Indigenous Rhizobia Research Articles

Genetic diversity of indigenous Bradyrhizobium nodulating promiscuous soybean [Glycine max (L) Merr.] varieties in Kenya: Impact of phosphorus and lime fertilization in two contrasting sites

While soybean is an exotic crop introduced in Kenya early last century, promiscuous (TGx) varieties which nodulate with indigenous rhizobia have only recently been introduced. Since farmers in Kenya generally cannot afford or access fertilizer or inoculants, the identification of effective indigenous Bradyrhizobium strains which nodulate promiscuous soybean could be useful in the development of inoculant strains. Genetic diversity and phylogeny of indigenous Bradyrhizobium strains nodulating seven introduced promiscuous soybean varieties grown in two different sites in Kenya was assayed using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 16S-23S rDNA intergenic spacer region and 16S rRNA gene sequencing. PCR-RFLP analysis directly applied on 289 nodules using Msp I distinguished 18 intergenic spacer groups (IGS) I–XVIII. Predominant IGS groups were I, III, II, IV and VI which constituted 43.9%, 24.6%, 8.3% 7.6% and 6.9% respectively of all the analyzed nodules from the two sites while IGS group VII, IX, X, XI, XII, XIV, XVI, XVII, XVIII each constituted 1% or less. The IGS groups were specific to sites and treatments but not varieties. Phylogenetic analysis of the 16S rRNA gene sequences showed that all indigenous strains belong to the genus Bradyrhizobium. Bradyrhizobium elkanii, Bradyrhizobium spp and Bradyrhizobium japonicum related strains were the most predominant and accounted for 37.9%, 34.5%, and 20.7% respectively while B. yuanmigense related accounted for 6.9% of all strains identified in the two combined sites. The diversity identified in Bradyrhizobium populations in the two sites represent a valuable genetic resource that has potential utility for the selection of more competitive and effective strains to improve biological nitrogen fixation and thus increase soybean yields at low cost.

Population size of indigenous Rhizobium leguminosarum biovar trifolii in long-term field experiments with sewage sludge cake, metal-amended liquid sludge or metal salts: Effects of zinc, copper and cadmium

There is conflicting evidence, and therefore continuing concern, as to whether metals in sewage sludge are deleterious to soil microbial processes and long-term agricultural productivity. Nine field experiments with sewage sludge cakes, three with metal-amended liquid sludges and three with inorganic metal salts were set up across Britain in 1994 to give individual metal dose–response treatments to try to answer this question. This study reports on the effects of Zn, Cu and Cd on the population size of Rhizobium leguminosarum biovar trifolii, a nitrogen fixing symbiont of white clover ( Trifolium repens), in soils from these experiments over 11 years. Significant ( P < 0.05) reductions in indigenous rhizobial numbers occurred on the Zn metal dose–response treatments at eight of the sludge cake sites in 2005, but few consistent effects were evident on the Cu or Cd metal dose–response treatments during the 11-year monitoring period. The soil total Zn concentrations where effects occurred were near to the UK statutory limit of 300 mg kg −1 for soils receiving sewage sludge. No significant reductions occurred in any treatments on the metal-amended liquid sludge or inorganic metal salt experiments in which the metals would be expected to be in a more bioavailable form, even after 11 years. The effects in the sludge cake experiments were related consistently with soil total Zn, with no recovery to date. The reductions in clover rhizobial numbers in the sludge cake experiments were due to Zn effects on free-living rhizobia in the soil, with gradual die-off over a long time with increasing soil total Zn concentrations. Currently, no consistent adverse effects on rhizobia have been seen at the UK limits for Cu and Cd of 135 and 3 mg kg −1, respectively.

