Strain USDA Research Articles

Integrated roles of BclA and DD-carboxypeptidase 1 in Bradyrhizobium differentiation within NCR-producing and NCR-lacking root nodules

Legumes harbor in their symbiotic nodule organs nitrogen fixing rhizobium bacteria called bacteroids. Some legumes produce Nodule-specific Cysteine-Rich (NCR) peptides in the nodule cells to control the intracellular bacterial population. NCR peptides have antimicrobial activity and drive bacteroids toward terminal differentiation. Other legumes do not produce NCR peptides and their bacteroids are not differentiated. Bradyrhizobia, infecting NCR-producing Aeschynomene plants, require the peptide uptake transporter BclA to cope with the NCR peptides as well as a specific peptidoglycan-modifying DD-carboxypeptidase, DD-CPase1. We show that Bradyrhizobium diazoefficiens strain USDA110 forms undifferentiated bacteroids in NCR-lacking soybean nodules. Unexpectedly, in Aeschynomene afraspera nodules the nitrogen fixing USDA110 bacteroids are hardly differentiated despite the fact that this host produces NCR peptides, suggesting that USDA110 is insensitive to the host peptide effectors and that nitrogen fixation can be uncoupled from differentiation. In agreement with the absence of bacteroid differentiation, USDA110 does not require its bclA gene for nitrogen fixing symbiosis with these two host plants. Furthermore, we show that the BclA and DD-CPase1 act independently in the NCR-induced morphological differentiation of bacteroids. Our results suggest that BclA is required to protect the rhizobia against the NCR stress but not to induce the terminal differentiation pathway.

Ureide essay to assess N2-fixation abilities of soybean (Glycine max) genotypes under different Bradyrhizobium strains

The high protein content of soybean (Glycine max) seeds results in high nitrogen demand, causing a huge nitrogen uptake during plant growth. As a legume crop, soybean can fix atmospheric N through symbiotic associations with Bradyrhizobia and perform well in African nitrogen poor soils. This study aimed at establishing the ability of promiscuous soybean genotypes to fix nitrogen and devise the relationship between nodule scores and amount of nitrogen fixed. Twelve soybean genotypes were inoculated with Bradyrhizobium japonicum Strain USDA 110 (specific) and Bradyrhizobium sp. Strain USDA 3456 (native) and raised in pots in a greenhouse. At the R3.5 growth stage, nodules were scored and xylem sap was extracted, which xylem sap was used to carry out ureide, amino-N, and nitrates assays. The relative abundance of ureide was used to devise the proportion of nitrogen fixed by each genotype. The proportion of nitrogen derived from atmospheric N2 (Ndfa) ranged from 47.9 to 78.8% under USDA 3456 and from 36.7 to 78.7% under USDA 110. A strong correlation was found between nodule scores, especially nodules’ effectiveness, and Ndfa. The genotypes Wondersoya (78.8%), Maksoy 2N (78.4%), Namsoy 3 (78.3%), and Maksoy 3N (75.7%) had high nitrogen-fixing ability in response to USDA 3456. Promiscuous soybean genotypes can fix nitrogen equally under both native and specific Bradyrhizobium types. Nodules’ effectiveness can be a good predictor of biological nitrogen fixation. This study highlighted that crop improvement to boost soybean production in Africa should target promiscuous varieties for better yield with less inputs.

