【作者】 吕成群；

【导师】 叶建仁； 武波；

【作者基本信息】 南京林业大学 ， 森林保护学， 2004， 博士

【摘要】 从广西不同立地条件下的7种相思树木林分中采集根瘤并分离纯化获得53个相思树种根瘤菌菌株，对其进行了较为全面、系统的研究。研究表明它们在平板上培养的菌落均具有典型的根瘤菌形态特征。在试管内和盆栽苗木上进行回接和互接种，所有菌株均能有效诱导结瘤。取接种后的苗木根瘤测定表明，它们都具有固氮酶活性。在利用柠檬酸盐、3-酮基乳糖、牛肉膏蛋白胨、淀粉水解、水解明胶、氮源和碳源等方面，它们均具有典型的根瘤菌生理生化特征。在BTB反应中有的菌株产酸，有的产碱。 相思树种根瘤菌具有广泛的生长适应性。在供试的53个菌株中，有许多菌株在9～39℃之间都能生长。一些菌株对酸碱条件的适应范围较广（pH值4.0～9.0），而有的菌株能适应较酸（pH值4.0）或较碱（pH值9.0）的生长环境。一些菌株在NaCl盐浓度0.2～2.0％条件下仍生长良好。有2个菌株对供试抗菌素均无抗性，4个菌株对所有供试抗菌素都具有抗性，其余菌株对抗菌素的抗性存在着明显程度上的差别。 分离、纯化的53株相思树种根瘤菌株，经代时测定和在平板上菌落形成天数测定，首次从相思根瘤菌中分出快生菌和慢生菌两种类型。其中有9个慢生菌株（代时6.2h以上），生长代时在10h以上的有4株。这些慢生根瘤菌与许多快生相思根瘤菌株那样具有良好的耐酸碱、温度和盐碱逆境的能力。 用分离纯化获得的53个相思树种根瘤菌株接种试管苗，用33个菌株接种盆栽苗，结果表明，接种后的试管苗平均结瘤率为53.5％、平均结瘤数2.4个、平均单株瘤重量为6.4mg、平均单个根瘤重量2.7mg、平均固氮酶活性2126n mol C₂H₄·g^-1瘤重·h^-1。不接种根瘤菌的试管苗均无结瘤。接种后的盆栽苗木与对照相比，平均结瘤率提高6.2％、单株结瘤数提高142.1％、单株瘤重提高113.9％、单个根瘤重增加48.6％、苗高生长量提高27.4％、地径生长量提高14.4％、单株总生物量提高26.5％、固氮酶活性提高40.5％、叶片含氮量增加20.8％、单株总固氮量增加51.4％。 用5株不同的相思根瘤菌菌株接种到厚荚相思苗木后的造林结果显示，ZG04菌株的综合效应最好，其树高生长量增长15.5％、地径生长量增长7.5％、叶绿素含量增加10.3％，这三项指标均居各菌株之首。 首次通过分子生物学方法对豆科树种根瘤菌的16S rDNA全序列进行测定和分析，相思树种根瘤菌株HJ06的16S rDNA序列分别与Rhizobium tropici、Rhizobium rhizogenes、Rhizobium genosp和Rhizobium leguminosarum16S rDNA序列的同源性都在98％。因此，菌株HJ06在分类上应属Rhizobium属。 首次从豆科树种根瘤菌中用PCR方法扩增出nifA基因片段，确定了相思树种根瘤菌株HJ06和ZG04的nifA基因片段DNA序列分别与Klebsiella pneumoniae的同源性达到99％；与Klebsiella oxytoca基因的NifF protein，NifL protein，NifA protein和NifB protein的同源性为99％。更多还原

【Abstract】 In this study , root nodules were selected from 7 kinds of Acacia spp. forests in various site conditions and 53 strains rhizobia were isolated and purified, and then rather complete and systematic research . The colony have typical rhizobia colony features on plate cultivation. When inoculating or cross-inoculating in test tubes and potted seedlings ,the nodules could appeare in its host plant and had nitrogenase activity . There were positive reaction about use of citrate, and negative reaction about use of 3-ketolactose, beef extract peptone, hydrolysis of starch in all test strains and liquefaction of gelatin in greater part of strains. (NH4)2HPO4 was used in all test strains and KNO3 was used in most of them as nitrogen sources. Both five monosaccharides and three disaccharides could be utilized as carbon sources except HM11 which is unable to use lactose . Some strains generate acids and other generate alkali in BTB tests and so litmus milk reactions.Acacia spp. rhizobia have wider rang of adaption for growth environment. Among the 53 strains for the tests, some strains could grow between 9~39癈. Some strains could grow on pH 4.0 -9.0 cultrue medium, while some nodule bacteria of the Acacia spp. can adapt slightly sour (pH 4.0) or alkaline (pH 9.0) growth environment conditions. Some strains could grow quite well in NaCl solution concentration 0.2% to 2.0%. 2 strains having no resistance to all the antibiotics used in the tests and 4 strains having resistance to all the antibiotics, other nodule bacteria of different Acacia spp. have obvious differences in resistance to antibiotics .After generation time determination and tests of colony formation days on plate for the 53 separated and purified strains of nodule bacteria from Acacia spp. species, fast-growers and slow-growers rhizobia came for the first time from nodule bacteria of the Acacia spp, 9 belonging to slow-growers rhizobia (generation time above 6.2 h) including 4 with growth generation time beyond 10 h. The results showed that the slow-growers rhizobia isolated from Acacia spp. with superior tolerance to stresses of acid, alkali, temperature and NaCl as many fast-growers rhizobia.The results of Acacia spp. seedlings inoculated or cross-inoculated with 53 strains Acacia spp. rhizobia in test tubes showed that average nodulation rate was 53.5%, nodulation amounts of single seedling was 2.4 , nodule weight of single seedling was 6.4mg , single nodule weight was 2.7mg , nitrogenase activity was 2126 nmol C2H4 g-1 h-1. The nodule could not appeare in its host seedlings without rhizobia in test tubes.When potted Acacia seedlings were inoculated with 33 strains of rhizobia separted and purified from the nodules of different Acacia spp, the average nodulation rate, nodulation amounts of single seedling, nodule weight of single seedling ,single nodule weight, seedling height growth, basal-diameter growth, total biomass of single seedling, nitrogenase activity , nitrogen contents in the leaves and total nitrogen fixationof single seedling separately increased by 6.2%, 142.1%, 113.9% , 48.6%,27.4%, 14.4%, 26.5%,40.5% ,20.8% and 51.4% over the control group .The results of afforestation by inoculation with nodule bacteria has indicated that ZG04 has best integrated effects, with tree height growth increasing by 15.5%, basal-diameter growth by 7.5% and chlorophyll contents by 10.3% ranking first among all the stains. After 5 strains of nodule bacteria of different Acacia spp. species were inoculated into Acacia crassccarp seedlings.For the first time, determination and analysis were made on 16S rDNA sequencing of nodule bacteria of bean trees by method of molecular biology. The results of determination and analysis on 16S rDNA sequencing of HJ06 showed that it has 98% isogenesis in nucleotide levels with Rhizobium tropici Rhizobium rhizogeness Rhizobium genosp and Rhizobium leguminosarum\6S rDNA, thus it with these species should belong to the same genus.For the first time, nifA gene segments was generated from nodule bacteria of bean trees species by PCR metho更多还原