稻鸭复合生态系统对水稻纹枯病控制机制研究
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摘要
本研究通过对稻田物理、化学和微生物生态环境方面研究,分别探讨了稻鸭复合生态系统中水稻基部叶鞘泥巴覆盖层、鸭子分泌物、鸭子携带微生物对该系统中水稻纹枯病发生发展的影响。对筛选出的具有良好防治效果的菌株A-168进行了鉴定,同时初步研究了其抑病机理。旨在阐明稻鸭复合生态种养模式提高水稻对纹枯病抗性的机理与调控因素,同时为协调粮食生产和环境保护之间的矛盾,寻求理想的新型防治技术和农业可持续发展道路提供理论基础。本试验主要研究结果如下:
1.水稻基部叶鞘覆盖泥巴层对水稻纹枯病的影响
通过模拟稻田养鸭物理生态环境,在稻基部叶鞘覆盖泥巴来,研究水稻基部叶鞘泥巴覆盖层对水稻纹枯病发生发展的影响。结果表明:稻鸭复合生态系统中水稻基部叶鞘泥巴层的覆盖,能显著遏制水稻纹枯病发生发展。封泥区平均病株率为17.22%,较空白处理44.50%低27.28%,比施药处理15.67%高出1.55%;其中,发病高峰期封泥处理病株率较空白处理低30%。成熟期封泥处理的病情指数平均为18.12,显著低于空白处理60.65,较施药处理病情指数仅高36%。从而证实了水稻基部叶鞘上泥巴的覆盖是影响稻鸭复合生态系统中水稻纹枯病流行的主要因素之一。
2.鸭子分泌物对水稻纹枯病的影响
室内平板抑菌活性测定及气质联用初步鉴定结果表明:各分泌物均表现出一定的抑菌活性。鸭粪80.0mg/mL浓度的乙酸乙酯萃取组分对水稻纹枯病病菌的24h菌丝抑制率达74.28%,其L_2流份经初步鉴定是酚酸类似物和含硫醇有机物,在80.0mg/mL的供试浓度下在24h时菌丝抑制率达到88.13%,表现出较高活性,确定了鸭粪中存在对水稻纹枯病菌有抑菌活性物质的假设。
3鸭子携带微生物生物活性分析
采用平板对峙生长法从鸭粪中分离到A-168、B-137、B-137Y三株活性良好的拮抗细菌菌株,各菌株24h抑菌圈半径分别可达16mm、11mm、13mm;田间小区防治效果以A-168菌株的抑制作用较为明显,喷药后10d和20d的防效分别是94.1%,46.7%,而井岗霉素喷药后10d和20d的防效分别为95.9%,65.8%。因此作者认为鸭粪中拮抗细菌的存在是导致稻鸭生态系统中纹枯病得到控制的原因之一。
4菌株A-168的鉴定及抑病机理的研究。
采用传统及16S rDNA分子鉴定方法,通过生理生化特征比对,系统发育树的建立,拮抗菌A-168与蜡状芽孢杆菌的模式菌株Bacillus cereus.NA10(FJ462699.1)的序列同源性为99%,鉴定为芽孢杆菌属蜡状芽单胞杆菌。抑病机理研究表明:菌株A-168同时存在拮抗、促生、定殖和诱导抗性四种抑病作用。与对照相比,菌液处理组水稻幼苗株高、根长、地上鲜重和地下鲜重分别增长15.25%、16.27%、19.90%和13.06%,表现出显著促生作用。测定中菌株A-168表现出较强定殖能力,能够通过某种途径迁移至植株地上部,占据病原菌的繁殖位点发挥防病作用。菌株A-168诱导后水稻叶片中各种抗病相关酶活性均高于对照处理。所有抗病相关酶活性变化均随着时间的延续而逐渐上升达到较高值,随后呈下降趋势,PAL、PO、PPO分别在接种后24、64和24h达到最高峰。
This research, based on the study of physical, chemical characteristics and microbiological ecological environment in paddy rice, had investigated the influences on the occurrence of rice sheath blight in rice-duck compound ecosystem, including the effects of the mud layer of the rice leaf sheath bases, duck secretions, as well as the microbes carried by ducks. Besides, preliminary explorations had done on the disease resistance mechanism of the strain A-168 which was identified to have good effects on controlling of rice sheath blight. The aim of this research was to illustrate the effective elements in rice-duck compound ecosystem which resulted in the improvement of the controlling of rice sheath blight, and to provide theoretical basis for seeking new-type control techniques in terms of controlling disease of rice and new way in the development of sustainable agriculture by coordinating the contradiction between food production and environmental protection. The main results of the research were as follows:
1. Effect of the Mud Layer of the Rice Leaf Sheath Bases on Rice Sheath Blight
By modeling the physical ecological environment of rice-duck compound ecosystem, the effects of the mud layer of the rice leaf sheath bases on rice sheath bight were investigated. The results showed that the mud layer had significant impacts on controlling the occurrence and development of rice sheath blight.
