Commercial Inoculant Strain Research Articles

Evolution of novel Mesorhizobium genospecies that competitively and effectively nodulate Cicer arietinum following inoculation with the Australian commercial inoculant strain M. ciceri CC1192

Abstract Background and aims Mesorhizobium ciceri CC1192 is the commercial inoculant strain for Cicer arietinum (chickpea) cultivation in Australia, including in the Ord River Irrigation Area (ORIA), where C. arietinum cropping began in 1985. Mesorhizobium strains are known to gain the capacity to nodulate legumes through acquisition of symbiosis Integrative and Conjugative Elements (ICEs), leading to the evolution of novel rhizobia. Here, we assess the impact of symbiosis ICE transfer and compare the genomic diversity and symbiotic effectiveness of C. arietinum nodulating rhizobia from the ORIA. Methods Nodule isolates collected from field cultivated C. arietinum were genotyped by RAPD-PCR, and representative strains from each genotype were whole genome sequenced and symbiotically phenotyped in glasshouse conditions to assess N2 fixation effectiveness against CC1192. Results A total of 68 nodule isolates, all harbouring the CC1192 symbiosis ICE (ICEMcSym1192), were analysed, with 12 identified as the inoculant strain, and 56 novel strains clustering into ten genotypes. These novel genotypes dominated as nodule occupants across the majority of sites sampled. Nine of the ten representative strains were as effective at N2 fixation as CC1192, with WSM4904 the only ineffective strain. Core genome phylogeny showed the ten strains represent four novel Mesorhizobium genospecies. Novel strains WSM4904 and WSM4906 shared 98.7% sequence identity, yet exhibited very different symbiotic phenotypes. Conclusions The CC1192 symbiosis ICE has transferred to a wide diversity of Mesorhizobium spp. in the ORIA. These evolved strains are competitive against CC1192 at nodulating C. arietinum, and the majority are effective symbiotic N2 fixers.

Soyabean response to rhizobial inoculation under contrasting smallholder agro-ecological conditions in Zimbabwe

Soyabean is grown widely with rhizobial inoculants on commercial farms in Zimbabwe, but its inoculation response in smallholder cropping environments is poorly characterized. Symbiotic interactions of six rhizobial strains (MAR 1305,1306,1326,1494, 1497 and 1515) from the Grasslands Rhizobium Collection were evaluated under contrasting soils and rainfall zones (natural region) in Zimbabwe. The rhizobial strains were evaluated on one specific and three promiscuous soyabean varieties (SC Saga and TGX (1740-2F/ 1987-628/ 1987-11E). The study was conducted over two rainfall seasons namely 2011-2012 and 2012-2013. Field experiments were set up as a randomized complete block design with two factors (soyabean variety and rhizobial strain), a negative control without inoculation and a positive control inoculated with commercial inoculant strain MAR 1491. All treatments were replicated three times. Nodulation was significantly (P< .0001) influenced by rhizobial strain inoculation with nodulation ranging from 28 to37 nodules per plant. Over the two seasons, soyabean inoculated with MAR 1305, 1306, 1494 and 1515 consistently gave higher grain yield higher (average of 600kg/ha) compared to that inoculated with commercial inoculant strain MAR 1491 (average of 400kg/ha). Nodulation of both promiscuous and specific varieties decreased with annual rainfall amounts in the order: Natural Region (NR)II> NRIII> NRIV. Rhizobial strains MAR 1305, 1306 and 1494 were superior in NR II and NRIII while rhizobial strain MAR 1515 was superior in NR IV. Our results show that identification of superior strains best suited to smallholder cropping environments and inoculation response are crucial for increasing soyabean productivity in these environments.

Genomic Diversity of Pigeon Pea (Cajanus cajan L. Millsp.) Endosymbionts in India and Selection of Potential Strains for Use as Agricultural Inoculants.

