桉树抗青枯病的鉴定及其抗病机制研究
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摘要
本实验通过采用不同的接种方法对18个桉树种和无性系进行抗抗病性测定,筛选出了优良的桉树抗青枯病树种和无性系,获得快捷准确的抗病性鉴定技术。同时,运用16S rDNA PCR-DGGE技术检测不同抗性桉树无性系中内生细菌种群多样性,探讨细菌多样性与抗病能力之间的关系,为桉树抗病机制探索及青枯病防治工作提供了新的线索和途径。
1桉树抗青枯病的鉴定技术
本研究采用菌液浸泡法、顶尖接种法对不同的桉树无性系盆栽苗和生根组培苗进行抗青枯病性能测定,并结合林间调查筛选桉树抗青枯病树种和无性系,并从根系分泌物和组织研磨液的角度研究其对桉树青枯病菌生长的抑制作用,探索不同抗性桉树无性系抗病性强弱与其根系分泌物和组织研磨液之间的关系。结果表明,供试的18个桉树无性系中,bd1、bd2、赤桉和南宁巨尾桉为抗病无性系;U6、南宁尾叶桉、雷9、钦32-29为中抗无性系;DH32-27、钦9、南宁广9、钦8、邓恩桉、钦32-22、雷2、巨桉、尾叶桉和钦广9为感病无性系。不同抗性桉树无性系根系分泌物和组织研磨液对青枯菌没有直接的抑制作用,但随着接种时间的延长,青枯病菌在抗病性强的无性系的根系分泌物及组织液中的增殖显著低于感病无性系,验证了通过茎段浸泡接种筛选出的桉树无性系的准确性,同时表明了根系分泌物和组织研磨液可以反映桉树不同无性系的抗病性强弱。
2不同抗性桉树无性系内生细菌多样性及其与抗病性的关系研究
利用PCR-DGGE(变性梯度凝胶电泳)与16S rDNA克隆文库的构建相结合的两种方法研究不同抗性桉树无性系(雷2,雷9,bd2)内生细菌多样性,分析其细菌种群结构,寻找优势菌群及特异性菌群,探讨细菌多样性与抗病能力之间的关系。结果表明,不同抗性桉树无性系DGGE图谱没有明显差异,各无性系条带数目相同为11条,其中2条为优势条带,将其回收测序,经Genebank比对后,为不可培养细菌;PCR产物的直接克隆3个不同抗性的桉树无性系共得到18个克隆子,对其测序结果进行分析发现,克隆产物均为同一种不可培养细菌,属于Cyanobacteria phylum。两种方法显示了该3种桉树无性系的内生细菌种群结构相同,其中以Uncultured bacterium clone为优势菌群,结果初步表明桉树无性系的抗青枯病能力与其内生菌种群之间没有必然的联系。
In this study, in order to obtain a concise, accurate detection and determination technique, the disease of resistance of Eucalyptus spp. against bacterial wilt caused by Ralstonia solanacearum was determined using different inoculation methods on 18 eucalypt species and clones, which from different sources and culturing by different reproductive mothods. The diversity of endophytic bacteria in clones with different resistance was investigated by using 16S rDNA PCR-DGGE to explore the relationship between the resistant ability and bacterial diversity, which might provide new clues and ways to understand the resistance mechanism and prevent the bacteria wilt.
1 The resistance ability of Eucalyptus spp. against bacterial wilt caused by Ralstonia solanacearum was determined by dipping inoculation of shoot cuttings and shoot tip inoculation using potted plantlets and tissue culture seedlings, as well as field disease survey of eucalypt plantation. The inhibition effects of root exudate and supernate of tissue triturate collected from different resistant clones of Eucalyptus against R. solanacearum were performed using the methods of liquid culture to explore the relationship of root exudate, tissue triturate and the levels of disease resistance. The results showed that the clones, bd1, bd2, E. camaldulensis and E. grandis×urophylla from Nanning were highly resistant to bacterial wilt; U6, E. urophylla from Nanning,Lei9 and Qin32-29 were the mid wilt-resistant clones; DH32-27, Qin9, Nanningguang9, Qin8, E. dunnii, Qin32-22, Lei2, E. grandis, E. urophylla and Qinguang9 were susceptible clones. The results also indicated that root exudates and the supernatant of triturated tissue had no direct inhibition to the growth of R. solanacearum. However, the proliferation number of R. solanacearum in root exudate and tissue triturate from wilt-susceptible clones was markedly higher than the ones from highly wilt-resistant clones, which was in accordance with the determination results of disease resistance using dipping inoculation and tip inoculation of shoot cuttings, indicating that the resistance ability of different clones could be manifested by the growth of R. solanacearum in root exudate and supernate of triturated tissue.
2 In order to make a further exploration about the relationship of endophytic bacteria population structure and the levels of disease resistance, the combination of PCR-DGGE (denaturing gradient gel electrophoresis) technology and 16S rDNA gene library construction method were used to study the endophytic bacteria diversity among wilt resistant and susceptible Eucalyptus clones(Lei 2, Lei 9, bd2), the relationship of bacterial population and the disease resistance ability were explored by analysing the dominant and specific bacteria group. The results showed that there was no significant difference among resistant and susceptible clones in DGGE profile, which had 11 bands with same number and position of each Eucalyptus clone. Through cutting and sequencing the two dominant bands sequence the Genebank comparion indicated that they were both uncultured bacterial clones. As for 16S rDNA gene library construction, 18 clones were obtained by cloning PCR products directly, the sequencing analysis showed that all the clones were of the same uncultural bacteria, belonging to Cyanobacteria phylum, which was in conformity with DGGE method. The result indicated that Lei2, Lei9, bd2 had same endophytic bactieria variety, the uncultured bacterium clone was the dominant group, which preliminary demonstrated there was no necessary link between the bacterial population structure and the levels of disease resistance.
