栲属植物生斑痣盘菌目的分类和皮下盘菌种内遗传多样性的ITS序列分析
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摘要
栲属植物在我国约有58种,主要分布于长江及其以南的温暖地区。我省的皖南山区、大别山区、及黄山风景区均有栲属植物的分布,主要有甜槠、栲树、罗浮栲、苦槠等。由于栲属植物具有很高的经济利用价值,科学有效的防治该栲属植物病害便显得格外重要。本论文采用较先进的菌物分类技术,从形态解剖学、个体发育、生态及地理分布等方面,对生长在栲属植物上的斑痣盘菌进行了较系统的分类研究。同时采用ITS技术对皮下盘菌的12个菌株进行了种内遗传多样性的研究,进一步弄清了各类群之间的系统发育关系。该研究在菌物分类学和植物保护学方面均具有重要的科学意义。
本文以Darke(r1967)的分类系统为基础,同时参考Kor(f1973)、Cannon & Minter(1986)、Kirk et al.(2001)等人的若干补充和修订,对所采样品进行分类和鉴定,共获得隶属于散斑壳属、齿裂菌属的2属7种,即钩丝散斑壳(Lophodermium unciforme)、贝壳杉散斑壳(Lophodermium agathidis)、显缘齿裂菌(Coccomyces limitatus)隐齿裂菌(Coccomyces occultus)、辐射状齿裂菌(Coccomyces radiatus)、小散斑壳(Lophodermium minor)和尖丝齿裂菌(Coccomyces mucronatus)。其中钩丝散斑壳(Lophodermium unciforme)为待发表新种,其模式标本保藏于安徽农业大学森保教研室(AAUFP)。对6个已知种进行了补充研究,使其形态结构特征得以完善。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。另外,根据主要形态特征,提出了栲属植物生斑痣盘菌的二叉式分类检索表。
本文亦通过组织分离法对采自安徽各地的悬钩子皮下盘菌(Hypoderma rubi)的鲜材料进行了分离培养,一共获得纯菌株12个。对各菌株进行DNA提取、rDNA-ITS区PCR扩增和序列测定,其序列测定结果均已被GenBank接受注册(GU138743、GU138751、GU138753、GU138749、GU138735、GU138750、GU138744、GU138746、GU138741、GU138736、GU138738、GU138739)。对12个菌株的ITS区的序列比较分析表明,悬钩子皮下盘菌ITS序列的长度为443bp~456bp,G+C含量为48.7%~51.5%;不同菌株间的ITS区序列变异较大,该菌种内遗传多样性丰富。同时利用MEGA4.1软件中的邻接法(neighbour-joining method , NJ)构建系统发育树。分析结果表明,寄主种类对悬钩子皮下盘菌种内遗传多样性会产生较大影响,木本植物和草本植物上的悬钩子皮下盘菌菌株可能是亲缘关系较远的两个类群;该菌种内遗传差异与其寄生部位和采集地无明显相关性。据统计,我国该菌的寄主植物涉及被子及裸子植物中的槭树科(Aceraceae)、漆树科(Anacardiaceae)、五加科(Araliaceae)、菊科(Compositae)、壳斗科(Fagaceae)、禾本科(Gramineae)等21科,共30属32种(变种)。因此,悬钩子皮下盘菌在与寄主的长期协同进化中,是否会产生更加丰富的变异类群,将成为一个值得探究的问题。
Castanopsis plants in China are about 58 species, mainly distributed in the Yangtze river and its south warm regions. South of Anhui Province, Dabie Mountain area and Huangshan of our province are the distribution of the Castanopsis plants, and there are mainly Castanopsis eyrei, Castanopsis fargesii, Castanopsis fabri, Castanopsis sclerophylla and so on. Because of Castanopsis species have high economic value, scientific and effective disease prevention and control of the Castanopsis species are particularly important. In this thesis, systematics of part of specie of Rhytismataceae on Castanopsis was studied from morphology, development biology, ecology, distribution etc. with the techniques of modern mycology. Moreover nucleotide sequences of the internal transcribed spacer (ITS) region unclear rubosomal DNA were used to infer phylogenetic relationships of 12 Hypoderma strains. This results have important scientific significance on the taxonomy of the fungi and plant protection aspects.
