Fusarium falciforme 在中国海南辣椒上引起镰刀菌枯萎病的首次报告。

IF 4.4 2区 农林科学 Q1 PLANT SCIENCES Plant disease Pub Date : 2024-10-16 DOI:10.1094/PDIS-09-24-1854-PDN
Xingliang Wang, Yue Huang, Na Yang, Xue Wang, Xiaoyu Shen, Lijuan Pei, Ying Wang, Hui Zhang
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As the lesions expanded, the whole plant gradually wilted and died. Ten diseased plants were randomly selected from the most severely affected area (667 m²). Diseased tissues (5 mm²) were subsequently removed from the lesion edges of these plants, surface sterilized in 75% ethanol for 30 s, and rinsed with sterile distilled water three times, finally cultured on potato dextrose agar (PDA) at 25 °C. Six fungal isolates were obtained using the single-spore isolation method (HN-01 to HN-06). Colonies produced white aerial mycelia with apricot pigments in the PDA medium. The spore morphology and size were observed and measured using synthetic nutrient-poor agar (SNA) medium. Macroconidia were hyaline, slightly curved in shape with 3 or 4 septa, measuring 28.6 to 41.4 × 3.2 to 6.2 μm (av. = 34.8 ± 3.32 × 4.6 ± 0.85 um, n = 20). Microconidia were elongated, oval with 0 or 1 septum, and measured 11.2 to 16.8 × 2.6 to 5.8 μm (av. = 13.5 ± 1.47 × 4.12 ± 1.03 um, n = 20). Chlamydospores were spherical, terminal or intercalary, solitary or chain-forming, with diameters ranging from 2.8 to 10.5 um (av. = 5.8 ± 2.31 um, n = 20). For molecular identification, genomic DNA from all six isolates was extracted using the cetyl trimethyl ammonium bromide (CTAB) method, and the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α), and RNA polymerase II beta subunit (RPB2) regions were amplified and sequenced using the primers ITS1/ITS4, EF-1/EF-2, and RPB2-5F/7cR (White et al. 1990; O'Donnell et al. 2010). The sequences were deposited in GenBank (ITS: PP779839, PP779840, PP779841, PP779842, PP779843, PP779844; EF1-α: PP797138, PP797139, PP797140, PP797141, PP797142, PP797143; RPB2: PP797144, PP797145, PP797146, PP797147, PP797148, PP797149). 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The experiment was repeated three times. After 10 days, Inoculated plants stem developed necrosis, browning, while the control group remained asymptomatic. The fungus reisolated from the artificially infected stems was identified through EF1-α and RPB2 sequence analysis as <i>F. falciforme</i>, thus fulfilling Koch's postulates. <i>Fusarium falciforme</i> has been previoulsy reported as causing various diseases on different hosts in several countries, including Korea (Kang et al., 2024), Malaysia (Balasubramaniam et al., 2023), and Mexico (Payán-Arzapalo et al., 2024). To the best of our knowledge, this is the initial report demonstrating that <i>F. falciforme</i> causes Fusarium wilt on peppers in China. 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This makes pepper the leading vegetable in terms of both planting area and economic value. In December 2023, a total of 70% disease incidence of Fusarium wilt was observed in a 1200 m² pepper seed breeding base in Sanya City, Hainan Province, China (18°38'60″ N, 109°16'51″ E). Symptoms initially appeared as wilting on upper leaves. Subsequently, the base of the stem started to necrosis, browning, and gradually spreading upward along the stem. As the lesions expanded, the whole plant gradually wilted and died. Ten diseased plants were randomly selected from the most severely affected area (667 m²). Diseased tissues (5 mm²) were subsequently removed from the lesion edges of these plants, surface sterilized in 75% ethanol for 30 s, and rinsed with sterile distilled water three times, finally cultured on potato dextrose agar (PDA) at 25 °C. Six fungal isolates were obtained using the single-spore isolation method (HN-01 to HN-06). Colonies produced white aerial mycelia with apricot pigments in the PDA medium. The spore morphology and size were observed and measured using synthetic nutrient-poor agar (SNA) medium. Macroconidia were hyaline, slightly curved in shape with 3 or 4 septa, measuring 28.6 to 41.4 × 3.2 to 6.2 μm (av. = 34.8 ± 3.32 × 4.6 ± 0.85 um, n = 20). Microconidia were elongated, oval with 0 or 1 septum, and measured 11.2 to 16.8 × 2.6 to 5.8 μm (av. = 13.5 ± 1.47 × 4.12 ± 1.03 um, n = 20). Chlamydospores were spherical, terminal or intercalary, solitary or chain-forming, with diameters ranging from 2.8 to 10.5 um (av. = 5.8 ± 2.31 um, n = 20). For molecular identification, genomic DNA from all six isolates was extracted using the cetyl trimethyl ammonium bromide (CTAB) method, and the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α), and RNA polymerase II beta subunit (RPB2) regions were amplified and sequenced using the primers ITS1/ITS4, EF-1/EF-2, and RPB2-5F/7cR (White et al. 1990; O'Donnell et al. 2010). The sequences were deposited in GenBank (ITS: PP779839, PP779840, PP779841, PP779842, PP779843, PP779844; EF1-α: PP797138, PP797139, PP797140, PP797141, PP797142, PP797143; RPB2: PP797144, PP797145, PP797146, PP797147, PP797148, PP797149). The sequences of all three genes showed 99 to 100% similarity with <i>Fusarium falciforme</i> and other closely related <i>Fusarium</i> species (ITS: PP735125, EF1-α: OP163897 and RPB2: MF467484). 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引用次数: 0

