Competition between inoculated and indigenous Rhizobium/Bradyrhizobium spp. strains for nodulation of grain and fodder legumes in Pakistan

Summary The competitive ability of inoculated and indigenous Rhizobium/Bradyrhizobium spp. to nodulate and fix N₂ in grain legumes (Glycine max, Vigna unguiculata, Phaseolus vulgaris) and fodder legumes (Vicia sativa, Medicago sativa, and Trifolium subterraneum) was studied in pots with two local soils collected from two different fields on the basis of cropping history. The native population was estimated by a most-probable-number plant infectivity test in growth pouches and culture tubes. The indigenous rhizobial/bradyrhizobial population ranged from 3 to 2×10⁴ and 0 to 4.4×10³ cells g^-1 in the two soils (the first with, the second without a history of legume cropping). Inoculated G. max, P. vulgaris, and T. subterraneum plants had significantly more nodules with a greater nodule mass than uninoculated plants, but N₂ fixation was increased only in G. max and P. vulgaris. A significant response to inoculation was observed in the grain legume P. vulgaris in the soil not previously used to grow legumes, even in the presence of higher indigenous population (>10³ cells g^-1 soil of Rhizobium leguminosarum bv phaseoli). No difference in yield was observed with the fodder legumes in response to inoculation, even with the indigenous Rhizobium sp. as low as <14 cells g^-1 soil and although the number and weight of nodules were significantly increased by the inoculation in T. subterraneum. Overall recovery of the inoculated strains was 38–100%, as determined by a fluorescent antibody technique. In general, the inoculation increased N₂ fixation only in 3 out of 12 legume species-soil combinations in the presence of an indigenous population of rhizobial/bradyrhizobial strains.     

Cobalt reduces nitrate inhibition of nodulation in mungbean (Vigna radiata)

 A cobalt-mediated decrease in ethylene production reduced the inhibition of nodulation by nitrate in Vigna radiata (mungbean). Nitrate increased the ethylene production in 5-day-old seedlings, while it caused a reduction in the nodulation status (nodule number and nodule weight) and nodule efficiency (acetylene reduction activity) in mungbean plants. The application of cobalt chloride inhibited nitrate-affected ethylene production and also decreased the inhibitory effect of nitrate on nodulation. The effect of cobalt was most marked on nodule number. Received: 6 August 1999     

Influence of a vesicular-arbuscular mycorrhizal fungus on the competitive ability of Bradyrhizobium spp. for nodulation of cowpea Vigna unguiculata (L.) Walp in non-sterilized soil

Summary We examined the influence of a vesicular-arbuscular (VAM) fungus (Glomus pallidum Hall) on the competitive ability of introduced and native Bradyrhizobium strains to nodulate cowpeas [Vigna unguiculata (L) Walp]. Our experiments in non-sterilized soil revealed that in the presence of VAM fungus, introduced Bradyrhizobium spp. strains become more competitive than native rhizobia. For example, strain JRC29 occupied 59.2% of the total nodules when inoculated alone, but this figure increased to 71.2% when JRC29 was used in dual inoculations with VAM fungus. A similar pattern of enhanced competitiveness for nodule formation was observed with the two other strains in the presence of the VAM fungus. Our results suggest that the competitiveness of rhizobia can be enhanced by co-inoculating with a selected strain of a VAM fungus.     

Metabolic and physiological characteristics of salt-tolerant strains of Bradyrhizobium spp.

 Biochemical and physiological characteristics of salt-tolerant (88 mM, 264 mM and 440 mM NaCl) strains of Bradyrhizobium were evaluated according to their capacity for using different carbon sources, growth rate, resistance to antibiotics, plasmid profile and exopolysaccharide production. Salt-tolerant strains significantly enhance their capacity to oxidize C sources (about 75–85 compounds) by increasing growth rate and exopolysaccharide production involved in adhesion, resulting in a greater adapting capacity to colonize unfavorable saline environments. However, salt stress could work as a curing agent and thus the gene stability would become critical for the biological nitrogen-fixation information present in plasmids, as is the case in Rhizobium. Received: 5 January 1998     

Competition between Rhizobium strain NGR234 and Bradyrhizobium strain CP283 for nodulation in Siratro investigated with the GUS reporter gene

