白术根腐病病原菌分离鉴定、生物学特性及植物源农药筛选

谷清义; 张耀洲; 吴晓亚; 黄雅琴; 乔新荣; 申君

doi:10.19303/j.issn.1008-0384.2024.03.010

白术根腐病病原菌分离鉴定、生物学特性及植物源农药筛选

doi: 10.19303/j.issn.1008-0384.2024.03.010

信阳农林学院药学院，河南　信阳　464000

基金项目: 河南省高等学校重点科研项目（21B210009）；信阳农林学院青年教师科研基金（QN2021060）；河南省科技攻关项目（222102110247）

详细信息

作者简介:
谷清义（1982 — ），男，硕士，助教，主要从事农药研究，E-mail：271490029@qq.com

通讯作者:
申君（1984 — ），女，博士，副教授，主要从事农药研究，E-mail：shenjun996@163.com

中图分类号: S436
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- 被引次数: 0
出版历程
- 收稿日期: 2023-10-25
- 修回日期: 2024-01-12
- 网络出版日期: 2024-03-28
- 刊出日期: 2024-03-28

Isolation, Identification, Biological Characteristics, and Fungicide Toxicity of Atractylodes Root Rot Pathogen

School of Pharmaceutical School of Pharmacy, Xinyang Agriculture and Forestry University, Xinyang , Henan 464000, China

摘要

摘要: 目的明确河南信阳地区白术根腐病病原菌种属，研究其生物学特性并筛选可用于防控其病原菌的植物源农药。方法从种植区采集病株，室内分离病原菌并纯化，利用形态学和多基因联合分析对病原菌进行鉴定，并对病原菌的生物学特性进行研究，利用菌丝生长速率法评价3种植物源杀菌剂对其菌落的抑制作用。结果该病原菌菌丝白色，孢子呈卵圆形，两端稍尖，大型分生孢子3～5隔，大小为（7～10） μm×（3～4） μm，小型分生孢子2隔或无格，大小为（3～5） μm×（1～2） μm，多基因联合分析表明该病原菌与藤仓镰刀菌（Fusarium fujikuroi）同源性高，结合形态学特征与分子生物学分析，将信阳白术根腐病病原菌鉴定为藤仓镰刀菌（Fusarium fujikuroi），这是藤仓镰刀菌引起白术根腐病的首次报道。生物学特性研究显示，该病原菌最适培养条件为温度28 ℃、pH为7，最适生长碳源为蔗糖，最适生长氮源为硝酸钾。3种植物源杀菌剂中，0.3%丁子香酚可溶液剂的EC₅₀为6.906 mg·L⁻¹，高于其他供试药剂，对病原菌的毒力最强。结论河南信阳地区白术根腐病病原菌为藤仓镰刀菌，该研究结果为信阳地区白术根腐病科学防控提供了依据。
- 白术 /
- 根腐病 /
- 藤仓镰刀菌 /
- 植物源杀菌剂
Abstract: Objective Pathogen that caused the root rot on Atractylodes macrocephala at Xinyang, Henan Province was isolated, identified, and studied for prevention and control of the disease. Method Morphological and multiple gene analyses were employed to identify the pathogen. Biological characteristics of the isolate were determined. Result The isolate had white hyphae, oval spores with slightly pointed ends, macroconidia sized (7–10) μm× (3–4) μm with 3–5 septa, and microconidia sized (3–5) μm × (1–2) μm with two or no compartments. It showed a high homology with Fusarium fujikuroi and was, for the first time, identified as the fungal pathogen responsible for the root rot disease on Atractylodes macrocephala. It thrived at 28 ℃, pH 7, sucrose for carbon, and potassium nitrate for nitrogen. Of the 3 plant-based fungicides tested, 0.3% eugenol soluble agent exerted the greatest toxicity on the isolate with an EC₅₀ of 6.906 mg·L⁻¹. Conclusion F. fuciformis was identified as the pathogen that caused the root rot disease on A. macrocephala in Henan. The optimal culture conditions for the fungus included 28 ℃, pH 7, sucrose as the carbon source, and potassium nitrate as the nitrogen source.
- Atractylodes macrocephala /
- root rot disease /
- Fusarium fujikura /
- botanical fungicides

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图 1 正常及患根腐病白术

图中1~6分别为正常植株、回接后患病植株、田间患病植株、正常根茎、回接后发病根茎、田间患病根茎。

Figure 1. Healthy and diseased A. macrocephala plants

1–6: Normal plants, diseased plants,diseased plant in field, normal rhizomes, diseased rhizomes and diseased rhizomes in field, respectively.

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图 2 病原菌的形态特征

A为菌落正面；B为菌落反面；C为菌丝；D为大、小型分生孢子。

Figure 2. Morphology of pathogen

A: Face of colony; B: back of colony; C: mycelium; D: macroconidium and microconidium.

