红豆树异黄酮类成分抑制番茄灰霉病菌的活性研究

潘镇泽; 傅佳蕊; 郑威; 耿帅丽; 张琳婧; 徐会有; 倪林

doi:10.19303/j.issn.1008-0384.2022.06.015

红豆树异黄酮类成分抑制番茄灰霉病菌的活性研究

doi: 10.19303/j.issn.1008-0384.2022.06.015

潘镇泽^1,,
傅佳蕊¹,
郑威¹,
耿帅丽¹,
张琳婧¹,
徐会有¹,
倪林^{1, 2, ,}

1.
福建农林大学植物保护学院，福建　福州　350002
2.
自然生物资源保育利用福建省高校工程研究中心，福建　福州　350002

基金项目: 全国农业教指委研究课题（2021-NYYB-12）；福建农林大学杰青人才项目（xjq202103）

详细信息

作者简介:
潘镇泽（1998−），男，硕士研究生，研究方向：活性天然产物的开发与利用（E-mail：panzhenze123@163.com）

通讯作者:
倪林（1986−），男，博士，副教授，硕士生导师，研究方向：活性天然产物的开发与利用（E-mail：nilin_fjau@126.com）

中图分类号: S 436
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出版历程
- 收稿日期: 2022-04-12
- 修回日期: 2022-05-21
- 网络出版日期: 2022-06-20
- 刊出日期: 2022-06-28

Inhibitory Activity of Isoflavones from Ormosia hosiei Seeds against Botrytis cinerea

1.
College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Fujian Colleges and Universities Engineering Research Institute of Conservation & Utilization of Natural Bioresources, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的研究红豆树提取物及其异黄酮对番茄灰霉病菌的抑菌作用，为植物源抑菌剂的开发和红豆树提取物利用奠定基础。方法采用生长速率法筛选红豆树异黄酮对番茄灰霉病菌的抑制作用，进一步测定异黄酮鹰嘴豆芽素A对番茄灰霉病菌的菌丝干重、细胞膜、还原糖含量、过氧化氢酶、过氧化物酶和超氧化物歧化酶等保护酶活性的影响，探讨抑制植物病原真菌的生理生化的作用，评价单体在离体番茄上的抑菌效果。结果鹰嘴豆芽素A对番茄灰霉病菌具有好的抑制效果，EC₅₀值为203.189 μg·mL^-1。番茄灰霉病菌经鹰嘴豆芽素A处理后，菌丝干重减少，细胞膜的通透性增加，菌体内还原糖含量减少，保护酶含量增多，并且在离体番茄上菌丝生长防治效果较好。结论红豆树种子乙酸乙酯活性部位富含生物碱和黄酮类化学成分，其中异黄酮成分是关键的抑菌活性成分。异黄酮成分发挥抑菌活性的作用方式，可能与其干扰菌丝生长、使菌丝细胞膜正常功能受损以及降低菌丝体内保护酶活性等有关。
- 红豆树 /
- 番茄灰霉病菌 /
- 抑菌活性 /
- 鹰嘴豆芽素A /
- 异黄酮
Abstract: Objective Antifungal activity of the isoflavones of Ormosia hosiei on Botrytis cinerea Pers was investigated for the development of a natural disease control agent on tomato plants. Methods Inhibitory effect of the isoflavones extract from seeds of O. hosiei on the pathogen, B. cinerea, that infects tomato plants was tested according to the in vitro fungal growth rate. Functions of the isoflavone monomer biochanin A on the changes of mycelial dry weight, cell membrane, reducing sugar content, and activities of catalase, peroxidase, superoxide dismutase, and other protective enzymes of B. cinerea were analyzed. Physiological, biochemical, and bacteriostatic effects of the monomer on tomatoes were observed in vitro. Results Biochanin A of O. hosiei significantly inhibited the growth of B. cinerea with an EC₅₀ of 203.189 μg·mL⁻¹. Its presence lowered the dry mycelia weight, cell membrane permeability, and reducing sugar content but rose the protective enzyme activities in hyphae of B. cinerea. Conclusions The ethyl acetate extract from the seeds of O. hosiei was rich in alkaloids and flavonoids. Of which, isoflavones was believed to be the key active antibacterial components that inhibited the fungal growth by interfering the growth, impairing the cell membrane functions, and reducing the protective enzyme activities of the hyphae.
- Ormosia hosiei /
- Botrytis cinerea /
- antifungal activity /
- biochanin A /
- isoflavones

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图 1 红豆树异黄酮单体化合物结构

Figure 1. Chemical structure of isoflavone monomer

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图 2 番茄灰霉病菌菌丝形态影响

Figure 2. Effect of biochanin A on hyphae morphology of B. cinerea

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图 3 鹰嘴豆芽素A对番茄灰霉病菌的干重影响

不同小写字母表示不同处理浓度之间差异显著（P<0.05）。

Figure 3. Effect of biochanin A on dry weight of B. cinerea

Different lowercase letters indicate significant differences among different treatment concentrations (P<0.05).

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图 4 鹰嘴豆芽素A对番茄灰霉病菌MDA的影响

不同小写字母表示同一处理不同时间之间差异显著（P<0.05）。图5~8同。

Figure 4. Effect of biochanin A on MDA of B. cinerea

Different lowercase letters indicate significant differences between different times of the same treatment (P<0.05). Same for Table 5–8.

