| 衡 周 1，孙小晴 1,2，黄俊霖 1，孙保娟 1，李植良 1，李 颖 1，李 涛 1.茄子褐纹病菌的分离鉴定及产孢条件的优化[J].广东农业科学,2020,47(6):56-62 |
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茄子褐纹病菌的分离鉴定及产孢条件的优化 |
| Isolation and Identification of Phomopsis vexans Harter and Optimization of Sporulation Conditions |
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| DOI：10.16768/j.issn.1004-874X.2020.06.008 |
| 中文关键词: 褐纹病菌 分子鉴定 液体培养 产孢 条件优化 |
| 英文关键词: Phomopsis vexans molecular identification liquid culture sporulation condition optimization |
| 基金项目:广东省自然科学基金（2020A151501337）；广东省农业科学院“十三五”学科团队项目（201630TD）；广东省农业科学院院长基金（201812）；广东省农业厅岗位专家项目（2018kczx06，2019KJ106，2019KJ110）；肇庆市科信局项目（2019N008） |
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| 中文摘要: |
| 【目的】由茄褐纹拟茎点霉（Phomopsis vexans Harter）引起的褐纹病是当前茄子产业的重要病害，孢子富集是深入研究褐纹病菌孢子萌发及其致病机理的基础。进一步简化当前复杂的产孢过程，提高产孢效率。【方法】采用组织分离法开展病原菌的分离纯化，利用形态学特征观察及 rDNA-ITS 序列分析对病原菌进行鉴定；使用液体培养的方式，对茄子褐纹病菌的产孢条件进行优化。【结果】分离获得的病原菌 rDNA-ITS 序列经NCBI 序列比对，与茄子褐纹病菌遗传关系最近，同源性达 99%；相同培养条件下培养 1 d 后，液体培养基中的菌丝生长量较大；28 ℃培养 1 d 24 ℃培养 3 d 后的产孢量为 1.2×1010 个 /mL，大于 24 ℃培养 4 d 的产孢量（6×107个 /mL）；不同培养温度产孢量分别为 2.5×107（20 ℃）、1.2×1010（22 ℃）、9.4×109（24 ℃）、8.5×108（26 ℃）个 /mL；相同培养条件下，25% 葡萄糖马铃薯液体（PDB）培养基产孢量最高（1.2×1010 个 /mL）。【结论】分离并鉴定了茄褐纹病病原菌，优化了褐纹病菌产孢条件。优化后的产孢条件为：在 150 r/min 摇床中，使用 25%葡萄糖 PDB 培养基 28℃培养 1 d 后 24℃培养 3 d。 |
| 英文摘要: |
| 【Objective】Phomopsis blight of eggplant caused by Phomopsis vexans Harter is an important disease in the current eggplant industry. A sufficient amount of spores is the material basis for studying the spore germination of P. vexans and its pathogenic mechanism. This research is aimed to further simplify the current complex sporulation process and improve the sporulation efficiency.【Method】The tissue separation method was used to isolate and purify the pathogen. The morphological characteristics observation and rDNA-ITS sequence analysis were used to identify the pathogen. The liquid culture method was used to optimize the sporulation conditions of P. vexans.【Result】The rDNA-ITS sequence of P. vexans was obtained. The sequence had the closest genetic relationship with P. vexans through NCBI and sequence alignment, with a homology of 99%. After cultivation for one day under the same condition, the mycelial growth in the liquid medium was better. The spore production after culture at 28 ℃ for one day then 24 ℃ for three days was 1.2 × 1010 spores/mL, which was larger than the spore production（6 × 107 spores/mL）after culture at 24 ℃ for four days. The spore production at different culture temperatures were 2.5×107（20 ℃）, 1.2×1010（22 ℃）, 9.4×109（24 ℃）and 8.5×108（26 ℃）spores/mL, respectively. Under the same culture condition, the 25% glucose PDB medium had the highest spore production（1.2×1010 spores/mL）.【Conclusion】The pathogens of P. vexans were isolated and identified, and the sporulation conditions were optimized. The optimized sporulation conditions were as follows: in a 150 r/min shaker, using the 25% glucose PDB medium to culture at 28 ℃ for one day, and then culture at 24 ℃ for three days. |
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