Nodulation and Symbiotic Nitrogen Fixation in the Biofuel Legume Tree Pongamia pinnata

The legume tree Pongamia pinnata (also called Millettia pinnata) is a non-food crop that can grow on marginal land not destined for the cultivation of food crops. It is an important candidate for the production of biofuel (bio-oil, biodiesel and aviation biofuel) from its oil-rich seeds. An important trait is its ability to grow in marginal, nitrogen-limited soils. This growth ability is attributed to the nitrogen-fixation activity of root nodules. Cutting both cotyledons in halves had little effect on shoot dry weight, nodule number and weight per plant at 8 weeks. These parameters were reduced when both cotyledons were removed; plants were visibly yellow. Surprisingly when no nutrients were supplied and with both cotyledons intact, plants grew as well as those with nutrients. To isolate rhizobial inoculants from soil, samples were collected from Meandu Mine, a coal-mining site near Kingaroy, Queensland (Australia), where pongamia is currently grown to rehabilitate the soil. Pongamia nodules were also collected from Mt. Coot-tha Botanic Gardens (Brisbane), where pongamia was introduced decades ago. Established ‘Baiting technique’ and ‘One-drop-one-nodule technique’ were applied to isolate potential rhizobia from the soil samples and nodules. All isolates were screened and characterised for symbiotic effectiveness and nitrogenase activity. A total of 21 putative rhizobial samples were isolated – five from soil samples and 16 from pongamia nodules. Out of these iso lates, seven were slow-growers while 14 were fast-growers. These isolates were coded as PR-UQ. Among the isolates, the fast-growers PR-UQ-03 and PR-UQ-05 were considered as the superior strains compared to the slow-growers PR-UQ-01 and PR-UQ-04, which were more effective than the rest of the isolates. The new isolates enhanced nodule number, shoot length and total plant dry weight and resulted in better plant growth than the available Bradyrhizobium japonicum strains CB1809, USDA110 and CB564. Acetylene reduction assay (short term) and Nitrogen Difference Analysis (long term) further confirmed that pongamia fixed more nitrogen upon inoculation with these rhizobia. Based on 16S rDNA sequence analysis, PR-UQ-01 and PR-UQ-04 are closely related to Bradyrhizobium elkanii and Bradyrhizobium pachyrizi, while PR-UQ-03 and PR-UQ-05 are both related to Rhizobium mesoamericanum. Cotyledons supply the seedling with nutrients for a considerable period of early seedling growth. The newly isolated, fast-growing rhizobia PR-UQ-03 and PR-UQ-05 and the slow-growing PR-UQ-01 and PR-UQ-04 are related to Rhizobium and Bradyrhizobium species and aid in the nodulation, nitrogen fixation and seedling/sapling growth of Pongamia pinnata.

Nodulation and Symbiotic Nitrogen Fixation in the Biofuel Legume Tree Pongamia pinnata

The legume tree Pongamia pinnata (also called Millettia pinnata) is a non-food crop that can grow on marginal land not destined for the cultivation of food crops. It is an important candidate for the production of biofuel (bio-oil, biodiesel and aviation biofuel) from its oil-rich seeds. An important trait is its ability to grow in marginal, nitrogen-limited soils. This growth ability is attributed to the nitrogen-fixation activity of root nodules. Cutting both cotyledons in halves had little effect on shoot dry weight, nodule number and weight per plant at 8 weeks. These parameters were reduced when both cotyledons were removed; plants were visibly yellow. Surprisingly when no nutrients were supplied and with both cotyledons intact, plants grew as well as those with nutrients. To isolate rhizobial inoculants from soil, samples were collected from Meandu Mine, a coal-mining site near Kingaroy, Queensland (Australia), where pongamia is currently grown to rehabilitate the soil. Pongamia nodules were also collected from Mt. Coot-tha Botanic Gardens (Brisbane), where pongamia was introduced decades ago. Established ‘Baiting technique’ and ‘One-drop-one-nodule technique’ were applied to isolate potential rhizobia from the soil samples and nodules. All isolates were screened and characterised for symbiotic effectiveness and nitrogenase activity. A total of 21 putative rhizobial samples were isolated – five from soil samples and 16 from pongamia nodules. Out of these iso lates, seven were slow-growers while 14 were fast-growers. These isolates were coded as PR-UQ. Among the isolates, the fast-growers PR-UQ-03 and PR-UQ-05 were considered as the superior strains compared to the slow-growers PR-UQ-01 and PR-UQ-04, which were more effective than the rest of the isolates. The new isolates enhanced nodule number, shoot length and total plant dry weight and resulted in better plant growth than the available Bradyrhizobium japonicum strains CB1809, USDA110 and CB564. Acetylene reduction assay (short term) and Nitrogen Difference Analysis (long term) further confirmed that pongamia fixed more nitrogen upon inoculation with these rhizobia. Based on 16S rDNA sequence analysis, PR-UQ-01 and PR-UQ-04 are closely related to Bradyrhizobium elkanii and Bradyrhizobium pachyrizi, while PR-UQ-03 and PR-UQ-05 are both related to Rhizobium mesoamericanum. Cotyledons supply the seedling with nutrients for a considerable period of early seedling growth. The newly isolated, fast-growing rhizobia PR-UQ-03 and PR-UQ-05 and the slow-growing PR-UQ-01 and PR-UQ-04 are related to Rhizobium and Bradyrhizobium species and aid in the nodulation, nitrogen fixation and seedling/sapling growth of Pongamia pinnata.

