Phenotypic, stress tolerance, and plant growth promoting characteristics of rhizobial isolates of grass pea.

Grass pea (Lathyrus sativus L.) is widely cultivated for food and feed in some developing countries including Ethiopia. However, due to its overexaggerated neuro-lathyrism alkaloid causing paralysis of limbs, it failed to attract attention of the research community and is one of the most neglected orphan crops in the world. But, the crop is considered an insurance crop by resource-poor farmers due to its strong abiotic stress tolerance and ability to produce high yields when all other crops fail due to unfavorable environmental conditions. This study was aimed at screening rhizobial isolates of grass pea and evaluating their symbiotic nitrogen fixation efficiency and tolerance to abiotic stresses. Fifty rhizobial isolates collected from grass pea nodules were isolated, screened, and characterized based on standard microbiological methods. The rhizobial isolates showed diversity in nodulation, symbiotic nitrogen fixation, and nutrient utilization. The 16S rRNA gene sequencing of 14 rhizobial isolates showed that two of them were identified as Rhizobium leguminosarum and the remaining twelve as Rhizobium species. Based on their overall performance, strains AAUGR-9, AAUGR-11, and AAUGR-14 that performed top and identified as Rhizobium species were recommended for field trials. This study screened and identified effective and competitive rhizobial isolates enriched with high nitrogen-fixing and abiotic stress tolerant traits, which contributes much to the application of microbial inoculants as alternative to chemical fertilizers.

In situ effects of Lathyrus sativus- PGPR to remediate and restore quality and fertility of Pb and Cd polluted soils.

Rehabilitation of heavy metals contaminated soils using association between legumes and beneficial rhizospheric microorganisms such as plant growth-promoting bacteria (PGPR) is a major challenge in agronomy. The present study focuses on assessing the impact of field inoculation with I1 (Rhizobium leguminosarum (M5) + Bacillus simplex + Luteibacter sp. + Variovorax sp.) and I5 (R. leguminosarum (M5) + Pseudomonas fluorescens (K23) + Luteibacter sp. + Variovorax sp.) on growth and phytoremediation potential of Lathyrus sativus plants as well as soil quality and fertility. The experimentation was carried out in mine tailings of northern Tunisia. Obtained Results indicated that the in situ inoculation with I1 and I5 significantly increased the shoots (47% and 22%) and roots dry weights (22% and 29%), as well as nodules number (48% and 31%), respectively, compared to uninoculated plants. The maximum Pb accumulation in the above-ground tissue was recorded in plants inoculated with I5 (1180.85 mg kg-1 DW). At the same time, we noticed a reduction in total Pb and Cd in the rhizosphere of inoculated plots mainly in those inoculated with I5 reaching 46% and 61%, respectively, compared to uninoculated plots. Likewise, I5 inoculum significantly enhanced soil total nitrogen (35%) and available phosphorus (100%), as well as ß-glucosidase (16%), urease (32%) and alkaline phosphatase (12%) activities. Here we demonstrate the usefulness of L. sativus inoculated with I5 inoculum formed by mixing efficient and heavy metals resistant PGPR to boost an efficient reclamation of Cd and Pb contaminated soils and, ultimately, to improve their quality and fertility.

Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia.

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.

Heavy metal accumulation in Lathyrus sativus growing in contaminated soils and identification of symbiotic resistant bacteria.

In this study, two populations of leguminous plants Lathyrus sativus were grown in four soils that were collected from sites differently contaminated by heavy metals. Evaluations included basic soil properties, concentrations of major nutrients and four metals (copper, zinc, lead and cadmium) in these soils. Investigation of Lathyrus sativus response to contamination showed that the increase of heavy metal concentration in soils affected biomass of plant, number of nodules and plant metal uptake. Heavy metal tolerance of 46 isolated bacteria from the root nodules was evaluated and demonstrated that the maximum concentration of Cd, Pb, Cu and Zn tolerated by strains were 0.8, 2.5, 0.2, and 0.5 mM, respectively. Twenty-two isolates were tested for their effects on plant biomass production and nodule formation and showed that only R. leguminosarum nodulated Lathyrus sativus, while some bacteria improved the shoot and root dry biomass. Sequences of their 16S rDNA gene fragments were also obtained and evaluated for tentative identification of the isolates which revealed different bacterial genera represented by Rhizobium sp, Rhizobium leguminosarum, Sinorhizobium meliloti, Pseudomonas sp, Pseudomonas fluorescens, Luteibacter sp, Variovorax sp, Bacillus simplex and Bacillus megaterium. The existence of Pb- and Cd-resistant genes (PbrA and CadA) in these bacteria was determined by PCR, and it showed high homology with PbrA and CadA genes from other bacteria. The tested resistant population was able to accumulate high concentrations of Pb and Cd in all plant parts and, therefore, can be classified as a strong metal accumulator with suitable potential for phytoremediation of Pb and Cd polluted sites. Heavy metal resistant and efficient bacteria isolated from root nodules were chosen with Lathyrus sativus to form symbiotic associations for eventual bioremediation program, which could be tested to remove pollutants from contaminated sites.