The average rate of infected plants in the mud-covered area was 17.22%, lower than the rate of 44.50% in blank control area by 27.28%, and only higher than the rate of 15.67% in pesticide application area by 1.55% ;The average index of diseased plants was 18.12 in ripening period, obviously lower than 60.65 in blank-contrast area. According to the above data, it proved that the mud layer of the rice leaf sheath bases is one of the major factors in controlling the rice sheath blight in rice-duck compound ecosystem.
2. Effect of Duck Secretions on Rice Sheath Blight
By means of laboratory plate inhibiting test and QP-2010 GC-MS, the results of preliminary identifications showed that duck secretions presented antibiotic effects. Ethyl acetate fractions extracting from duck manure with the concentration of 80.0mg/mL had 74.28% inhibiting rates to rice sheath blight during 24 hours in which the inhibiting rate of the second fraction (L2), reached 88.13%. With a higher antibacterial activity, the chemical elements of the second fraction (L2) were identified as phenolic acid analogues and mercaptan organics by preliminary research. Based on the above results, the experiment confirmed the proposition about the existence of antibacterial substance in the duck manure.
3. The Analysis of Biological Activities on the Duck-Carrying Microbes
By the method of paring culture on PDA, three antagonistic strains with good activity , A-168, B-137 and B-137Y were extracted from duck manure, of which the 24-hour inhibition zone radius were 16mm, 11mm, 13 mm respectively. Among those A-168 showed more significantly antagonistic effects in field experiment. In the test, the control efficacy of A-168 was 94.1% after 10 days and 95.9% after 20 days, while the control efficacy of Jinggangmycin was 46.7% and 65.8% under the same condition. Based on the above results, the paper reported that the existence of antagonistic strains in duck manure partly contributed to the effective controlling of rice sheath blight in rice-duck compound ecosystem.
4. With the application of traditional and 16S rDNA molecular identification methods to compare the physiological biochemical characteristics and construct the phylogenetic trees, the percentage nucleotide sequence homology between A-168 and cereus. NA10 (FJ462699.1) was 99% which proved that the bacillus was the bacillus cereus.
The research about the mechanisms of disease resistance of strains suggested that there were four disease suppressive functions concerning antagonism , growth promoting effect, colonization and induced resistance, coexisted in A-168. Rice seedlings after being processed by bacteria liquid, presented obvious growth promoting effect with the increasing rate of 15.25%、16.27%、19.90% and 13.06% respectively in plan height, root length, fresh weight of shoot and fresh weight of root. Besides, A-168 also showed stronger colonized ability by transferring itself to the ground parts of plants in a certain way to fill in the reproductive site of disease germs and exert its influences on resisting diseabse. After being induced by A-168 strain, the activity of various disease resistance related enzymes in rice leaves were higher than the blank control treatment. The activity of disease resistance related enzymes fluctuated with time, increasing gradually until reaching the peak and then beginning to decrease, of which PAL,PO,PPO reached their peak respectively in 24、64 and 24hours after inoculation.
1.水稻基部叶鞘覆盖泥巴层对水稻纹枯病的影响
通过模拟稻田养鸭物理生态环境,在稻基部叶鞘覆盖泥巴来,研究水稻基部叶鞘泥巴覆盖层对水稻纹枯病发生发展的影响。结果表明:稻鸭复合生态系统中水稻基部叶鞘泥巴层的覆盖,能显著遏制水稻纹枯病发生发展。封泥区平均病株率为17.22%,较空白处理44.50%低27.28%,比施药处理15.67%高出1.55%;其中,发病高峰期封泥处理病株率较空白处理低30%。成熟期封泥处理的病情指数平均为18.12,显著低于空白处理60.65,较施药处理病情指数仅高36%。从而证实了水稻基部叶鞘上泥巴的覆盖是影响稻鸭复合生态系统中水稻纹枯病流行的主要因素之一。
2.鸭子分泌物对水稻纹枯病的影响
室内平板抑菌活性测定及气质联用初步鉴定结果表明:各分泌物均表现出一定的抑菌活性。鸭粪80.0mg/mL浓度的乙酸乙酯萃取组分对水稻纹枯病病菌的24h菌丝抑制率达74.28%,其L_2流份经初步鉴定是酚酸类似物和含硫醇有机物,在80.0mg/mL的供试浓度下在24h时菌丝抑制率达到88.13%,表现出较高活性,确定了鸭粪中存在对水稻纹枯病菌有抑菌活性物质的假设。
3鸭子携带微生物生物活性分析
采用平板对峙生长法从鸭粪中分离到A-168、B-137、B-137Y三株活性良好的拮抗细菌菌株,各菌株24h抑菌圈半径分别可达16mm、11mm、13mm;田间小区防治效果以A-168菌株的抑制作用较为明显,喷药后10d和20d的防效分别是94.1%,46.7%,而井岗霉素喷药后10d和20d的防效分别为95.9%,65.8%。因此作者认为鸭粪中拮抗细菌的存在是导致稻鸭生态系统中纹枯病得到控制的原因之一。
4菌株A-168的鉴定及抑病机理的研究。
采用传统及16S rDNA分子鉴定方法,通过生理生化特征比对,系统发育树的建立,拮抗菌A-168与蜡状芽孢杆菌的模式菌株Bacillus cereus.NA10(FJ462699.1)的序列同源性为99%,鉴定为芽孢杆菌属蜡状芽单胞杆菌。抑病机理研究表明:菌株A-168同时存在拮抗、促生、定殖和诱导抗性四种抑病作用。与对照相比,菌液处理组水稻幼苗株高、根长、地上鲜重和地下鲜重分别增长15.25%、16.27%、19.90%和13.06%,表现出显著促生作用。测定中菌株A-168表现出较强定殖能力,能够通过某种途径迁移至植株地上部,占据病原菌的繁殖位点发挥防病作用。菌株A-168诱导后水稻叶片中各种抗病相关酶活性均高于对照处理。所有抗病相关酶活性变化均随着时间的延续而逐渐上升达到较高值,随后呈下降趋势,PAL、PO、PPO分别在接种后24、64和24h达到最高峰。