Pigeon pea (Cajanus cajan L. Millsp. ) is a legume crop resilient to climate change due to its tolerance to drought. It is grown by millions of resource-poor farmers in semiarid and tropical subregions of Asia and Africa and is a major contributor to their nutritional food security. Pigeon pea is the sixth most important legume in the world, with India contributing more than 70% of the total production and harbouring a wide variety of cultivars. Nevertheless, the low yield of pigeon pea grown under dry land conditions and its yield instability need to be improved. This may be done by enhancing crop nodulation and, hence, biological nitrogen fixation (BNF) by supplying effective symbiotic rhizobia through the application of “elite” inoculants. Therefore, the main aim in this study was the isolation and genomic analysis of effective rhizobial strains potentially adapted to drought conditions. Accordingly, pigeon pea endosymbionts were isolated from different soil types in Southern, Central, and Northern India. After functional characterisation of the isolated strains in terms of their ability to nodulate and promote the growth of pigeon pea, 19 were selected for full genome sequencing, along with eight commercial inoculant strains obtained from the ICRISAT culture collection. The phylogenomic analysis [Average nucleotide identity MUMmer (ANIm)] revealed that the pigeon pea endosymbionts were members of the genera Bradyrhizobium and Ensifer. Based on nodC phylogeny and nod cluster synteny, Bradyrhizobium yuanmingense was revealed as the most common endosymbiont, harbouring nod genes similar to those of Bradyrhizobium cajani and Bradyrhizobium zhanjiangense. This symbiont type (e.g., strain BRP05 from Madhya Pradesh) also outperformed all other strains tested on pigeon pea, with the notable exception of an Ensifer alkalisoli strain from North India (NBAIM29). The results provide the basis for the development of pigeon pea inoculants to increase the yield of this legume through the use of effective nitrogen-fixing rhizobia, tailored for the different agroclimatic regions of India.

Large-scale screening of rhizobacteria to enhance the chickpea-Mesorhizobium symbiosis using a plant-based strategy

Rhizobacteria able to express multiple plant growth-promoting (PGP) traits has been frequently failed to enhance the chickpea-Mesorhizobium symbiosis possibly because a relatively large amount of organic acids exudated by chickpea root to form an acid rhizosphere reduced the survival and colonisation of plant growth-promoting rhizobacteria (PGPR). Therefore, this study was initiated to screen rhizobacteria that would enhance the chickpea-Mesorhizobium symbiosis using a plant-basedstrategyA total of 743 Bacillus-, Burkholderia- and Pseudomonas-like bacteria were isolated from 74 agricultural soil samples collected across Australia. Only 167 isolates were selected, based upon their individual performance in a seedling growth bioassay, to test their effect on nodulation and growth of chickpea in sterile growth pouches. Also, their ability to produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indole acetic acid (IAA) was investigated in relation to their mode of action. The diversity of the 167 isolates was investigated using 16S rDNA sequencing. Of 743 isolates, 697 (83%) were able to produce IAA in the presence of L-tryptophan. ACC deaminase production was detected in 57 isolates (7.7%). Commercial inoculant strain Mesorhizobium ciceri CC1192 produced 7 μg mL−1 IAA and lacked ACC deaminase activity. In further investigation, the promotion of seedling root elongation by rhizobacteria predicted their effect on nodulation and growth of chickpea after dual inoculation in aseptic conditions. The effect of rhizobacteria on seedling root growth as well as shoot growth and nodulation was associated with the capacity of the isolates to produce IAA and ACC deaminase. These isolates were superior in plant growth promotion than those PGPR unable to produce PGP traits. The synthesis of IAA along with ACC deaminase activity by rhizobacteria gave an added advantage in promoting the ability of rhizobia to form an efficient chickpea-Mesorhizobium symbiosis as measured by biomass production and nodulation. Most of these rhizobacteria belonged to the genus Burkholderia. The use of a plant-based first-stage screening strategy in combination with assays for in vitro production of IAA and ACC deaminase enabled the identification of efficient PGPR that were able to enhance the legume-rhizobia symbiosis.

Evolution of diverse effective N2-fixing microsymbionts of Cicer arietinum following horizontal transfer of the Mesorhizobium ciceri CC1192 symbiosis integrative and conjugative element.

