Potential of indigenous bradyrhizobia versus commercial inoculants to improve cowpea (Vigna unguiculata L. walp.) and green gram (Vigna radiata L. wilczek.) yields in Kenya

Limited information is available on reduced cowpea (Vigna unguiculata L. Walp.) and green gram (Vigna radiata L.Wilczek.) yields in Kenya. Declining soil fertility and absence or presence of ineffective indigenous rhizobia in soils are assumptions that have been formulated but still require to be demonstrated. In this study, soils were collected from legume growing areas of Western (Bungoma), Nyanza (Bondo), Eastern (Isiolo), Central (Meru) and Coast (Kilifi) provinces in Kenya to assess indigenous rhizobia in soils nodulating cowpea and green gram under greenhouse conditions. Our results showed that highest nodule fresh weights of 4.63 and 3.32?g plant^?1 for cowpea and green gram were observed in one soil from Isiolo and another from Kilifi, respectively, suggesting the presence of significant infective indigenous strains in both soils. On the other hand, the lowest nodule fresh weights of 2.17 and 0.72?g plant^?1 were observed in one soil from Bungoma for cowpea and green gram, respectively. Symbiotic nitrogen (N) fixation by cowpea and green gram was highest in Kilifi soil with values of 98% and 97%, respectively. A second greenhouse experiment was undertaken to evaluate the performance of commercial rhizobial inoculants with both legumes in Chonyi soil (also from Coast province) containing significant indigenous rhizobia [>13.5?×?10³ Colony Forming Units (CFU) g^?1]. Rhizobial inoculation did not significantly (P?相似文献   

Antibiotic-resistant mutants identified from nodules of uninoculated soybeans grown in a strongly acidic soil

Two hypotheses that antibiotic-resistant nodule isolates from uninoculated soybeans grown in a strongly acidic soil were naturally occurring rhizobia which had acquired resistance to spectinomycin and streptomycin or were contaminants from adjacent, inoculated treatments, were tested in laboratory experiments. Soybean nodule isolates (166) as well as 48 cowpea and 89 Rhizobium japonicum strains were used in tests of resistance to six concentrations (0–500 μg ml^?1) of kanamycin, spectinomycin and streptomycin, tolerance of stresses of pH 4.6, with or without 50 μm Al, and serological cross-reactivity.More strains from the strongly acidic soil were resistant to the antibiotics than from slightly acidic soils, but resistance to antibiotics did not necessarily entail resistance to pH 4.6 or to 50 μm Al. Twenty-three nodule isolates which were resistant to spectinomycin or streptomycin cross-reacted with antisera of the inoculum strains, indicating that they were contaminants. None of 59 antibiotic-sensitive nodule isolates from uninoculated plants and none of 31 from inoculated plants cross-reacted with the antisera. All 53 antibiotic-resistant isolates from nodules of plants in inoculated plots cross-reacted with test antisera, indicating stability of the antibiotic markers.Cowpea rhizobia were generally more resistant to the antibiotics and more tolerant of pH 4.6 and 50 μm Al than were R. japonicum. Among strains of R. japonicum, slow growers were more resistant to antibiotics than moderately fast growers.     

Growth and yield responses of some cowpea accessions to cucumber mosaic virus infection

Effects of cucumber mosaic virus (CMV) on the yield and yield components of six cowpea genotypes (IT04K-217-5, IT07K-251-3-3, IT07K-299-4, IT07K-299-6, IT99K-1060 and Ife Brown) were evaluated in a screenhouse. The experiment was conducted as inoculated and uninoculated control using completely randomised design with five replications. Seedlings were inoculated with CMV at 10 days after sowing by sap transmission and maintained under screenhouse conditions. Disease incidence, disease severity, yield and yield components were recorded. Data were subjected to analysis of variance and significance determined at p = 0.05. All the CMV-inoculated plants exhibited typical leaf chlorosis and curling symptoms of CMV infection. Disease severity (4) and absorbance of virus concentration (2.9) were highest in the inoculated leaves of Ife Brown, whereas the cowpea genotype IT07K-299-6 exhibited the lowest disease severity (2.2) and virus titre (1.7). The lowest reductions in leaf number (6.2%), plant height (3%) and seed weight (2.6%) were found in IT07K-299-6. The cowpea genotype IT07K-299-6 is considered the most tolerant to CMV infection and its tolerance genes could be exploited for germplasm improvement in cowpea breeding programmes.     

