Extensive Local Geographic Variation in Locoweed Toxin Produced by a Fungal Endophyte.

Legumes are notorious for coevolutionary arms races where chemical defenses are employed to ward off herbivores-particularly insect seed predators. Locoweeds are legumes containing the toxic alkaloid swainsonine which can poison livestock, but its role as a deterrent for insects is unknown. Swainsonine is produced by the fungal endophyte Alternaria section Undifilum, and the chemical composition of the toxin has been well characterized. Despite this knowledge, the ecological roles and evolutionary drivers of swainsonine toxins in locoweeds remain uncertain. Here, we quantify swainsonine concentrations and herbivory levels in the hyper-diverse locoweed Astragalus lentiginosus to evaluate its role as an evolved chemical defense. We found that A. lentiginosus shows considerable variation in swainsonine concentrations according to variety, in particular showing presence/absence variation at both population and local geographic scales. Surprisingly, herbivory levels from presumed generalist insects emerging from fruits showed no correlation with swainsonine concentrations. Conversely, seed and fruit herbivory levels linked to specialist Acanthoscelides seed beetles increased with concentrations of swainsonine-suggesting a possible coevolutionary arms race. Our results highlight that variation in endophyte-produced toxin systems may not follow classical expectations for geographic variation and ecological roles of plant chemicals. We discuss the implications of these results on plant-endophytic toxin systems and coevolutionary dynamics more broadly, highlighting a considerable need for more research in these systems.

Integration of Transcriptomics and WGCNA to Characterize Trichoderma harzianum-Induced Systemic Resistance in Astragalus mongholicus for Defense against Fusarium solani.

Beneficial fungi of the genus Trichoderma are among the most widespread biocontrol agents that induce a plant's defense response against pathogens. Fusarium solani is one of the main pathogens that can negatively affect Astragalus mongholicus production and quality. To investigate the impact of Trichoderma harzianum on Astragalus mongholicus defense responses to Fusarium solani, A. mongholicus roots under T. harzianum + F. solani (T + F) treatment and F. solani (F) treatment were sampled and subjected to transcriptomic analysis. A differential expression analysis revealed that 6361 differentially expressed genes (DEGs) responded to T. harzianum induction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the 6361 DEGs revealed that the genes significantly clustered into resistance-related pathways, such as the plant-pathogen interaction pathway, phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway, isoflavonoid biosynthesis pathway, mitogen-activated protein kinase (MAPK) signaling pathway, and plant hormone signal transduction pathway. Pathway analysis revealed that the PR1, formononetin biosynthesis, biochanin A biosynthesis, and CHIB, ROS production, and HSP90 may be upregulated by T. harzianum and play important roles in disease resistance. Our study further revealed that the H2O2 content was significantly increased by T. harzianum induction. Formononetin and biochanin A had the potential to suppress F. solani. Weighted gene coexpression network analysis (WGCNA) revealed one module, including 58 DEGs associated with T. harzianum induction. One core hub gene, RPS25, was found to be upregulated by T. harzianum, SA (salicylic acid) and ETH (ethephon). Overall, our data indicate that T. harzianum can induce induced systemic resistance (ISR) and systemic acquired resistance (SAR) in A. mongholicus. The results of this study lay a foundation for a further understanding of the molecular mechanism by which T. harzianum induces resistance in A. mongholicus.

Nitrogen-fixing bacteria promote growth and bioactive components accumulation of Astragalus mongholicus by regulating plant metabolism and rhizosphere microbiota.

