【作者】 聂勇；

【导师】 黄勃；

【作者基本信息】 安徽农业大学 ， 微生物学， 2013， 博士

【摘要】 耳霉属真菌是虫霉门重要类群之一，在其系统学中占有特殊的重要地位。它是生态环境中重要的分解者，有些种还可用于害虫生物防治。王承芳等在2008年11月至2009年4月间，选择安徽和山东地区的自然保护区进行耳霉的分离和鉴定，共分离到耳霉菌株100余株，并发表了4株中国耳霉新记录种。本研究采用高效的耳霉分离方法，拟在我国东北、华东、华中、华南及西南地区选择自然保护区进行采样分离，来丰富我国耳霉菌种资源并对耳霉属的系统发育研究提供菌种材料。由于耳霉菌株在形态学上的高度异质性，且形态结构较为简单，使得仅仅依据形态学特征难以准确将耳霉鉴定到种级水平；近年来，发现朝上耳霉（ConidiobolusadiaeretusDrechsler）既产生小分生孢子，又产生毛管孢子，这导致基于次生分生孢子产生的类型对耳霉属三个亚属的划分产生了疑问，因此，需要开发一些新的分类特征和运用分子生物学手段来解决耳霉属种的分类地位和确定耳霉属亚属的分类地位。本研究拟运用耳霉一些稳定的形态学分类特征、新开发的耳霉生理和细胞学特性（最高生长温度和初生分生孢子细胞核平均数）及多基因位点的分子系统发育分析方法，准确鉴定中国耳霉分离菌株，澄清King耳霉分类系统中模式菌株的分类地位，并确定耳霉三个亚属的分类地位。通过对耳霉同种不同菌株和不同种的最高生长温度的比较，发现同种不同菌株间具有相同或相差不超过1℃的最高生长温度值，大部分不同种间具有相差超过2℃的最高生长温度值，也有少数不同种之间具有相同的最高生长温度值，揭示最高生长温度是一个稳定的特性，可以作为耳霉种级鉴定的分类学指标；通过对耳霉同种不同菌株和不同种之间的初生分生孢子细胞核平均数的比较，发现同种不同菌株间具有相同或相近的细胞核平均数，大部分不同种间细胞核平均数差别较大，证明了初生分生孢子细胞核平均数与初生分生孢子大小相关性强，成正比关系，因而这种细胞学性状也是一个鉴定种的较为重要的分类特征。通过nuc-LSU、mt-SSU、EF-1α和RPB2这4个基因位点的分子系统发育分析，发现耳霉在DNA水平上异质度高，形成了3个主要分支，且各分支类群之间遗传距离较远，再结合耳霉次生分生孢子类型和这三个类群具有较强的关联性，我们将耳霉属原有的3个亚属上升为属的分类地位：德拉霉属（DelacroixiaSacc.&P.Syd.）、毛管霉属（CapillidiobolusY.Nie&B.Huang）、耳霉属（ConidiobolusBrefeld）；其中，在德拉霉属的基部产生3个独立的分支，形态学观察这3个分支所包含的种都未产生小分生孢子，由于这3个分支在系统发育树上的位置特殊，但没有明确的属的分类特征，因此，这3个分支的分类地位待定，暂时划为广义耳霉属（Conidiobolussensulato)处理。此外虽然朝上毛管霉(Capillidiobolus adiaeretus)和班加罗尔毛管霉(CapillidioboluS bangalorensis Srin.&Thirum.)在毛管霉属中形成独立的分支,但他们还是位于产生毛管孢子类群的基部,和毛管孢子类群关系较近,同时考虑在他们都产生毛管孢子,虽然朝上耳霉也可产生小分生孢子,我们仍将这两个种划为毛管霉属来处理。对基于数值分类法的King分类系统中的部分耳霉模式菌株的分类地位进行了修订,通过形态学特征、初生分生孢子细胞核平均数、最高生长温度和分子系统发育分析,恢复了8个耳霉的种的分类地位,并将其中4个种组合进不同的属中：Capillidiobolus rugosus(Drechsler)Y.Nie&B.Huang.Delacroixia lichenicola(Srin.&Thirum.)Y.Nie&B.Huang、D.megalotocus var.indicus(Srin.&Thirum.)Y.Nie&B. Huang、D.mycophagus(Srin.&Thirum.)Y.Nie&B.Huang；其余4个种作为广义耳霉属处理：Conidiobolus nodosus Srin.&Thirum.、C. terrestris Srin.&Thirum.、C. undulates Srin.&Thirum.、C. parvus Drechsler。对15个耳霉种进行了新组合：Delacroixia macrosporus(Srin.&Thirum.)Y.Nie&B.Huang、D. mycophilus(Srin.&Thirum.)Y.Nie&B.Huang、D.khandalensis(Srin.&Thirum.)Y.Nie&B.Huang、D. humicola(Srin.&Thirum.)Y.Nie&B.Huang、D.polytocus(Drechsler)Y.Nie&B. Huang、D.incongruus (Drechsler)Y.Nie&B.Huang、D.firmipilleus(Drechsler)Y.Nie&B.Huang、D.megalotocus(Drechsler)Y.Nie&B.Huang、D.brefeldianus(Couch)Y. Nie&B.Huang.Capillidiobolus heterosporus(Drechsler)Y.Nie&B.Huang、C.lobatus (Srin.&Thirum.)Y.Nie&B.Huang、C. rhysosporus(Drechsler) Y.Nie&B.Huang、C. pumilus(Drechsler)Y.Nie&B.Huang、C. adiaeretus(Drechsler) Y.Nie&B.Huang、C. bangalorensis(Srin.&Thirum.)Y.Nie&B.Huang.本研究共分离得到250余株中国广义耳霉菌株,对这些中国分离株运用形态学特征,新分类性状以及分子数据进行了分类地位的确认,共鉴定出24个种,1个待鉴定种,其中包括12个新种：霍山毛管霉(Capillidiobolus huoshanensis Y.Nie&B. Huang)、黄孢毛管霉(C.flavosporus Y.Nie&B.Huang)、中国耳霉(C. sinensis Y. Nie&B.Huang)、牯牛降耳霉(Conidiobous guniujiangensis Y. Nie&B.Huang)、双孢耳霉(C. bisporus Y. Nie&B.Huang)、束梗耳霉(C. stilbella Y. Nie&B.Huang)、李氏德拉霉(Delacroixia lii Y. Nie&B.Huang)、石台德拉霉(C. shitaiensis Y. Nie&B.Huang).鼎湖山德拉霉(D. dinghushanensis Y. Nie&B.Huang)、琅琊山德拉霉(D. langyashanensis Y. Nie&B.Huang)、汉博德拉霉(D. humberii Y. Nie&B. Huang)和德雷克斯勒德拉霉(C. drechslerii Y. Nie&B.Huang);1个新变种：冠德拉霉长柔毛变种(D. coronatus var. longvillosus Y.Nie&B.Huang);3个新纪录种：皱孢毛管霉(Capillidiobolus rhysosporus Drechsler)、褶孢毛管霉(C. rugosus Drechsler)和毛耳霉(Conidiobolus lachnodes Drechsler);8个已知种：有味耳霉(Conidiobolus osmodes Drechsler)、块状耳霉(C. thromboides Drechsler)、大乳突耳霉(C. macropapillatus C. F. Wang&B. Huang)、近隔接合孢耳霉(C. iuxtagenitus S. D. Waters&Callaghan)、异形孢毛管霉(Capillidiobolus heterosporus Drechsler)、冠德拉霉(Delacroixia coronatus (Costantin) Sacc.&P. Syd.)、牢盖德拉霉(D. firmipilleus Drechsler)和布尔弗雷德德拉霉(D. brefeldianus Couch);和1株待鉴定种：RCEF5845。更多还原

