产几丁质酶木霉的诱变选育及对玉米纹枯病的生物防治
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摘要
玉米纹枯病属世界性土传病害,在我国各玉米产区普遍发生。近年来,其危害逐年加重,纹枯病已成为我国西南地区玉米生产最严重的病害之一。目前,常规育种难以选择到多抗性的植株,土传病害防治困难,化学防治具有很大的局限性,本试验通过对木霉菌的筛选和改良,以期能从生物防治途径控制病害的发生,降低危害程度。试验结果表明：
1.从雅安四川农业大学农场采集发生纹枯病的玉米叶鞘分离得到病原菌,经鉴定为立枯丝核菌(Rhizoctonia solani),属于AG-1-IA菌丝融合群。用组织分离法从有绿色孢子附着的玉米叶鞘上分离获得生长速度较快的木霉菌株40个,其中哈茨木霉24株、康氏木霉7株、绿色木霉5株、拟康氏木霉4株。木霉生长速度测定结果表明,大多数木霉生长速度快、生命力强,能够迅速占领生长的空间和营养。所有木霉发酵滤液对立枯丝核菌的生长都有抑制作用,效果最好的是T19,抑制率为61.67%。因此,选择在木霉菌株T19进行改良。
2.采用UV、UV+LiCl和DES三种不同的诱变方法来获得具有稳定遗传性的高产几丁质酶木霉菌株。确定了对T19的紫外诱变最佳时间为120s、氯化锂诱变最佳计量为0.4%、硫酸二乙酯诱变最佳时间为40分钟。其中使用紫外光合氯化锂复合诱变获得的突变菌株UV+LiCl-1的酶活力最高,为1.21 U/ml,是出发菌株的2.52倍,且遗传稳定性良好。
3.采用正交试验设计,研究了培养基碳源、氮源等营养物质以及发酵培养温度、时间等条件对突变菌株UV+LiCl-1产几丁质酶的影响。结果表明,产酶培养基的最佳配比为：胶体几丁质10.0g/L、蛋白胨2.0g/L、KH2PO4 2.0g/L、MgSO4.7H2O 0.6g/L。优化发酵的条件为：起始pH6.0、温度28℃、接种量7%、诱导5d。在此条件下,变异菌株获得的最高几丁质酶活性进一步提高到1.78U/ml,是出发菌株的3.71倍。
4.采用硫酸铵(NH4)2SO4分级沉淀法提取几丁质酶液,得到提取木霉几丁质酶的最佳(NH4)2SO4浓度为75%。将粗酶液加入PD培养基中,当几丁质酶含量为10%时,对立枯丝核菌菌丝干重的抑制率达到了68.14%,抑制效果明显。
5.木霉菌株T19和UV+LiCl-1都能够在玉米根际土壤、根表及叶鞘上定殖。T19在根际土壤、根表土壤和玉米叶鞘菌落形成单位分别为16.67×104个/g、5.56x104个/g、8.33×104个/g；而突变菌株UV+LiCl-1的菌落形成单位分别为34.44×104个/g、13.33×104个/g、10.67×104个/g,较野生菌株的定殖能力有所提高。
6.试验中用于生物防治的2个木霉菌株对玉米纹枯病都有一定的防治效果。突变菌株UV+LiCl-1的防效达到了75.7%,显著高于野生菌株T19(65.06%),较好地控制了玉米纹枯病的发生。
Maize sheath blight is a main disease of corn which is destructive soil-borne disease and can cause significant yield loss. Presently, it is difficult to select resistant plants using conventional breeding, soil-borne diseases are hard to be controlled and chemical control meets dilemma. So in this experiment, we screened and improved the Trichoderma strains in order to favor for the disease biocontrol. The results showed that:
1. According to the test of anastomosis groups of Rhizoctonia solani, the pathogen of maize sheath blight, which was isolated from diseased sheath of maize in suburb of Yaan, Sichuan, was grouped into Rhizoctonia solani AG-1-IA.40 Trichoderma strains were isolated from the maize rhizosphere soils in suburb of Ya'an and identified. There were 24 isolates of T.harzianum,7 isolates of T.koningii,5 isolates of T.viride,4 isolates of T.pseudokoningii respectively. The test of hyphal growth rate showed that most of Trichoderma strains grew rapidly, and were the stronger competitor for space and nutrition resource. All the fermentation filtrates of Trichoderma strains could inhibite the growth of R.solani. T19 showed the highest inhibition rate,61.67%. Therefore, we selected it to perform the further improvement.