Potential of efficient and resistant plant growth-promoting rhizobacteria in lead uptake and plant defence stimulation in Lathyrus sativus under lead stress.

The ability of plant growth-promoting rhizobacteria (PGPR) to enhance Lathyrus sativus tolerance to lead (Pb) stress was investigated. Ten consortia formed by mixing four efficient and Pb-resistant PGPR strains were assessed for their beneficial effect in improving Pb (0.5 mM) uptake and in inducing the host defence system of L. sativus under hydroponic conditions based on various physiological and biochemical parameters. Lead stress significantly decreased shoot (SDW) and root (RDW) dry weight, but PGPR inoculation improved both dry weights, with highest increases in SDW and RDW of plants inoculated with I5 (R. leguminosarum (M5) + P. fluorescens (K23) + Luteibacter sp. + Variovorax sp.) and I9 (R. leguminosarum (M5) + Variovorax sp. + Luteibacter sp. + S. meliloti) by 151% and 94%, respectively. Additionally, inoculation significantly enhanced both chlorophyll and soluble sugar content, mainly in I5 inoculated leaves by 238% and 71%, respectively, despite the fact that Pb decreased these parameters. We also found that PGPR inoculation helps to reduce oxidative damage and enhances antioxidant enzyme activity, phenolic compound biosynthesis, carotenoids and proline content. PGPR inoculation increased Pb uptake in L. sativus, with highest increase in shoots of plants inoculated with I5 and I7, and in roots and nodules of plants inoculated with I1. Moreover, PGPR inoculation enhanced mineral homeostasis for Ca, Cu and Zn under Pb stress, mainly in plants inoculated with I1, I5, I7 and I9. Results of our study suggest the potential of efficient and Pb-resistant PGPR in alleviating harmful effects of metal stress via activation of various defence mechanisms and enhancing Pb uptake that promotes tolerance of L. sativus to Pb stress.

Effect of Pb-resistant plant growth-promoting rhizobacteria inoculation on growth and lead uptake by Lathyrus sativus.

In search of efficient and resistant plant growth-promoting rhizobacteria (PGPR) strains with multiple activities, a total of twelve bacterial belonging to R. leguminosarum, S. meliloti, Pseudomonas sp., P. fluorescens, Luteibacter sp., Variovorax sp., B. simplex, and B. megaterium were isolated from root nodules of grass pea (Lathyrus sativus L.) grown in contaminated soils. Upon screening, all test strains were able to synthesize indoleacetic acid; more than 90% were siderophore producers and 75% showed varying levels of phosphate solubilizing ability. The gaseous metabolite biosynthesis showed that 42% of strains were cyanogenic. The lead (Pb) bioaccumulation differs with incubation times between cell wall and cytoplasm. Indeed, the most part of Pb was adsorbed to cell surface. A pot experiment was conducted for investigating the capability of combined bacteria to promote plant growth of Lathyrus sativus under controlled conditions. Subsequently, the performance of symbiosis Lathyrus sativus-PGPR (I4: R. leguminosarum (M5) + B. simplex + Luteibacter sp. + Variovorax sp.) was investigated under lead stress using hydroponic culture to elucidate the effect of bacterial inoculation on Pb uptake as well as plant growth. Results showed that under 0.5 mM Pb, inoculation with I4 significantly increased shoots and roots biomass by 59% and 56%, respectively, and improved Pb uptake in both shoots and roots by 39% and 47%, respectively, as compared to uninoculated plants. The inoculation of Lathyrus sativus with efficient and Pb resistant PGPR is a promising symbiosis that having significant potential to improve phytoremediation of Pb-polluted soils.

Lathyroxins A and B, Phytotoxic Monosubstituted Phenols Isolated from Ascochyta lentis var. lathyri, a Fungal Pathogen of Grass Pea ( Lathyrus sativus).

