The putative vacuolar processing enzyme gene TaVPE3cB is a candidate gene for wheat stem pith-thickness.

KEY MESSAGE: The vacuolar processing enzyme gene TaVPE3cB is identified as a candidate gene for a QTL of wheat pith-thickness on chromosome 3B by BSR-seq and differential expression analyses. The high pith-thickness (PT) of the wheat stem could greatly enhance stem mechanical strength, especially the basal internodes which support the heavier upper part, such as upper stems, leaves and spikes. A QTL for PT in wheat was previously discovered on 3BL in a double haploid population of 'Westonia' × 'Kauz'. Here, a bulked segregant RNA-seq analysis was applied to identify candidate genes and develop associated SNP markers for PT. In this study, we aimed at screening differentially expressed genes (DEGs) and SNPs in the 3BL QTL interval. Sixteen DEGs were obtained based on BSR-seq and differential expression analyses. Twenty-four high-probability SNPs in eight genes were identified by comparing the allelic polymorphism in mRNA sequences between the high PT and low PT samples. Among them, six genes were confirmed to be associated with PT by qRT-PCR and sequencing. A putative vacuolar processing enzyme gene TaVPE3cB was screened out as a potential PT candidate gene in Australian wheat 'Westonia'. A robust SNP marker associated with TaVPE3cB was developed, which can assist in the introgression of TaVPE3cB.b in wheat breeding programs. In addition, we also discussed the function of other DEGs which may be related to pith development and programmed cell death (PCD). A five-level hierarchical regulation mechanism of stem pith PCD in wheat was proposed.

Bradyrhizobium cenepequi sp. nov., Bradyrhizobium semiaridum sp. nov., Bradyrhizobium hereditatis sp. nov. and Bradyrhizobium australafricanum sp. nov., symbionts of different leguminous plants of Western Australia and South Africa and definition of three novel symbiovars.

Bradyrhizobium is a heterogeneous bacterial genus capable of establishing symbiotic associations with a broad range of legume hosts, including species of economic and environmental importance. This study was focused on the taxonomic and symbiovar definition of four strains - CNPSo 4026T, WSM 1704T, WSM 1738T and WSM 4400T - previously isolated from nodules of legumes in Western Australia and South Africa. The 16S rRNA gene phylogenetic tree allocated the strains to the Bradyrhizobium elkanii supergroup. The multilocus sequence analysis (MLSA) with partial sequences of six housekeeping genes - atpD, dnaK, glnII, gyrB, recA and rpoB - did not cluster the strains under study as conspecific to any described Bradyrhizobium species. Average nucleotide identity and digital DNA-DNA hybridization values were calculated for the four strains of this study and the closest species according to the MLSA phylogeny with the highest values being 95.46 and 62.20 %, respectively; therefore, both being lower than the species delineation cut-off values. The nodC and nifH phylogenies included strains WSM 1738T and WSM 4400T in the symbiovars retamae and vignae respectively, and also allowed the definition of three new symbiovars, sv. cenepequi, sv. glycinis, and sv. cajani. Analysis of morphophysiological characterization reinforced the identification of four novel proposed Bradyrhizobium species that are accordingly named as follows: Bradyrhizobium cenepequi sp. nov. (CNPSo 4026T=WSM 4798T=LMG 31653T), isolated from Vigna unguiculata; Bradyrhizobium semiaridum sp. nov. (WSM 1704T=CNPSo 4028T=LMG 31654T), isolated from Tephrosia gardneri; Bradyrhizobium hereditatis sp. nov. (WSM 1738T=CNPSo 4025T=LMG 31652T), isolated from Indigofera sp.; and Bradyrhizobium australafricanum sp. nov. (WSM 4400T=CNPSo 4015T=LMG 31648T) isolated from Glycine sp.

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.

Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements.

