Zheng H H, Wu X H
Department of Plant Pathology, China Agricultural University, Beijing 100193, China.
Plant Dis. 2013 Sep;97(9):1246. doi: 10.1094/PDIS-08-12-0763-PDN.
Potato (Solanum tuberosum L.) is grown worldwide as a major food crop. Potato early blight is an important disease caused by Alternaria solani (4). In 2011, diseased potato leaves with blight symptoms were collected from 21 sites (incidence averaged 60% for about 2,000 ha of potato fields examined) in Gansu Province, northwest China. Small pieces of tissue taken from the margin between healthy and diseased tissues were surface-disinfected in 0.3% NaOCl for 2 min, rinsed with sterilized, distilled water, then placed on potato dextrose agar (PDA) at 25°C in the dark. Two of 24 Alternaria isolates from single-spore cultures were identified preliminarily as A. tenuissima, and the remaining isolates as A. solani or A. alternata, based on morphological traits. Colony appearance on potato carrot agar (PCA) was loosely cottony under a day/night cycle of 8 h fluorescent light/16 h dark at 22°C for 7 days (3). The isolates were characterized by formation of unbranched conidial chains up to 12 conidia in length, with one or two lateral branches forming occasionally. Conidia were typically ovoid to obclavate, and ranged from 20.4 to 42.4 × 7.7 to 13.2 μm. Transverse septa and longitudinal septa of conidia varied from 1 to 6 and 0 to 2, respectively. Short conidiophores arose singly and were 15.1 to 76.8 μm long by 2.4 to 6.2 μm wide. The internal transcribed spacer (ITS) region of rDNA and partial coding sequence of a histone gene were amplified from genomic DNA of the two A. tenuissima isolates using the ITS1/ITS4 and H3-1a/H3-1b primers (2), respectively. The ITS sequences of the two isolates (GenBank Accession Nos. JX495165 and JX495166) were 100% identical to those of A. tenuissima strains sdau 07-100 and BL08-3 (GQ871507 and AB470887), as well as to other Alternaria species, but the partial histone gene sequences (JX495167 and JX495168) were 99% identical to those of A. tenuissima isolates CR27, MA1, MA6, and CN-L-01 (AF404622, AF404633, AF404634, and EF371552, respectively) with less similarity to those of other Alternaria spp. Therefore, the isolates were identified as A. tenuissima based on morphological and molecular characteristics. Pathogenicity tests were conducted by inoculating detached leaves (30 per isolate) from 45-day-old plants of potato cv. Favorita with 20 μl drops (one drop per leaf) of a conidial suspension containing 10 conidia/ml in sterilized, distilled water. Thirty control leaves were inoculated similarly with sterilized, distilled water. Inoculated leaves were incubated in chambers at 25°C and 90% RH with a 12-h photoperiod/day. After 7 days, symptoms on the inoculated leaves were similar to those naturally occurring on the original plants, and the two cultures were reisolated consistently from those leaves, and the species identity was confirmed by morphological and molecular characteristics, fulfilling Koch's postulates. The control leaves remained asymptomatic and Alternaria was not isolated from those leaves. Alternaria blight of potato caused by A. tenuissima was previously detected in Iran (1). To our knowledge, this is the first report of A. tenuissima causing blight on potatoes in China. References: (1) S. T. Ardestani et al. Iran. J. Plant Pathol. 45:83, 2010. (2) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (3) E. G. Simmons. Alternaria. An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands, 2007. (4) J. E. van der Waals et al. Plant Dis. 88:959, 2004.
马铃薯（Solanum tuberosum L.）作为一种主要粮食作物在全球广泛种植。马铃薯早疫病是由链格孢菌（Alternaria solani）引起的一种重要病害（4）。2011年，在中国西北部甘肃省的21个地点采集了有疫病症状的患病马铃薯叶片（在所检测的约2000公顷马铃薯田中，发病率平均为60%）。从健康组织与患病组织之间的边缘取小块组织，在0.3%次氯酸钠中进行表面消毒2分钟，用无菌蒸馏水冲洗，然后置于马铃薯葡萄糖琼脂（PDA）上，于25°C黑暗条件下培养。根据形态学特征，从单孢子培养物中获得的24个链格孢分离株中有2个初步鉴定为细极链格孢（A. tenuissima），其余分离株鉴定为茄链格孢（A. solani）或链格孢（A. alternata）。在22°C、8小时荧光光照/16小时黑暗的昼夜循环条件下，于马铃薯胡萝卜琼脂（PCA）上培养7天，菌落外观呈疏松棉絮状（3）。这些分离株的特征是形成不分枝的分生孢子链，长度可达12个分生孢子，偶尔形成一两个侧枝。分生孢子通常为卵形至倒棍棒形，大小为20.4至42.4×7.7至13.2μm。分生孢子的横隔和纵隔分别为1至6个和0至2个。短分生孢子梗单生，长15.1至76.8μm，宽2.4至6.2μm。分别使用引物ITS1/ITS4和H3 - 1a/H3 - 1b从两个细极链格孢分离株的基因组DNA中扩增rDNA的内部转录间隔区（ITS）和一个组蛋白基因的部分编码序列（2）。两个分离株的ITS序列（GenBank登录号：JX495165和JX495166）与细极链格孢菌株sdau 07 - 100和BL08 - 3（GQ871,507和AB470,887）以及其他链格孢属物种的序列100%相同，但组蛋白基因部分序列（JX495167和JX495168）与细极链格孢分离株CR27、MA1、MA6和CN - L - 01（分别为AF404,622、AF404,633、AF404,634和EF371,552）的序列99%相同，与其他链格孢属物种的序列相似性较低。因此，根据形态学和分子特征，这些分离株被鉴定为细极链格孢。通过用含有10个分生孢子/毫升的分生孢子悬浮液（20微升滴液，每片叶子一滴）接种45日龄马铃薯品种费沃瑞塔（cv. Favorita）植株的离体叶片（每个分离株接种30片）进行致病性测试，无菌蒸馏水作对照，接种30片。接种后的叶片在25°C、90%相对湿度、12小时光周期/天的培养箱中培养。77天后，接种叶片上的症状与原始植株上自然发生的症状相似，并且从这些叶片上始终能重新分离到这两种培养物，通过形态学和分子特征确认了物种身份，满足柯赫氏法则。对照叶片无症状，未从这些叶片上分离到链格孢。细极链格孢引起的马铃薯早疫病此前在伊朗有过报道（1）。据我们所知，这是中国首次报道细极链格孢引起马铃薯早疫病。参考文献：（1）S. T. Ardestani等人，《伊朗植物病理学杂志》45:83，2010年。（2）N. L. Glass和G. C. Donaldson，《应用与环境微生物学》61:1323，1995年。（3）E. G. Simmons，《链格孢属。鉴定手册》，荷兰乌得勒支CBS真菌生物多样性中心，2007年。（4）J. E. van der Waals等人，《植物病害》88:959，2004年。
