain M2-3 was gram-negative, pectolytic on CVP, grew at 37°C and 5% NaCl. The bacterium was positive for phosphatase activity, erythromycin sensitivity, indole production, gelatin liquefaction, malonic utilization, and acid production from, melibiose, raffinose, and arabinose. The bacterium was negative for sucrose, α-methyl glucoside, sorbitol, trehalose, lactose, and sodium citrate (Fujimoto et al. 2018;),although sucrose and lactose did not provide the expected results, there are exception in all species. The genome of strain M2-3 was sequenced and deposited in the NCBI database under accession numbers: CP077422. An Average Nucleotide Identity (ANI) analysis showed that M2-3 clustered with other D. dadantii strains and has a 98.39% identity with D. dadantii strain DSM 18020 (CP023467). The housekeeping genes (recA, dnaX, acnA, gapA, icd, mdh, mtlD and pgi) were amplified with primer pairs designed previously(Fujimoto et al. 2018; Ma et al. 2007) and sequenced. A multilocus sequence analysis (MLSA) was performed by concatenating the 8 gene sequences and constructing a maximum likelihood phylogenetic tree using PhyloSuite version 1.2.1 (Zhang et al. 2020) and IQ-tree version 1.6.8 (Nguyen et al. 2015) software. Strain M2-3 was clustered together with Dickeya dadantii. For the pathogenicity test, three plants per treatment, totaling nine plants, were used. Bacterial suspensions (1×10^8 CFU/mL) were made in a 10mM PBS buffer. 10 μL of M2-3, D. dadantii type strain 18020 (positive control), and buffer (negative control) were injected into the plant stems near the base. Water stains appeared at the site of inoculation after 2 days and they gradually became black and rotten. The leaves became yellow and wilted, and the petiole base rotted within 5 days of inoculation completing the Koch postulate. According to average nucleotide identity and housekeeping gene sequence analysis, strain M2-3 was identified as Dickeya dadantii. Previous studies have reported several pathogens that cause potato blackleg in China, including P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense (Li-ping et al. 2020; Wang et al. 2021). To the best of our knowledge, this study is the first to report potato blackleg disease caused by Dickeya dadantii in Fujian Province, China. This finding suggests that this pathogen may cause a threat to potato production in Fujian Province.