Abstract：
Rosemary (Rosmarinus officinalis L.) is an aromatic, evergreen, medicinally important shrub and widely used for cooking, tea, cosmetics as well as medicinal materials. It is grown in many countries including China that had more than 9300 hm2 of commercial cultivation area in 2021. In March 2020, a leaf spot disease sporadic occurred in field rosemarry plants in Nanyang City (32º51´ N, 111º36´ E), Henan Province, China. The disease outbreaked in September with a disease incidence of 57-83%. Symptoms initially appeared as small brown leaf spots that gradually expanded into dark blackbrown irregular lesions. Most of the spots started from the leaf tip or leaf margin, and gradually spread to the leaf base, resulting in heavy defoliation especially on rainy days. Diseased leaf segments (1×3 mm) were surface-sterilized by dipping in 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar, then incubated at 28°C in the dark for 5 days. Twelve fungal isolates with the same morphological characteristics were obtained from nine affected leaves. The fungal colonies were initially white and turned gray brown with flocculent aerial mycelia and a whorled back. Conidia were frequently born in a long chain, with a short beak, brown or light-brown, 13.2 to 48. 7 (average 26.1) × 4.0 to 13.1 (average 8.0) μm in size (n=148) with 0 to 8 transverse and 0 to 3 longitudinal/oblique septa. Phenotypic features of the isolates agreed with those of Alternaria alternata (Simmons et al. 2007). Two isolates Aa1 and Aa2 were randomly selected for molecular and pathogenicity tests. DNA was extracted from mycelia. Partial sequences of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) were amplified using the primer pairs ITS1/ITS4 and EFI-728F/EFI-986R (Wei et al. 2022), respectively. The GenBank accession nos. were OK036714 and OK036715 for ITS, and ON951980 and ON951981 for TEF1-α of Aa1 and Aa2, respectively, with a maximal identity of greater than 99% to multiple A. alternata strains. In the neighbour joining phylogenetic tree of the amplified ITS and TEF1-α sequences both Aa1 and Aa2 clustered with A. alternata strains, clearly separating them from other Alternaria spp. For pathogenicity test, conidial suspensions (1×106 spores /mL) of Aa1 and Aa2 were separately sprayed on healthy one-year-old rosemary plants (n=3) with their leaves slightly wounded with a sterilized needle. Control plants (n=3) were sprayed with sterile water. Both inoculated and control plants were incubated at 90% RH, 28 °C. After 14 days, all the inoculated leaves showed black brown lesions similar to those on naturally affected field plants, whereas controls remained symptomless. Fungal cultures with the same phenotypic features as the inocula were constantly re-isolated from the infected leaves. A. alternata was reported as pathogen causing foliar necrosis on rosemary in Italy (Perello et al.1995) and leaf spot (or leaf blight) on multiple plant species such as Actaea dahurica (Hai et al. 2022), and Ligustrum japonicum (Wei et al. 2022) in China. This is the first report of A. alternata causing leaf black spot on rosemary in China.
摘要：
迷迭香（迷迭香）是一种芳香，常绿,重要的药用灌木，广泛用于烹饪，茶，化妆品和药材。它在包括中国在内的许多国家种植，2021年商业种植面积超过9300hm2。2020年3月，南阳市田间迷迭香属植物发生一次零星叶斑病（32º51'N，111º36'E),河南省,中国。该病在9月爆发，发病率为57-83％。症状最初表现为小的棕色叶斑，逐渐扩展为深黑棕色不规则病变。大多数斑点从叶尖或叶缘开始，并逐渐扩散到叶基，导致严重落叶，尤其是在雨天。通过浸入1％次氯酸钠1分钟对患病的叶段（1×3mm）进行表面灭菌，用无菌蒸馏水冲洗三次，并铺在马铃薯葡萄糖琼脂上，然后在黑暗中在28°C下孵育5天。从9个受影响的叶片中获得了12个具有相同形态特征的真菌分离株。真菌菌落最初是白色的，变成灰棕色，有絮凝的气生菌丝体和轮生。分生孢子经常出生在一条长链中，短喙,棕色或浅棕色，13.2至48。7（平均26.1）×4.0至13.1（平均8.0）μm（n=148），具有0至8个横向和0至3个纵向/倾斜间隔。分离株的表型特征与链格孢菌的表型特征一致(Simmons等人。2007).随机选择两个分离株Aa1和Aa2进行分子和致病性测试。从菌丝体中提取DNA。使用引物对ITS1/ITS4和EFI-728F/EFI-986R扩增了内部转录间隔区（ITS）和翻译延伸因子1-α（TEF1-α）的部分序列（Wei等人。2022),分别。GenBank登录号。ITS的OK036714和OK036715，和ON951980和ON951981分别为Aa1和Aa2的TEF1-α，与多个交替A菌株的最大同一性大于99%。在扩增的ITS和TEF1-α序列的相邻连接系统发育树中，Aa1和Aa2均与交替菌株成簇，清楚地将它们与其他链格孢属物种分开。对于致病性测试，将Aa1和Aa2的分生孢子悬浮液（1×106孢子/mL）分别喷洒在健康的一岁迷迭香植物（n=3）上，用无菌针头将其叶子轻伤。用无菌水喷洒对照植物(n=3)。接种和对照植物均在90％RH下孵育，28°C。14天后,所有接种的叶子都显示出与自然受影响的田间植物相似的黑褐色病变，而对照组保持无症状。与接种物具有相同表型特征的真菌培养物不断从感染的叶片中重新分离。据报道，在意大利的迷迭香（Perello等人，1995年）和在多种植物物种上引起叶斑病（或叶枯病）的病原体，例如Actaeadahurica（Hai等人。2022),和日本女贞子(Wei等人。2022）在中国。这是迷迭香叶斑病在中国首次报道。
