拮抗微生物对列当的防除作用及机理
发布时间：2018-07-16 23:36
【摘要】：根寄生杂草列当对全球许多地区农业生产造成严重危害。目前,有效防除列当仍是一个世界性难题。微生物防治是防除列当的有效途径之一,但关于列当生防微生物的研究目前多集中于列当病原菌上。本研究以筛选自来源于健康土壤的上万株微生物资源库中对多种作物病害病原菌有拮抗作用的放线菌和真菌为试验材料,通过培养皿内种子萌发抑制试验筛选向日葵列当和瓜列当的生防菌株;通过皿内共培养试验结合盆栽及田间试验对筛选出的生防菌株防除列当的效果进行了验证并对生防菌株防除列当的机理进行了研究;通过盆栽试验研究了列当生防菌株对寄主作物生长的影响。此外,本研究还通过皿内种子萌发试验从供试拮抗微生物中筛选具有诱导列当种子萌发潜能的菌株。本研究所得主要结论如下:(1)向日葵列当和瓜列当拮抗微生物的筛选。本研究筛选出强烈抑制向日葵列当种子萌发的放线菌和真菌各1株,分别为淡紫褐链霉菌(Streptomyces enissocaesilis,509)和灰黄青霉(Penicillium griseofulvum,CF3);同时还筛选出强烈抑制瓜列当种子萌发的放线菌2株(密旋链霉菌S.pactum,12#;黄白链霉菌S.albidoflavus,T4)和真菌1株,CF3。在3.5 mg ml-1浓度下,509和CF3的无细胞发酵滤液对向日葵列当及12#、T4和CF3的无细胞发酵滤液对瓜列当种子萌发的抑制率均达到75%以上。在50.0 mg ml-1浓度下,509和CF3的菌体甲醇浸提液对向日葵列当种子萌发的抑制率分别为14.8%和100.0%;而12#、T4和CF3的菌体甲醇浸提液对瓜列当种子萌发的抑制率为50.7%~100.0%。在皿内共培养试验中,509和CF3的无细胞发酵滤液均能够显著抑制向日葵列当种子的萌发。CF3的无细胞发酵滤液在皿内共培养8天后对瓜列当种子萌发的抑制率仍高达47.5%~80.3%。(2)放线菌防除列当作用研究。盆栽试验中,施加1.0 g kg-1的509菌剂使收获期向日葵列当的出土数量和寄生总数较对照分别降低了39.2%~47.5%和39.3%~62.4%。在杨凌盆栽试验中,施加1.0 g kg-1的509菌剂使生长中期和收获期向日葵列当的总干重与对照相比分别降低了46.9%和36.7%。施加12#菌剂使盆栽试验收获期瓜列当的出土数量、出土率和总干重较对照分别降低了85.7%、75.7%和55.4%。(3)真菌防除列当作用研究。杨凌盆栽试验中,施加1.0 g kg-1的CF3菌剂使收获期向日葵列当的寄生总数和总干重较对照分别降低了25.2%和46.0%。施加CF3菌剂使盆栽试验收获期瓜列当的出土数量和出土率较对照分别降低了76.2%和85.3%。进一步研究发现展青霉素是CF3抑制列当种子萌发的主要活性物质。展青霉素和CF3无细胞发酵滤液的乙酸乙酯萃取物在1.0 mg ml-1浓度下均能够完全抑制向日葵列当和瓜列当种子的萌发。(4)拮抗微生物对列当寄主的促生作用研究。在有列当寄生的条件下,施加509和CF3菌剂显著促进了盆栽向日葵的生长,增加了向日葵的生物量及籽粒干重。其中,拌土施加509和CF3菌剂使杨凌盆栽试验收获期向日葵的株高增加了39.1%~63.4%,地上干重增加了114.7%~179.0%。施加12#和CF3菌剂使盆栽试验收获期番茄产量与对照相比分别增加了51.6%和84.9%。在无列当寄生条件下,施加1.0 g kg-1的509和CF3菌剂使盆栽试验收获期向日葵的产量较对照分别增加了79.2%和46.6%,而施加12#菌剂使收获期番茄产量增加了57.3%。(5)拮抗微生物诱导列当种子萌发研究。从供试拮抗微生物中分别筛选出能够诱导瓜列当种子萌发的放线菌(链霉菌Streptomyces sp.,Act13;球孢链霉菌球孢亚种S.globisporus subsp.globisporus,D141;加利利链霉菌S.galilaeus,G37)3株,能够诱导向日葵列当种子萌发的放线菌(球孢链霉菌S.globisporus,C28和链霉菌Streptomyces sp.,S8)和真菌(木霉Trichoderma spp.;M1,M2)各2株。试验结果表明Act13、D141和G37的无细胞发酵滤液在0.35 mg ml-1浓度下可分别诱导14.7%、14.9%和15.4%的瓜列当种子萌发。C28的无细胞发酵滤液在3.5×10-4 mg ml-1浓度下可诱导13.4%的向日葵列当种子萌发。S8、M1和M2的菌体甲醇浸提液在50.0 mg ml-1浓度下分别诱导了23.4%、17.0%和15.5%的向日葵列当种子萌发。(6)拮抗微生物防除列当机理研究。试验结果表明拮抗微生物主要通过以下3种机理来防除列当:(1)通过改变寄主根区土壤中微生物区系来抑制列当的寄生和生长。盆栽试验中,向日葵列当的总数及出土数与向日葵根区土壤中放线菌和细菌的数量及比例呈显著负相关关系。瓜列当的出土数和出土率与放线菌与真菌的数量之比(A/F)、细菌与真菌的数量之比(B/F)呈负相关关系。施加1.0 g kg-1的509菌剂使盆栽试验向日葵根区土壤中放线菌的数量较对照增加了76.7%~439.9%。施加CF3、509和12#也显著增加了寄主根区土壤中A/F和B/F。(2)通过增强寄主植物的防御酶系统抗性来阻碍列当的寄生和生长。固原盆栽试验中向日葵列当的总数、鲜重及干重均与向日葵根系中多酚氧化酶PPO活力呈显著负相关。施加509菌剂使寄主向日葵根系中PPO活力与对照相比增加了43.1%。(3)通过抑制列当种子的萌发、芽管的伸长,影响芽管的形态来直接防除列当。在0.35 mg ml-1浓度下,509和12#的无细胞发酵滤液使向日葵列当芽管长度较对照分别减少了86.2%和89.6%,而CF3和12#的无细胞发酵滤液也使瓜列当芽管长度分别缩短了68.8%和55.6%。经509无细胞发酵滤液和CF3菌体甲醇浸提液处理后,部分向日葵列当的芽管发生了褐变。
[Abstract]:Root parasitic weeds have caused serious harm to agricultural production in many parts of the world. At present, effective prevention and control is still a worldwide problem. Microbial control is one of the effective ways to prevent and control, but the research on Biocontrol microorganism is mainly concentrated on the pathogenic bacteria. This study is to screen from healthy soil. Strains of actinomycetes and fungi, which have antagonistic effects on the pathogens of various crop diseases, were used as experimental materials to screen the biocontrol strains of sunflower and cucumber by seed germination inhibition test in a Petri dish. The effect was verified and the mechanism of the anti-bacterial strain was studied. The effect of the Biocontrol Strain on the growth of the host crops was studied by a pot experiment. In