BACKGROUND: The excessive application of chemical fertilizers in the cultivation of Astragalus mongholicus Bunge results in a reduction in the quality of the medicinal plant and compromises the sustainable productivity of the soil. PGPB inoculant is a hot topic in ecological agriculture research. In the cultivation of Astragalus mongholicus, the screened nitrogen-fixing bacteria can promote plant growth, however, whether it can promote the accumulation of main bioactive components remains unknown. In this study, mixed inoculants containing 5 strains of growth promoting bacteria (Rhizobium T16 , Sinorhizobium T21 , Bacillus J1 , Bacillus G4 and Arthrobacter J2) were used in the field experiment. The metabolic substances in the root tissues of Astragalus mongholicus were identified during the harvest period by non-targeted metabolomics method, and the differential metabolites between groups were identified by statistical analysis. Meanwhile, high-throughput sequencing was performed to analyze the changes of rhizosphere soil and endophytic microbial community structure after mixed microbial treatment. RESULTS: The results of non-targeted metabolism indicated a significant increase in the levels of 26 metabolites after treatment including 13 flavonoids, 3 saponins and 10 other components. The contents of three plant hormones (abscisic acid, salicylic acid and spermidine) also increased after treatment, which presumed to play an important role in regulating plant growth and metabolism. Studies on endosphere and rhizosphere bacterial communities showed that Rhzobiaceae, Micromonosporaceae, and Hypomicrobiaceae in endophytic, and Oxalobactereae in rhizosphere were significantly increased after treatment. These findings suggest their potential importance in plant growth promotion and secondary metabolism regulation. CONCLUSIONS: This finding provides a basis for developing nitrogen-fixing bacteria fertilizer and improving the ecological planting efficiency of Astragalus mongholicus.

Progress on metabolites of Astragalus medicinal plants and a new factor affecting their formation: Biotransformation of endophytic fungi.

It is generally believed that the main influencing factors of plant metabolism are genetic and environmental factors. However, the transformation and catalysis of metabolic intermediates by endophytic fungi have become a new factor and resource attracting attention in recent years. There are over 2000 precious plant species in the Astragalus genus. In the past decade, at least 303 high-value metabolites have been isolated from the Astragalus medicinal plants, including 124 saponins, 150 flavonoids, two alkaloids, six sterols, and over 20 other types of compounds. These medicinal plants contain abundant endophytic fungi with unique functions, and nearly 600 endophytic fungi with known identity have been detected, but only about 35 strains belonging to 13 genera have been isolated. Among them, at least four strains affiliated to Penicillium roseopurpureum, Alternaria eureka, Neosartorya hiratsukae, and Camarosporium laburnicola have demonstrated the ability to biotransform four saponin compounds from the Astragalus genus, resulting in the production of 66 new compounds, which have significantly enhanced our understanding of the formation of metabolites in plants of the Astragalus genus. They provide a scientific basis for improving the cultivation quality of Astragalus plants through the modification of dominant fungal endophytes or reshaping the endophytic fungal community. Additionally, they open up new avenues for the discovery of specialized, green, efficient, and sustainable biotransformation pathways for complex pharmaceutical intermediates.

Organic amendments increased Chinese milk vetch symbiotic nitrogen fixation by enriching Mesorhizobium in rhizosphere.

Symbiotic nitrogen fixation of Chinese milk vetch (Astragalus sinicus L.) can fix nitrogen from the atmosphere and serve as an organic nitrogen source in agricultural ecosystems. Exogenous organic material application is a common practice of affecting symbiotic nitrogen fixation; however, the results of the regulation activities remain under discussion. Studies on the impact of organic amendments on symbiotic nitrogen fixation have focused on dissolved organic carbon content changes, whereas the impact on dissolved organic carbon composition and the underlying mechanism remain unclear. In situ pot experiments were carried out using soils from a 40-year-old field experiment platform to investigate symbiotic nitrogen fixation rate trends, dissolved organic carbon concentration and component, and diazotroph community structure in roots and in rhizosphere soils following long-term application of different exogenous organic substrates, i.e., green manure, green manure and pig manure, and green manure and rice straw. Remarkable increases in rate were observed in and when compared with that in green manure treatment, with the greatest enhancement observed in the treatment. Moreover, organic amendments, particularly pig manure application, altered diazotroph community composition in rhizosphere soils, therefore increasing the abundance of the host-specific genus Mesorhizobium. Furthermore, organic amendments influence the diazotroph communities through two primary mechanisms. Firstly, the components of dissolved organic carbon promote an increase in available iron, facilitated by the presence of humus substrates. Secondly, the elevated content of dissolved organic carbon and available iron expands the niche breadth of Mesorhizobium within the rhizosphere. Consequently, these alterations result in a modified diazotroph community within the rhizosphere, which in turn influences Mesorhizobium nodulation in the root and symbiotic nitrogen fixation rate. The results of the present study enhance our understanding of the impact of organic amendments on symbiotic nitrogen fixation and the underlying mechanism, highlighting the key role of dissolved organic carbon composition on diazotroph community composition in the rhizosphere.