Rhizobium gallicum as an efficient symbiont for bean cultivation

Rhizobia are soil bacteria that fix atmospheric nitrogen in symbiosis with legumes in specialized organs called nodules. The legumes thus acquire the autonomy to grow in nitrogen-deficient soils. When nitrogen fixation by indigenous rhizobia is limited, field inoculation with efficient and competitive strains is an economically feasible way to increase production. When the inoculant is made from native strains of rhizobia the success of inoculation should be increased, since local strains are better adapted than commercial inoculants. Here, a Rhizobium gallicum strain, 8a3, previously selected for its competitiveness and symbiotic effectiveness with common bean under laboratory conditions, was tested in field trials in Tunisia. The experiments were conducted in six fields using three common bean cultivars. The majority of the fields showed a low density of the native rhizobia and inefficient nodulation by Sinorhizobium meliloti, a known symbiont of Medicago. Our results show that inoculation with R. gallicum strain 8a3 induced an increase in nodule numbers accompanied by a more than twofold increase in shoot dry yield. Monitoring of the nodulation occupancy through the fingerprinting of the repetitive extragenic palindromic sequences (REP-PCR) showed that strain 8a3 was competitive even in the soil showing a high population density of indigenous R. gallicum, and occupied more than 40% of the nodules. Moreover, in vitro antibiosis assays indicated that strain 8a3 produces antimicrobial activity on agar medium against indigenous common bean rhizobia, including the inefficient strains of S. meliloti. These results point out the benefits that could be achieved by selecting efficient and competitive strains among natural populations of rhizobia.

Diversidade genética e eficiência simbiótica de rizóbios noduladores de acácia-negra de solos do Rio Grande do Sul

A acácia-negra é a terceira espécie florestal mais cultivada no Brasil. Além de sua importância econômica, é utilizada na recuperação de áreas degradadas, nas quais o solo geralmente apresenta pH baixo e altos teores de Al. O presente trabalho objetivou avaliar a diversidade genética de rizóbios naturais de solos do Rio Grande do Sul e selecionar isolados eficientes na fixação de N2 em condições de pH baixo. Um total de 50 isolados de Bradyrhizobium sp. foi obtido, os quais, juntamente com as estirpes recomendadas BR 3067 e BR 3068, foram caracterizados com o marcador BOX A 1-R. O padrão de bandas dos isolados foi utilizado na construção de um dendrograma, a partir do qual se calculou o índice de diversidade de Shannon. Dez isolados foram testados quanto à tolerância a pH baixo e à presença de Al, selecionando-se oito para o teste de eficiência simbiótica em casa de vegetação. Observou-se diversidade genética elevada entre os isolados, com a formação de 10 grupos, a partir do ponto de corte de 70 % de similaridade e com o índice de diversidade de 4,30. A presença de Al não afetou os isolados avaliados, que tiveram seu crescimento reduzido em pH 4,5. Quanto à eficiência simbiótica, os isolados T6-16 e V-7 foram os mais eficientes, assemelhando-se à estirpe recomendada BR 3068.

Long term evaluation of field-released genetically modified rhizobia.

This is the report of the first open field release of genetically modified microorganisms (GMMs) in Italy. It covers ten years of monitoring, and follows in-field GMM dynamics from strain release to disappearance below detection limits, as well as assessment of impact on resident microorganisms. The bacteria released belong to the nitrogen fixing legume endosymbiont Rhizobium leguminosarum bv. viciae, and were engineered with non-agronomically-proficient traits, in order to assess their behavior and fate without GMM-specific positive feedback from the plant. A DNA cassette containing mercury resistance and ss-galactosidase genes was introduced in either plasmid-borne or chromosomally integrated versions, in order to test the resulting strain stability. A synthetic promoter was used to drive the lacZ gene, conferring high catabolic activity to the GMM. Two different wild-type Rhizobium backgrounds were tested, comparing a non-indigenous vs. an indigenous, highly competitive strain. The latter had much greater persistence, since it was able to survive and establish at technically detectable levels for over four years after release. Selection factors, such as reiterated presence of the plant host, or lactose substrate supply, enhanced long-term survival to different extents. The lactose treatment showed that even a single trophic supplementation can surpass the benefits of symbiotic interaction for a period of several years. Concerning impact, the GMMs did not alter substantially the other soil community general microbiota. However, there were some significant differences in microbiota as a consequence of the Rhizobium inoculation. This effect was observed with either the WT or GMM, and was more evident in the release of the indigenous Rhizobium. Moreover, as the indigenous GMM had its parental, dominant wild-type in the same soil, it was possible to evaluate to what extent the GMM version could result in parent displacement ("self-impact"), and how much the two rhizobia would additively contribute to nodulation.