1桉树抗青枯病的鉴定技术
本研究采用菌液浸泡法、顶尖接种法对不同的桉树无性系盆栽苗和生根组培苗进行抗青枯病性能测定,并结合林间调查筛选桉树抗青枯病树种和无性系,并从根系分泌物和组织研磨液的角度研究其对桉树青枯病菌生长的抑制作用,探索不同抗性桉树无性系抗病性强弱与其根系分泌物和组织研磨液之间的关系。结果表明,供试的18个桉树无性系中,bd1、bd2、赤桉和南宁巨尾桉为抗病无性系;U6、南宁尾叶桉、雷9、钦32-29为中抗无性系;DH32-27、钦9、南宁广9、钦8、邓恩桉、钦32-22、雷2、巨桉、尾叶桉和钦广9为感病无性系。不同抗性桉树无性系根系分泌物和组织研磨液对青枯菌没有直接的抑制作用,但随着接种时间的延长,青枯病菌在抗病性强的无性系的根系分泌物及组织液中的增殖显著低于感病无性系,验证了通过茎段浸泡接种筛选出的桉树无性系的准确性,同时表明了根系分泌物和组织研磨液可以反映桉树不同无性系的抗病性强弱。
2不同抗性桉树无性系内生细菌多样性及其与抗病性的关系研究
利用PCR-DGGE(变性梯度凝胶电泳)与16S rDNA克隆文库的构建相结合的两种方法研究不同抗性桉树无性系(雷2,雷9,bd2)内生细菌多样性,分析其细菌种群结构,寻找优势菌群及特异性菌群,探讨细菌多样性与抗病能力之间的关系。结果表明,不同抗性桉树无性系DGGE图谱没有明显差异,各无性系条带数目相同为11条,其中2条为优势条带,将其回收测序,经Genebank比对后,为不可培养细菌;PCR产物的直接克隆3个不同抗性的桉树无性系共得到18个克隆子,对其测序结果进行分析发现,克隆产物均为同一种不可培养细菌,属于Cyanobacteria phylum。两种方法显示了该3种桉树无性系的内生细菌种群结构相同,其中以Uncultured bacterium clone为优势菌群,结果初步表明桉树无性系的抗青枯病能力与其内生菌种群之间没有必然的联系。
In this study, in order to obtain a concise, accurate detection and determination technique, the disease of resistance of Eucalyptus spp. against bacterial wilt caused by Ralstonia solanacearum was determined using different inoculation methods on 18 eucalypt species and clones, which from different sources and culturing by different reproductive mothods. The diversity of endophytic bacteria in clones with different resistance was investigated by using 16S rDNA PCR-DGGE to explore the relationship between the resistant ability and bacterial diversity, which might provide new clues and ways to understand the resistance mechanism and prevent the bacteria wilt.
1 The resistance ability of Eucalyptus spp. against bacterial wilt caused by Ralstonia solanacearum was determined by dipping inoculation of shoot cuttings and shoot tip inoculation using potted plantlets and tissue culture seedlings, as well as field disease survey of eucalypt plantation. The inhibition effects of root exudate and supernate of tissue triturate collected from different resistant clones of Eucalyptus against R. solanacearum were performed using the methods of liquid culture to explore the relationship of root exudate, tissue triturate and the levels of disease resistance. The results showed that the clones, bd1, bd2, E. camaldulensis and E. grandis×urophylla from Nanning were highly resistant to bacterial wilt; U6, E. urophylla from Nanning,Lei9 and Qin32-29 were the mid wilt-resistant clones; DH32-27, Qin9, Nanningguang9, Qin8, E. dunnii, Qin32-22, Lei2, E. grandis, E. urophylla and Qinguang9 were susceptible clones. The results also indicated that root exudates and the supernatant of triturated tissue had no direct inhibition to the growth of R. solanacearum. However, the proliferation number of R. solanacearum in root exudate and tissue triturate from wilt-susceptible clones was markedly higher than the ones from highly wilt-resistant clones, which was in accordance with the determination results of disease resistance using dipping inoculation and tip inoculation of shoot cuttings, indicating that the resistance ability of different clones could be manifested by the growth of R. solanacearum in root exudate and supernate of triturated tissue.
2 In order to make a further exploration about the relationship of endophytic bacteria population structure and the levels of disease resistance, the combination of PCR-DGGE (denaturing gradient gel electrophoresis) technology and 16S rDNA gene library construction method were used to study the endophytic bacteria diversity among wilt resistant and susceptible Eucalyptus clones(Lei 2, Lei 9, bd2), the relationship of bacterial population and the disease resistance ability were explored by analysing the dominant and specific bacteria group. The results showed that there was no significant difference among resistant and susceptible clones in DGGE profile, which had 11 bands with same number and position of each Eucalyptus clone. Through cutting and sequencing the two dominant bands sequence the Genebank comparion indicated that they were both uncultured bacterial clones. As for 16S rDNA gene library construction, 18 clones were obtained by cloning PCR products directly, the sequencing analysis showed that all the clones were of the same uncultural bacteria, belonging to Cyanobacteria phylum, which was in conformity with DGGE method. The result indicated that Lei2, Lei9, bd2 had same endophytic bactieria variety, the uncultured bacterium clone was the dominant group, which preliminary demonstrated there was no necessary link between the bacterial population structure and the levels of disease resistance.
引文
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