Integrated soil fertility management enhances population and effectiveness of indigenous cowpea rhizobia in semi-arid eastern Kenya

Legume biological nitrogen fixation is an environmentally friendly and economical means that can reduce low resource farmer dependence on expensive chemical nitrogen (N) fertilizers. We investigated the effect of two cowpea (Vigna unguiculata (L.) Walp) varieties (IT95K-52-34, an international variety from IITA and Kang’au, a local variety) under an integrated soil fertility management trial on indigenous symbiotic rhizobia in a semi-arid farmer's field in eastern Kenya. The ox-ploughed field trial had the following treatments: an unamended control, manure applied at 2.5 t ha⁻¹, triple superphosphate (TSP as (P₂O₅, 0:46:0) at 15 kg ha⁻¹; and a combination of manure and TSP applied at the single rates. Soil samples were collected from each treatment during planting and harvesting of the cowpea crop and used in most probable number (MPN) plant infection assays with the two cowpea varieties as traphosts in Leonard jar growth systems and grown under glasshouse conditions. Generally, soil amendments enhanced cowpea rhizobial populations which varied from 4.89 × 10² rhizobia g⁻¹ soil to 1.074 × 10³ rhizobia g⁻¹ soil. The highest shoot biomass accumulation occurred on cowpea variety IT95K-52-34 plants inoculated with soils from the manure applied plots. We isolated 150 fast- and slow-growing cowpea rhizobia. Contrary to most previous studies, the bulk (97%) of the isolates was fast growing which grouped into 9 types on the growth characteristics on yeast extract agar (YEMA). The study indicated that ISFM was important for rhizobia population build up over a cowpea-growing season.     

Isolation,selection and evaluation of Rhizobium under controlled conditions

Abstract

Rhizobia were screened in growth chamber and greenhouse tests with the host plants: bean (Phaseolus vulgaris L.), lentil (Lens esculenta Moench.), cowpea (Vigna unguiculata (L.) Walp) and peanut (Arachis hypogaea L.). Rhizobial isolates varied in effectiveness, time to nodulation, and host plant specifities. Initial screening procedures in plastic growth pouches allowed selection of infective strains of Rhizobium. These tests enabled the selection of a small number of highly effective isolates for more critical evaluation. Highly significant correlations (p=0.01) were obtained between shoot dry weight and total nitrogen, suggesting that the technique was applying sufficient selection pressure to differentiate variation in N₂ fixation among strains of Rhizobium. Results indicate that highly effective rhizobia can be efficiently selected under controlled conditions following a step‐wise procedure.     

Abundance and characterization of cowpea miscellany Rhizobium from sudanese soils

Sudan is the fourth largest exporter of groundnuts in the world, yet little is known concerning the plant-rhizobial symbiosis. A study was made on the abundance of groundnut-nodulating rhizobia in the soils of Sudan as related to soil properties and the duration since groundnuts were last planted. Also, physiological, serological and nitrogen-fixing characteristics of Sudanese rhizobia are reported. All but one of 32 sites contained more than 300 rhizobia g^?1 soil capable of forming nodules on siratro (Macroptilium atropurpureum). Several of these soils had never been planted to groundnut. A correlation matrix indicated no relationship was present between soil rhizobial populations and any of the measured soil properties, or between soil rhizobial populations and the time since groundnuts were last planted in the rotation. Individual isolates of Rhizobium from six legumes: groundnut (Arachis hypogaea), mung bean (Vigna radiata), lubia (Dolichos lablab), cowpea (Vigna unguiculata), pigeonpea (Cajanus cajan) and bambara groundnut (Voandzeia subterranea) were obtained from four locations in Sudan. All isolates were able to nodulate each of the six legumes when grown in sterile vermiculite. The isolates grew in 0.1% NaCl-amended media, but growth was variable in 2.0% amended media. Most isolates grew after exposure to moist heat for 15 min at 50°C. Optimum pH for growth was, in general, between pH 6 and 8. Agglutination reactions indicated isolates from groundnuts, as well as isolates from other legumes, belonged to several serological groupings. Some isolates formed a large number of nodules on a Sudanese groundnut cultivar, whereas other isolates formed only few nodules.     

