Chapara Venkataramana, Al Salman Dua'a, Azizi Abdolbaset, Del Rio Mendoza Luis E
NDSU, Langdon Research Extension Center, 9280, 107Th Avenue NE, Langdon, North Dakota, United States, 58249;
North Dakota State University, Plant Pathology, Fargo, North Dakota, United States;
Plant Dis. 2023 Aug 23. doi: 10.1094/PDIS-06-23-1089-PDN.
North Dakota leads the U.S.A. in canola ( L.) production (5) and approximately one-third of the acreage is located in the northeastern portion of the state. A field survey conducted at the end of the 2021 growing season in northeastern North Dakota revealed the presence of a single field with approximately 15% plants exhibiting whitish discoloration symptoms in the lower third of the stems. The epidermis on the discolored areas was peeling, and the exposed tissues were dark grey to dark-brown. Stem samples taken to the laboratory were surface disinfested with a 5% aqueous solution of NaOCl for 60 sec and rinsed thrice with sterile water. Under aseptic conditions, small stem pieces were plated on V-8 medium amended with 150 mg each of penicillin and streptomycin per liter of medium and incubated at 22 C under 16 h light daily. Under the microscope, conidiophores were verticillate, hyaline, and had three branches. Conidia were single-celled, hyaline, and measured on average 9.2 + 1.8 µm. Microsclerotia were irregularly shaped. These features match the description of (Stark) Karapapa Bainbr. & Heale, (4). Genomic DNA was extracted from a single-spore culture of an isolate as described by Azizi et al. (1). PCR assays were conducted twice on two independent DNA samples extracted from the same isolate using species-specific primer set VlspF1 and VlspR4 (2) with denaturation at 95 C for 3 min, followed by 35 cycles of amplification at 94 C for 1 min then 56 C for 30 sec and 72 C for 1 min, followed by a final period at 72 C for 5 min. The sequenced PCR product, which had 100% homology with GenBank reference samples KY704097 and HE972063, was assigned GenBank accession number OR088215. Pathogenicity tests were conducted in greenhouse. Briefly, twenty seeds of the canola cv. Westar were incubated on a sterilized wet paper towel for five days at 22 C. The seedlings were carefully lifted, and their root tips cut with scissors. Ten wounded seedlings were immersed in a spore suspension with 2.07 x 10 spores per ml for 30 minutes and the other ten in distilled water (controls). The plants were transplanted into pots (10x10x13 cm) containing Sunshine Mix # 1 potting mix (Fison Horticulture, Vancouver, B. C.). The study was conducted twice, with individual plants as replications. Three weeks later, 20% of inoculated plants had died and at physiological maturity, the rest of them had stunted growth and blackened internal stem tissues while external stem symptoms resembled those found in the field. All control plants reached maturity without symptoms. The pathogen re-isolated from inoculated plants were morphologically identical to the one retrieved from the field. These results confirmed the isolate as . This is the first report of Verticillium stripe on canola in the US. In North America, the disease was first reported in Manitoba, Canada, in 2014 but subsequent surveys showed it is widespread in Canada (3). Identification of genetic resistance against this disease is required to ameliorate the threat this disease represents to US canola production.
北达科他州的油菜籽(L.)产量在美国居首(5),该州约三分之一的种植面积位于其东北部。2021年生长季末在北达科他州东北部进行的一项田间调查发现,有一块田地里约15%的植株茎干下部三分之一处出现发白变色症状。变色区域的表皮正在脱落,暴露的组织呈深灰色至深褐色。采集到实验室的茎干样本用5%的次氯酸钠水溶液进行表面消毒60秒,并用无菌水冲洗三次。在无菌条件下,将小的茎段接种到每升培养基添加150毫克青霉素和链霉素的V - 8培养基上,于22℃、每天光照16小时的条件下培养。在显微镜下,分生孢子梗轮生、透明且有三个分支。分生孢子单细胞、透明,平均大小为9.2 + 1.8微米。微菌核形状不规则。这些特征与(斯塔克)卡拉帕帕·贝恩布尔和希利(4)的描述相符。按照阿齐兹等人(1)所述方法从一个分离株的单孢子培养物中提取基因组DNA。使用物种特异性引物对VlspF1和VlspR4(2)对从同一分离株提取的两个独立DNA样本进行两次PCR检测:95℃变性3分钟后,进行35个循环的扩增(94℃1分钟、56℃30秒、72℃1分钟),最后72℃延伸5分钟。测序得到的PCR产物与GenBank参考样本KY704097和HE972063具有100%的同源性,被赋予GenBank登录号OR088215。在温室中进行致病性测试。简要过程如下:将20粒油菜品种韦斯塔(Westar)种子在灭菌的湿纸巾上于22℃培养5天。小心提起幼苗并用剪刀剪去根尖。10株受伤幼苗浸入每毫升含2.07×10个孢子的孢子悬浮液中30分钟,另外10株浸入蒸馏水中(作为对照)。将这些植株移栽到装有阳光混合一号盆栽基质(菲森园艺公司生产,温哥华,不列颠哥伦比亚省)的花盆(10×10×13厘米)中。该研究进行了两次,以单株植物作为重复样本。三周后,20%的接种植株死亡,在生理成熟期,其余植株生长发育受阻,茎干内部组织变黑,而茎干外部症状与田间发现的症状相似。所有对照植株正常成熟且无病症表现。从接种植株上重新分离得到的病原菌在形态上与从田间分离得到的病原菌相同。这些结果证实该分离株为(某种病原菌)。这是美国油菜上出现黄萎病条斑的首次报道。在北美,该病于2014年在加拿大马尼托巴省首次报道,但随后的调查表明它在加拿大广泛分布(3)。需要鉴定对这种病害具有遗传抗性的品种以减轻该病对美国油菜生产造成的威胁。