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图 3 基于ITS序列构建的系统发育树

Figure 3. Phylogenetic tree based on ITS sequence

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图 4 基于TUB2序列构建的系统发育树

Figure 4. Phylogenetic tree based on TUB2 sequence

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图 5 基于GAPDH序列构建的系统发育树

Figure 5. Phylogenetic tree based on GAPDH sequence

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图 6 基于ACT序列构建的系统发育树

Figure 6. Phylogenetic tree based on ACT sequence

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图 7 不同温度对病原菌生长的影响

A：10、15、25、28、30 ℃条件下培养5 d的菌丝长度；不同字母表示处理间差异达显著水平（P＜0.05），图8～10同。B：1~5分别为10、15、25、28、30 ℃条件下培养5 d的菌落大小。

Figure 7. Effect of temperatures on growth of pathogen

A: Mycelial length cultured at 10, 15, 25, 28, and 30°C for 5 d. Data with different letters indicate significant differences at P＜0.05. Same for Figs. 8–10. B: 1–5 show colonies cultured at 10, 15, 25, 28, and 30 ℃, respectively, for 5 d.

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图 8 不同pH对病原菌生长的影响

A：pH 5、6、7、8、9条件下培养5 d的菌丝长度；B：1~5分别为pH 5、6、7、8、9条件下培养5 d的菌落大小。

Figure 8. Effect of pH on growth of pathogen

A: Mycelial length cultured at pH 5, 6, 7, 8, and 9 for 5 d. B: 1–5 show colonies cultured at pH 5, 6, 7, 8, and 9, respectively, for 5 d.

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图 9 不同碳源对病原菌生长的影响

A：淀粉、乳糖、果糖、蔗糖、葡萄糖、麦芽糖条件下培养5 d的菌丝长度；B： 1~6分别为淀粉、乳糖、果糖、蔗糖、葡萄糖、麦芽糖条件下培养5 d的菌落大小。

Figure 9. Effect of carbon sources on growth of pathogen

A: Mycelial length cultured on soluble starch, lactose, fructose, sucrose, glucose, and maltose, respectively, for 5 d. B: 1–6 show colonies cultured on soluble starch, lactose, fructose, sucrose, glucose, and maltose, respectively, for 5 d.

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图 10 不同氮源对病原菌生长的影响

A：硫酸铵、尿素、硝酸钾、硝酸铵、谷氨酸、丙氨酸条件下培养5 d的菌丝长度；B：1~6分别为硫酸铵、尿素、硝酸钾、硝酸铵、谷氨酸、丙氨酸条件下培养5 d的菌落大小。

Figure 10. Effect of nitrogen sources on growth of pathogen

A: Mycelial length cultured on sodium nitrate, urea, potassium nitrate, ammonium nitrate, glutamine, and alanine, respectively, for 5 d. B: 1–6 show colonies cultured sodium nitrate, urea, potassium nitrate, ammonium nitrate, glutamine, and alanine, respectively, for 5 d.

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表 1 引物信息

Table 1. Information on primer

基因 Gene	引物 Primer	序列 Sequence
ITS	ITS1	5'-TCCGTAGGTGAACCTGCGG-3′
	ITS4	5′-TCCTCCGCTTATTGATATGC-3′
ACT	ACT-512F	5′-ATGTGCAAGGCCGGTTTCGC-3′
	ACT-783R	5′-TACGAGTCCTTCTGGCCCAT-3′
TUB-2	T1	5′-GCCGTCAACGACCCCTTCATTGA-3′
	Bt2	5′-ACCCTCAGTGTAGTGACCCTTGGC-3′
GAPDH	GDF1	5′-GCCGTCAACGACCCCTTCATTGA-3′
	GDR1	5′-GGGTGGAGTCGTACTTGAGCATGT-3′

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表 2 3种植物源农药室内毒力测定浓度设置

Table 2. Concentrations of 3 plant-based pesticides applied for toxicity test in laboratory

供试药剂 Pesticide	药剂质量浓度 Pesticide concentration/（μg·mL⁻¹）
1%蛇床子素EW 1% osthole EW	200、 100、 50、 25、 12.5
0.5%小檗碱AS 0.5% berberine AS	250、 125、 62.5、 31.25、15.625
0.3%丁子香酚SL 0.3% eugenol SL	60、 30、 15、 7.5、 3.75
CK	—

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表 3 3种植物源杀菌剂对白术根腐病病原菌的室内毒力测定

Table 3. Toxicity of plant-based fungicides on Fusarium fujikuroi in laboratory

药剂 Pesticide	毒力回归方程 Toxicity regression equation	R²	EC₅₀ 值 EC₅₀ value/(mg·L⁻¹)
0.3% 丁子香酚SL 0.3% eugenol SL	y=1.2062x+3.9877	0.9919	6.906
0.5% 小檗碱AS 0.5% berberine AS	y=1.0629x+2.7199	0.9907	139.637
1% 蛇床子素EW 1% osthole EW	y=1.2181x+3.5096	0.9909	16.749

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