addition, the study also screened the strains with the potential of the Seed Germination from the tested antagonist microorganism in the dish seed germination test. The following conclusions are as follows: (1) screening of the antagonistic microorganisms from the sunflower and the cucumber. This study screened out 1 strains of actinomycetes and fungi which strongly inhibited the germination of sunflower, Streptomyces enissocaesilis, 509, and Penicillium griseofulvum, CF3, respectively. When 2 strains of actinomycetes (Streptomyces S.pactum, 12#; Streptomyces yellow and white fungus S.albidoflavus, T4) and 1 strains of fungi were germinated, CF3. at the concentration of 3.5 mg ml-1, 509 and CF3 without cell-free filtrating filtrate of sunflower, 12#, T4 and CF3 on the germination of melon seeds reached more than 75%. In 50 mg The inhibitory rates of methanol extracts from 509 and CF3 on the germination of sunflower seeds were 14.8% and 100%, respectively, while the inhibition rate of methanol extracts from 12#, T4 and CF3 was 50.7%~100.0%. in the co culture test of 50.7%~100.0%. in the dish, and the 509 and CF3 filtrate free filtrates could significantly inhibit the sunflower seeded species. The inhibitory rate of the seedless fermentation filtrate of.CF3 on seed germination was still up to 47.5%~80.3%. (2) of actinomycetes in the germination of seed germination of the seedless fermentation filtrate of the seed of.CF3. In pot experiment, the number of unearthed and parasitism of the sunflower in the harvest period decreased by 39.2%~47.5% and 3, respectively, by applying the 509 agent of 1 g kg-1. In the Yangling pot experiment, the total dry weight of 1 g kg-1 was applied to the total dry weight of 1 g kg-1 and the total dry weight of sunflower in the mid growth and harvest period was reduced by 46.9% and 36.7%., respectively. The amount of unearthed melon in the harvest period of the pot experiment was reduced by 85.7%, 75.7% and 55.4%. (3), respectively. In the pot experiment in Yangling, the 1 g kg-1 inoculants were applied to reduce the parasitic total and total dry weight of sunflower in the harvest period by 25.2% and 46.0%. by applying CF3 bacteria, respectively. The amount of unearthed soil and the unearthed rate of the melon in the harvest period were reduced by 76.2% and 85.3%., respectively, by 76.2% and 85.3%. respectively. It was found that penicillin was the main active substance for CF3 inhibition of seed germination. The ethyl acetate extract of penicillin and CF3 without cell fermentation filtrate could completely inhibit the germination of sunflower and cucumber seeds at the concentration of 1 mg ml-1. (4) study on the growth promoting effect of antagonistic microorganisms on the host. The application of 509 and CF3 inoculants significantly promoted the growth of sunflower in potted plants and increased the biomass and dry weight of sunflower. Among them, 509 and CF3 inoculants in the mixed soil increased the plant height of the sunflower in the harvest period of Yangling, and the dry weight of the ground was increased by 114.7%~ 179.0%., and 12# and CF3 were applied to the harvest period of the pot experiment. Compared with the control, tomato yield increased by 51.6% and 84.9%., respectively, under the parasitic conditions. The 1 g kg-1 509 and CF3 inoculants increased the yield of sunflower in the harvest period by 79.2% and 46.6% respectively, while the 12# inoculant increased the yield of the harvested tomato with 57.3%. (5) antagonistic microorganisms to induce the seed germination. 