论文摘要
内生菌对作物健康生长起着重要作用。本文分离、鉴定了大量小麦内生菌,研究了小麦内生菌的多样性组成及其促生植物潜能和抗菌抗糖尿病潜能。1.从陕西、山西、河北、北京4省区采集到不同生长期的小麦植株样品15个,从根、茎、叶中分离到细菌600株,放线菌2株、真菌16株。经过初步鉴定,新分离细菌系统发育地位属于Achromobacter,Bacillus,Cedaceae,Citrobacter,Enterobacter,Flavobacterium,Flexibacter,Klebsiella,Kosakonia,Microbacterium,Ochrobactrum,Paenibacillus,Providencia,Pseudomonas,Rahnella,Rheinheimera,Rhizobium,Serratia,Sphingobacterium,Sphingobium,Stenotrophomonas,Xenophilus,Variovorax等23属58种。其中12属21种检测到了nif H基因。2.高通量测序结果显示小麦植株中内生细菌有500个属,从高到低排列主要类群为Streptomyces,Sinorhizobium,Promicromonospora,Pantoea,Massilia,Cellulosimicrobium,Ohtaekwangia,Devosia,Kribbela,Micromonospora,Rhizobium,Nonomuraea,Kluyvera,Pseudomonas,Sphingomonas,Glutamicibacter,Niastella,Klebsiella。采用nif H基因高通量测序检测到的小麦内生固氮菌100个属。16S rRNA高通量测序检测到大量放线菌,nif H基因高通量测序检测到了大量稀有固氮菌群,而基于传统培养技术的多样性研究检测到大量常见固氮菌群。3.对72株细菌,2株放线菌和15种真菌进行了促进植物生长和胁迫潜能测试分析。菌株固氮酶活（18.16±0.82581.73±0.71nmol/mg蛋白·h）,1-氨基环丙烷-1-羧酸脱氨酶（ACC deaminase;3.97±0.020.03±0.011μmolα丁酮酸/h·mg）,IAA(1.00±0.149.37±0.005μgml-1)和PSI（2.17±0.025.16±0.36）活性。超过80%的分离株在铁载体,NH3和脲酶试验中表现出阳性结果;只有13菌株产生了HCN。而在非生物胁迫测试中,Trichoderma菌株在高盐（10%）,重金属(300μgml-1)和干旱（35%PEG）胁迫下表现出强耐受性,并对测试的抗生素（制霉菌素,酮康唑和伊曲康唑）具有抗性;菌株Talaromyces funiculosus耐高温达到550C。盆栽试验显示,与对照相比,7株小麦内生固氮菌使小麦、玉米株高、干重和SPAD值增加。4.发现并命名了一株新的细菌Paenibacillusrhizophilus 7197T。该菌株含有不同类型的脂肪酸(anteiso-C15:0,C14:0,C16:0等)、极性脂（DPG,PG,PE等）、呼吸醌MK-7,Meso-DAP。含有4个nif H基因,显示出高固氮酶活性（2526.41±34.9 nmol/mg蛋白·h）。ACC deaminase（1.53±0.01μmolα-丁酮酸/h·mg）,IAA(26.12±0.004μg ml-1),PSI（2.08±0.36）,NH3,铁载体阳性,抗非生物胁迫[温度（400C）、盐度（2.5%NaCl）、干旱（20%PEG）、重金属和抗生素]。盆栽试验对小麦和玉米植物表现出生长促进作用（株高、干重和SPAD值高于对照处理）。
论文目录
摘要AbstractCHAPTER Ⅰ INTRODUCTION 1.1 BACKGROUND 1.2 ENDOPHYTES 1.3 ENTRY OF ENDOPHYTES IN TO THE HOST PLANTS 1.4 BENEFICIAL FUNCTIONS OF ENDOPHYTES FOR PLANTS 1.4.1 ENHANCE PHYTOSTIMULATION 1.4.2 NITROGEN FIXATION 1.4.3 PHOSPHATE SOLUBILIZATION ABILITY OF ENDOPHYTES 1.4.4 IRON HOMEOSTASIS 1.4.5 ROLE IN INDUCED SYSTEMIC TOLERANCE 1.5 GENES RELATED TO NITROGEN FIXATION 1.6 MIRACULOUS DIAZOTROPHIC BACTERIA 1.6.1 BIOLOGICAL NITROGEN FIXATION OF DIAZOTROPHS 1.6.2 SYMBIOTIC NITROGEN FIXERS PIONEERS FOR BNF 1.6.2.1 IMPORTANCE OF SYMBIOTIC NITROGEN FIXATION IN LEGUMES 1.6.2.2 ROLE OF SYMBIOTIC NITROGEN FIXATION IN NON-LEGUMES 1.6.3 EFFECT OF ASSOCIATIVE DIAZOTROPHIC NITROGEN FIXERS 1.6.4 ENDOPHYTIC DIAZOTROPHIC