西瓜枯萎病拮抗菌株筛选及其活性物质的研究
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摘要
西瓜枯萎病是世界各主要西瓜产区最为严重的病害之一。本课题以西瓜枯萎病的病原菌为筛选模型,采用对峙培养法筛选西瓜枯萎病拮抗菌株,并研究拮抗菌株所产生的活性物质。首先采集合肥、马鞍山等地健康西瓜的根际土壤,进行菌株的分离纯化,建立了细菌菌种库;再用对峙培养法筛选出西瓜枯萎病拮抗菌株,获得拮抗作用很强的菌株FM4;然后对FM4进行了详细的研究:包括菌株鉴定、活性物质的分离纯化及其理化性质研究、发酵条件优化,并初步尝试利用N~+注入法对FM4进行诱变育种。主要结论如下:
1.采集安庆、合肥和马鞍山地区健康西瓜的根际土壤,进行菌株分离纯化,获得了784株不同类型的菌株,对峙培养结果表明,对西瓜枯萎病有拮抗作用的有29株。其中,菌株FM4拮抗作用很强。
2.根据《伯杰细菌鉴定手册》和《常见细菌系统鉴定手册》的有关细菌分类鉴定标准,FM4菌株的形态特征、培养特征及生理生化性质与微球菌属的主要鉴别特征基本一致。由此,初步认定FM4菌株属于为微球菌属。
3.采用醇沉和Sephadex G-150凝胶层析法对拮抗菌株FM4的活性物质进行分离纯化,从中分离得到一个拮抗蛋白质,分子量约120kDa。经质谱初步鉴定,可知该蛋白与来源于Brevibacillus brevis的Middle cell wall protein precursor (MWP)有较高的相似度。实验表明,该抗菌物质具有拮抗活性稳定、耐热性好、pH值稳定及抗菌谱广的特性,在25-100℃范围内随着处理温度的提高,抑菌能力呈稳定趋势,几乎不变化。对不同酸碱度的试验表明抗菌物的活性pH范围较宽,从pH 2-10,检测皆有活性。活性物质对大肠埃希氏菌、金黄色葡萄球菌、毕氏酵母菌、肠膜明串珠菌、里氏木霉菌皆具有抑制作用。在西瓜枯萎病的防治中有着广阔的应用前景。
4.本研究采用单因素加正交设计试验法和响应曲面法,对拮抗菌株FM4分别进行了培养基优化和培养条件的优化。获得了最佳的培养基配方和培养条件:葡萄糖1.00%,蛋白胨2.23%,磷酸氢二钾0.01%,七水硫酸镁0.05%。最佳的培养条件为:培养时间22h,培养温度28.5℃,初始pH为7.50,摇床转速170.00r/min。与原始发酵条件相比,发酵液抑菌圈直径提高了42.85%。
5.采用N~+注入法进行高产菌株的初步选育。现代育种理论认为诱变微生物致死率在70%～85%左右时正突变率较高,实验表明,选择10 keV,40×2.6×1013N~+/cm2作为该菌株的离子注入参数,致死率在81.25%左右。第一轮N~+注入后,筛选得到9株突变株,分别为:75、76、81、82、83、89、90、188和190.经过摇瓶复筛和菌株传代稳定性实验后,最后得到一株突变株190,其抑菌圈直径比出发菌株的增加了52.38%,且传代稳定。
Watermelon wilt disease is a soil epidemic which affects the growth of watermelon badly.The experiment was aim to obtain some high antagonistic strains and study their antibiotic substances.Firstly,strains library was established from healthy watermelon rhizosphere's soil .Secondly,some high antagonistic strains was obtained against fusarium oxysporum f.sp.melons from library by“bacterium to bacterium”biological control and the most antifunction strain-FM4 was picked out.At last,we did some research for FM4 in some aspects,e.g. resistance、identification、active substance isolation and purification、screening shake flask fermentation medium and fermentation conditions、utilization N~+ injection to obtain high-product strain.Main conclusions are as follows:
1.Our laboratory had obtained the 784 different types of bacteria by collecting soils from healthy watermelon rhizosphere, and then combining gradient dilution method and scribing isolation method in AnQing、HeFei、MaAnShan.Confrontation each other experiment show that there are 29 strains among them with antifunction .
2.The morphous,cultivation characteristic,biology character and physiological and biochemistry nature of the FM4 were systematically evaluated according to“Bergey’s ermination Bacteriology”and“Identification of Common Bacterial Systems Manual”,The FM4 was preliminary identified as Micrococcus.
3.By experiment research, we obtained a antifunction protein ,about 120kd,through ethanol precipitation and Sephadex G-150 .by mass spectrum initial identification, it show that this protein have a high semblance with Middle cell wall protein precursor (MWP),from Brevibacillus brevis.the antibiotic substance have higher stabilization at antibacterium、far-range temperature and pH. At 25-100℃, with the treatment temperature increasing, antimicrobial activity was stable trend, almost no change. PH of the tests showed almost antibacterial activity at a wide pH range, from pH2-10. Active substances has inhibitory effect on Escherichia coli, Staphylococcus aureus, Pichia yeast, Leuconostoc bacteria, Richter Trichoderma.
4. This research used one-factor plus orthogonal design and RSM design exepirment,and finally identified the most suitab1e 1aboratory culture medium . the laboratory culture medium was: glucose 1.00%, peptone 2.23%, dipotassium hydrogen phosphate 0.01%, epsom salt 0.05 %. The most fementation condition was : initial pH7.50,170r/min,28.45℃,21.16h.