Produção de amilase por rizóbios, usando farinha de pupunha como substrato

Amylases are among the most important industrial enzymes and are of great significance in present-day biotechnology. Although they can be derived from various sources, enzymes from microbial sources are generally the most required by industry. Species of the genus Bacillus are considered to be the main sources of amylases, although screening for new microbial sources is increasing all over the world. The objective of the present study was to evaluate the production of amylase by indigenous rhizobia, using peach palm flour as substrate. In this study, a completely randomized experimental design was adopted with three replicates. Pearson's correlation coefficients were calculated for medium pH, extracellular protein, cellular biomass, mean colony diameter (MCD), mean halo diameter (MHD), enzymatic index (EI) and amylolytic activity variables from the bacteria selected. Out of the 19 rhizobia strains with amylolytic activity on modified YMA, seven (36.8%) strains showed EI > 2,1, and they were considered as good producers of amylase. The IE presented by bacteria INPA R-987, R-950 and R-915B were significantly lower (p < 0.01) than those shown by the INPA R-926, R-975 and R-957. The amylolytic activity varied significantly (p < 0.01) among the rhizobia strains. INPA R-975 and R-926 strains presented, respectively, the highest (1.00 U.min-1.mL-1) and the lowest (0.31 U.min1.mL-1) activity average. In general terms, the extracellular protein was correlated positively with EI (r = 0.52*; p < 0.05) and MHD (r = 0.55*; p < 0.05). The cellular biomass showed positive correlations with amylolytic activity (r = 0.55*; p < 0.05) and MHD (r = 0.54*; p < 0.05), and a negative with final pH medium (r = 0.93**; p < 0.01).

Effects of Cd, Zn, or both on soil microbial biomass and activity in a clay loam soil

We investigated Cd, Zn, and Cd + Zn toxicity to soil microbial biomass and activity, and indigenous Rhizobium leguminosarum biovar trifolii, in two near neutral pH clay loam soils, under long-term arable and grassland management, in a 6-month laboratory incubation, with a view to determining the causative metal. Both soils were amended with Cd- or Zn-enriched sewage sludge, to produce soils with total Cd concentrations at four times (12 mg Cd g−1 soil), and total Zn concentrations (300 mg Zn kg−1 soil) at the EU upper permitted limit. The additive effects of Cd plus Zn at these soil concentrations were also investigated. There were no significant differences in microbial biomass C (BC), biomass ninhydrin N (BN), ATP, or microbial respiration between the different treatments. Microbial metabolic quotient (defined as qCO2 = units of CO2–C evolved unit−1 biomass C unit−1 time) also did not differ significantly between treatments. However, the microbial maintenance energy (in this study defined as qCO2-to-μ ratio value, where μ is the growth rate) indicated that more energy was required for microbial synthesis in metal-rich sludge-treated soils (especially Zn) than in control sludge-treated soils. Indigenous R.leguminosarum bv. trifolii numbers were not significantly different between untreated and sludge-treated grassland soils after 24 weeks regardless of metal or metal concentrations. However, rhizobial numbers in the arable soils treated with metal-contaminated sludges decreased significantly (P Cd > Cd + Zn.

Soybean [Glycine max (L.) Merrill] rhizobial diversity in Brazilian oxisols under various soil, cropping, and inoculation managements

In this study, soybean nodules were collected from 12 sites in the State of Mato Grosso, in the Brazilian Cerrados, where both exotic soybean [Glycine max (L.) Merrill] and bradyrhizobial strains have been introduced from 1 to 18 years before. All soils were originally devoid of rhizobia capable of effectively nodulating soybean and varied in terms of chemical and physical properties, inoculation procedures, and cropping systems. Rhizobial genetic diversity was assessed on 240 isolates by rep-PCR fingerprinting with BOX primer, and indices of diversity (abundance-based coverage estimator and traditional and modified Shannon indices) were applied to the profiles obtained. The genetic diversity was much greater than expected, as after the introduction of a maximum of four strains, up to 13 profiles were identified, some sharing many similar bands with the inoculant strains, but others quite distinct from the putative parental genotypes. The increase in the number of rep-PCR profiles could be attributed to genetic variability due to the stressful tropical environmental conditions, but also might indicate that indigenous rhizobia become capable of nodulating the host legume. After the third year of cropping with the host legume, inoculation did not affect rhizobial diversity. A high content of clay decreased diversity in comparison with that seen in a sandy soil, probably due to reduced aeration. Diversity was higher under the no-tillage system when compared to the conventional tillage management, highlighting the importance of maintaining crop residues in tropical soils. Understanding the ecology of exotic rhizobia after being introduced into new cropping areas represents a first step towards the establishment of better strategies of inoculation, which in turn may result in sustainability and higher plant yields.