Samples of Rhytismataceae on Castanopsis were systematically studied according to the taxonomic principles Darker (1967), and also refer to Korf (1973), Cannon & Minter (1986), Kirk et al. (2001). 7 species among 2 genera (Lophodermium and Coccomyces) were identified. Enumerates as follows: Lophodermium unciforme, Lophodermium agathidis, Coccomyces limitatus, Coccomyces occultus, Coccomyces radiatus, Lophodermium minor, Coccomyces mucronatus. Among them Lophodermium unciforme was a new specie to be published, type specimens of the new specie is preserved in the Forest Protection Laboratory of Anhui Agricultural University, (AAUFP), hefei, China. The six known species were complementary studied, to perfect their morphological characteristics. Morphological and anatomical characteristics of the description and illustration, host and habitat records, and their similar groups, parasites, and pathogenicity are discussed. And according to the main morphological characteristics, dichotomous keys to these species were complied.
In this thesis, Fresh materials of Hypoderma rubi collected from Anhui Province were isolated by tissue isolation technique, and 12 pure strains were obtained. DNA of all the strains were extracted from the dry mycelia, rDNA-ITS regions were amplified by PCR and then sequenced. The sequencing results of rDNA-ITS regions were to be registered in GenBank (GU138743, GU138751, GU138753, GU138749, GU138735, GU138750, GU138744, GU138746, GU138741, GU138736, GU138738, GU138739). The ITS sequence analysis results showed that the length range of ITS sequences of H. rubi was from 443bp to 456bp, and the percentage of G+C contents of the ITS sequences varied from 48.7% to 51.5%. There were obvious variations in the ITS sequences of different strains and showed abundant genetic diversity among them. A phylogenetic tree was constructed by neighbour-joining method using MEGA4.1. The results indicated that the intraspecific genetic diversity of H. rubi was related to hosts obviously, and strains from woody plants and herbaceous plants probably were two distant relationship groups. In addition, the intraspecific genetic differences of H. rubi were independent of parasitic sites and sample collection area. According to statistics, the strains of the host plants involved in the angiosperms and gymnosperms. For example, Aceraceae, Anacardiaceae, Araliaceae, Asteraceae, Fagaceae, Gramineae and other 21 families, 30 genera and 32 species (varieties). Therefore, Hypoderma rubi in collaboration with the host of the long-term evolution, will produce a richer variation of groups, will be a question worth exploring.
本文以Darke(r1967)的分类系统为基础,同时参考Kor(f1973)、Cannon & Minter(1986)、Kirk et al.(2001)等人的若干补充和修订,对所采样品进行分类和鉴定,共获得隶属于散斑壳属、齿裂菌属的2属7种,即钩丝散斑壳(Lophodermium unciforme)、贝壳杉散斑壳(Lophodermium agathidis)、显缘齿裂菌(Coccomyces limitatus)隐齿裂菌(Coccomyces occultus)、辐射状齿裂菌(Coccomyces radiatus)、小散斑壳(Lophodermium minor)和尖丝齿裂菌(Coccomyces mucronatus)。其中钩丝散斑壳(Lophodermium unciforme)为待发表新种,其模式标本保藏于安徽农业大学森保教研室(AAUFP)。对6个已知种进行了补充研究,使其形态结构特征得以完善。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。另外,根据主要形态特征,提出了栲属植物生斑痣盘菌的二叉式分类检索表。
本文亦通过组织分离法对采自安徽各地的悬钩子皮下盘菌(Hypoderma rubi)的鲜材料进行了分离培养,一共获得纯菌株12个。对各菌株进行DNA提取、rDNA-ITS区PCR扩增和序列测定,其序列测定结果均已被GenBank接受注册(GU138743、GU138751、GU138753、GU138749、GU138735、GU138750、GU138744、GU138746、GU138741、GU138736、GU138738、GU138739)。对12个菌株的ITS区的序列比较分析表明,悬钩子皮下盘菌ITS序列的长度为443bp~456bp,G+C含量为48.7%~51.5%;不同菌株间的ITS区序列变异较大,该菌种内遗传多样性丰富。同时利用MEGA4.1软件中的邻接法(neighbour-joining method , NJ)构建系统发育树。分析结果表明,寄主种类对悬钩子皮下盘菌种内遗传多样性会产生较大影响,木本植物和草本植物上的悬钩子皮下盘菌菌株可能是亲缘关系较远的两个类群;该菌种内遗传差异与其寄生部位和采集地无明显相关性。据统计,我国该菌的寄主植物涉及被子及裸子植物中的槭树科(Aceraceae)、漆树科(Anacardiaceae)、五加科(Araliaceae)、菊科(Compositae)、壳斗科(Fagaceae)、禾本科(Gramineae)等21科,共30属32种(变种)。因此,悬钩子皮下盘菌在与寄主的长期协同进化中,是否会产生更加丰富的变异类群,将成为一个值得探究的问题。
