茄科植物内生细菌对几种病原真菌的作用及抗性物质分析
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摘要
植物病害是影响农业生产的主要因素之一,随着保护地蔬菜的发展和种植制度的调整,蔬菜病害的发生及危害有逐年加重趋势。疫病、病毒病、青枯病等主要病害的发生,每年给茄科蔬菜的生产造成严重的损失。目前化学农药仍是防治植物病害的主要措施,但存在着易造成残留污染、诱发病原菌抗药性和伤害非靶标生物等不利影响。利用植物内生细菌控制植物病害具有极大的发展潜力,从内生菌中寻找和发现新的活性化合物是植物内生菌研究的一个热点,植物内生菌作为一种新的微生物资源引起了广泛的关注。
本研究通过对茄科植物内生细菌的分离,并针对茄子黄萎病、番茄枯萎病、蔬菜苗期立枯病等发生面积大、防治困难的病害,筛选在植物体内定殖能力强,防病与促生作用结合的内生细菌;分析内生细菌所产生的抗菌物质及部分特性;探讨内生细菌的拮抗作用和内生性与防病作用的关系,为植物内生细菌的研究提供理论依据。其结果如下:
1、从泰安、新泰等7县(市)采集番茄、茄子、辣椒、烟草等茄科植物,分离到内生细菌共149株,其中根中47株,茎中56株,叶中33株,果中13株。筛选出对茄子黄萎病菌(Verticillium dahliae)、番茄枯萎病菌(Fusarium oxysporum f.sp.cinerea)、番茄早疫病菌(Alternaria solani)、辣椒疫病病菌(Phytophthora capsici)、蔬菜苗立枯病菌(Rhizoctonia solani)、烟草赤星病菌(Alternaria alternata)、小麦根腐病菌(Bipolaris sorokiniana)、枣叶斑病菌(Alternaria sp.)具有较好抑菌活性的内生细菌菌株共18株。
2、应用传统细菌鉴定方法对18株拮抗内生细菌进行了初步鉴定,经细菌培养性状和生理生化特性测定结果表明,所筛选的拮抗内生细菌主要为芽孢杆菌群(Bacillus sp.),占分离数的55.6%;少数为假单胞菌群(Pseudomonas sp.)和黄单胞菌群(Xanthomonas sp.)。对其中1株具有较强活性的内生细菌L-6菌株进行16S rDNA分子鉴定,通过同源性比较,确定内生拮抗细菌L-6为多粘类芽孢杆菌(Paenibacilluspolymyxa)。关于在辣椒植株体内分离到内生细菌为多粘类芽孢杆菌(Paenibacillus palymyxa),本研究尚属首次报道。
3、采用抗药性标记法对L-6菌株进行了内生性研究。通过灌根与浸种试验,表明L-6菌株可定殖于根、茎和叶片组织内,并在一定时间内可维持相当数量,10d时的菌落定殖量看不出明显减少。
4、促生作用试验结果表明,内生细菌L-6发酵液能促进辣椒种子根的萌发和根部的伸长生长;经发酵处理后,辣椒体内的POD酶活性和根内SOD酶活性均有不同程度的提高。
5、内生拮抗菌株L-6发酵液对番茄枯萎病菌(Fusariumoxysporum)、番茄早疫病菌(Alternaria solani)等病原真菌的菌丝生长和孢子萌发有较强的抑制作用,病原菌菌丝生长缓慢,表现畸形、扭曲,严重的可致使病原菌菌丝胞壁溶解,原生质外溢,抑制病原菌孢子萌发。
6、对内生拮抗菌株L-6分泌抗菌物质进行分析认为,L-6菌株产生的抗菌物质主要为脂溶性抗生素。薄层层析(Thin layerchromatography,TLC)及紫外吸收光谱检测结果表明,其抗生素的活性组分为在323nm处有稳定吸收峰的单一化合物。对该组分的性质研究发现,菌株L-6分泌的这一抗菌活性组分具有较好的热稳定性和酸碱稳定性,但对紫外照射稳定性差。
Plant diseases are one of the main factors to disturb agricultural production,in recent years,vegetables are infracted severitily by plant diseases as dependency vegetable's develop and crop system's regulate. Epidemic disease,virus disease,bacterial wilt and other main plant diseases can make losses to nightshade vegetables' production. Chemical pesticides are the main measures to control plant disease presently,which can make environmental pollution,pathogens drug-resistant and do harm to non-target organisms.Abundant of research showed that there are plentiful of endophytic bacteria existing in plants bodies which show good efforts to plant diseases,which has become the hotspot in Plant Pathology.There will be good future for endophytic bacteria in Plant Pathology,for their good effort and variety of biological functions to plant diseases.
