黑龙江省大豆核盘菌生物学特性和生物防治的研究
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摘要
核盘菌Sclerotinia sclerotiorum (Lib) de Bary是一种子囊真菌,其寄主范围和地理分布十分广泛,是一种危害相当严重的世界性植物病害真菌。近几年来核盘菌病害,俗称菌核病,在东北大豆产区发病日趋严重,本研究的主要目的是对黑龙江省不同地区的菌核病病原进行形态学、生物学、致病性系统研究,以及用绿色木霉防治该病的室内和田间试验研究,以期获得东北不同地区菌核病病原的主要差异和在不同地区的绿色木霉防治大豆菌核病的效果,为大豆安全生产奠定理论基础。主要的工作及结果如下:
完整地揭示了黑龙江大豆核盘菌的生物学特性及核盘菌侵染寄主对农艺性状所造成的不良影响;提供了研究核盘菌侵染寄主的多种观察方法。
通过对东北不同生态区的菌核病病原的形态学特性分析表明菌核病的菌落特征有差异,毒力相近。海伦市采集的菌核形成了闭合的圆环菌落特征;采自佳木斯市的菌核首先形成不均匀的离散的菌落特征,而后在培养皿边缘或近边缘形成菌核粒组成的环形菌落特征。海伦地区的菌丝丰度明显高于佳木斯地区,海伦地区采集的菌核大小大于佳木斯地区采集的。离体草酸试验表明两个地区的分离物的毒力可能相近。
离体叶片接种和盆栽试验表明大豆离体叶片接种核盘菌病原30天后整个叶片全部被核盘菌丝分解;盆栽条件下大豆两片真叶出现菌丝体,16天下部叶片出现菌丝团。染病的大豆籽粒百粒重下降,粗脂肪含量、粗蛋白含量均有下降趋势,粗脂肪含量下降幅度大于粗蛋白含量。氨基酸总量、氨峰值也有相同趋势。不同地区的核盘菌生理小种病情指数差异显著,致病力差异显著,且病情指数与病斑长度呈正相关。以成株期的鉴定结果作为品种抗菌核病的指标较为合适。
采用实验室与田间相结合的方法,全面研究生防菌防治大豆菌核病的效果,为生物防治核盘菌提供了具有可操作性的实验模式。
室内外试验表明绿色木霉对核盘菌有明显的抑制作用;风沙土上施用生防剂防治大豆菌核病的效果好于其它土壤。随着大豆生长时间周期推移,生防菌剂的作用效果逐渐下降;在黑土、风沙土施用不同量的生防菌剂,随着用量的增加,大豆菌核病发病率呈下降趋势;风沙土中,生防菌剂能够有效地降低田间发病率,但在增产效果上幅度不大。
摸索出一整套针对核盘菌丝制备原生质体并进行转化的分子生物学操作体系,搭建了生物防治机理研究的验证平台。
由于绿色木霉和核盘菌的菌丝在微观下很难辨认,它们之间的抑制作用很难明确,为此构建了含有绿色荧光蛋白(GFP)基因表达载体pPGT。并采用PEG介导的原生质体转化法成功地把绿色荧光蛋白基因GFP转入病原真菌核盘菌中。通过Southern杂交分析表明,GFP基因已经整合到核盘菌基因组DNA中,从而证明绿色荧光蛋白基因在核盘菌中成功表达,并且能够稳定遗传。通过核盘菌和绿色木霉的对峙试验在荧光显微镜下的观察证明了绿色木霉对核盘菌的抑制作用。
针对东北不同生态区菌核病病原的生物学特性和致病性的研究,为进一步研究菌核病的发生规律和利用抗病大豆品种防治菌核病提供基础材料,同时利用绿色木霉菌防治大豆菌核病,为东北的大豆安全生产提供了必要的措施。
Sclerotinia sclerotiorum (Lib) de Bary is a Ascomycete with a wide host rangeS and geographical distribution, which is one of the most non-specific, omnivoroes and successful plant pathogens. In resent years, the outbreak of Sclerotinia sclerotiorum, also called sclerotium, more and more severe in northeast soybean plant areas. The objective of the study was to identify morphology, biochemistry and pathogenicity of the sclerotium isolated from different areas in northeast of China and the biocontrol effect of Trichoderma spp. to sclerotium by antagonistic on plates and field experiments, to show the main various sclerotium isolates and biocontrol effect of Trichoderma viride Pers. in northeast different areas. The results provided theory basis for soybean safety product. Main experiments and acquired results like these:
The results showed Sclerotinia sclerotiorum (Lib) de Bary chacteristics and the negative effects of agronomic traits for inoculated hosts in northeat soybean areas; It supplied mang research methods for sclerotium inoculated hosts.
Analysis results of Sclerotinia sclerotiorum morphology character collected from different norheast ecological zones showed that it was discrepancy and toxicity similar. Sclerotium acquired from Hailun city can form close roundly colony, however, acquired from Jiamu Si city form uneven and discrete colony first, and then form ring-shaped colony by sclerotium at the marginal zone or near edge of culture dish. Hypha abundance of Hailun city was higher than acquired Jiamu Si city significantly, and sclerotia acquired from Hailun city are bigger than Jiamu Si city. Results of exsomatize oxalic acid test showed that isolates toxicity from two areas may be similar.
