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。     

抗药性根瘤菌基因工程菌的构建与应用

用三亲本杂交的方法将外源抗药性质粒导入到高效固氮的根瘤菌 Tal377中,外源质粒在 Tal377中能正常表达,但不影响其原有的固氮结瘤能力。利用此抗药性根瘤菌所携带的抗药性,在接种此根瘤菌的同时结合施加一定浓度的抗菌素,抗药性根瘤菌与出发菌株相比表现出一定程度地提高其结瘤固氮能力和占瘤率。     

土壤中大豆根瘤菌优势类群以及与寄主共生关系的多样性

我国大豆种植区使用人工选育制成的根瘤菌剂接种效果不稳定。研究指出南方、北方和黄淮流域的6个省土壤中分布的主要Bradrhizobium.japonicum是DH444、USDA110、LL120、005和C224血清型的菌株,主要的Rhizobium.fredii是2048、USDA217、DE1611、2120和2077血清型的菌株。它们占每个取样地点分离物总数的60.3-81.6％。植物感染结瘤法测得大豆种植地点土著根瘤菌菌数在104/克土以上。盆栽试验表明大豆根瘤菌与大豆品种共生时有较强的亲和选择性和共生效应的多样性,其有效结瘤和固氮效率与根瘤菌个体菌株和大豆品种极相关。大多数土著大豆根瘤菌是低或中效固氮的菌株,因而认为选育抗土著根瘤菌而有利于人工接种菌株结瘤的大豆品种和强竞争性的高效菌株仍是提高我国大豆生产的有效途径。     

根瘤菌与促生菌双接种对大豆生长和土壤酶活的影响

【目的】慢生大豆根瘤菌和胶质类芽孢杆菌单一菌株固氮或促生效果及机理已有较多研究,但两者双接种对作物的作用和增产机理尚未有所报道。本研究以慢生大豆根瘤菌5136与胶质类芽孢杆菌3016为研究对象,通过田间小区试验研究根瘤菌与促生菌不同施用模式对大豆生长和土壤酶活的影响,以期为开发新型高效复合菌剂提供理论依据。【方法】试验设对照(T1)、接种胶质类芽孢杆菌3016菌剂(T2)、接种慢生大豆根瘤菌5136菌剂(T3),胶质类芽孢杆菌3016和慢生大豆根瘤菌5136双接种(T4)和常规施肥(T5)5个处理,分别于大豆不同生育期调查大豆的农艺性状和结瘤状况,测定土壤酶活性,用BOX-PCR技术监测慢生大豆根瘤菌5136的占瘤率。【结果】1)在大豆成熟期,双接种(T4)处理的大豆单株分枝数、单株粒数、收获指数和产量均为最高,分别比T1高11.3%、9.7%、41.0%和9.3%,且单株空荚数最低,比T1降低了44.0%。2)在花荚期,双接种(T4)处理的占瘤率为25.4%,比T3处理高8.0%,且单株根瘤数和单株根瘤干重均为最高,分别比T1高41.6%和47.1%;说明双接种处理下,胶质类芽孢杆菌3016能够促进慢生大豆根瘤菌5136结瘤固氮。3)接种微生物菌剂均可不同程度地提高土壤酶活性,以双接种(T4)处理的效果最为显著,在大豆成熟期,土壤过氧化氢酶、脲酶和蔗糖酶活性均为最高,分别比对照高12.9%、8.9%和9.4%。4)相关性分析表明,土壤酶活性与大豆收获指数显著正相关或极显著正相关(P0.01或P0.05),其中过氧化氢酶与产量显著正相关;单株根瘤数和单株根瘤干重均与收获指数和蔗糖酶活性呈极显著正相关,与产量呈显著正相关。【结论】慢生大豆根瘤菌和胶质类芽孢杆菌双接种可以促进大豆生长,显著增加大豆的单株分枝数、单株粒数、收获指数和占瘤率,降低单株空荚数,增加大豆产量,同时可显著提高相关土壤酶活性,是一种节本增效的农艺措施。     

接种根瘤菌对鸡骨草幼苗生长、结瘤和固氮能力的影响

用来自不同产地的鸡骨草根瘤菌接种盆栽幼苗,室内培养70 d后,测定植株生长和结瘤情况及植株含氮量。结果表明,接种根瘤菌对鸡骨草的株高、植株生物量、氮素相对积累量和固氮效率有明显的促进作用;不同来源的根瘤菌对鸡骨草生长和固氮的影响不同,来自玉林的根瘤菌菌株有较高的固氮效率。接种根瘤菌能促进鸡骨草生长,增加氮素累积,从而可以减少氮肥的使用,有利于充分发挥生物固氮的作用来提高原料药材的质量。     