New Rhizobium leguminosarum bv. trifolii isolates: Evaluation of competitiveness for clover nodule occupancy

An interrelationship between introduced and indigenous rhizobia focused on their competitiveness in nodulation was evaluated in a soil pot experiment. Clover seeds were inoculated by six different gradual concentrations of inoculum of two effective Rhizobium leguminosarum bv. trifolii isolates (inoculation strains 1/2 and 14/2). At the beginning of flowering, clover plants were removed from the pots, and nodules from each pot representing different degrees of inoculum level were taken for reisolate cultivation. The PCR technique was used for the identification of rhizobial reisolates, random amplified polymorphic DNA product patterns were acquired and analysed. Nodule occupation by inoculation strains or indigenous Rhizobium leguminosarum bv. trifolii strains was assessed by comparing the number of nodules formed by inoculation or indigenous strains and inoculation strains competitiveness was calculated. Nodule occupancy by the inoculation strain 1/2 increased sharply with the increase in inoculum dose, whereas in inoculation strain 14/2 more nodules were formed gradually starting from low inoculum level. Competitiveness of inoculation strain 1/2 was calculated as low and was documented by an absence of nodule occupancy in four inoculation levels. On the other hand, competitiveness of the inoculation strain 14/2 was considerably higher, and even in low inoculum dose this strain was more competitive than native rhizobia. Although both the inoculation strains 1/2 and 14/2 were found highly efficient in nitrogen fixation, only the strain 14/2 was able to manifest this characteristic due to the higher competitiveness when applied in lower doses.

Genetic diversity and symbiotic efficiency of population of rhizobia of Phaseolus vulgaris L. in Brazil

The genetic diversity and symbiotic efficiency among indigenous rhizobia isolates obtained from native field with or without organic fertilization and superficial mineral fertilization were investigated. Eighty-six indigenous rhizobia were isolated from these fields using four common bean varieties as trap-host. The common bean varieties Mexico 309 and Rio Tibagi selected the most efficient rhizobia strains because they showed the best yields and N contents results. The genetic characterization of 36 rhizobia isolates was evaluated by using electrophoretic profiles of amplification products using primers ERIC1-R and ERIC-2. Our results demonstrated that besides the large diversity in the indigenous rhizobial community, the genotype of the trap-host probably influences the selection of the most efficient strains.

Optimal biostimulation strategy for phenol degradation with indigenous rhizobium Ralstonia taiwanensis

This study provides a first attempt from a perspective of Gaden's classification of fermentation and phase–plane to put forward phenol degradation using various augmented nutrient media for biostimulation. It aimed to identify the most promising nutrient source(s) to attenuate synergistic interactions with phenol for optimal phenol degradation. Therefore, the growth association of phenol degradation using various nutrient media in place of combined toxic interactions was established via Gaden's classification scheme of fermentation and phase–plane analysis. In cultures grown on medium bearing dual carbon sources (glycerol and phenol) or phenol alone, phenol was found to be firstly biodegraded for microbial growth (i.e., growth-associated degradation). In contrast, when yeast extract or acetate was supplemented, a diauxic growth behavior was observed as the augmented nutrient was primarily utilized while phenol degradation was repressed. Moreover, using glycerol as the nutrient source, phenol degradation seemed to be enhanced simultaneously during the consumption of glycerol for cellular growth after ca. 2 h response lag in growth. Although gluconic acid could enhance cell growth as well as phenol degradation, the phenol degradation performance was still not as good as that of glycerol. Thus, biostimulation with glycerol appeared to show the most favorable metabolic characteristics against phenol toxicity on Ralstonia taiwanensis, leading to better degradation efficiency of the toxic pollutant. Phase–plane trajectories also clearly confirmed that glycerol was the optimal biostimulating nutrient source for phenol degradation.