Efficient biosynthesis of β-alanine with a tandem reaction strategy to eliminate amide by-product in the nitrilase-catalyzed hydrolysis

An efficient biosynthesis of β-alanine from 3-aminopropionitrile at high concentration has been developed using a one-pot bienzymatic cascade of a nitrilase and an amidase. The nitrilase BjNIT3397 from Bradyrhizobium japonicum strain USDA110 catalyzes the hydrolysis of 3-aminopropionitrile to β-alanine at the concentration up to 3.0mol/L with the formation 23% of 3-aminopropanamide. In order to eliminate the by-product 3-aminopropanamide, we cloned and characterized a new amidase from Pseudomonas nitroreducens through gene mining. Under the optimal conditions (50mmol/L Na2HPO4-NaH2PO4 buffer, pH 6.0, 40°C), 2.0mol/L (176g/L) of 3-aminopropanamide was completely hydrolyzed within 12h. A tandem reaction system was then established to eliminate the by-product 3-aminopropanamide and increase the production of β-alanine to 90% isolated yield with 15.02g/(L.h) space-time-yield. These results demonstrated that the tandem reaction strategy was an effective method of eliminating the amide by-products in the nitrilase-catalyzed hydrolysis at high substrate concentration.

Screening soybean genotypes for promiscuous symbiotic association with <i>Bradyrhizobium</i> strains

The current low soybean (Glycine max L. Merrill) yields in Sub-Saharan Africa can be alleviated by developing promiscuous genotypes. The research trend in Africa is towards developing promiscuous varieties for less labour and high yields in soybean production. A greenhouse experiment was conducted at Makerere University Agricultural Research Institute, Kabanyolo (MUARIK) with the aim of screening of soybean germplasm for promiscuous symbiotic association with Bradyrhizobium sp. in order to identify genotypes with potential to be used as parents to initiate a breeding programme focusing on promiscuous nodulation. The response of 65 soybean genotypes from different origins to cowpea-type inoculant, Bradyrhizobium sp. strain USDA 3456 were evaluated. Soybean seeds inoculated with both Bradyrhizobium sp. strain USDA 3456 and Bradyrhizobium japonicum strain USDA 110 were planted in buckets filled with steam-sterilised soil. Significant differences in the number of nodules, effective nodules, fresh and dry weight of nodules were observed among genotypes (P<0.001). Twelve of the 65 genotypes were highly responsive to Bradyrhizobium sp., with over 10 nodules per plant, over 50 mg dry weight of nodules, and 50% effective nodules. A highly positive correlation was shown between dry weight of nodules and nodule number. This study proposes the soybean genotypes NamII, WonderSoya, Bulindi 48C, NamSoy 4M, MakSoy 3N, NamSoy 3, K-Local, Kabanyolo 1, UG 5, Soprano, MakSoy 2N, and MakSoy 5N as potential parental materials for subsequent breeding work.Keywords: Glycine max, nodules, promiscuous, Uganda

Identification of the Hydrogen Uptake Gene Cluster for Chemolithoautotrophic Growth and Symbiosis Hydrogen Uptake in Bradyrhizobium Diazoefficiens.

