Grouping of Bradyrhizobium USDA strains by sequence analysis of 168 rDNA and 168-238 rDNA internal transcribed spacer region

In order to select appropriate Bradyrhizobium USDA reference strains for primary grouping of indigenous soybean bradyrhizobia, we systematically constructed phylogenetic trees of 20 USDA strains based on DNA sequence analysis and PCR-restriction fragment length polymorphism (RFLP) targeted to 16S rDNA and the internal transcribed spacer (ITS) region between 16S and 23S rDNAs. The phylogenetic trees of 16S rDNA showed 3 major groups, cluster USDA 110 (USDA 62, 110, 122, 125, and 129), cluster USDA 6 (USDA 4, 6^T, 38, 115, 123, 127, 135, and 3622^T) and cluster B. elkanii (USDA 31, 46, 61, 76^T, 94, and 130), as well as the phylogenetically independent strain USDA 124. The topology of the ITS trees was almost similar to that of 16S rDNA, although the positions of two extra-slow-growing strains, USDA 135 and USDA 3622^T were variable among the ITS sequences, PCR-RFLP of the ITS region and 16S rDNA. Only two strains, USDA 110 and USDA 122, harbored hup genes and they fell into the USDA 110 cluster. These results suggest that PCR-RFLP analysis of 16S rDNA and the 16S-23S rDNA ITS region may be useful for the grouping of bradyrhizobia and for the first screening of hup-positive strains. Based on the above results, we propose a minimum set of USDA strains reflecting Bradyrhizobium diversity that includes B. japonicum USDA 6^T, B. japonicum USDA 110, B. japonicum USDA 124, and B. elkanii USDA 76^T. In addition, an extra-slow-growing strain with the serotype USDA 135 might be necessary for genomic diversity analysis of bradyrhizobia, because their phylogenetic positions were variable.     

Diversity and geographical distribution of indigenous soybean-nodulating bradyrhizobia in Japan

Abstract

Genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in Japan were investigated based on restriction fragment length polymorphism analysis of PCR product (PCR-RFLP) analysis of the 16S?23S rDNA internal transcribed spacer (ITS) region using Bradyrhizobium USDA strains as reference strains. Soil samples were collected from five field sites in Hokkaido, Fukushima, Kyoto, Miyazaki and Okinawa in Japan. A total of 300 isolates were derived from three Rj-genotype soybean cultivars, Akishirome (non-Rj), CNS (Rj ₂ Rj ₃) and Fukuyutaka (Rj ₄), and five field site combinations. The PCR products of the ITS region were digested with HaeIII, HhaI, MspI and XspI. Electrophoresed patterns were analyzed for phylogenetic relationship using Bradyrhizobium reference strains. Results revealed 22 RFLP patterns and 11 clusters. The RFLP patterns of the seven clusters were similar or identical to Bradyrhizobium japonicum USDA 6, 38, 110, 115, 123 and Bradyrhizobium elkanii USDA 76 and 94. Four minor clusters were independent from the clusters of the reference strains. The isolation ratio revealed the major clusters at each field site. These results suggested that major clusters of indigenous bradyrhizobia might be in the order Bj123, Bj38, Bj110, Bj6 and Be76 from the northern to southern regions in Japan.     

Diversity,phylogeny and host specificity of soybean and peanut bradyrhizobia

A study on the diversity, phylogeny, and host specificity of soybean (Glycine max L.) and peanut (Arachis hypogaea L.) bradyrhizobia was conducted based on the 16S ribosomal RNA (rRNA) restriction fragment length polymorphisms (RFLPs), 16S rRNA sequencing, and 16S–23S rRNA intergenetic spacer (IGS) RFLP assays. Based on 16S rRNA RFLP assay, tested bradyrhizobia were divided into five genotypes, which could be further clustered into five groups by IGS RFLP assays. According to the 16S rRNA sequencing, strains of IGS-II, IV, and V were phylogenetically related to Bradyrhizobium liaoningense, Bradyrhizobium japonicum, and Bradyrhizobium elkanii, while strains of IGS-Ic and IGS-III related to Bradyrhizobium yuanmingense and Bradyrhizobium canariense, respectively. All isolates could crossly nodulate Phaseolus vulgaris, forming small white nodules. Strains of IGS-II originally isolated from peanut could efficiently nodulate Glycine soja, and two strains isolated from soybean could also nodulate peanut.     

Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability

Summary Bacteria isolated from the root zones of field-grown soybean plants [Glycine max (L.) Merr.] were examined in a series of glasshouse experiments for an ability to affect nodulation competition among three strains of Bradyrhizobium japonicum (USDA 31, USDA 110, and USDA 123). Inocula applied at planting contained competing strains of B. japonicum with or without one of eleven isolates of rhizosphere bacteria. Tap-root nodules were harvested 28 days after planting, and nodule occupancies were determined for the bradyrhizobia strains originally applied. Under conditions of low iron availability, five isolates (four Pseudomonas spp. plus one Serratia sp.) caused significant changes in nodule occupancy relative to the corresponding control which was not inoculated with rhizosphere bacteria. During subsequent glasshouse experiments designed to verify and further characterize these effects, three fluorescent Pseudomonas spp. consistently altered nodulation competition among certain combinations of bradyrhizobia strains when the rooting medium did not contain added iron. This alteration typically reflected enhanced nodulation by USDA 110. Two of these isolates produced similar, although less pronounced, effects when ferric hydroxide was added to the rooting medium. The results suggest that certain rhizosphere bacteria, particularly fluorescent Pseudomonas spp., can affect nodulation competition among strains of R. japonicum. An additional implication is that iron availability may be an important factor modifying interactions involving the soybean plant, B. japonicum, and associated microorganisms in the host rhizosphere.Paper No. 10648 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA     

土壤中大豆根瘤菌优势类群以及与寄主共生关系的多样性

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

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.     

Rhizobiophage effects on nodulation,nitrogen fixation,and yield of field-grown soybeans (Glycine max L. Merr.)

Summary Previous laboratory and greenhouse studies have shown that phages significantly reduce soil populations of homologous rhizobia. Reductions in nodulation and N₂ fixation have also been observed. The purpose of the current study was to examine the effect of a phage specific ofBradyrhizobium japonicum USDA 117 on nodulation, nodule occupancy, N₂ fixation and soybean growth and yield under field conditions. The phage was inoculated in combination withB. japonicum USDA 117 and/orB. japonicum USDA 110 (resistant strain) into a rhizobia-free sandy loam soil and planted toGlycine max (L.) Merr. Williams. When the phage was applied to soil inoculated withB. japonicum USDA 117 alone, significant reductions in nodule weight and number, shoot weight, foliar N, nitrogenase activity, and seed index were observed. When, however, the soil also contained the non-homologous strain,B. japonicum USDA 110, no significant effects on any of these parameters were found. Nodule occupancy by competing strains ofB. japonicum USDA 110 and USDA 117 was also affected by the phage. In soil which did not contain the phage, 46% and 44% of the identified nodules were occupied by USDA 110 and 117, respectively. When the phage was present in the soil, nodule occupancy byB. japonicum USDA 117 was reduced to 23%, while occupancy byB. japonicum USDA 110 was increased to 71%. These results suggest that nodulation by selected strains of rhizobia can be restricted and nodulation by more effective, inoculated strains can be increased through the introduction of a homologous phage to soils.     

Serological properties and intrinsic antibiotic resistance of soybean bradyrhizobia isolated from Thailand

A group of Bradyrhizobium strains isolated from soybean plants in Thailand did not correspond to any known DNA homology groups of Bradyrhizobium japonicum and Bradyrhizobium elkanii reported by Hollis et al. (J. Gen. Microbiol., 123, 215–222, 1981). To clarify the phenotypic characteristics of the group, serological properties and intrinsic antibiotic resistance (IAR) profile of 94 Thai strains were compared with those of USDA and Japanese strains. Indirect ELISA tests for each Thai strain were performed agaiIl.st polyclonal antisera prepared against 15 USDA standard serotype strains of B. japonicum and B. elkanii. Among the 94 Thai strains tested, 36 which were previously identified as B. elkanii, with the exception of one strain, were strongly responsive to an antiserum prepared against USDA 31. The remaining 58 strains, with the exception of two strains, showed multiple cross reactions which were peculiar to the Thai strains. These serological reaction patterns did not correspond to any known serogroups labeled as B. japonicum and B. elkanii. In the IAR test, the taxonomically unknown Thai soybean bradyrhizobia exhibited a high level of resistance to neomycin (50 µg/mL), polymyxin (50 µg/mL), nalidixic acid (15 µg/mL), and kanamycin (15 µg/mL). Kanamycin could thus be useful in combination with neomycine and nalidixic acid for distinguishing between the unknown Thai strains and strains of B. japonicum and B. elkanii. Our results demonstrated that the unknown Thai strains were serologically and IAR-phenotypically remote from both B. japonicum and B. elkanii.     