Castanopsis plants in China are about 58 species, mainly distributed in the Yangtze river and its south warm regions. South of Anhui Province, Dabie Mountain area and Huangshan of our province are the distribution of the Castanopsis plants, and there are mainly Castanopsis eyrei, Castanopsis fargesii, Castanopsis fabri, Castanopsis sclerophylla and so on. Because of Castanopsis species have high economic value, scientific and effective disease prevention and control of the Castanopsis species are particularly important. In this thesis, systematics of part of specie of Rhytismataceae on Castanopsis was studied from morphology, development biology, ecology, distribution etc. with the techniques of modern mycology. Moreover nucleotide sequences of the internal transcribed spacer (ITS) region unclear rubosomal DNA were used to infer phylogenetic relationships of 12 Hypoderma strains. This results have important scientific significance on the taxonomy of the fungi and plant protection aspects.
Samples of Rhytismataceae on Castanopsis were systematically studied according to the taxonomic principles Darker (1967), and also refer to Korf (1973), Cannon & Minter (1986), Kirk et al. (2001). 7 species among 2 genera (Lophodermium and Coccomyces) were identified. Enumerates as follows: Lophodermium unciforme, Lophodermium agathidis, Coccomyces limitatus, Coccomyces occultus, Coccomyces radiatus, Lophodermium minor, Coccomyces mucronatus. Among them Lophodermium unciforme was a new specie to be published, type specimens of the new specie is preserved in the Forest Protection Laboratory of Anhui Agricultural University, (AAUFP), hefei, China. The six known species were complementary studied, to perfect their morphological characteristics. Morphological and anatomical characteristics of the description and illustration, host and habitat records, and their similar groups, parasites, and pathogenicity are discussed. And according to the main morphological characteristics, dichotomous keys to these species were complied.
In this thesis, Fresh materials of Hypoderma rubi collected from Anhui Province were isolated by tissue isolation technique, and 12 pure strains were obtained. DNA of all the strains were extracted from the dry mycelia, rDNA-ITS regions were amplified by PCR and then sequenced. The sequencing results of rDNA-ITS regions were to be registered in GenBank (GU138743, GU138751, GU138753, GU138749, GU138735, GU138750, GU138744, GU138746, GU138741, GU138736, GU138738, GU138739). The ITS sequence analysis results showed that the length range of ITS sequences of H. rubi was from 443bp to 456bp, and the percentage of G+C contents of the ITS sequences varied from 48.7% to 51.5%. There were obvious variations in the ITS sequences of different strains and showed abundant genetic diversity among them. A phylogenetic tree was constructed by neighbour-joining method using MEGA4.1. The results indicated that the intraspecific genetic diversity of H. rubi was related to hosts obviously, and strains from woody plants and herbaceous plants probably were two distant relationship groups. In addition, the intraspecific genetic differences of H. rubi were independent of parasitic sites and sample collection area. According to statistics, the strains of the host plants involved in the angiosperms and gymnosperms. For example, Aceraceae, Anacardiaceae, Araliaceae, Asteraceae, Fagaceae, Gramineae and other 21 families, 30 genera and 32 species (varieties). Therefore, Hypoderma rubi in collaboration with the host of the long-term evolution, will produce a richer variation of groups, will be a question worth exploring.
引文
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