Tripartite integrative and conjugative elements (ICE3) are a novel form of ICE that exist as three separate DNA regions integrated within the genomes of Mesorhizobium spp. Prior to conjugative transfer the three ICE3 regions of M. ciceri WSM1271 ICEMcSym1271 combine and excise to form a single circular element. This assembly requires three coordinated recombination events involving three site-specific recombinases IntS, IntG and IntM. Here, we demonstrate that three excisionases-or recombination directionality factors-RdfS, RdfG and RdfM are required for ICE3 excision. Transcriptome sequencing revealed that expression of ICE3 transfer and conjugation genes was induced by quorum sensing. Quorum sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a "master controller" of ICE3 assembly and excision. The dependence of all three excisive reactions on RdfS ensures that ICE3 excision occurs via a stepwise sequence of recombination events that avoids splitting the chromosome into a non-viable configuration. These discoveries expose a surprisingly simple control system guiding molecular assembly of these novel and complex mobile genetic elements and highlight the diverse and critical functions of excisionase proteins in control of horizontal gene transfer.

Bradyrhizobium archetypum sp. nov., Bradyrhizobium australiense sp. nov. and Bradyrhizobium murdochi sp. nov., isolated from nodules of legumes indigenous to Western Australia.

The genus Bradyrhizobium is considered as the probable ancestor lineage of all rhizobia, broadly spread in a variety of ecosystems and with remarkable diversity. A polyphasic study was performed to characterize and clarify the taxonomic position of eight bradyrhizobial strains isolated from indigenous legumes to Western Australia. As expected for the genus, the 16S rRNA gene sequences were highly conserved, but the results of multilocus sequence analysis with four housekeeping genes (dnaK, glnII, gyrB and recA) confirmed three new distinct clades including the following strains: (1) WSM 1744T, WSM 1736 and WSM 1737; (2) WSM 1791T and WSM 1742; and (3) WSM 1741T, WSM 1735 and WSM 1790. The highest ANI values of the three groups in relation to the closest type strains were 92.4, 92.3 and 93.3 %, respectively, below the threshold of species circumscription. The digital DNA-DNA hybridization analysis also confirmed new species descriptions, with less than 52 % relatedness with the closest type strains. The phylogeny of the symbiotic gene nodC clustered the eight strains into the symbiovar retamae, together with seven Bradyrhizobium type strains, sharing from 94.2-98.1 % nucleotide identity (NI), and less than 88.7 % NI with other related strains and symbiovars. Morpho-physiological, phylogenetics, genomic and symbiotic traits were determined for the new groups and our data support the description of three new species, Bradyrhizobium archetypum sp. nov., Bradyrhizobium australiense sp. nov. and Bradyrhizobium murdochi sp. nov., with WSM 1744T (=CNPSo 4013T=LMG 31646T), WSM 1791T (=CNPSo 4014T=LMG 31647T) and WSM 1741T (=CNPSo 4020T=LMG 31651T) designated as type strains, respectively.

Bradyrhizobium agreste sp. nov., Bradyrhizobium glycinis sp. nov. and Bradyrhizobium diversitatis sp. nov., isolated from a biodiversity hotspot of the genus Glycine in Western Australia.

Strains of the genus Bradyrhizobium associated with agronomically important crops such as soybean (Glycine max) are increasingly studied; however, information about symbionts of wild Glycine species is scarce. Australia is a genetic centre of wild Glycine species and we performed a polyphasic analysis of three Bradyrhizobium strains-CNPSo 4010T, CNPSo 4016T, and CNPSo 4019T-trapped from Western Australian soils with Glycine clandestina, Glycine tabacina and Glycine max, respectively. The phylogenetic tree of the 16S rRNA gene clustered all strains into the Bradyrhizobium japonicum superclade; strains CNPSo 4010T and CNPSo 4016T had Bradyrhizobium yuanmingense CCBAU 10071T as the closest species, whereas strain CNPSo 4019T was closer to Bradyrhizobium liaoningense LMG 18230T. The multilocus sequence analysis (MLSA) with five housekeeping genes-dnaK, glnII, gyrB, recA and rpoB-confirmed the same clusters as the 16S rRNA phylogeny, but indicated low similarity to described species, with nucleotide identities ranging from 93.6 to 97.6% of similarity. Considering the genomes of the three strains, the average nucleotide identity and digital DNA-DNA hybridization values were lower than 94.97 and 59.80 %, respectively, with the closest species. In the nodC phylogeny, strains CNPSo 4010T and CNPSo 4019T grouped with Bradyrhizobium zhanjiangense and Bradyrhizobium ganzhouense, respectively, while strain CNPSo 4016T was positioned separately from the all symbiotic Bradyrhizobium species. Other genomic (BOX-PCR), phenotypic and symbiotic properties were evaluated and corroborated with the description of three new lineages of Bradyrhizobium. We propose the names of Bradyrhizobium agreste sp. nov. for CNPSo 4010T (=WSM 4802T=LMG 31645T) isolated from Glycine clandestina, Bradyrhizobium glycinis sp. nov. for CNPSo 4016T (=WSM 4801T=LMG 31649T) isolated from Glycine tabacina and Bradyrhizobium diversitatis sp. nov. for CNPSo 4019T (=WSM 4799T=LMG 31650T) isolated from G. max.