2. Three approaches of physical mutagenesis, UV、UV+LiCl and DES, were used to obtain high yielding chitinase Rhizoctonia solani strain with stable inheritance. The result showed that the best UV mutagenic time is 120s, the LiCl strengths is 0.4% and DES mutagenic time is 40min. The chitinase activity of UV+LiCl-1, with stable heritage, increased to 1.21 U/ml which is 2.52 times compared with that the initial strain T19.
3. The optimum medium and fermentation condition for UV+LiCl-1 have been established through orthogonal test design. Under the established optimal culture conditions which incoulding colloidal chitin 10.0g/L, peptone 2.0g/L, KH2PO4 2.0g/L, MgSO4.7H2O 0.6g/L and in the established optimal medium which incoulding temperature 28℃, fermentation time 5d, pH 6.0, inoculum size 7%. The chitinase activity of UV+LiCl-1 increased to 1.78U/ml which is 3.71 times compared with that the initial strain under the culture condition before optimization.
4. The methord of ammonium sulfate grading precipitation was used to extract the chitinase solution. The best concentration of (NH4)2SO4, which was used to extract the Trichoderma chitinase, was 75%. Put the chitinase into the PD medium, when the concentration of the chitinase was 10%, the inhibition rate against the dry weight of Rhizoctonia mycelial was up to 68.14%, the inhibition effect is obvious.
5. Trichoderma strain T19 and UV+LiCl-1 were able to colonize in maize rhizosphere soils, the surface of its roots and the leaf sheath. The colony forming units (CFU) of the T19 were 16.67×104/g,5.56×104/g, and 8.33×104/g, respectively. And the CFU of the mutant UV+LiCl-1 were 34.44×104/g,13.33×104/g, and 10.67×104/g, respectively, which was higher than the colonization ability of wild strains.
6. All of the two biocontrol's Trichoderma strains have a certain effect against maize sheath blight in this experiment. The effect of mutant strain UV+LiCl-1 was 75.7%, which was significantly higher than the effect 65.06% of original strain T19. It has conrroled the incidence of maize sheath blight better.
1.从雅安四川农业大学农场采集发生纹枯病的玉米叶鞘分离得到病原菌,经鉴定为立枯丝核菌(Rhizoctonia solani),属于AG-1-IA菌丝融合群。用组织分离法从有绿色孢子附着的玉米叶鞘上分离获得生长速度较快的木霉菌株40个,其中哈茨木霉24株、康氏木霉7株、绿色木霉5株、拟康氏木霉4株。木霉生长速度测定结果表明,大多数木霉生长速度快、生命力强,能够迅速占领生长的空间和营养。所有木霉发酵滤液对立枯丝核菌的生长都有抑制作用,效果最好的是T19,抑制率为61.67%。因此,选择在木霉菌株T19进行改良。
2.采用UV、UV+LiCl和DES三种不同的诱变方法来获得具有稳定遗传性的高产几丁质酶木霉菌株。确定了对T19的紫外诱变最佳时间为120s、氯化锂诱变最佳计量为0.4%、硫酸二乙酯诱变最佳时间为40分钟。其中使用紫外光合氯化锂复合诱变获得的突变菌株UV+LiCl-1的酶活力最高,为1.21 U/ml,是出发菌株的2.52倍,且遗传稳定性良好。
3.采用正交试验设计,研究了培养基碳源、氮源等营养物质以及发酵培养温度、时间等条件对突变菌株UV+LiCl-1产几丁质酶的影响。结果表明,产酶培养基的最佳配比为：胶体几丁质10.0g/L、蛋白胨2.0g/L、KH2PO4 2.0g/L、MgSO4.7H2O 0.6g/L。优化发酵的条件为：起始pH6.0、温度28℃、接种量7%、诱导5d。在此条件下,变异菌株获得的最高几丁质酶活性进一步提高到1.78U/ml,是出发菌株的3.71倍。
4.采用硫酸铵(NH4)2SO4分级沉淀法提取几丁质酶液,得到提取木霉几丁质酶的最佳(NH4)2SO4浓度为75%。将粗酶液加入PD培养基中,当几丁质酶含量为10%时,对立枯丝核菌菌丝干重的抑制率达到了68.14%,抑制效果明显。
5.木霉菌株T19和UV+LiCl-1都能够在玉米根际土壤、根表及叶鞘上定殖。T19在根际土壤、根表土壤和玉米叶鞘菌落形成单位分别为16.67×104个/g、5.56x104个/g、8.33×104个/g；而突变菌株UV+LiCl-1的菌落形成单位分别为34.44×104个/g、13.33×104个/g、10.67×104个/g,较野生菌株的定殖能力有所提高。
6.试验中用于生物防治的2个木霉菌株对玉米纹枯病都有一定的防治效果。突变菌株UV+LiCl-1的防效达到了75.7%,显著高于野生菌株T19(65.06%),较好地控制了玉米纹枯病的发生。
