苹果腐烂病菌拮抗放线菌的筛选、鉴定及发酵条件的优化
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摘要
苹果腐烂病是由Valsa mali Miyable et Yamada引起的一种真菌性病害。中国于1916年在辽宁南部发现该病,1948-1949年及1960年曾两度在辽南地区大流行,造成了严重的经济损失。随着化学农药引起的环境污染以及人们公认的3R(抗性、残毒、再猖獗)问题的出现,利用自然环境中的拮抗微生物防治植物病害愈来愈受到人们的重视。本文旨在从广东省中山市采集的土样中分离、筛选出对苹果腐烂病菌有较强拮抗作用的放线菌,并对拮抗菌株进行初步鉴定及对其发酵条件进行研究,以期为苹果腐烂病的防治提供新的解决方案。主要研究内容及结果如下:
1对从广东省采集的土样进行热处理及化学处理,以降低常规放线菌的出现几率,然后通过稀释平板法对原土样及处理土样同时进行分离,共获得放线菌293株。其中,未处理、热处理及苯酚处理的土样分离得到的菌株数量分别为214、22、57株,试验结果表明,经过一定处理的土样分离得到菌株的数量明显减少,同时稀有放线菌的数量可能有所增加。
2以苹果腐烂病菌为指示菌对分离菌株进行筛选,平板对峙法初筛得到有抑制作用的菌株61株,其中抑菌率在50%以上的14株,占总数的4.78%;管碟法进一步对拮抗效果明显的菌株进行复筛,试验数据表明,菌株Z-6的发酵滤液对苹果腐烂病菌抑制作用最显著,抑菌圈直径达32.87 mm。为了测定该菌株对苹果腐烂病的防治效果,本文还进行了苹果离体枝条防治效果试验,结果表明,该菌株的防治效果达32.9%。
3采用插片培养的方法,以观察菌株的培养特征、形态特征尤其是孢子及孢子丝的形态特征为主,生理生化特性、抑菌谱测定为辅的传统方法,初步鉴定菌株Z-6为链霉菌属(Streptomyces spp.)的金色类群(Aureus)。同时,抑菌谱测定结果表明,菌株Z-6不仅对苹果腐烂病菌抑制效果明显,对其他16种供试病原真菌同样有一定程度的抑制作用。尤其对小麦全蚀病菌、番茄灰霉病菌和稻瘟病菌效果明显,抑制率均在70%以上。
4经平板对峙培养发现,苹果腐烂病菌生长缓慢,周围有红褐色物质产生,说明菌株Z-6产生或分泌某种物质从而抑制病原菌的生长。挑取病原菌菌苔边缘菌丝镜检,发现菌丝表现为念珠状、畸形、扭曲,顶端中空、膨胀,形成大量囊状体。在发酵滤液对病原菌丝生长的测定中,菌株Z-6发酵原液基本完全抑制病原菌菌丝的生长,其作用强度随无菌滤液稀释倍数的增加而减弱。稀释1000倍液时基本无抑制作用。综上所述,菌株Z-6对病原菌的作用机理是产生抗菌物质直接破坏病原菌菌丝正常生长。
5为了提高菌株Z-6抑菌活性物质的产量,增强其对苹果腐烂病菌的拮抗效果,本文通过正交设计试验,对菌株Z-6的发酵培养基的成分及发酵条件进行优化。最终确定培养基最佳配方为:葡萄糖35.5g/L,玉米粉30.0 g/L,黄豆饼粉27.7g/L,碳酸钙3.0 g/L,淀粉10.0g/L,氯化铵3.0g/L;其最佳培养条件为:菌龄为120h,接种量4个(φ5 mm),发酵时间72h,培养基初始pH 6.0,培养基装量80 mL(250mL三角瓶),发酵温度28℃,摇床转速180 r/min。
Apple canker caused by Valsa mali Miyable et Yamada is an important fungal disease, Which discovered in 1916 in china. It has happened more and more seriously in south of Liaoning area in 1948-1949 and1960 and has already caused great economic losses. Along with the appearance of environmental pollution caused by chemical pesticide and 3R including Resistance, Residue, Resurgence, Utilizing natural antagonistic microorganisms is a potential safe tool for crop protection and offers a promising alternative. The most aim of this experiment is to isolate and screen the antagonistic actinomyces from the soil , Which collected from zhongshan city, guangdong province, In the test, we make further research on the identifing, optimizing of the fermention condition of the antagonistic actinomyces. The main content and results are as follows:
1 In order to decrease the common actinomyces, The soil was treated by thermotherapy and chemotherapy method. In this study, Two hundred and ninty-three strains of actinomyces were isolated with dilution isolation method. The quantities of the soil unsettled, thermotherapy and chemotherapy treatment is hundred and fourteen, twenty-two, fifty-seven respectively. The experimental data indicates that the quantities of actinomyces isolated from treated soil decreased obviously, At the same time, The rare actinomyces may be increase.
