除草微生物禾长蠕孢菌种诱变改良及分子鉴定
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摘要
草害是制约农作物产量的主要因子之一。目前,杂草的防治主要依赖化学除草剂,但其对环境的负面影响日益受到人们的关注,减少化学除草剂的使用是未来农业的发展趋势。发展微生物除草剂,实现杂草的生物防治,可以大幅度减少化学除草剂的使用,实现农业的可持续发展。
禾长蠕孢稗草专化型(Helminthosporium gramineum Rabenh f.sp.echinochloae,简称HGE),是本实验室从感病的稗草上分离获得、由中国科学院微生物研究所完成菌种鉴定。HGE对稗草的致病力强,对水稻安全,是稗草生物防治的潜力菌。为进一步提高该菌种的孢子产量和代谢产物产量,采用紫外诱变对该菌种进行了改良。
经过筛选和评价,获得适合液体发酵和固体发酵的突变菌株M1和M3。其中突变菌株M1菌落生长疏松,气生菌丝多,菌落呈灰白色至白色,与出发菌株HGE有显著区别,该突变菌株生长速度快,菌丝体和发酵液毒素产量分别达到7.0g/L和15.98mg/L。另一突变菌株M3菌落生长紧密,气生菌丝少,菌落呈黑色,与出发菌株HGE相似。突变菌株M3的孢子产量达到1.21×10~7/皿,比出发菌株提高21%。两突变菌株均具有良好的遗传稳定性,对水稻、玉米等作物安全。
利用RAPD-PCR方法对出发菌株和突变菌株M1和M3进行了分子鉴定,初步说明突变菌株的遗传物质发生了改变。对出发菌株和突变菌株的固体发酵和液体发酵工艺进行了初步研究,确定了相应的最佳发酵条件。其中固体发酵的最佳条件是稗草基质培养基中添加生长因子③,以2菌块/克稗草干物质接种,27℃培养箱中黑暗培养10天。突变菌株M1菌丝体产量的最佳发酵条件组合为:3号培养基,初始pH值为7,发酵温度为24℃,150r/min振荡培养6d;毒素产量的最佳发酵条件组合为:4号培养基,初始pH值为7,发酵温度为26℃,静置培养14d。
Weeds interference is a serious constraint in agricultural production. Presently, the use of chemical herbicides was an important measure to save the cost and improve the efficiency of the agriculture. But the concern is now being expressed throughout the world about the environmental impact, and effects of the widespread use of chemical herbicides. This has fuelled the current upsurge of interest in biological control of weeds using pathogens. It is increasingly being considered as an alternative to chemical control strategies. This is especially true for sustainable agriculture, where adverse effects of agrochemicals on the environment must be minimized or avoided.The plant pathogenic fungus Helminthosporium gramineum Rabenh f.sp.echinochloae, abbreviated as HGE, had been isolated from diseased barnyardgrass (Echinochloa crus-galli (L.) Beauv.) plants. The isolate showed high pathogenicity and specificity towards barnyardgrass. The rice tested was not susceptible to the fugus. Thus HGE is well suited to be developed as a mycoherbicide for barnyardgrass control. In this project, the conidia of HGE were treated by ultraviolet radiation to improve the yield of conidia and metabolized products.From the mutants, we selected mutant Ml and mutant M3 for which may be utilized in the fermentation.The colonies of Ml are white, loose and with more mycelia, which was obviously different from HGE. The M1 grew faster with a higher mycelia and toxin yields. The dry-weight of mycelia was 7.0g/L and the toxin yield was 15.98 mg/L. The colonies of M3 are black, compact and with few mycelia. which was similar to HGE. The mutant M3 produced 1.21 × 10~7 conidia on a fresh potato dextrose agar plate after culturing for two weeks at 28℃ in dark. The conidia yield was 21 percent higher than that of HGE. They both have better pathogenicity and genetic stablitity, and were safe to crop plants such as rice and maize.The change of hereditary material between M1,M3 and HGE can be proved by RAPD-PCR. The solid substrates that use barnyardgrass plants and rice as primary ingredient for mass conidia production were studied respectively. For mass conidia production, it was better to use barnyardgrass plants as primary ingredient. The substrate were inoculated with mycelial plug and incubated for 10 days at 27℃ in dark. Submerged culture experiments were conducted to determine the optimal medium for rapid producing mycelia and metabolized products of the mycoherbicide. The highest mycelial yield of Ml occurred in No. 3 medium, of which the pH was adjusted to 7. The cultures were grown on a shaker adjusted to 150 rpm at 24℃ for 6 days. The No. 4 medium is good for producing toxin. The pH was adjusted to 7 and the cultures wre incubated at 26℃ for 2 weeks.