Maize sheath blight is a main disease of corn which is destructive soil-borne disease and can cause significant yield loss. Presently, it is difficult to select resistant plants using conventional breeding, soil-borne diseases are hard to be controlled and chemical control meets dilemma. So in this experiment, we screened and improved the Trichoderma strains in order to favor for the disease biocontrol. The results showed that:
1. According to the test of anastomosis groups of Rhizoctonia solani, the pathogen of maize sheath blight, which was isolated from diseased sheath of maize in suburb of Yaan, Sichuan, was grouped into Rhizoctonia solani AG-1-IA.40 Trichoderma strains were isolated from the maize rhizosphere soils in suburb of Ya'an and identified. There were 24 isolates of T.harzianum,7 isolates of T.koningii,5 isolates of T.viride,4 isolates of T.pseudokoningii respectively. The test of hyphal growth rate showed that most of Trichoderma strains grew rapidly, and were the stronger competitor for space and nutrition resource. All the fermentation filtrates of Trichoderma strains could inhibite the growth of R.solani. T19 showed the highest inhibition rate,61.67%. Therefore, we selected it to perform the further improvement.
2. Three approaches of physical mutagenesis, UV、UV+LiCl and DES, were used to obtain high yielding chitinase Rhizoctonia solani strain with stable inheritance. The result showed that the best UV mutagenic time is 120s, the LiCl strengths is 0.4% and DES mutagenic time is 40min. The chitinase activity of UV+LiCl-1, with stable heritage, increased to 1.21 U/ml which is 2.52 times compared with that the initial strain T19.
3. The optimum medium and fermentation condition for UV+LiCl-1 have been established through orthogonal test design. Under the established optimal culture conditions which incoulding colloidal chitin 10.0g/L, peptone 2.0g/L, KH2PO4 2.0g/L, MgSO4.7H2O 0.6g/L and in the established optimal medium which incoulding temperature 28℃, fermentation time 5d, pH 6.0, inoculum size 7%. The chitinase activity of UV+LiCl-1 increased to 1.78U/ml which is 3.71 times compared with that the initial strain under the culture condition before optimization.
4. The methord of ammonium sulfate grading precipitation was used to extract the chitinase solution. The best concentration of (NH4)2SO4, which was used to extract the Trichoderma chitinase, was 75%. Put the chitinase into the PD medium, when the concentration of the chitinase was 10%, the inhibition rate against the dry weight of Rhizoctonia mycelial was up to 68.14%, the inhibition effect is obvious.
5. Trichoderma strain T19 and UV+LiCl-1 were able to colonize in maize rhizosphere soils, the surface of its roots and the leaf sheath. The colony forming units (CFU) of the T19 were 16.67×104/g,5.56×104/g, and 8.33×104/g, respectively. And the CFU of the mutant UV+LiCl-1 were 34.44×104/g,13.33×104/g, and 10.67×104/g, respectively, which was higher than the colonization ability of wild strains.
6. All of the two biocontrol's Trichoderma strains have a certain effect against maize sheath blight in this experiment. The effect of mutant strain UV+LiCl-1 was 75.7%, which was significantly higher than the effect 65.06% of original strain T19. It has conrroled the incidence of maize sheath blight better.
引文
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