Peanut (Arachis hypogaea) response to inoculation with Bradyrhizobium sp. in soils of Argentina

AbstractSoil bacteria (rhizobia) of the genus Bradyrhizobium form symbiotic relationships with peanut root cells and fix atmospheric nitrogen by converting it to nitrogenous compounds. Inoculation of peanut with rhizobia can enhance the plant’s ability to fix nitrogen from the air and thereby reduce the requirement for nitrogen fertiliser. We evaluated three Bradyrhizobium sp. strains for effect on root nodulation and on pod yield of peanut in Argentina soils, using laboratory and field experiments. Of these, strain C‐145 was the most effective in laboratory studies. In‐furrow inoculation with this strain produced increased nodule number, relative to seed inoculation. However, pod yield was not increased significantly by either type of inoculation. In view of the inconsistent response of peanut to inoculation, we examined the effect of indigenous strains of bradyrhizobia. The high degree of nodulation and nitrogen fixation produced by indigenous rhizobia were sufficient for maximal yield under the field and inoculation conditions used in this study. The data are important for future investigation of alternative inoculant strains and conditions for improving peanut production.

Diversity of a Soybean Bradyrhizobial Population Adapted to an Indian Soil

Soybean (Glycine max) is an introduced crop in India. Over the years it has been regularly inoculated with indigenous rhizobia. In this study genetic diversity has been studied at a site where soybean has been regularly grown with inoculation. Rhizobia were plant trapped using soybean varieties as host, and fingerprinted using BOX-PCR. BOX-PCR genomic fingerprints of 69 isolates from the nodules of 4 soybean varieties Pusa22, Bragg, PK1041 and PK1142 showed a high level of genetic diversity. The population profiles of the 69 isolates clustered them into 10 groups. Root nodule isolates from the four varieties were Bradyrhizobium japonicum types, growing in 4–7 days with typical colonies which were found to be genetically distinct from the USDA and SEMIA strains of B. japonicum and B. elkanii. Also the genotype of the host plant seemed to be one of the factors determining the diversity. The high diversity could be attributed both to lateral transfer of genetic material between inoculant and indigenous strains and to genomic rearrangements during the adaptation to the Indian soils.

Evidence of selection for effective nodulation in the Trifolium spp. symbiosis with Rhizobium leguminosarum biovar trifolii

The pasture-breeding program to improve production in the natural grasslands in Uruguay has acknowledged that indigenous Rhizobium strains are incompatible with introduced Mediterranean clovers. In an attempt to understand and overcome this problem, a cross-row experiment was set up in 1999 in a basaltic, acid soil in Glencoe, Uruguay, to follow the survival and performance of 9 exotic strains of Rhizobium leguminosarum bv. trifolii. This paper reports on the ability of the introduced strains to compete for nodule occupancy of Mediterranean clover hosts and impacts of the introduced strains on the productivity of the indigenous Uruguayan clover Trifolium polymorphum. Strain WSM1325 was a superior inoculant and remained highly persistent and competitive for the effective symbiosis with the Mediterranean hosts, T. purpureum and T. repens, in the Uruguayan environment in the third year of the experiment. The Mediterranean hosts (T. purpureum and T. repens) nodulated with the introduced strains but did not nodulate with any indigenous R. leguminosarum bv. trifolii typed from nodules of T. polymorphum. Conversely, there were no nodules on the Uruguayan host T. polymorphum that contained introduced R. leguminosarum bv. trifolii. These results reveal the establishment of effective symbioses between strains of R. leguminosarum bv. trifolii and clover even though the soil contained ineffective R. leguminosarum bv. trifolii for all hosts. We believe our results are the first reported example of ‘selective’ nodulation for an effective symbiosis in situ with annual and perennial clovers in acid soils.

Characteristics of rhizobia nodulating beans in the central region of Minnesota

Until recently, beans (Phaseolus vulgaris L.) grown in Minnesota were rarely inoculated. Because of this, we hypothesized that bean rhizobia collected in Minnesota would either share characteristics identifiable with Rhizobium etli of Mesoamerican or Andean origin, introduced into the region as seed-borne contaminants, or be indigenous rhizobia from prairie species, such as Dalea spp. The latter organisms have been shown to nodulate and fix N2 with Phaseolus vulgaris. Rhizobia recovered from the Staples, Verndale, and Park Rapids areas of Minnesota were grouped according to the results of BOXA1R-PCR fingerprint analysis into 5 groups, with only one of these having banding patterns similar to 2 of 4 R. etli reference strains. When representative isolates were subject to fatty acid - methyl ester analysis and 16S rRNA gene sequence analysis, the results obtained differed. 16S rRNA gene sequences of half the organisms tested were most similar to Rhizobium leguminosarum. Rhizobia from Dalea spp., an important legume in the prairie ecosystem, did not play a significant role as the microsymbiont of beans in this area. This appears to be due to the longer time needed for them to initiate infection in Phaseolus vulgaris. Strains of Rhizobium tropici IIB, including UMR1899, proved tolerant to streptomycin and captan, which are commonly applied as seed treatments for beans. Local rhizobia appeared to have very limited tolerance to these compounds.