This research, based on the study of physical, chemical characteristics and microbiological ecological environment in paddy rice, had investigated the influences on the occurrence of rice sheath blight in rice-duck compound ecosystem, including the effects of the mud layer of the rice leaf sheath bases, duck secretions, as well as the microbes carried by ducks. Besides, preliminary explorations had done on the disease resistance mechanism of the strain A-168 which was identified to have good effects on controlling of rice sheath blight. The aim of this research was to illustrate the effective elements in rice-duck compound ecosystem which resulted in the improvement of the controlling of rice sheath blight, and to provide theoretical basis for seeking new-type control techniques in terms of controlling disease of rice and new way in the development of sustainable agriculture by coordinating the contradiction between food production and environmental protection. The main results of the research were as follows:
1. Effect of the Mud Layer of the Rice Leaf Sheath Bases on Rice Sheath Blight
By modeling the physical ecological environment of rice-duck compound ecosystem, the effects of the mud layer of the rice leaf sheath bases on rice sheath bight were investigated. The results showed that the mud layer had significant impacts on controlling the occurrence and development of rice sheath blight.
The average rate of infected plants in the mud-covered area was 17.22%, lower than the rate of 44.50% in blank control area by 27.28%, and only higher than the rate of 15.67% in pesticide application area by 1.55% ;The average index of diseased plants was 18.12 in ripening period, obviously lower than 60.65 in blank-contrast area. According to the above data, it proved that the mud layer of the rice leaf sheath bases is one of the major factors in controlling the rice sheath blight in rice-duck compound ecosystem.
2. Effect of Duck Secretions on Rice Sheath Blight
By means of laboratory plate inhibiting test and QP-2010 GC-MS, the results of preliminary identifications showed that duck secretions presented antibiotic effects. Ethyl acetate fractions extracting from duck manure with the concentration of 80.0mg/mL had 74.28% inhibiting rates to rice sheath blight during 24 hours in which the inhibiting rate of the second fraction (L2), reached 88.13%. With a higher antibacterial activity, the chemical elements of the second fraction (L2) were identified as phenolic acid analogues and mercaptan organics by preliminary research. Based on the above results, the experiment confirmed the proposition about the existence of antibacterial substance in the duck manure.
3. The Analysis of Biological Activities on the Duck-Carrying Microbes
By the method of paring culture on PDA, three antagonistic strains with good activity , A-168, B-137 and B-137Y were extracted from duck manure, of which the 24-hour inhibition zone radius were 16mm, 11mm, 13 mm respectively. Among those A-168 showed more significantly antagonistic effects in field experiment. In the test, the control efficacy of A-168 was 94.1% after 10 days and 95.9% after 20 days, while the control efficacy of Jinggangmycin was 46.7% and 65.8% under the same condition. Based on the above results, the paper reported that the existence of antagonistic strains in duck manure partly contributed to the effective controlling of rice sheath blight in rice-duck compound ecosystem.
4. With the application of traditional and 16S rDNA molecular identification methods to compare the physiological biochemical characteristics and construct the phylogenetic trees, the percentage nucleotide sequence homology between A-168 and cereus. NA10 (FJ462699.1) was 99% which proved that the bacillus was the bacillus cereus.
The research about the mechanisms of disease resistance of strains suggested that there were four disease suppressive functions concerning antagonism , growth promoting effect, colonization and induced resistance, coexisted in A-168. Rice seedlings after being processed by bacteria liquid, presented obvious growth promoting effect with the increasing rate of 15.25%、16.27%、19.90% and 13.06% respectively in plan height, root length, fresh weight of shoot and fresh weight of root. Besides, A-168 also showed stronger colonized ability by transferring itself to the ground parts of plants in a certain way to fill in the reproductive site of disease germs and exert its influences on resisting diseabse. After being induced by A-168 strain, the activity of various disease resistance related enzymes in rice leaves were higher than the blank control treatment. The activity of disease resistance related enzymes fluctuated with time, increasing gradually until reaching the peak and then beginning to decrease, of which PAL,PO,PPO reached their peak respectively in 24、64 and 24hours after inoculation.
引文
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