Alternatives to peat as a carrier for rhizobia inoculants: Solid and liquid formulations

Many of the microbial inoculants all over the world are based on solid peat formulations. This has been mostly true for well developed legume inoculants based on selected rhizobial strains, due to peat bacterial protection properties. Six carriers (bagasse, cork compost, attapulgite, sepiolite, perlite and amorphous silica) were evaluated as alternatives to peat. Compost from the cork industry and perlite were superior to peat in maintaining survival of different rhizospheric bacteria. Other tested materials were discarded as potential carriers for soybean rhizobia. Also, different liquid culture media have been assayed employing mannitol or glycerol as C sources. Some media maintained more than 10⁹ cfu ml^?1 of Sinorhizobium (Ensifer) fredii SMH12 or Bradyrhizobium japonicum USDA110 after 3 months of storage. Rhizobial survival on pre-inoculated seeds with both solid and liquid formulations previously cured for 15 days led to a higher bacterial numbers in comparison with recently made inoculants. An additional curing time of solid inoculants up to 120 days had a beneficial effect on rhizobial survival on seeds. The performance of different formulations of two highly effective soybean rhizobia strains was assayed under field conditions. Soybean inoculated with cork compost, perlite and liquid formulations produced seed yields that were not significantly different to those produced by peat-based inoculants.     

Enhanced Growth and nodule occupancy of red kidney bean and soybean inoculated with soil aggregate-based inoculant

Volcanic ash soil, which is widely distributed in Japan, contains a large amount of well-structured soil aggregates. By using these aggregates as carrier materials, we prepared (brady)rhizobial inoculants for red kidney bean (Phaseolus vulgaris) and soybean (Glycine max). Autoclaved soil aggregates were inoculated with Rhizobium tropici CIATS99R or Bradyrhizobium japonicum USDA110R, incubated for 15 or 21 d at 30°C, slowly air-dried at 20°C to prepare the aggregate-based inoculants, and stored at various temperatures. The populations of CIATS99R and USDA110R in the aggregate-based inoculants were maintained during several months of storage at 20°C. When the aggregate-based inoculants were mixed with soil, CIATS99R and USDA110R cells showed a remarkably improved survival in soils compared with those mixed with soil without carrier material. The effect of the aggregate-based inoculants on the growth of red kidney bean and soybean was examined in pot experiments. By placing a small amount of the inoculant just beneath the seeds at the time of sowing, plant growth was significantly enhanced compared with the use of traditional peat-based inoculant. In addition, nodule formation on the upper part of soybean roots and nodule occupancy by the inoculated strain were remarkably enhanced by the aggregate-based inoculant. It is suggested that soil aggregates might be suitable carrier materials for preparing cheap and effective (brady)rhizobial inoculants.     

Growth and survival of cowpea bradyrhizobia in various carrier materials

Summary Pakistan does not yet have the technology for commercial production ofRhizobium andBradyrhizobium inoculum. Therefore, investigations were undertaken to evaluate the suitability of different materials like compost, sawdust, rice husks, sugar cane, filter mud, and peat asBradyrhizobium carriers. The growth and survival of bradyrhizobia (strain TAL 441 of the cowpea type) was studied in sterilized and unsterilized carriers mixed with loam and enriched with lucerne meal and sucrose. Three different sterilization methods (autoclaving, gamma irradiation, and dry heating of the carriers) were used. The growth and survival of bradyrhizobia in the inoculants were studied at two different storage temperatures, 4° and 20°C. After 2–21 months of inoculation, maximum survival of rhizobia (7.6 × 10⁹ cells g^–1) was observed in autoclaved filter mud containing loam-lucerne meal and sucrose. The survival of rhizobia in autoclaved peat was 3.4x 109 cells g-t. The maximum viable number of rhizobia per seed of mungbean (Vigna radiata) was 7.7 × 10⁸ in gamma-irradiated compost and least (1 × 10⁷ cells seed^–1) in rice husks.     