3 strains of actinomycetes (Streptomyces Streptomyces sp., Act13, S.globisporus subsp.globisporus, D141, S.galilaeus, G37) of Streptomyces Galilee were screened from the tested antagonist microorganism to induce seed germination respectively, which could guide the actinomycetes (Streptomyces spores S.) as seed germination of sunflower (Streptomyces spores S.). 2 strains of globisporus, C28 and Streptomyces Streptomyces sp., S8) and fungi (Trichoderma Trichoderma spp.; M1, M2). The results showed that the cell-free filtrate of Act13, D141 and G37 could induce 14.7%, 14.9% and 15.4% of the seedless fermentation filtrate of seed germination respectively in the concentration of 3.5 * 10-4. The 13.4% sunflower seed germination was.S8, the methanol extract of M1 and M2 induced 23.4%, 17% and 15.5% of sunflower seed germination at the concentration of 50 mg ml-1. (6) the antagonistic microorganism defense mechanism was studied. The experimental results showed that the antagonistic microorganisms were mainly through the following 3 mechanisms to prevent and treat: (1) through change. Parasitism and growth of the soil in the soil of the main root zone were sent to inhibit the parasitism and growth of the column. In the pot experiment, the total number and unearthed number of sunflower were negatively correlated with the number and proportion of actinomycetes and bacteria in the soil of the sunflower root region. The number of unearthed soil and the ratio of unearthed soil to the number of actinomycetes and fungi (A/F), bacteria and true The number of bacteria was negatively correlated with the ratio of B/F. The number of 509 inoculants with 1 g kg-1 increased the number of actinomycetes in the soil of the sunflower root zone increased by 76.7%~439.9%. and CF3509 and 12# significantly increased A/F and B/F. (2) in the soil of the main root region. The total number, fresh weight and dry weight of sunflower in Guyuan pot experiment were all negatively correlated with the activity of polyphenol oxidase PPO in sunflower root system. The effect of 509 inoculant on PPO activity in the root of sunflower was increased by 43.1%. (3) by inhibiting the germination of the seed, the elongation of the bud tube and the shape of the bud tube. Under the concentration of 0.35 mg ml-1, 509 and 12# cell-free filtrates reduced the length of the sunflower bud tube by 86.2% and 89.6% respectively, while the CF3 and 12# cell-free filtrates shortened the length of the melon bud tube by 68.8% and 55.6%. through 509 fine cell fermentation filtrate and CF3 bacterial extract solution. After that, some of the sunflower bud tubes were browning.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S45
，
本文编号：2128060