Rhizobia are soil bacteria capable of forming N2-fixing symbioses with legumes, with highly effective strains often selected in agriculture as inoculants to maximize symbiotic N2 fixation. When rhizobia in the genus Mesorhizobium have been introduced with exotic legumes into farming systems, horizontal transfer of symbiosis Integrative and Conjugative Elements (ICEs) from the inoculant strain to soil bacteria has resulted in the evolution of ineffective N2-fixing rhizobia that are competitive for nodulation with the target legume. In Australia, Cicer arietinum (chickpea) has been inoculated since the 1970's with Mesorhizobium ciceri sv. ciceri CC1192, a highly effective strain from Israel. Although the full genome sequence of this organism is available, little is known about the mobility of its symbiosis genes and the diversity of cultivated C. arietinum-nodulating organisms. Here, we show the CC1192 genome harbors a 419-kb symbiosis ICE (ICEMcSym1192) and a 648-kb repABC-type plasmid pMC1192 carrying putative fix genes. We sequenced the genomes of 11 C. arietinum nodule isolates from a field site exclusively inoculated with CC1192 and showed they were diverse unrelated Mesorhizobium carrying ICEMcSym1192, indicating they had acquired the ICE by environmental transfer. No exconjugants harboured pMc1192 and the plasmid was not essential for N2 fixation in CC1192. Laboratory conjugation experiments confirmed ICEMcSym1192 is mobile, integrating site-specifically within the 3' end of one of the four ser-tRNA genes in the R7ANS recipient genome. Strikingly, all ICEMcSym1192 exconjugants were as efficient at fixing N2 with C. arietinum as CC1192, demonstrating ICE transfer does not necessarily yield ineffective microsymbionts as previously observed.Importance Symbiotic N2 fixation is a key component of sustainable agriculture and in many parts of the world legumes are inoculated with highly efficient strains of rhizobia to maximise fixed N2 inputs into farming systems. Symbiosis genes for Mesorhizobium spp. are often encoded chromosomally within mobile gene clusters called Integrative and Conjugative Elements or ICEs. In Australia, where all agricultural legumes and their rhizobia are exotic, horizontal transfer of ICEs from inoculant Mesorhizobium strains to native rhizobia has led to the evolution of inefficient strains that outcompete the original inoculant, with the potential to render it ineffective. However, the commercial inoculant strain for Cicer arietinum (chickpea), M. ciceri CC1192, has a mobile symbiosis ICE (ICEMcSym1192) which can support high rates of N2 fixation following either environmental or laboratory transfer into diverse Mesorhizobium backgrounds, demonstrating ICE transfer does not necessarily yield ineffective microsymbionts as previously observed.

Evaluation of thermogravimetric analysis as a rapid tool for the detection of rhizobacteria biostimulants used in precision agriculture

Six-week-old root samples were evaluated using high resolution (maximum resolution [MaxRes]) thermogravimetric analyses (TGA) of the cell wall compositions of Gram-positive (Bacillus mucilaginosus, Bacillus amyloliquefaciens, and Bacillus subtilis) and Gram-negative (Burkholderia sp., Rahnella aquatilis strain H 2.6, and R. aquatilis strain RC 2.5) root colonizing plant growth promoting rhizobacteria (PGPR) commercial inoculant strains (biostimulants) applied to pot grown wheat plants. TGA discriminated the strains within the two types of rhizobacterial cohorts and thus provided a rapid non-molecular means for the detection of PGPR inoculant biostimulants within hours of root sampling. The latter was due to the greater degree of definition of TGA fingerprints of individual thermal weight loss events occurring over a degradation range, and heightened the corresponding peak temperature divergences within strains of either type of bacteria themselves for their unequivocal identification. Confirmation of biostimulant rhizobacteria identity in concomitant root samples was achieved through either cultural methods or direct tissue PCR molecular protocols within 5 days and 2 days of sampling, respectively. The results suggested that MaxRes TGA could serve as a rapid, inexpensive stand-alone tool or as combinatorial utility alongside pyrolysis gas chromatography mass spectra, and Fourier transform infrared analytics for the early detection of PGPR biostimulants in precision farming.

Cross-host compatibility of commercial rhizobial strains for new and existing pasture legume cultivars in south-eastern Australia