By separating endophytic bacteria from nightshade vegetables,this study screens antagonistic bacteria which can colonize in plant favorably and do good in promoting health and disease prevention.To these endophytic antagonistic bacteria,we study their antibacterial substances and the relations of antagonism and endogenous.For these research,we can provide a theoretical basis to the research of endophytic bacteria. The results are as follows.
1.There were 149 endophytic bacteria separated from nightshade vegetables,while 47 were from roots,56 were from caudexes,33 were from leaves and 13 were from fruits.We screened 18 antagonistic bacteria showed good efforts to Verticillium dahliae,Fusarium oxysporum,Alternaria solani,Phytophthora capsici,Rhizoctonia solani, Alternaria alternata,Bipolaris sorokiniana and Alternaria sp.from those 149 endophytic bacteria.
2.With the observation of figuration characters,the physiological and biochemical tests,we found that most of these antagonistic bacteria were Bacillus sp.,the percent was 55.6%.Left strains were Pseudomonas sp.and Xanthomonas sp.Combined the 16S rDNA gene analysis of strain L-6,we identified L-6 to be Paenibacillus polymyxa.There was no report to isolate Paenibacillus polymyxa from capsicum,and we are the first.
3.We Used the measure of Drug-resistant marker to strain L-6 in order to study its endogenous.By the study of watering roots and soaking seeds of capsicum,we found strain L-6 can colonize in roots, caudexes and leaves,and maintain aconsiderable number for a long time fre the colonization number reduce less in 10 days.
4.The suspension of strain L-6 can promote capsicum roots' elongation evidently,improve the activity of peroxidase(POD) in capsicum body and the activity of superoxidedimutase(SOD) in capsicum roots.
5.The broth of strain L-6 can show good inhibition to the growth of mycelium and the bourgeon of spores of Fusarium oxysporum and Alternaria solani et al.,which made the mycelium become contorted and abnormality and even the mycelium's cell wall ablated,reduce the bourgeon of spores.
6.The results of thin layer chromatography(TLC) and UV detection showed that L-6 strain's active substance has the absorbing peaks steady in 323nm.By study its characters,we found that L-6' active santibiotics showed good heat resistance,good antibacterial stability and weak anti-UV stability.
本研究通过对茄科植物内生细菌的分离,并针对茄子黄萎病、番茄枯萎病、蔬菜苗期立枯病等发生面积大、防治困难的病害,筛选在植物体内定殖能力强,防病与促生作用结合的内生细菌;分析内生细菌所产生的抗菌物质及部分特性;探讨内生细菌的拮抗作用和内生性与防病作用的关系,为植物内生细菌的研究提供理论依据。其结果如下:
1、从泰安、新泰等7县(市)采集番茄、茄子、辣椒、烟草等茄科植物,分离到内生细菌共149株,其中根中47株,茎中56株,叶中33株,果中13株。筛选出对茄子黄萎病菌(Verticillium dahliae)、番茄枯萎病菌(Fusarium oxysporum f.sp.cinerea)、番茄早疫病菌(Alternaria solani)、辣椒疫病病菌(Phytophthora capsici)、蔬菜苗立枯病菌(Rhizoctonia solani)、烟草赤星病菌(Alternaria alternata)、小麦根腐病菌(Bipolaris sorokiniana)、枣叶斑病菌(Alternaria sp.)具有较好抑菌活性的内生细菌菌株共18株。
2、应用传统细菌鉴定方法对18株拮抗内生细菌进行了初步鉴定,经细菌培养性状和生理生化特性测定结果表明,所筛选的拮抗内生细菌主要为芽孢杆菌群(Bacillus sp.),占分离数的55.6%;少数为假单胞菌群(Pseudomonas sp.)和黄单胞菌群(Xanthomonas sp.)。对其中1株具有较强活性的内生细菌L-6菌株进行16S rDNA分子鉴定,通过同源性比较,确定内生拮抗细菌L-6为多粘类芽孢杆菌(Paenibacilluspolymyxa)。关于在辣椒植株体内分离到内生细菌为多粘类芽孢杆菌(Paenibacillus palymyxa),本研究尚属首次报道。
3、采用抗药性标记法对L-6菌株进行了内生性研究。通过灌根与浸种试验,表明L-6菌株可定殖于根、茎和叶片组织内,并在一定时间内可维持相当数量,10d时的菌落定殖量看不出明显减少。
4、促生作用试验结果表明,内生细菌L-6发酵液能促进辣椒种子根的萌发和根部的伸长生长;经发酵处理后,辣椒体内的POD酶活性和根内SOD酶活性均有不同程度的提高。
5、内生拮抗菌株L-6发酵液对番茄枯萎病菌(Fusariumoxysporum)、番茄早疫病菌(Alternaria solani)等病原真菌的菌丝生长和孢子萌发有较强的抑制作用,病原菌菌丝生长缓慢,表现畸形、扭曲,严重的可致使病原菌菌丝胞壁溶解,原生质外溢,抑制病原菌孢子萌发。