The use of the GUS reporter gene was found to be very suitable for studying the competitiveness of the Rhizobium strain NGR234 and Bradyrhizobium strain CP283 for nodulation in siratro. However, the expression from the transposon mTn5SSgusA20 declined in the nodules of old plants, particularly the nodules at 35 d after inoculation and onward. Siratro inoculated with both gusA-marked NGR234 and CP283 strains showed a similar nodulation and acetylene reduction activity (ARA) to those of their parental strains. No major changes in nodulation and symbiotic properties in these marked strains were observed. When the Rhizobium strain NGR234 and Bradyrhizobium strain CP283 were inoculated separately to siratro, both of them were found equally effective for nodulation in the plant. But when the Rhizobium strain NGR234, and gusAmarked Bradyrhizobium strain CP283 were co-inoculated to the plants in a ratio of 1:1, the strain NGR234 occupied 75% of the nodules, while, the strain CP283 occupied only 25%, irrespective of the growth stages and inoculum concentrations. Similar results were also observed in the plants in which the Bradyrhizobium strain CP283 and gusA-marked Rhizobium strain NGR234 had been co-inoculated. Thus, the Rhizobium strain NGR234 was more competitive for nodulation in siratro.     

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.     

Antagonistic effect of Pseudomonas spp. on pathogenic fungi and enhancement of growth of green gram (Vigna radiata)

species were isolated from the rhizosphere of green gram [Vigna radiata (L.) Wilczek] and some of the rhizobacterial isolates were found to have a wide range of antifungal activity inhibiting growth of the phytopathogenic fungi Aspergillus sp., Curvularia sp., Fusarium oxysporum and Rhizoctonia solani in culture. These isolates also showed slight inhibition of the growth of a Bradyrhizobium strain (Vigna) in a spot test which was mainly a result of nutrient competition as culture supernatants of the Pseudomonas isolates did not inhibit the growth of bradyrhizobia but inhibited the growth of fungi. The rhizobacterial isolates produced siderophores in Fe-deficient succinate medium. However, the inhibition of fungal growth by different Pseudomonas isolates in Luria Bertani and King's medium B which were not limiting in Fe³⁺ ions suggested that, besides siderophores, other antifungal compounds (antibiotics) produced by these rhizobacteria were involved in antagonism. On coinoculation of green gram with Pseudomonas strains MRS13 and MRS16 and Bradyrhizobium sp. (Vigna) strain S24, there was a significant increase in nodule weight, plant dry weight and total plant N as compared to inoculation with Bradyrhizobium strain S24 alone, suggesting that the nodule-promoting effects of Pseudomonas sp. lead to an increase in symbiotic N fixation and plant growth. Received: 27 October 1997     

Competition between species of Rhizobium for nodulation of Glycine max

Glycine max cv. Malayan is a promiscuously nodulating cultivar which formed nodules with 6 out of 9 strains of Rhizobium spp of diverse origin and all strains of R. japonicum tested. No generalizations can be made as to the probability of strains isolated from a particular host being infective on Malayan as only some isolated from Centrosema pubescens, and Cajanus cajan were able to form nodules. In competition with R. japonicum at 30°C all 20 strains of Rhizobium spp isolated from Malayan grown in Nigeria formed fewer than 50% of the nodules and 14 strains fewer than 25%. Competition was influenced by root temperature. Three strains of Rhizobium spp were poor competitors with R. japonicum between 24° and 33°C but at 36°C they formed more nodules (74–88%) than R. japonicum. Another strain of Rhizobium spp formed the majority of the nodules between 27° and 36°C whereas R. japonicum formed the most at 24°C.     

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。     

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。     

Genetic diversity and relationship between Bradyrhizobium strains isolated from blackgram and cowpea

The genetic diversity of bradyrhizobial strains associated with blackgram and cowpea grown in two different agricultural soils (non-saline and saline) along the coastline of Tamil Nadu has been analysed. Phenotypically indistinguishable isolates were analysed for DNA polymorphism using random amplification of polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of 16S rDNA and nifD. Although these bacteria belong to a group with a broad host range, RAPD analysis showed a considerable level of genetic diversity among the strains isolated from different host plants. Soil pH and salinity seem to have an effect on the selection of natural populations as revealed by PCR-RFLP of 16S rDNA. A combination of PCR-RFLP genotyping with nodulation studies indicates that monocropping of blackgram and the salinity of the soil have made ineffective rhizobia the dominant genotype, thereby creating an ecological burden on their other compatible hosts. A group of strains and a type strain sharing three different 16S PCR-RFLP types were shown to have the same set of symbiotic genes as inferred from the PCR-RFLP pattern of nifD. Another group of cowpea rhizobia that were found to be effective nitrogen fixers and sharing distinct 16S profiles were found to have a different set of symbiotic genes.     

