斑痣盘菌科的分类和针叶树生散斑壳属等随机扩增多态性DNA分析
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摘要
斑痣盘菌科(Rhytismataceae)是世界菌物资源中的一个重要组成部分。本文采用现代菌物学研究技术,从形态解剖学、个体发育学、生态与地理分布、培养性状等方面对斑痣盘菌科部分种进行了较为系统的分类研究。同时采用RAPD技术对26个针叶树生散斑壳属(Lophodermium Chevall.)和2个相关属(Ploioderma destruens,Soleellachinensis)菌株部分种内及近似种间的遗传多样性进行了分析研究,进一步弄清了各分类群之间的系统发育关系。
以Darker(1967)的分类系统为基础,同时参考Korf(1973)、Cannon & Minter(1986)、Kirk et al.(2001)等人的若干补充和修订,对源于皖南山区和大别山区的斑痣盘菌科菌物进行分类研究。共查明4属8种,其中包括栎皮下盘菌(Hypodermaquerci)、栲散斑壳(Lophodermium castanopsis)、短孢齿裂菌(Coccomyces brevisporus)3个新种;对5个已知种进行了补充研究,使其形态结构、个体发育等特征得以完善,同时追记了它们的寄主新记录和地理新分布。对各种均提供了形态解剖学特征的描述与图解、寄主及生境的记载,并对其相近类群、寄生性与致病性等加以讨论。编制了各属的二叉式分种检索表。
利用组织分离法对采自安徽的菌物活材料进行分离纯化,获得针叶树生散斑壳属(Lophodermium)及其近似属10个分离株。通过定期观察、记载各菌株的培养性状和拍摄纯培养物彩色照片,发现各菌株在MA平板培养基上的生长速度、菌落形状、颜色和结构以及子实体形成与否及成熟期等方面存在一定的差异,少数菌株产生分生孢子器和线(环)纹,所有菌株均不产生子囊果,仅个别菌株分泌水溶性色素而使培养基变色。
利用氯化苄法对26个针叶树生散斑壳属(Lophodermium)和2个舟皮盘菌属及小鞋孢盘菌属菌株进行DNA提取。通过对模板用量、随机引物用量、Mg离子用量、dNTP用量、Taq酶用量和反应退火温度及时间、延伸温度及时间、循环次数等参数进行优化,建立了该类菌物RAPD-PCR反应的最佳体系,即在25μl反应体积中,DNA模板(100ng/μl)1.0gμl,随机引物(12.5μM)1.0μl,MgCl_2(25mM)2.0μl,dNTPs(10mM)2.0μl,Taq酶(5U/μl)0.2μl,10×PCR buffer 2.5μl,ddH_2O 16.3μl。扩增程序为:先95℃预变性5min,循环50次,每个循环包括94℃变性1min,36℃退火100s,72℃延伸140s,最后一次循环后在72℃延伸10min。
采用上述优化的扩增体系对26个针叶树生散斑壳属及其相关属菌株进行RAPD分析。利用9个随机引物共扩增出111个位点,其中多态性位点数104个,占总数的94%,扩增片段长度大多数集中在250-2000bp之间。采用UPGMA方法构建RAPD聚类树状图。分析结果表明:针叶树上的散斑壳属(Lophodermium)各分离株和近似菌株之间遗传距离变异范围在1.98-5.35之间。在遗传距离4.91处,将针叶树上26个分离株分成A、B、C、D、E、F 6个类群。同种或近似种的菌株基本上能很好地聚在一起,不同种之间也能较好地区分;这与表型性状分类的结果基本一致。由此可见,RAPD技术在种内和近似种间系统学研究中具有较好的适用性。
通过将表型性状与RAPD分析结果进行比较,进一步验证了子囊果大小、线纹的有无及特征、寄主种类等在针叶树生散斑壳属部分种内及近似种间区分方面的重要性。同时从另外一个侧面也证明了子囊孢子的类型这一特征在针叶树生散斑壳属与相关属区分方面是十分重要的。
Rhytismataceae is an important part in fungi resources of the world.Systematics of part of species of Rhytismataceae was studied from morphology,development biology, ecology,distribution,culture characters etc.with the techniques of modern mycology.And phylogenetic relationships of intraspecies and similar interspecies of Lophodermium and its two similar genera(Ploioderma,Soleella)strains on conifiers were studied.It is clear further to the phylogenetic relationships among these groups.
Some samples from the mountains of south and west Anhui and were systematically studied with the techniques of modern classification.Morphology structure and development biology of these fungi were roundly examined according to taxonomic principles of Darker(1967)、Korf(1973)、Cannon & Minter(1986)、Kirk et al.(2001). 8 species among 4 genera were identified.Among the taxa there were 3 new species including Hypoderma querci sp.nov.,Lophodermium castanopsis sp.nov.and Coccomyces brevisporus sp.nov.;the studies of 5 known species involved in adding characters of morphology,development biology,new record of host and geographical distribution.Morphological characteristics were described and illustrated,and hosts and distribution were recorded for all the species,and their similar groups,parasititism and nosogenesis were discussed.Dichotomous key of the species was compiled.
