植物提取物对草莓灰霉病菌等2种采后病原真菌的

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3.0 陈辉 2024-11-19 9 4 3.39MB 57 页 15积分

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摘要

以草莓灰霉病菌 Botrytis cinerea 和桃褐腐病菌 Monilinia fructicola 为供试菌，

在植物材料质量浓度为 20mg/mL 条件下，分别用不同极性溶剂不同提取方法提取

银杏、香樟、蒲公英、牵牛花、牵牛子、夹竹桃等 6 种植物获得的植物提取物,采

用菌丝生长速率法测定了其对草莓灰霉病菌和桃褐腐病菌的抑制活性，进而筛选

出抑菌活性最强的植物提取物，并测定其对供试病原菌孢子萌发的抑制作用；通

过药液培养法测定不同浓度下该植物提取物对菌丝体细胞膜透性、内含物渗漏及

细胞壁降解酶活性的影响，初步探讨该提取物对供试菌的抑菌活性及相关机理；

并采用活体组织法研究了该提取物对采后草莓、桃发病率、病斑直径及抗病相关

性酶活力的影响。研究结果如下：

1. 在植物材料质量浓度为 20mg/mL 条件下，虽然同种溶剂不同提取方法对病

原菌的抑制活性相差不大，但间歇振荡法效果最好，因此确定间歇振荡法为提取

方法。

6种植物不同极性溶剂提取物对草莓灰霉病菌和桃褐腐病菌均有一定的抑制

作用，香樟叶石油醚提取物、香樟叶乙酸乙酯提取物、香樟叶 95%乙醇提取物和

蒲公英乙酸乙酯提取物对 2种病原菌的抑菌率均在 70%以上；其中香樟叶石油醚

提取物抑菌率大于 90%。经线性回归计算 50%抑制时对病原菌的有效浓度最低的

为石油醚香樟提取物，EC50 值分别为 1.89 mg/mL 和2.99 mg/mL。

2. 当香樟叶提取物浓度大于 5mg/mL 时，香樟叶提取物能强烈的抑制 2种病

原菌（草莓灰霉病菌 Botrytis cinerea 和桃褐腐病菌 Monilinia fructicola）分生孢子

萌发，抑制率均大于 70%；20mg/mL 的处理浓度对孢子萌发抑制效果显著，抑制

率均在 90%以上；10mg/mL 的处理浓度效果次之，但抑制率也在 80%以上。

3. 当香樟叶提取物浓度为 20mg/mL 时，能显著破坏病菌细胞膜的选择通透

性，导致培养液中电解质、可溶性糖和可溶性蛋白含量增加，并且能显著抑制菌

丝体产生的多聚半乳糖醛酸酶（PG）、甲胶甲基半乳糖醛酸酶(PMG)和果胶甲基

反式消除酶（PMTE）三种细胞壁降解酶活性，且酶活抑制率与处理浓度成正比。

4. 利用针刺法，在常温下测定草莓果实和桃果实接种提取物后对相应病害的

防治效果。试验表明，香樟叶提取物能有效抑制采后草莓果实和桃果实相应病害

的发病率和病斑直径，其防治效果与香樟叶的处理浓度成正比，

100mg/mL 浓度处

理的果实完全抑制病菌对草莓灰霉病和桃褐腐病的发生。

5. 观测接种香樟叶提取物和病原菌对相应果品抗病相关性酶苯丙氨酸解氨酶

（PAL）、多酚氧化酶（PPO）和过氧化物酶（POD）活性的影响。结果表明，仅

刺伤和仅接种病原菌处理果实，均能提高 PAL、PPO 和POD 的活性，但果实接种

香樟叶提取物+病原菌后，PAL、PPO 和POD 的活性进一步升高，并在整个试验

过程中保持较高水平。说明香樟叶提取物对果实病害的抗性，不仅与提取物能破

坏菌丝体本身有关，还可能与提取物能诱导果实自身防御酶活性，提高果实抗病

性有关。

关键词：香樟叶提取物草莓灰霉病菌桃褐腐病菌抑菌机理生物

防治

ABSTRACT

In order to study antifungal ability of extracts from Ginkgo,Cinamomum

camphora,Dandelion, Morning glory,Pharbitidis Seed,Oleander by different solvents

