慢生型大豆根瘤菌putA基因的功能分析

重组质粒pGX300上的putA基因是从慢生型大豆根瘤菌GX201中克隆到的编码脯氨酸脱氢酶的基因。通过Tn5gusA5定位诱变方法构建了putA基因的突变株GX20120，实验表明，GX20120的结瘤时间推迟，竞争结瘤能力降低。     

慢生型大豆根瘤菌菌株GX201的脯氨酸脱氢酶基因(putA)的分子克隆

根据已报道的脯氨酸脱氢酶基因（ｐｕｔＡ）序列,通过ＰＣＲ方法,从慢生型大豆根瘤菌菌株ＧＸ２０１的总ＤＮＡ中扩增到—ＰＣＲ产物。序列分析表明,该ＰＣＲ产物的长度为４１８ｂｐ,与已报道的ｐｕｔＡ基因具有９３．５％的同源性。以广谱寄主范围质粒ｐＬＡＦＲ３为载体,在大肠杆菌ＤＨ５α中构建了ＧＸ２０１的基因文库,并以该ＰＣＲ产物为探针,从基因文库中筛选到一重组质粒ｐＧＸＮ３００。     

慢生型大豆根瘤菌nfeC基因的定位

在前期工作中 ,从慢生型大豆根瘤菌菌株 GX2 0 1的基因文库中筛选到一个含 nfe C基因同源片段的重组质粒 p GXN2 0 1。在本工作中 ,将 nfe C基因同源片段定位在 4 kb Cla I Xba I片段上。对该片段的部分序列分析表明与已报道的 nfe C基因具有 95%的同源性     

根瘤菌结瘤基因与竞争结瘤基因的研究进展

根瘤菌的结瘤基因与竞争结瘤基因在根瘤菌与结瘤的过程中起着关键的作用。随着研究的深入,已经发现,分离和定位了数十个结瘤基因和几个与竞争结瘤有关的基因,本文将简要地介绍了这方面的研究成果。     

GUS基因标记法对慢生花生根瘤菌竞争结瘤和接种效果的研究

应用GUS基因标记技术,可简便、快速、准确、原位、直观地确定标记花生根瘤菌株形成的根瘤,从而方便地研究标记菌株与土著根瘤菌的竞争结瘤能力。无氮水培试验表明,标记菌株gusA4-5、gusA2-9分别与土著菌混和接种占瘤率为71.4%、77.0%。盆栽试验表明,接种供试菌株Spr4-5、Spr2-9占瘤率分别为57.9%、63.0%,比对照极显著增产52.5%、22.7%;接种Spr4-5比Spr2-9极显著增产24.2%。初步说明两个供试菌株的竞争结瘤力比土著根瘤菌强,菌株Spr2-9强于Spr4-5;Spr4-5比Spr2-9有效性高,是结瘤适量,竞争结瘤能力强的高效菌株。     

安徽省北部大豆根瘤菌的分离,血清学分析及田间自然结瘤调查

对安徽省北部分离的２７株快生大豆根瘤菌和３８株慢生大豆根瘤菌的血清学分析及田间自然结瘤的血清学调查表明：００５血清型系安徽北部夏大豆产区占据优势的血清型，１８组样品和再现频率为１００％；３６株分离物中２６株属００５血清型，占总分离物的７２．５％。２０４８血清型出现频率为８３．３％；在３组样品中占瘤率达４１－５１。６％。２１７血清型占瘤率普遍低于１６％。讨论了自然结瘤调查中采样地点，大豆品种和土壤类     

根瘤菌结瘤基因的表达调控研究概况

根瘤菌结瘤基因的表达调控在根瘤菌与植物的共生结瘤过程中起着十分重要的作用。随着研究的深入,发现根瘤菌的结瘤过程不仅与根瘤菌结瘤基因表达调控有关,而且与寄主植物的信号分子如黄酮类物质有关。根瘤菌结瘤基因的表达调控有一个复杂的过程,本文将简要地介绍这方面的研究成果。     

快,慢型大豆根瘤菌质粒接合重组的初步研究

通过含有Ｒ６８．４５质粒的大肠杆菌将快生型大豆根瘤ＡＣＣＣ１５０６７菌株的大质粒转到慢生型大豆根瘤菌６１Ａ７６菌株中去，共接合转移出２２个接合子菌株，对其中的１２个菌株进行了与８３－１１９大豆的共生接种试验，其效果是部分接合子菌株超过供体菌和受体菌对接合子ＢＦ２－Ａ１菌株进行了质粒检测。田间应用亦有增产趋势。     

山西省快生型大豆根瘤菌资源调查和鉴定

从山西省主要大豆产区的不同土壤和大豆品种中分离得到的38个快生型大豆根瘸菌株的鉴定表明,这些分离物的IAR除了氨苄青霉素外,均较慢生型为低。38个菌株被分为4个血清型,其中2个为新发现的,命名为2077和2120型。细胞成分N%含量为2.01-3.78,C%含量为50.52-55.53%,N/C值<10。所检测的7个菌株都有1-2个大质粒,且每个均有112 Md的大质粒。分离株的共生效应和结瘤竞争由于大豆品种不同而有显著差异。本研究表明,我国大豆起源地之一的山西省,快生型大豆根瘸菌的分布广泛,分离频率较高,菌株类型也多。     

