内蒙芹菜连做障碍微生物修复研究
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摘要
内蒙古太仆寺旗被誉为“中国芹菜第一乡”,它位于内蒙古锡林郭勒盟南部,芹菜种植面积2000多hm2,产值约3亿元。近几年来由于连茬种植,芹菜根腐病大面积发生,一般田块发病率达30%以上,重田块绝收,损失惨重,化学防治效果差,该病害已成为限制当地芹菜种植业发展的瓶颈。为了有效解决该病害的发生,本试验筛选得到对芹菜根腐病具有拮抗作用的高效放线菌菌株,并探讨其对芹菜的防病促生作用。主要得出以下结果:
1.对内蒙古芹菜病株与健株根区、根表土壤及根内的细菌、真菌和放线菌的数量及组成进行了比较分析,研究表明根腐病的发生与根区和根表土壤中微生物区系异常密切相关。就根区土壤而言,发病初期病株细菌和真菌的数量分别较健株有所减少,而发病中、晚期,病株细菌和真菌数量分别较健株有所增加。就根表土壤而言,发病初期细菌数量较健株减少,而发病中期和晚期,细菌数量分别较健株大幅提高。发病初期、中期和晚期根表土壤真菌数量均高于健株。发病初期、中期和晚期,根区和根表土壤中B/F均大幅度减少,表明病原真菌所占比例大幅度提高,土壤由细菌型向真菌型转变。
2.从内蒙古太仆寺旗芹菜种植地染病植株及根区土壤、根表土壤中分离得到病原菌,经过形态学和分子生物学鉴定,确定其为接骨木镰刀菌(Fusarium sambucinum)、链格孢菌(Alternaria alternata)、尖孢镰刀菌萎蔫专化型(Fusarium oxysporum f. sp. Vasinfectum)、芬芳镰刀菌(Fusarium redolen)、黄瓜萎蔫病菌(Plectosphaerella cucumerina)。
3.利用琼脂块法和生长速率法,从621株放线菌中筛选得到5株对芹菜根腐病原菌有较强拮抗作用的生防放线菌,其原始编号分别为A11、A12、A7、A28及A49号。对其进行16S rDNA序列分析,将其确定为肉质链霉菌(Streptomyces carnosus)、密旋链霉菌(Streptomyces pactum),球孢链霉菌(Streptomyces globisporus)、球孢链霉菌的亚种(Streptomyces globisporus subsp. globisporus )及卡那霉素链霉菌。(Streptomyces kanamyceticus)。
4.研究了培养基中加入CaCl2和微波处理的方法对钙质土壤放线菌总数、链霉菌属、小单孢菌属、未鉴定属数量及对拮抗性放线菌的影响。发现向高氏1号培养基上加入CaCl2后大部分供试土壤的放线菌总数及拮抗放线菌数量增加,强拮抗性及广谱拮抗性菌株增加。同样,微波处理后绝大部分供试钙质土壤在GA、GACa及HA 3种培养基上的放线菌总数、链霉菌数、小单孢菌及未鉴定放线菌数量均有明显增加,表明微波处理土壤对放线菌分离时出菌量的提高具有显著作用。
5.盆栽试验表明,单接3株病原菌时,芹菜叶片和根系PPO酶活性、叶片及根系可溶性蛋白含量、芹菜叶片的POD活性、丙二醛含量及芹菜根系活力均较对照显著降低,而芹菜叶片细胞膜的相对透性和伤害率均较对照有所提高。而3株生防放线菌单接时,且在适宜接种量处理下,能显著提高叶片和根系的PPO酶活性、叶片及根系可溶性蛋白含量、芹菜叶片的POD活性以及降低芹菜叶片细胞膜的相对透性和伤害率。当3株生防放线菌与3株病原菌混接时,芹菜叶片和根系PPO酶活性、叶片及根系可溶性蛋白含量、芹菜叶片的POD活性和叶片丙二醛含量及芹菜根系活力都较单接病原菌时显著升高,而芹菜叶片细胞膜的相对透性和伤害率均有所降低。进而证明,3株生防放线菌有助于提高芹菜的诱导抗性,抑制连作病害发生。
The Taibus County located in the southern part of Xilin Gol League is known as“the Chinese celery first township”where the celery planting-area is about 2000hm2and the business about celery can produce about 3 million yuan per year. For the continuous cropping, a kind of celery disease named root-rot often happened recently over large area. The happening frequency of this kind disease is generally more than 30%, and the more serious situation is the completely loss in harvest on the heavy infected region. The traditional chemical pesticide has little effective on this kind disease. The happening of this disease has limited the development of local celery planting. In order to solve this problem effectively, the experiments study on screening higher efficient antagonistic actinomyces, and discusses that it can whether promote the growth of plant, and control the disease of plant or not. The main results are as follow:
1.The occurrence of root rot disease in is closely related to the abnormal of microflora in celery rhizosphere soil and root surface soil in Inner Mongolia. In the rgizosphere soil, the number of bacteria, fungus of diseased plant in initial stage is less than healthy plant, while the number of bacteria, fungus of diseased plant in middle and late stage is much more than healthy plant. In the root surface soil, the number of bacteria of diseased plant in initial stage is less than healthy plant, while that in middle and late stage is much more than healthy plant. In initial, middle and late stage, the number of fungus of diseased plant is much more than healthy plant. In the reduction of the ratio of B / F in the rgizosphere soil and the root surface soil and the proportion of pathogenic fungi increasing and lead to the disease. The type of soil change form bacterium to fungi.
