湖北潜江灰潮土中费氏中华根瘤菌(Sinorhizobium fredii)多样性的研究

用5种代表性大豆为捕集植物从湖北潜江灰潮土中经盆栽试验分离筛选出50株费氏中华根瘤菌,对它们的生长速度、耐盐性、pH生长范围、天然抗药性、碳氮源利用和共生效应等同时进行了比较研究,证实了同一土壤环境中费氏中华根瘤菌群体的多样性。     

新疆土著大豆根瘤菌的表型多样性

选用分离自新疆昌吉市郊土壤的大豆根瘤菌61株和参比菌5株,对它们进行唯一碳氮源、抗生素抗性和抗逆性等表型性状分析,结果表明所有菌株在70.1％相似水平上分为快生大豆根瘤菌和慢生大豆根瘤菌2群,其中快生大豆根瘤菌在81.4％相似水平上又分为2个亚群,40株供试的新疆快生大豆根瘤菌与新疆中华根瘤菌聚为一群;7株供试菌聚为一小群,抗逆性强。所有供试快生菌株都与费氏中华根瘤菌相似性低,所以新疆快生大豆根瘤菌可能是与费氏中华根瘤菌相独立的一个种。     

pH对土壤中土著快、慢生大豆根瘤菌结瘤的影响

1　引　　言土壤 ｐＨ对根瘤菌结瘤的影响一直是微生物学和微生物生态学研究的内容之一[4] .在对大豆根瘤菌的研究中 ,早期的研究主要集中于生长慢、产碱的大豆慢生根瘤菌 (Ｂｒａｄｙｒｈｉｚｏｂｉｕｍｊａｐｏｎｉｃｕｍ) [1,2 ] .1982年 ,Ｋｅｙｓｅｒ等[3] 报道了一类生长快、产酸的大豆根瘤菌 ,并命名为费氏中华根瘤菌 (Ｓｉｎｏｒｈｚｏｂｉｕｍ ｆｒｅｄｉ ｉ) .由于它们在生理特性方面存在着明显的差异 ,其结瘤能力以及环境的生物、物理和化学等因素对结瘤的影响一直受到广泛的重视 .本文研究了偏酸、偏碱的 ｐＨ对费氏中华根瘤菌…     

费氏中华根瘤菌共生质粒扩增对结瘤因子组分和共生固氮能力的影响

费氏中华根瘤菌(Sinorhizobium fredii)YC4能在大豆(Glycine max)和野大豆(G．soja)上形成正常固氮的根瘤．人工培养条件下用^14C标记的薄层层析(TLC)法检测根瘤菌产生的结瘤因子(LCOs)的结果表明,与其它4株费氏中华根瘤菌相比,YC4产生的LCOs含有较多的疏水性基团．从YC4菌株中分离到1株共生质粒发生了扩增的自发突变株YSC3,其产生的LCOs中含有较野生型菌株多的1个疏水性组分,28℃培养条件下产生的LCOs量亦较YC4显著增加．结瘤试验结果表明,YSC3菌株只能在大豆和野大豆上形成无效的根瘤．     

一株能在苜蓿上结瘤的费氏中华根瘤菌

费氏中华根瘤菌 (Sinorhizobiumfredii) 0 4 2BS分离自新疆的苜蓿根瘤 ,通过交叉结瘤试验 ,发现它既可在苜蓿上又可在大豆上结瘤固氮。 1 6SrDNAPCR RFLP分析表明 ,0 4 2BS与费氏中华根瘤菌模式菌株USDA2 0 5的 4种限制性酶切图谱完全一致。其G +Cmol%为 60 0 ,与费氏中华根瘤菌USDA2 0 5和USDA1 91的DNA同源性分别为 84 9%和89 6% ,表明 0 4 2BS属于费氏中华根瘤菌。应用绿色荧光蛋白基因标记 0 4 2BS ,得到重组菌株 0 4 2BSG。将其接种保定苜蓿和北引 1号大豆 ,并重新分离出根瘤菌 ,利用激光共聚焦荧光显微镜检测到标记基因的表达 ,从而确证了 0 4 2BS能在苜蓿和大豆上结瘤。而且 ,0 4 2BS对不同苜蓿品种的结瘤能力不同     

鲁黄1号大豆与根瘤菌的共生匹配性

大豆可与中华根瘤菌属及慢生根瘤菌属的多种根瘤菌共生固氮.研究大豆品种与不同种根瘤菌之间的共生匹配性,对获得高效根瘤菌用于接种,提高大豆的产量及品质有重要的理论和实践意义.本研究使用黄淮海地区的优质高蛋白大豆品种鲁黄1号从当地土壤内捕捉并分离纯化到27株根瘤菌.经持家基因recA的序列分析,发现其中18株属于中华根瘤菌属,9株属于慢生根瘤菌属.选用两个属的代表菌株各一株(Sinorhizobium fredii S6和Bradyrhizobium sp. S10),分别在蛭石、土壤盆栽及大田试验条件下,研究这两株菌单独及混合接种对鲁黄1号大豆的生长、结瘤、固氮活力、产量、种子蛋白含量及含油量的影响.结果表明： 与S10菌株相比,S6菌株对大豆的促生能力更强,对提高产量和品质的效果更好,从而确定S6为与鲁黄1号大豆相匹配的高效根瘤菌,可作为黄淮海地区推广种植鲁黄1号大豆时接种高效根瘤菌的菌种资源.
     