新开垦土壤上构建玉米/蚕豆-根瘤菌高效固氮模式

为了在新开垦土壤上构建高效种植模式,本文采用温室盆栽和大田试验相结合的方法,选用4种根瘤菌接种方式(保水剂拌种、清水拌种、三叶期灌根和种子丸衣化)接种4种不同蚕豆根瘤菌(NM353、CCBAU、G254和QH258),分析接菌后新开垦土壤上玉米/蚕豆间作体系的生产潜力、地上部氮素吸收和结瘤特性以及生物固氮等方面的优势,拟为该体系筛选出高效的根瘤菌及其接种技术。结果表明:接种NM353后,玉米/蚕豆间作体系中蚕豆籽粒产量比单作平均增加152.84%,而玉米保持相对稳产;以保水剂拌种的方式接种NM353的间作蚕豆地上部氮素积累量最高,蚕豆结瘤数、瘤重、固氮比例和固氮量均高于本试验中其他3种方式接种的根瘤菌。在盛花期和盛花鼓粒期,接种NM353蚕豆的固氮比例比接种CCBAU的分别高19.1%和11.1%,在各个生育时期两者固氮量之间差异均达显著水平;接种NM353与接种其他菌种间固氮量和固氮比例差异更显著。因此,在新开垦土壤上,用保水剂拌种的方式对间作蚕豆接种NM353根瘤菌,构建玉米/蚕豆-根瘤菌高效固氮体系,为新开垦土壤合理开发利用的可持续发展模式。     

胡枝子根瘤菌优良菌种筛选及应用

介绍胡枝子根瘤菌的生理生化特性，共生固氮性能及连续三年的草场接种效果试验。人工接种根瘤菌可以使胡枝子早结瘤、多结瘤。是提高胡枝子共生固氮作用、增加产草量的有效措施。     

Mo与花生根瘤菌的复合菌剂对盛花期花生生长的影响

通过盆栽试验分析了"Mo+花生根瘤菌Spr2-9"复合菌剂对花生盛花期生长的影响.结果表明:(1)单接种Spr2-9(R1)能明显或显著增加植株干重、叶绿素和全氮量,分别比相应对照增加10.0%,14.6%,37.0%.(2)复合菌剂处理(R2,R3,R4)的占瘤率随钼浓度增大而增大,平均单瘤重随钼浓度增大显著降低,而总瘤数、全氮、叶绿素含量、植株干重随钼浓度的变化不显著.(3)钼能显著促进盛花期无菌处理(2,3,4)植株和根瘤的生长,平均单瘤重、叶绿素含量、植株干重、全氮量等随钼浓度增大显著增高,但对总根瘤数的影响不大.(4)等钼量的复合菌剂和无菌处理间,除无菌处理的单瘤重显著或明显高于复合菌剂外,其它都是复合菌剂处理高于无菌处理.表明供试根瘤菌是高效菌株,"Mo+供试根瘤菌"的复合菌剂对竞争结瘤、根瘤及植株生长有明显作用.     

马占相思根瘤菌结瘤固氮特性的分析

相思属（Acacia）树种是速生的热带、亚热带豆科植物。对现在大面积种植的相思树品种——马占相思的根瘤菌进行分离和筛选，获得菌株若干株，对它们的结瘤固氮特性和对马占相思、大叶相思及厚荚相思生长的影响等进行了初步研究。结果表明。无氮条件下接种马占相思根瘤菌，能显著促进这3个树种的株重、株高和生物量的增长；苗圃接种马占相思根瘤菌。马占相思苗高增加27．6％，地径增加14．8％，植株鲜重增加32．6％，结瘤数增加83．8％。     

芽孢杆菌与根瘤菌复合包衣对大豆结瘤固氮能力的影响

为了探究不同芽孢杆菌与根瘤菌复合包衣对大豆结瘤固氮的影响,通过盆栽试验,设置不接菌对照(CK)、根瘤菌单独包衣(R)、阿氏芽孢杆菌(RB)和胶质类芽孢杆菌(RP)2种促生菌分别与根瘤菌双包衣以及 3种菌混合包衣(RBP)共5个处理,测定大豆根瘤数量、干重、根瘤固氮酶活性及植株氮、磷、钾含量,筛选根瘤菌剂最佳组合。结果表明,接种菌剂的处理均能够促进大豆结瘤固氮,其中RB处理效果最好,能够获得最高的大豆植株干重、根瘤数量、根瘤干重、固氮酶活性、全氮含量,比CK分别提高51.80%、14.44%、53.33%、44.36%、25.61%,3种菌包衣并没有比双接种促生效果更好。相关性分析表明,根瘤干重与固氮酶活性、根瘤数量及植株干重都呈显著正相关关系,说明根瘤的质量可能比数量更能准确评价共生固氮效率。此结果为根瘤菌剂应用于机械化种植提供可能性,为进一步推广根瘤菌剂的规模化生产应用提供了科学依据。     