NITROGEN FIXERS 1.6.5 FREE LIVING NITROGEN FIXERS 1.7 NATURAL PRODUCTS FROM ENDOPHYTES 1.7.1 ANTIBIOTICS 1.7.2 ANTIVIRAL COMPOUNDS 1.7.3 ANTICANCER COMPOUNDS 1.7.4 ANTIOXIDANT PROPERTY 1.7.5 ANTIDIABETIC ACTIVITY 1.7.6 IMMUNOSUPPRESIVE COMPOUNDS FROM ENDOPHYTES 1.8 MICROBIAL DIVERSITY RESEARCH METHODS AND PROGRESS 1.9 JUSTIFICATION OF THE STUDY 1.10 RESEARCH PLAN 1.11 SCIENTIFIC QUESTIONS 1.12 HYPOTHESIS 1.13 OBJECTIVESCHAPTER Ⅱ MATERIALS AND METHODS 2.1 PLANT SAMPLING AND ISOLATION OF ENDOPHYTES 2.2 MOLECULAR IDENTIFICATION OF THE ISOLATED ENDOPHYTES 2.3 ANALYSIS OF THE DIVERSITY OF WHEAT ENDOPHYTIC NITROGEN FIXING BACTERIA 2.3.1 ENDOPHYTIC BACTERIA16S r DNA V5-V7 REGION AMPLIFICATION 2.4 ANALYSIS OF THE DIVERSITY OF nif HCONTAINING DIAZOTROPHS 2.5 DATA PROCESSING METHODS 2.6 GROWTH PROMOTING AND STRESS TOLERANCE PROPERTIES OF WHEAT ENDOPHYTES 2.6.1 EVALUATION OF SOME IMPORTANT PGP PROPERTIES 2.6.1.1 QUALITATIVE AND QUANTITATIVE ESTIMATION OF ACC DEMINASEACTIVITY 2.6.1.2 QUALITATIVE AND QUANTITATIVE SCREENING OF NITROGEN FIXING PROPERTY OF THE ISOLATED ENDOPHYTIC BACTERIA 2.6.1.3 QUANTIFICATION OF IAA 2.6.1.4 SIDEROPHORE PRODUCTION 2.6.1.5 QUALITATIVE SCREENING OF PHOSPHATE SOLUBILIZATION POTENCY· 2.6.1.6 AMMONIA（NH3）PRODUCTION 2.6.1.7 CYANOGENESIS-HYDROGEN CYANIDE（HCN）PRODUCTION 2.6.1.8 UREASE TEST 2.6.1.9 CATALASE TEST 2.6.2 ABIOTIC STRESS TOLERANCE PROPERTIES 2.6.2.1 SALT TOLERANCE TEST 2.6.2.2 HEAVY METAL RESISTANCE TEST 2.6.2.3 DROUGHT TOLERANCE TEST 2.6.2.4 TEMPERATURE TOLERANCE TEST 2.6.2.5 ANTIBIOTIC SENSITIVITY TEST 2.6.3 EFFECT OF ENDOPHYTIC INOCULATION ON WHEAT AND MAIZE GROWTH 2.7 ANTIDIABETIC AND ANTIMICROBIAL POTENTIALS OF WHEAT ENDOPHYTES· 2.7.1 SECONDARY METABOLITE EXTRACTION 2.7.2 PHYTOCHEMICAL ANALYSIS 2.7.2.1 TEST FOR PHENOLS 2.7.2.2 TEST FOR TERPENOIDS 2.7.2.3 TEST FOR FLAVONOIDS 2.7.2.4 TEST FOR SAPONINS 2.7.2.5 TEST FOR TANNINS 2.7.2.6 TEST FOR ALKALOIDS 2.7.2.7 TEST FOR CARBOHYDRATES 2.7.2.8 TEST FOR STEROIDS 2.7.3 ANTIDIABETIC SCREENING OF THE ENDOPHYTIC METABOLITES 2.7.3.1 α-AMYLASE INHIBITION ASSAY 2.7.3.2 α-GLUCOSIDASE INHIBITION ASSAY 2.7.4 ANTIMICROBIAL TEST OF CRUDE METABOLITES OF ENDOPHYTES 2.7.4.1 TEST STRAINS AND CULTURE MEDIA 2.7.4.2 ANTIFUNGAL ASSAY 2.7.4.3 ANTIBACTERIAL ASSAYT（Paenibacillus rhizophilus）'> 2.8 CHARACTERIZATION OF THE ISOLATED