Living materials of the fungi from the mountains of south and west Anhui were isolated by tissue isolation technics,and 10 strains of Lophodermium and its similar genera on conifiers were obtained.Culture characters were studied by observing,recording and screening the chromophotographs of pure cultures periodically.The result showed that there were definite difference in growth rate,shape,structure and color of colonies and mature period of fruiting bodies in MA culture media.Few of the species formed pycnidia, and none produced ascomata during the culture.Only very few of the strains produced coloring matter that make culture media colored.
Template DNA of 26Lophodermium strains and 2 Ploioderma destruens and Soleella chinensis strains on conifiers were extracted by Benzyl Chloride.The RAPD-PCR amplified system was optimized from aspects of the dosage of template DNA,prime,Mg~(2+), dNTPs and Taq enzyme,and anneal temperature and time,elongation temperature and time, cycle number.The amplified diagrams were quite well.The final reaction system(25ul) included ddH_2O 14.2ul,10×Buffer 2.5,Mg~(2+)(25mmol.L~(-1))2ul,dNTPs(10mmol.l~(-1))1.5ul, primer(12.5umol.L~(-1))0.6ul,Taq enzyme(5U.ul~(-1))0.2ul,template DNA(100ng.ul~(-1))1ul. Its amplified procedure was pre-denature at 95℃for 5min.cycle 50 times,and every cycle included denature at 94℃for lmin,anneal at 36℃for 100s,elongation at 72℃for 140s. And the latest cycle elongation time is 10min.
24 Lophodermium and 2 related genera strains on conifiers were amplified by the optimal procedure,which amplified 111 sites including 104 polymorphic sites,accounting for 94%.The length of most amplified fragments ranged from 250 to 2000bp.Its genetic distance cluster figure was constracted using UPGMA method.From the clustering figure we can find that the distance of genetic ranged from 1.98 to 5.35.26 strains of Lophodermium and 2 related strains on conifiers were classified to 6 groups(A,B,C,D,E and F)in the genetic distance 4.91.The strains of intraspecies and similar interspecies can cluster very well,and the strains of different species also can distinct quite well.From the results we can conclude that RAPD technique has good applicability in the study of relationships of intraspecies and similar interspecies.
Compared with phenotypic character and RAPD analysis results,we can conform the importance of the phenotypic characteritics such as zone lines,ascomata and hosts etc.in the classification of intraspecies and similar interspecies of Lophodermium on conifiers. And the type of ascopores was proved being important in distinction of Lophodermium and its similar genera on conifiers from another ascept.
以Darker(1967)的分类系统为基础,同时参考Korf(1973)、Cannon & Minter(1986)、Kirk et al.(2001)等人的若干补充和修订,对源于皖南山区和大别山区的斑痣盘菌科菌物进行分类研究。共查明4属8种,其中包括栎皮下盘菌(Hypodermaquerci)、栲散斑壳(Lophodermium castanopsis)、短孢齿裂菌(Coccomyces brevisporus)3个新种;对5个已知种进行了补充研究,使其形态结构、个体发育等特征得以完善,同时追记了它们的寄主新记录和地理新分布。对各种均提供了形态解剖学特征的描述与图解、寄主及生境的记载,并对其相近类群、寄生性与致病性等加以讨论。编制了各属的二叉式分种检索表。
利用组织分离法对采自安徽的菌物活材料进行分离纯化,获得针叶树生散斑壳属(Lophodermium)及其近似属10个分离株。通过定期观察、记载各菌株的培养性状和拍摄纯培养物彩色照片,发现各菌株在MA平板培养基上的生长速度、菌落形状、颜色和结构以及子实体形成与否及成熟期等方面存在一定的差异,少数菌株产生分生孢子器和线(环)纹,所有菌株均不产生子囊果,仅个别菌株分泌水溶性色素而使培养基变色。
利用氯化苄法对26个针叶树生散斑壳属(Lophodermium)和2个舟皮盘菌属及小鞋孢盘菌属菌株进行DNA提取。通过对模板用量、随机引物用量、Mg离子用量、dNTP用量、Taq酶用量和反应退火温度及时间、延伸温度及时间、循环次数等参数进行优化,建立了该类菌物RAPD-PCR反应的最佳体系,即在25μl反应体积中,DNA模板(100ng/μl)1.0gμl,随机引物(12.5μM)1.0μl,MgCl_2(25mM)2.0μl,dNTPs(10mM)2.0μl,Taq酶(5U/μl)0.2μl,10×PCR buffer 2.5μl,ddH_2O 16.3μl。扩增程序为:先95℃预变性5min,循环50次,每个循环包括94℃变性1min,36℃退火100s,72℃延伸140s,最后一次循环后在72℃延伸10min。
采用上述优化的扩增体系对26个针叶树生散斑壳属及其相关属菌株进行RAPD分析。利用9个随机引物共扩增出111个位点,其中多态性位点数104个,占总数的94%,扩增片段长度大多数集中在250-2000bp之间。采用UPGMA方法构建RAPD聚类树状图。分析结果表明:针叶树上的散斑壳属(Lophodermium)各分离株和近似菌株之间遗传距离变异范围在1.98-5.35之间。在遗传距离4.91处,将针叶树上26个分离株分成A、B、C、D、E、F 6个类群。同种或近似种的菌株基本上能很好地聚在一起,不同种之间也能较好地区分;这与表型性状分类的结果基本一致。由此可见,RAPD技术在种内和近似种间系统学研究中具有较好的适用性。
通过将表型性状与RAPD分析结果进行比较,进一步验证了子囊果大小、线纹的有无及特征、寄主种类等在针叶树生散斑壳属部分种内及近似种间区分方面的重要性。同时从另外一个侧面也证明了子囊孢子的类型这一特征在针叶树生散斑壳属与相关属区分方面是十分重要的。