Assembly and transfer of tripartite integrative and conjugative genetic elements.

Integrative and conjugative elements (ICEs) are ubiquitous mobile genetic elements present as "genomic islands" within bacterial chromosomes. Symbiosis islands are ICEs that convert nonsymbiotic mesorhizobia into symbionts of legumes. Here we report the discovery of symbiosis ICEs that exist as three separate chromosomal regions when integrated in their hosts, but through recombination assemble as a single circular ICE for conjugative transfer. Whole-genome comparisons revealed exconjugants derived from nonsymbiotic mesorhizobia received three separate chromosomal regions from the donor Mesorhizobium ciceri WSM1271. The three regions were each bordered by two nonhomologous integrase attachment (att) sites, which together comprised three homologous pairs of attL and attR sites. Sequential recombination between each attL and attR pair produced corresponding attP and attB sites and joined the three fragments to produce a single circular ICE, ICEMcSym1271 A plasmid carrying the three attP sites was used to recreate the process of tripartite ICE integration and to confirm the role of integrase genes intS, intM, and intG in this process. Nine additional tripartite ICEs were identified in diverse mesorhizobia and transfer was demonstrated for three of them. The transfer of tripartite ICEs to nonsymbiotic mesorhizobia explains the evolution of competitive but suboptimal N2-fixing strains found in Western Australian soils. The unheralded existence of tripartite ICEs raises the possibility that multipartite elements reside in other organisms, but have been overlooked because of their unusual biology. These discoveries reveal mechanisms by which integrases dramatically manipulate bacterial genomes to allow cotransfer of disparate chromosomal regions.

Evolutionary persistence of tripartite integrative and conjugative elements.

Integrative and conjugative elements (ICEs) are generally regarded as regions of contiguous DNA integrated within a bacterial genome that are capable of excision and horizontal transfer via conjugation. We recently characterized a unique group of ICEs present in Mesorhizobium spp., which exist as three entirely separate but inextricably linked chromosomal regions termed α, ß and γ. These regions occupy three different recombinase attachment (att) sites; however, they do not excise independently. Rather, they recombine the host chromosome to form a single contiguous region prior to excision and conjugative transfer. Like the single-part ICE carried by M. loti R7A (ICEMlSymR7A), these "tripartite" ICEs (ICE3s) are widespread throughout the Mesorhizobium genus and enable strains to form nitrogen-fixing symbioses with a variety of legumes. ICE3s have likely evolved following recombination between three separate ancestral integrative elements, however, the persistence of ICE3 structure in diverse mesorhizobia is perplexing due to its seemingly unnecessary complexity. In this study, examination of ICE3s revealed that most symbiosis genes are carried on the large α fragment. Some ICE3-ß and γ regions also carry genes that potentially contribute to the symbiosis, or to persistence in the soil environment, but these regions have been frequently subjected to recombination events including deletions, insertions and recombination with genes located on other integrative elements. Examination of a new ICE3 in M. ciceri Ca181 revealed it has jettisoned the genetic cargo from its ß region and recruited a serine recombinase gene within its γ region, resulting in replacement of one of the three ICE3 integration sites. Overall the recombination loci appear to be the only conserved features of the ß and γ regions, suggesting that the tripartite structure itself provides a selective benefit to the element. We propose the ICE3 structure provides enhanced host range, host stability and resistance to destabilization by tandem insertion of competing integrative elements. Furthermore, we suspect the ICE3 tripartite structure increases the likelihood of gene capture from integrative elements sharing the same attachment sites.

