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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.

13 páginas, 6 tablas, Microbial inoculants are gaining prominence in technologically advanced agri-systems due to the need for alternatives to the most pollutant agricultural inputs. The objective of this work was to improve the agronomic performance of the rhizobial inoculants for common bean, based on the superior native strain Rlp-LCS0306 of Rhizobium leguminosarum bv. phaseoli (R), through co-inoculation with non-rhizobial partners, namely the autochthonous isolate RVPB2-2 from Pseudomonas brassicacearum subsp. neoaurantiaca (P) and the type strain of Azotobacter chroococcum. It has been reported that co-inoculation improves nodulation, nodule functions and plant growth, although there is a lack of field testing in technologically advanced agri-systems. This work bridges this gap. In the field trial which was carried out in two different environments, the consortium R + P was the most successful, because it increased the N2 fixation by 51.7 kg ha−1 (87 %) and the yield by 1337 kg ha−1 (59 %), compared with the uninoculated and unfertilised control. In addition, the increased yield observed following inoculation with the above indicated consortium was 16.7 %, compared with the single rhizobia inoculation, and this increase was also superior to that observed with other consortia. The superiority of the R + P consortium could partially be explained because in this study, there was an increased tendency for improved nodule biomass and function following co-inoculation. While this increase was not deemed to be statistically significant, it is noteworthy that nodule biomass increased by 25 % in average and N-fixed by more than 20 %, which, in turn, could be explained by the indole-3-acetic acid (IAA) production and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of the P strain. However, further delineation of the system is required in order to explain the yield improvement exerted by the consortium. Here, we observed, i) the strong plant growth-promoting potential displayed by the P strain; ii) the colonisation of the nodules by the P strain; and iii) the strategy of colonisation of complementary spaces inside the nodules by P (intercellular) and the rhizobia (intracellular), by confocal microscopy., This work was supported by the Ministry of Economy and Competitiveness (Spain) under the National R&D Plan, subprogram RETOS-COLABORACION [Grant number RTC 2016-5834-5]; Junta de Castilla y León (Spain) [Grant number LE029A10-210−2]; M.B. received a pre-doctoral fellowship from AECID (Spain) (Agencia Española de Cooperación Internacional para el Desarrollo); A.J-G. received a pre-doctoral fellowship from the Ministry of Science, Innovation and Universities (Spain).

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 &lt, 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.

The regular configuration of fungal basidiomycetous fronts in fairy rings allows a thorough scrutiny of associated soil changes. Here, we studied the soil chemistry and the microbiota modifications induced by the passage of Calocybe gambosa fairy rings in a botanical garden. Significant changes in soil physicochemical properties and microbiota were observed in correspondence of active fungal mycelium, where an accumulation of nutrients, an increase in hydrophobicity, and a decrease in pH were recorded. In addition, on one hand, a sharp decline of fungal diversity in soil occurred in association with C. gambosa colonization. On the other hand, the bacterial community showed a variable taxon-specific response, with taxa within Bacteroidetes and Proteobacteria being able to thrive in association with C. gambosa.