and different extract methods on Botrytis cinerea and Monilinia fructicola, the toxicity

action of plant extracts to B. cinerea and M. fructicola were evaluated by using mycelial

growth rate method at the extracts concentration of 20mg/mL in vitro. To explore the

antimicrobial activity of the selected plant extract on B. cinerea,M. fructicola and the

modes of action. The best inhibiting effect extracts was evaluated by spore germination

on slide glass in vitro. Penetrability of cell membranes, mycelial leakage of sugar,

protein and cell wall degrading enzymes was tested by liquid cultivates method. In

addition, the effects of the selected plant extract on disease incidences、lesion diameters

and enzymes activity which related with disease resistance were tested in vivo. The

main conclusions are as follows:

1. As far as antifungal activity was concerned ，the antifungal activity of every

method extract was similar by the same solvent，but interval oscillation method were a

little better. At concentration of 20mg/mL, all of them had ability to inhibit the mycelial

growth of B. cinerea and M. fructicola, and the inhibitory rate of cinamomum camphora

leaves extracts by petroleum ether, cinamomum camphora extracts leaves by ethyl

acetate, Cinamomum camphora leaves extracts by 95% ethanol and Dandelion extract

by ethyl acetate had more than 70% on all tested fungi, especially, the extracts of

cinamomum camphora leaves by petroleum ether had the best inhibiting effect above

90%. By line regression, cinamomum camphora leaves extracts by petroleum ether had

best antifungal ability to B. cinerea and M. fructicola, 50% of effective concentration

(EC50) were 1.89 mg/mL and 2.99 mg/mL。

2. The cinamomum camphora leaves extracts concentration higher 5mg/mL had

good antifungal properties; they all have more than 70% inhibiting rates to the spore

germination of 2 kinds of fungi. At the concentration of 20mg/mL, the extracts of

cinamomum camphora leaves had more than 90% inhibiting rates. At the concentration

of 10mg/mL, the extracts of cinamomum camphora leaves also showed good antifungal

properties but with less extent, they all had more than 80% inhibiting rates.

3. At the concentration of 20mg/mL, The cinamomum camphora leaves extracts

could disrupt the penetrability of cell membranes that resulted in the increase of soluble

sugar, protein and electrolyte in liquid cultivate, and inhibit the cell wall degrading

enzymes(PG、PMG、PMTE) activities, the efficacy was positively correlated with the

concentration.

4. The controlling effects of cinamomum camphora leaves extracts screened

previously on fruit rots were determined in strawberry and peach fruit during storage. In

vivo, the cinamomum camphora leaves extracts could significantly reduce disease

incidences and lesion diameters, and the inhibition of fruit decay was closely correlated

with extracting solution concentrations, it could control B. cinerea rot of strawberry

friut and M. fructicola rot of peach fruit completely at 100mg/mL.

5. Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine

ammonium-lyase (PAL) activities induced by inoculation with cinamomum camphora

leaves extracts and pathogen fungi were studied in strawberry fruit and peach fruit. The

results showed fruit inoculated with artificial wound alone or pathogen fungi alone

could stimulate PAL, PPO and POD activity. But, the activities of PPO,POD and PAL in

fruit increased significantly when inoculated with cinamomum camphora leaves extracts

and pathogen fungi and maintained at a high level throughout the experiment. The

results suggest that the activation of these defense enzymes might involve in the action

of cinamomum camphora leaves extracts against pathogen fungi.

Key words: Cinamomum camphora leaves extracts, strawberry, Botrytis

cinerea, peach, Monilinia fructicola, Mechanism of inhibition,

biological control

摘要

ABSTRACT

第一章绪论.....................................................................................................................1

§1.1 研究背景与意义................................................................................................1

§1.2 水果采后主要病原真菌....................................................................................2

§1.2.1 灰葡萄孢菌（Botrytis cinerea）............................................................ 2

§1.2.2 褐腐病菌（Monilinia fructicola）......................................................... 2

§1.2.3 青霉（Penicillium expansum）.............................................................. 2

§1.2.4 链格孢菌（Alternaria alternata）......................................................... 2

§1.2.5 根霉菌（Rhizopus）.............................................................................3