【Abstract】 Conidiobolus is one of the most important genus, and has a special systematic position in the phylum Entomophthoromycota. The Conidiobolus is a key decomposer in ecosystem, and some species can be used as the agents for controlling pest. From November2008to April2009, more than100Conidiobolus isolates from Anhui and Shandong provinces were isolated by Wang et al., and4new records of Conidooblus in China were published. To investigate the species diversity in the genus Conidiobolus from China and provide much more materials for systematic research, an efficient method improved by our team was used to isolate Conidiobolus isolates from Nature Reserves in Northeast, East, Central, South and Southwest of China. It is difficult to identify exactly the Conidiobolus strains at species level only based on the morphological character for its high heterogeneity and simple morphological structure. Recent studies showed that Conidiobolus adiaeretus Drechsler produced either microconida or capilliconida, which means that the division of three subgenera in the genus Conidiobolus is unreasonable. Some new developed classification features combined with molecular method should be used to identify Conidiobolus strains and to clarify phylogenetic relations within the genus Conidiobolus. In this research, we will exactly identify the Conidiobolus strains isolated from China, revise the King’s classification and resolve the taxonomic placement of three subgenera based on the new taxonomic characters (the maximum growth temperature and the average numbers of nucleus in primary conidia), important morphological features and nucleotide sequence data from multiloci.By comparing the maximum growth temperature among different Conidiobolus strains of the same species and among different Conidiobolus species, each species possessed a stable and specific maximum growth temperature, which can be distinguishing Conidiobolus species. The optimum growth temperature showed variation within a single species and obvious overlap between species, which are of little value in taxonomy. By comparing the average numbers of nucleus in primary conidia among different Conidiobolus strains of the same species and among different Conidiobolus species, each species had similar average numbers of nucleus in primary conidia, which are of important taxonomic value. We also found that the average numbers of nucleus in primary conidia had proportional relations with the size of primary conidia.Based on the molecular phylogenetic analysis of4gene loci:nuc-LSU, mt-SSU, EF-la and RPB2, a high level of heterogeneity was detected in the genus Conidiobolus. Combined far gentic distance among every clade in phylogenetic tree and the stable morphylogical character of the type of secondary conidia production, three subgenera in the genus Conidiobolus were rised to the status of the genus. A phylogenetic analysis assigned the Conidiobolus strains to three main lineages corresponding to the restored genus Delacroixia Sacc.