Engineered humic acid-coated goethite (HA-Goe) colloids find increasing application in in situ remediation of metal(loid)-polluted groundwater. Once introduced into the subsurface, the colloids interact with groundwater altering their physicochemical properties. In comparison to freshly synthesized, unreacted HA-Goe colloids, such alterations could reduce the adsorption affinity towards metal(loid)s and also result in altered ecotoxicological effects. In our study, HA-Goe colloids were exposed to two groundwaters (low vs. high concentrations of metal(loid)s) from two metal(loid)-contaminated sites for 87 days. We investigated (i) the course of HA-Goe ecotoxicity (Daphnia magna immobilization tests), (ii) HA-Goe adsorption properties (multi-element solutions containing As, Cu, Zn, Ni and Co), and (iii) changes in the chemical composition as well as in the mineral and aggregate properties of HA-Goe. The adsorption affinity of HA-Goe decreased in the order As ≈ Cu ≫ Zn > Ni ≈ Co. The metal(loid) adsorption occurred rapidly after mixing prior to the first sampling, while the duration of ongoing exposition to groundwater had no effect on the adsorption of these metal(loid)s. We neither observed a desorption of humic acids from the goethite surface nor alterations in the mineralogy, crystallinity, and surface properties of HA-Goe. Standardized Daphnia magna immobilization tests showed an increased number of mobile organisms with increasing exposure time of HA-Goe to both groundwaters. The decrease in HA-Goe-mediated immobilization of D. magna was strongest within the first 30 d. We attribute this to a shift to smaller sizes due to the breakdown of large HA-Goe aggregates, particularly within the first 30 d. The breakdown of these μm-sized aggregates may result mainly from the repeated shaking of the HA-Goe suspensions. Our study confirms within this particular setting that the tested HA-Goe colloids are suitable for the long-term immobilization of metal(loid)s, while lethal effects on D. magna were negligible.

A biofertilizer (BF) based on the plant growth promoting rhizobacterium (PGPR) Bacillus siamensis was produced using anaerobic digestate (AD) as the main ingredient of the growth medium, alongside a carbon source from residual origin. The use of residues for the growth of PGPR reduces the production costs of biofertilizers, but makes an assessment of the possible toxicity of residues for the bacteria or plants necessary. Therefore, the growth medium of PGPR was first optimized using the response surface methodology (RSM), followed by phytotoxicity tests and a field trial of the BF in a sweet pepper (Capsicum annuum L.) crop at two different locations. AD at 50% dilution, supplemented with 2.3% sugar beet molasses, was the optimum growth medium for producing the BF, with a bacterial concentration of 109 cfu mL−1. In the field trial, the treatments inoculated with BF and fertilized with decreased mineral N (80%) produced significantly better yields per ha than the controls with decreased N (80%) and full N (100%) without BF. This indicates improved efficiency of N use by the crop, as a consequence of the use of BF.

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.

12 páginas, 5 tablas, 1 figura, The legume nodules are a rich source not only of rhizobia but also of endophytic bacteria exhibiting plant growth-promoting mechanisms with potential as plant biostimulants. In this work we analyzed the genomes of Phyllobacterium endophyticum PEPV15 and Rhizobium laguerreae PEPV16 strains, both isolated from Phaseolus vulgaris nodules. In silico analysis showed that the genomes of these two strains contain genes related to N-acyl-homoserine lactone (AHL) and cellulose biosynthesis, involved in quorum sensing and biofilm formation, which are essential for plant colonization. Several genes involved in plant growth promotion such as those related to phosphate solubilization, indole acetic acid production, siderophore biosynthesis and nitrogen fixation were also located in both genomes. When strains PEPV15 and PEPV16 were inoculated in lettuce and carrot in field assays, we found that both significantly increased the yield of lettuce shoots and carrot roots by more than 20% and 10%, respectively. The results of this work confirmed that the genome mining of genes involved in plant colonization and growth promotion is a good strategy for predicting the potential of bacterial strains as crops inoculants, opening new horizons for the selection of bacterial strains with which to design new, effective bacteria-based plant biostimulants., This work was supported by JCyL (Junta de Castilla y León, Spanish Regional government) grant SA183A11-2 to R.R. J.D.F.-F are granted with a FEMS grant FEMS-RG-2016-0213

