【作者】 陈汝；

【导师】 姜远茂；

【作者基本信息】 山东农业大学 ， 果树学， 2012， 博士

【摘要】 本文通过盆栽试验，采用微生物稀释平板培养法对平邑甜茶、八棱海棠、楸子、新疆野苹果、东北山荆子5种苹果砧木土壤根际微生物数量、土壤酶活性及其相关性进行研究；以2年生红富士/平邑甜茶为试材，研究生草和覆盖对苹果根际土壤养分及微生物数量和群落代谢功能的影响；通过采集12个省（市）的81个苹果园土壤样品，采用微生物培养法研究了我国苹果主产区果园土壤微生物特征；利用26S rRNA的D1/D2区域序列分析并结合形态学特征对红富士苹果园的酵母菌多样性进行了研究，并基于形态、生理生化特征以及18S rRNA、ITS和26S rRNA基因D1/D2区基因序列对子囊菌酵母新种和担子菌酵母新种进行鉴定。主要结果如下：1.不同苹果砧木土壤根际微生物各生理类群数量差异显著，并且都以细菌占绝对优势，放线菌次之，真菌最少。不同苹果砧木土壤根际蔗糖酶、磷酸酶、脲酶和过氧化氢酶活性各异。皮尔逊相关分析表明，土壤微生物数量与土壤酶活性之间存在相关关系。苹果砧木根际土壤微生物多样性指数依次为新疆野苹果>平邑甜茶>八棱海棠>楸子>东北山荆子。2.与传统清耕栽培相比，生草和覆盖处理均能显著提高幼龄苹果根际土壤养分含量；各处理幼龄苹果根际土壤微生物以细菌占优势、放线菌次之、真菌最少，生草和覆盖处理的根际土壤细菌、真菌、放线菌、自生固氮菌、纤维素降解菌和微生物总数量均显著高于清耕。土壤微生物数量与土壤养分含量之间存在显著相关性。生草和覆盖均显著增强土壤微生物利用碳源的能力，提高土壤微生物群落的功能多样性，效果依次为白三叶草>黑麦草>秸秆覆盖>清耕。3.土壤微生物各生理类群在我国苹果主产区果园中的优势不同，细菌在各苹果园土壤微生物中的比例均最高。Pearson相关分析表明，土壤微生物多样性指数与土壤有机质含量显著正相关。不同省区苹果园土壤微生物多样性指数存在差异，依次为：黑龙江>云南>新疆>山东>北京>宁夏>山西>辽宁>河北>河南>甘肃>陕西。对各地苹果园土壤微生物数量进行聚类分析可将各省苹果园分为三类：黑龙江、辽宁；新疆、云南、陕西、甘肃、山西、宁夏；河北、山东、北京、河南，与我国根据气候和生态适宜标准划分的苹果主产区状况基本吻合。4.红富士果园中富含丰富的酵母资源，北京和山东两果园的酵母菌种构成明显差异，并分别具有不同的优势属。北京苹果园共分离酵母菌129株，鉴定为13属21种，优势属为Pichia（4个种），Cryptococcus（3个种），Pseudozyma（3个种），子囊菌占较大优势，分布于8属12种，占总种数的57.1%。山东苹果园共分离酵母291株，鉴定为13属26种，优势属为Candida（6个种），Pichia（4个种）和Cryptococcus（3个种），并且子囊菌占较大优势，分布于7属17种，占总种数的65.4%。5.从泰安苹果园的土壤中分离到三株子囊菌酵母(TA11TR-1T, TA11TR-4和TA11TR-6)，根据表型特征及18S rRNA，ITS和26S rRNA基因D1/D2区基因序列分析表明，属于Kazachstania属。命名为Kazachstania taianensis sp. nov.（模式菌株TA11TR-1T=AS2.4160T=CBS11405T）。通过18S及D1/D2序列N-J比对得，K.taianensis sp. nov.与Kazachstania sinensis, Kazachstania naganishii和Kazachstaniatelluris在同一个分支上。ITS-5.8S rDNA区总长度为983bp，长于已分离到的其它子囊菌酵母。6.从泰安苹果园中分离到4株担子菌酵母(Y13-1T, Y2-1, Y6-3和Y8-2)，基于ITS和26S rRNA基因D1/D2区基因序列进行系统聚类分析，在系统树上这四个菌株聚类在Tremellomycetes的Kwoniella分支上，明显区别于它们的最近缘种。与Cryptococcuscuniculi最接近，但远离于Kwoniella heveanensis。因此定为新种，命名为Kwoniellashandongensis sp. nov.(模式菌株Y13-1T=CGMCC2.04458T=CBS12478T)。MycoBank编号为MB564868。更多还原