Bradyrhizobium centrolobii and Bradyrhizobium macuxiense sp. nov. isolated from Centrolobium paraense grown in soil of Amazonia, Brazil.

Thirteen Gram-negative, aerobic, motile with polar flagella, rod-shaped bacteria were isolated from root nodules of Centrolobium paraense Tul. grown in soils from the Amazon region of Brazil. Growth of strains was observed at temperature range 20-36 °C (optimal 28 °C), pH ranges 5-11 (optimal 6.0-7.0), and 0.1-0.5%NaCl (optimal 0.1-0.3%). Analysis of 16S rRNA gene placed the strains into two groups within Bradyrhizobium. Closest neighbouring species (98.8%) for group I was B. neotropicale while for group II were 12 species with more than 99% of similarity. Multi-locus sequence analysis (MLSA) with dnaK, glnII, recA, and rpoB confirmed B. neotropicale BR 10247T as the closest type strain for the group I and B. elkanii USDA 76T and B. pachyrhizi PAC 48T for group II. Average Nucleotide Identity (ANI) differentiated group I from the B. neotropicale BR 10247T (79.6%) and group II from B. elkanii USDA 76T and B. pachyrhizi PAC 48T (88.1% and 87.9%, respectively). Fatty acid profiles [majority C16:0 and Summed feature 8 (18:1ω6c/18:1ω7c) for both groups], DNA G + C content, and carbon compound utilization supported the placement of the novel strains in the genus Bradyrhizobium. Gene nodC and nifH of the new strains have in general low similarity with other Bradyrhizobium species. Both groups nodulated plants from the tribes Crotalarieae, Dalbergiae, Genisteae, and Phaseoleae. Based on the presented data, two novel species which the names Bradyrhizobium centrolobii and Bradyrhizobium macuxiense are proposed, with BR 10245T (=HAMBI 3597T) and BR 10303T (=HAMBI 3602T) as the respective-type strains.

Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

Bradyrhizobium ingae sp. nov., isolated from effective nodules of Inga laurina grown in Cerrado soil.

Root-nodule bacteria were isolated from Inga laurina (Sw.) Willd. growing in the Cerrado Amazon region, State of Roraima, Brazil. The 16S rRNA gene sequences of six strains (BR 10250(T), BR 10248, BR 10249, BR 10251, BR 10252 and BR 10253) showed low similarities with currently described species of the genus Bradyrhizobium. Phylogenetic analyses of sequences of five housekeeping genes (dnaK, glnII, gyrB, recA and rpoB) revealed Bradyrhizobium iriomotense EK05(T) to be the closest type strain (97.4% sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [with the major components C16:0 and summed feature 8 (C18:1ω6c/C18:1ω7c)], the slow growth rate and carbon compound utilization patterns supported the assignment of our strains to the genus Bradyrhizobium. Results from DNA-DNA hybridizations and physiological traits differentiated our strains from the closest related species of the genus Bradyrhizobium with validly published names. Sequences of symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nifH) grouped together with those of B. iriomotense EK05(T) and Bradyrhizobium sp. strains BR 6610 (used as a commercial inoculant for Inga marginata in Brazil) and TUXTLAS-10 (previously observed in Central America). Based on these data, the six strains represent a novel species, for which the name Bradyrhizobium ingae sp. nov. is proposed. The type strain is BR 10250(T) ( = HAMBI 3600(T)).

Bradyrhizobium neotropicale sp. nov., isolated from effective nodules of Centrolobium paraense.