Effect of Deep Placement of N Fertilizers and Different Inoculation Methods of Bradyrhizobia on Growth, N2 Fixation Activity and N Absorption Rate of Field-grown Soybean Plants

The effects of deep placement (supplied at 20 cm depth from soil surface below plants) of 100 kg N ha−1 of N fertilizers, urea, coated urea or calcium cyanamide (lime nitrogen) on the growth, nitrogen fixation activity, nitrogen absorption rate and seed yield of soybean (Glycine max L. Merr.) plants were examined by comparing them with control plots without deep placement of N fertilizer in sandy dune field. In addition, three different inoculation methods of bradyrhizobia were used for each N treatment: (1) transplantation of 10-day-old seedling in a paper pot with vermiculite inoculated with Bradyrhizobium japonicum USDA110, (2) direct transplantation of inoculated 10-day-old seedlings, and (3) transplantation of 10-day-old seedlings in a non-inoculated paper pot. The deep placement of N fertilizers, especially calcium cyanamide and coated urea, markedly increased the growth and total N accumulation in shoot, roots and nodules, which resulted in an increase in seed yield. Daily N2 fixation activity and N absorption rate were estimated by relative abundance of ureide-N analysed from the concentration of N constituents (ureide-N, amide-N and nitrate-N) in root bleeding xylem sap and increase in total N accumulation in whole plants at R1, R3, R5 and R7 stages. The total amount of N2 fixation was about 50 % higher in the plants with calcium cyanamide and coated urea deep placements compared with control plants. Deep placement of slow release fertilizers kept nodule dry weight higher in the maturing stage of seed, possibly through abundant supply of photoassimilate to the nodules by supporting leaf area and activity until late reproductive stages. The results indicate that deep placement of calcium cyanamide or coated urea enhances N2 fixation activity, which ultimately increases the seed yield. The promotive effect was observed with the seedlings transplanted in paper pot with inoculum of bradyrhizobia within any treatments, although nodulation by indigenous rhizobia was observed in the plants transplanted with non-inoculated paper pot.

Seleção de rizóbios nativos para guandu, caupi e feijão-de-porco nos tabuleiros costeiros de Sergipe

A inoculação de estirpes de rizóbios em sementes de feijão-de-porco (Canavalia ensiformis), caupi (Vigna unguiculata) e guandu (Cajanus cajan), recomendadas para outras regiões do País, não tem resultado em incrementos nas taxas de fixação biológica de N2 nem no crescimento dessas leguminosas em solos dos tabuleiros costeiros de Sergipe. Os objetivos deste trabalho foram avaliar a eficiência simbiótica de rizóbios nativos dos tabuleiros costeiros associados a essas leguminosas e a tolerância deles a estresses. Das 17 estirpes de rizóbios isoladas e analisadas em casa de vegetação, quatro foram selecionadas para o guandu, sete para o caupi e três para o feijão-de-porco. O número e a massa de nódulos secos por planta correlacionaram-se com a massa da parte aérea seca, a área foliar e o N total acumulado nas folhas das três leguminosas. Os mesmos rizóbios foram eficientes para o caupi e para o guandu. Três estirpes do guandu (R35, R43 e R45) e duas do caupi (R10 e R17) foram caracterizadas in vitro e todas apresentaram tolerância às concentrações elevadas de ácido nalidíxico, cloranfenicol e tetraciclina, porém, foram sensíveis à estreptomicina e à kanamicina. Todas as estirpes cresceram a 35ºC e, exceto a R17, toleraram o alumínio (10 mg L-1).

