Abstract：
Pomegranate (Punica granatum) is an important fruit crop for therapeutic and food applications. In June 2022, brown spots were observed on the fruit surface of pomegranate cultivar named Guangyan in Mengzi (23°20\'6\'\'N,103°25\'5\'\'E), Yunnan, China. The early spots appeared as circular or irregular lesions, measuring 1~1.5 mm in diameter. They were light brown with a clear boundary between disease and healthy tissues. Over time, these spots developed into polygonal lesions covering the entire fruit surface. Eventually, the diseased fruits decayed, and more than 50% of fruits were infected in pomegranate orchards. The tissues from the interface between health and disease were cut down, immersed in 75% ethanol for 15 s, then 5% NaOCl disinfecting for 2 min, washed three times with sterile water, and the PDA cultured at 26 °C in an incubator under dark conditions. Twenty-five samples were collected for pathogen isolation, ten fungal isolates were obtained by single spore germination, and these isolates had similar morphological characters. The colonies were white with 81 mm diameter at 7 days of incubation, containing undulate edges with dense aerial mycelium. After 14 days, the black conidiomata formed superficially, gathering into black droplets. Conidiogenous cells were hyaline, short, and filiform. Conidia were fusiform, straight or slightly curved, and comprised five cells, 24.12 to 34.53 (x̄=29.78) μm × 4.21 to 12.15 (x̄=8.68) μm (n=50). The three median cells were 13.13 to 25.22 μm (x̄=18.54), dark brown, whose septa and periclinal walls were darker than the other two cells. The apical cells showed two to four appendages, 12.31 to 29.15 (x̄=21.56) μm. Only a single appendage was found on the basal cell, 2.34 to 7.16 μm. Based on morphological features, these isolates were identified as Neopestalotiopsis clavispora (Maharachchikumbura et al., 2012, 2014). Molecular identification of isolate YNSL-3 was performed by amplification and sequencing of ITS4/ITS5, BT2A/ BT2B and EF1-728F/EF-2, respectively (White et al. 1990, Glass et al.1995, Carbone et al. 1999, O\'Donnell et al. 1998). These base sequences were deposited in GenBank with accession numbers OQ891378 (ITS), OR088917 (Tef) and OR513439(Tub), respectively. BLAST searches of the sequences revealed 100% (478/478 bp), 100% (484/484 bp), and 94.67% (426/450 bp) homology with those of N. clavispora NM16311a from GenBank (LC209216, LC209220, and LC209221), respectively. Phylogenetic analysis (IQ-TREE) by maximum likelihood method showed that the isolate YNSL-3 was clustered with N. clavispora. The pathogenicity was tested with the isolate of YNSL-3, YNSL-5 and YNSL-8 by detached assay. The fruit surface of pomegranate cultivar Guangyan was wounded with a sterilized needle. The mycelial blocks (5mm2) of isolates cultured on PDA for 7 days were attached to the points of inoculation. Controls were inoculated with sterile PDA agar. All inoculated fruits were maintained in a growth chamber at 26°C with 75% relative humidity. The test was performed thrice. The brown lesions were observed after 7 days, whereas the controls showed no symptoms. The same pathogens reisolated were identical to the original isolates based on morphological characterization and molecular analyses. N. clavispora could cause different diseases in many plants (Rajashekara et al. 2023, Loredana et al. 2020). To our knowledge, this is the first report of fruit brown spot on Punica granatum caused by N. clavispora in China. This finding will help improve management strategies against the fruit brown spots on P. granatum in China.
摘要：
石榴（Punicagranatum）是用于治疗和食品应用的重要水果作物。2022年6月，在蒙自（23°20\'6\'\'N，103°25\'5\'\'E)，云南,中国。早期斑点表现为圆形或不规则病变，直径1~1.5毫米。它们为浅棕色，在疾病和健康组织之间有明确的界限。随着时间的推移,这些斑点发展成多边形病变，覆盖整个水果表面。最终,患病的果实腐烂了，石榴园感染了50%以上的水果。健康和疾病之间的界面上的组织被切割下来，浸入75%乙醇中15秒，然后5%NaOCl消毒2分钟，用无菌水冲洗三次，并且PDA在黑暗条件下在培养箱中在26°C下培养。收集了25个样本进行病原体分离，通过单孢子萌发获得10株真菌，这些分离株具有相似的形态特征。孵育7天时菌落呈白色，直径为81mm，含波状边缘，具密集的气生菌丝。14天后,黑色分生孢子表面形成，聚集成黑色的水滴。分生孢子细胞是透明的，短,和丝状。分生孢子是梭形的,笔直或略微弯曲，由五个细胞组成，24.12至34.53(x炭黑=29.78)μm×4.21至12.15(x炭黑=8.68)μm(n=50)。三个中位数细胞为13.13至25.22μm（x²=18.54），深棕色,其隔垫和外围壁比其他两个细胞更暗。顶端细胞显示两到四个附属物，12.31至29.15(x²=21.56)μm。在基底细胞上只发现了一个附属物,2.34至7.16μm。根据形态特征，这些分离株被鉴定为新雌虫(Maharachchikumbura等.,2012、2014)。分离株YNSL-3的分子鉴定分别通过ITS4/ITS5，BT2A/BT2B和EF1-728F/EF-2的扩增和测序进行（White等人。1990年，Glass等人，1995年，Carbone等人。1999年，奥唐奈等人。1998).这些碱基序列存放在GenBank中，登录号为OQ891378（ITS），OR088917(Tef)和OR513439(浴缸),分别。BLAST序列搜索显示100%(478/478bp)，100%(484/484bp)，和94.67%（426/450bp）的同源性，与来自GenBank（LC209216，LC209220和LC209221）的N.clavisporaNM16311a的同源性，分别。通过最大似然方法进行的系统发育分析（IQ-TREE）表明，分离株YNSL-3与N。通过分离测定法用YNSL-3，YNSL-5和YNSL-8的分离物测试致病性。石榴品种广燕的果实表面用灭菌针受伤。将在PDA上培养7天的分离物的菌丝体块(5mm2)附着于接种点。对照用无菌PDA琼脂接种。将所有接种的果实保持在26°C和75%相对湿度的生长室中。测试进行三次。7天后观察到棕色病变，而对照组没有症状。根据形态学表征和分子分析，重新分离的相同病原体与原始分离株相同。N.clavispora可能在许多植物中引起不同的疾病(Rajashekara等人。2023年，Loredana等人。2020）。据我们所知,这是中国首例由丁香引起的石榴果实褐斑的报道。这一发现将有助于改善中国石榴果实褐斑的管理策略。