Yield and N assimilation of winter wheat (Triticum aestivum L., var. Norstar) inoculated with rhizobacteria

The effects of bacterial inoculants on the growth of winter wheat were studied in a growth chamber. Azospirillum brasilense, Azotobacter chroococcum, Bacillus polymyxa, Enterobacter cloacae, or a mixture of the four rhizobacteria were the inoculants tested. Inoculation effects on yield, yield components, and N-derived from fertilizer (Ndff) were assessed. The response of plants inoculated with individual bacteria was inconsistent and varied with treatment. At the first harvest (58 days after planting-DAP) plants inoculated with the mixture exhibited increases in plant dry weight, total-N and Ndff. At the second harvest (105 DAP), plants inoculated with A. brasilense and the mixture exhibited increases in shoot biomass, whereas at maturity (170 DAP), the inoculated plants showed no differences in total-N or shoot dry matter yield, as compared to the uninoculated controls. Inoculation with A. brasilense, however, increased the Ndff in the shoots, and B. polymyxa tended to enhance grain yield. Practical use of these rhizobacteria as inoculants for winter wheat may have limited value until such time as we better understand factors which influence rhizosphere competence of bacterial inoculants.     

Characterization of a cowpea (Vigna unguiculata) rhizobiophage and its effect on cowpea nodulation and growth

A cowpea rhizobiophage (JRW 3 phage) from Jamaican soil was isolated and characterized. The phage has a polyhedral head and a non-contractile tail; maximum adsorption of the phage to the host occurred after 5 min. A one-step growth experiment revealed that the latent period, rise period and burst size of JRW3 phage were 12 h, 16 h, and 28 plaque-forming units/cell, respectively. The JRW 3 phage was highly sensitive to heat, but survived well between pH 5 and 8. The phage was stable in EDTA, though completely inactivated in sodium citrate. Host range analysis showed that 7 of the 40Rhizobium andBradyrhizobium strains tested were sensitive to phage infection. The phage significantly reduced nodule numbers and shoot dry weight of cowpea plants when inoculated with rhizobia in combination with the phage.     

Combined application of inoculant,phosphorus and organic manure improves grain yield of cowpea

ABSTRACT

Low concentrations of P and organic manure in savanna soils limit cowpea response to rhizobia. The study was conducted to determine the combined effect of P and organic manure on cowpea response to rhizobia in a factorial experiment arranged in randomized complete block design with three replications on smallholder farmers’ fields in northern Ghana in 2015. The factors were two levels of Bradyrhizobium inoculant, two levels of P fertilizer, three treatments of manure (fertisoil, cattle manure, and no manure). Addition of Bradyrhizobium inoculant to P and fertisoil significantly increased shoot biomass yield from 1677 kg ha^?1 in the plots without Bradyrhizobium inoculation to 1913 kg ha^?1. Likewise, the addition of Bradyrhizobium inoculant to P and cattle manure significantly increased shoot biomass from 1437 kg ha^?1 to 1813 kg ha^?1. Grain yield increases of 1427 and 1278 kg ha^?1 were obtained over the control when either fertisoil or cattle manure and P, respectively, were added to Bradyrhizobium inoculant. The value cost ratio for adding Bradyrhizobium inoculant to phosphorus and fertisoil was two indicating that it could be attractive to risk-averse smallholder farmers. The study demonstrated the potential of the combined application of organic matter and P to improve cowpea response to Bradyrhizobium inoculation.     

Inoculation with Native Bradyrhizobia Strains Improved Growth of Cowpea Plants Cultivated on a Saline Soil

Selection of osmotolerant rhizobia gained importance because of increasing soil salinity in many regions. A field experiment in the Cauto Valley was conducted to determine the effect of seed inoculation with two native strains of Bradyrhizobium sp. on biological nitrogen (N)-fixation of cowpea (Vigna unguiculata) on a salt affected soil. Inoculation with both strains resulted in higher numbers of nodules, specific N-fixation, and yields of cowpeas than the control without inoculation. The strain VIBA-1 (Bradyrhizobium liaoningense) was partly superior to VIBA-2 (Bradyrhizobium yuanmingense). This study indicates that the strains inoculated are competitive against other natural strains and able to nodulate cowpea.     

Cowpea rhizobia: Comparison of plant infection and plate counts

Enumeration of Rhizobium in soil is usually accomplished by the most probable number (MPN) plant infection method. The accuracy of MPN counts as compared to plate counts was determined for five strains of cowpea rhizobia. Host plants included cowpea (Vigna unguiculata (L.) Walp.), siratro (Macroptilium atropurpureum (DC.) Urb.) and peanut (Arachis hypogaea L.). Plastic growth pouches were used primarily for cowpea and siratro while plastic cups containing vermiculite were used for peanut. The number of rhizobia determined by the MPN method using cowpea, siratro and peanut underestimated the population from 10- to 100-fold. A control experiment using soybeans (Glycine max (L.) Merrill) indicated that the MPN method was accurate for R. japonicum. Experimentors using the MPN method should be aware of its accuracy for particular Rhizobiutn-legume combinations.     