Impacts of continuous cropping on the rhizospheric and endospheric microbial communities and root exudates of Astragalus mongholicus.

Astragalus mongholicus is a medicinal plant that is known to decrease in quality in response to continuous cropping. However, the differences in the root-associated microbiome and root exudates in the rhizosphere soil that may lead to these decreases are barely under studies. We investigated the plant biomass production, root-associated microbiota, and root exudates of A. mongholicus grown in two different fields: virgin soil (Field I) and in a long-term continuous cropping field (Field II). Virgin soil is soil that has never been cultivated for A. mongholicus. Plant physiological measurements showed reduced fresh and dry weight of A. mongholicus under continuous cropping conditions (i.e. Field II). High-throughput sequencing of the fungal and bacterial communities revealed differences in fungal diversity between samples from the two fields, including enrichment of potentially pathogenic fungi in the roots of A. mongholicus grown in Field II. Metabolomic analysis yielded 20 compounds in A. mongholicus root exudates that differed in relative abundance between rhizosphere samples from the two fields. Four of these metabolites (2-aminophenol, quinic acid, tartaric acid, and maleamate) inhibited the growth of A. mongholicus, the soil-borne pathogen Fusarium oxysporum, or both. This comprehensive analysis enhances our understanding of the A. mongholicus microbiome, root exudates, and interactions between the two in response to continuous cropping. These results offer new information for future design of effective, economical approaches to achieving food security.

Identification and distribution of a fungal endosymbiotic Alternaria species (Alternaria section Undifilum sp.) in Astragalus garbancillo tissues.

Plants belonging to the genera Astragalus, Oxytropis, Ipomoea, Sida, and Swainsona often contain the toxin swainsonine (SW) produced by an associated fungal symbiont. Consumption of SW-containing plants causes a serious neurological disorder in livestock, which can be fatal. In this study, a fungal endophyte, Alternaria section Undifilum, was identified in Astragalus garbancillo seeds, using polymerase chain reaction (PCR) followed by direct sequencing. In seeds, the SW concentrations were about 4 times higher than in other parts of the plant. Furthermore, microscopic examination demonstrated that the fungus mycelium grows inside the petioles and stems, on the outer surface and inside the mesocarp of the fruit, in the mesotesta and endotesta layers of the seed coat, and inside the endosperm of the seeds. Our results support the notion that the SW-producing fungus is vertically transmitted in the host plant A. garbancillo.

The Mesorhizobium huakuii transcriptional regulator AbiEi plays a critical role in nodulation and is important for bacterial stress response.

BACKGROUND: Bacterial abortive infection (Abi) systems are type IV toxin-antitoxin (TA) system, which could elicit programmed cell death and constitute a native survival strategy of pathogenic bacteria under various stress conditions. However, no rhizobial AbiE family TA system has been reported so far. Here, a M. huakuii AbiE TA system was identified and characterized. RESULTS: A mutation in M. huakuii abiEi gene, encoding an adjacent GntR-type transcriptional regulator, was generated by homologous recombination. The abiEi mutant strain grew less well in rich TY medium, and displayed increased antioxidative capacity and enhanced gentamicin resistance, indicating the abiEi operon was negatively regulated by the antitoxin AbiEi in response to the oxidative stress and a particular antibiotic. The mRNA expression of abiEi gene was significantly up-regulated during Astragalus sinicus nodule development. The abiEi mutant was severely impaired in its competitive ability in rhizosphere colonization, and was defective in nodulation with 97% reduction in nitrogen-fixing capacity. The mutant infected nodule cells contained vacuolation and a small number of abnormal bacteroids with senescence character. RNA-seq experiment revealed it had 5 up-regulated and 111 down-regulated genes relative to wild type. Of these down-regulated genes, 21 are related to symbiosis nitrogen fixation and nitrogen mechanism, 16 are involved in the electron transport chain and antioxidant responses, and 12 belong to type VI secretion system (T6SS). CONCLUSIONS: M. huakuii AbiEi behaves as a key transcriptional regulator mediating root nodule symbiosis.

