刺梨是一种起源于中国西南部的玫瑰灌木(Fu等人。2020)。2022年9月至10月,贵州省发生刺梨腐病,中国,在5公顷至50公顷的主要刺梨生产区内,发病率为10%至15%。症状表现为果实表面的黑色和棕色病变,是凹的,软,臭味,周围是灰棕色组织.从每个家庭随机收集三株受感染的刺梨灌木,放在透明的塑料袋中,病原体分离是在实验室进行的。用0.5%NaOCl对感染的刺梨果实进行表面灭菌2分钟,用无菌水冲洗五次,干了。将坏死组织和健康组织之间的边缘的症状组织切成5×5mm的碎片,置于马铃薯葡萄糖琼脂(PDA)上,并在黑暗中在28ºC下孵育5天。将从样品生长的真菌的菌丝尖端转移到新的PDA平板上并孵育直到它们产生分生孢子。总共获得了5种具有相似形态特征的真菌分离株。通过单孢子纯化获得的菌落为浅紫色至深紫色,具有丰富的气生菌丝体。大分生孢子相对细长,呈曲线状,为零至三个间隔。微分生孢子出现卵形至梨形,尺寸为5.2至17.2×2.1至3.3μm(n=50)。形态特征与环状镰刀菌一致(Yilmaz等人。2021)。使用DN14十六烷基三甲基溴化铵植物基因组快速提取试剂盒提取了两个代表性分离株(赵昌林1621和1622)的基因组DNA(AidlabBiotechnologiesCo.,Ltd,北京)。使用引物EF1-983F/EF1-2218R通过聚合酶链反应扩增TEF1和RPB2基因(Rehner等,2005),bRPB2-6F/bRPB2-7.1R(Matheny等人,2002),分别。所有序列都保存在GenBank(TEF1,PP236860,PP236861;RPB2,PP767864,PP767865)中。对TEF1和RPB2序列的BLAST搜索显示,TEF1序列与环生F.分离株CBS258.54具有99.89%(937/938bp)的同一性;RPB2序列具有99.86%(737/738bp)的同一性,以分离CBS267.93。在系统发育树中,分离株(赵昌林1621和1622)与环生F.形态学和多基因系统发育分析表明,该分离株为环生F.为了完成科赫的假设,五个成熟,健康的刺梨果实用1%NaClO溶液表面消毒1分钟,用无菌水冲洗,25℃干燥30分钟。将从两个分离株(赵昌林1621和1622)收集的分生孢子悬浮液(106孢子/ml)喷洒到刺梨果实上,和对照处理用无菌蒸馏水喷雾。所有刺梨果实在25ºC和80%相对湿度下孵育。实验有五个重复。经过7天的孵化,所有接种的水果都表现出与最初在最初感染的植物上观察到的相似的症状。通过形态学特征和分子分析对同一病原体进行了重新分离和鉴定,履行科赫的假设。因此,导致刺梨腐烂的病原体被确定为环叶F.(H.张等人,2024).据我们所知,这是在中国首次报道引起刺梨腐烂病。环孢菌具有广泛的宿主,据报道可以感染多种作物,水果,和蔬菜(培根和纳尔逊1994)。本研究为进一步研究和开发刺梨的病害防治方法及提高刺梨的经济效益奠定了基础。
Rosa roxburghii Tratt is a rosaceous shrub originating from southwest China (Fu et al. 2020). From September to October 2022, R. roxburghii rot occurred in Guizhou Province, China, within a major R. roxburghii production area covering from 5 ha to 50 ha, with an incidence rate of 10 to 15%. Symptoms manifested as black and brown lesions on the fruit surface, which were concave, soft, foul-smelling, and surrounded by grayish-brown tissue. Three infected R. roxburghii shrubs were randomly collected from each household, placed in transparent plastic bags, and pathogen isolation was conducted in a laboratory. Infected R. roxburghii fruits were surface-sterilized with 0.5% NaOCl for 2 min, rinsed five times with sterile water, and dried. Symptomatic tissues from the margin between necrotic and healthy tissues were cut into 5 × 5 mm pieces, placed onto potato dextrose agar (PDA), and incubated at 28ºC in the dark for 5 days. Hyphal tips of fungi growing from the samples were transferred onto new PDA plates and incubated until they produced conidia. A total of five fungal isolates with similar morphological characteristics were obtained. The colony obtained by single-spore purification was light purple to dark purple with abundant aerial mycelium. Macroconidia were relatively slender with a curve and zero to three septate. Microconidia appeared obovoid to pyriform, with sizes of 5.2 to 17.2 × 2.1 to 3.3 μm (n = 50). The morphological characteristics were consistent with Fusarium annulatum (Yilmaz et al. 2021). Genomic DNA of two representative isolates (Zhaochanglin 1621 and 1622) was extracted using the DN14 cetyltrimethylammonium bromide rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing). The TEF1 and RPB2 gene were amplified by Polymerase Chain Reaction using primers EF1-983F/EF1-2218R (Rehner et al, 2005), bRPB2-6F/bRPB2-7.1R (Matheny et al, 2002), respectively. All sequences were deposited in GenBank (TEF1, PP236860, PP236861; RPB2, PP767864, PP767865). BLAST searches of the TEF1, and RPB2 sequences revealed the TEF1 sequences had 99.89% (937/938 bp) identity with F. annulatum isolate CBS 258.54; and the RPB2 sequences had 99.86% (737/738 bp) identity to isolate CBS 267.93. In the phylogenetic tree, the isolates (Zhaochanglin 1621 and 1622) clustered with the representative strains of F. annulatum. The morphology and multi-gene phylogenetic analysis indicated that is the isolates were F. annulatum. To complete Koch\'s postulates, five mature, healthy R. roxburghii fruits were surface disinfected with 1% NaClO solution for 1 min, rinsed with sterile water, and dried at 25℃ for 30 min. A conidial suspension (106 spores/ml) collected from two isolates (Zhaochanglin 1621 and 1622) was sprayed onto R. roxburghii fruits, and the control treatments were sprayed with sterile distilled water. All R. roxburghii fruits were incubated at 25 ºC with 80% relative humidity. The experiment had five replicates. After 7 days of incubation, all the inoculated fruits showed similar symptoms to those initially observed on the originally infected plants. The same pathogen was reisolated and identified by morphological character ization and molecular analysis, fulfilling Koch\'s postulates. Thus, the pathogen causing rot of R. roxburghii was determined to be F. annulatum (H. Zhang et al, 2024). To our knowledge, this is the first report of F. annulatum causing R. roxburghii rot disease in China. F.annulatum has a wide range of hosts and has been reported to infect a wide range of crops, fruits, and vegetables (Bacon and Nelson 1994). This study lays a foundation for further study and developing disease control methods and the improvement of the economic benefits of R. roxburghii.