烟草青枯病的田间检测及拮抗菌在土壤与菌肥中的生长动态研究
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摘要
烟草青枯罗尔氏菌(Ralstonia solanacearum)是一种土传病原细菌,在生产上造成严重危害,至今没一种安全有效的药剂防治,因此开发高效的微生物菌肥对它进行生物防治,具有十分重大的现实意义。本实验室利用拮抗菌011和2-Q-9研制一种防治烟草青枯病的高效微生物菌肥,本实验针对该产品的使用进行了一些基础研究。
土壤青枯菌检测中选择性培养基的配置,用100 mL肉汁冻培养基加2.5×10-2μg/mL结晶紫和2.5×10-2μg/mL红四氮唑作为基本培养基,依次加入7种抗菌素最佳药量。抗菌素最佳浓度分别为：氨苄西林胶囊19.996×10-5μg/mL、氧氟沙星片9.990×10-7μg/mL、罗红霉素片2.998×10-6μg/mL、头孢拉定胶囊3.000×10-6μg/mL、乙酰螺旋霉素片1.998×10-5μg/mL、克拉霉素分散片2.999×10-5μg/mL、阿奇霉素胶囊1.999×10-6μg/mL。
选择性培养基对湘西,永州,郴州三地的土壤检测结果表明,湘西土样中含菌量最多,永州次之,郴州相对较少。土壤含菌量与青枯菌发病情况呈正相关,其中加菌肥土样中菌量少于未加菌肥土样中菌量。
拮抗菌与枯饼发酵菌肥的互作测定中,拮抗菌2-Q-9与枯饼发酵菌m1、DB2及发酵液无拮抗作用,说明2-Q-9能与枯饼菌肥中的主要微生物协同生长,用2-Q-9与发酵菌来制作微生物菌肥是可行的；拮抗菌011与枯饼发酵菌m1、DB2及发酵液有拮抗作用,初步确定用011与发酵菌m1、DB2来制作微生物菌肥是不可行的。
烟草拮抗菌突变菌株011和2-Q-9在烟草高效活性菌肥中的定殖测定结果表明,拮抗菌在菌肥中20到30 d内,菌量增加较快,30 d后菌量逐渐减少。表明011和2-Q-9能在菌肥中短期定殖,但不适合长期定殖。
烟草拮抗菌突变菌株011和2-Q-9在土壤中均可成功的回收到,实验结果表明,011菌株在15 d到45 d之间,未灭菌的土壤中回收到的菌量多于灭菌土壤；2-Q-9菌株在15 d到75 d,未灭菌的土壤中回收到的菌量明显多于灭菌土壤；在整个生长过程中,60 d后菌量明显减少；说明011和2-Q-9菌株可以与土壤中微生物协同共生,60 d后为第二次施用防治菌肥的关键时期。
通过小区和大田施用微生物菌肥结果表明,小区试验中011、2-Q-9拮抗菌菌肥和2株拮抗菌混和菌肥的防治效果分别为71.20%、49.28%和83.39%,说明混合菌肥处理防治效果优于对应的单菌菌肥；大田实验结果表明,2株拮抗菌混和菌肥施用在郴州和永州的防效分别为90%和37.71%；大田和小区防效均高于常用的农用链霉素。
Ralstonia solanacearum is a soil-borne pathogenic bacteria, which does serious harm to the production of tobacco. It was significant to develop a efficient microbial fertilizer for biological control to tobacco bacterial wilt, because of no safe and effective bactericide. In our laboratory, a kind of highly effective microbial fertilizer, preventing tobacco bacterial wilt, was developed by using antagonistic bacteria 011 and 2-Q-9. Some basic researches to the use of the product were done in this experiment.
The optimized and the collocation of selective medium for detecting Pseudomonas solanacearum in the soil was that:beef extract medium 100 mL; crystal violet2.5×10-2μg/mL; TTC2.5×10-2μg/mL; ampicillin capsules 9.996×10-5μg/mL; ofloxacin tablets 9.990×10-7μg/mL; roxithromycin tablets,2.998×10-6μg/mL; cefradine capsules3.000×10-6μg/mL; acetylspiramycin tablets,1.998x×10-5μg/mL; clarithromycin dispersible tablets 2.999×10-5μg/mL; azithromycin capsules 1.999×10-6μg/mL.