Dynamic content changes of cordycepin and adenosine and transcriptome in Cordyceps kyushuensis Kob at different fermentation stages.

20% (w/w) Astragali radix was added to the rice medium to cultivate C. kyushuensis Kob. The fermentation product was collected at mycelium stage, coloring stage, stromata-forming initial stage and fruiting body stage of C. kyushuensis Kob. The dynamic content changes of cordycepin and adenosine were detected at different fermentation stages. In the rice medium with Astragalus radix, both cordycepin and adenosine reached the highest content value on the 30th day of fermentation, 17.31 mg/g and 0.94 mg/g, respectively, which were 8.6 times and 2.0 times of that in rice medium at the same stage. At the same time, transcriptomics technology was used to analyze C. kyushuensis Kob during these four periods.

Telomerase activators from 20(27)-octanor-cycloastragenol via biotransformation by the fungal endophytes.

Cycloastragenol [20(R),24(S)-epoxy-3ß,6α,16ß,25-tetrahydroxycycloartane] (CA), the principle sapogenol of many cycloartane-type glycosides found in Astragalus genus, is currently the only natural product in the anti-aging market as telomerase activator. Here, we report biotransformation of 20(27)-octanor-cycloastragenol (1), a thermal degradation product of CA, using Astragalus species originated endophytic fungi, viz. Penicillium roseopurpureum, Alternaria eureka, Neosartorya hiratsukae and Camarosporium laburnicola. Fifteen new biotransformation products (2-16) were isolated, and their structures were established by NMR and HRESIMS. Endophytic fungi were found to be capable of performing hydroxylation, oxidation, ring cleavage-methyl migration, dehydrogenation and Baeyer-Villiger type oxidation reactions on the starting compound (1), which would be difficult to achieve by conventional synthetic methods. In addition, the ability of the metabolites to increase telomerase activation in Hekn cells was evaluated, which showed from 1.08 to 12.4-fold activation compared to the control cells treated with DMSO. Among the compounds tested, 10, 11 and 12 were found to be the most potent in terms of telomerase activation with 12.40-, 7.89- and 5.43-fold increase, respectively (at 0.1, 2 and 10 nM concentrations, respectively).

Mesorhizobium jarvisii is a dominant and widespread species symbiotically efficient on Astragalus sinicus L. in the Southwest of China.

In order to identify rhizobia of Astragalus sinicus L. and estimate their geographic distribution in the Southwest China, native rhizobia nodulating A. sinicus were isolated and their genetic diversity were studied at 13 sites cultivated in four Chinese provinces. A total of 451 rhizobial isolates were trapped with A. sinicus plants from soils and classified into 8 different genotypes defined by PCR-based restriction fragment length polymorphism (RFLP) of 16S-23S rRNA intergenic spacer (IGS). Twenty-one representative strains were further identified into three defined Mesorhizobium species by phylogenetic analyses of 16S rRNA genes and housekeeping genes (glnII and atpD). M. jarvisii was dominant accounting for 76.3% of the total isolates, 22.8% of the isolates were identified as M. huakuii and five strains belonged to M. qingshengii. All representatives were assigned to the symbiovar astragali by sharing high nodC sequence similarities of more than 99%. Furthermore, the biogeography distribution of these rhizobial genotypes and species was mainly affected by contents of available phosphorus, available potassium, total salts and pH in soils. The most remarkable point was the identification of M. jarvisii as a widespread and predominant species of A. sinicus in southwest of China. These results revealed a novel geographic pattern of rhizobia associated with A. sinicus in China.

Reduction mechanism of Cd accumulation in rice grain by Chinese milk vetch residue: Insight into microbial community.