Responses to rhizobial inoculation by two promiscuous soybean cultivars in soils of the Southern Guinea savanna zone of Nigeria

TGX soybean lines were bred at IITA Ibadan for promiscuity with indigenous rhizobia in Nigerian soils. Two cultivars, TGX1456-2E and TGX1660-19F, were tested in a 2-year trial for their response to rhizobial inoculation in five farmers' fields within a 60-km radius of Minna town, in the Southern Guinea savanna zone of Nigeria. Using the ELISA method, the competitiveness and persistence of the two elite strains of rhizobia contained in the inoculant mixture were also studied. There was a close relationship between nodulation and the size of resident rhizobial populations, with wide variation in nodulation across the various sites irrespective of the treatments. Cultivar effect on height and nodule number was significant only in the first cropping season of the trial. The inoculant strains appeared to be less competitive, but more effective, than the indigenous populations. The proportions of the nodules occupied by the inoculant strains were 17% in the first cropping season, and 24% in the second. Inoculation with rhizobia increased the percent arbuscular mycorrhizal infection by an average of 50%. Although grain yield varied between sites, no significant cultivar effect was observed. However, inoculation increased grain yield by 40% in the first cropping season, while no such yield differences occurred in the second season. The proportion of nitrogen derived from N2 fixation ranged from 27% to 50% in both cropping seasons, and this was dependent on crop management on farmers' fields, rather than any cultivar or inoculation effect.

토양수분 차이가 대두 근류균의 질소고정에 미치는 영향

대두의 질소고정 근류균들 중 우리나라 토착균, USDA110, USDA122, RJl-29, R2l4 등으로 종자분의접종한 삼남콩을 폿트에 재배하여 개화기에 한발처리한 것과 관행으로 폿트에 재배한 것과의 근류균별 질소고정력 조사와 콩의 제요인에 미치는 근류균의 영향, 한발조건시 근류균 질소고정력과 협중과의 상관관계, 식물체 호흡량과 엽면적과의 상관관계 등을 시험한 결과를 요약하면 다음과 같다. 관행재배에서는 근류균의 질소고정력이 R2l4와 토착균이 높게 나타났으나, 개화기에 한발처리를 한 구에서는 토착근류균 보다 R2l4와 Rll-29의 균들이 질소고정력을 높게 나타내었다. 토착근류균에 비하여 USDA122와 RJl-29의 근류균을 접종한 구가 한발조건에서 식물체 호흡량을 적게 유지하여 엽면적 및 협중의 상대지수를 높게 나타내었다. 한발조건에서 근류균 질소고정력과 협중 상대지수와의 상관관계는 r=0.85'로서 유의적인 정의 상관관계를 나타내었다. 한발조건에서 식물체 호흡량과 엽면적 상대지수와의 상관관계는 r=-0.89'로서 유의적인 부의 상관관계를 나타내었다. The object of this study was to investigate nitrogen fixation activity of rhizobium inoculated at seed coat when drought condition was applied in flowering period of soybean c.v. Samnamkong. The rhizobia used in this experiment were indigenous rhizobium, R2l4, RJl-29, USDA110 and USDA122. The experiment was done with 1/2000 Wagner pots in laboratory and greenhouse and was tested in completely randomized design with four replications. Nitrogen fixation activity in coventional culture was the highest in R2l4 and indigenous rhizobium among the five rhizobia strains. As given drought condition from flowering to maturity, nitrogen fixation activity was higher in R2l4 and RJl-29 than indigenous rhizobium. Leaf area and relative index (drought/convention) of pod weight were higher in USDA122, RJl-29 and R2l4 than indigenous rhizobium as given drought condition from flowering to maturity. High positive correlation was observed between nitrogen fixation activity of rhizobium and relative index(drought/convention) of pod weight. High negative correlation was observed between respiration of plant and relative index (drought/convention) of leaf area.

In vivo cloning strategy for Rhizobium plasmids.

We have developed a simple system to clone indigenous Rhizobium plasmids into E. coli. The strategy consists of three matings: the first is to insert Tn5 in the plasmid to be cloned, the second incorporates the integrative vector into the inserted Tn5 in the native Rhizobium plasmid, and the last mating transfers the target plasmid directly into E. coli. This mating-based system was successfully used to clone plasmids of Rhizobium species with sizes ranging from 150 to 270 kb. In addition, a 500-kb fragment of a 600-kb megaplasmid was also cloned. This strategy could be used for cloning indigenous replicons of other gram-negative bacteria into a different host.