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图 5 鹰嘴豆芽素A对番茄灰霉病菌还原糖的影响

Figure 5. Effect of biochanin A on reducing sugar of B. cinerea

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图 6 鹰嘴豆芽素A对番茄灰霉病菌的CAT酶影响

Figure 6. Effect of biochanin A on CAT activity of B. cinerea

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图 7 鹰嘴豆芽素A对番茄灰霉病菌的POD酶影响

Figure 7. Effect of biochanin A on POD activity of B. cinerea

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图 8 鹰嘴豆芽素A对番茄灰霉病菌的SOD酶影响

Figure 8. Effect of biochanin A on SOD activity of B. cinerea

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图 9 鹰嘴豆芽素A在离体番茄果实上对番茄灰霉病的治疗作用

Figure 9. Efficacy of biochanin A on B. cinerea-inoculated tomatoes

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图 10 鹰嘴豆芽素A在离体番茄果实上对番茄灰霉病的预防作用

Figure 10. Preventive effect of biochanin A on tomatoes inoculated by B. cinerea

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表 1 红豆树异黄酮单体化合物

Table 1. Isoflavone monomer of O. hosiei

序号 No.	化合物名称 Compound name	序号 No.	化合物名称 Compound name
1	鹰嘴豆芽素A Biochanin A	11	染料木素-7-O-β-D-呋喃芹糖基-(1→6)-O-β-D-吡喃葡萄糖苷 ambocin
2	大豆素 Daidzein	12	5,7-二羟基-4′-甲氧基异黄酮-7-O-β-D-木糖-(1→6)-O-β-D-吡喃葡萄糖苷 Kakkanin
3	染料木素 Genistein	13	4′-甲氧基异黄酮-7-O-β-D-木糖-(1→6)-O-β-D-吡喃葡萄糖苷 Kushenol O
4	异樱黄素 Isoprunetin	14	4′-甲氧基异黄酮-7-O-β-D-芹糖-(1→6)-O-β-D-吡喃葡萄糖苷7-hydroxy-4′-methoxylisoflavone-7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside
5	2′,4′,5,7-四羟基异黄酮 2′,4′,5,7-tetrahydroxyisoflavone	15	3′,4′,7-三羟基-5-甲氧基异黄酮 3',4',7-trihydroxy-5-me-thoxyisoflavone
6	4′,8-二甲氧基-7-O-β-D- 葡萄糖基异黄酮 4′,8-dimethoxyl-7-O-β-D-glucopyranosyl isoflavone	16	染料木苷-5,4′-二甲醚 Genistin-5, 4′-methyl ether
7	降紫香苷 Sissotrin	17	澳白檀苷Lanceolarin
8	芒柄花苷 Ononin	18	鹰嘴豆芽素A-7-O-芸香糖苷 biochanin A-7-O-rutinoside
9	异樱黄素-7-O-β-D-吡喃葡萄糖苷 Isoprunetin-7-O-β-D-glucoside	19	染料木苷 Genistin
10	圆荚草双糖苷 Sphaerobioside	20	2′,4′,7-三羟基-5-甲氧基异黄酮 2',4',7-trihydroxy-5-me-thoxyisoflavone

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表 2 红豆树提取物及单体化合物对番茄灰霉病菌的抑制作用

Table 2. Inhibitory effects of O. hosiei extract and monomer against B. cinerea

序号 No.	化合物名称 Compound name	抑菌率 Antibacterial rate/%
1	鹰嘴豆芽素A Biochanin A	46.74±0.43 b
4	异樱黄素 Isoprunetin	42.14±0.29 c
15	3′,4′,7-三羟基-5-甲氧基异黄酮 3′,4′,7-trihydroxy-5-me-thoxyisoflavone	25.54±0.73 e
16	染料木苷-5,4′-二甲醚 Genistin-5, 4′-methyl ether	29.76±0.35 d
阳性对照 Positive control	丁子香酚 Eugenol	57.79±0.08 a

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表 3 两种化合物对番茄灰霉病菌的毒力测定

Table 3. Toxicity of two compounds on B. cinerea

样品 Sample	毒力曲线 Virulence curve	相关系数 Correlation coefficient (R²)	EC₅₀ / (μg·mL⁻¹）	95%置信度 Confidence level/（μg·mL⁻¹）
丁子香酚（CK） Eugenol(CK)	y=0.787x−1.766	0.989	175.739	128.365～279.500
异樱黄素 Isosakura flavin	y=0.635x−1.504	0.998	232.599	153.616～503.442
鹰嘴豆芽素 Chickpea sprout	y=0.783x−1.807	0.994	203.189	145.968~341.329

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表 4 鹰嘴豆芽素A在离体番茄果实上对番茄灰霉病菌的防治效果

Table 4. Control efficacy of biochanin A against B. cinerea on tomatoes

处理编号 No.	样品质量浓度 Concentration of samples/（mg·mL⁻¹）	先接菌后喷药 First inoculate, then spray		先喷药后接菌 First spray, then inoculate
处理编号 No.	样品质量浓度 Concentration of samples/（mg·mL⁻¹）	病斑面积 Lesion area/cm²	防治效果 Control efficiency/%	病斑面积 Lesion area/cm²	防治效果 Control efficiency/%
A	0(CK)	2.56±0.14 a	—	2.52±1.13 a	—
B	0.5	1.90±0.12 b	26.06	2.15±0.13 a	14.99
C	1.0	0.51±0.05 c	80.04	0.64±0.14 b	74.62
D	2.0	0.21±0.07 d	91.80	0.29±0.09 b	88.60

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