2. From Inner Mongolia Taibus we isolated some fungi form infected plants and rhizosphere soil and root surface of soil. After morphological and molecular biology, we determine these are Fusarium sambucinum, Alternaria alternate, Fusarium oxysporum f. sp. Vasinfectum, Fusarium redolen, Plectosphaerella cucumerina.
3. Using agar block and the growth rate method, we were screened five strong antagonistic former 621 actinomycetes. These stains number are A11, A12, A7, A28 and A49. After we analyzing its 16S rDNA sequence and turned out these are Streptomyces carnosus, Streptomyces pactum , Streptomyces globisporus , Streptomyces globisporus subsp. Globisporus and Streptomyces kanamyceticu.
4.we studyed on the medium by adding CaCl2 and the method of microwave treatment have effect on the total number of actinomycetes, Streptomyces, Micromonospora, not identified kinds and the antagonistic actinomycetes. After adding CaCl2 to the medium, we found that the total number of actinomycetes, antagonistic actinomycetes and strong broad-spectrum of antagonistic strains increased. Similarly, the microwave treatment of tested soil in the GA, GACa and HA 3 kinds of media, we found out the total number of actinomycetes, Streptomyces, Micromonospora and unidentified genus were significantly increased, which show that the microwave could increase the amount of actinomycetes significantly.
5. Pot experiments showed that when inoculating three pathogens, celery leaves and roots PPO enzyme activity and soluble protein content, POD activity and MDA content in leaves were significantly lower than the CK, while the cell membrane permeability and injury rate celery leaves higher than the CK. when inoculating three antagonistic actinomycetes, celery leaves and roots PPO enzyme activity and soluble protein content, POD activity and MDA content in leaves were significantly higher than the CK, while the cell membrane permeability and injury rate celery leaves lower than the CK. when mixed inoculating three pathogens and three antagonistic actinomycetes, celery leaves and roots PPO enzyme activity and soluble protein content, POD activity and MDA content in leaves were significantly higher than inoculated pathogens, while the cell membrane permeability and injury rate celery leaves lower than inoculated pathogens. Further proof, three actinomycetes contribute to the induction of celery resistance and inhibit the disease occurred.
1.对内蒙古芹菜病株与健株根区、根表土壤及根内的细菌、真菌和放线菌的数量及组成进行了比较分析,研究表明根腐病的发生与根区和根表土壤中微生物区系异常密切相关。就根区土壤而言,发病初期病株细菌和真菌的数量分别较健株有所减少,而发病中、晚期,病株细菌和真菌数量分别较健株有所增加。就根表土壤而言,发病初期细菌数量较健株减少,而发病中期和晚期,细菌数量分别较健株大幅提高。发病初期、中期和晚期根表土壤真菌数量均高于健株。发病初期、中期和晚期,根区和根表土壤中B/F均大幅度减少,表明病原真菌所占比例大幅度提高,土壤由细菌型向真菌型转变。
2.从内蒙古太仆寺旗芹菜种植地染病植株及根区土壤、根表土壤中分离得到病原菌,经过形态学和分子生物学鉴定,确定其为接骨木镰刀菌(Fusarium sambucinum)、链格孢菌(Alternaria alternata)、尖孢镰刀菌萎蔫专化型(Fusarium oxysporum f. sp. Vasinfectum)、芬芳镰刀菌(Fusarium redolen)、黄瓜萎蔫病菌(Plectosphaerella cucumerina)。
3.利用琼脂块法和生长速率法,从621株放线菌中筛选得到5株对芹菜根腐病原菌有较强拮抗作用的生防放线菌,其原始编号分别为A11、A12、A7、A28及A49号。对其进行16S rDNA序列分析,将其确定为肉质链霉菌(Streptomyces carnosus)、密旋链霉菌(Streptomyces pactum),球孢链霉菌(Streptomyces globisporus)、球孢链霉菌的亚种(Streptomyces globisporus subsp. globisporus )及卡那霉素链霉菌。(Streptomyces kanamyceticus)。
4.研究了培养基中加入CaCl2和微波处理的方法对钙质土壤放线菌总数、链霉菌属、小单孢菌属、未鉴定属数量及对拮抗性放线菌的影响。发现向高氏1号培养基上加入CaCl2后大部分供试土壤的放线菌总数及拮抗放线菌数量增加,强拮抗性及广谱拮抗性菌株增加。同样,微波处理后绝大部分供试钙质土壤在GA、GACa及HA 3种培养基上的放线菌总数、链霉菌数、小单孢菌及未鉴定放线菌数量均有明显增加,表明微波处理土壤对放线菌分离时出菌量的提高具有显著作用。
5.盆栽试验表明,单接3株病原菌时,芹菜叶片和根系PPO酶活性、叶片及根系可溶性蛋白含量、芹菜叶片的POD活性、丙二醛含量及芹菜根系活力均较对照显著降低,而芹菜叶片细胞膜的相对透性和伤害率均较对照有所提高。而3株生防放线菌单接时,且在适宜接种量处理下,能显著提高叶片和根系的PPO酶活性、叶片及根系可溶性蛋白含量、芹菜叶片的POD活性以及降低芹菜叶片细胞膜的相对透性和伤害率。当3株生防放线菌与3株病原菌混接时,芹菜叶片和根系PPO酶活性、叶片及根系可溶性蛋白含量、芹菜叶片的POD活性和叶片丙二醛含量及芹菜根系活力都较单接病原菌时显著升高,而芹菜叶片细胞膜的相对透性和伤害率均有所降低。进而证明,3株生防放线菌有助于提高芹菜的诱导抗性,抑制连作病害发生。