&P. Syd., the estabished newly genus CapillidiobolusY. Nie&B. Huang and the emended genus Conidiobolus Brefeld. Clade I consists of four subgroups incuding subgroup A, subgroup B, subgroup C and subgroup D corresponding to the genus Delacroixia with microconidia, Conidiobolus iuxtagenitus, two Conidiobolus species and three small conidial species, respectively. The species from subgroup B, subgroup C and subgroup D do not produced microconidia and located at the base of the Clade I, and were put in Conidiobolus sensu lato. Although C. adiaeretus and C. bangalorensis Srin.&Thirum. formed a independent clade in the genus Capillidiobolus, they shared closer phylogentic relationship with Capillidiobolus than the species from other two genera. Therefore, these two species should classificated into the genus Capillidiobolus while C. adiaeretus could produce both microconidia and capilliconidia. The King’s classification was revised based on molecular data and two new taxonomic features.8homonyms in Conidiobolus were restored as species level,4of them were combined into the new genus. They are Capillidiobolus rugosus (Drechsler) Y. Nie&B. Huang, Delacroixia lichenicola (Srin.&Thirum.) Y. Nie&B. Huang, D. megalotocus var. indicus (Srin.&Thirum.) Y. Nie&B. Huang, D. mycophagus (Srin.&Thirum.) Y. Nie&B. Huang, Conidiobolus nodosus Srin.&Thirum., C. terrestris Srin.&Thirum., C. undulates Srin.&Thirum., C. parvus Drechsler.15new combinations were made i.e. Delacroixia macrosporus (Srin.&Thirum.) Y. Nie&B. Huang, D. mycophilus (Srin.&Thirum.) Y. Nie&B. Huang, D. khandalensis (Srin.&Thirum.) Y. Nie&B. Huang, D. humicola (Srin.&Thirum.) Y. Nie&B. Huang, D. polytocus (Drechsler) Y. Nie&B. Huang, D. incongruus (Drechsler) Y. Nie&B. Huang, D. firmipilleus (Drechsler) Y. Nie&B. Huang, D. megalotocus (Drechsler) Y. Nie&B. Huang, D. brefeldianus (Couch) Y. Nie&B. Huang, Capillidiobolus heterosporus (Drechsler) Y. Nie&B. Huang, C. lobatus (Srin.&Thirum.) Y. Nie&B. Huang, C. rhysosporus (Drechsler) Y. Nie&B. Huang, C. pumilus (Drechsler) Y. Nie&B. Huang, C. adiaeretus (Drechsler) Y. Nie&B. Huang, C. bangalorensis (Srin.&Thirum.) Y. Nie&B. Huang.During the investigation of Conidiobolus sensu lato in China in2009-2013, we have isolated more than250strains. Based on the careful morphological identification and molecular data,24taxa at species or variety level were described and illustrated. Among them,13taxa are new species or new variety. They are Capillidiobolus huoshanensis Y. Nie&B. Huang, C. flavosporus Y. Nie&B. Huang, C. sinensis Y. Nie&B. Huang, Conidiobolus guniujiangensis Y. Nie&B. Huang, C. bisporus Y. Nie&B. Huang, C. stilbella Y. Nie&B. Huang, Delacroixia lii Y. Nie&B. Huang, D. shitaiensis Y. Nie&B. Huang, D. dinghushanensis Y. Nie&B. Huang, D. langyashanensis Y. Nie&B. Huang, D. humberii Y. Nie&B. Huang, D. Drechslerii Y. Nie&B. Huang and Delacroixia coronatus var. longvillosus Y. Nie&B. Huang.3species are newly recorded for China. The3new records are:Capillidiobolus rhysosporus Drechsler, C. rugosus Drechsler, Conidiobolus lachnodes Drechsler.8species having been previously reported from China are redescribed and discussed, i. e. Conidiobolus osmodes Drechsler, C. thromboides Drechsler, C. macropapillatus C. F. Wang&B. Huang, C. iuxtagenitus S. D. Waters&Callaghan, Capillidiobolus heterosporus Drechsler, Delacroixia coronatus (Costantin) Sacc.&P. Syd., D. firmipilleus Drechsler and D. brefeldianus Couch. The strain RCEF5845needs to be instensively studied.更多还原