Two novel symbiovars of Bradyrhizobium yuanmingense, americaense and caribense, the symbiovar tropici of Bradyrhizobium pachyrhizi and the symbiovar cajani of Bradyrhizobium cajani are microsymbionts of the legume Cajanus cajan in Dominican Republic.

Cajanus cajan L. (guandul) is commonly cultivated in Dominican Republic where this legume is a subsistence crop. Here we identified through MALDI-TOF MS several rhizobial strains nodulating C. cajan in two Dominican locations as Bradyrhizobium yuanmingense. The phylogenetic analysis of recA and glnII housekeeping genes showed that these strains belong to a wide cluster together with the type strain of B. yuanmingense and other C. cajan nodulating strains previously isolated in Dominican Republic. The comparison of genomes from strains representative of different lineages within this cluster support the existence of several genospecies within B. yuanmingense, which is the major microsymbiont of C. cajan in Dominican Republic where it is also nodulated by Bradyrhizobium cajani and Bradyrhizobium pachyrhizi. The analysis of the symbiotic nodC gene showed that the C. cajan nodulating strains from the B. yuanmingense complex belong to two clusters with less than 90% similarity between them. The strains from these two clusters showed nodC gene similarity values lower than 90% with respect to the remaining Bradyrhizobium symbiovars and then they correspond to two new symbiovars for which we propose the names americaense and caribense. The results of the nodC gene analysis also showed that C. cajan is nodulated by the symbiovar tropici, which has been found by first time in this work within the species Bradyrhizobium pachyrhizi. These results confirmed the high promiscuity degree of C. cajan, which is also nodulated by the symbiovar cajani of Bradyrhizobium cajani in Dominican Republic.

Mechanisms involved in drought stress tolerance triggered by rhizobia strains in wheat.

Rhizobium spp. is a well-known microbial plant biostimulant in non-legume crops, but little is known about the mechanisms by which rhizobia enhance crop productivity under drought stress. This work analyzed the mechanisms involved in drought stress alleviation exerted by Rhizobium leguminosarum strains in wheat plants under water shortage conditions. Two (LBM1210 and LET4910) of the four R. leguminosarum strains significantly improved the growth parameters (fresh and dry aerial weight, FW and DW, respectively), chlorophyll content, and relative water content (RWC) compared to a non-inoculated control under water stress, providing values similar to or even higher for FW (+4%) and RWC (+2.3%) than the non-inoculated and non-stressed control. Some other biochemical parameters and gene expression explain the observed drought stress alleviation, namely the reduction of MDA, H2O2 (stronger when inoculating with LET4910), and ABA content (stronger when inoculating with LBM1210). In agreement with these results, inoculation with LET4910 downregulated DREB2 and CAT1 genes in plants under water deficiency and upregulated the CYP707A1 gene, while inoculation with LBM1210 strongly upregulated the CYP707A1 gene, which encodes an ABA catabolic enzyme. Conversely, from our results, ethylene metabolism did not seem to be involved in the alleviation of drought stress exerted by the two strains, as the expression of the CTR1 gene was very similar in all treatments and controls. The obtained results regarding the effect of the analyzed strains in alleviating drought stress are very relevant in the present situation of climate change, which negatively influences agricultural production.

Diversity and Genetic Structure of Scarlet Plume (Euphorbia fulgens), an Endemic Plant of Mexico.

Euphorbia fulgens is an ornamental species cultivated in Europe and endemic to Mexico; its ecological, genetic, and evolutionary aspects are not known. The objectives of this study were to determine its distribution, describe the places it inhabits, and analyze the diversity and genetic structures of wild populations of E. fulgens. A bibliographic review of the herbarium specimens and a field evaluation were carried out to develop a potential distribution map based on a multi-criteria analysis of the climatic and topographic variables. Three populations (forty-five individuals) from pine-oak and cloud forests located in the Southern Sierra of Oaxaca were analyzed using ten microsatellite loci. The analysis was conducted using Arlequin v. 3.5, Mega v. 10, and Structure v. 2.3 programs. Eight loci were polymorphic, and a total of thirty-eight alleles were obtained. The average number of alleles per polymorphic locus was 4.6. The average heterozygosity of the three populations was high (Ho = 0.5483), and genetic differentiation between populations were low, with a high genetic flow, suggesting that it could be an ancestral population that became fragmented and was just beginning to differentiate genetically. The information generated on this restricted distribution species can be used in conservation programs pertaining to human activities that endanger the habitats where it is found.

