木薯(Manihotesculenta)是大妖科的多年生作物,由于其植物学和经济价值在中国广泛种植。2022年11月,湛江发现木薯叶斑病,广东,中国(21.17°N,110.18°E),100%的发病率。感染植物上约80%的叶子被斑点覆盖。典型的症状最初表现为不规则的浸水性病变,随着疾病的进展而变成棕色和发白,病变逐渐扩大并合并,导致叶子枯萎,干燥和最后的秋天。从病变边缘切除组织(4至5毫米),在3%H2O2溶液中灭菌3分钟,用无菌水冲洗三次,置于马铃薯葡萄糖琼脂(PDA)培养基(含50mg/L青霉素)上,并在25-28°C下孵育。获得了十个具有相似形态的单个菌丝分离株,并进一步纯化为单个分生孢子继代培养物。在25-28℃的黑暗中孵育四天后,菌落呈灰色白色,稀疏的气生菌丝体,菌落直径达到70.4mm。呈球状或不规则状出现的黑色棘突,成熟时出现错误,通常带有乳脂状的白色,培养30天后从前列腺中挤出的分生孢子卷云。分生孢子是透明的,光滑,无分支。阿尔法分生孢子是双点状的,透明玻璃,椭圆体,无菌,尺寸5.1~7.5×1.9~3.4µm(平均6.2×2.8µm,n>50)。β分生孢子丰富,丝状体,透明玻璃,光滑,呈钩状弯曲,截断基底,尺寸为18.5-26.4×0.6-1.2μm(平均23.4×1.0μm,n=40)。未观察到γ分生孢子。形态特征与Diaportheueckeri相似(Udayanga等人。2015).内部转录间隔区(ITS),大亚基(LSU)rRNA序列,肌动蛋白(ACT),钙调素(CAL),组蛋白H3(他的),翻译延伸因子1-α(TEF1-α),和代表性分离株CCAS-MS-6(ACCC35497)的β-微管蛋白(TUB)基因进行扩增并使用引物对进行测序:ITS5/ITS4,LR0R/LR5,ACT-512F/ACT-783R,CAL228F/CAL737R,CYLH3F/H3-1b,EF1-728F/EF1-986R和Bt2a/Bt2b(Gao等人2017;Udayanga等人2014)。所有序列都保存在GenBank(OR361671、OR361672和OR365605-9)中。BLAST搜索显示与Diaportheueckeri(表1)的序列高度相似。CAL的级联数据的最大似然分析,HIS,ITS,使用Mega11的TEF和TUB将CCAS-MS-6放置在D.ueckeri进化枝中。因此,这种真菌被鉴定为D.ueckeri.使用三株一岁健康植物在盆中进行致病性测试。每株植物的两片15天龄的叶子用75%的酒精清洗,每片叶子上有三个地方受伤,其中一片叶子上的部位被PDA上15天大培养物的真菌栓覆盖,另一片叶子上的站点带有PDA插头作为对照。将所有植物保持在环境温度(约28°C)并用含有无菌湿棉的塑料袋覆盖以保持湿度。接种后七天,所有接种部位都显示出坏死的症状,而对照组没有任何症状。从有症状的接种叶中重新分离出根据形态学和分子标准鉴定的相同真菌。在中国,据报道,ueckeri在桉树上引起疾病,茶树,和石路含笑(高等人2016年;廖等人2023年;易等人2018年),这是关于M.esculenta的第一份报告.疾病病因的定义是制定有效管理策略的前提。
Cassava (Manihot esculenta) is a perennial crop of the family
Euphorbiaceae, widely cultivated due to its phytopharmacological and economic values in China. In November 2022, a leaf spot disease on cassava was observed in Zhanjiang, Guangdong, China (21.17° N, 110.18° E), with 100% disease incidence. About 80 % of leaves were covered with spots on the infected plants. Typical symptoms initially appeared as irregular water-soaked lesions that became brown and whitish with the progress of the disease, lesions gradually expanded and coalesced, causing leaf withering, drying and final fall. Tissues (4 to 5 mm) were excised from the margin of lesions, sterilized in 3% H2O2 solution for 3 min, rinsed three times with sterile water, placed on potato dextrose agar (PDA) medium (containing 50mg/L penicillin), and incubated at 25-28 °C. Ten single hypha isolates with similar morphology were obtained and further purified as single conidium subcultures. The colony was grey whitish with sparse aerial mycelium and colony diameter reached 70.4 mm after four days incubation at 25-28℃ in the dark. Black pycnidia occurring as clusters were spherical or irregular, erumpent at maturity, often with a creamy whitish, conidial cirrus extruding from ostiole after 30-days incubation. Conidiophores were hyaline, smooth, unbranched. Alpha conidia were bi-guttulate, hyaline, ellipsoidal, aseptate, with dimensions of 5.1~7.5×1.9~3.4µm (mean 6.2×2.8 µm, n>50). Beta conidia were abundant, filiform, hyaline, smooth, curved in a hooked shape, with a truncate base and dimensions of 18.5-26.4 × 0.6-1.2μm (mean 23.4 × 1.0 μm, n= 40) . Gamma conidia were not observed. The morphological characteristics were similar to those of Diaporthe ueckeri (Udayanga et al. 2015). The internal transcribed spacer (ITS) region, large subunit (LSU) rRNA sequence, actin (ACT), calmodulin (CAL), histone H3 (HIS), translation elongation factor 1-alpha (TEF1-α), and β-tubulin (TUB) genes of a representative isolate CCAS-MS-6 (ACCC 35497) were amplified and sequenced using primer pairs: ITS5/ITS4, LR0R/LR5, ACT-512F/ACT-783R, CAL228F/CAL737R, CYLH3F/ H3-1b, EF1-728F/ EF1-986R and Bt2a/Bt2b (Gao et al 2017;Udayanga et al 2014). All sequences were deposited in GenBank (OR361671, OR361672, and OR365605-9). BLAST search showed high similarities with sequences of Diaporthe ueckeri (Tab 1). Maximum likelihood analyses of the concatenated data of CAL, HIS, ITS, TEF and TUB using Mega 11 placed CCAS-MS-6 in the D. ueckeri clade. Thus, the fungus was identified as D. ueckeri. Three one-year old healthy plants were used for pathogenicity tests in pots. Two 15-day old leaves of each plant were cleaned with 75% alcohol, three sites on each leaf were wounded, and sites on one of the leaf were covered with fungal plugs from 15-day-old cultures on PDA, and sites on the other leaf with PDA plugs as a control. All plants were kept at ambient temperature (about 28℃) and covered with plastic bags containing sterile wet cotton to maintain the humidity. Seven days after inoculation, all inoculated sites showed symptoms of necrosis, while control sites showed no symptoms. The same fungus identified on the basis of morphological and molecular criteria was reisolated from symptomatic inoculated leaves. In China, D. ueckeri had been reported to cause diseases on Eucalyptus citriodora, Camellia sinensis, and Michelia shiluensis (Gao et al 2016; Liao et al 2023; Yi et al 2018), this is the first report on M. esculenta. The definition of the disease etiology is a prerequisite to develop effective management strategies.