Perennial legumes have potential to increase pasture productivity in the high rainfall zone (600–850 mm) of south-eastern Australia through their ability to use summer rainfall and fix nitrogen (N2). Various perennial legumes are being evaluated for this environment; however, little information exists on legume–rhizobia cross-host compatibility and its consequences for biological N2 fixation. This is especially important when legumes are sown into fields with a background of competitive rhizobia such as WSM1325 or sown as a pasture mix with different host–symbiont pairs. We studied the effectiveness and cross-host compatibility of five commercial rhizobial strains for a range of pasture legumes (nine species, 18 cultivars) under controlled environment conditions, and further evaluated nodule occupancy and competitiveness of a newly established pasture (13 species, 20 cultivars) in the field, by determining nodulation and production (biomass and N2 fixation). Three of the commercial inoculant strains formed root nodules with multiple legume species; commonly however, less N2 was fixed in cases where the inoculant was not the recommended strain for the legume species. Within a legume species, cultivars could differ in their ability to form effective root nodules with multiple rhizobial strains. White clover cvv. Trophy, Haifa and Storm, strawberry clover cv. Palestine, and Talish clover cv. Permatas formed effective nodules with both TA1 and WSM1325 rhizobial strains. White clover cultivars that could not form an effective symbiosis with the common background strain WSM1325 fixed less N2. The white clover × Caucasian clover hybrid formed effective symbiosis with strain TA1 but not with other commercial strains. Some species such as birdsfoot trefoil, Talish clover, sulfur clover and tetraploid Caucasian clover formed ineffective symbiosis in the field. Until resolved, this will likely inhibit their further development as pasture plants for similar permanent pasture environments.

Complete Genome Sequences of Trifolium spp. Inoculant Strains Rhizobium leguminosarum sv. trifolii TA1 and CC275e: Resources for Genomic Study of the Rhizobium-Trifolium Symbiosis.

Rhizobium leguminosarum symbiovar trifolii strains TA1 and CC275e are nitrogen-fixing microsymbionts of Trifolium spp. and have been used as commercial inoculant strains for clovers in pastoral agriculture in Australia and New Zealand. Here we present the complete genome sequences of both strains, resolving their multipartite genome structures and allowing for future studies using genomic approaches.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Spontaneous Mutations in the Nitrate Reductase Gene napC Drive the Emergence of Eco-friendly Low-N2O-Emitting Alfalfa Rhizobia in Regions with Different Climates.

We have recently shown that commercial alfalfa inoculants (e.g., Sinorhizobium meliloti B399), which are closely related to the denitrifier model strain Sinorhizobium meliloti 1021, have conserved nitrate, nitrite, and nitric oxide reductases associated with the production of the greenhouse gas nitrous oxide (N2O) from nitrate but lost the N2O reductase related to the degradation of N2O to gas nitrogen. Here, we screened a library of nitrogen-fixing alfalfa symbionts originating from different ecoregions and containing N2O reductase genes and identified novel rhizobia (Sinorhizobium meliloti INTA1-6) exhibiting exceptionally low N2O emissions. To understand the genetic basis of this novel eco-friendly phenotype, we sequenced and analyzed the genomes of these strains, focusing on their denitrification genes, and found mutations only in the nitrate reductase structural gene napC. The evolutionary analysis supported that, in these natural strains, the denitrification genes were inherited by vertical transfer and that their defective nitrate reductase napC alleles emerged by independent spontaneous mutations. In silico analyses showed that mutations in this gene occurred in ssDNA loop structures with high negative free energy (-ΔG) and that the resulting mutated stem-loop structures exhibited increased stability, suggesting the occurrence of transcription-associated mutation events. In vivo assays supported that at least one of these ssDNA sites is a mutational hot spot under denitrification conditions. Similar benefits from nitrogen fixation were observed when plants were inoculated with the commercial inoculant B399 and strains INTA4-6, suggesting that the low-N2O-emitting rhizobia can be an ecological alternative to the current inoculants without resigning economic profitability.

Increasing biological nitrogen fixation by white clover-rhizobia symbiosis

Biological nitrogen fixation (BNF) is the process of converting atmospheric nitrogen to ammonia through legume–rhizobia symbiosis. The nitrogen fixed by rhizobia in root nodules is available for plant use. This process can be harnessed to improve N fertility on farm. Field surveys across New Zealand (NZ), within a farm and within paddocks, have revealed large spatial variability of rhizobial population size and symbiotic effectiveness with white clover. These results indicate that naturalised rhizobia may not be supporting optimal BNF. Over 500 strains of clover-nodulating rhizobia were isolated from NZ pasture soils, with more than 90 demonstrating greater N-fixation capacity with white clover than the commercial inoculant strain TA1. Seven NZ isolates were tested for nodule occupancy and all seven had significantly higher occupancy rates than TA1 in an in vitro assay, indicating increased competitiveness of those strains. In addition, novel seed-coating technology improved the survival of TA1 and isolate S10N9 from 1 month to more than 4 months compared with a standard coating formulation. There is potential to increase the symbiotic capacity of white clover in pastures through use of more effective and competitive rhizobial strains, along with their improved survival on seed provided by a new coating technology.