Population size,distribution, and symbiotic characteristics of indigenous Bradyrhizobium spp. that nodulate TGx soybean genotypes in Africa

Soils of many potential soybean fields in Africa are characterized by low levels of biological nitrogen fixation (BNF) activities and often cannot support high soybean yields without addition of inorganic N fertilizers or external application of soybean rhizobia. The most probable number (MPN) technique was used to determine the bradyrhizobial populations that nodulate TGx soybean genotypes (a cross between nonpromiscuous North American soybean genotypes and promiscuous Asian soybean genotypes), cowpea or North American soybean cv. Clark IV, in soils from 65 sites in 9 African countries. The symbiotic effectiveness of isolates from these soils was compared to that of Bradyrhizobium japonicum strain USDA110. The bradyrhizobial population sizes ranged from 0 to 10⁴ cells g⁻¹ soil. Bradyrhizobium sp. (TGx) populations were detected in 72% and B. japonicum (Clark) in 37% of the soil samples. Bradyrhizobium sp. (TGx) populations were generally low, and significantly less than that of the cowpea bradyrhizobial populations in 57% of the samples. Population sizes of less than 10 cells g⁻¹ soil were common as these were detected in at least 43% of the soil samples. B. japonicum (Clark) occurred in higher population densities in research sites compared to farmers’ fields. Bradyrhizobium sp. (TGx) populations were highly correlated with biotic but not abiotic factors. The frequent incidence of low Bradyrhizobium sp. (TGx) populations is unlikely to support optimum BNF enough for high soybean yields while the presence of B. japonicum (Clark) in research fields has the potential to compromise the selection pressure anticipated from the indigenous Bradyrhizobium spp. (Vigna) populations. Bradyrhizobium isolates could be placed in four symbiotic phenotype groups based on their effectiveness on a TGx soybean genotype and the North American cultivar Clark IV. Symbiotic phenotype group II isolates were as effective as B. japonicum strain USDA110 on both soybean genotypes while isolates of group IV were effective on the TGx soybean genotype but not on the Clark IV. The group IV isolates represent a unique subgroup of indigenous bradyrhizobia that can sustain high soybean yields when available in sufficient population densities.     

Characterization of local rhizobia in Thailand and distribution of malic enzymes

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD⁺-dependent (DME: EC 1.1.1.39) and NADP⁺-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was performed to survey the distribution of the malic enzyme genes among the local rhizobia, which are similar to those of B. japonicum. DNA probes (ME1 for DME and ME2 for TME) were prepared by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes. Southern blot analysis with ME1 as a probe showed a single band in about half of the isolates, especially in B. japonicum and R. leguminosarum strains, suggesting the wide distribution of such DME genes among local rhizobia. In contrast, Southern blot analysis with ME2 as a probe detected a single band only in five B. japonicum strains, suggesting that the TME genes, which are similar to those of B. japonicum, would be unique in a group of B. japonicum.     

高效亲和的花生寄主植物-根瘤菌株组合

本文针对栽培花生，从大、小共生体双方研究了寄主植物－根瘤菌株组合的亲和性。结果指出：（１）在温室水培盆栽条件下，用菌株１４７－３接种的寄主植物结瘤、固氮能力育成品种〉普通型；（２）血清学鉴定出５个不同血清型菌株，其竞争力或回收率与寄主品种、根瘤菌株和土著菌数密切相关；（３）在温室与田间条件下，不同花生寄主一根瘤菌株组合存在着广谱或特异共生亲和性与非亲和性的差异，并鉴定出高效、广谱亲和的品种徐州６－     

Quantification of nitrogen fixation by the peanut/Rhizobium symbiotic system in a virgin sandy soil

Nitrogen balance studies were conducted to quantify the nitrogen fixed by peanut/Rhizobium symbiotic system under field conditions in a sandy soil. Large scale inoculation with three NifTAL strains of cowpea rhizobia, 1000, 169, 1371 was done using two inoculation techniques: peat-based inoculant or injection of inoculant with irrigation water through an injection tank attached to the central pivot system. The results show nitrogen fixation amounting up to 186 kg N ha^?1 in peat-based inoculant and 171 kg N ha^?1 in liquid inoculant injected through the irrigation system. However, no significant differences in yield response were recorded between both inoculation techniques.     

