2022年7月,在重庆市祁江区的花生感染茎上检测到枯萎和变色(106.56°E,29.41°N),中国,发病率高达5%。这些花生茎有典型的炭疽病症状,有不规则的灰棕色斑点和深棕色边缘,沉没,和坏死。高温高湿有利于病原菌的生长。为了分离病原体,我们收集了10个典型的受感染花生,并从每个有症状的茎上切下一块,表面用0.5%NaClO灭菌1分钟,和75%的乙醇持续30秒,然后用无菌蒸馏水冲洗三次,并在灭菌的滤纸上干燥。将这些碎片在25°C下在黑暗中在马铃薯葡萄糖琼脂(PDA)上孵育。从每个菌落的菌丝尖端获得纯培养物。发现从每个感染的茎中分离出具有相同菌落形态的分离株。代表性分离株(L7)用于形态学表征,分子分析,系统发育分析,和致病性测试。殖民地看起来是白色到灰色,有白色边缘和空中菌丝,殖民地的反面是灰色到棕色。分生孢子是圆柱形的,无菌,两端呈钝角到略带圆形,13.4至18.8×4.2至5.8μm(n=50)。形态特征通常与炭疽菌物种复合物的形态特征一致(Cannon等人。,2012).对于分子鉴定,使用CTAB方法和β-微管蛋白(TUB2)的部分序列提取基因组DNA,肌动蛋白(ACT)基因,几丁质合成酶(CHS)和甘油醛-3-磷酸脱氢酶(GAPDH)基因扩增和测序使用引物T1/T2,ACT-512F/ACT-783R,CHS-79F/CHS-345R,和GDF1/GDR1分别(Damm等人。,2012;Dowling等人。,2020)。使用BLAST,TUB2,ACT,CHS和GAPDH基因序列(GenBank登录号OR714793、OP168707、OP168708和OR714794分别为100%(429bp中的429bp),99.22%(258个基点中的256个基点),99.64%(276bp中的277bp)和100%(253bp中的253bp)与C.gloeosporioidesCBS:112999(JQ005587,JQ005500,JQ005326和JQ005239)相同,分别。使用邻居连接算法,系统发育分析是基于公开的TUB2、ACT、CHS和GAPDH基因。鉴定的分离株(L7)与C.gloeosporioides密切相关。为了评估致病性,用无菌牙签将十个花生(Zhonghua12)幼苗(2周)的茎炸伤,并接种菌丝体塞(直径5mm)或20μl分生孢子悬浮液(105/ml)。将未定殖的琼脂塞或20μl无菌蒸馏水作为对照。接种后,将花生在黑暗中保持在28°C,湿度为80%的潮湿室内24小时,随后转移到潮湿的房间,12小时的明暗周期,持续6天,在所有接种的花生上观察到类似的症状。对照保持无症状。C.gloeosporioides从患病的茎中重新分离,并使用TUB2,ACT的形态学特征和序列分析进行确认,CHS和GAPDH。据报道,在中国的花生叶上有由C.truncum和C.fructicola引起的炭疽病(Gong等人。,2023年;Yu等人。,2019)。据我们所知,这是重庆市首次报道的由C.gloeosporioides引起的花生茎上的炭疽病。我们的报告将为该疾病的流行病学和管理研究提供重要信息。
In July 2022, dieback and discoloration were detected on infected stems of peanut in Qijiang District of Chongqing (106.56°E,29.41°N), China, with an incidence up to 5%. These peanut stems had disease symptoms typical of anthracnose with irregular gray-brown spots with dark brown edges, sunken, and necrotic. High temperature and high humidity were favorable for the growth of the pathogen. To isolate the pathogen, we collected 10 typical infected peanuts and cut one piece from each of symptomatic stems, surface sterilized with 0.5% NaClO for 1 min, and 75% ethanol for 30 s, then rinsed three times with sterile distilled water and dried on sterilized filter paper. These pieces were incubated on potato dextrose agar (PDA) at 25°C in the dark. Pure cultures were obtained from hyphal tips of each colony. It was found that isolates with the same colony morphology were isolated from each infected stem. A representative isolate (L7) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. The colonies appeared white to gray, with white margins and aerial hyphae, and the reverse of the colonies was gray to brown. Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, 13.4 to 18.8 × 4.2 to 5.8 μm (n=50). Morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides species complex (Cannon et al., 2012). For molecular identification, genomic DNA was extracted using a CTAB method and partial sequences of β-tubulin (TUB2), actin (ACT) genes, chitin synthase (CHS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced using primers T1/T2, ACT-512F/ACT-783R, CHS-79F/CHS-345R, and GDF1/GDR1, respectively (Damm et al., 2012; Dowling et al., 2020). Using the BLAST, TUB2, ACT, CHS and GAPDH gene sequences (GenBank accession No. OR714793, OP168707, OP168708 and OR714794, respectively) were100% (429 bp out of 429 bp), 99.22% (256 bp out of 258 bp), 99.64% (276 bp out of 277 bp) and 100% (253 bp out of 253 bp) identical to C. gloeosporioides CBS:112999 (JQ005587, JQ005500, JQ005326, and JQ005239), respectively. Using Neighbor-Joining algorithm, phylogenetic analysis was conducted based on the concatenated sequences of published TUB2, ACT, CHS and GAPDH genes. The identified isolate (L7) was closely related to C. gloeosporioides. To evaluate pathogenicity, the stems of ten peanut (Zhonghua12) seedlings (2 weeks) were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) or 20 μl of conidial suspension (105/ml) were inoculated. Non-colonized agar plugs or 20 μl of sterile distilled water were treated as control. After inoculation, the peanuts were kept in a moist chamber at 28°C with 80% humidity in the dark for 24 h, and subsequently transferred to the moist chamber with 12 h light and darkness cycle for 6 days, similar symptoms were observed on all inoculated peanuts. Controls remained asymptomatic. C. gloeosporioides was reisolated from the diseased stems and confirmed using morphological features and sequence analysis of TUB2, ACT, CHS and GAPDH. Anthracnose caused by C. truncatum and C. fructicola has been reported on peanut leaves in China (Gong et al., 2023; Yu et al., 2019). To our knowledge, this is the first report of anthracnose on peanut stem caused by C. gloeosporioides in Chongqing. Our report will provide crucial information for studying on epidemiology and management of this disease.