Two hundred and sixty-one bacterial isolates associated with banana tree roots in organic systems were isolated from 19 farms located in four different provinces of the Dominican Republic. The isolates were analysed by means of ARDRA plus RAPD, and as a consequence 114 of them were selected for identification by means of complete 16S rRNA sequencing and phylogenetic reconstruction. The 114 isolates belonged to 20 different genera, with Bacillus , Pseudomonas , Enterobacter and Stenotrophomonas the prevailing genera. Of these, 65 isolates showed more than 99.5% similarity with a type strain, and they were assigned to 34 different species from 16 genera; 29 isolates could constitute new species and the remaining 20 isolates belonged to groups containing more than one species with identical 16S rRNA genes, and therefore they could not be assigned to any species. This result showed a higher number of bacterial taxa associated with banana tree roots than previously described. Additionally, we found seven bacterial species with significant in vitro plant growth promoting (PGP) activity, which had not been previously described as PGP bacteria in any crop. Field trials with isolates pre-selected based on their in vitro PGP activity showed that one strain of the species Pseudomonas plecoglossicida improved fruit yield and controlled the incidence of the disease black sigatoka caused by Mycosphaerella fijiensis . This activity was tentatively attributed to induced systemic resistance mechanisms. Bacterial diversity was analysed among the 261 isolates based on the Shannon index of diversity (H), calculated from the ARDRA profiles. Interestingly, the majority of the bacterial diversity was found within farms (86% of the total), being higher than the bacterial diversity between farms (14%). Moreover, the differences in the average H Index between provinces were very low. Consequently the biodiversity of the bacterial communities was little influenced by the soil characteristics. These results could work in favour of the efficient adaptation of the bacterial strains selected for use as plant probiotics in a range of soils in the region analysed.

Botrytis cinerea is a polyphagous necrotrophic fungus and is the causal agent of grey mold diseases in more than 1400 different hosts. This fungus causes serious economic losses in both preharvest and post-harvest—mainly in grape, strawberry, and tomato crops—and is the second most important pathogen worldwide, to our knowledge. Beneficial bacteria and fungi are efficient biocontrol agents against B. cinerea through direct mechanisms, such as parasitism, antibiosis, and competition, but also indirectly through the activation of systemic plant resistance. The interaction between plants and these microorganisms can lead to the development of defensive responses in distant plant organs, which are highly effective against foliar, flower, and fruit pathogens, such as B. cinerea. This review aimed to explore the systemic plant defense responses against B. cinerea by compiling all cases reported (to the best of our knowledge) on the use of beneficial bacteria and fungi for agriculture, a subject not yet specifically addressed.

Replacing legume fertilisation using mineral nitrogen (N) (conventional technology, CT) with rhizobial/bradyrhizobial inoculation (inoculation technology, IT) is currently encouraged worldwide due to environmental concerns. However, the environmental effect of such inoculants has scarcely been addressed using a systematic methodology like life cycle analysis (LCA). The objective of this work was to assess, in tropical developing countries, the agronomic and environmental performance of rhizobial inoculants. We worked with two contrasting pulses, common bean, which is conventionally grown using an intensive mineral fertilisation strategy, and Pigeon pea, which is subject to a deficit fertilisation strategy. From the agronomic point of view, the replacement of CT by IT improved Pigeon pea yield by 30%, while increasing common bean yield by a not significant 2%. The Global Warming Potential (GWP) and Cumulative Energy Demand (CED) of inoculant production for 1 ha is less than 1% of the GWP and CED corresponding to the production of the mineral N fertiliser used in CT. The high input-high output crop (common bean) caused less environmental damage than the less productive and low input crop (Pigeon pea), indicating that an improvement in the yield would reduce agriculture’s overall environmental impact. Considering the five most impacted categories (CML-IA methodology), the substitution of CT by IT in the common bean resulted in an average reduction of environmental burdens by 19% per ha and 21% per t; in Pigeon pea, the average reduction was 12% per ha and 32% per t. However, phytosanitary treatments and irrigation accounted for higher impacts than N. Thus, improvement in environmental performance must include other technologies to optimise plant protection and irrigation and other strategies to increase yield, such as the optimisation of P, K fertilisation.