The hydrogen uptake (Hup) system of Bradyrhizobium diazoefficiens recycles the H2 released by nitrogenase in soybean nodule symbiosis, and is responsible for H2-dependent chemolithoautotrophic growth. The strain USDA110 has two hup gene clusters located outside (locus I) and inside (locus II) a symbiosis island. Bacterial growth under H2-dependent chemolithoautotrophic conditions was markedly weaker and H2 production by soybean nodules was markedly stronger for the mutant of hup locus I (ΔhupS1L1) than for the mutant of hup locus II (ΔhupS2L2). These results indicate that locus I is primarily responsible for Hup activity.

Growth Rate of and Gene Expression in Bradyrhizobium diazoefficiens USDA110 due to a Mutation in blr7984, a TetR Family Transcriptional Regulator Gene.

Previous transcriptome analyses have suggested that a gene cluster including a transcriptional regulator (blr7984) of the tetracycline repressor family was markedly down-regulated in symbiosis. Since blr7984 is annotated to be the transcriptional repressor, we hypothesized that it is involved in the repression of genes in the genomic cluster including blr7984 in symbiotic bacteroids. In order to examine the function and involvement of the blr7984 gene in differentiation into bacteroids, we compared the free-living growth/symbiotic phenotype and gene expression between a blr7984-knockout mutant and the wild-type strain of Bradyrhizobium diazoefficiens USDA110. The mutant transiently increased the cell growth rate under free-living conditions and nodule numbers over those with the wild-type strain USDA110. The expression of three genes adjacent to the disrupted blr7984 gene was strongly up-regulated in the mutant in free-living and symbiotic cells. The mutant also induced the expression of genes for glutathione S-transferase, cytochrome c oxidases, ABC transporters, PTS sugar transport systems, and flagella synthesis under free-living conditions. bll7983 encoding glutathione S-transferase was up-regulated the most by the blr7984 disruption. Since redox regulation by glutathione is known to be involved in cell division in prokaryotes and eukaryotes, the strong expression of glutathione S-transferase encoded by the bll7983 gene may have caused redox changes in mutant cells, which resulted in higher rates of cell division.

Survival and Competitiveness of Bradyrhizobium japonicum Strains 20 Years after Introduction into Field Locations in Poland.

It was previously demonstrated that there are no indigenous strains of Bradyrhizobium japonicum forming nitrogen-fixing root nodule symbioses with soybean plants in arable field soils in Poland. However, bacteria currently classified within this species are present (together with Bradyrhizobium canariense) as indigenous populations of strains specific for nodulation of legumes in the Genisteae tribe. These rhizobia, infecting legumes such as lupins, are well established in Polish soils. The studies described here were based on soybean nodulation field experiments, established at the Poznań University of Life Sciences Experiment Station in Gorzyń, Poland, and initiated in the spring of 1994. Long-term research was then conducted in order to study the relation between B. japonicum USDA 110 and USDA 123, introduced together into the same location, where no soybean rhizobia were earlier detected, and nodulation and competitive success were followed over time. Here we report the extra-long-term saprophytic survival of B. japonicum strains nodulating soybeans that were introduced as inoculants 20 years earlier and where soybeans were not grown for the next 17 years. The strains remained viable and symbiotically competent, and molecular and immunochemical methods showed that the strains were undistinguishable from the original inoculum strains USDA 110 and USDA 123. We also show that the strains had balanced numbers and their mobility in soil was low. To our knowledge, this is the first report showing the extra-long-term persistence of soybean-nodulating strains introduced into Polish soils and the first analyzing the long-term competitive relations of USDA 110 and USDA 123 after the two strains, neither of which was native, were introduced into the environment almost 2 decades ago.