Inhibition of growth of Bradyrhizobium japonicum bacteroid by spermidine and spermine in yeast extract

Abstract

Bacteroids are defined as the symbiotic forms of Rhizobium or Bradyrhizobium cells in the root nodules of their legume host. The differentiation to bacteroids involves various physiological changes and may be associated with some genetical changes. Single-colony isolates from a nodule formed by a Bradyrhizobium strain often differed in their effectiveness and intrinsic antibiotic resistance (Weaver and Wright 1987; Ozawa unpublished data). Detailed analysis of the changes in the gene structure requires the isolation of individual bacteroids from a nodule. However it has been reported that the viability of bacteroids in culture is very low and only a small fraction of a bacteroid population could produce colonies on yeast extract-mannitol (YEM) agar (Sutton et al. 1977). Bergersen (1974) concluded that the reversion of bacteroids to the vegetative, growing form is very rare.     

丛枝菌根真菌与根瘤菌接种对大豆根瘤分布及磷素吸收的影响

在温室条件下,采用石英砂盆栽试验研究了大豆(冀豆6号)接种丛枝菌根真菌（Glomous mosseae）与根瘤菌 （Bradyrhizobium japonicum）对根瘤的形成、分布以及磷素吸收效率的影响。结果表明,大豆生长至开花期（接种后56 d）,与单接种根瘤菌处理相比,双接种AM真菌和根瘤菌显著增加大豆生物量、氮、磷含量、根系上的总根瘤数。单接种根瘤菌条件下,总根瘤数的48.4%分布在主根上,51.6%分布在侧根上;根瘤菌与AM真菌双接种时,总根瘤数的32.5%分布在主根上,67.5%分布在侧根上。双接种处理的侧根根瘤的固氮酶活性显著高于单接种处理的。双接种条件下大豆侧根中AMF侵染增强,尤其是结根瘤侧根上的AM真菌的侵染率高于未结瘤的侧根的菌根侵染率。接种后28 d单接种菌根真菌处理显著高于双接种处理的植株磷的吸收效率;而56 d 时趋势相反。以上结果表明,AM真菌侵染改变根瘤在大豆根系上的分布,根瘤数量、分布与结根瘤侧根上AM真菌的侵染强度存在正相关关系。     

Soybean preference for Bradyrhizobium japonicum for nodulation

For examining the probability of increase in the occupation ratio of inoculated rhizobium in nodules, various Rj-soybean cultivars including the Rj ₂ Rj ₃ Rj ₄-lines of soybean were grown in a field of the Kyushu University Farm. Bradyrhizobium japonicum USDA110 that carries uptake hydrogenase (Hup⁺) was used as an inoculum. The relative efficiency of nitrogen fixation generally increased by the inoculation. However, there were no significant differences in the effects among the genotypes of the host plants. The occupation ratio of serogroup USDA110 in the nodules on the taproot of the inoculated plants was in the range of 77–100%, suggesting that the B. japonicum strain USDA110 infected taproots immediately after inoculation. The occupation ratios in the nodules on the lateral roots were 53–67, 40–86, 63–83, and 62–77% in inoculated plants of the non-Rj-, Rj ₂ Rj ₃-, Rj ₄-, and Rj ₂ Rj ₃ Rj ₄-genotypes, respectively, and they decreased in all the genotypes with the progression of growth. At the time of the first sampling, the occupation ratios on the lateral roots of these Rj ₂ Rj ₃ Rj ₄-genotypes showed values intermediate between those of IAC-2 (Rj ₂ Rj ₃) and Hill (Rj ₄) , which were the parent cultivars of the Rj ₂ Rj ₃ Rj ₄-lines, B340, B349, and C242. The reduction in the occupation ratio of the serogroup USDA110 for about 1 month after the first sampling was the lowest (0.13–0.16) in the Rj ₂ Rj ₃ Rj ₄-genotypes, excluding B349, followed by the non-Rj- and Rj ₂ Rj ₃-genotypes and highest (0.52–0.69) in the Rj ₄-genotypes, excluding Hill. Therefore, it was considered that the population of compatible rhizobia with host soybean plants increased in the rhizosphere with the progression of the development and growth. The results showed that with the expansion of the root area of host plants, the occupation ratio of type A rhizobia including the serogroup USDA110 was high. Therefore, the Rj ₂ Rj ₃ Rj ₄-genotypes were superior to other Rj-genotypes in terms of the inoculation effects of nodulation type A rhizobium, B. japonicum USDA110. However, the preference of the Rj ₂ Rj ₃ Rj ₄-genotype for serogroup USDA110 is not sufficient to rule out the competition with the other serogroups in this study. Therefore, the study should be centered on the isolation of more efficient (Hup⁺) and highly compatible rhizobial strains with the Rj ₂ Rj ₃ Rj ₄- genotypes.     