Morphological Variation in Scarlet Plume (Euphorbia fulgens Karw ex Klotzsch, Euphorbiaceae), an Underutilized Ornamental Resource of Mexico with Global Importance.

Morphological variation is useful in conservation and genetic improvement programs. Euphorbia fulgens, a range-restricted local endemic species of Mexico, is used locally during the altars in the festivities of different saints and is also cultivated as an ornamental plant mainly in Europe. Thus, in the present study, morphological variation was evaluated in wild populations and cultivated populations. Characterization of 90 individuals from three wild populations (the only ones recorded to date) was done by measuring 30 morphological traits both vegetative and reproductive. Thereafter, seeds were collected, and established under greenhouse conditions, and 39 morphometric variables were evaluated in adult plants. An analysis of variance (ANOVA) was done for wild and cultivated groups independently, and when significant differences were found, Tukey's comparison of means was applied (p < 0.05). To identify the traits responsible for the differences between wild and cultivated groups, a linear discriminant analysis (LDA) was conducted. Morphological variation was found among wild populations, and this variation decreased in cultivated populations, mainly in reproductive structures. The LDA separated the wild populations from the cultivated groups, according to inflorescence length, petiole length/blade length ratio, and leaf roundness. The variables that determined the separation of individuals between wild and cultivated populations were cyme number, foliar Feret diameter, and inflorescence length, variables that can be important for breeding strategies and artificial selection.

Bacillus as a source of phytohormones for use in agriculture.

Microbial plant biostimulants (MPBs) are capable of improving the productivity and quality of crops by activating plant physiological and molecular processes, representing an efficient tool in sustainable agriculture. Through phytohormone production, MPBs are capable of regulating plant physiological processes, increasing the productivity and quality of crops, in addition to being an efficient alternative in the industrial production of phytohormones. Bacillus is a bacterial genus with various species on the market being used as biopesticides, due to their ability to produce antimicrobial, nematicidal and insecticidal compounds. The capability of Bacillus species to protect plants against pests and/or pathogens also entails the triggering or increase of plant defense responses. Furthermore, a relevant number of species from the genus Bacillus provoke plant growth promotion by different mechanisms such as increasing the tolerance of their host plants under abiotic stress conditions or improving plant nutrition. In several cases, the plant response is mediated by the bacterial production of phytohormones. In the present work, all studies from recent decades where the production of phytohormones by Bacillus species are reported, highlighting their role in host plants and the mechanisms by which they are capable of increasing plant growth, promoting their development, and improving their response to different stresses. KEY POINTS: • Different Bacillus-species are known as agricultural biopesticides. • Bacillus role as biostimulants is being increasingly addressed. • Bacillus represents a good source of phytohormones of agricultural interest.

Formulation of a Highly Effective Inoculant for Common Bean Based on an Autochthonous Elite Strain of Rhizobium leguminosarum bv. phaseoli, and Genomic-Based Insights Into Its Agronomic Performance.

Common bean is a poor symbiotic N-fixer, with a low response to inoculation owing to its promiscuous nodulation with competitive but inefficient resident rhizobia. Consequently, farmers prefer to fertilize them rather than rely on their capacity for Biological Nitrogen Fixation (BNF). However, when rhizobial inoculants are based on autochthonous strains, they often have superior BNF performance in the field due to their genetic adaptations to the local environment. Nevertheless, there is scant information at the genomic level explaining their superiority or on how their genomes may influence the inoculant performance. This information is especially important in technologically advanced agri-systems like Europe, where environmental concerns and increasingly stringent fertilizer regulations are encouraging a return to the use of rhizobial inoculants, but based upon strains that have been thoroughly characterized in terms of their symbiotic performance and their genetics. The aim of this study was to design an inoculant formulation based on a superior autochthonous strain, Rhizobium leguminosarum bv. phaseoli LCS0306, to assess its performance in the field, and to determine the genomic features contributing to the high effectiveness of its symbiosis with common bean. Plants inoculated with the autochthonous strain LCS0306 fixed significantly more nitrogen than those with the allochthonous strains R. phaseoli ATCC 14482T and R. etli CFN42T, and had grain yield similar to the nitrogen-fertilized controls. Inoculation with LCS0306 was particularly efficacious when formulated with a carrier based upon a mixture of perlite and biochar. Whole genome comparisons revealed no differences in the classical symbiotic genes of strain LCS0306 within the symbiovar phaseoli. However, its symbiotic superior performance might be due to its genomic versatility, as it harbors a large assortment of genes contributing to fitness and competitiveness. It is concluded that inoculation with elite rhizobia formulated with perlite-biochar carriers might constitute a step-change in the sustainable cultivation of common bean in Spanish soils.