MicroRNA167-Directed Regulation of the Auxin Response Factors GmARF8a and GmARF8b Is Required for Soybean Nodulation and Lateral Root Development.

Legume root nodules convert atmospheric nitrogen gas into ammonium through symbiosis with a prokaryotic microsymbiont broadly called rhizobia. Auxin signaling is required for determinant nodule development; however, the molecular mechanism of auxin-mediated nodule formation remains largely unknown. Here, we show in soybean (Glycine max) that the microRNA miR167 acts as a positive regulator of lateral root organs, namely nodules and lateral roots. miR167c expression was up-regulated in the vasculature, pericycle, and cortex of soybean roots following inoculation with Bradyrhizobium japonicum strain USDA110 (the microsymbiont). It was found to positively regulate nodule numbers directly by repressing the target genes GmARF8a and GmARF8b (homologous genes of Arabidopsis [Arabidopsis thaliana] AtARF8 that encode auxin response factors). Moreover, the expression of miR167 and its targets was up- and down-regulated by auxin, respectively. The miR167-GmARF8 module also positively regulated nodulation efficiency under low microsymbiont density, a condition often associated with environmental stress. The regulatory role of miR167 on nodule initiation was dependent on the Nod factor receptor GmNFR1α, and it acts upstream of the nodulation-associated genes nodule inception, nodulation signaling pathway1, early nodulin40-1, NF-YA1 (previously known as HAEM activator protein2-1), and NF-YA2. miR167 also promoted lateral root numbers. Collectively, our findings establish a key role for the miR167-GmARF8 module in auxin-mediated nodule and lateral root formation in soybean.

Symbiosis island shuffling with abundant insertion sequences in the genomes of extra-slow-growing strains of soybean bradyrhizobia.

Extra-slow-growing bradyrhizobia from root nodules of field-grown soybeans harbor abundant insertion sequences (ISs) and are termed highly reiterated sequence-possessing (HRS) strains. We analyzed the genome organization of HRS strains with the focus on IS distribution and symbiosis island structure. Using pulsed-field gel electrophoresis, we consistently detected several plasmids (0.07 to 0.4 Mb) in the HRS strains (NK5, NK6, USDA135, 2281, USDA123, and T2), whereas no plasmids were detected in the non-HRS strain USDA110. The chromosomes of the six HRS strains (9.7 to 10.7 Mb) were larger than that of USDA110 (9.1 Mb). Using MiSeq sequences of 6 HRS and 17 non-HRS strains mapped to the USDA110 genome, we found that the copy numbers of ISRj1, ISRj2, ISFK1, IS1632, ISB27, ISBj8, and IS1631 were markedly higher in HRS strains. Whole-genome sequencing showed that the HRS strain NK6 had four small plasmids (136 to 212 kb) and a large chromosome (9,780 kb). Strong colinearity was found between 7.4-Mb core regions of the NK6 and USDA110 chromosomes. USDA110 symbiosis islands corresponded mainly to five small regions (S1 to S5) within two variable regions, V1 (0.8 Mb) and V2 (1.6 Mb), of the NK6 chromosome. The USDA110 nif gene cluster (nifDKENXSBZHQW-fixBCX) was split into two regions, S2 and S3, where ISRj1-mediated rearrangement occurred between nifS and nifB. ISs were also scattered in NK6 core regions, and ISRj1 insertion often disrupted some genes important for survival and environmental responses. These results suggest that HRS strains of soybean bradyrhizobia were subjected to IS-mediated symbiosis island shuffling and core genome degradation.

Predominant populations of indigenous soybean-nodulating Bradyrhizobium japonicum strains obtained from organic farming systems in Minnesota.