Ascochyta lentis var. lathyri has recently been reported to be the causal agent of Ascochyta blight of grass pea ( Lathyrus sativus), a disease characterized by the appearance of necrotic lesions of leaves and stems. Considering the novelty of the pathogen and the possible involvement of secondary metabolites in symptom appearance, a study was carried out to ascertain the capability of this fungus to produce bioactive metabolites. Some phytotoxic phenols were isolated from the culture filtrates of the fungus. In particular, two new phytotoxic metabolites, named lathyroxins A and B, were characterized by spectroscopic methods as 4-(2-hydroxy-3,3-dimethoxypropyl)phenol and 3-(4-hydroxyphenyl)propane-1,2-diol, respectively, and the R absolute configuration of C-2 of their 2-dimethoxy- and 2,3-diol-propyl side chain was assigned. Moreover, other well-known fungal metabolites, namely, p-hydroxybenzaldehyde, p-methoxyphenol, and tyrosol, were also identified. Lathyroxins A and B showed interesting phytotoxic properties, being able to cause necrosis on leaves and to inhibit seed germination and rootlet elongation. Moreover, both of the new metabolites had no effect against bacteria, arthropods, and nematodes.

Analysis of rhizobial endosymbionts of Vicia, Lathyrus and Trifolium species used to maintain mountain firewalls in Sierra Nevada National Park (South Spain).

Forest fires lead to the annual disappearance of many natural formations that require the creation of firewall areas. They can be maintained by enriching their pastures with attractive plants for grazing livestock, mainly legumes, which have a high protein content and low dependence on N fertilizers due to their ability to establish nitrogen-fixing symbiosis with rhizobia. In this study, the rhizobia isolated from the nodules of six legumes from the genera Vicia, Lathyrus and Trifolium were analysed in a firewall zone established in Lanjarón (Granada) close to the Sierra Nevada National Park (Spain). The results showed a high genetic diversity of the isolated strains that had 3, 16, 14 and 13 different types of rrs, recA, atpD and glnII genes, respectively. All strains were phylogenetically close to the species from the Rhizobium leguminosarum group, although they were not identified as any of them. The isolated strains belonged to the symbiovars viciae and trifolii but high phylogenetic diversity was found within both symbiovars, since there were 16 and 14 nodC gene types, respectively. Some of these strains clustered with strains isolated in other countries and continents, but others formed atpD, recA, glnII and nodC clusters and lineages only found to date in this study.

Rhizobium anhuiense as the predominant microsymbionts of Lathyrus maritimus along the Shandong Peninsula seashore line.

Beach pea [Lathyrus maritimus Bigelow, or Lathyrus japonicus subsp. maritimus (L.) P.W. Ball] is a wild legume distributed on the seashore line, and the rhizobia nodulating with this plant have been reported only rarely. In order to reveal the diversity of beach pea rhizobia on the seashore line of Shandong Peninsula, China, a total of 124 bacterial strains were isolated from the root nodules of beach pea plants collected from five sites. All the isolates were divided into five recA types after screening by recA gene sequence analysis and they consisted of Rhizobium anhuiense covering 122 symbiotic isolates in three recA types, as well as two single isolates Rhizobium sp. and Rhizobium lusitanum representing distinct recA types. The recA genotype III of R. anhuiense (103 isolates) represented by strain YIC11270 was dominant at all five sampling sites. Identical symbiotic genes (nodC and nifH) were detected in the three recA genotypes of R. anhuiense isolates that were closely related to those of the pea and faba rhizobia. This study clarified that R. anhuiense was the main symbiont for beach pea rhizobia on the seashore line of Shandong Peninsula. The low level genetic diversity of beach pea rhizobia revealed by both MLSA and the symbiotic genes might be related to the strong selection pressure produced by the saline-alkaline environment and the host plants.

Proteolysis in tempeh-type products obtained with Rhizopus and Aspergillus strains from grass pea (Lathyrus sativus) seeds.