Competitive abilities of common field isolates and a commercial strain of Rhizobium leguminosarum bv. trifolii for clover nodule occupancy

We previously reported that commercial Rhizobium leguminosarum bv. trifolii inoculants failed to outcompete naturalized strains for nodule occupation of clover sown into an alkaline soil [Aust. J. Agric. Res. 53 (2002) 1019]. Two field isolates that dominated nodule occupancy at the field site were labeled with a PnifH-gusA marker. Marked strains were chosen on the basis that they were equally competitive and fixed similar amounts of nitrogen in comparison to their parental strain. The minitransposon insertions were cloned and sequence analysis revealed that neither lesion disrupted the integrity of any known gene. The marked strains were then used to follow nodule occupancy of Trifolium alexandrinum in competition against the commercial inoculant TA1 under a range of experimental conditions. In co-inoculation experiments in sand-vermiculite, TA1 outcompeted each marked field isolate for nodule occupancy. However, using TA1-inoculated seed sown into alkaline soil containing a marked field strain, it was demonstrated that by increasing the cell number of marked rhizobia in the soil and reducing the cell number of the commercial inoculant, the proportion of nodules occupied by TA1 was reduced. These studies indicate that the ability of the field isolates to dominate nodule occupancy in the alkaline field soils was most likely caused by poor commercial inoculant survival providing the advantage for naturalized soil rhizobia to initiate nodulation.     

A supernodulating mutant isolated from soybean cultivar enrei

Abstract

Symbiotic nitrogen fixation in nodules of legumes depends on the complex interaction between the legume plant and (Brady)Rhizobium bacteria. Nodule formation and nitrogen fixation are closely regulated by both the host plant and the microsymbiont. Plant mutants with altered symbiotic performance are considered to be useful to gain a better understanding of the plant—microbe interactions in the legume—(Brady)Rhizobium symbiosis (Jacobsen 1984; Carroll et al 1985a, b; Park and Buttery 1988; Duc and Messager 1989; Gremaud and Harper 1989). Recently, Carroll et al. (1985a, b) have isolated the supernodulating mutants of the soybean cv. “Bragg,” which display a very large number of nodules and “nitrate-tolerant-symbiotic” (nts) characteristics. More recently, Gremaud and Harper (1989) have also isolated similar mutants from the soybean cv. “Williams.” These mutants not only provide materials that are useful for investigatings on the interaction in the nodule formation processes but also for agricultural practice. In particular, the nitrate-tolerance of these supernodulating mutants (Carroll et al. 1985b; Gremaud and Harper 1989) is useful for their cultivation in Japan where the level of soil nitrogen in fields is generally high. However, the cultivars previously used for the isolation of these mutants cannot adapt easily in Japanese climate due to different Maturity Group. Therefore, we attempted to isolate mutants with altered symbiotic phenotypes from the soybean cultivar “Enrei,” one of the most common cultivars in Japan.     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

某些生物因子对苕子根瘤菌菌剂和土著根瘤菌竞争结瘤的影响

早在本世纪三十年代,国外已发现根瘤菌竞争结瘤的现象,并对三叶草根瘤菌品系之间的竞争结瘤进行了系统的研究。认为同时接种两个不同的根瘤菌品系,它们的结瘤率是取决于各个品系入侵竞争能力的大小,并引出了竞争优势种的概念。之后,随着免疫学、遗传标记和抗药标记等方法的相继应用,对竞争结瘤问题进行了深入的研究。     

Effects of bacterial inoculant biofertilizers on growth,yield and nutrition of rice in Australia

Inoculant biofertilizer application increased fertilizer nitrogen (N) use efficiency in Vietnam in some previous field experiments. Similar results may be obtained in Australia. With this view in mind, a greenhouse experiment and two field experiments were conducted using a Vietnamese inoculant biofertilizer (BioGro) and several other plant growth promoting (PGP) bacteria. In the greenhouse trial, bacterial inoculations increased shoot and root weights of rice plants significantly. In the field experiments, particularly with Rhizobium leguminosarum, similar effects including significant differences in nitrogen uptake in vegetative matter were observed at the panicle initiation (PI) stage. However, these effects were not significant on grain yield at harvest and it is concluded that the much longer period of growth for Australian rice may allow compensation between treatments. Re-inoculation of plants at the PI stage, and lower application rates of N fertilizer in at least two splits are suggested for future field experiments.     