Selection of type A and type B strains for improving symbiotic effectiveness on non-Rj and Rj4 genotype soybean varieties

The selection of effective rhizobia for higher efficiency nitrogen fixation is one of the most important steps for inoculant production. Therefore, this experiment was conducted to select the most effective type A and type B strains for specific Rj-gene harboring soybean varieties and to test the symbiotic effectiveness of selected strains on different Rj-gene harboring soybean varieties. Screening experiments using the specific soybean varieties were done with a completely randomized design and three replications in this study. Evaluation of the effective Myanmar Bradyrhizobium strains for plant growth, nodulation and N₂ fixation were studied in pot experiments using sterilized vermiculite in the Phytotron (controlled-environmental condition). Then, a pot experiment was conducted using Futsukaichi soil in the screen house (natural environmental condition). The N₂ fixation ability of soybean was evaluated by acetylene reduction activity (ARA) and the relative ureide index method. In the first screening experiment, type A and type B strains with higher nitrogen fixation and proper nodulation on their respective soybean cultivars were selected for the next screening. In the second screening, Bradyrhizobium elkanii AHY3-1 (type A), Bradyrhizobium japonicum SAY3-7 (type A), B. elkanii BLY3-8 (type B) and B. japonicum SAY3-10 (type B) isolates, which showed higher nitrogen fixation and nodulation in Yezin-3 (Rj₄) and Yezin-6 (non-Rj), were selected for the next experiment. In the third screening experiment, SAY3-7 and BLY3-8, which had higher nitrogen fixing potential and proper nodulation, were selected as effective isolates. These two isolates were compatible with non-Rj and Rj₄ soybean varieties for nodulation and nitrogen fixation. Based on the results of the screening experiment, these two strains were tested for their symbiotic efficacy in Futsukaichi soil. This study shows that inoculation treatment of SAY3-7 and BLY3-8 significantly increased plant growth, nodulation, and N₂ fixation at the V6, R3.5 and R8 stages in Yezin-3 (Rj₄) and/or Yezin-6 (non-Rj), and the seed yield at R8 stage, in Yezin-3 (Rj₄) and Yezin-6 (non-Rj) soybean varieties compared with the control treatment. It can be concluded that SAY3-7 and BLY3-8 are suitable for inoculant production because of their higher nitrogen fixation ability, proper nodulation and better productivity of Myanmar soybean cultivars.     

Genetic variability of soybean bradyrhizobia populations under different soil managements

Due to their ecologic and economic importance, bradyrhizobia have been extensively studied in recent years. Since 1992, Bradyrhizobium elkanii SEMIA 587 and SEMIA 5019 and Bradyrhizobium japonicum SEMIA 5079 and SEMIA 5080 have been widely used in most Brazilian soybean fields. The objective of this work was to estimate the genetic variability of bradyrhizobial isolates recovered from soils under rhizobial inoculation and different soil managements. Only 25% of the isolates demonstrated high similarities to the original strains, and a strong correlation was obtained between the bradyrhizobial genetic variability and soil management. A high level of genetic diversity was observed both within isolates (H = 5.46) as well as among the different soil practices. Soil under no-tillage presented a higher bradyrhizobia diversity compared with bradyrhizobia isolated from soil under conventional tillage. Serological characterization also indicated that B. elkanii strains SEMIA 587 and SEMIA 5019 were more competitive and presented a higher nodular occupancy capacity than strains belonging to B. japonicum species in Southern Brazilian soils.     