Influence of inoculation with bradyrhizobium japonicum and glomus claroideum on seed yield of soybean under greenhouse and field conditions

Soybean plants [Glycine max (L.) Men., cv. Polanka], inoculated with the VAM fungus Glomus claroideum Schenck and Smith and Bradyrhizobium japonicum strain D344, were grown in pots and in the field. The VAM fungus positively influenced N2 fixation, nodulation and N, P, K, and Mg concentrations in the leaves. In pots, (at green pods formation) VAM inoculated plants produced a 24% greater biomass as compared with non‐inoculated plants colonized by native VAM populations. Under field conditions characterized by a high level of P and N, the seed yield of VAM inoculated plants increased in comparison with non‐ and only rhizobia‐inoculated soybean (+28% and 17%, respectively). Glomus claroideum was capable of competing with the native VAM populations both in the greenhouse and in the field experiment.     

Genetic variability in dinitrogen fixation between cowpea [Vigna unguiculata (L.) Walp] cultivars determined using the nitrogen-15 isotope dilution technique

 N fixed in 16 cultivars of cowpea [Vigna unguiculata (L.) Walp] inoculated with effective Bradyrhizobium strains collected from the West African MIRCEN culture collection was measured by ¹⁵N isotope dilution technique. In all plant parts, significant differences in the percentage of N derived from the atmosphere (%Ndfa) and the amount of Ndfa occurred between the cultivars. Ndoute variety exhibited the highest %Ndfa (74.33% in shoots; 60.90% in roots) and accumulated more fixed N (960 mg N plant^–1 and 38 mg N plant^–1 in shoots and roots, respectively). Therefore this cultivar should be selected as the highest N-fixing cowpea cultivar. It also should be used in a breeding programme to contribute to the development of cultivars that could stimulate an intensive use of cowpea in many different cropping systems in Africa with a view to maintaining soil fertility. Received: 14 June 1999     

Genetic diversity and phylogeny of indigenous rhizobia from cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp.]

16S rRNA RFLP, 16S rRNA sequencing, 16S-23S rRNA Intergenetic Spacer (IGS) RFLP and G-C rich random amplified polymorphic DNA (RAPD) assays were conducted to genetically characterise indigenous cowpea [Vigna unguiculata (L.) Walp.] rhizobia from different geographic regions of China. Isolated cowpea rhizobia comprised six 16S rRNA genospecies. Genotype I was composed of 14 isolated strains and the reference strains of B. japonicum and B. liaoningense. This group was divided into two sub-groups respectively related to B. japonicum and B. liaoningense by 16S rRNA sequencing, IGS restriction fragment length polymorphism and RAPD assays. Genotype II composed of 27 isolates from a variety of geographic regions. Four different assays confirmed this group was genetically distinct from B. japonicum and B. liaoningense and probably represent an uncharacterised species. Strains isolated from Hongan, Central China and B. elkanii were grouped to genotype III. Strain DdE4 was solely clustered into genotype IV and related to Rhizobium leguminosarum. Genotypes V and VI consisted of six fast-growing isolates and clustered with reference strain of Sinorhizobium fredii. Comparing with the miscellaneous slow-growing isolates, fast-growing isolates mainly isolated from cowpea cultivar Egang I exhibited strict microbe–host specificity except SjzZ4. Nucleotide sequences reported were deposited in the GenBank with the accession numbers DQ786795–DQ786804.     

Co-inoculation of Acacia mangium with Glomus intraradices and Bradyrhizobium sp. in aeroponic culture

Acacia mangium grown in aeroponic culture was co-inoculated with selected strains of Bradyrhizobium sp. and Glomus intraradices. A single-step technique using alginate as an embedding and sticking agent for an inoculum composed of arbuscular mycorrhiza (AM)-infected sheared roots was used to infect plants. This method resulted in the successful establishment of AM in 100% of the inoculated plants after 7 weeks. The results indicated that dual microbial inoculation with Glomus intraradices strain S-043 and Bradyrhizobium strain AUST 13C stimulated the growth of A. mangium in aeroponic culture. The effects of single and dual microbial inoculations were also evaluated at two levels of P in the nutrient medium. A concentration of 5 mg P kg^–1 stimulated the development of AM without affecting plant development or establishment of Bradyrhizobium symbiosis. In contrast, saplings supplemented with a higher concentration of P (25 mg kg^–1) alone or co-inoculated with Bradyrhizobium had lower AM frequencies.