Intrinsic antibiotic resistance in relation to colony morphology in three populations of West African cowpea rhizobia

Rhizobia isolated from cowpeas (Vigna unguiculata (L.) Walp.) grown in three west African soils were examined for intrinsic resistance to five antibiotics and were scored for one of two colony morphologies. Half of the strains tested had a “wet”, slimy colony morphology and half had small discrete “dry”, non-slimy colonies. The populations as a whole were resistant to gentamicin (87%) but varied in their resistance to streptomycin, rifampicin, kanamycin and penicillin. Thirteen patterns of resistance were found for the 128 strains screened and strains within the same pattern usually had the same colony type. The most common pattern (32%) was resistance to all five antibiotics. Associations between resistances were random within populations and colony type. Each population was diverse, expressing from 6 to 8 patterns of resistance, but one population was relatively homogeneous, with 68% of its members exhibiting the same pattern. Correlations between intrinsic antibiotic resistance and colony type were demonstrated both for the west African cowpea rhizobia and for a broader group of cowpea miscellany rhizobia. The method was practical, rapid and reliable for identification of groups within populations.     

Quantitative PCR assays to enumerate Rhizobium leguminosarum strains in soil also target non viable cells and overestimate those detected by the plant infection method

The plant infection method is commonly used to estimate the Most Probable Number (MPN) of soil rhizobia. Here, a qPCR method was set-up and validated with newly developed ANU (strain specific) and RHIZ (more general) primers to quantify the specific Rhizobium leguminosarum bv. trifolii ANU843 strain or general R. leguminosarum strains. Detection limits of qPCR protocols in soil were 1.2 × 10⁴ (ANU) and 4.2 × 10³ (RHIZ) cells per g soil. The qPCR assay appears robust and accurate in freshly inoculated soils but overestimated MPN for indigenous soil rhizobia. An incubation experiment showed that qPCR detected added DNA or non viable cells in soils up to 5 months after addition and incubation at 20 °C in moist conditions.     

Numbers of clover rhizobia needed for crown nodulation and early growth of clover in soil

Low soil populations of Rhizobium leguminosarum biovar trifolii indicate a need for inoculating clovers (Trifolium sp.) at planting. The number of rhizobia in soil varies considerably from field to field and the number needed for nodulation on the upper taproot and for vigorous seedling development is not known. Two experiments were undertaken using arrowleaf clover (T. vesiculosum Savi) and crimson clover (T. incarnatum L.) grown in pots filled with soil. Two soils were used; one contained 10 indigenous rhizobia g^-1 and the other contained fewer than three. The treatments consisted of amending each soil with two strains of inoculant rhizobia to contain from 10 to approximately 1×10⁶ rhizobia g^-1 followed by planting to clover. The number of nodules near the top of the root increased as the number of rhizobia in the soil increased to the highest inoculum level. A low number (approximately 1×10³ to 1×10⁴) of rhizobia was sufficient for maximal N content of seedlings. It seems that soil containing 100 or fewer rhizobia g^-1 may respond to inoculation with increased crown nodulation and seedling vigor.     

