First Report of Rhizopus arrhizusi causing rot of Dictyophora rubrovolvata in China.

IF 4.4 2区 农林科学 Q1 PLANT SCIENCES Plant disease Pub Date : 2024-10-09 DOI:10.1094/PDIS-08-24-1767-PDN
Shiqi Tu, Feng Zhou, Yin Zhang, YiFan Chen, ShengQian Chao, Beibei Lü, Hailong Yu
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Abstract

Dictyophora rubrovolvata, as an edible fungus with high medicinal value, is widely cultivated in several provinces in China (Hang et al. 2012). However, between December 2023 and March 2024, a rot disease occurred in the main production area in Fengxian District, Shanghai, China (N30°93', E121°49'). The disease incidence was 25% in the affected 1.33-ha growing area. High temperatures (>25℃) and poor ventilation provide favorable conditions for the spread of this disease. The disease mainly occurs at the stage of fruiting bodies formation of D. rubrovolvata. When the epidermis is damaged and broken, it becomes infested with mold, which then produces a layer of moldy rot with pus. The infected D. rubrovolvata tissues at the edge of the lesions were isolated, surface sterilized and cultured on potato dextrose agar (PDA) at 30 ℃ under dark conditions. Pure cultures were obtained by single-spore isolation. After 3 days, isolates were transferred to Czapek Yeast agar (CYA) (Samson et al, 2014). On CYA, the fungal colony consisted of white flocculent hyphae. Scanning electron microscopy analysis showed that the mycelium was white, and the internodes of the stolons formed characteristic pseudoroots, from which upwardly clustered erect, unbranched sporocarp peduncles expanded apically to form rounded sporocarp sacs, within which sporocarp spores were produced. (Hariprasath P, 2019). To confirm the identity of the pathogen, the genomic fragments for the internal transcribed spacer (ITS) and intergenic spacer (IGS) gene of the isolate were amplified by PCR (White et al. 1990; Liu XY. 2008). The resulting sequence was deposited in GenBank with accession PP951880 and PQ001670, respectively. PCR results and morphological observations indicated the isolated strain was a pure culture and the strain was designated as DIC01. Comparative results showed that the sequences with accession numbers MT603964.1 and DQ990323.1 showed high homology of 99.15% and 98.96% to the ITS and IGS sequences of Rhizopus arrhizusi DIC01, respectively. Phylogenetic analysis with ITS and IGS genes of the isolated strain and 7 Rhizopus spp. strains were performed using MEGAX with Neighbor-Joining (NJ) method. Based on the results of growth habits, morphological observations, and phylogenetic analysis, the pathogen was identified as R. arrhizusi. A spore suspension of the R. arrhizusi DIC01 (1 x107 conidia/mL) was inoculated back to healthy D. rubrovolvata. Five healthy fruit bodies of D. rubrovolvata were injected, and another five healthy morels were treated with potato dextrose broth (PDB) as controls. D. rubrovolvata was incubated at 25°C and 90% relative humidity without ventilation for 5 days. The pathogen successfully infected the D. rubrovolvata, which developed white moldy lesions similar to those of natural diseases. The controls remained healthy without any symptoms. The pathogen was reisolated from the affected lesions and identified as R. arrhizusi DIC01 based on its morphological characteristics and phylogenetic marker genes. R. arrhizusi has been reported to cause endothelial cell damage and mycelial invasion into blood vessels, leading to thrombosis and tissue necrosis. (Hariprasath P, 2019). To our knowledge, this is the first report of R. arrhizusi causing rot disease of D. rubrovolvata. This study confirmed that R. arrhizusi is the pathogenic fungus responsible for rotting disease in D. rubrovolvata farms in Fengxian, Shanghai.

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在中国首次报道了由根瘤菌(Rhizopus arrhizusi)引起的红豆杉(Dictyophora rubrovolvata)腐烂病。
红豆杉(Dictyophora rubrovolvata)是一种药用价值极高的食用菌,在中国多个省份广泛种植(Hang 等,2012 年)。然而,在 2023 年 12 月至 2024 年 3 月期间,中国上海市奉贤区(N30°93',E121°49')的主产区发生了腐烂病。发病面积为 1.33 公顷,发病率为 25%。高温(>25℃)和通风不良为该病的传播提供了有利条件。该病主要发生在 D. rubrovvolata 子实体形成阶段。当表皮受损和破损时,就会被霉菌侵染,然后产生一层带有脓液的霉烂层。分离病斑边缘受感染的红铃虫组织,进行表面灭菌,在 30 ℃ 黑暗条件下置于马铃薯葡萄糖琼脂(PDA)上培养。通过单孢分离获得纯培养物。3 天后,将分离物转移到 Czapek 酵母菌琼脂(CYA)上(Samson 等人,2014 年)。在 CYA 上,真菌菌落由白色絮状菌丝组成。扫描电子显微镜分析表明,菌丝是白色的,匍匐茎节间形成特征性的假根,从假根向上簇生直立、不分枝的孢子囊梗,顶部膨大形成圆形的孢子囊,孢子囊内产生孢子。(Hariprasath P,2019 年)。为确认病原体的身份,通过 PCR 扩增了分离株的内部转录间隔(ITS)和基因间间隔(IGS)基因的基因组片段(White 等,1990 年;Liu XY,2008 年)。所得序列分别以 PP951880 和 PQ001670 的登录号存入 GenBank。聚合酶链式反应结果和形态学观察结果表明,分离出的菌株是纯培养物,该菌株被命名为 DIC01。比较结果表明,登录号为 MT603964.1 和 DQ990323.1 的序列与根瘤菌 DIC01 的 ITS 和 IGS 序列的同源性分别为 99.15%和 98.96%。利用 MEGAX 和 Neighbor-Joining (NJ) 方法对分离菌株和 7 株根瘤菌的 ITS 和 IGS 基因进行了系统进化分析。根据生长习性、形态观察和系统发育分析的结果,确定病原体为 R. arrhizusi。将 R. arrhizusi DIC01 的孢子悬浮液(1 x107 分生孢子/毫升)接种回健康的 D. rubrovolvata。注射 5 个健康的 D. rubrovolvata 果体,另用马铃薯葡萄糖肉汤(PDB)处理 5 个健康的羊肚菌作为对照。在 25°C、相对湿度为 90%、不通风的条件下培养 D. rubrovolvata 5 天。病原体成功感染了 D. rubrovolvata,它出现了与自然病害类似的白色霉斑。对照组仍然健康,没有出现任何症状。根据病原体的形态特征和系统发育标记基因,从受害病斑中重新分离出病原体,并确定其为 R. arrhizusi DIC01。据报道,R. arrhizusi 可造成内皮细胞损伤,菌丝侵入血管,导致血栓形成和组织坏死。(Hariprasath P, 2019)。据我们所知,这是第一份关于 R. arrhizusi 导致 D. rubrovolvata 腐烂病的报告。本研究证实,R. arrhizusi 是导致上海奉贤红富士农场红富士腐烂病的病原真菌。
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来源期刊
Plant disease
Plant disease 农林科学-植物科学
CiteScore
5.10
自引率
13.30%
发文量
1993
审稿时长
2 months
期刊介绍: Plant Disease is the leading international journal for rapid reporting of research on new, emerging, and established plant diseases. The journal publishes papers that describe basic and applied research focusing on practical aspects of disease diagnosis, development, and management.
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