Soybean preference for Bradyrhizobium japonicum for nodulation

For the increase of the occupation ratio of inoculum strain in the competition with indigenous rhizobia, the relationship between Rj-genotypes of soybean and the preference of Rj-cultivars for various types of rhizobia for nodulation was investigated by using the Rj ₂ Rj ₄-genotype of soybean isolated from the cross between the Rj ₂ Rj ₃-cultivar IAC-2 and Rj ₄-one Hill (Ishizuka et al. 1993: Soil Sci. Plant Nutr., 39, 79-86). Firstly, these Rj ₂ Rj ₄-genotypes were found to harbor the Rj ₃-gene. The Rj ₂Rj₃Rj₄-genotypes of soybean were considered to exhibit a more narrow microsymbiont range for nodulation than the Rj ₂ Rj ₃-and Rj₄-cultivars. Therefore, rhizobia were isolated from the nodules of various Rj-genotypes of soybeans grown in soils, and the preference of the Rj ₂ Rj ₃ Rj ₄-genotype for indigenous rhizobia was examined. The nodule occupancy of serotype 110 was significantly higher in the bacteroids of the nodules from the Rj ₂ Rj ₃ Rj ₄-rgenotypes than in those from the other genotypes, non Rj-, Rj ₂ Rj _3-, and Rj ₄-cultivars. These results demonstrated that the Rj ₂ Rj ³ Rj ⁴-genotype prefers more actively serogroup USDA110 to the others of rhizobia. Thus, Rj ₂ Rj ₃ Rj ₄-genotype is superior to non- Rj-, Rj ₂ Rj ₃-, and Rj ₄-genotypes for the formation of efficient nodules for nitrogen fixation.     

Hydrogenase Activity of Soybean Nodules Doubly Infected with Bradyrhizobium japonicum and B. elkanii

Rhizobitoxine (2-amino-4-(2-amino-3-hydropropoxy)-trans-but-3-erioic acid) is a phytotoxin produced by some strains of Bradyrhizobium species. Rhizobitoxine-producing strains often induce chlorosis in new leaves of soybean as a result of the synthesis of the toxin in nodules (Owens and Wright 1964; Owens et al. 1972). Some of the B. japonicum bacteroids possessing the hydrogen uptake (Hup) system are capable of ATP production by recycling H₂ evolved from nitrogenase (Evans et al. 1987). Adequate uptake hydrogenase activity in soybean bacteroids often enhances plant growth, as well as the efficiency of energy utilization during nitrogen fixation (Evans et al. 1987).     

Soil aggregate as a favorable habitat for Bradyrhizobium japonicum strains

Abstract

Rhizobial cells are present in soils as saprophytes after the decay of host plant nodules, and must survive in the soil until the next encounter with the infection sites of the host plant root. Biotic and abiotic environmental factors affect the population size of these rhizobia in the soil (Vincent 1977). Precise estimation of the population size of the native and the introduced rhizobia in the soil is necessary to study the conditions for the successful nodule formation by introduced strains.     

Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elkanii strains

 In a previous study soybean Bradyrhizobium strains, used in Brazilian studies and inoculants over the last 30 years, and strains adapted to the Brazilian Cerrados, a region frequently submitted to environmental and nutritional stresses, were analyzed for 32 morphological and physiological parameters in vivo and in vitro. A cluster analysis allowed the subdivision of these strains into species Bradyrhizobium japonicum, Bradyrhizobium elkanii and a mixed genotype. In this study, the bacteria were analyzed for nodulation, N₂ fixation capacity, nodule occupancy and the ability to increase yield. The goal was to find a relationship between the strain groups and the symbiotic performance. Two strains of Brazilian B. japonicum showed higher rates of N₂ fixation and nodule efficiency (mg of N mg^–1 of nodules) under axenic conditions. These strains also showed greater yield increases in field experiments when compared to B. elkanii strains. However, no differences were detected between B. japonicum and B. elkanii strains when comparing nodule occupancy capacity. The adapted strains belonging to the serogroup B. elkanii SEMIA 566, most clustered in a mixed genotype, were more competitive than the parental strain, and some showed a higher capacity of N₂ fixation. Some of the adapted strains, such as S-370 and S-372, have shown similar N₂ fixation rates and nodulation competitiveness to two Brazilian strains of B. japonicum. This similarity demonstrates the possibility of enhancing N₂ fixing ability, after local adaptation, even within B. elkanii species. Differences in the DNA profiles were also detected between the parental SEMIA 566 and the adapted strains by analyses with the ERIC and REP-PCR techniques. Consequently, genetic, morphological and physiological changes can be a result of adaptation of rhizobia to the soil. This variability can be used to select strains capable of increasing the contribution of N₂ fixation to soybean nutrition. Received: 28 May 1997     

Nodulation and competitiveness of gusA-marked Bradyrhizobium japonicum A1017 in soybean

Attempts to improve the symbiotic nitrogen fixation with effective (Brady) rhizobium strains do not always succeed under field conditions due to the lower nodulation competitiveness of the introduced strains than that of the indigenous rhizobia (Triplett and Sadowsky 1992). An introduced strain needs to be marked for monitoring its nodule occupancy under competitive nodulation conditions.