Co-selection of compatible rhizobia and vesicular-arbuscular mycorrhizal fungi for cowpea in sterilized and non-sterilized soils

Summary We selected two isolates of Rhizobium for cowpea (Vigna unguiculata) with sterilized soil tests and two different isolates by non-sterilized soil testing. The four rhizobia were then paired individually with either Glomus pallidum, Glomus aggregatum, or Sclerocystis microcarpa in separate, sterilized, or non-sterilized soil experiments. The purpose of the experiments was to determine the effect of soil sterilization on the selection of effective cowpea rhizobia, and to see whether these rhizobia differed in their effects on cowpea growth when paired with various vesicular-arbuscular mycorrhizal (VAM) fungi. Our experiments showed that the rhizobia selected in sterilized soil tests produced few growth responses in the cowpea compared to the other introduced rhizobia, irrespective of pairing with VAM fungi in sterilized or non-sterilized soil. In contrast, the two rhizobia initially selected by non-sterilized soil testing significantly improved cowpea growth in non-sterilized soil, especially when paired with G. pallidum. Our results suggest that it is important to select for effective rhizobia in non-sterilized soil, and that pairing these rhizobia with specific, coselected VAM fungi can significantly improve the legume growth response.     

Role of rhizobial exopolysaccharides in crack entry/intercellular infection of peanut

The early stages of the symbiosis in legumes, when rhizobia enter the plant root, penetrate into the root cortex and spread inside nodules, vary among legume species. The most studied infection mechanism involves infection thread formation while the most simple occur by crack entry/intercellular spreading mode as in Arachis hypogaea L. (peanut). Using a peanut nodulating rhizobia strain impaired in the exopolysaccharide production we have examined the nature of its symbiotic deficiency. Our observations indicate that the inoculation of Arachis hypogaea L. with this exopolysaccharide deficient mutant results in the formation of nodule-like structures and very few nitrogen-fixing nodules. These data contribute to our understanding of exopolysaccharides role in the plant-rhizobia interactions leading to the formation of determinate nodules, particularly relevant for legumes that are infected without infection thread formation.     

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   

Cropping history affects nodulation and symbiotic efficiency of distinct hairy vetch (Vicia villosa Roth.) genotypes with resident soil rhizobia

Compatible rhizobia strains are essential for nodulation and biological nitrogen fixation (BNF) of hairy vetch (Vicia villosa Roth, HV). We evaluated how past HV cultivation affected nodulation and BNF across host genotypes. Five groups of similar HV genotypes were inoculated with soil dilutions from six paired fields, three with 10-year HV cultivation history (HV+) and three with no history (HV?), and used to determine efficiency of rhizobia nodulation and BNF. Nodulation was equated to nodule number and mass, BNF to plant N and Rhizobium leguminosarum biovar viceae (Rlv) soil cell counts using qPCR to generate an amplicon of targeted Rlv nodD genes. Both HV cultivation history and genotype affected BNF parameters. Plants inoculated with HV+ soil dilutions averaged 60 and 70 % greater nodule number and mass, respectively. Such plants also had greater biomass and tissue N than those inoculated with HV? soil. Plant biomass and tissue N were strongly correlated to nodule mass (r ²?=?0.80 and 0.50, respectively), while correlations to nodule number were low (r ²?=?0.50 and 0.31, respectively). Although hairy vetch rhizobia occur naturally in soils, past cultivation of HV was shown in this study to enhance nodulation gene-carrying Rlv population size and/or efficiency of rhizobia capable of nodulation and N fixation.     

Population size and N2-fixing activity of native peanut rhizobia in soils of Thailand

Summary The objective of this study was to assess the number and effectiveness of peanut rhizobia in soils of the major peanut-growing areas of Thailand. Three cropping areas, (1) continuously cropped with peanuts, (2) continuously cropped with non-legumes, and (3) non-cultivated fields, were chosen in each region. Peanut rhizobia were found in the soil at 38 to 55 sites sampled. Cultivated fields with a peanut cultivation history contained (as estimated by most probable numbers) an average of 1.6×10³ cells g^-1 of soil. The numbers of peanut rhizobia in most of the fallow fields and some of the noncultivated shrub or forest locations were much the same as at the sites where Arachis hypogaea was cultivated. In contrast, there were no or few (28–46 cells g^-1 soil) peanut rhizobia in the majority of fields continuously cultivated with sugarcane, cassava, corn, and pineapple. It appears that in these areas the indigenous peanut rhizobial populations are not adequate in number for a maximal nodulation of peanuts. A total of 343 Bradyrhizobium isolates were tested for effectiveness and were found to vary widely in their ability to fix N₂. In some areas the majority of rhizobia were quite effective while in others they were less effective than the inoculum strain THA 205 recommended in Thailand.     