Chinese milk vetch is an efficient approach to reduce Cd accumulation in rice, nevertheless, its reduction mechanism is not well understood. In this study, we investigated the rice grain Cd, soil properties and microbial community in a Cd-polluted paddy field amended with milk vetch residue (MV) or without (CK) during rice growth period. We found that milk vetch residue averagely decreased the Cd content in rice grain by 45%. Decrease of Cd in rice mainly attributed to the inhibition of Cd activation by milk vetch residue at heading stage probably by the formation of HA-Cd (Humic Acid) and CdS. Increased pH and organic matter (OM) promoted the reduction of available Cd. In addition, nonmetric multidimensional scaling (NMDS) analysis revealed that microbial community structure was significantly different between MV and CK treatment (r = 0.187, p = 0.002), and the core functions of differentially abundant genera were mainly associated with N-cycling, organic matter degradation and sulfate-reducing. The application of milk vetch residue increased the abundance of sulfate-reducing bacteria (SRB) by 8-112% during the rice growth period, which may involve in promoting the transformation of Cd to a more stably residual Cd (CdS). Canonical correspondence analysis (CCA) and mantel test analysis indicated that available K (p = 0.004) and available N (p = 0.005) were the key environmental factors of shaping the SRB. Altogether, changes in soil properties affected microbial structure and functional characteristics, especially the response of SRB in MV treatment would provide valuable insights into reducing the bioavailability of Cd in soil.

A Methionine Sulfoxide Reductase B Is Required for the Establishment of Astragalus sinicus-Mesorhizobium Symbiosis.

Methionine sulfoxide reductase B (MsrB) is involved in oxidative stress or defense responses in plants. However, little is known about its role in legume-rhizobium symbiosis. In this study, an MsrB gene was identified from Astragalus sinicus and its function in symbiosis was characterized. AsMsrB was induced under phosphorus starvation and displayed different expression patterns under symbiotic and nonsymbiotic conditions. Hydrogen peroxide or methyl viologen treatment enhanced the transcript level of AsMsrB in roots and nodules. Subcellular localization showed that AsMsrB was localized in the cytoplasm of onion epidermal cells and co-localized with rhizobia in nodules. Plants with AsMsrB-RNAi hairy roots exhibited significant decreases in nodule number, nodule nitrogenase activity and fresh weight of the aerial part, as well as an abnormal nodule and symbiosome development. Statistical analysis of infection events showed that plants with AsMsrB-RNAi hairy roots had significant decreases in the number of root hair curling events, infection threads and nodule primordia compared with the control. The content of hydrogen peroxide increased in AsMsrB-RNAi roots but decreased in AsMsrB overexpression roots at the early stage of infection. The transcriptome analysis showed synergistic modulations of the expression of genes involved in reactive oxygen species generation and scavenging, defense and pathogenesis and early nodulation. In addition, a candidate protein interacting with AsMsrB was identified and confirmed by bimolecular fluorescence complementation. Taken together, our results indicate that AsMsrB plays an essential role in nodule development and symbiotic nitrogen fixation by affecting the redox homeostasis in roots and nodules.

[Screening and identification of antagonistic Bacillus against Astragalus membranaceus root rot and its effect on microorganism community in root zone soil].

The Astragalus membranaceus root rot disease,a soil-borne disease,has become increasingly severe in Shanxi province.This study was aimed at getting antagonistic Bacillus with excellent bio-control effects,and determining its effects on bacterial communities in root zone soil. With Fusarium solani and F. acuminatum as the target,antagonistic Bacillus was selected through such tests as living body dual culture,antifungal effect of bacteria-free filtrate,mycelia growth inhibition in vitro and control effect in detached roots,and identified with morphology,physio-biochemical characteristics and 16 S r DNA sequence analysis. The results showed that the Bacillus strain SXKF16-1 had obvious antifungal effect. The diameter of inhibition zone of its bacteria-free filtrate to F. solani and F. acuminatum was( 25. 90±1. 18) mm and( 25. 86±1. 85) mm respectively,and showed a lasting inhibition effect to mycelia growth. The disease index of the protective treatment and that of the cure treatment in detached roots test to F. solani and F. acuminatum were( 37. 50±8. 58),( 41. 67±4. 90) and( 25. 00±8. 33),( 38. 89±9. 62) respectively,both being significantly different( P<0. 05) from that of the control. The strain SXKF16-1 was identified as Bacillus atrophaeus. The B. atrophaeus SXKF16-1 showed significantly inhibition effect to pathogen causing root rot and could increase the bacterial diversity in root zone soil. It has potential to be developed as a special biocontrol agent.