The result of detecting tobacco bacterial wilt in soil from Xiangxi, Yongzhou, Chenzhou by the selective medium demonstrated that the number of Pseudomonas solanacearum in soil samples from Xiangxi was larger than that from Yongzhou, and that from Chenzhou was relatively small. There was a positive correlation between disease index and the number of bacteria, and the amount of tobacco bacteria in the soil with antagonistic bacterial fertilizer was smaller than that in common soil.
The results of the interaction test between antagonistic bacteria and blight bacteria fermentation of manure showed that:there was no antagonism between antagonistic bacteria 2-Q-9, blight bacteria fermentation m1,DB2 and the fermentation broth, which indicated 2-Q-9 and main microbe in the microbial fertilizer could grow collaboratively, and it was feasible to make microbial fertilizer by using 2-Q-9 and fermentative bacteria; there was antagonism between antagonistic bacteria 01 land fermentation bacteria m1, DB2 and the fermentation broth, and it was primarily identified that using antagonistic bacteria 011 and fermentation bacteria m1, DB2 to produce microbial fertilizer is not feasible.
The colonization test of tobacco antagonistic mutant strain 011 and 2-Q-9 in tobacco efficient fertilizer showed that the number of antagonistic bacteria increased rapidly from 20 to 30 days, but the amount decreased'after 30 days. It showed that the strains 011 and 2-Q-9 could colonise in short time in the fertilizer, but were not suitable for long-term colonization.
Tobacco antagonistic mutant strain 011 and 2-Q-9 can be successfully recovered from the soil, the results showed that the amount of the strains 011 recovered from unsterilized soil was larger than that from sterilized soil from15 to 45 days; and the amount of the strains 2-Q-9 recovered from unsterilized soil was larger than that from sterilized soil from15 to 75 days; during the whole growth process, the number of bacteria significantly decreased after 60 days.The results indicated that strains 011 and 2-Q-9 could collaborate with soil microbes, and the critical period of using fertilizer next time was the 60-day.
Through the application of microbial fertilizer in the small district and in field, the results showed that in the small district the control efficiency of antagonistic bacteria fertilizer with strains 011, strains 2-Q-9 and the mixture of both strains were 71.20%, 49.28% and 83.39% in the small district, respectively. Which indicated that the control effect of mixed fertilizer is better than that of single bacterial fertilizer; in field, the control efficiency of the mixed fertilizer in Chenzhou and Yongzhou were 90% and 37.71%; The control efficiency were higher than common agricultural streptomycin in both small districts and fields.
土壤青枯菌检测中选择性培养基的配置,用100 mL肉汁冻培养基加2.5×10-2μg/mL结晶紫和2.5×10-2μg/mL红四氮唑作为基本培养基,依次加入7种抗菌素最佳药量。抗菌素最佳浓度分别为：氨苄西林胶囊19.996×10-5μg/mL、氧氟沙星片9.990×10-7μg/mL、罗红霉素片2.998×10-6μg/mL、头孢拉定胶囊3.000×10-6μg/mL、乙酰螺旋霉素片1.998×10-5μg/mL、克拉霉素分散片2.999×10-5μg/mL、阿奇霉素胶囊1.999×10-6μg/mL。
选择性培养基对湘西,永州,郴州三地的土壤检测结果表明,湘西土样中含菌量最多,永州次之,郴州相对较少。土壤含菌量与青枯菌发病情况呈正相关,其中加菌肥土样中菌量少于未加菌肥土样中菌量。
拮抗菌与枯饼发酵菌肥的互作测定中,拮抗菌2-Q-9与枯饼发酵菌m1、DB2及发酵液无拮抗作用,说明2-Q-9能与枯饼菌肥中的主要微生物协同生长,用2-Q-9与发酵菌来制作微生物菌肥是可行的；拮抗菌011与枯饼发酵菌m1、DB2及发酵液有拮抗作用,初步确定用011与发酵菌m1、DB2来制作微生物菌肥是不可行的。
烟草拮抗菌突变菌株011和2-Q-9在烟草高效活性菌肥中的定殖测定结果表明,拮抗菌在菌肥中20到30 d内,菌量增加较快,30 d后菌量逐渐减少。表明011和2-Q-9能在菌肥中短期定殖,但不适合长期定殖。
烟草拮抗菌突变菌株011和2-Q-9在土壤中均可成功的回收到,实验结果表明,011菌株在15 d到45 d之间,未灭菌的土壤中回收到的菌量多于灭菌土壤；2-Q-9菌株在15 d到75 d,未灭菌的土壤中回收到的菌量明显多于灭菌土壤；在整个生长过程中,60 d后菌量明显减少；说明011和2-Q-9菌株可以与土壤中微生物协同共生,60 d后为第二次施用防治菌肥的关键时期。
通过小区和大田施用微生物菌肥结果表明,小区试验中011、2-Q-9拮抗菌菌肥和2株拮抗菌混和菌肥的防治效果分别为71.20%、49.28%和83.39%,说明混合菌肥处理防治效果优于对应的单菌菌肥；大田实验结果表明,2株拮抗菌混和菌肥施用在郴州和永州的防效分别为90%和37.71%；大田和小区防效均高于常用的农用链霉素。
Ralstonia solanacearum is a soil-borne pathogenic bacteria, which does serious harm to the production of tobacco. It was significant to develop a efficient microbial fertilizer for biological control to tobacco bacterial wilt, because of no safe and effective bactericide. In our laboratory, a kind of highly effective microbial fertilizer, preventing tobacco bacterial wilt, was developed by using antagonistic bacteria 011 and 2-Q-9. Some basic researches to the use of the product were done in this experiment.