Effect of fertilizers, lime, and inoculation with rhizobia and mycorrhizal fungi on the growth of four leguminous tree species in a low-fertility soil

The aim of this study was to evaluate the effects of lime, fertilizers, mycorrhizal fungi, and selected rhizobia strains on the growth of four woody legume species, Albizia lebbeck (L.) Benth., Enterolobium contortisiliquum (Vell.) Morong., Leucaena leucocephala (Lam.) de Wit, and Sesbania virgata (Cav.) Pers. in a low-fertility soil. The experiment was conducted under greenhouse condition in plastic pots (4 kg). Eight treatments and eight replicates per treatment were performed in a completely randomized design. The treatments were: (1) complete treatment (C) (NPK fertilization?+?micronutrients?+?liming?+?MR that is inoculation with mycorrhizal fungi and rhizobia); (2) C minus N (C???N that is as C without the addition of N); (3) C???N???M (as C???N without inoculation with arbuscular mycorrhizal fungi (AMF)); (4) C???N??R (as C???N without inoculation of rhizobia); (5) C???N???liming (as C???N without liming); (6) C???N???micro (as C???N without addition of micronutrients); (7) C???N???P (as C???N without addition of P); (8) control without fertilization, liming, and without inoculation with AMF and rhizobia. After 4 months of growth, we determined the yield of individual plants, nodulation, mycorrhizal colonization, and nutrient contents. Phosphorus was the most limiting nutrient for plant growth, followed by nitrogen. L. leucocephala and S. virgata had the most robust response to the addition of micronutrients and liming, showing an increase in nutrient content, plant height, and root and shoot dry matter. When compared to the single inoculation, the dual inoculation increased growth of all plants, except that of A. lebbeck, which did not respond to either rhizobia or mycorrhizal fungi inoculation.     

Survival of rhizobia in soil is sensitive to elevated zinc in the absence of the host plant

The survival of free-living rhizobia in soil is sensitive to elevated heavy metals in soil and can explain adverse effects of metals on symbiotic nitrogen fixation in soils. A survival experiment was set-up to derive critical cadmium (Cd) and zinc (Zn) concentrations in a range of field-contaminated soils in the absence of their host plant (Trifolium repens L.). Soils applied with metal salts or sewage sludge >10 years ago were sampled and were inoculated with Rhizobium leguminosarum bv. trifolii (10⁸ cells g⁻¹ soil) and incubated outdoors for up to 6 months. The most probable number (MPN) decreased 1-2 orders of magnitude in uncontaminated soils during the incubation. There was no significant effect of total metal concentrations on rhizobia survival in soils contaminated with Cd salts or with high Ni/Cd sewage sludge with highest Cd concentrations between 18 and 118 mg Cd kg⁻¹. In contrast, survival was strongly affected in soils contaminated by sewage sludge, where Zn was the principal metal contaminant. Neither total Cd nor soil solution Cd was large enough to attribute these effects to Cd when compared with the soil series, where Cd salts had been applied. The MPN decreased at least one order of magnitude above total Zn concentrations of 233 mg Zn kg⁻¹ (soil pH 5.6) and 876 mg Zn kg⁻¹ (soil pH 6.3). The EC50s of log MPN were 204 and 604 mg Zn kg⁻¹, respectively, and were lower than those for the symbiotic nitrogen fixation measured in the pot trial on the same soils (respectively 602 and 737 mg Zn kg⁻¹). This study corroborates the evidence that symbiotic nitrogen fixation is affected by Zn in the field when Zn decreases the free-living population of rhizobia to below a critical threshold.     