禾长蠕孢稗草专化型(Helminthosporium gramineum Rabenh f.sp.echinochloae,简称HGE),是本实验室从感病的稗草上分离获得、由中国科学院微生物研究所完成菌种鉴定。HGE对稗草的致病力强,对水稻安全,是稗草生物防治的潜力菌。为进一步提高该菌种的孢子产量和代谢产物产量,采用紫外诱变对该菌种进行了改良。
经过筛选和评价,获得适合液体发酵和固体发酵的突变菌株M1和M3。其中突变菌株M1菌落生长疏松,气生菌丝多,菌落呈灰白色至白色,与出发菌株HGE有显著区别,该突变菌株生长速度快,菌丝体和发酵液毒素产量分别达到7.0g/L和15.98mg/L。另一突变菌株M3菌落生长紧密,气生菌丝少,菌落呈黑色,与出发菌株HGE相似。突变菌株M3的孢子产量达到1.21×10~7/皿,比出发菌株提高21%。两突变菌株均具有良好的遗传稳定性,对水稻、玉米等作物安全。
利用RAPD-PCR方法对出发菌株和突变菌株M1和M3进行了分子鉴定,初步说明突变菌株的遗传物质发生了改变。对出发菌株和突变菌株的固体发酵和液体发酵工艺进行了初步研究,确定了相应的最佳发酵条件。其中固体发酵的最佳条件是稗草基质培养基中添加生长因子③,以2菌块/克稗草干物质接种,27℃培养箱中黑暗培养10天。突变菌株M1菌丝体产量的最佳发酵条件组合为:3号培养基,初始pH值为7,发酵温度为24℃,150r/min振荡培养6d;毒素产量的最佳发酵条件组合为:4号培养基,初始pH值为7,发酵温度为26℃,静置培养14d。
Weeds interference is a serious constraint in agricultural production. Presently, the use of chemical herbicides was an important measure to save the cost and improve the efficiency of the agriculture. But the concern is now being expressed throughout the world about the environmental impact, and effects of the widespread use of chemical herbicides. This has fuelled the current upsurge of interest in biological control of weeds using pathogens. It is increasingly being considered as an alternative to chemical control strategies. This is especially true for sustainable agriculture, where adverse effects of agrochemicals on the environment must be minimized or avoided.The plant pathogenic fungus Helminthosporium gramineum Rabenh f.sp.echinochloae, abbreviated as HGE, had been isolated from diseased barnyardgrass (Echinochloa crus-galli (L.) Beauv.) plants. The isolate showed high pathogenicity and specificity towards barnyardgrass. The rice tested was not susceptible to the fugus. Thus HGE is well suited to be developed as a mycoherbicide for barnyardgrass control. In this project, the conidia of HGE were treated by ultraviolet radiation to improve the yield of conidia and metabolized products.From the mutants, we selected mutant Ml and mutant M3 for which may be utilized in the fermentation.The colonies of Ml are white, loose and with more mycelia, which was obviously different from HGE. The M1 grew faster with a higher mycelia and toxin yields. The dry-weight of mycelia was 7.0g/L and the toxin yield was 15.98 mg/L. The colonies of M3 are black, compact and with few mycelia. which was similar to HGE. The mutant M3 produced 1.21 × 10~7 conidia on a fresh potato dextrose agar plate after culturing for two weeks at 28℃ in dark. The conidia yield was 21 percent higher than that of HGE. They both have better pathogenicity and genetic stablitity, and were safe to crop plants such as rice and maize.The change of hereditary material between M1,M3 and HGE can be proved by RAPD-PCR. The solid substrates that use barnyardgrass plants and rice as primary ingredient for mass conidia production were studied respectively. For mass conidia production, it was better to use barnyardgrass plants as primary ingredient. The substrate were inoculated with mycelial plug and incubated for 10 days at 27℃ in dark. Submerged culture experiments were conducted to determine the optimal medium for rapid producing mycelia and metabolized products of the mycoherbicide. The highest mycelial yield of Ml occurred in No. 3 medium, of which the pH was adjusted to 7. The cultures were grown on a shaker adjusted to 150 rpm at 24℃ for 6 days. The No. 4 medium is good for producing toxin. The pH was adjusted to 7 and the cultures wre incubated at 26℃ for 2 weeks.
引文
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