2 Using confronted culture method we had separated sixty-one actinomyces antagonistic to Valsa mali. In which inhibition rate of fourteen strains are above 50%, 4.78% of total. Using cylinder plate method we discovered one actinomyces of the strain Z-6 in the following selection , The results showed that this strain inhibited Valsa mali growing obviously, Inhibition zone account for 32.87 mm. In order to inditify the effection of the strain Z-6, We did experiment in further, In the mensuration of excised twigs Z-6 also showed evident effection. The inhibition rate is 32.9%.
3 According to the cultural, morphological characteristics, such as spore and spore chains, as well as physiological, biochemical characteristics and inhibition spectrum test, The strain Z-6 cultured using filling-in bits culture was identified as the type of Aureus, belonging to the genus of Streptomyces. At the same, In the following test of antagonistic spectrum of the strain Z-6, The strain could not only inhibit the growth of the Valsa Mali, but also effect other sixteen plant pathogenic fungi, especially on Gaeumannomyces graminis, Botrytis cinerea, and Pyricularia grisea, Which Inhibition rate are abve 70%.
4 The test on confronted culture showed that the pathogen grow slowly and the red substance appear around, It indicate the strain product or excrete some substance inhibite the pathogen growing. Through obserbation under the microscope we know that there were a lot of vesicles like prayer beads appearing in the apex and middle of the mycelia of both pathogens, dilate, deforaiedand formed empty vesicles. a series of diluted culture supernatant of the strain Z-6 from zero to one thousand time had been tested and the results showed that the hypha can hardly grow at zero time, one thousand time was completely lose inhibition effect, In conclusion, The inhibition mechanism is Producting anti-fungal substance to restrain the pathogen nutural growing.
5 In order to increase the production of the fungicidal substance, the liquid media composition was optimized and the fermentation condition was primarily studied. The optimum fermentation medium of the strain Z-6 was composed of com powder 30.0g/L, soybean cake powder 27.7 g/L, glucose 35.5 g/L, starch 10.0 g/L, CaCO_3 3.0 g/L, NH_4Cl 3.0g/L. The optimum fermentation condition was the initial pH of medium is 6.0, fermentation tempertarure 28°C, inoculum size is four (Φ5 mm), liquid volue 50 mL(in 250 mL flask), strain culture time 120 h, 210 r/min for 108 h.
1对从广东省采集的土样进行热处理及化学处理,以降低常规放线菌的出现几率,然后通过稀释平板法对原土样及处理土样同时进行分离,共获得放线菌293株。其中,未处理、热处理及苯酚处理的土样分离得到的菌株数量分别为214、22、57株,试验结果表明,经过一定处理的土样分离得到菌株的数量明显减少,同时稀有放线菌的数量可能有所增加。
2以苹果腐烂病菌为指示菌对分离菌株进行筛选,平板对峙法初筛得到有抑制作用的菌株61株,其中抑菌率在50%以上的14株,占总数的4.78%;管碟法进一步对拮抗效果明显的菌株进行复筛,试验数据表明,菌株Z-6的发酵滤液对苹果腐烂病菌抑制作用最显著,抑菌圈直径达32.87 mm。为了测定该菌株对苹果腐烂病的防治效果,本文还进行了苹果离体枝条防治效果试验,结果表明,该菌株的防治效果达32.9%。
3采用插片培养的方法,以观察菌株的培养特征、形态特征尤其是孢子及孢子丝的形态特征为主,生理生化特性、抑菌谱测定为辅的传统方法,初步鉴定菌株Z-6为链霉菌属(Streptomyces spp.)的金色类群(Aureus)。同时,抑菌谱测定结果表明,菌株Z-6不仅对苹果腐烂病菌抑制效果明显,对其他16种供试病原真菌同样有一定程度的抑制作用。尤其对小麦全蚀病菌、番茄灰霉病菌和稻瘟病菌效果明显,抑制率均在70%以上。
4经平板对峙培养发现,苹果腐烂病菌生长缓慢,周围有红褐色物质产生,说明菌株Z-6产生或分泌某种物质从而抑制病原菌的生长。挑取病原菌菌苔边缘菌丝镜检,发现菌丝表现为念珠状、畸形、扭曲,顶端中空、膨胀,形成大量囊状体。在发酵滤液对病原菌丝生长的测定中,菌株Z-6发酵原液基本完全抑制病原菌菌丝的生长,其作用强度随无菌滤液稀释倍数的增加而减弱。稀释1000倍液时基本无抑制作用。综上所述,菌株Z-6对病原菌的作用机理是产生抗菌物质直接破坏病原菌菌丝正常生长。
5为了提高菌株Z-6抑菌活性物质的产量,增强其对苹果腐烂病菌的拮抗效果,本文通过正交设计试验,对菌株Z-6的发酵培养基的成分及发酵条件进行优化。最终确定培养基最佳配方为:葡萄糖35.5g/L,玉米粉30.0 g/L,黄豆饼粉27.7g/L,碳酸钙3.0 g/L,淀粉10.0g/L,氯化铵3.0g/L;其最佳培养条件为:菌龄为120h,接种量4个(φ5 mm),发酵时间72h,培养基初始pH 6.0,培养基装量80 mL(250mL三角瓶),发酵温度28℃,摇床转速180 r/min。