NOVEL ENDOPHYTIC BACTERIAUM7197T（Paenibacillus rhizophilus） 2.8.1 GENOME SEQUENCING 2.8.2 16S rRNA GENE SEQUENCING AND ANALYSIS 2.8.3 MORPHOLOGICAL CHARACTERISTICS 2.8.4 PHENOTYPICAL AND BIOCHEMICAL CHARACTERISTICS 2.8.5 NITROGENASE ACTIVITY OF7197T 2.8.6 CYTOCHEMICAL COMPONENTST'> 2.8.7 Growth Promoting and Abiotic Stress Tolerance Properties of 7197TT ON WHEAT AND MAIZE GROWTH'> 2.8.8 INOCULATION EFFECT OF7197T ON WHEAT AND MAIZE GROWTH 2.9 STATISTICAL ANALYSISCHAPTER Ⅲ ANALYSIS OF THE DIVERSITY OF WHEAT ENDOPHYTIC NITROGEN FIXING BACTERIA 3.1 INTRODUCTION 3.2 RESULTS AND ANALYSIS 3.2.1 DIVESRSITY OF WHEAT ENDOPHYTIC NITROGEN FIXING BACTERIA WITH CULTURE DEPENDENT METHOD 3.2.2 ANALYSIS OF DIVERSITY OF WHEAT ENDOPHYTIC NITROGEN FIXING BACTERIA BY CULTURE INDEPENDENT METHOD 3.2.2.1 DATA QUALITY CONTROL AND OTU BASIS ANALYSIS 3.2.2.2 GENUS ABUNDANCE HEAT-MAP 3.2.2.3 ANALYSIS OFΑLPHA（α）DIVERSITY OF N-FIXING ENDOPHYTIC BACTERIA IN WHEAT 3.2.2.4 ANALYSIS OF BETA（β）DIVERSITY OF N-FIXING ENDOPHYTIC BACTERIA IN WHEAT 3.2.2.5 ANALYSIS OF N-FIXING ENDOPHYTIC BACTERIA CORE FLORA 3.2.2.6 ANALYSIS OF N FIXING ENDOPHYTIC BACTERIA ON TAXONOMIC LEVELS· 3.3 Comparison of Biodiversity of Endophytic Bacteria with Culture Dependent and Culture Independent Methods 3.4 SUMMARY OF THE CHAPTERCHAPTER Ⅳ DIVERSITY OF nif H CONTAINING WHEAT DIAZOTROPHS 4.1 INTRODUCTION 4.2 RESULTS AND ANALYSIS 4.2.1 DIVERSITY OF nif H CONTAINING DIAZOTROPHS WITH CULTURE DEPENDENT METHOD 4.2.2 ANALYSIS OF DIVERSITY OF WHEAT ENDOPHYTIC NITROGENFIXINGDIAZOTROPHSIN WHEAT BY CULTURE INDEPENDENT METHOD 4.2.2.1 DATA QUALITY CONTROL AND OTU BASIS ANALYSIS 4.2.2.2 GENUS ABUNDANCE HEAT-MAP 4.2.2.3 ANALYSIS OFΑLPHA（α）DIVERSITY OF NITROGEN FIXING DIAZOTROPHS IN WHEAT 4.2.2.4 ANALYSIS OF BETA（β）DIVERSITY OF NITROGEN FIXING DIAZOTROPHS IN WHEAT 4.2.2.5 ANALYSIS OF CORE FLORA OF ENDOPHYTIC NITROGEN FIXING DIAZOTROPHS 4.2.2.6 ANALYSIS OF ENDOPHYTIC DIAZOTROPHS ON TAXONOMIC LEVELS 4.3 COMPARISON OF DIAZOTROPHBIODIVERSITYWITH CULTURE DEPENDENTAND CULTURE INDEPENDENT METHODS 4.4 SUMMARY OF THE CHAPTERCHAPTER Ⅴ GROWTH PROMOTING AND STRESS TOLERANCE PROPERTIES OF WHEAT ENDOPHYTES 5.1 INTRODUCTION 5.2 RESULTS 5.2.1 PGP TRAITS OF THE ISOLATED ENDOPHYTIC MICROBES 5.2.1.1 QUALITATIVE AND QUANTITATIVE SCREENING FOR NITROGENFIXATION