根瘤菌可溶性蛋白和酯酶的电泳图谱分析

我们分析行了二十株根瘤菌可溶性蛋白和酯酶的电泳图谱。菌株中14株分离自野大豆的快生型大豆根瘤菌和3株慢生型大豆根瘤菌;另外3株根瘤菌分别分离于苜蓿、豌豆和三叶草。根据它们电泳谱带的Rf值,计算了各根瘤菌可溶性蛋白图谱间的相似性系数。快生型大豆根瘤菌的电泳图谱均不同于慢生型大豆根瘤菌和其他根瘤菌。鉴于各菌株有其独特图谱,以此作为根瘤菌分类和鉴定的依据,是较可靠的方法。     

黄土高原地区大豆根瘤菌的遗传多样性和系统发育

【目的】研究黄土高原地区大豆根瘤菌的遗传多样性和系统发育。【方法】采用BOX-PCR、16S rDNAPCR-RFLP、16S-23S IGS PCR-RFLP和16S rRNA基因序列分析方法对分离自我国黄土高原地区4个省的15个地区的130株大豆根瘤菌及部分参比菌株进行了遗传多样性和系统发育分析。【结果】BOX-PCR反映的菌株多样性最丰富,形成的遗传群最多,16S rDNA PCR-RFLP方法在属、种水平上聚群较好,16S-23S IGSPCR RFLP反映的多样性介于BOX-PCR和16S rDNA PCR-RFLP之间,能够较好地反映出属、种和亲缘关系很近的菌株间的差异,3种方法聚类分析结果基本一致,可将所有供试菌株分为两大类群,中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium)。从系统发育来看,供试的快生大豆根瘤菌为费氏中华根瘤菌(Sinorhizobium fredii),慢生大豆根瘤菌为日本慢生大豆根瘤菌(Bradyrhizobium japonicum)和辽宁慢生根瘤菌(Bradyrhizobium liaoningense)。【结论】我国黄土高原地区大豆根瘤菌具有较丰富的遗传多样性,S.fredii优势种,慢生大豆根瘤菌仅占10%,同时,分离到2株B.liaoningense。     

费氏中华根瘤菌腺嘌呤缺陷突变株的构建与筛选

构建出费氏中华根瘤菌嘌呤合成酶基因purL的基因置换载体pHN701,purL内部NotⅠXhoⅠ片段被luxAB基因取代,造成正常基因的破坏。用该载体对野生型费氏中华根瘤菌HH103进行purL的基因置换,筛选到腺嘌呤缺陷型突变株P825。波动实验和连续转接试验结果表明该突变菌株表型十分稳定。purL表达载体pBBRPG在P825中可恢复其在基本培养基上的生长情况,证明突变株确实为purL单基因破坏。盆栽结瘤实验结果表明,该突变株只能侵染大豆根系形成不固氮的根瘤。     

侵染3株模式豆科根瘤菌的噬菌体生物学特性

豆科作物根瘤菌被噬菌体浸染后,在一定程度上会引起根瘤菌数目和结瘤量的降低,进而导致共生固氮作用弱化和作物产量的显著下降.然而,目前关于根瘤菌噬菌体的相关研究报道较少.本研究以3株模式根瘤菌,即慢生型大豆根瘤菌、中华大豆根瘤菌和中华苜蓿根瘤菌为宿主,于黑土农田土壤中采用双层平板培养法从每个宿主细菌分离3株噬菌体,共分离获得9株根瘤菌噬菌体,对其形态结构及生物学特征进行综合分析.结果表明:侵染苜蓿根瘤菌噬菌体(SMM)和慢生型根瘤菌噬菌体(BDM)属于肌尾噬菌体科,而侵染中华根瘤菌噬菌体(SSS)隶属于长尾噬菌体科.9株噬菌体的最佳感染复数均在0.001~1.0的变化范围内.一步生长曲线结果显示,BDM的潜伏期和爆发期明显长于SMM和SSS,但获得的裂解量最小.根瘤菌噬菌体在30~40℃和中性pH条件下侵染活性最大.对比发现,侵染同一宿主的噬菌体生物学特征虽存在一定差异,但分异度远小于不同宿主噬菌体间的差异.     