Apple canker caused by Valsa mali Miyable et Yamada is an important fungal disease, Which discovered in 1916 in china. It has happened more and more seriously in south of Liaoning area in 1948-1949 and1960 and has already caused great economic losses. Along with the appearance of environmental pollution caused by chemical pesticide and 3R including Resistance, Residue, Resurgence, Utilizing natural antagonistic microorganisms is a potential safe tool for crop protection and offers a promising alternative. The most aim of this experiment is to isolate and screen the antagonistic actinomyces from the soil , Which collected from zhongshan city, guangdong province, In the test, we make further research on the identifing, optimizing of the fermention condition of the antagonistic actinomyces. The main content and results are as follows:
1 In order to decrease the common actinomyces, The soil was treated by thermotherapy and chemotherapy method. In this study, Two hundred and ninty-three strains of actinomyces were isolated with dilution isolation method. The quantities of the soil unsettled, thermotherapy and chemotherapy treatment is hundred and fourteen, twenty-two, fifty-seven respectively. The experimental data indicates that the quantities of actinomyces isolated from treated soil decreased obviously, At the same time, The rare actinomyces may be increase.
2 Using confronted culture method we had separated sixty-one actinomyces antagonistic to Valsa mali. In which inhibition rate of fourteen strains are above 50%, 4.78% of total. Using cylinder plate method we discovered one actinomyces of the strain Z-6 in the following selection , The results showed that this strain inhibited Valsa mali growing obviously, Inhibition zone account for 32.87 mm. In order to inditify the effection of the strain Z-6, We did experiment in further, In the mensuration of excised twigs Z-6 also showed evident effection. The inhibition rate is 32.9%.
3 According to the cultural, morphological characteristics, such as spore and spore chains, as well as physiological, biochemical characteristics and inhibition spectrum test, The strain Z-6 cultured using filling-in bits culture was identified as the type of Aureus, belonging to the genus of Streptomyces. At the same, In the following test of antagonistic spectrum of the strain Z-6, The strain could not only inhibit the growth of the Valsa Mali, but also effect other sixteen plant pathogenic fungi, especially on Gaeumannomyces graminis, Botrytis cinerea, and Pyricularia grisea, Which Inhibition rate are abve 70%.
4 The test on confronted culture showed that the pathogen grow slowly and the red substance appear around, It indicate the strain product or excrete some substance inhibite the pathogen growing. Through obserbation under the microscope we know that there were a lot of vesicles like prayer beads appearing in the apex and middle of the mycelia of both pathogens, dilate, deforaiedand formed empty vesicles. a series of diluted culture supernatant of the strain Z-6 from zero to one thousand time had been tested and the results showed that the hypha can hardly grow at zero time, one thousand time was completely lose inhibition effect, In conclusion, The inhibition mechanism is Producting anti-fungal substance to restrain the pathogen nutural growing.
5 In order to increase the production of the fungicidal substance, the liquid media composition was optimized and the fermentation condition was primarily studied. The optimum fermentation medium of the strain Z-6 was composed of com powder 30.0g/L, soybean cake powder 27.7 g/L, glucose 35.5 g/L, starch 10.0 g/L, CaCO_3 3.0 g/L, NH_4Cl 3.0g/L. The optimum fermentation condition was the initial pH of medium is 6.0, fermentation tempertarure 28°C, inoculum size is four (Φ5 mm), liquid volue 50 mL(in 250 mL flask), strain culture time 120 h, 210 r/min for 108 h.
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