§1.2.6 炭疽病菌（Colletotrichum）................................................................. 3

§1.3 控制水果采后病害常见的方法........................................................................3

§1.3.1 物理方法..................................................................................................3

§1.3.2 化学杀菌剂..............................................................................................5

§1.3.3 生物防治..................................................................................................5

§1.4 植物源杀菌剂用于水果采后病害控制的研究进展........................................7

§1.5 本课题的研究目的和内容................................................................................9

第二章植物提取物对 2种采后病原真菌抑菌活性筛选 ...........................................11

§2.1 引言..................................................................................................................11

§2.2 材料与方法......................................................................................................11

§2.2.1 供试材料................................................................................................11

§2.2.2 供试菌种................................................................................................11

§2.2.3 实验仪器与试剂....................................................................................12

§2.2.4 试验方法................................................................................................12

§2.2.4 统计分析................................................................................................13

§2.3 结果与分析......................................................................................................14

§2.3.1 供试植物提取物对草莓灰霉病菌的抑制作用....................................14

§2.3.2 供试植物提取物对桃褐腐病菌的抑制作用.......................................17

§2.4 讨论..................................................................................................................20

§2.5 本章小结..........................................................................................................20

第三章香樟叶提取物抑菌作用机理研究...................................................................21

§3.1 引言..................................................................................................................21

§3.2 材料与方法......................................................................................................21

§3.2.1 供试材料................................................................................................21

§3.2.2 供试菌种................................................................................................21

§3.2.3 实验仪器与试剂...................................................................................21

§3.2.4 试验方法................................................................................................22

§3.2.5 统计分析................................................................................................25

§3.3 结果与分析......................................................................................................25

§3.3.1 香樟叶提取物对病原菌孢子萌发抑制................................................25

§3.3.2 香樟叶提取物对病原菌菌丝体细胞膜透性的影响............................25

§3.3.3 香樟叶提取物对病原菌菌丝体内含物渗漏的影响............................26

§3.3.4 香樟叶提取物对病原菌菌丝体细胞壁降解酶活性的影响................27

§3.4 讨论..................................................................................................................29

§3.5 本章小结..........................................................................................................30

第四章香樟叶提取物对果品采后病害的防治作用及抗病性相关酶的活性的影响

.........................................................................................................................................32

§4.1 引言..................................................................................................................32

§4.2 材料与方法......................................................................................................32

§4.2.1 供试材料................................................................................................32

§4.2.2 供试菌种................................................................................................32

§4.2.3 实验设备与试剂...................................................................................32

§4.2.4 供试果品................................................................................................32

§4.2.5 试验方法................................................................................................32

§4.2.6 统计分析................................................................................................33

§4.3 结果与分析......................................................................................................33

§4.3.1 香樟叶提取物对采后实验果品病害的防治效果................................33

§4.3.2 香樟叶提取物对采后实验果品 PAL 活性的影响 .............................. 36

§4.3.3 香樟叶提取物对采后实验果品 PPO 活性的影响 .............................. 36

§4.3.4 香樟叶提取物对采后实验果品 POD 活性的影响 ..............................37

§4.4 讨论..................................................................................................................38

§4.5 本章小结..........................................................................................................38

第五章结论与展望.......................................................................................................40

§5.1 主要结论..........................................................................................................40

§5.2 展望与建议......................................................................................................41

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摘要：

摘要以草莓灰霉病菌Botrytiscinerea和桃褐腐病菌Moniliniafructicola为供试菌，在植物材料质量浓度为20mg/mL条件下，分别用不同极性溶剂不同提取方法提取银杏、香樟、蒲公英、牵牛花、牵牛子、夹竹桃等6种植物获得的植物提取物,采用菌丝生长速率法测定了其对草莓灰霉病菌和桃褐腐病菌的抑制活性，进而筛选出抑菌活性最强的植物提取物，并测定其对供试病原菌孢子萌发的抑制作用；通过药液培养法测定不同浓度下该植物提取物对菌丝体细胞膜透性、内含物渗漏及细胞壁降解酶活性的影响，初步探讨该提取物对供试菌的抑菌活性及相关机理；并采用活体组织法研究了该提取物对采后草莓、桃发病率、病斑直径及抗...

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