The Taibus County located in the southern part of Xilin Gol League is known as“the Chinese celery first township”where the celery planting-area is about 2000hm2and the business about celery can produce about 3 million yuan per year. For the continuous cropping, a kind of celery disease named root-rot often happened recently over large area. The happening frequency of this kind disease is generally more than 30%, and the more serious situation is the completely loss in harvest on the heavy infected region. The traditional chemical pesticide has little effective on this kind disease. The happening of this disease has limited the development of local celery planting. In order to solve this problem effectively, the experiments study on screening higher efficient antagonistic actinomyces, and discusses that it can whether promote the growth of plant, and control the disease of plant or not. The main results are as follow:
1.The occurrence of root rot disease in is closely related to the abnormal of microflora in celery rhizosphere soil and root surface soil in Inner Mongolia. In the rgizosphere soil, the number of bacteria, fungus of diseased plant in initial stage is less than healthy plant, while the number of bacteria, fungus of diseased plant in middle and late stage is much more than healthy plant. In the root surface soil, the number of bacteria of diseased plant in initial stage is less than healthy plant, while that in middle and late stage is much more than healthy plant. In initial, middle and late stage, the number of fungus of diseased plant is much more than healthy plant. In the reduction of the ratio of B / F in the rgizosphere soil and the root surface soil and the proportion of pathogenic fungi increasing and lead to the disease. The type of soil change form bacterium to fungi.
2. From Inner Mongolia Taibus we isolated some fungi form infected plants and rhizosphere soil and root surface of soil. After morphological and molecular biology, we determine these are Fusarium sambucinum, Alternaria alternate, Fusarium oxysporum f. sp. Vasinfectum, Fusarium redolen, Plectosphaerella cucumerina.
3. Using agar block and the growth rate method, we were screened five strong antagonistic former 621 actinomycetes. These stains number are A11, A12, A7, A28 and A49. After we analyzing its 16S rDNA sequence and turned out these are Streptomyces carnosus, Streptomyces pactum , Streptomyces globisporus , Streptomyces globisporus subsp. Globisporus and Streptomyces kanamyceticu.
4.we studyed on the medium by adding CaCl2 and the method of microwave treatment have effect on the total number of actinomycetes, Streptomyces, Micromonospora, not identified kinds and the antagonistic actinomycetes. After adding CaCl2 to the medium, we found that the total number of actinomycetes, antagonistic actinomycetes and strong broad-spectrum of antagonistic strains increased. Similarly, the microwave treatment of tested soil in the GA, GACa and HA 3 kinds of media, we found out the total number of actinomycetes, Streptomyces, Micromonospora and unidentified genus were significantly increased, which show that the microwave could increase the amount of actinomycetes significantly.
5. Pot experiments showed that when inoculating three pathogens, celery leaves and roots PPO enzyme activity and soluble protein content, POD activity and MDA content in leaves were significantly lower than the CK, while the cell membrane permeability and injury rate celery leaves higher than the CK. when inoculating three antagonistic actinomycetes, celery leaves and roots PPO enzyme activity and soluble protein content, POD activity and MDA content in leaves were significantly higher than the CK, while the cell membrane permeability and injury rate celery leaves lower than the CK. when mixed inoculating three pathogens and three antagonistic actinomycetes, celery leaves and roots PPO enzyme activity and soluble protein content, POD activity and MDA content in leaves were significantly higher than inoculated pathogens, while the cell membrane permeability and injury rate celery leaves lower than inoculated pathogens. Further proof, three actinomycetes contribute to the induction of celery resistance and inhibit the disease occurred.
引文
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