Bradyrhizobium from organic fields in Minnesota were isolated and genotyped to assess diversity of soybean-bradyrhizobia in organic farming systems that can be used to improve soybean productivity. Soil samples were collected from 25 organic fields in Minnesota during May to July 2012. Soybean (cv. Lambert) was used as a host to trap indigenous bradyrhizobia in each sample. Genetic diversity of Bradyrhizobium strains (n=733) was determined using the horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (HFERP) DNA fingerprinting technique and the soybean-bradyrhizobia were classified into 79 different genotypes. Of these, 15 dominant genotypes were found and were highly similar (>92% fingerprint similarity) to serotypes USDA 127 (40.4%), USDA 4 (31.8%) and USDA 123 (15.5%), which were the three main populations of soybean-bradyrhizobia in organic fields. Bradyrhizobium japonicum serogroup USDA 4 strains were found to make up a previously unrecognized, predominant rhizobial population in the organic farming soils examined. The relative abundance of strain USDA 4 was negatively correlated with that of USDA 127 and this relationship may be influenced by the levels of NO3 -N and other soil edaphic factors. The local community of bradyrhizobia can be affected by applying inoculant bacteria to organic fields. Based on these results, soybean production in organic farms may be improved by displacing strains similar to USDA 4 with those better at nitrogen fixation and competitive ability than indigenous strains.

Revision of the taxonomic status of type strains of Mesorhizobium loti and reclassification of strain USDA 3471T as the type strain of Mesorhizobiumerdmanii sp. nov. and ATCC 33669T as the type strain of Mesorhizobiumjarvisii sp. nov.

The species Mesorhizobim loti was isolated from nodules of Lotus corniculatus and its type strain deposited in several collections. Some of these type strains, such as those deposited in the USDA and ATCC collections before 1990, are not coincident with the original strain, NZP 2213T, deposited in the NZP culture collection. The analysis of the 16S rRNA gene showed that strains USDA 3471T and ATCC 33669T formed independent branches from that occupied by Mesorhizobium loti NZP 2213T and related to those occupied by Mesorhizobium opportunistum WSM2075T and Mesorhizobium huakuii IFO 15243T, respectively, with 99.9 % similarity in both cases. However, the analysis of concatenated recA, atpD and glnII genes with similarities lower than 96, 98 and 94 %, respectively, between strains USDA 3471T and M. opportunistum WSM2075T and between strains ATCC 33669T and M. huakuii IFO 15243T, indicated that the strains USDA 3471T and ATCC 33669T represent different species of the genus Mesorhizobium. These results were confirmed by DNA-DNA hybridization experiments and phenotypic characterization. Therefore, the two strains were reclassified as representatives of the two species Mesorhizobium erdmanii sp. nov. (type strain USDA 3471T = CECT 8631T = LMG 17826t2T) and Mesorhizobium jarvisii sp. nov. (type strain ATCC 33669T = CECT 8632T = LMG 28313T).

Nitrilase-catalyzed hydrolysis of 3-aminopropionitrile at high concentration with a tandem reaction strategy for shifting the reaction to β-alanine formation

Given the importance of β-alanine, the nitrilase BjNIT3397 from Bradyrhizobium japonicum strain USDA110 was examined toward the hydrolysis of 3-aminopropionitrile. It has been found that nitrilase BjNIT3397 effectively hydrolyzed 3-aminopropionitrile with substrate concentration up to 3M (210g/L) at the pH 7.3 and temperature 30°C. With the increase of substrate concentration from 0.6 to 3M, 3-aminopropanamide was formed and its percentage in the products was increased up to 33%. In order to reduce the formation of 3-aminopropanamide, aspartate ammonia-lyase and fumaric acid were added into the reaction system to consume the byproduct ammonia. As expected, the reaction was shifted toward the formation of β-alanine, resulting in the decrease of 3-aminopropanamide from 33% to 3%. Therefore, a tandem reaction strategy was developed to effectively prevent the formation of 3-aminopropanamide. This might also offer a possibility of producing β-alanine and l-aspartic acid in one process.

