广东月露瓜镰刀菌果腐病菌的鉴定、生物学特性及对杀菌剂敏感性测定

Identification, Biological characteristics and fungicide selection of the pathogen causing Yuelu melon Fusarium fruit rot in Guangdong

摘要

【目的】明确广东月露瓜镰刀菌果腐病菌的种类及生物学特性，筛选防治药剂。【方法】自广州市增城区采集病害样本，采用组织分离法分离病原菌；结合形态特征和多基因系统发育分析确定病原菌的分类地位；测定温度、pH、光照、碳源、氮源和杀菌剂对病原菌生长的影响。【结果】分离菌株在PDA上成熟时淡黄色，背面米黄色。产孢细胞瓶梗状，单生或复生，在低营养合成琼脂（SNA）上，长2.72~49.69 µm（ n=80）；在康乃馨叶片琼脂（CLA）上，长3.63~52.65 µm（ n=80）。大型分生孢子镰刀形，顶端渐尖稍弯曲，1~5个分隔，在SNA上大小为（17.45~46.78）µm×（2.62~4.93）µm（ n=80），在CLA上大小为（15.84~44.27）µm×（2.71~4.95）µm（ n=80）。厚垣孢子球形。基于 CaM、 EF-1α和 RPB2基因序列联合构建系统发育树，确定病原菌是伯氏镰刀菌（ Fusarium pernambucanum）。病原菌最适生长温度为26~28 ℃，pH为7~9。碳源葡萄糖、可溶性淀粉和有机氮源蛋白胨、牛肉浸粉和酵母膏均利于菌丝生长和产孢。咯菌腈和咪鲜胺对病菌有较好的抑制作用，EC₅₀分别为0.014 7 mg·L^-1和0.637 3 mg·L^-1。【结论】广东月露瓜镰刀菌果腐病菌为伯氏镰刀菌（ Fusarium pernambucanum），病原菌适宜相对较高温度及中性至偏碱环境，对咯菌腈和咪鲜胺敏感。本研究结果可为病害诊断和防治提供理论依据。

Abstract

【Objective】The Yuelu melon is a premium melon variety. After being introduced and cultivated in limited quantities in Hainan, Fujian, and Guangdong provinces, it has demonstrated significant economic value. In 2025, a Fusarium fruit rot disease affecting Yuelu melon was discovered in Guangzhou, with an incidence rate ranging from 5% to 8%. The disease altered the aroma, flavor, and quality of the melon, thereby diminishing its nutritional and commercial value. During cultivation, symptoms were observed exclusively within the fruit, with no visible external manifestations, which significantly increased the challenges associated with disease detection and management. The study aimed to identify the pathogen and elucidate its suitable growth conditions, including temperature, pH, light, as well as carbon and nitrogen sources. In addition, the toxicity of various fungicides to the pathogen was evaluated to assess their efficacy, thereby providing insights for effective disease control. 【Methods】From April to June 2025, a survey was conducted to assess the incidence and severity of fruit rot in Yuelu melon at the Xiangfeng Special Fruit Industrial Park in Zengcheng District, Guangzhou City. Field symptoms were documented photographically. Diseased tissue samples were collected and, following surface disinfection, they were cultured on potato dextrose agar (PDA) at 26 ℃ for 3 days using the tissue isolation method. Mycelia from the margins of developing colonies were then transferred to PDA plates. Purified strains were obtained through single-spore isolation and stored at 4 ℃ for subsequent use. Pathogenicity was confirmed through two inoculation methods. The first involved mycelia inoculation: The mycelia surfaces of the tested strains were adhered to the puncture site of the melon fruits, with blank agar discs used as controls. The second method employed spore suspension inoculation: 100 µL of spore suspension (5×10 ⁶ spores · mL ^-1) was injected into the fruit from the tail end. All treated fruits were incubated at 26 ℃. Once softening or clear symptoms developed, the pathogens were isolated and purified again for verification in accordance with Koch’s postulates. The strains were inoculated on potato dextrose agar (PDA), synthetic nutrient-poor agar (SNA), and carnation leaf agar (CLA) in the dark at 26 ℃ for 10 days. Mycelia, conidiophores, conidia attachment patterns, conidia and chlamydospores were observed under an optical microscope, and the size of conidiophores and conidia were measured ( n=80). Genomic DNA was extracted from the tested strains, and the calmodulin ( CAM), translation elongation factor 1-alpha ( EF1-α), and the second largest subunit of RNA polymerase Ⅱ ( RPB2) gene regions were amplified via PCR. The products were sequenced by Shanghai Sangon Biotech Co., Ltd. The resulting sequences were submitted to the NCBI database to obtain accession numbers. Concurrently, BLAST analyses were performed on NCBI to identify and retrieve sequences of closely related species from GenBank. Sequence alignment was conducted using Mega 7.0 software, followed by manual splicing of the aligned datasets. A phylogenetic tree was constructed using the concatenated sequences of CAM, EF1-α, and RPB2 through the neighbor-joining method implemented in MEGA 7.0 to confirm species identification. Furthermore, the tested strains were cultured in an incubator under varying growth conditions, including temperature, pH, light intensity, carbon sources, and nitrogen sources, to determine the optimal culture environment for mycelia growth and spore production. Subsequently, to evaluate the sensitivity of the pathogen to various fungicides, eight commercial fungicides were tested. Mycelia discs were inoculated onto potato dextrose agar (PDA) plates supplemented with each fungicide at designated concentrations and incubated at 26 ℃. Colony diameters were measured after 5 days of incubation. 【Results】The external appearance of diseased fruits showed no visible symptoms in the field. However, their tail ends felt soft to the press, and internal examination revealed brown, water-soaked rot, occasionally accompanied by a thin layer of white mold on the diseased areas. A total of 27 Fusarium spp. isolates with similar morphological characteristics were obtained. Pathogenicity tests revealed that artificial inoculation with strains YLG-4, YLG-13, and YLG-21 produced symptoms consistent with those observed under field conditions. On PDA medium, the colonies were initially white and later turned light yellow with cotton-like and abundant aerial mycelia. Conidiogenous cells were phialidic, 2.72 to 49.69 µm ( n=80) on SNA medium and 3.63 to 52.65 µm ( n=80) on CLA medium. Macroconidia were falcate, slightly curved, tapering apically with 1 to 5 septa, (17.45-46.78) µm×(2.62-4.93) µm ( n=80) on SNA medium, (15.84-44.27) µm×(2.71-4.95) µm ( n=80) on CLA medium, borne false heads from monophialides and polyphialides. Chlamydospores were spherical on both media.

关键词

月露瓜 / 果腐病 / 病原鉴定 / 伯氏镰刀菌 / 生物学特性 / 药剂筛选

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基金资助

国家重点研发计划(2024YFD1701202)

深圳市可持续发展专项(KCXFZ20230731100700001)

广东省农业科学院科技创新支撑农业强省建设专项(QSJS202628)