摘要

辣椒(Capsicum annuum L.)是一种重要的蔬菜作物,具有重要的营养和经济价值(Pang 等,2023 年)。中国的辣椒种植面积约占蔬菜种植总面积的 8-10%,产值约为 2500 亿元。这使得辣椒在种植面积和经济价值方面都成为蔬菜中的佼佼者。2023 年 12 月,在中国海南省三亚市(北纬 18°38'60″,东经 109°16'51″)的一个 1200 平方米的辣椒种子繁育基地,发现镰刀菌枯萎病的发病率高达 70%。症状最初表现为上部叶片枯萎。随后,茎基部开始坏死、变褐,并逐渐沿茎向上蔓延。随着病斑扩大,整个植株逐渐枯萎死亡。从受灾最严重的地区(667 平方米)随机抽取了 10 株病株。随后从这些植株的病斑边缘取下病变组织(5 平方毫米),在 75% 的乙醇中表面消毒 30 秒,并用无菌蒸馏水冲洗三次,最后在 25 °C 的马铃薯葡萄糖琼脂(PDA)上培养。采用单孢分离法获得了六种真菌分离物(HN-01 至 HN-06)。菌落在 PDA 培养基中产生白色气生菌丝体,带有杏色素。使用贫养分合成琼脂(SNA)培养基对孢子形态和大小进行了观察和测量。大锥体呈透明状,形状略微弯曲,有 3 或 4 个隔膜,大小为 28.6 至 41.4 × 3.2 至 6.2 μm(平均值 = 34.8 ± 3.32 × 4.6 ± 0.85 um,n = 20)。微囊拉长,椭圆形,有 0 或 1 个隔膜,大小为 11.2-16.8 × 2.6-5.8 μm(平均值 = 13.5 ± 1.47 × 4.12 ± 1.03 um,n = 20)。衣孢子呈球形,顶生或闰生,单生或成链,直径范围为 2.8 至 10.5 微米(平均值 = 5.8 ± 2.31 微米,n = 20)。为了进行分子鉴定,使用十六烷基三甲基溴化铵(CTAB)法提取了所有 6 个分离株的基因组 DNA,并使用引物 ITS1/ITS4、EF-1/EF-2 和 RPB2-5F/7cR 对 rDNA 内部转录间隔区(ITS)、翻译延伸因子 1-α (EF1-α)和 RNA 聚合酶 II beta 亚基(RPB2)区进行了扩增和测序(White et al.1990;O'Donnell 等人,2010)。序列已存入 GenBank(ITS:PP779839, PP779840, PP779841, PP779842, PP779843, PP779844; EF1-α:PP797138、PP797139、PP797140、PP797141、PP797142、PP797143;RPB2:PP797144、PP797145、PP797146、PP797147、PP797148、PP797149)。这三个基因的序列与镰刀菌和其他近缘镰刀菌(ITS:PP735125;EF1-α:OP163897;RPB2:MF467484)的相似度为 99%至 100%。根据所有分离物的 ITS、EF1-α 和 RPB2 序列以及其他近缘镰刀菌种的序列,构建了最大似然系统发生树。根据形态学和系统发育特征,所有分离株都被鉴定为镰刀菌(Xu 等,2023 年;Wang 等,2023 年)。利用三个代表性分离株对 10 个辣椒近交系 A23-41(接种处理 5 个,对照 5 个)进行了致病性测试:HN-01、HN-02 和 HN-03。将浓度为 106 个孢子/毫升的孢子悬浮液共 20 ul 注入茎干土壤表面附近,而对照处理则接种 20 μl 无菌水。接种后,将植物置于相对湿度为 80-90%、温度为 25/20 °C(昼/夜)的恒温室中。实验重复三次。10 天后,接种的植株茎干出现坏死、褐变,而对照组仍无症状。通过 EF1-α 和 RPB2 序列分析,从人工感染的茎秆中重新分离出的真菌被鉴定为镰刀菌,因此符合科赫假说。据报道,镰刀菌(Fusarium falciforme)曾在多个国家的不同寄主上引起多种病害,包括韩国(Kang 等人,2024 年)、马来西亚(Balasubramaniam 等人,2023 年)和墨西哥(Payán-Arzapalo 等人,2024 年)。据我们所知,这是 F. falciforme 在中国引起辣椒镰刀菌枯萎病的首次报道。研究结果可为今后研究该病害的发生、预防和管理提供依据。
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First Report of Fusarium falciforme Causing Fusarium Wilt on pepper in Hainan, China.