Microbial Transformation of Cycloastragenol and Astragenol by Endophytic Fungi Isolated from Astragalus Species.

Biotransformation of Astragalus sapogenins (cycloastragenol (1) and astragenol (2)) by Astragalus species originated endophytic fungi resulted in the production of five new metabolites (3, 7, 10, 12, 14) together with 10 known compounds. The structures of the new compounds were established by NMR spectroscopic and HRMS analysis. Oxygenation, oxidation, epoxidation, dehydrogenation, and ring cleavage reactions were observed on the cycloartane (9,19-cyclolanostane) nucleus. The ability of the compounds to increase telomerase activity in neonatal cells was also evaluated. After prescreening studies to define potent telomerase activators, four compounds were selected for subsequent bioassays. These were performed using very low doses ranging from 0.1 to 30 nM compared to the control cells treated with DMSO. The positive control cycloastragenol and 8 were found to be the most active compounds, with 5.2- (2 nM) and 5.1- (0.5 nM) fold activations versus DMSO, respectively. At the lowest dose of 0.1 nM, compounds 4 and 13 provided 3.5- and 3.8-fold activations, respectively, while cycloastragenol showed a limited activation (1.5-fold).

Host Genotype and Precipitation Influence of Fungal Endophyte Symbiosis and Mycotoxin Abundance in a Locoweed.

Many plant endophytes produce mycotoxins, but how host genetic variation influences endophyte colonization and mycotoxin production under natural conditions is poorly understood. This interaction has not been fully considered in many previous studies which used controlled experiments with agronomic or model plant species. Here, we investigated this interaction in a naturally occurring forb (a locoweed species) Oxytropis ochrocephala, its symbiotic endophyte Alternaria oxytropis, and the mycotoxin swainsonine. Host genetic variation was characterized by microsatellite markers. Endophyte infection rate and swainsonine levels were determined by PCR and HPLC, respectively. Genetic markers defined two distinct host populations and revealed that host genetics were significantly correlated with geographical location, elevation, and precipitation. As the host diverged, symbiotic interactions were reduced or failed to produce detectable swainsonine in one host population. Host genotype and precipitation had a significant impact in shaping swainsonine production at the population level. This study highlights the effect of host genotype in influencing this interaction in locoweeds.

Detection of swainsonine-producing endophytes in Patagonian Astragalus species.

Swainsonine has been identified as the toxin in legumes belonging to the genera Astragalus and Oxytropis throughout the world including China, North America, and South America. Several South American Astragalus species have been reported to contain swainsonine; however, data is lacking to support the presence of a fungal symbiont in South American Astragalus species as has been shown for North American and Chinese Astragalus and Oxytropis species. The objective of this study was to investigate several South American species that have been reported to contain swainsonine for the presence of the fungal symbiont using culturing and PCR. Swainsonine was detected in field collections of A. pehuenches, A. illinii and A. chamissonis but not A. moyanoi, which is consistent with reports of toxicity regarding these species. The symbiont Alternaria section Undifilum was detected by PCR in all three species that contained swainsonine but not in A. moyanoi. A fungal symbiont was isolated from seeds of Astragalus pehuenches and A. illinii. The isolated symbiont from both respective species produced swainsonine in vitro, and was demonstrated to belong to the genus Alternaria section Undifilum by analysis of the nuclear ribosomal DNA. It is highly likely that Alternaria section Undifilum isolates will be associated with other South American Astragalus species that are reported to contain swainsonine.

Antioxidation and symbiotic nitrogen fixation function of prxA gene in Mesorhizobium huakuii.