Results of exsomatized leaves inoculation and pot experiments showed that whole soybean leaves would be decomposed by Sclerotinia sclerotiorum (Lib) de Bary hypha after inoculated 30d; pot experiments results indicated that mycelium appears when soybean has two leaves and after 16d hypha block appears. Kernel weight, coarse fat and coarse prote content of infected soybean all declined, and coarse content declined more than coarse protein content. Amino acid gross and ammonia peak value have the same trend with them. Disease severity index (DSI) and pathopoiesia of physiological race of Sclerotinia sclerotiorum (Lib) de Bary were various greatly among different areas, besides disease severity index (DSI) and lesion length has positive correlation. It is suitable that identify at the stage of growth up as the standard of species resistance to the disease.
Roundly researched on the biocontrol effects of biocontrol bacteria on soybean sclerotium by combined methods of laboratory and yield supplied maneuverability experimental model for sclerotium biocontrol.
Results of pot experiments and on PDA plates showed that Trichoderma viride Pers. can inhibit Sclerotinia sclerotiorum (Lib) de Bary significantly; the effect of application biocontrol agent on wind-sand soil was better than other types of soil. With the soybean growth, the effect of biocontrol agent declined gradually; Application of potassium fertilizer can inhibit the growth of Sclerotinia sclerotiorum (Lib) de Bary. With the increase application of biocontrol agent, incidence of the disease declined on black soil and wind-sand soil, it can reduce the incidence of field significantly, but hasn’t great effect to yield on wind-sand soil.
Groping a molecular biology operationg system for protoplast preparated by Sclerotinia sclerotiorum (Lib) de Bary hypha and translated, built examination platform for biocontrol mechanism studies.
It was difficult to identify the hypha of Trichoderma viride Pers or Sclerotinia sclerotiorum (Lib) de Bary, and illustrate their inhibition, so that composing gene expressing carrier pPGT which has GFP. It was succeeded to transfer the GFP gene to pathogen fungal Sclerotium by the method of protoplast transformation with PEG. Results of Southern hybridize indicated that GFP gene has been integrated to Sclerotium genesis DNA. It proved that GFP gene could be expressed in Sclerotium successfully, and could inherit steadily. Antagonistic experiments proved that Trichoderma viride Pers could inhibit Sclerotinia sclerotiorum (Lib) de Bary observed under the fluorescence microscope.
Base on the study of pathogen isolates morphology characters and pathopoiesia in different northeast ecological zones, supplied more materials for the studies of sclerotium occur discipline and soybean resist disease breeding. The biocontrol study of Trichoderma viride Pers to soybean sclerotium provided necessary measures for northeast soybean safety products.
完整地揭示了黑龙江大豆核盘菌的生物学特性及核盘菌侵染寄主对农艺性状所造成的不良影响;提供了研究核盘菌侵染寄主的多种观察方法。
通过对东北不同生态区的菌核病病原的形态学特性分析表明菌核病的菌落特征有差异,毒力相近。海伦市采集的菌核形成了闭合的圆环菌落特征;采自佳木斯市的菌核首先形成不均匀的离散的菌落特征,而后在培养皿边缘或近边缘形成菌核粒组成的环形菌落特征。海伦地区的菌丝丰度明显高于佳木斯地区,海伦地区采集的菌核大小大于佳木斯地区采集的。离体草酸试验表明两个地区的分离物的毒力可能相近。
离体叶片接种和盆栽试验表明大豆离体叶片接种核盘菌病原30天后整个叶片全部被核盘菌丝分解;盆栽条件下大豆两片真叶出现菌丝体,16天下部叶片出现菌丝团。染病的大豆籽粒百粒重下降,粗脂肪含量、粗蛋白含量均有下降趋势,粗脂肪含量下降幅度大于粗蛋白含量。氨基酸总量、氨峰值也有相同趋势。不同地区的核盘菌生理小种病情指数差异显著,致病力差异显著,且病情指数与病斑长度呈正相关。以成株期的鉴定结果作为品种抗菌核病的指标较为合适。
采用实验室与田间相结合的方法,全面研究生防菌防治大豆菌核病的效果,为生物防治核盘菌提供了具有可操作性的实验模式。
室内外试验表明绿色木霉对核盘菌有明显的抑制作用;风沙土上施用生防剂防治大豆菌核病的效果好于其它土壤。随着大豆生长时间周期推移,生防菌剂的作用效果逐渐下降;在黑土、风沙土施用不同量的生防菌剂,随着用量的增加,大豆菌核病发病率呈下降趋势;风沙土中,生防菌剂能够有效地降低田间发病率,但在增产效果上幅度不大。
摸索出一整套针对核盘菌丝制备原生质体并进行转化的分子生物学操作体系,搭建了生物防治机理研究的验证平台。
由于绿色木霉和核盘菌的菌丝在微观下很难辨认,它们之间的抑制作用很难明确,为此构建了含有绿色荧光蛋白(GFP)基因表达载体pPGT。并采用PEG介导的原生质体转化法成功地把绿色荧光蛋白基因GFP转入病原真菌核盘菌中。通过Southern杂交分析表明,GFP基因已经整合到核盘菌基因组DNA中,从而证明绿色荧光蛋白基因在核盘菌中成功表达,并且能够稳定遗传。通过核盘菌和绿色木霉的对峙试验在荧光显微镜下的观察证明了绿色木霉对核盘菌的抑制作用。
针对东北不同生态区菌核病病原的生物学特性和致病性的研究,为进一步研究菌核病的发生规律和利用抗病大豆品种防治菌核病提供基础材料,同时利用绿色木霉菌防治大豆菌核病,为东北的大豆安全生产提供了必要的措施。
