魔芋魔芋,通常被称为巫毒百合,是中国西南地区广泛种植的经济作物(Gao等人。2022年)。2022年8月,在富源(25.67°N;104.25°E)的一个田地(1公顷)中观察到叶斑病症状,云南,中国,造成了巨大的经济损失。棕色病变,发病率从20%到40%不等,通常有白色或灰色的中心,并被黄色的光环包围。斑点的微观观察显示变形物种Cercosporachevalieri。分生孢子为50-150×4-7μm,圆柱形,无分支,光滑的壁,浅棕色和聚集在密集的束簇中,由棕色的基质产生。分生孢子细胞被整合,终端或插层,浅棕色到棕色和共生增殖。分生孢子位点变厚变暗,直径2-3μm。分生孢子是单独形成的,球形圆柱形,90-160×5-7μm,平均值为130×6μm(n=30),6-11隔片,薄壁,光滑,透明或亚透明,直的或弯曲的,有钝的先端和斜截形的基部,有增厚和变暗的肺门。这些形态特征与C.chevalieri的形态特征相匹配,白芍叶斑病的致病因子(Braun等人。2014年;Saccardo等人。1913).使用来自病变的无菌水中的分生孢子悬浮液接种水琼脂,将发芽的分生孢子转移到马铃薯葡萄糖琼脂(PDA)中,并在27°C下孵育7天。使用PDA诱导孢子形成不成功,以及麦芽提取物琼脂,马铃薯蔗糖琼脂和合成营养贫乏的琼脂。选择十个分离株中的两个进行分子鉴定和致病性测定。提取来自两个纯分离物(KUNCC22-12536和KUNCC22-12537)的基因组DNA进行PCR,并用内部转录间隔区的引物(ITS:ITS1/ITS4)进行扩增,钙调素(CMD:CAL228F/CAL2Rd),平移伸长因子1-α(TEF1-α:728F/986R),肌动蛋白(ACT:512F/783R),组蛋白H3(HIS3:CYLH3F/CYLH3R),β-微管蛋白基因(TUB2:BT-1F/BT-1R)和甘油醛-3-磷酸脱氢酶(GAPDH:Gpd1/Gpd2),分别(Vaghefi等人。2021)。新生成的ITS序列(OP719153/OP719154),CMD(OP740904/OP740905),TEF1-α(OP740910/OP740911),ACT(OP740902/OP740903),HIS3(OP740908/OP740909),TUB2(OP740912/OP740913),C.chevalieri的GAPDH(OP740906/OP740907)已提交给GenBank。到目前为止,GenBank数据库中没有C.chevalieri的序列数据。不出所料,大多数基因(TEF1-α,ACT,CMD,HIS,TUB2和GAPDH)与Cercospora属物种中的最佳命中表现出91%至95%的同一性。系统发育树显示,从魔芋叶斑中获得的两个分离株中检索到的序列在Cercospora中聚集在一起,形成了强烈支持的进化枝。为了测试科赫的假设,将在盆中生长的十株4个月大的健康魔芋植物用于温室中的致病性测试。每株植物的一叶接种菌丝体栓,用无菌PDA塞接种一片叶子。将这些植物装入塑料袋中72小时。只有接种菌丝体塞的叶子会产生棕色病变,在接种的叶子上10到14天后出现。用无菌PDA塞子处理的对照植物保持无症状。重复该实验两次,得到相同的结果。从感染的叶片中重新分离出山核桃,并根据ITS区域的形态学和Sanger测序进行鉴定。据我们所知,这是C.chevalieri在魔芋上引起叶斑病的第一份报告,也是中国对该物种的第一份报告(Braun等人。2014),它为这种疾病的诊断和管理提供了关键信息。
Amorphophallus konjac, commonly called voodoo lily, is a cash crop widely cultivated in southwest China (Gao et al. 2022). In August 2022, leaf spot symptoms were observed in a field (1 ha) located at Fuyuan (25.67°N; 104.25°E), Yunnan, China, resulting in substantial economic losses. Brown lesions, with an incidence ranging from 20 to 40%, typically had a whitish or gray center and were surrounded by yellow halos. Microscopic observations of the spots revealed anamorphic species Cercospora chevalieri. Conidiophores were 50-150 × 4-7 μm, cylindrical, unbranched, smooth-walled, pale brown and aggregated in dense fascicles arising from a brown stroma. The conidiogenous cells were integrated, terminal or intercalary, pale brown to brown and proliferated sympodially. The conidiogenous loci were thickened and darkened, and 2-3 μm in diam. The conidia were formed singly, obclavate-cylindrical, 90-160 × 5-7 μm, with an average of 130 × 6 μm (n = 30), 6-11 septa, thin-walled, smooth, hyaline or subhyaline, straight or curved with an obtuse apex and obconically truncate base, with thickened and darkened hilum. These morphological characteristics matched those of C. chevalieri, the causal agent of leaf spot on A. paeoniifolius (Braun et al. 2014; Saccardo et al. 1913). A conidial suspension in sterile water from lesions was used to inoculate water agar, and germinated conidia were transferred to potato dextrose agar(PDA) and incubated at 27°C for 7 days. Induction of sporulation was unsuccessful using PDA, as well as malt extract agar, potato sucrose agar and synthetic nutrient-poor agar. Two out of ten isolates were selected for molecular identification and pathogenicity assay. Genomic DNA from two pure isolates (KUNCC22-12536 and KUNCC22-12537) was extracted for PCR and amplified with primers for the internal transcribed spacers (ITS: ITS1/ITS4), calmodulin (CMD: CAL228F/CAL2Rd), translation elongation factor 1-alpha (TEF1-α: 728F/986R), actin (ACT: 512F/783R), histone H3 (HIS3: CYLH3F/CYLH3R), beta-tubulin gene (TUB2: BT-1F/BT-1R) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH: Gpd1/Gpd2), respectively (Vaghefi et al. 2021). The newly generated sequences for ITS (OP719153/OP719154), CMD(OP740904/OP740905), TEF1-α (OP740910/OP740911), ACT (OP740902/OP740903), HIS3 (OP740908/OP740909), TUB2 (OP740912/OP740913), GAPDH (OP740906/OP740907) of C. chevalieri were submitted to GenBank. So far, no sequence data of C. chevalieri were available in the GenBank database. As expected, most genes (TEF1-α, ACT, CMD, HIS, TUB2 and GAPDH) showed 91 to 95% identity to their best hits within species of the genus Cercospora. The phylogenetic tree showed that sequences retrieved from two isolates obtained from the A. konjac leaf spots clustered together within Cercospora forming a strongly supported clade. To test Koch\'s postulates, ten four-month-old healthy A. konjac plants grown in pots were used for a pathogenicity test in a greenhouse. One leaf of each plant was inoculated with mycelial plugs, and one leaf was inoculated with a sterile PDA plug. These plants were enclosed in plastic bags for 72 h. Only leaves inoculated with mycelium plugs produced brown lesions, which appeared after 10 to 14 days on inoculated leaves. Control plants treated with sterile PDA plugs remained asymptomatic. This experiment was repeated twice with the same results. C. chevalieri was reisolated from infected leaves and identified based on morphology and Sanger sequencing of the ITS region. To our knowledge, this is the first report of C. chevalieri causing leaf spot on A. konjac and the first report of this species from China (Braun et al. 2014), which provides key information for diagnosis and management of this disease.