Diversity and phylogeny of soybean rhizobia in central India

Soybean is the most important crop legume with the highest share of biological nitrogen fixation among cultivated legumes. During the early years following the introduction of soybean cultivation in India, the effects of rhizobial inoculation were impressive, but they have declined over time due to naturalization of the introduced strains. We have characterized the diversity of soybean rhizobia, mainly those from central India, for phenotypic features, such as utilization of carbon sources and intrinsic resistance to antibiotics. The PCR-amplified 16S rRNA gene products of the strains were sequenced to study genetic diversity and phylogenetic relationships. We found that the rhizobia comprised both slow and fast growers, with the former having a higher Shannon–Weiner Diversity Index (H = 2.93 and 3.00 for carbon utilization and intrinsic antibiotic resistance, respectively) than the latter (H = 2.62 and 1.90, respectively). There were two 16S rRNA sequence types among the slow growers—Bradyrhizobium spp. (99.4–99.8 % sequence homology) and Rhizobium radiobacter (96.1–99.7 %). In contrast, the fast growing strains belonged exclusively to R. radiobacter (98.9–99.7 %). Bradyrhizobium japonicum strain USDA 110, which was originally introduced on a large scale in Indian soils more than four decades ago, shared 34–81 % phenotypic and 63–83 % genotypic similarity with the other Indian rhizobial isolates characterized. There was conservation of 16S rRNA gene sequences among rhizobia in various soybean-growing areas and the evolution of native rhizobial strains among slow and fast growers. These results on the biodiversity of soybean rhizobia are important for strain selection, which is crucial for the design of successful inoculation programs.

Symbiotic properties of Bradyrhizobium sp. (Lupinus) assayed on serradella plants

Physiological and symbiotic properties of <i>Bradyrhizobium</i> sp. (<i>Lupinus</i>) nodule isolates were compared to the standard slow-growing <i>Bradyrhizobium</i> sp. (<i>Lupinus</i>) strain USDA 3045. Lupine nodules isolates showed typical characteristics for bradyrhizobial strains and nodulated small seed legume, serradella (<i>Ornithopus sativus</i>), in tube test. We observed a permanent physiological segregation of the effective (Fix') and ineffective (Fix-) symbiotic phenotype for all tested bradyrhizobial strains during the growth of serradella in plant tube test. The ultrastructural differences between Fix* and Fix serradella nodules were observed. Rapid and visible nodulation as well as easy assay of the reduction of acetylene make serradella a convenient system for studies of <i>Bradyrhizobium</i> sp. (<i>Lupinus</i>) strains in laboratory conditions.

Sequencing the complete mitochondrial genome of Eimeria mitis strain USDA 50 (Apicomplexa: Eimeriidae) suggests conserved start positions for mtCOI- and mtCOIII-coding regions

Four complete mitochondrial (mt) sequences from a single-oocyst-derived line of Eimeria mitis USDA 50 were obtained (three from cloned whole-genome PCR products, one from directly sequenced whole-genome PCR product). The mt genome is 6,408bp long with three genes (CytB, cytochrome c oxidase subunit I (COI) and cytochrome c oxidase subunit III (COIII)) and many rDNA fragments (large subunit rDNA 13, small subunit rDNA 10); organisation was identical to other Eimeria sp. mt genomes. Conserved start codon positions for both COI and COIII are suggested for all Eimeria mt genomes; these start codon positions exist and may also be conserved, in related apicomplexan parasites. Within the three separate cloned PCR products of near-complete mt genomes, there were 26 nucleotide differences (collectively) compared to the directly sequenced mt genome. These changes appear to be base misincorporations during PCR. Direct sequencing of long PCR amplification products may be more likely to generate accurate mt genomic sequences than cloning and subsequent sequencing.