Rhizobial symbiosis efficiency increases in common bean under sparingly soluble calcium-phosphorus

The use of phosphate-solubilizing rhizobia as a rhizobial inoculant improves plant nutrient uptake (nitrogen (N) and phosphorus (P)) and subsequently crop yield stability. Three common bean varieties namely Coco blanc, Wafa, and Rebia were inoculated with the Rhizobium strain “Ar02” and grown under 250 μmol P as KH₂PO₄ Pi and 250 μmol P as Ca₃HPO₄ (Ca-P). Rebia showed the highest root biomass increase (35%) both under Pi and Ca-P supplies, while Wafa's root biomass significantly decreased under Ca-P condition. Application of Ca₃PO₄ stimulated acid phosphatase activities in shoots (50%), roots (45%) and nodules (49%) of Coco blanc variety as compared to Rebia and Wafa. Moreover, phenols content was enhanced in Wafa roots as compared to Coco blanc roots. N content increased in shoots (14% under Pi and Ca-P supplies) and nodules (6% under Ca-P supplies) of Coco blanc. P and K nutrition largely varied in response to P supplies through all plant parts.     

Competitive interaction among strains of Rhizobium leguminosarum bv. phaseoli in the nodulation of kidney beans (Phaseolus vulgaris L.)

We examined the competitiveness of five effective Rhizobium leguminosarum biovar phaseoli strains in the nodulation of kidney beans (Phaseolus vulgaris L.), either alone or in pairwise combination, against the indigenous strains. The results showed that the introduced Rhizobium sp. strains (B2, B17, B36, T2, or CIAT 652) occupying 64–79% of the total nodules (as single inocula) were more competitive in nodulation than the native rhizobia. However, the competitiveness of the individual Rhizobium sp. strain either increased or decreased when used in a pairwise combination of double-strain inocula. For example, strain B17, although quite competitive against the indigenous population (68% nodule occupancy), became poorly competitive in the presence of strain B2 (reduced from 68 to 2.5%). A similar reduction in nodule occupancy by strain B17 was observed in the presence of B36 or CIAT 652, indicating that two competitive strains may not always be compatible. These results suggest that it is important to co-select competitive as well as compatible rhizobia for multistrain inoculant formulation.     

Survey of genes identified in Sinorhizobium meliloti spp., necessaryfor the development of an efficient symbiosis

The symbiosis between the soil bacteria Rhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium or Bradyrhizobium and leguminous plants is characterised by a specific multistep signal exchange. Only when a compatible rhizobial strain encounters its leguminous host, nodules will be formed on the roots of the host. During infection of this nodule, the microsymbiont evolves into a bacteroid form which, when provided with plant-derived carbon sources, is able to convert atmospheric nitrogen to ammonia that subsequently is supplied to the plant. The developmental programme underlying nodule organogenesis and functioning has been studied intensively for several decades. In this review, several observed plant phenotypes resulting from an ineffective symbiosis between plants and mutant rhizobial strains are represented. Besides the influence of the bacterial nodulation, nitrogen fixation and surface polysaccharide genes on symbiosis, the role of other genes important for the formation of effective nitrogen fixing nodules will be explained.     

Inoculation with the native Rhizobium gallicum 8a3 improves osmotic stress tolerance in common bean drought-sensitive cultivar

Abstract

Symbiotic nitrogen fixation potential in common bean is considered to be low in comparison with other grain legumes. However, it may be possible to improve the nitrogen fixation potential of common bean using efficient rhizobia. In order to improve osmotic stress tolerance of a drought-sensitive common bean cultivar (COCOT) consumed in Tunisia, plants were inoculated either by the reference strain Rhizobium tropici CIAT 899 or by inoculation with rhizobia isolated from native soils Rhizobium gallicum 8a3. Fifteen days after sowing, osmotic stress was applied by means of 25 mM mannitol (low stress level) or by 75 mM mannitol (high stress level). Fifteen days after treatment plants were harvested and different physiological and biochemical parameters were analysed. Results showed no significant differences between the studied symbioses under control conditions. However after exposure to osmotic stress our results showed better tolerance of COCOT to osmotic stress when inoculated with the native R. gallicum 8a3. This can be partially explained by better water-use efficiency in both leaves and nodules, better relative water content in nodules and better efficiency in utilization of rhizobial symbiosis as compared with COCOT-CIAT 899 symbiosis. Hence, the present study suggested the better use of native soil isolated strains for the inoculation of common bean in order to improve its performance and nitrogen fixation potential under stressful conditions.     

Competition for nodule formation between effective and ineffective strains of Rhizobium meliloti

Ineffective mutants of four effective strains of Rhizobium meliloti were isolated and tested for their ability to compete with effective parents or antibiotic resistant mutants in the formation of nodules on Medicago sativa. In 5 out of the 6 cases studied, ineffective mutants were no different to effective strains of the same origin in their competitive ability. A difference in selection for infection by the host plant was observed between equally effective strains as well as between ineffective strains. Except for one pair of strains, the more-competitive effective strains (resistant or not to antibiotics) had the same origin as the more-competitive ineffective strains. For such strains the ability to compete with other strains to form nodules was a characteristic of each parent strain. Competitiveness was independent of effectiveness and had been retained during mutation.