Rhytismataceae is an important part in fungi resources of the world.Systematics of part of species of Rhytismataceae was studied from morphology,development biology, ecology,distribution,culture characters etc.with the techniques of modern mycology.And phylogenetic relationships of intraspecies and similar interspecies of Lophodermium and its two similar genera(Ploioderma,Soleella)strains on conifiers were studied.It is clear further to the phylogenetic relationships among these groups.
Some samples from the mountains of south and west Anhui and were systematically studied with the techniques of modern classification.Morphology structure and development biology of these fungi were roundly examined according to taxonomic principles of Darker(1967)、Korf(1973)、Cannon & Minter(1986)、Kirk et al.(2001). 8 species among 4 genera were identified.Among the taxa there were 3 new species including Hypoderma querci sp.nov.,Lophodermium castanopsis sp.nov.and Coccomyces brevisporus sp.nov.;the studies of 5 known species involved in adding characters of morphology,development biology,new record of host and geographical distribution.Morphological characteristics were described and illustrated,and hosts and distribution were recorded for all the species,and their similar groups,parasititism and nosogenesis were discussed.Dichotomous key of the species was compiled.
Living materials of the fungi from the mountains of south and west Anhui were isolated by tissue isolation technics,and 10 strains of Lophodermium and its similar genera on conifiers were obtained.Culture characters were studied by observing,recording and screening the chromophotographs of pure cultures periodically.The result showed that there were definite difference in growth rate,shape,structure and color of colonies and mature period of fruiting bodies in MA culture media.Few of the species formed pycnidia, and none produced ascomata during the culture.Only very few of the strains produced coloring matter that make culture media colored.
Template DNA of 26Lophodermium strains and 2 Ploioderma destruens and Soleella chinensis strains on conifiers were extracted by Benzyl Chloride.The RAPD-PCR amplified system was optimized from aspects of the dosage of template DNA,prime,Mg~(2+), dNTPs and Taq enzyme,and anneal temperature and time,elongation temperature and time, cycle number.The amplified diagrams were quite well.The final reaction system(25ul) included ddH_2O 14.2ul,10×Buffer 2.5,Mg~(2+)(25mmol.L~(-1))2ul,dNTPs(10mmol.l~(-1))1.5ul, primer(12.5umol.L~(-1))0.6ul,Taq enzyme(5U.ul~(-1))0.2ul,template DNA(100ng.ul~(-1))1ul. Its amplified procedure was pre-denature at 95℃for 5min.cycle 50 times,and every cycle included denature at 94℃for lmin,anneal at 36℃for 100s,elongation at 72℃for 140s. And the latest cycle elongation time is 10min.
24 Lophodermium and 2 related genera strains on conifiers were amplified by the optimal procedure,which amplified 111 sites including 104 polymorphic sites,accounting for 94%.The length of most amplified fragments ranged from 250 to 2000bp.Its genetic distance cluster figure was constracted using UPGMA method.From the clustering figure we can find that the distance of genetic ranged from 1.98 to 5.35.26 strains of Lophodermium and 2 related strains on conifiers were classified to 6 groups(A,B,C,D,E and F)in the genetic distance 4.91.The strains of intraspecies and similar interspecies can cluster very well,and the strains of different species also can distinct quite well.From the results we can conclude that RAPD technique has good applicability in the study of relationships of intraspecies and similar interspecies.
Compared with phenotypic character and RAPD analysis results,we can conform the importance of the phenotypic characteritics such as zone lines,ascomata and hosts etc.in the classification of intraspecies and similar interspecies of Lophodermium on conifiers. And the type of ascopores was proved being important in distinction of Lophodermium and its similar genera on conifiers from another ascept.
引文
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