Temperature Effects on Bradyrhizobium spp. Growth and Symbiotic Effectiveness with Pigeonpea and Cowpea

Temperature is a limiting factor on legume-Bradyrhizobium symbiosis of subtropical plants in the temperate region. Twelve strains of Bradyrhizobium spp. that nodulate pigeonpea [Cajanus cajan (L.) Millsp], and cowpea [Vigna unguiculata (L.) Walp], were evaluated for tolerance to three temperature regimes (20°C/10°C, 30°C/20°C, and 38°C/25°C day/night temperature) by determining their growth following exposure to the regimes. The five most temperature-tolerant strains were further evaluated for symbiotic effectiveness with pigeonpea and cowpea under controlled temperatures. These strains were USDA 3278, USDA 3362, USDA 3364, USDA 3458, and USDA 3472. Plant heights of both crops were generally independent of Bradyrhizobium strains and were dependent mainly on temperature regimes. Plant heights were the shortest at the lowest temperature. At the lowest temperature regime, biological nitrogen (N) fixation by pigeonpea was almost completely inhibited. Cowpea genotype IT82E-16 inoculated with USDA 3458 formed the most effective symbiosis. The 30°C/20°C temperature regime was optimum for effective symbiotic association in both crops, and also for Bradyrhizobium survival.     

Comparative efficieny of Rhizobium/Bradyrhizobium spp. strains in nodulating Cajanus cajan in relation to characteristic metabolic enzyme activities

The comparative symbiotic properties of Rhizobium spp. and Bradyrhizobium spp. strains infecting pigeon pea were evaluated. Bradyrhizobium strains (Cajanus) were found to be superior to Rhizobium strains (Cajanus) and the superiority was ascertained to be due to the higher enzyme activity of the tricarboxylic acid (TCA) cycle in comparison to Rhizobium spp. strains. Moreover, metabolic superiority or rapid growth rate does not necessarily correlate with symbiotic effectiveness. The symbiotic performance of isolates varied with the host cultivar. The dry matter accumulation could be correlated with the total acetylene reduction activities rather than nodule number or nodule fresh weight per plant. Received: 3 March 1993     

Competition among strains of Bradyrhizobium and vesicular-arbuscular mycorrhizae for groundnut (Arachis hypogaea L.) root infection and their effect on plant growth and yield

Summary Strains of Bradyrhizobium influenced root colonization by a species of vesicular-arbuscular mycorrhizae (VAM), and species of VAM influenced root nodulation by strains of Bradyrhizobium in pot experiments. In a field experiment, the effects of VAM on competition amongst inoculated bradyrhizobia were less evident, but inoculation with Bradyrhizobium strains increased root colonization by VAM. Certain VAM/Bradyrhizobium inoculum strain combinations produced higher nodule numbers. Plants grown without Bradyrhizobium and VAM, but supplied with ammonium nitrate (300 g ml^–1) and potassium phosphate (16 g ml^–1), produced higher dry-matter yields than those inoculated with both symbionts in the pot experiment. Inoculation with either symbiont in the field did not result in higher pod and haulm yields at harvest.ICRISAT Journal Article No. 886     

Selection of Rhizobium strains on their competitive ability for nodulation

The shoot dry weight of alfalfa inoculated with an effective strain of Rhizobium meliloti mixed with an ineffective strain in different ratios was found to be directly proportional to the log of the number of effective nodules. Consequently the comparison of the shoot dry weight of plants inoculated with a mixture of effective and ineffective strains with the shoot dry weight of plants inoculated with the effective strain should allow the estimation of the relative competitiveness of the effective strains. To check this. the competitiveness of 14 antibiotic-resistant strains of R. leguminosarum was evaluated in this way and compared with the ability of the strains to form nodules when inoculated to seeds of Vicia faba planted in a soil containing indigenous R. leguminosarum. The percentage of recovery of the inoculum strains in the nodules of field-grown fababeans was positively correlated with the competitiveness of the strains as estimated by the greenhouse test. This simple way of evaluating the nodulating competitiveness of strains of rhizobia being indicative of their competitive behaviour with indigenous rhizobia in the field could therefore be useful for screening a large number of strains for competitiveness.     

A proposal for re-evaluating the most probable number procedure for estimating numbers of Bradyrhizobium spp.

The reliability of the most probable number (MPN) method for estimating bradyrhizobial numbers was evaluated by comparison with the plate count procedure. MPN estimates increased with time of nodulation scoring after seedling inoculation through 6 weeks of incubation. Ratios of MPN to plate counts increased as the bradyrhizobial cell suspension concentration increased. The MPN method could not detect Bradyrhizobium japonicum numbers at concentrations of 10³ colony forming units (CFU) ml^-1 and below. A proposal for re-evaluating MPN estimates is discussed.     