CHARACTERISATION OF SOYBEAN RHIZOBIAL STRAINS FROM JAVA AND SUMATRA

To get insight in the structure of soybean rhizobial population native to Indonesian soils, a thorough survey of the occurrence of the soybean rhizobia were conducted in several locations in Java and Sumatra. A total of 51 different isolates of rhizobial strains were characterised phenotypically based on their symbiotic properties, and genetically using amplified ribosomal DNA restriction analysis (ARDRA). Based on their nodulation capacity on both soybean and the native legume mungbean, these rhizobial strains could be divided into a group of 16 strains specific for soybean only and another group of 35 promiscuous strains that nodulated both leguminous plants. Based on ARDRA of PCRamplified 16S rDNA and 16S-23S rDNA spacer fragments, the rhizobial strains isolated from Java differed with those from Sumatra. Six Java isolates and only one Sumatra isolate were classified as Bradyrhizobium japonicum and these similar to that of B. japonicum strain USDA 110. All these B. japonicum strains were highly specific for soybean. One isolate from Java showed a rather unique position. The remaining strains from Java (20), which were symbiotically promiscuous strains, were clustered in another group. This group and another group containing most Sumatra isolates were distinct from B. japonicum USDA 110 and therefore it is tempting to speculate that these represent indigenous soybean rhizobial bacteria. Application of agricultural practices, such as enhancement of rhizobial population, to increase soybean production is still essential and noteworthy in Sumatra.

CHARACTERISATION OF SOYBEAN RHIZOBIAL STRAINS FROM JAVA AND SUMATRA

To get insight in the structure of soybean rhizobial population native to Indonesian soils, a thorough survey of the occurrence of the soybean rhizobia were conducted in several locations in Java and Sumatra. A total of 51 different isolates of rhizobial strains were characterised phenotypically based on their symbiotic properties, and genetically using amplified ribosomal DNA restriction analysis (ARDRA). Based on their nodulation capacity on both soybean and the native legume mungbean, these rhizobial strains could be divided into a group of 16 strains specific for soybean only and another group of 35 promiscuous strains that nodulated both leguminous plants. Based on ARDRA of PCRamplified 16S rDNA and 16S-23S rDNA spacer fragments, the rhizobial strains isolated from Java differed with those from Sumatra. Six Java isolates and only one Sumatra isolate were classified as Bradyrhizobium japonicum and these similar to that of B. japonicum strain USDA 110. All these B. japonicum strains were highly specific for soybean. One isolate from Java showed a rather unique position. The remaining strains from Java (20), which were symbiotically promiscuous strains, were clustered in another group. This group and another group containing most Sumatra isolates were distinct from B. japonicum USDA 110 and therefore it is tempting to speculate that these represent indigenous soybean rhizobial bacteria. Application of agricultural practices, such as enhancement of rhizobial population, to increase soybean production is still essential and noteworthy in Sumatra.

Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobium diazoefficiens sp. nov.

Bradyrhizobium japonicum was described from soybean root-nodule bacterial isolates. Since its description, several studies have revealed heterogeneities among rhizobia assigned to this species. Strains assigned to B. japonicum group Ia have been isolated in several countries, and many of them are outstanding soybean symbionts used in inoculants worldwide, but they have also been isolated from other legume hosts. Here, we summarize published studies that indicate that group Ia strains are different from the B. japonicum type strain USDA 6(T) and closely related strains, and present new morphophysiological, genotypic and genomic evidence to support their reclassification into a novel species, for which the name Bradyrhizobium diazoefficiens sp. nov. is proposed. The type strain of the novel species is the well-studied strain USDA 110(T) ( =IAM 13628(T) =CCRC 13528(T) =NRRL B-4361(T) =NRRL B-4450(T) =TAL 102(T) =BCRC 13528(T) =JCM 10833(T) =TISTR 339(T) =SEMIA 5032(T) =3I1B110(T) =ACCC 15034(T) =CCT 4249(T) = NBRC 14792(T) = R-12974(T) = CNPSo 46(T)).