Pepper (Capsicum annuum L.) is a significant vegetable crop, valued for its nutritional and economic importance (Pang et al. 2023). Pepper cultivation in China accounts for about 8-10% of the total vegetable planting area, contributing an output value of approximately 250 billion yuan. This makes pepper the leading vegetable in terms of both planting area and economic value. In December 2023, a total of 70% disease incidence of Fusarium wilt was observed in a 1200 m² pepper seed breeding base in Sanya City, Hainan Province, China (18°38'60″ N, 109°16'51″ E). Symptoms initially appeared as wilting on upper leaves. Subsequently, the base of the stem started to necrosis, browning, and gradually spreading upward along the stem. As the lesions expanded, the whole plant gradually wilted and died. Ten diseased plants were randomly selected from the most severely affected area (667 m²). Diseased tissues (5 mm²) were subsequently removed from the lesion edges of these plants, surface sterilized in 75% ethanol for 30 s, and rinsed with sterile distilled water three times, finally cultured on potato dextrose agar (PDA) at 25 °C. Six fungal isolates were obtained using the single-spore isolation method (HN-01 to HN-06). Colonies produced white aerial mycelia with apricot pigments in the PDA medium. The spore morphology and size were observed and measured using synthetic nutrient-poor agar (SNA) medium. Macroconidia were hyaline, slightly curved in shape with 3 or 4 septa, measuring 28.6 to 41.4 × 3.2 to 6.2 μm (av. = 34.8 ± 3.32 × 4.6 ± 0.85 um, n = 20). Microconidia were elongated, oval with 0 or 1 septum, and measured 11.2 to 16.8 × 2.6 to 5.8 μm (av. = 13.5 ± 1.47 × 4.12 ± 1.03 um, n = 20). Chlamydospores were spherical, terminal or intercalary, solitary or chain-forming, with diameters ranging from 2.8 to 10.5 um (av. = 5.8 ± 2.31 um, n = 20). For molecular identification, genomic DNA from all six isolates was extracted using the cetyl trimethyl ammonium bromide (CTAB) method, and the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α), and RNA polymerase II beta subunit (RPB2) regions were amplified and sequenced using the primers ITS1/ITS4, EF-1/EF-2, and RPB2-5F/7cR (White et al. 1990; O'Donnell et al. 2010). The sequences were deposited in GenBank (ITS: PP779839, PP779840, PP779841, PP779842, PP779843, PP779844; EF1-α: PP797138, PP797139, PP797140, PP797141, PP797142, PP797143; RPB2: PP797144, PP797145, PP797146, PP797147, PP797148, PP797149). The sequences of all three genes showed 99 to 100% similarity with Fusarium falciforme and other closely related Fusarium species (ITS: PP735125, EF1-α: OP163897 and RPB2: MF467484). A maximum likelihood phylogenetic tree was constructed based on the ITS, EF1-α, and RPB2 sequences of all isolates, along with other closely related Fusarium species. Based on morphological and phylogenetic characteristics, all isolates were identified as F. falciforme (Xu et al. 2023; Wang et al. 2023). Ten Pepper inbred line A23-41 (5 for the inoculation treatment and 5 for the control) were tested for pathogenicity using three representative isolates: HN-01, HN-02, and HN-03. A total of 20 ul of spore suspension with concentration 106 spores/ml was injected near the soil surface of the stem, while the control treatment was inoculated with 20 μl of sterile water. The plants were placed in a climatic chamber at RH80-90% and 25/20 °C (day/night) after inoculation. The experiment was repeated three times. After 10 days, Inoculated plants stem developed necrosis, browning, while the control group remained asymptomatic. The fungus reisolated from the artificially infected stems was identified through EF1-α and RPB2 sequence analysis as F. falciforme, thus fulfilling Koch's postulates. Fusarium falciforme has been previoulsy reported as causing various diseases on different hosts in several countries, including Korea (Kang et al., 2024), Malaysia (Balasubramaniam et al., 2023), and Mexico (Payán-Arzapalo et al., 2024). To the best of our knowledge, this is the initial report demonstrating that F. falciforme causes Fusarium wilt on peppers in China. The study results can provide the basis for future research on the occurrence, prevention, and management of this disease.

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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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