The optimized and the collocation of selective medium for detecting Pseudomonas solanacearum in the soil was that:beef extract medium 100 mL; crystal violet2.5×10-2μg/mL; TTC2.5×10-2μg/mL; ampicillin capsules 9.996×10-5μg/mL; ofloxacin tablets 9.990×10-7μg/mL; roxithromycin tablets,2.998×10-6μg/mL; cefradine capsules3.000×10-6μg/mL; acetylspiramycin tablets,1.998x×10-5μg/mL; clarithromycin dispersible tablets 2.999×10-5μg/mL; azithromycin capsules 1.999×10-6μg/mL.
The result of detecting tobacco bacterial wilt in soil from Xiangxi, Yongzhou, Chenzhou by the selective medium demonstrated that the number of Pseudomonas solanacearum in soil samples from Xiangxi was larger than that from Yongzhou, and that from Chenzhou was relatively small. There was a positive correlation between disease index and the number of bacteria, and the amount of tobacco bacteria in the soil with antagonistic bacterial fertilizer was smaller than that in common soil.
The results of the interaction test between antagonistic bacteria and blight bacteria fermentation of manure showed that:there was no antagonism between antagonistic bacteria 2-Q-9, blight bacteria fermentation m1,DB2 and the fermentation broth, which indicated 2-Q-9 and main microbe in the microbial fertilizer could grow collaboratively, and it was feasible to make microbial fertilizer by using 2-Q-9 and fermentative bacteria; there was antagonism between antagonistic bacteria 01 land fermentation bacteria m1, DB2 and the fermentation broth, and it was primarily identified that using antagonistic bacteria 011 and fermentation bacteria m1, DB2 to produce microbial fertilizer is not feasible.
The colonization test of tobacco antagonistic mutant strain 011 and 2-Q-9 in tobacco efficient fertilizer showed that the number of antagonistic bacteria increased rapidly from 20 to 30 days, but the amount decreased'after 30 days. It showed that the strains 011 and 2-Q-9 could colonise in short time in the fertilizer, but were not suitable for long-term colonization.
Tobacco antagonistic mutant strain 011 and 2-Q-9 can be successfully recovered from the soil, the results showed that the amount of the strains 011 recovered from unsterilized soil was larger than that from sterilized soil from15 to 45 days; and the amount of the strains 2-Q-9 recovered from unsterilized soil was larger than that from sterilized soil from15 to 75 days; during the whole growth process, the number of bacteria significantly decreased after 60 days.The results indicated that strains 011 and 2-Q-9 could collaborate with soil microbes, and the critical period of using fertilizer next time was the 60-day.
Through the application of microbial fertilizer in the small district and in field, the results showed that in the small district the control efficiency of antagonistic bacteria fertilizer with strains 011, strains 2-Q-9 and the mixture of both strains were 71.20%, 49.28% and 83.39% in the small district, respectively. Which indicated that the control effect of mixed fertilizer is better than that of single bacterial fertilizer; in field, the control efficiency of the mixed fertilizer in Chenzhou and Yongzhou were 90% and 37.71%; The control efficiency were higher than common agricultural streptomycin in both small districts and fields.
引文
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