ABILITY OF THE ISOLATED ENDOPHYTES 5.2.1.2 QUALITATIVE AND QUANTITATIVE ESTIMATION OF ACC DEAMINASE ACTIVITY 5.2.1.3 IAA PRODUCTION 5.2.1.4 SIDEROPHORE PRODUCTION 5.2.1.5 PHOSPHATE SOLUBILIZATION POTENCY 5.2.1.6 AMMONIA PRODUCTION 5.2.1.7 CYANOGENESIS 5.2.1.8 UREASE TEST 5.2.1.9 CATALASE TEST 5.2.2 ABIOTIC STRESS TOLERANCE 5.2.2.1 SALT TOLERANCE TEST 5.2.2.2 HEAVY METAL RESISTANCE TEST 5.2.2.3 DROUGHT RESISTANCE TEST 5.2.2.4 TEMPERATURE TOLERANCE TEST 5.2.2.5 ANTIBIOTIC SENSITIVITY TEST 5.3 INOCULATION EFFECT OF ENDOPHYTES ON WHEAT AND MAIZE 5.4 SUMMARY OF THE CHAPTERCHAPTER Ⅵ ANTIDIABETIC AND ANTIMICROBIAL POTENTIALS OF WHEATENDOPHYTES 6.1 INTRODUCTION 6.2 RESULTS 6.2.1 PHYTOCHEMICAL ANALYSIS 6.2.2 ANTIDIABETIC ACTIVITY OF THE MICROBIAL EXTRACTS 6.2.3 ANTIMICROBIAL ACTIVITY OF THE MICROBIAL EXTRACTS 6.3 SUMMARY OF THE CHAPTERCHAPTER Ⅶ CHARACTERIZATION OF THE ISOLATED NOVEL ENDOPHYTIC BACTERIUM7197T（Paenibacillus rhizophilus） 7.1 INTRODUCTION 7.2 RESULTS AND ANALYSIS 7.2.1 PHYLOGENETIC ANALYSIS OF16S r RNA GENE SEQUENCES 7.2.2 DETERMINATION OF DNA G+C MOL%AND DNA HOMOLOGY 7.2.3 MORPHOLOGICAL CHARACTERISTICS 7.2.4 PHENOTYPICAL AND BIOCHEMICAL CHARACTERISTICS 7.2.5 NITROGENASE ACTIVITY OF7197T 7.2.6 CYTOCHEMICAL COMPONENTS 7.2.6.1ANALYSIS OF FATTY ACID COMPONENTS 7.2.6.2 ANALYSIS OF POLAR VALUE COMPONENTS 7.2.6.3 ANALYSIS OF BISMATH COMPONENT 7.2.6.4 ANALYSIS OF AMINO ACID COMPOSITION IN CELL WALL 7.3 GROWTH PROMOTING AND STRESS TOLERANCE PROPERTIES OF7197T 7.4 INOCULATION EFFECT OF7197T ON WHEAT AND MAIZE PLANTS 7.5 SUMMARY OF THE CHAPTERCHAPTER Ⅷ DISCUSSION 8.1 ANALYSIS OF DIVERSITY OF WHEAT ENDOPHYTIC NITROGEN FIXING BACTERIA. 8.2 ANALYSIS OF DIVERSITY OF nif H CONTAINING WHEAT DIAZOTROPHS 8.3 GROWTH PROMOTING AND STRESS TOLERANCE PROPERTIES OF WHEAT ENDOPHYTES 8.4 ANTIDIABETIC AND ANTIMICROBIAL POTENTIALS OF WHEAT ENDOPHYTEST （Paenibacillus rhizophilus）'> 8.5 Characterization of the Isolated Novel Endophytic Bacterium 7197T （Paenibacillus rhizophilus）CONCLUSIONREFERENCESAPPENDIXACKNOWLEDGEMENTSRESUME
文章来源
类型: 博士论文
作者: Farhana Alam Ripa
导师: 曹卫东
关键词: 小麦内生菌,固氮菌,生物多样性,促进植物生长
来源: 中国农业科学院
年度: 2019
分类: 基础科学,农业科技
专业: 生物学,农业基础科学,农作物
单位: 中国农业科学院
分类号: S512.1;S182
总页数: 209
文件大小: 6229K
下载量: 306
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标签：小麦内生菌论文; 固氮菌论文; 生物多样性论文; 促进植物生长论文;