Fermentation quality and aerobic stability of mulberry silage prepared with lactic acid bacteria and propionic acid.

The objective of this study was to examine the fermentation quality and aerobic stability of mulberry (Morus alba L.) silage prepared with lactic acid bacteria (LAB) and propionic acid (PA). The selected LAB strains Lactobacillus (L.) plantarum LC365281 (L1) and L. brevis LC365282 (L2), and commercial inoculant strains L. plantarum Gaofuji (GF) and L. buchneri Fresh (FR), and PA were used as additives for silage preparation. Silage treatments were designed as control, L1, L2, GF, FR, PA, PA+L1, PA+L2, PA+GF, or PA+FR. After 30days of ensiling, the fermentation quality of silages treated with PA+L1 was improved, with a lower (p<0.05) pH and NH3 -N content than those of other treatments. During the aerobic exposure, the PA+LAB-treated silages displayed an aerobic stability with stable pH value and lactic acid content. The results confirm that L. plantarum L1 and PA were the best additive combination for ensiling mulberry.

Genetic diversity and symbiotic effectiveness of Phaseolus vulgaris-nodulating rhizobia in Kenya

Phaseolus vulgaris (common bean) was introduced to Kenya several centuries ago but the rhizobia that nodulate it in the country remain poorly characterised. To address this gap in knowledge, 178 isolates recovered from the root nodules of P. vulgaris cultivated in Kenya were genotyped stepwise by the analysis of genomic DNA fingerprints, PCR-RFLP and 16S rRNA, atpD, recA and nodC gene sequences. Results indicated that P. vulgaris in Kenya is nodulated by at least six Rhizobium genospecies, with most of the isolates belonging to Rhizobium phaseoli and a possibly novel Rhizobium species. Infrequently, isolates belonged to Rhizobium paranaense, Rhizobium leucaenae, Rhizobium sophoriradicis and Rhizobium aegyptiacum. Despite considerable core-gene heterogeneity among the isolates, only four nodC gene alleles were observed indicating conservation within this gene. Testing of the capacity of the isolates to fix nitrogen (N2) in symbiosis with P. vulgaris revealed wide variations in effectiveness, with ten isolates comparable to Rhizobium tropici CIAT 899, a commercial inoculant strain for P. vulgaris. In addition to unveiling effective native rhizobial strains with potential as inoculants in Kenya, this study demonstrated that Kenyan soils harbour diverse P. vulgaris-nodulating rhizobia, some of which formed phylogenetic clusters distinct from known lineages. The native rhizobia differed by site, suggesting that field inoculation of P. vulgaris may need to be locally optimised.

High spatial variation in population size and symbiotic performance of Rhizobium leguminosarum bv. trifolii with white clover in New Zealand pasture soils.

Biological nitrogen fixation through the legume-rhizobia symbiosis is important for sustainable pastoral production. In New Zealand, the most widespread and valuable symbiosis occurs between white clover (Trifolium repens L.) and Rhizobium leguminosarum bv. trifolii (Rlt). As variation in the population size (determined by most probable number assays; MPN) and effectiveness of N-fixation (symbiotic potential; SP) of Rlt in soils may affect white clover performance, the extent in variation in these properties was examined at three different spatial scales: (1) From 26 sites across New Zealand, (2) at farm-wide scale, and (3) within single fields. Overall, Rlt populations ranged from 95 to >1 x 108 per g soil, with variation similar at the three spatial scales assessed. For almost all samples, there was no relationship between rhizobia population size and ability of the population to fix N during legume symbiosis (SP). When compared with the commercial inoculant strain, the SP of soils ranged between 14 to 143% efficacy. The N-fixing ability of rhizobia populations varied more between samples collected from within a single hill country field (0.8 ha) than between 26 samples collected from diverse locations across New Zealand. Correlations between SP and calcium and aluminium content were found in all sites, except within a dairy farm field. Given the general lack of association between SP and MPN, and high spatial variability of SP at single field scale, provision of advice for treating legume seed with rhizobia based on field-average MPN counts needs to be carefully considered.