Peroxiredoxins (Prxs) play an essential role in the antioxidant activity and symbiotic capacity of Mesorhizobium huakuii. A mutation in the M. huakuii prxA gene (encoding a Prx5-like peroxiredoxin) was generated by homologous recombination. The mutation of prxA did not affect M. huakuii growth, but the strain displayed decreased antioxidative capacity under organic cumene hydroperoxide (CUOOH) conditions. The higher resistance of the prxA mutant strain compared with the wild-type strain to more than 1 mmol/L H2 O2 was associated with a significantly higher level of glutathione reductase activity and a significantly lower level of intracellular hydrogen peroxide content. Real-time quantitative PCR showed that under 1 mmol/L H2 O2 conditions, expression of the stress-responsive genes katG and katE was significantly upregulated in the prxA mutant. Although the prxA mutant can form nodules, the symbiotic ability was severely impaired, which led to an abnormal nodulation phenotype coupled to a 53.25% reduction in nitrogen fixation capacity. This phenotype was linked to an absence of bacteroid differentiation and deregulation of the transcription of the symbiotic genes nifH, nifD, and fdxN. Expression of the prxA gene was induced during symbiosis. Thus, the PrxA protein is essential for antioxidant capacity and symbiotic nitrogen fixation, playing independent roles in bacterial differentiation and cellular antioxidative systems.

Biodiversity of rhizobia present in plant nodules of Biserrula pelecinus across Southwest Spain.

Biodiversity studies of native Mesorhizobium spp. strains able to nodulate the annual herbaceous legume Biserrula pelecinus L. in soils from Southwest Spain have been carried out. One or two isolates per plant, 30 in total, were randomly selected for further characterization. There was no association between the presence of mesorhizobia nodulating-B. pelecinus and the chemical or textural properties of the soils. The isolates were tested for their symbiotic effectiveness on this forage legume under greenhouse conditions and characterized on the basis of physiological parameters: carbon source utilisation (API 50CH), 16S rRNA sequencing and ERIC-PCR, lipopolysaccharide, protein and plasmid profiles. Our results show that in spite of the great diversity found among the native isolates, most of them belong to the genus Mesorhizobium, the exception being strain B24 which sequence matches 97.52% with Neorhizobium huautlense; this is the first description of a Neorhizobium strain effectively nodulating-biserrula plants. Results of a field trial indicated that some of these isolates could be recommended as inoculants for this legume. B24=DSM 28743=CECT 8815; ENA (HF955513) 16S rRNA sequences of isolates B13, B18, B26, B30 and B1 are deposited at ENA under numbers LS999402 to LS999406, respectively.

Astragalus algarbiensis is nodulated by the genistearum symbiovar of Bradyrhizobium spp. in Morocco.

Astragalus algarbiensis is a wild herbaceous legume growing in Maamora, the most important cork oak forest in northern Africa. It is a plant of great importance as fodder in silvopastoral systems, and in the restoration of poor and degraded soils. The purpose of this study was to describe the biodiversity of rhizobia nodulating this plant and determine their identity. Out of 80 bacterial isolates, 56 strains isolated from root nodules of A. algarbiensis were characterized. ERIC-PCR fingerprinting grouped the strains in two main clusters containing 29 and 27 isolates, respectively, and the amplified ribosomal DNA restriction analysis (ARDRA) generated two different ribotypes. Based on both the ERIC-PCR and ARDRA results, representative strains As21 and As36 were selected for further genetic studies. The nearly complete 16S rRNA gene sequences of As21 and As36 showed that they were closely related to Bradyrhizobium cytisi CTAW11T with similarity values of 99.84% and 99.77%, respectively. Concatenation of atpD, recA, gyrB and dnaK housekeeping gene sequences indicated that strains As21 and As36 had a 95.22% similarity but they showed values of 95.80% and 94.97% with B. cytisi CTAW11T, respectively. The sequencing of the symbiotic nodC gene of the two strains revealed 97.20% and 97.76% identities, respectively, with that of B. cytisi CTAW11T isolated from Cytisus villosus growing in the Moroccan Rif Mountains. Furthermore, the phylogenic analysis showed that the strains isolated from A. algarbiensis clustered with B. cytisi and B. rifense within the bradyrhizobia genistearum symbiovar and may constitute two novel genospecies.