Repeated sequence RSα is diagnostic for Bradyhizobium japonicum and Bradyrhizobium elkanii

The genome of Bradyrhizobium japonicum and B. elkanii contains multiple copies of the repeated DNA sequence RSα. A collection of 18 B. japonicum, 4 B. elkanii and 72 other bacterial strains was screened by polymerase chain reaction (PCR) using a pair of primers specific for RSα. Only strains of B. japonicum and B. elkanii gave the predicted amplification product. Restriction analysis of PCR products obtained from different strains of B. japonicum showed that the RSα sequence was generally conserved. The usefulness of RSα as a specific probe for Bradyrhizobium strains capable of nodulating soybean was also demonstrated. Received: 11 May 1995     

Yield and N accumulation responses of late‐season determinate soybean to irrigation and inoculation with various strains ofBradyrhizobium japonicum

Abstract

Cobb and Coker 488, late‐season (maturity group VIII) cultivars of soybean [Glycine max(L.) Merr], were grovn under irrigated and non‐irrigated conditions on a Norfolk loamy sand in a two‐year field experiment. Each cultivar was inoculated withBradyrhizobium japonicumstrains [USDA 3I1b110; Brazil 587; NifTAL 184 and 102 (NifTAL cultures of Brazil 587 and USDA 110, respectively); and North Carolina 1001, 1004, 1005, 1010, and 1029). Drought conditions were present both years, and irrigation significantly increased the overall yield (2.49 vs 1.92 Mg ha^‐1). Coker 488 was significantly higher in seed yield than Cobb (2.55 vs 2.02 Mg ha^‐1). Strain ofB.japonicumalso affected seed yields. NC1010‐inoculated soybean was significantly higher in seed yield rank than all other soybean at the P<0.01 level, when compared by single degree of freedom contrast (sdfc). The yield ranking of soybean inoculated with NC1001 was significantly lower than soybean inoculated with all other strains, when compared by sdfc (P<0.10). Other strains differed in responses which ranged from good to poor inoculants under specific water management conditions. For instance, under nonirrigated conditions, soybean inoculated with strains ofB.japonicumfrom North Carolina was significantly higher in seed yield than those inoculated with the cultures of USDA 110, B587, or the control, when compared by sdfc (P>0.03, 0.05, 0.06, respectively). Since soybean inoculated with either strain of USDA 110 was generally high in yield rank under irrigated conditions, their response to irrigation was large relative to soybean inoculated with the NC strain (P<0.04). Neither seed nitrogen nor xylem water potential was highly correlated to seed yield. Since seed yield and N content were not highly correlated, the amount of N accumulated in soybean dry mass and that removed in seed were not highly correlated. Thus, the amount of N returned to the soil would be affected by management combinations of late‐season determinate soybean cultivar,B.japonicumstrain, and irrigation     

Field performance of three Bradyrhizobium japonicum strains with two soybean (Glycine max. L.) cultivars

Summary A field experiment was condutced in a clay loam soil to study the performance of three Bradyrhizobium japonicum strains; USDA 110, USDA 138 and TAL 379, in relation to their N₂-fixing potential and competitiveness on two soybean cultivars (Clark and Calland). Inoculation of soybean cultivars with these strains, either singly or in combination, induced significant increases in plant dry weight, N₂ fixation and seed yields. However, no significant differences were found between the rhizobial strains and/or their mixtures in N₂ fixation and increased seed yield for both cultivars. The two soybean cultivars gave similar responses to inoculation. No significant differences in seed yield were observed between Clark and Calland cultivars. The interaction between inoculant strain and soybean cultivar was not significant. The competition between strains for nodulation was assessed. Strain USDA 110 was the most competitive, followed by USDA 138. Strain TAL 379 was always less competitive on both cultivars. The incidence of double-strain occupancy of nodules varied from 8% to 40%.     

Occurrence and nature of mixed infections in nodules of field-grown soybeans (Glycine max)

Summary Mixed infections of Bradyrhizobium japonicum strains in early and late nodules of four soybean cultivars were studied in a field soil. Nodule occupants were identified by immunofluorescence using serogroup specific antibodies prepared against B. japonicum strains USDA 110, USDA 123, and USDA 138. Double infection was determined directly by combined examination of the same microscopic field by fluorescence and phase contrast microscopy. Double strain occupancy was observed consistently, and its occurrence did not differ substantially in pouch, soil pot, and field experiments, ranging in incidence from 12% to 32%. No significant differences in the incidence or nature of double infection could be attributed to cultivar, seed inoculation, or plant maturity. Strains reactive to strain USDA 123-fluorescent antibody were dominant in both singly and doubly infected nodules irrespective of cultivar, plant age, or seed inoculation with strain USDA 110.Paper no. 15092 in the Scientific Journal Series of the Minnesota Agricultural Experiment Station, St. Paul     

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.     

Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elkanii strains

 In a previous study soybean Bradyrhizobium strains, used in Brazilian studies and inoculants over the last 30 years, and strains adapted to the Brazilian Cerrados, a region frequently submitted to environmental and nutritional stresses, were analyzed for 32 morphological and physiological parameters in vivo and in vitro. A cluster analysis allowed the subdivision of these strains into species Bradyrhizobium japonicum, Bradyrhizobium elkanii and a mixed genotype. In this study, the bacteria were analyzed for nodulation, N₂ fixation capacity, nodule occupancy and the ability to increase yield. The goal was to find a relationship between the strain groups and the symbiotic performance. Two strains of Brazilian B. japonicum showed higher rates of N₂ fixation and nodule efficiency (mg of N mg^–1 of nodules) under axenic conditions. These strains also showed greater yield increases in field experiments when compared to B. elkanii strains. However, no differences were detected between B. japonicum and B. elkanii strains when comparing nodule occupancy capacity. The adapted strains belonging to the serogroup B. elkanii SEMIA 566, most clustered in a mixed genotype, were more competitive than the parental strain, and some showed a higher capacity of N₂ fixation. Some of the adapted strains, such as S-370 and S-372, have shown similar N₂ fixation rates and nodulation competitiveness to two Brazilian strains of B. japonicum. This similarity demonstrates the possibility of enhancing N₂ fixing ability, after local adaptation, even within B. elkanii species. Differences in the DNA profiles were also detected between the parental SEMIA 566 and the adapted strains by analyses with the ERIC and REP-PCR techniques. Consequently, genetic, morphological and physiological changes can be a result of adaptation of rhizobia to the soil. This variability can be used to select strains capable of increasing the contribution of N₂ fixation to soybean nutrition. Received: 28 May 1997     

A significant proportion of indigenous rhizobia from India associated with soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) distinctly belong to <Emphasis Type="Italic">Bradyrhizobium</Emphasis> and <Emphasis Type="Italic">Ensifer</Emphasis> genera

The diversity among 269 rhizobia isolated from naturally occurring root nodules of soybean collected from two different agro-ecological regions of India, based on RFLP and sequences of the intergenic spacer (IGS) between the 16S and 23S rRNA genes, growth rate, and indole acetic acid production, revealed their significant, site-dependent genomic diversity. Among these bacteria, nine IGS genotypes were identified with two endonucleases. They were distributed into five divergent lineages by sequence analysis of each IGS representative strain, i.e., (1) comprising IGS genotypes I, II, III, and reference Bradyrhizobium yuanmingense; (2) with genotype IV and strains of unclassified bradyrhizobia genomic species; (3) including genotypes V, VI, and Bradyrhizobium liaoningense; (4) with IGS genotype VII and Bradyrhizobium elkanii strains; and (5) comprising IGS genotypes VIII, IX, and different Ensifer genus bacteria. Host-specificity test revealed that all rhizobia-nodulated soybean and cowpea and only part of them formed nodules on Arachis hypogeae and Cajanus cajan. The great diversity of soybean nodulators observed in this study emphasises that Indian soil is an important reservoir of nitrogen-fixing rhizobia.     

Phylogeny and nitrogen fixation potential of Bradyrhizobium species isolated from the legume cover crop Pueraria phaseoloides (Roxb.) Benth. in Eastern Cameroon

Pueraria is an herbaceous, perennial legume crop originating in Asia. Pueraria phaseoloides (Roxb.) Benth. (tropical kudzu) is frequently introduced into production systems and is used as green manure, a cover crop and a forage plant, making it important economically. We used P. phaseoloides as a trap crop to study and characterize soil rhizobia in Eastern Cameroon. Bacteria were isolated from fresh nodules collected from field-grown P. phaseoloides roots. The 16S-23S rRNA internal transcribed spacer (ITS) sequences from 30 bacterial isolates were amplified by polymerase chain reaction (PCR) and the reaction products were sequenced. Phylogenetic analysis revealed that all isolates were ascribed to the genus Bradyrhizobium and were grouped into three clusters of Bradyrhizobium sp. strains, one cluster of B. yuanmingense strains, and one cluster of B. elkanii strains. Acetylene reduction assay (ARA) results indicated that the B. yuanmingense strains had significantly higher nitrogen fixation potential and that they could be used as inoculants to enhance nitrogen fixation in Pueraria grown in Eastern Cameroon.     

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.