Burkholderia fungorum promotes common bean growth in a dystrophic oxisol

Burkolderia fungorum and Azospirillum brasilense are species commonly found in soils, and have been shown to be plant growth-promoting rhizobacteria (PGPR). This study examined the effect on the growth of the common bean plant of three strains belonging to B. fungorum and A. brasilense species inoculated alone or co-inoculated with Rhizobium tropici CIAT 899T (commercial inoculant strain). The ability of PGPR strains to improve bean growth in the presence of mineral nitrogen was also studied. CIAT 899T co-inoculated with B. fungorum UFLA 04-155 and UFLA 04-226 and A. brasilense BR 11001T did not show any benefits for bean growth. However, inoculation with B. fungorum UFLA 04-155 alone resulted in increased dry weight of the nodules and shoots, increased foliar contents of P, Mg, Cu, Fe, K, Ca, S, and Zn, and fostered nodulation by the indigenous rhizobia community. Plant growth promotion by B. fungorum UFLA 04-226 was stimulated in the presence of nitrogen fertilizer in experiments using potted plants. The results indicate the potential ability of B. fungorum to promote growth of the common bean plant under natural conditions.

English

Lack of indigenous soil rhizobia that colonize and nodulate soybean (Glycine max L. Merrill) roots is a common problem of South African soils. Bradyrhizobium japonicum strain WB74 has long been used as an effective commercial inoculant strain for soybean since its introduction in 1998. This paper investigates the major limiting factors involved in the nodulation efficiency of B. japonicum WB74 that affect soybean growth and yield in South Africa. Methods including analysis of soil physicochemical properties, farmers&rsquo; management practices and quality control tests of locally manufactured inoculant products were employed. Inoculant&rsquo;s strain verification was conducted using phylogenetic analysis of the 16S ribosomal RNA of the Bradyrhizobium strains in each of the inoculant product. The major findings of this study is that nodulation failure of introduced B. japonicum is caused by a combination of several limiting factors such as acidic nature of the soils as well as poor soil nutrition status especially that of phosphorous. Nodulation failure was also prominent in the soybean farms where there was no proper soil management practice. Results of the viability and shelf life studies also indicate that nodulation failure is caused by using sub-standard soybean inoculants products available on the market. Key words: Bradyrhizobium japonicum WB74, inoculants, nodulation, quality control.

Genome sequence of the Medicago-nodulating Ensifer meliloti commercial inoculant strain RRI128.

Ensifer meliloti strain RRI128 is an aerobic, motile, Gram-negative, non-spore-forming rod. RRI128 was isolated from a nodule recovered from the roots of barrel medic (Medicago truncatula) grown in the greenhouse and inoculated with soil collected from Victoria, Australia. The strain is used in commercial inoculants in Australia. RRI128 nodulates and forms an effective symbiosis with a diverse range of lucerne cultivars (Medicago sativa) and several species of annual medic (M. truncatula, Medicago littoralis and Medicago tornata), but forms an ineffective symbiosis with Medicago polymorpha. Here we describe the features of E. meliloti strain RRI128, together with genome sequence information and annotation. The 6,900,273 bp draft genome is arranged into 156 scaffolds of 157 contigs, contains 6,683 protein-coding genes and 87 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Genome sequence of the lupin-nodulating Bradyrhizobium sp. strain WSM1417

Bradyrhizobium sp. strain WSM1417 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen (N2) fixing root nodule of Lupinus sp. collected in Papudo, Chile, in 1995. However, this microsymbiont is a poorly effective N2 fixer with the legume host Lupinus angustifolius L.; a lupin species of considerable economic importance in both Chile and Australia. The symbiosis formed with L. angustifolius produces less than half of the dry matter achieved by the symbioses with commercial inoculant strains such as Bradyrhizobium sp. strain WSM471. Therefore, WSM1417 is an important candidate strain with which to investigate the genetics of effective N2 fixation in the lupin-bradyrhizobia symbioses. Here we describe the features of Bradyrhizobium sp. strain WSM1417, together with genome sequence information and annotation. The 8,048,963 bp high-quality-draft genome is arranged in a single scaffold of 2 contigs, contains 7,695 protein-coding genes and 77 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.