【Abstract】 Using microbial dilution plate counting cultivation method, the rhizosphere soilmicrobial quantity and enzyme activity and their relations of five kinds of apple rootstocks (M.hupehensis Rehd., M. micromalus Makino., M. prunifolia(Willd) Borkh., M. sievesii(Ledeb.)Roemer. and M. baccata Borkh.) were analyzed in a short-term pot experiment. One fieldexperiment was conducted to investigate the effects of grassing (Trifolium repens and Loliumperenne) and mulching (straw mulch) on rhizosphere soil microbial quantity and functionaldiversity in young ‘Fuji’apple orchard by using plate count method and Biolog micro-platetechnique. Soil samples were collected from81apple orchards in12different provinces andcities. The microbial characteristics of apple orchard in major production areas of China werestudied by microbial cultivation. Yeast diversity of the Fuji apple orchard was studied by26SrDNA Dl/D2domain sequence analysis and the combination of morphology. Based onphenotypic and physiological characteristics and sequence analyses of the18S rRNA gene,internal transcribed spacer (ITS) regions and26S rRNA gene D1/D2domain. The nameKazachstania taianensis sp. nov. and Kwoniella shandongensis sp. nov. are proposed. Themain results were followed:1. The number of microbial physiological groups in rhizosphere soil under differentvarieties of apple rootstocks were significantly differences, and bacteria was dominant,followed by actinomycetes, fungi at least. Different varieties of apple rootstocks rhizospheresoil invertase, phosphatase, urease and catalase activity varied. Pearson correlation analysisindicated that certain correlation existenced between soil microbial amount and soil enzymeactivities. The microbial biodiversity indices in rhizosphere soil were different among applerootstocks, were as follows: M. sievesii(Ledeb.) Roemer> M. hupehensis Rehd.> M.micromalus Makino.> M. prunifolia(Willd) Borkh.> M. baccata Borkh.2. Compared with traditional farming method, grassing and mulching measures couldsignificantly improve the soil nutrient content and soil microorganisms in rhizosphere of young apple, and bacteria was dominant, followed by actinomycetes, fungi at least, andbacteria, fungi, actinomycetes, nitrogen-fixing bacteria, cellulose-degrading bacteria and totalmicroorganisms quantity was significantly higher than the conventional farming control.Pearson correlation analysis showed that significant correlation were existenced between soilmicrobial quantity and soil nutrient content. Grassing and mulching measures can alsoincrease the capacity to utilize carbon substrate and the biodiversity and evenness of soilmicrobial community, were as follows: Trifolium repens> Lolium perenne> straw mulch>clean tillage.3. The advantages of soil microbial physiological groups were different in each appleorchard in different regions of China. Bacteria was in the highest proportion in the appleorchard. Pearson correlation analysis indicated that significantly positive correlationexistenced between soil microbial diversity index and soil organic matter content. Soilmicrobial diversity index was different in each apple orchard between different provinces wasas follows: Heilongjiang> Yunnan> Xinjiang> Shandong> Beijing> Ningxia> Shanxi>Liaoning> Hebei> Henan> Gansu> Shanxi. Cluster analysis the number of microorganismsof different apple orchards can be divided into three groups: Heilongjiang, Liaoning; Xinjiang,Yunnan, Shaanxi, Gansu, Shanxi, Ningxia; Hebei, Shandong, Beijing, Henan, consisted withthe status of the major apple producing areas divided by the climate and ecological suitabilitystandards.4. Yeast is abundant in the Fuji orchard. The difference of yeast species between the twoorchards is significant, which contain different dominant genera yeast, respectively. A total of129yeast strains belonging to21species in13genera were isolated from samples collectedfrom Fuji apple orchards in Beijing. The dominant genera identified were Pichia (4species),Cryptococcus (4species) and Pseudozyma (3species). Among the yeast species identified,57.1%were ascomycetous yeasts belonging to12species in8genera. A total of291yeaststrains belonging to26species in13genera were isolated from samples collected from Fujiapple orchards in Shandong province. The dominant genera identified were Candida (6species), Pichia (4species) and Cryptococcus (3species). Ascomycetous yeast species werealso dominant, accounting for65.4%(17species belonging to7genera) of the speciesidentified from the Shangdong strains. 5. Three teleomorphic ascomycetous yeast strains (TA11TR-1T, TA11TR-4andTA11TR-6) isolated from the orchard soil from Tai’an, Shandong province, China, wererevealed to represent a novel species within the genus Kazachstania based on phenotypiccharacterization and sequence analyses of the18S rRNA gene, internal transcribed spacer(ITS) regions and26S rRNA gene D1/D2domain. The name Kazachstania taianensis sp. nov.(type strain TA11TR-1T=AS2.4160T=CBS11405T) is proposed. K. taianensis sp. nov. wasclustered in a branch together with Kazachstania sinensis, Kazachstania naganishii and theKazachstania telluris complex with moderate bootstrap support in the neighbor-joining treeconstructed from combined18S and D1/D2sequences. The new species possessed specialITS1(338bp) and ITS2(488bp) sequences. The total length of the ITS-5.8S rDNA regionof the species was983bp, being much longer than those of other ascomycetous yeast speciesdescribed so far.6. Four basidiomycetous yeast strains (Y13-1T, Y2-1, Y6-3and Y8-2) isolated from anapple orchard in Tai’an, Shandong province, China. Phylogenetic analysis based on26S rRNAgene D1/D2domains and ITS regions revealed that those strains were located in theKwoniella clade in the Tremellomycetes and were closely related to Cryptococcus cuniculiout Kwoniella heveanensis and clearly distinct from the latter species. Therefore, a newspecies, named as Kwoniella shandongensis sp. nov.(type strain Y13-1T=CGMCC2.04458T=CBS12478T) is proposed. The MycoBank number of the new species is MB564868.更多还原