Root nodule bacteria were isolated from Centrolobium paraense Tul. grown in soils from the Amazon region, State of Roraima (Brazil). 16S rRNA gene sequence analysis of seven strains (BR 10247(T), BR 10296, BR 10297, BR 10298, BR 10299, BR 10300 and BR 10301) placed them in the genus Bradyrhizobium with the closest neighbours being the type strains of Bradyrhizobium paxllaeri (98.8 % similarity), Bradyrhizobium icense (98.8 %), Bradyrhizobium lablabi (98.7 %), Bradyrhizobium jicamae (98.6 %), Bradyrhizobium elkanii (98.6 %), Bradyrhizobium pachyrhizi (98.6 %) and Bradyrhizobium retamae (98.3 %). This high similarity, however, was not confirmed by the intergenic transcribed spacer (ITS) 16S-23S rRNA region sequence analysis nor by multi-locus sequence analysis. Phylogenetic analyses of five housekeeping genes (dnaK, glnII, gyrB, recA and rpoB) revealed Bradyrhizobium iriomotense EK05(T) ( = LMG 24129(T)) to be the most closely related type strain (95.7 % sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [major components being C16 : 0 and summed feature 8 (18 : 1ω6c/18 : 1ω7c)], DNA G+C content, slow growth rate and carbon compound utilization patterns, supported the placement of the novel strains in the genus Bradyrhizobium. Results of DNA-DNA relatedness studies and physiological data (especially carbon source utilization) differentiated the strains from the closest recognized species of the genus Bradyrhizobium. Symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nifH) placed the novel species in a new branch within the genus Bradyrhizobium. Based on the current data, these seven strains represent a novel species for which the name Bradyrhizobium neotropicale sp. nov. is proposed. The type strain is BR 10247(T) ( = HAMBI 3599(T)).

Bradyrhizobium manausense sp. nov., isolated from effective nodules of Vigna unguiculata grown in Brazilian Amazonian rainforest soils.

Root nodule bacteria were trapped within cowpea (Vigna unguiculata) in soils with different cultivation histories collected from the Amazonian rainforest in northern Brazil. Analysis of the 16S rRNA gene sequences of six strains (BR 3351(T), BR 3307, BR 3310, BR 3315, BR 3323 BR and BR 3361) isolated from cowpea nodules showed that they formed a distinct group within the genus Bradyrhizobium, which was separate from previously identified type strains. Phylogenetic analyses of three housekeeping genes (glnII, recA and rpoB) revealed that Bradyrhizobium huanghuaihaiense CCBAU 23303(T) was the most closely related type strain (96% sequence similarity or lower). Chemotaxonomic data, including fatty acid profiles (predominant fatty acids being C16 : 0 and summed feature 8), the slow growth rate and carbon compound utilization patterns supported the assignment of the strains to the genus Bradyrhizobium. The results of DNA-DNA hybridizations, antibiotic resistance and physiological tests differentiated these novel strains from the most closely related species of the genus Bradyrhizobium with validly published names. Symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nifH) grouped the novel strains of the genus Bradyrhizobium together with Bradyrhizobium iriomotense strain EK05(T), with 94% and 96% sequence similarity, respectively. Based on these data, these six strains represent a novel species for which the name Brabyrhizobium manausense sp. nov. (BR 3351(T) = HAMBI 3596(T)), is proposed.

Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the Southern African crotalarioid clade Lotononis s.l.

BACKGROUND AND AIMS: The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. METHODS: Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. KEY RESULTS: Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. CONCLUSIONS: Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.

Current Progress and Future Prospect of Wheat Genetics Research towards an Enhanced Nitrogen Use Efficiency.