Laboratory prescreening of Bradyrhizobium japonicum for low pH, Al and Mn tolerance can be used to predict their survival in acid soils

We examined whether strains of Bradyrhizobium japonicum selected for growth on acid media in vitro would also survive and grow better in acid soils. Four agar screening media for acid-tolerant rhizobia, which differed in the number of acid soil stresses imposed (pH, low calcium (Ca) and phosphorus (P), high aluminum (Al) and manganese (Mn)), were assessed for their effects on the survival of 14 Indonesian strains and two commercial strains of B. japonicum. Survival of B. japonicum in the agar media was compared with that in two acid soils. A repeat stab inoculation method which provided a declining range of inoculum cell number to 103 cells per stab was used to assess the daily growth of the strains on the screening media at 5 pH levels (3.8, 4.2, 4.5, 5.0, and 6.8). The growth and survival of the 16 strains were then measured at days 1, 8, 18, and 28 after inoculation in two acid soils (pH 4.24 and 4.35) sterilized using γ-irradiation at 5.0 Mrad. Selectivity of the agar media improved as more acid stress factors were incorporated in the media. Those strains of Bradyrhizobium identified as acid, Al and Mn-tolerant in acidic agar media, also had better survival in the low pH soils. There was no relationship between acid or alkali production on agar media and acid tolerance on agar or in soil. There was no apparent relationship between symbiotic performance and acid tolerance, and one acid-tolerant strain was as effective as the commercial inoculant strain CB1809. The most acid-tolerant strain was also the most ineffective.

Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes

Lotus species are legumes with potential for pastures in soils with low-fertility and environmental constraints. The aim of this work was to characterize bacteria that establish efficient nitrogen-fixing symbiosis with the forage species Lotus uliginosus. A total of 39 isolates were obtained from nodules of L. uliginosus naturally growing in two different locations of Portugal. Molecular identification of the isolates plus the commercial inoculant strain NZP2039 was performed by REP-PCR, 16S rRNA RFLP, and 16S rRNA, glnII and recA sequence analyses. Limited genetic diversity was found among the L. uliginosus symbionts, which showed a close phylogenetic relationship with the species Bradyrhizobium japonicum. The symbiotic nifH, nodA and nodC gene sequences were closely related with the corresponding genes of various Bradyrhizobium strains isolated from Lupinus and other genistoid legumes and therefore were phylogenetically separated from other Lotus spp. rhizobia. The L. uliginosus bradyrhizobia were able to nodulate and fix nitrogen in association with L. uliginosus, could nodulate Lotus corniculatus with generally poor nitrogen-fixing efficiency, formed nonfixing nodules in Lotus tenuis and Lupinus luteus roots and were unable to nodulate Glycine soja or Glycine max. Thus, L. uliginosus rhizobia seem closely related to B. japonicum biovar genistearum strains.

Three native Pseudomonas fluorescens strains tested under growth chamber and field conditions as biocontrol agents against damping-off in alfalfa

Alfalfa ( Medicago sativa) is one of the most important crops used in Uruguay for livestock feeding. Seedling diseases, particularly damping-off, are a critical factor which limits its establishment. Three native Pseudomonas fluorescens strains, UP61.2, UP143.8 and UP148.2, previously isolated from Lotus corniculatus, were evaluated to determine their efficacy as biological control agents for alfalfa seedling diseases in the field. Their compatibility with the alfalfa- Sinorhizobium meliloti symbiosis was also assessed. In growth chamber conditions seed inoculation with Pseudomonas strains did not affect different parameters of alfalfa-rhizobium symbiosis as shown by nodulation rate and shoot dry weight of plants. The presence of the commercial inoculant strains of S. meliloti did not impair colonization by the P. fluorescens and vice versa. In field trials the dynamics of rhizobial rhizospheric populations were not affected by the presence of P. fluorescens. Each P. fluorescens strain successfully colonized alfalfa roots at adequate densities for biocontrol activity. Results showed that P. fluorescens strains provided a 10–13% increase in the number of established plants relative to the control, an intermediate result compared to the fungicide treatment (24%). The alfalfa above-ground biomass was increased by 13% and 15–18% in the presence of the fungicide and P. fluorescens strains, respectively. Therefore, results from this study demonstrated that the three P. fluorescens strains provided effective control against soil-borne pathogens and suggest a potential use in the development of a commercial inoculant to be applied for the control of legume seedling diseases.