Rhizobium and Phyllobacterium bacterial inoculants increase bioactive compounds and quality of strawberries cultivated in field conditions.

Strawberries (Fragaria × ananassa Duch.) are widely demanded by the consumers because they contain several bioactive compounds, mainly vitamin C and anthocyanins, which may be increased by biofertilization with some plant growth promoting bacteria. In this work we have analysed two bacterial strains, PEPV15 and PEPV16, from genera Phyllobacterium and Rhizobium, respectively, which under microcosms conditions were able to promote the strawberry growth, increasing the content of some bioactive compounds, such as vitamin C or organic acids. Here we have analysed the effect on bioactive compounds in strawberries from plants biofertilized with the strains PEPV15 and PEPV16 in field conditions. Under these conditions, the anthocyanin content was increased when plants were biofertilized with the strain PEPV15 and the pelargonidin-3-O-rutinoside content significantly increased. Besides, citric acid, vitamin C and epicatechin contents were significantly higher when either of the two strains was used as biofertilizer. Our results showed that the inoculation with Phyllobacterium and Rhizobium strains is a good agronomical practice, which improve the content of several bioactive compounds of strawberries increasing the beneficial effects on human health.

Bradyrhizobium cajani sp. nov. isolated from nodules of Cajanus cajan.

Two slow-growing strains, AMBPC1010T and AMBPC1011, were isolated from nodules of Cajanus cajan in the Dominican Republic. 16S rRNA gene analysis placed these strains within the genus Bradyrhizobium, being phylogenetically equidistant to several species of this genus. Analysis of the recA and atpD genes showed that the strains isolated belong to a cluster containing the strains Bradyrhizobium ottawaense OO99T, 'Bradyrhizobium americanum' CMVU44 and Bradyrhizobium daqingense CCBAU 15774T, and presented similarity values lower than 96 % for both genes with respect to the strains nodulating C. cajan. DNA-DNA hybridization analysis showed averages of 36, 40 and 39 % relatedness with respect to the representative strains of Bradyrhizobium ottawaense, 'Bradyrhizobium americanum' and Bradyrhizobium daqingense, respectively. Phenotypic characteristics also differed from those of the most closely related species of the genus Bradyrhizobium. Therefore, based on the data obtained in this study, we propose to classify the strains AMBPC1010T (=LMG 29967T=CECT 9227T) and AMBPC1011 into a novel species named Bradyrhizobium cajani sp. nov.

Delftia rhizosphaerae sp. nov. isolated from the rhizosphere of Cistus ladanifer.

A bacterial strain, designated RA6T, was isolated from the rhizosphere of Cistus ladanifer. Phylogenetic analyses based on 16S rRNA gene sequence placed the isolate into the genus Delftia within a cluster encompassing the type strains of Delftia lacustris, Delftia tsuruhatensis, Delftia acidovorans and Delftia litopenaei, which presented greater than 97 % sequence similarity with respect to strain RA6T. DNA-DNA hybridization studies showed average relatedness ranging from of 11 to 18 % between these species of the genus Delftia and strain RA6T. Catalase and oxidase were positive. Casein was hydrolysed but gelatin and starch were not. Ubiquinone 8 was the major respiratory quinone detected in strain RA6T together with low amounts of ubiquinones 7 and 9. The major fatty acids were those from summed feature 3 (C16 : 1ω7c/C16 : 1 ω6c) and C16 : 0. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain RA6T should be considered as a representative of a novel species of genus Delftia, for which the name Delftia rhizosphaerae sp. nov. is proposed. The type strain is RA6T (=LMG 29737T= CECT 9171T).

New biofuel alternatives: integrating waste management and single cell oil production.