To improve the yield and quality of wheat is of great importance for food security worldwide. One of the most effective and significant approaches to achieve this goal is to enhance the nitrogen use efficiency (NUE) in wheat. In this review, a comprehensive understanding of the factors involved in the process of the wheat nitrogen uptake, assimilation and remobilization of nitrogen in wheat were introduced. An appropriate definition of NUE is vital prior to its precise evaluation for the following gene identification and breeding process. Apart from grain yield (GY) and grain protein content (GPC), the commonly recognized major indicators of NUE, grain protein deviation (GPD) could also be considered as a potential trait for NUE evaluation. As a complex quantitative trait, NUE is affected by transporter proteins, kinases, transcription factors (TFs) and micro RNAs (miRNAs), which participate in the nitrogen uptake process, as well as key enzymes, circadian regulators, cross-talks between carbon metabolism, which are associated with nitrogen assimilation and remobilization. A series of quantitative genetic loci (QTLs) and linking markers were compiled in the hope to help discover more efficient and useful genetic resources for breeding program. For future NUE improvement, an exploration for other criteria during selection process that incorporates morphological, physiological and biochemical traits is needed. Applying new technologies from phenomics will allow high-throughput NUE phenotyping and accelerate the breeding process. A combination of multi-omics techniques and the previously verified QTLs and molecular markers will facilitate the NUE QTL-mapping and novel gene identification.

Complete genome sequence of Rhizobium leguminosarum bv. viciae SRDI969, an acid-tolerant, efficient N2-fixing microsymbiont of Vicia faba.

We report the complete genome sequence of Rhizobium leguminosarum bv. viciae SRDI969, an acid-tolerant, efficient nitrogen-fixing microorganism of Vicia faba. The 6.8 Mbp genome consists of a chromosome and four plasmids, with the symbiosis and nitrogen fixation genes encoded on the chromosome.

Competition in the Phaseolus vulgaris-Rhizobium symbiosis and the role of resident soil rhizobia in determining the outcomes of inoculation.

Background and Aims: Inoculation of legumes with effective N2-fixing rhizobia is a common practice to improve farming profitability and sustainability. To succeed, inoculant rhizobia must overcome competition for nodulation by resident soil rhizobia that fix N2 ineffectively. In Kenya, where Phaseolus vulgaris (common bean) is inoculated with highly effective Rhizobium tropici CIAT899 from Colombia, response to inoculation is low, possibly due to competition from ineffective resident soil rhizobia. Here, we evaluate the competitiveness of CIAT899 against diverse rhizobia isolated from cultivated Kenyan P. vulgaris. Methods: The ability of 28 Kenyan P. vulgaris strains to nodulate this host when co-inoculated with CIAT899 was assessed. Rhizosphere competence of a subset of strains and the ability of seed inoculated CIAT899 to nodulate P. vulgaris when sown into soil with pre-existing populations of rhizobia was analyzed. Results: Competitiveness varied widely, with only 27% of the test strains more competitive than CIAT899 at nodulating P. vulgaris. While competitiveness did not correlate with symbiotic effectiveness, five strains were competitive against CIAT899 and symbiotically effective. In contrast, rhizosphere competence strongly correlated with competitiveness. Soil rhizobia had a position-dependent numerical advantage, outcompeting seed-inoculated CIAT899 for nodulation of P. vulgaris, unless the resident strain was poorly competitive. Conclusion: Suboptimally effective rhizobia can outcompete CIAT899 for nodulation of P. vulgaris. If these strains are widespread in Kenyan soils, they may largely explain the poor response to inoculation. The five competitive and effective strains characterized here are candidates for inoculant development and may prove better adapted to Kenyan conditions than CIAT899.

Physiological and cannabinoid responses of hemp (Cannabis sativa) to rock phosphate dust under tropical conditions.

Growing a high-value crop such as industrial hemp (Cannabis sativa L.) in post-mining environments is economically and environmentally attractive but faces a range of biotic and abiotic challenges. An opportunity to investigate the cultivation of C. sativa presented itself as part of post-mining activities on Christmas Island (Australia) to profitably utilise disused phosphate (PS) quarries. Challenges to plant growth and cadmium (Cd) uptake were addressed in this study using potted plants under fully controlled conditions in a growth chamber. A complete nutritional spectrum, slow-release fertiliser was applied to all plants as a control treatment, and two levels of rock PS dust, a waste product of PS mining that contains 35% phosphorus (P) and 40ppm of naturally occurring Cd, were applied at 54 and 162gL-1 . After 12weeks, control plants (no PS dust) significantly differed in phenological development, with no flower production, lower aboveground biomass and reduced photosynthesis efficiency than those with P applied as rock dust. Compared with the controls, the 54gL-1 level of P dust increased shoot biomass by 38%, while 162gL-1 increased shoot biomass by 85%. The concentration of Δ9 -tetrahydrocannabinol also increased with the higher P levels. Cd uptake from PS dust by C. sativa was substantial and warrants further investigation. However, there was no increase in Cd content between the 54 and 162gL-1 application rates in seed and leaf. Results indicate that hemp could become a high-value crop on Christmas Island, with the readily available rock PS dust providing a source of P.