6、对内生拮抗菌株L-6分泌抗菌物质进行分析认为,L-6菌株产生的抗菌物质主要为脂溶性抗生素。薄层层析(Thin layerchromatography,TLC)及紫外吸收光谱检测结果表明,其抗生素的活性组分为在323nm处有稳定吸收峰的单一化合物。对该组分的性质研究发现,菌株L-6分泌的这一抗菌活性组分具有较好的热稳定性和酸碱稳定性,但对紫外照射稳定性差。
Plant diseases are one of the main factors to disturb agricultural production,in recent years,vegetables are infracted severitily by plant diseases as dependency vegetable's develop and crop system's regulate. Epidemic disease,virus disease,bacterial wilt and other main plant diseases can make losses to nightshade vegetables' production. Chemical pesticides are the main measures to control plant disease presently,which can make environmental pollution,pathogens drug-resistant and do harm to non-target organisms.Abundant of research showed that there are plentiful of endophytic bacteria existing in plants bodies which show good efforts to plant diseases,which has become the hotspot in Plant Pathology.There will be good future for endophytic bacteria in Plant Pathology,for their good effort and variety of biological functions to plant diseases.
By separating endophytic bacteria from nightshade vegetables,this study screens antagonistic bacteria which can colonize in plant favorably and do good in promoting health and disease prevention.To these endophytic antagonistic bacteria,we study their antibacterial substances and the relations of antagonism and endogenous.For these research,we can provide a theoretical basis to the research of endophytic bacteria. The results are as follows.
1.There were 149 endophytic bacteria separated from nightshade vegetables,while 47 were from roots,56 were from caudexes,33 were from leaves and 13 were from fruits.We screened 18 antagonistic bacteria showed good efforts to Verticillium dahliae,Fusarium oxysporum,Alternaria solani,Phytophthora capsici,Rhizoctonia solani, Alternaria alternata,Bipolaris sorokiniana and Alternaria sp.from those 149 endophytic bacteria.
2.With the observation of figuration characters,the physiological and biochemical tests,we found that most of these antagonistic bacteria were Bacillus sp.,the percent was 55.6%.Left strains were Pseudomonas sp.and Xanthomonas sp.Combined the 16S rDNA gene analysis of strain L-6,we identified L-6 to be Paenibacillus polymyxa.There was no report to isolate Paenibacillus polymyxa from capsicum,and we are the first.
3.We Used the measure of Drug-resistant marker to strain L-6 in order to study its endogenous.By the study of watering roots and soaking seeds of capsicum,we found strain L-6 can colonize in roots, caudexes and leaves,and maintain aconsiderable number for a long time fre the colonization number reduce less in 10 days.
4.The suspension of strain L-6 can promote capsicum roots' elongation evidently,improve the activity of peroxidase(POD) in capsicum body and the activity of superoxidedimutase(SOD) in capsicum roots.
5.The broth of strain L-6 can show good inhibition to the growth of mycelium and the bourgeon of spores of Fusarium oxysporum and Alternaria solani et al.,which made the mycelium become contorted and abnormality and even the mycelium's cell wall ablated,reduce the bourgeon of spores.
6.The results of thin layer chromatography(TLC) and UV detection showed that L-6 strain's active substance has the absorbing peaks steady in 323nm.By study its characters,we found that L-6' active santibiotics showed good heat resistance,good antibacterial stability and weak anti-UV stability.
引文
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