BACKGROUND: Tempeh is a food product obtained from legumes by means of solid-state fermentation with Rhizopus sp. Our previous research proved that mixed-culture inoculum may also be successfully applied. The objective of present research was to study the proteolytic activity of R. microsporus var. chinensis and A. oryzae during tempeh-type fermentation of grass pea seeds, and the effect of inoculum composition on the protein level and in vitro protein bioavailability in products. METHODS: Fermentation substrate were soaked and cooked grass pea seeds. Material was mixed with single- or mixed-culture inoculum, and incubated in perforated plastic bags at 30°C for 32 hrs. In the products, the proteolytic activity (pH 3, 5 and 7), glucosamine, total protein and free amino acids levels, as well as protein in vitro bioavailability and degree of protein hydrolysis were obtained. RESULTS: The significant correlation was found between glucosamine content and proteolytic activity in grass pea seeds fermented with Rhizopus or Aspergillus. The activities of Rhizopus proteases were higher than Aspergillus ones, which corresponded with the degree of seed protein hydrolysis. Both strains showed the highest activity of protease at pH 3. Tempeh made with pure culture of Rhizopus had 37% protein of 69% in-vitro bioavailability. Mixed-culture fermentation improved nutritional parameters of products only when the dose of Aspergillus spores in the inoculum was equal and lower than that of Rhizopus. This process resulted in higher in-vitro bioavailability of protein, slightly more efficient protein hydrolysis and higher level of free amino acids, as compared to standard tempeh. CONCLUSIONS: The activity of A. oryzae in tempeh-type fermentation is beneficial as long as it does not dominate the activity and/or growth of Rhizopus strain.

[Characteristics of Natural Selection in Populations of Nodule Bacteria (Rhizobium leguminosarum) Interacting With Different Host Plants].

Using high throughput sequencing of the nodA gene, we studied the population dynamics of Rhizobium leguminosarum (bv. viciae, bv. trifolii) in rhizospheric and nodular subpopulations associated with the leguminous plants representing different cross-inoculation groups (Vicia sativa, Lathyrus pratensis of the vetch/vetchling/pea group and Trifolium hybridum of the clover group). The "rhizosphere-nodules" transitions result in either an increase or decrease in the frequencies of 10 of the 23 operational taxonomic units (OTUs) (which were identified with 95% similarity) depending on the symbiotic specificity and phylogenetic positions of OTUs. Statistical and bioinformatical analysis of the population structures suggest that the type of natural selection responsible for these changes may be diversifying at the whole-population level and frequency-dependent at the OTU-specific level, ensuring the divergent evolution of rhizobia interacting with different host species.

Allelic diversity in the transcriptomes of contrasting rust-infected genotypes of Lathyrus sativus, a lasting resource for smart breeding.

BACKGROUND: Grass pea (Lathyrus sativus L.) is a valuable resource for potentially durable partial resistance to rust. To gain insight into the resistance mechanism and identify potential resistance genes, we generated the first comprehensive transcriptome assemblies from control and Uromyces pisi inoculated leafs of a susceptible and a partially rust-resistant grass pea genotype by RNA-seq. RESULTS: 134,914 contigs, shared by both libraries, were used to analyse their differential expression in response to rust infection. Functional annotation grouped 60.4% of the contigs present in plant databases (37.8% of total) to 33 main functional categories, being "protein", "RNA", "signalling", "transport" and "stress" the most represented. Transcription profiles revealed considerable differences in regulation of major phytohormone signalling pathways: whereas Salicylic and Abscisic Acid pathways were up-regulated in the resistant genotype, Jasmonate and Ethylene pathways were down-regulated in the susceptible one. As potential Resistance-genes we identified a mildew resistance locus O (MLO)-like gene, and MLO-related transcripts. Also, several pathogenesis-related genes were up-regulated in the resistant and exclusively down regulated in the susceptible genotype. Pathogen effectors identified in both inoculated libraries, as e.g. the rust Rtp1 transcript, may be responsible for the down-regulation of defence-related transcripts. The two genotypes contained 4,892 polymorphic contigs with SNPs unevenly distributed between different functional categories. Protein degradation (29.7%) and signalling receptor kinases (8.2%) were the most diverged, illustrating evolutionary adaptation of grass pea to the host/pathogens arms race. CONCLUSIONS: The vast array of novel, resistance-related genomic information we present here provides a highly valuable resource for future smart breeding approaches in this hitherto under-researched, valuable legume crop.

Assessing genotypic diversity and symbiotic efficiency of five rhizobial legume interactions under cadmium stress for soil phytoremediation.