Concerns about greenhouse gas emissions have increased research efforts into alternatives in bio-based processes. With regard to transport fuel, bioethanol and biodiesel are still the main biofuels used. It is expected that future production of these biofuels will be based on processes using either non-food competing biomasses, or characterised by low CO2 emissions. Many microorganisms, such as microalgae, yeast, bacteria and fungi, have the ability to accumulate oils under special culture conditions. Microbial oils might become one of the potential feed-stocks for biodiesel production in the near future. The use of these oils is currently under extensive research in order to reduce production costs associated with the fermentation process, which is a crucial factor to increase economic feasibility. An important way to reduce processing costs is the use of wastes as carbon sources. The aim of the present review is to describe the main aspects related to the use of different oleaginous microorganisms for lipid production and their performance when using bio-wastes. The possibilities for combining hydrogen (H2) and lipid production are also explored in an attempt for improving the economic feasibility of the process.

Pseudomonas helmanticensis sp. nov., isolated from forest soil.

A bacterial strain, OHA11(T), was isolated during the course of a study of phosphate-solubilizing bacteria occurring in a forest soil from Salamanca, Spain. The 16S rRNA gene sequence of strain OHA11(T) shared 99.1% similarity with respect to Pseudomonas baetica a390(T), and 98.9% similarity with the type strains of Pseudomonas jessenii, Pseudomonas moorei, Pseudomonas umsongensis, Pseudomonas mohnii and Pseudomonas koreensis. The analysis of housekeeping genes rpoB, rpoD and gyrB confirmed its phylogenetic affiliation to the genus Pseudomonas and showed similarities lower than 95% in almost all cases with respect to the above species. Cells possessed two polar flagella. The respiratory quinone was Q9. The major fatty acids were C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH). The strain was oxidase-, catalase- and urease-positive, positive for arginine dihydrolase but negative for nitrate reduction, ß-galactosidase production and aesculin hydrolysis. It was able to grow at 31 °C and at pH 11. The DNA G+C content was 58.1 mol%. DNA-DNA hybridization results showed values lower than 49% relatedness with respect to the type strains of the seven closest related species. Therefore, the combined genotypic, phenotypic and chemotaxonomic data support the classification of strain OHA11(T) to a novel species of the genus Pseudomonas, for which the name Pseudomonas helmanticensis sp. nov. is proposed. The type strain is OHA11(T) ( = LMG 28168(T) = CECT 8548(T)).

Fontibacillus phaseoli sp. nov. isolated from Phaseolus vulgaris nodules.

A bacterial strain, designated BAPVE7BT, was isolated from root nodules of Phaseolus vulgaris in Spain. Phylogenetic analysis based on its 16S rRNA gene sequence placed the isolate into the genus Fontibacillus with Fontibacillus panacisegetis KCTC 13564T its closest relative with 97.1 % identity. The isolate was observed to be a Gram-positive, motile and sporulating rod. The catalase test was negative and oxidase was weak. The strain was found to reduce nitrate to nitrite and to produce ß-galactosidase but the production of gelatinase, caseinase, urease, arginine dehydrolase, ornithine or lysine decarboxylase was negative. Acetoin production and aesculin hydrolysis were found to be positive. Growth was observed to be supported by many carbohydrates and organic acids as carbon source. MK-7 was identified as the predominant menaquinone and the major fatty acid (43.7 %) as anteiso-C15:0, as occurs in the other species of the genus Fontibacillus. Strain BAPVE7BT displayed a complex lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, four glycolipids, four phospholipids, two lipids, two aminolipids and an aminophospholipid. Mesodiaminopimelic acid was detected in the peptidoglycan. The G+C content was determined to be 45.6 mol% (Tm). Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain BAPVE7BT should be considered a new species of genus Fontibacillus, for which the name Fontibacillus phaseoli sp. nov. is proposed (type strain, LMG 27589T, CECT 8333T).

Analysis of rhizobial strains nodulating Phaseolus vulgaris from Hispaniola Island, a geographic bridge between Meso and South America and the first historical link with Europe.

Hispaniola Island was the first stopover in the travels of Columbus between America and Spain, and played a crucial role in the exchange of Phaseolus vulgaris seeds and their endosymbionts. The analysis of recA and atpD genes from strains nodulating this legume in coastal and inner regions of Hispaniola Island showed that they were almost identical to those of the American strains CIAT 652, Ch24-10 and CNPAF512, which were initially named as Rhizobium etli and have been recently reclassified into Rhizobium phaseoli after the analysis of their genomes. Therefore, the species R. phaseoli is more abundant in America than previously thought, and since the proposal of the American origin of R. etli was based on the analysis of several strains that are currently known to be R. phaseoli, it can be concluded that both species have an American origin coevolving with their host in its distribution centres. The analysis of the symbiovar phaseoli nodC gene alleles carried by different species isolated in American and European countries suggested a Mesoamerican origin of the α allele and an Andean origin of the γ allele, which is supported by the dominance of this latter allele in Europe where mostly Andean cultivars of common beans have been traditionally cultivated.