[Abstract]:Root parasitic weeds have caused serious harm to agricultural production in many parts of the world. At present, effective prevention and control is still a worldwide problem. Microbial control is one of the effective ways to prevent and control, but the research on Biocontrol microorganism is mainly concentrated on the pathogenic bacteria. This study is to screen from healthy soil. Strains of actinomycetes and fungi, which have antagonistic effects on the pathogens of various crop diseases, were used as experimental materials to screen the biocontrol strains of sunflower and cucumber by seed germination inhibition test in a Petri dish. The effect was verified and the mechanism of the anti-bacterial strain was studied. The effect of the Biocontrol Strain on the growth of the host crops was studied by a pot experiment. In addition, the study also screened the strains with the potential of the Seed Germination from the tested antagonist microorganism in the dish seed germination test. The following conclusions are as follows: (1) screening of the antagonistic microorganisms from the sunflower and the cucumber. This study screened out 1 strains of actinomycetes and fungi which strongly inhibited the germination of sunflower, Streptomyces enissocaesilis, 509, and Penicillium griseofulvum, CF3, respectively. When 2 strains of actinomycetes (Streptomyces S.pactum, 12#; Streptomyces yellow and white fungus S.albidoflavus, T4) and 1 strains of fungi were germinated, CF3. at the concentration of 3.5 mg ml-1, 509 and CF3 without cell-free filtrating filtrate of sunflower, 12#, T4 and CF3 on the germination of melon seeds reached more than 75%. In 50 mg The inhibitory rates of methanol extracts from 509 and CF3 on the germination of sunflower seeds were 14.8% and 100%, respectively, while the inhibition rate of methanol extracts from 12#, T4 and CF3 was 50.7%~100.0%. in the co culture test of 50.7%~100.0%. in the dish, and the 509 and CF3 filtrate free filtrates could significantly inhibit the sunflower seeded species. The inhibitory rate of the seedless fermentation filtrate of.CF3 on seed germination was still up to 47.5%~80.3%. (2) of actinomycetes in the germination of seed germination of the seedless fermentation filtrate of the seed of.CF3. In pot experiment, the number of unearthed and parasitism of the sunflower in the harvest period decreased by 39.2%~47.5% and 3, respectively, by applying the 509 agent of 1 g kg-1. In the Yangling pot experiment, the total dry weight of 1 g kg-1 was applied to the total dry weight of 1 g kg-1 and the total dry weight of sunflower in the mid growth and harvest period was reduced by 46.9% and 36.7%., respectively. The amount of unearthed melon in the harvest period of the pot experiment was reduced by 85.7%, 75.7% and 55.4%. (3), respectively. In the pot experiment in Yangling, the 1 g kg-1 inoculants were applied to reduce the parasitic total and total dry weight of sunflower in the harvest period by 25.2% and 46.0%. by applying CF3 bacteria, respectively. The amount of unearthed soil and the unearthed rate of the melon in the harvest period were reduced by 76.2% and 85.3%., respectively, by 76.2% and 85.3%. respectively. It was found that penicillin was the main active substance for CF3 inhibition of seed germination. The ethyl acetate extract of penicillin and CF3 without cell fermentation filtrate could completely inhibit the germination of sunflower and cucumber seeds at the concentration of 1 mg ml-1. (4) study on the growth promoting effect of antagonistic microorganisms on the host. The application of 509 and CF3 inoculants significantly promoted the growth of sunflower in potted plants and increased the biomass and dry weight of sunflower. Among them, 509 and CF3 inoculants in the mixed soil increased