5. According to the survival rate, 40×2.6×10~(13) ions/cm~2 N~+ with 10 KeV energy was chosen as a suitable mutation factor. This thesis enriched the protecting methods in the ion beam implantation mutation.After first round N~+ injection, we isolated 9 mutant strains,named:75、76、81、82、83、89、90、188 and 190.Through shake flask re-screening and generational stability experiment.Finally,we get one mutant strain-190,its vitality was correspondent to initial strain after going down to the future generation by twelve times.
1.采集安庆、合肥和马鞍山地区健康西瓜的根际土壤,进行菌株分离纯化,获得了784株不同类型的菌株,对峙培养结果表明,对西瓜枯萎病有拮抗作用的有29株。其中,菌株FM4拮抗作用很强。
2.根据《伯杰细菌鉴定手册》和《常见细菌系统鉴定手册》的有关细菌分类鉴定标准,FM4菌株的形态特征、培养特征及生理生化性质与微球菌属的主要鉴别特征基本一致。由此,初步认定FM4菌株属于为微球菌属。
3.采用醇沉和Sephadex G-150凝胶层析法对拮抗菌株FM4的活性物质进行分离纯化,从中分离得到一个拮抗蛋白质,分子量约120kDa。经质谱初步鉴定,可知该蛋白与来源于Brevibacillus brevis的Middle cell wall protein precursor (MWP)有较高的相似度。实验表明,该抗菌物质具有拮抗活性稳定、耐热性好、pH值稳定及抗菌谱广的特性,在25-100℃范围内随着处理温度的提高,抑菌能力呈稳定趋势,几乎不变化。对不同酸碱度的试验表明抗菌物的活性pH范围较宽,从pH 2-10,检测皆有活性。活性物质对大肠埃希氏菌、金黄色葡萄球菌、毕氏酵母菌、肠膜明串珠菌、里氏木霉菌皆具有抑制作用。在西瓜枯萎病的防治中有着广阔的应用前景。
4.本研究采用单因素加正交设计试验法和响应曲面法,对拮抗菌株FM4分别进行了培养基优化和培养条件的优化。获得了最佳的培养基配方和培养条件:葡萄糖1.00%,蛋白胨2.23%,磷酸氢二钾0.01%,七水硫酸镁0.05%。最佳的培养条件为:培养时间22h,培养温度28.5℃,初始pH为7.50,摇床转速170.00r/min。与原始发酵条件相比,发酵液抑菌圈直径提高了42.85%。
5.采用N~+注入法进行高产菌株的初步选育。现代育种理论认为诱变微生物致死率在70%～85%左右时正突变率较高,实验表明,选择10 keV,40×2.6×1013N~+/cm2作为该菌株的离子注入参数,致死率在81.25%左右。第一轮N~+注入后,筛选得到9株突变株,分别为:75、76、81、82、83、89、90、188和190.经过摇瓶复筛和菌株传代稳定性实验后,最后得到一株突变株190,其抑菌圈直径比出发菌株的增加了52.38%,且传代稳定。
Watermelon wilt disease is a soil epidemic which affects the growth of watermelon badly.The experiment was aim to obtain some high antagonistic strains and study their antibiotic substances.Firstly,strains library was established from healthy watermelon rhizosphere's soil .Secondly,some high antagonistic strains was obtained against fusarium oxysporum f.sp.melons from library by“bacterium to bacterium”biological control and the most antifunction strain-FM4 was picked out.At last,we did some research for FM4 in some aspects,e.g. resistance、identification、active substance isolation and purification、screening shake flask fermentation medium and fermentation conditions、utilization N~+ injection to obtain high-product strain.Main conclusions are as follows:
1.Our laboratory had obtained the 784 different types of bacteria by collecting soils from healthy watermelon rhizosphere, and then combining gradient dilution method and scribing isolation method in AnQing、HeFei、MaAnShan.Confrontation each other experiment show that there are 29 strains among them with antifunction .
2.The morphous,cultivation characteristic,biology character and physiological and biochemistry nature of the FM4 were systematically evaluated according to“Bergey’s ermination Bacteriology”and“Identification of Common Bacterial Systems Manual”,The FM4 was preliminary identified as Micrococcus.
3.By experiment research, we obtained a antifunction protein ,about 120kd,through ethanol precipitation and Sephadex G-150 .by mass spectrum initial identification, it show that this protein have a high semblance with Middle cell wall protein precursor (MWP),from Brevibacillus brevis.the antibiotic substance have higher stabilization at antibacterium、far-range temperature and pH. At 25-100℃, with the treatment temperature increasing, antimicrobial activity was stable trend, almost no change. PH of the tests showed almost antibacterial activity at a wide pH range, from pH2-10. Active substances has inhibitory effect on Escherichia coli, Staphylococcus aureus, Pichia yeast, Leuconostoc bacteria, Richter Trichoderma.
4. This research used one-factor plus orthogonal design and RSM design exepirment,and finally identified the most suitab1e 1aboratory culture medium . the laboratory culture medium was: glucose 1.00%, peptone 2.23%, dipotassium hydrogen phosphate 0.01%, epsom salt 0.05 %. The most fementation condition was : initial pH7.50,170r/min,28.45℃,21.16h.
5. According to the survival rate, 40×2.6×10~(13) ions/cm~2 N~+ with 10 KeV energy was chosen as a suitable mutation factor. This thesis enriched the protecting methods in the ion beam implantation mutation.After first round N~+ injection, we isolated 9 mutant strains,named:75、76、81、82、83、89、90、188 and 190.Through shake flask re-screening and generational stability experiment.Finally,we get one mutant strain-190,its vitality was correspondent to initial strain after going down to the future generation by twelve times.
引文
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