采用PCR-RFLP技术在不同水平上鉴定大豆根瘤菌

采用16S rRNA基因PCR扩增与限制性酶切片段多态性分析(RFLP)技术对选自弗氏中华根瘤菌(S.fredii)、大豆慢生根瘤菌(B.japonicum)和埃氏慢生根瘤菌(B.elkanii)的19株代表菌进行了比较分析,根据用3种限制性内切酶的RFLP分析结果,可将供试菌株分为S.fredii,B.japonicum, B.elkanii Ⅱ和B.elkanii Ⅱa等4种基因型。各类菌株之间没有交叉,因此本研究采用的PCR-RFLP技术不失为一种快速鉴别大豆根瘤菌的新方法。采用本技术已将分离自中国的22株快生菌和19株慢生菌分别鉴定为S.fredii和B.japonicum。对供试参比菌株和野生型菌株进行的16S～23S基因间隔DNA(IGS)的PCR-RFLP分析结果表明：S.fredii和B.japonicum菌株的IGS长度不同,所有供试S.fredii菌株的IGS为2.1 kb,而供试B.japonicum菌株则为2.0 kb。依据RFLP的差异,可将来自中国两个不同地区的S.fredii株区分为2个基因型,而来自中国东北黑龙江地区的19株B.japonicum菌株则可分为11个基因型。对上述野生型菌株还进行了REP-PCR和ERIC-PCR分析并确定其具有菌株水平的特异性。     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.     

16S rDNA-RFLP分析新疆快生大豆根瘤菌的分类地位

采用１６Ｓ ｒＤＮＡ－ＲＦＬＰ技术,对自新疆土壤中捕捉的３４株快生大豆根瘤菌及相关已知种的模式菌株进行了比较分析。从酶切图谱类型和在结果表明,所有新分离的菌株与Ｓ．ｘｉｎｊｉａｎｇｅｎｓｉｓ的图谱类型基本一致,而与Ｓ．ｆｒｅｄｉｉ的图谱类型有明显差异,与Ｓ．ｍｅｌｉｌｏｔｉ,Ｓ．ｓａｈｅｌｉ,Ｓ．ｍｅｄｉｃａｅ,Ｓ．ｔｅｒａｎｇａ也不相同。３４株新分离的菌株全部与Ｓ．ｘｉｎｇｊｉａｎｇｅｎｓｉｓｉ     

Structural analysis of the capsular polysaccharide from Sinorhizobium fredii HWG35

We have determined the structure of a capsular polysaccharide from Sinorhizobium fredii HWG35. This polysaccharide was isolated following the standard protocols applied for lipopolysaccharide isolation. On the basis of monosaccharide analysis, methylation analysis, mass spectrometric analysis, one-dimensional (1)H and (13)C NMR, and two-dimensional NMR experiments, the structure was shown to consist of a polymer having the following disaccharide repeating unit: -->6)-2,4-di-O-methyl-alpha-d-Galp-(1-->4)-beta-d-GlcpA-(1-->. Strain HWG35 produces a capsular polysaccharide that does not show the structural motif (sugar-Kdx) observed in those S. fredii strains that, while effective with Asiatic soybean cultivars, are unable to form nitrogen-fixing nodules with American soybean cultivars. Instead, the structure of the capsular polysaccharide of S. fredii HWG35 is in line with those produced by strains HH303 (rhamnose and galacturonic acid) and B33 (4-O-methylglucose-3-O-methylglucuronic acid), two S. fredii strains that form nitrogen-fixing nodules with both groups of soybean cultivars. Hence, in these three strains that effectively nodulate American soybean cultivars, the repeating unit of the capsular polysaccharide is composed of two hexoses, one neutral (methylgalactose, rhamnose, or methylglucose) and the other acidic (glucuronic, galacturonic, or methylglucuronic acid).     

Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean.

It has been postulated that nodulation outer proteins (Nops) avoid effective nodulation of Sinorhizobium fredii USDA257 to nodulate with American soybeans. S. fredii HH103 naturally nodulates with both Asiatic (non-commercial) and American (commercial) soybeans. Inactivation of the S. fredii HH103 gene rhcJ, which belongs to the tts (type III secretion) cluster, abolished Nop secretion and decreased its symbiotic capacity with the two varieties of soybeans. S. fredii strains HH103 and USDA257, that only nodulates with Asian soybeans, showed different SDS-PAGE Nop profiles, indicating that these strains secrete different sets of Nops. In coinoculation experiments, the presence of strain USDA257 provoked a clear reduction of the nodulation ability of strain HH103 with the American soybean cultivar Williams. These results suggest that S. fredii Nops can act as either detrimental or beneficial symbiotic factors in a strain-cultivar-dependent manner. Differences in the flavonoid-mediated expression of rhcJ with respect to nodA were also detected. In addition, one of the Nops secreted by strain HH103 was identified as NopA.