the plant height of the sunflower in the harvest period of Yangling, and the dry weight of the ground was increased by 114.7%~ 179.0%., and 12# and CF3 were applied to the harvest period of the pot experiment. Compared with the control, tomato yield increased by 51.6% and 84.9%., respectively, under the parasitic conditions. The 1 g kg-1 509 and CF3 inoculants increased the yield of sunflower in the harvest period by 79.2% and 46.6% respectively, while the 12# inoculant increased the yield of the harvested tomato with 57.3%. (5) antagonistic microorganisms to induce the seed germination. 3 strains of actinomycetes (Streptomyces Streptomyces sp., Act13, S.globisporus subsp.globisporus, D141, S.galilaeus, G37) of Streptomyces Galilee were screened from the tested antagonist microorganism to induce seed germination respectively, which could guide the actinomycetes (Streptomyces spores S.) as seed germination of sunflower (Streptomyces spores S.). 2 strains of globisporus, C28 and Streptomyces Streptomyces sp., S8) and fungi (Trichoderma Trichoderma spp.; M1, M2). The results showed that the cell-free filtrate of Act13, D141 and G37 could induce 14.7%, 14.9% and 15.4% of the seedless fermentation filtrate of seed germination respectively in the concentration of 3.5 * 10-4. The 13.4% sunflower seed germination was.S8, the methanol extract of M1 and M2 induced 23.4%, 17% and 15.5% of sunflower seed germination at the concentration of 50 mg ml-1. (6) the antagonistic microorganism defense mechanism was studied. The experimental results showed that the antagonistic microorganisms were mainly through the following 3 mechanisms to prevent and treat: (1) through change. Parasitism and growth of the soil in the soil of the main root zone were sent to inhibit the parasitism and growth of the column. In the pot experiment, the total number and unearthed number of sunflower were negatively correlated with the number and proportion of actinomycetes and bacteria in the soil of the sunflower root region. The number of unearthed soil and the ratio of unearthed soil to the number of actinomycetes and fungi (A/F), bacteria and true The number of bacteria was negatively correlated with the ratio of B/F. The number of 509 inoculants with 1 g kg-1 increased the number of actinomycetes in the soil of the sunflower root zone increased by 76.7%~439.9%. and CF3509 and 12# significantly increased A/F and B/F. (2) in the soil of the main root region. The total number, fresh weight and dry weight of sunflower in Guyuan pot experiment were all negatively correlated with the activity of polyphenol oxidase PPO in sunflower root system. The effect of 509 inoculant on PPO activity in the root of sunflower was increased by 43.1%. (3) by inhibiting the germination of the seed, the elongation of the bud tube and the shape of the bud tube. Under the concentration of 0.35 mg ml-1, 509 and 12# cell-free filtrates reduced the length of the sunflower bud tube by 86.2% and 89.6% respectively, while the CF3 and 12# cell-free filtrates shortened the length of the melon bud tube by 68.8% and 55.6%. through 509 fine cell fermentation filtrate and CF3 bacterial extract solution. After that, some of the sunflower bud tubes were browning.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S45
，
本文编号：2128060
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