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.     

Root exudates as pre-invasive factors in the modulation of chick pea varieties

Five chick pea (Cicer arietinum L.) varieties when inoculated with an effective Rhizobium strain showed significant variations in nodule number. The root exudates of the plant varieties were analysed for sugars, TCA cycle intermediates and amino acids. Xylose, citrate, alanine and phenylalanine were detected in exudates of one or two varieties, in addition to compounds common to all varieties such as glucose, glucosamine, ribose, serine, homoserine and glutamic acid. However, the concentration of all these compounds in the exudates collectively was inadequate to support Rhizobium growth under aseptic conditions. Several of the exuded compounds were chemotactic at very low concentrations. The rhizobia showed taxes toward growing roots of all the varieties in agar plates. Competition experiments showed that the rhizobia have a multiple-chemotactic system like that of Escherichia coli. It appears that root exudates accumulate Rhizobium along the growing roots through chemotaxis. The minor differences in exudate composition among varieties is not a factor responsible for variation in nodule number.     

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.     

Use of fungicide-resistant rhizobia for legume inoculation

Isolates of Rhizobium phaseoli resistant to spergon (2,3,5,6-tetrachloro-l,4-benzoquinone), Rhizobium meliloti resistant to thiram (tetramethylthiuram disulfide) and of a cowpea Rhizobium resistant to phygon (2,3-dichloro-l,4-naphthoquinone) were obtained by culturing the bacteria in media with increasing concentrations of these fungicides. The cultures grew in media with 200 μg thiram ml^?1, 150 μg spergon ml^?1 or 400 μg phygon ml^?1. Spergon-tolerant R. phaseoli was sensitive to thiram, and thiram-tolerant R. meliloti was sensitive to spergon. The dry weights of beans, alfalfa and cowpeas and the amount of N₂ fixed were the same for plants inoculated with the fungicide-resistant or the sensitive parent rhizobia. However, when the three parent Rhizobium strains were applied to seeds treated with the three fungicides, the plants that developed were stunted, chlorotic, grew poorly and fixed little or no N₂. By contrast, beans, alfalfa or cowpea plants derived from seeds coated with spergon, thiram or phygon and inoculated with the resistant rhizobia grew as well and fixed as much N₂ as legumes derived from seeds not treated with the pesticides. These findings provide the basis for a simple method for simultaneously allowing for N₂ fixation and seed protection of legumes.     

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.     

Colonization of rhizospheres and nodulation of two Vigna species by rhizobia inoculated onto seed: Influence of soil

Changes in the populations of Rhizobium strains CB756str, CB985 and CB1024strspc in the rhizospheres of cowpea (Vigna unguiculata) and black gram (V. mungo) grown at three sites were evaluated. The population dynamics of the three rhizobia varied with soil type but the strain responses on the two legumes were generally similar. Most noticeable was the ability of CB756str to grow in the sandy soil (Beerwah) but not in the heavy clays (Narayen and Emerald). In contrast, the levels of CB1024strspc and CB985 generally increased in the clay soils.Nodulation (% due to the inoculum strain) did not always reflect events within the rhizosphere. Although not suited to Narayen, CB756str formed a similar proportion of the nodule population of black gram as CB1024strspc but this may have been due to higher seed inoculum levels of CB756str. At Emerald nodulation by all three strains of rhizobia was poor regardless of the success in colonization of the rhizosphere. Successful competition for nodule sites by native rhizobia may contribute to this discrepancy between Narayen and Emerald although lower seed inoculum levels at Emerald may also have been important.Nodule decay was consistently associated with a large increase in the number of rhizobia per root system. This is likely to be important in the survival of strains into the following season.Comparisons of nodulation by parent and mutant rhizobia suggested that resistance to antibotics may have slightly reduced nodule forming ability for CB1024strspc on black gram at Emerald.