In the framework of soil phytoremediation using local legume plants coupled with their native root-nodulating bacteria to increase forage yields and preserve contaminated soils in arid regions of Tunisia, we investigated the diversity of bacteria from root nodules of Lathyrus sativus, Lens culinaris, Medicago marina, M. truncatula, and M. minima and the symbiotic efficiency of these five legume symbiosis under Cadmium stress. Fifty bacterial strains were characterized using physiological and biochemical features such heavy metals resistant, and PCR-RFLP of 16S rDNA. Taxonomically, the isolates nodulating L. sativus, and L. culinaris are species within the genera Rhizobium and the ones associated to Medicago sp, within the genera Sinorhizobium. The results revealed also that the cadmium tolerance of the different legumes-rhizobia interaction was as follows: M. minima < M. truncatula < M. marina < L. sativus < L. culinaris indicating that the effect of Cadmium on root nodulation and biomass production is more deleterious on M. minima-S. meliloti and M. truncatula-S. meliloti than in other symbiosis. Knowledge on genetic and functional diversity of M. marina, L. sativus and L. culinaris microsymbiotes is very useful for inoculant strain selection and can be selected to develop inoculants for soil phytoremediation.

Factors affecting ß-ODAP content in Lathyrus sativus and their possible physiological mechanisms.

A neuroexcitatory non-protein amino acid, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), present in the seeds of the hardy legume crop grass pea (Lathyrus sativus L.), was considered responsible for human lathyrism. The levels of ß-ODAP were reported to vary in different tissues during plant development, and to be affected by a wide range of environmental stresses. In this paper, dynamic changes in ß-ODAP level at specific stages of plant development as well as the influences of various environmental factors, including nutrient deficiency, drought, salinity, toxic heavy metals, and Rhizobium symbiosis on ß-ODAP levels were analyzed, highlighting the relationship between changes in ß-ODAP concentrations and Rhizobium growth. Possible mechanisms underlying ß-ODAP accumulation are proposed and future research is suggested.

[Phylogenetic analysis of symbiotic genes of nodule bacteria in the plants of the genus Lathyrus (L.) (Fabaceae)].

The comparative analysis of the symbiotic genes nifD, nifH, nodA of wild-growing Lathyrus L. species (Fabaceae) connected by genes sequences of 16S aRNA to Rhizobium leguminosarum bv. viceae, Rhizobium tropici, Agrobacterium sp., and Phyllobacterium sp. was carried out. It was demonstrated that all tested genes of strains taken for analysis had high degree of homology with analogous genes of Rhizobium leguminosarum bv. viceae. It was suggested that symbiotic genes were introduced into Rhizobium tropici, Agrobacterium sp., and Phyllobacterium sp. strains by means of horizontal gene transfer over from Rhizobium leguminosarum bv. viceae strain. The recombinant strains were formed, capable to nodulate Lathyrus L. species that earlier was not considered characteristic for these plants.

Genotypic and phenotypic diversity of rhizobia isolated from Lathyrus japonicus indigenous to Japan.

Sixty-one rhizobial strains from Lathyrus japonicus nodules growing on the seashore in Japan were characterized and compared to two strains from Canada. The PCR-based method was used to identify test strains with novel taxonomic markers that were designed to discriminate between all known Lathyrus rhizobia. Three genomic groups (I, II, and III) were finally identified using RAPD, RFLP, and phylogenetic analyses. Strains in genomic group I (related to Rhizobium leguminosarum) were divided into two subgroups (Ia and Ib) and subgroup Ia was related to biovar viciae. Strains in subgroup Ib, which were all isolated from Japanese sea pea, belonged to a distinct group from other rhizobial groups in the recA phylogeny and PCR-based grouping, and were more tolerant to salt than the isolate from an inland legume. Test strains in genomic groups II and III belonged to a single clade with the reference strains of R. pisi, R. etli, and R. phaseoli in the 16S rRNA phylogeny. The PCR-based method and phylogenetic analysis of recA revealed that genomic group II was related to R. pisi. The analyses also showed that genomic group III harbored a mixed chromosomal sequence of different genomic groups, suggesting a recent horizontal gene transfer between diverse rhizobia. Although two Canadian strains belonged to subgroup Ia, molecular and physiological analyses showed the divergence between Canadian and Japanese strains. Phylogenetic analysis of nod genes divided the rhizobial strains into several groups that reflected the host range of rhizobia. Symbiosis between dispersing legumes and rhizobia at seashore is discussed.

Transgenic grasspea (Lathyrus sativus L.): factors influencing agrobacterium-mediated transformation and regeneration.

A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD(600) congruent with 0.6 and diluted to a cell density of 10(9) cells ml(-1) for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 mug ml(-1)). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l(-1) indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T(1) seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.