Microvirga lupini sp. nov., Microvirga lotononidis sp. nov. and Microvirga zambiensis sp. nov. are alphaproteobacterial root-nodule bacteria that specifically nodulate and fix nitrogen with geographically and taxonomically separate legume hosts.

Strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from nitrogen-fixing nodules of the native legumes Listia angolensis (from Zambia) and Lupinus texensis (from Texas, USA). Phylogenetic analysis of the 16S rRNA gene showed that the novel strains belong to the genus Microvirga, with ≥ 96.1% sequence similarity with type strains of this genus. The closest relative of the representative strains Lut6(T) and WSM3557(T) was Microvirga flocculans TFB(T), with 97.6-98.0% similarity, while WSM3693(T) was most closely related to Microvirga aerilata 5420S-16(T), with 98.8% similarity. Analysis of the concatenated sequences of four housekeeping gene loci (dnaK, gyrB, recA and rpoB) and cellular fatty acid profiles confirmed the placement of Lut6(T), WSM3557(T) and WSM3693(T) within the genus Microvirga. DNA-DNA relatedness values, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of Lut6(T), WSM3557(T) and WSM3693(T) from each other and from other Microvirga species with validly published names. The nodA sequence of Lut6(T) was placed in a clade that contained strains of Rhizobium, Mesorhizobium and Sinorhizobium, while the 100% identical nodA sequences of WSM3557(T) and WSM3693(T) clustered with Bradyrhizobium, Burkholderia and Methylobacterium strains. Concatenated sequences for nifD and nifH show that the sequences of Lut6(T), WSM3557(T) and WSM3693(T) were most closely related to that of Rhizobium etli CFN42(T) nifDH. On the basis of genotypic, phenotypic and DNA relatedness data, three novel species of Microvirga are proposed: Microvirga lupini sp. nov. (type strain Lut6(T) =LMG 26460(T) =HAMBI 3236(T)), Microvirga lotononidis sp. nov. (type strain WSM3557(T) =LMG 26455(T) =HAMBI 3237(T)) and Microvirga zambiensis sp. nov. (type strain WSM3693(T) =LMG 26454(T) =HAMBI 3238(T)).

Semi-quantitative analysis of cannabinoids in hemp (Cannabis sativa L.) using gas chromatography coupled to mass spectrometry.

BACKGROUND: Hemp (Cannabis sativa L.) is a producer of cannabinoids. These organic compounds are of increasing interest due to their potential applications in the medicinal field. Advances in analytical methods of identifying and quantifying these molecules are needed. METHOD: This study describes a new method of cannabinoid separation from plant material using gas chromatography-mass spectrometry (GC-MS) as the analytical tool to detect low abundance cannabinoids that will likely have implications for future therapeutical treatments. A novel approach was adopted to separate trichomes from plant material to analyse cannabinoids of low abundance not observed in raw plant extract. Required plant sample used for analysis was greatly reduced compared to other methods. Derivatisation method was simplified and deconvolution software was utilised to recognise unknown cannabinoid compounds of low abundance. RESULTS: The method produces well-separated spectra and allows the detection of major and minor cannabinoids. Ten cannabinoids that had available standards could be identified and quantified and numerous unidentified cannabinoids or pathway intermediates based on GC-MS spectra similarities could be extracted and analysed simultaneously with this method. CONCLUSIONS: This is a rapid novel extraction and analytical method from plant material that can identify major and minor cannabinoids using a simple technique. The method will be of use to future researchers seeking to study the multitude of cannabinoids whose values are currently not understood.