Inoculation with Bradyrhizobium japonicum enhances the organic and fatty acids content of soybean (Glycine max (L.) Merrill) seeds.

Soybean (Glycine max (L.) Merrill) is one of the most important food crops for human and animal consumption, providing oil and protein at relatively low cost. The least expensive source of nitrogen for soybean is the biological fixation of atmospheric nitrogen by the symbiotic association with soil bacteria, belonging mainly to the genus Bradyrhizobium. This study was conducted to assess the effect of the inoculation of G. max with Bradyrhizobium japonicum on the metabolite profile and antioxidant potential of its seeds. Phenolic compounds, sterols, triterpenes, organic acids, fatty acids and volatiles profiles were characterised by different chromatographic techniques. The antioxidant activity was evaluated against DPPH, superoxide and nitric oxide radicals. Inoculation with B. japonicum induced changes in the profiles of primary and secondary metabolites of G. max seeds, without affecting their antioxidant capacity. The increase of organic and fatty acids and volatiles suggest a positive effect of the inoculation process. These findings indicate that the inoculation with nodulating B. japonicum is a beneficial agricultural practice, increasing the content of bioactive metabolites in G. max seeds owing to the establishment of symbiosis between plant and microorganism, with direct effects on seed quality.

Phaseolus vulgaris is nodulated in northern Spain by Rhizobium leguminosarum strains harboring two nodC alleles present in American Rhizobium etli strains: biogeographical and evolutionary implications.

In this study a collection of rhizobial strains were isolated from effective nodules of Phaseolus vulgaris in a wide region of northern Spain, which is the major producer region of this legume in Spain. The analysis of their core genes, rrs, atpD, and recA, and the 16S-23S intergenic spacer showed that all isolates belong to the phylogenetic group of Rhizobium leguminosarum and some of them were identical to those of strains nodulating Vicia or Trifolium. None of the isolates was identified as Rhizobium etli; however, all of them carry the nodC alleles alpha and gamma harboured by American strains of this species. These alleles were also found in strains nodulating P. vulgaris in southern Spain identified as R. etli. These results suggest that R. etli was carried from America to Spain with common bean seeds, but that they could have found difficulties persisting in the soils of northern Spain, probably because of the climatic conditions. The symbiotic genes of this species could have been transferred, after the arrival of P. vulgaris, to strains of R. leguminosarum already present in northern Spanish soils.

Characterization of different compost extracts using Fourier-transform infrared spectroscopy (FTIR) and thermal analysis.

Compost extract or "compost tea" is a liquid extract of compost obtained by mixing compost and water for a defined period of time. Compost tea contains nutrients and a range of different organisms and is applied to the soil or directly to plants with the principal aim of suppressing certain plant diseases. In addition, the application of compost tea supplies nutrients and organic matter to the soil. Thermal analysis and Fourier transform infrared spectroscopy (FTIR) are two widely applied analytical techniques for establishing the stability of compost, and although numerous studies have evaluated the capacity of compost tea to suppress plant diseases, there are no studies employing these techniques to characterize compost-tea. For the present study, 12 compost extracts were produced under varying conditions in a purpose-built reactor. Two different composts, an stable compost produced from manure and an unstable compost produced from municipal solid waste, respectively, two aeration systems (aerated and non-aerated extracts) and three temperatures (10, 20 and 30 degrees C) were used in these experiments. The extracts were freeze-dried and subsequently analysed, together with the two composts, by means of FTIR and thermal analysis. Extracts produced from high stability compost, independently of the conditions of aeration and temperature, showed very similar results. In contrast, differences among extracts produced from the unstable compost were more noticeable. However, the different conditions of aeration and temperature during the production of the extracts only explained partially these differences, since the transformations undergone by compost over the 3 months that the experiments lasted were also reflected in the composition of the extracts. In spite of everything, extraction process favoured the degradation of easily oxidizable organic matter, which was more abundant in unstable compost. This degradation was more intense for non-aerated processes, probably due to the longer duration of these (10 days) with respect to aerated extractions (2 days). The effect of temperature was not clear in these experiments, although high temperatures could increase micro organism activity and consequently favour the degradation of easily oxidizable organic matter.