Nanhaiaspeciosa,俗称Niudali,是一种药用木本藤本植物,属于豆科。因其烹饪和药用特性而受到重视,它被广泛种植,在中国广西壮族自治区占地约5,973hm2。据报道,该植物的可食用块茎具有抗菌和抗氧化作用(Luo等人。,2023年;舒等人。,2020)。2021年7月,榆林的牛达利种植园,广西,中国(北纬22°64;东经110°29)出现叶斑病症状,在46,690平方米的区域内,发病率超过40%。最初,小圆形,叶子上出现淡黄色斑点,随后演变成被黄色光环包围的深棕色病变,最终导致树叶枯萎。收集表现出典型症状的叶片用于病原体调查。用无菌水彻底洗涤叶子,并从病变周边切除小组织碎片(5×5mm)。这些碎片用75%乙醇和1%NaClO进行表面灭菌,用无菌水冲洗三次,随后在28°C黑暗中在马铃薯葡萄糖琼脂(PDA)上培养7天。通过单孢子分离,获得了7个形态性状相似的分离株。在28°C黑暗中在PDA上孵育7天后,菌落上表面呈白色至灰色,有丰富的气生菌丝,而底面则呈深黑色。分生孢子,圆柱形或球形,是直的,浅棕色,测得30.1-128.9μm×4.8-15.0μm(n=50)。形态特征与Corynesporasp。的形态特征相匹配。(Wang等人。2021)。对于分子鉴定,使用基因组DNA和引物ITS1/ITS4和EF1-688F/EF1-1251R对分离株N5-2进行了DNA序列分析。序列(ITS:OP550425;TEF1-α:OQ117118)存放在GenBank,对于TEF1-α,与C.cassiicola(OP981637)具有98%的同一性,对于ITS,与C.cassiicola(OP957070)具有99%的同源性。基于连接的ITS和TEF1-α,使用MEGA7.0的最大似然系统发育分析将分离物与C.cassiicola聚类。因此,根据其形态和分子特征,该真菌被鉴定为C.cassiicola。在1岁的南海区幼苗的致病性测试中,将叶子轻轻划伤并用菌丝体栓(5mm)接种。对照幼苗接受PDA塞。选择每株植物五片叶和每种处理五株植物用于评估。所有幼苗均保持在温室中(12/12h光/暗循环,25±2°C,90%湿度)。经过7天的潜伏期,所有接受真菌接种的叶子都表现出与田间观察到的症状一致的症状,而对照植物保持无症状。在连续三次试验中,真菌成功地从感染的叶子中重新分离,履行科赫的假设。虽然C.cassiicola在各种植物物种上诱导叶斑是有据可查的,包括Jasminumnudiflorum,星盘,Acanthusilicifolius,丁香属物种(Hu等人,2023年;刘等人。,2023年;谢等人。,2021年;王等人。,2021),本研究是中国首次报道的南海区叶斑病。在Nanhaiaspeciosa中鉴定该病原体对未来的流行病学调查具有重要意义,并为控制Nanhaiaspeciosa的叶斑病提供了有价值的参考。
Nanhaia speciosa, commonly known as Niudali, is a medicinal woody vine belonging to the Leguminosae family. Valued for its culinary and medicinal properties, it is extensively cultivated, covering approximately 5,973 hm2 in the Guangxi Zhuang Autonomous Region of China. The edible tubers of this plant are reported to possess antibacterial and antioxidant effects (Luo et al., 2023; Shu et al., 2020). In July 2021, a Niudali plantation in Yulin, Guangxi, China (22°64\'N; 110°29\'E) exhibited leaf spot symptoms, with an incidence rate exceeding 40% across a 46,690 m2 area. Initially, small circular, pale yellow spots appeared on the leaves, which subsequently evolved into dark brown lesions surrounded by yellow halos, ultimately leading to foliage wilting. Leaves exhibiting typical symptoms were collected for pathogen investigation. The leaves were thoroughly washed with sterile water and small tissue fragments (5×5 mm) were excised from the lesion periphery. These fragments were surface-sterilized with 75% ethanol and 1% NaClO, rinsed three times with sterile water, and subsequently cultured on potato dextrose agar (PDA) at 28 °C in darkness for 7 days. Through single-spore isolation, seven isolates with similar morphological traits were obtained. After 7 days of incubation on PDA at 28 °C in dark, the colonies exhibited a white to grey coloration on the upper surface with abundant aerial hyphae, while the underside appeared dark black. The conidia, cylindrical or obclavate in shape, were straight, pale brown, and measured 30.1-128.9 μm × 4.8-15.0 μm (n=50). The morphological characteristics matched those of Corynespora sp.(Wang et al. 2021). For molecular identification, the isolate N5-2 underwent DNA sequence analysis using genomic DNA and primers ITS1/ITS4 and EF1-688F/EF1-1251R. The sequences (ITS: OP550425; TEF1-α: OQ117118) were deposited in GenBank, exhibiting 98% identity to C. cassiicola (OP981637) for TEF1-α and 99% homology to C. cassiicola (OP957070) for ITS. Based on the concatenated ITS and TEF1-α, a maximum likelihood phylogenetic analyses using MEGA7.0 clustered the isolate with C. cassiicola. Consequently, the fungus was identified as C. cassiicola based on its morphological and molecular features. In the pathogenicity test on 1-year-old Nanhaia speciosa seedlings, leaves were gently scratched and inoculated with mycelial plugs (5 mm). Control seedlings received PDA plugs. Five leaves per plant and five plants per treatment were selected for assessment. All seedling were maintained in a greenhouse (12/12h light/dark cycle, 25 ± 2°C, 90% humidity). After a 7-day incubation period, all leaves subjected to fungal inoculation exhibited symptoms consistent with those observed in the field, while control plants remained symptom-free. The fungus was successfully reisolated from the infected leaves in three successive trials, fulfilling Koch\'s postulates. While C. cassiicola is well-documented for inducing leaf spots on various plant species, including Jasminum nudiflorum, Strobilanthes cusia, Acanthus ilicifolius, Syringa species (Hu et al., 2023; Liu et al., 2023; Xie et al., 2021; Wang et al., 2021), this study represents the first report of C. cassiicola causing leaf spots on Nanhaia speciosa in China. The identification of this pathogen in Nanhaia speciosa has significant implications for future epidemiological investigations and serves as a valuable reference for controlling leaf spot disease in Nanhaia speciosa.