Evaluation of effective Myanmar Bradyrhizobium strains isolated from Myanmar soybean and effects of coinoculation with Streptomyces griseoflavus P4 for nitrogen fixation

In order to substitute the use of chemical fertilizers in legume production, there is a need for the production of rhizobial inoculants which are capable of being used as biofertilizers. To achieve this, an effective symbiotic nitrogen (N) fixation between legumes and root nodule bacteria will be essential. Evaluation of effective Myanmar Bradyrhizobium (Jordan 1982) strains isolated from Myanmar soybean (Glycine max L. Merr.) and effects of coinoculation with Streptomyces griseoflavus Krainsky 1914 P4 for N fixation were studied in pot experiments using sterilized vermiculite and Hoagland solution in the Phytotron (25°C and 70% relative humidity) with completely randomized design and three replicates. N fixation ability of soybean was evaluated by acetylene reduction activity (ARA) by gas chromatography. It was found that MAS23 showed a relatively high degree of stability and a high level of ARA per plant on both Yezin-3 and Yezin-6 soybean varieties. In the symbiotic relationship between Bradyrhizobium strains and P4 experiments, the treatments consisted of six Bradyrhizobium strains (MAS23, MAS33, MAS34, MAS43, MAS48 and USDA110) and Streptomyces griseoflavus P4 were evaluated with four Myanmar soybean varieties (Yezin-3, Yezin-6, Hinthada and Shan Sein). In the Yezin-3 soybean variety, the best treatment for ARA per plant was found in the dual inoculation of P4 and MAS34. In the Yezin-6 soybean variety, the highest nodule dry weight was found in dual inoculation of P4 with MAS34 but the highest ARA per plant was observed in the dual inoculation of P4 and MAS23. On the other hand, single inoculation of MAS43 and coinoculation of P4 with MAS48 were significantly higher in N fixation of Hinthada, and coinoculation of P4 with MAS33 was significant improvement of ARA per plant (P < 0.05) in Shan Sein soybean.     

Potential for enhancement of root growth and nodulation of soybean co-inoculated with Azospirillum and Bradyrhizobium in laboratory systems

The potential enhancement of root growth and nodulation in vegetable soybean (AGS190) was studied with application of Azospirillum brasilense (Sp7) and A. lipoferum (CCM3863) co-inoculated with two Bradyrhizobium japonicum strains (TAL102 and UPMR48). Significant root growth stimulation and nodulation were observed in Azospirillum as well as during its co-inoculation with Bradyrhizobium. Nodule formation is linked with the initiation of new roots; nodules were almost absent even in Bradyrhizobium inoculated plant due to the absence of new roots development in clipped rooted seedlings. Total root length, root number, specific root length, root dry matter, root hair development and shoot dry matter were significantly increased by Azospirillum alone and its co-inoculum. Co-inoculated plants significantly influenced the number of nodules and its fresh weight. A. brasilense seemed to perform better in root growth and nodule development compared to A. lipoferum.     

Competition for nodule occupancy between introduced and native strains of Rhizobium leguminosarum biovar trifolii

The aim of this work was to evaluate the competitive ability between Rhizobium leguminosarum bv trifolii strain U204 used as commercial inoculants in Uruguay for Trifolium repens L. and Trifolium pratense L. and two native strains isolated from inoculated pastures of T. pratense. T126 is an efficient nitrogen fixer and a melanin producer strain; T70 is inefficient and a melanin non-producer strain; and U204 is very efficient in both hosts but is a melanin non-producer strain. Competitiveness between the strains was determined in experiments in pots and in growth pouches under controlled conditions. In the last experiment, we evaluated pH of plant nutrient solution and inoculum ratios. Plant dry weight was determined, and the identification of nodule bacteria was done using melanin production and DNA fingerprinting (GTG₅-PCR). The U204 symbiotic efficiency was not affected by the co-inoculation with the others two native strains. The T70 strain was a poor competitor when was co-inoculated with one of the effective strains in both experiments. Our results confirmed a “selective nodulation” because an effective symbiosis occurred preferentially over an ineffective one in Trifolium species. The native effective strain competed with U204 for nodule formation in both clovers species, but the nodule occupancy depended on the inoculum ratio. The pH of nutritive solution did not affect competition ability of the studied strains. It may be possible to isolate efficient, competitive, and genetically different native rhizobial strains to be used as inoculant strains for clover pastures in Uruguay. Both (GTG)₅-PCR and melanin production were useful methods to identify nodulating bacteria in competition studies.