Sclerotinia sclerotiorum (Lib) de Bary is a Ascomycete with a wide host rangeS and geographical distribution, which is one of the most non-specific, omnivoroes and successful plant pathogens. In resent years, the outbreak of Sclerotinia sclerotiorum, also called sclerotium, more and more severe in northeast soybean plant areas. The objective of the study was to identify morphology, biochemistry and pathogenicity of the sclerotium isolated from different areas in northeast of China and the biocontrol effect of Trichoderma spp. to sclerotium by antagonistic on plates and field experiments, to show the main various sclerotium isolates and biocontrol effect of Trichoderma viride Pers. in northeast different areas. The results provided theory basis for soybean safety product. Main experiments and acquired results like these:
The results showed Sclerotinia sclerotiorum (Lib) de Bary chacteristics and the negative effects of agronomic traits for inoculated hosts in northeat soybean areas; It supplied mang research methods for sclerotium inoculated hosts.
Analysis results of Sclerotinia sclerotiorum morphology character collected from different norheast ecological zones showed that it was discrepancy and toxicity similar. Sclerotium acquired from Hailun city can form close roundly colony, however, acquired from Jiamu Si city form uneven and discrete colony first, and then form ring-shaped colony by sclerotium at the marginal zone or near edge of culture dish. Hypha abundance of Hailun city was higher than acquired Jiamu Si city significantly, and sclerotia acquired from Hailun city are bigger than Jiamu Si city. Results of exsomatize oxalic acid test showed that isolates toxicity from two areas may be similar.
Results of exsomatized leaves inoculation and pot experiments showed that whole soybean leaves would be decomposed by Sclerotinia sclerotiorum (Lib) de Bary hypha after inoculated 30d; pot experiments results indicated that mycelium appears when soybean has two leaves and after 16d hypha block appears. Kernel weight, coarse fat and coarse prote content of infected soybean all declined, and coarse content declined more than coarse protein content. Amino acid gross and ammonia peak value have the same trend with them. Disease severity index (DSI) and pathopoiesia of physiological race of Sclerotinia sclerotiorum (Lib) de Bary were various greatly among different areas, besides disease severity index (DSI) and lesion length has positive correlation. It is suitable that identify at the stage of growth up as the standard of species resistance to the disease.
Roundly researched on the biocontrol effects of biocontrol bacteria on soybean sclerotium by combined methods of laboratory and yield supplied maneuverability experimental model for sclerotium biocontrol.
Results of pot experiments and on PDA plates showed that Trichoderma viride Pers. can inhibit Sclerotinia sclerotiorum (Lib) de Bary significantly; the effect of application biocontrol agent on wind-sand soil was better than other types of soil. With the soybean growth, the effect of biocontrol agent declined gradually; Application of potassium fertilizer can inhibit the growth of Sclerotinia sclerotiorum (Lib) de Bary. With the increase application of biocontrol agent, incidence of the disease declined on black soil and wind-sand soil, it can reduce the incidence of field significantly, but hasn’t great effect to yield on wind-sand soil.
Groping a molecular biology operationg system for protoplast preparated by Sclerotinia sclerotiorum (Lib) de Bary hypha and translated, built examination platform for biocontrol mechanism studies.
It was difficult to identify the hypha of Trichoderma viride Pers or Sclerotinia sclerotiorum (Lib) de Bary, and illustrate their inhibition, so that composing gene expressing carrier pPGT which has GFP. It was succeeded to transfer the GFP gene to pathogen fungal Sclerotium by the method of protoplast transformation with PEG. Results of Southern hybridize indicated that GFP gene has been integrated to Sclerotium genesis DNA. It proved that GFP gene could be expressed in Sclerotium successfully, and could inherit steadily. Antagonistic experiments proved that Trichoderma viride Pers could inhibit Sclerotinia sclerotiorum (Lib) de Bary observed under the fluorescence microscope.
Base on the study of pathogen isolates morphology characters and pathopoiesia in different northeast ecological zones, supplied more materials for the studies of sclerotium occur discipline and soybean resist disease breeding. The biocontrol study of Trichoderma viride Pers to soybean sclerotium provided necessary measures for northeast soybean safety products.
引文
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