The Isla del Coco National Park, located on the Pacific coast of Costa Rica, is rich in biodiversity and has a high concentration of pelagic species. This high marine biodiversity makes the Isla del Coco National Park (PNIC) a very attractive place for illegal fishers. We analyzed a dataset covering 8 years (2003–2010) of patrol records from PNIC with the aim of determining, a) the spatial-temporal distribution of illegal fishing, b) other areas that could be prone to illegal fishing but are currently undetected, c) the most profitable areas for this activity and d) the economic trade-offs of this illegal activity in relation to potential gains and the costs. Residuals Autocovariate Generalized Additive Models (RAC-GAMs) were used to model the illegal fishing activity's spatial distribution in relation to topographic, biological and temporal (quarter of the year) variables. The final RAC-GAM showed that bathymetry, distance from the coast, slope of the seabed, and yellowfin tuna and silky shark abundance were the most important predictors of this activity. Predictive maps suggest a major trend in the abundance of illegal fishing between the second and third quarters of the year in waters surrounding a seamount within the Park. Maps of the most profitable areas highlighted a specific risk location that should be intensively monitored. Overall, the potential gains from this activity outweigh the potential costs of being caught. Our findings provide useful information that can be used to optimize enforcement, deter illegal fishing and, consequently, increasing the conservation of the protected species.

Pigeonpea (Cajanus cajan) originated in India and is now a major source of protein in countries in tropical and subtropical regions worldwide. However, little is known about the effect on crop yields of inoculation with appropriate rhizobia strains. The objective of this study was to explore the possibility of improving pigeonpea yields in the Caribbean region, by inoculating with selected N-fixing symbionts. To achieve the study objective, we selected three elite strains of the genus Bradyrhizobium isolated from root nodules of pigeonpea in the Dominican Republic, we formulated them using innovative carriers, and finally we tested the formulated inoculant on eight farms located in the Dominican Republic. Three different carriers (sewage sludge, pine bark biochar and poultry litter) plus perlite as control were tested for the formulation. The first step was to test bacterial survival in the different carriers at different times up to one year (shelf-life). Pine bark biochar and the control resulted in the best shelf-life and were selected for the field experiment. The experimental design comprised two pigeonpea cultivars and five fertilisation strategies: Inoculation with three Bradyrhizobium strains without nitrogen (N) fertilisation, plus two non-inoculated controls one of them fertilised with 125 and 210 kg N ha−1 in non-irrigated and irrigated fields respectively. Nodule occupancy by the inoculated bacterial strain depended solely on the level of soil native nodulating bacteria and not on the bacterial strain or the carrier. Inoculation produced, on average, a significant yield increase compared to the non-inoculated non-N-fertilised control. Furthermore, the yield with the inoculated treatments did not significantly differ from the yield of the N-fertilised control. However, either inoculation or N-fertilisation were ineffective in two out of the eight fields with more than 104 nodulating bacteria g soil−1, compared to the non-inoculated non-N-fertilised control. Yield was neither significantly affected by the bacterial strain nor by the cultivar-strain interaction, and therefore, the two cultivars can be considered promiscuous. Neither carrier had a significant effect on yield. Thus, inoculants based on strains of Bradyrhizobia isolated from the root nodules of pigeonpea, formulated with pine bark biochar or perlite as carrier, are recommended as a good strategy to ensure food security in tropical agroecosystems with pigeonpea.

Common bean (Phaseolus vulgaris L.) crops hold the potential to obtain higher yields by enhancing their biological nitrogen fixation (BNF) with Rhizobium. However in contrast to other legumes, common bean has shown a lack of positive response to inoculation with Rhizobium in many cases. This has led to a limited use of rhizobial inoculants in this crop, especially in Europe. The adaptation of bacterial strains to the rhizosphere is a key factor in the success of any inoculant, especially in a promiscuous legume such as common bean. This research aimed at increasing common bean yields via inoculation with effective indigenous Rhizobium leguminosarum strains. Three highly effective strains (LCS0306, LBM1123 and ZBM1008) which were selected according to their effectiveness at BNF in hydroponic conditions were separately inoculated onto common bean in a field experiment. The experiment was carried out under three environments and three tillage systems: conventional-tillage (CONVT), no-tillage (NT) and a cover-crop (CC). The grain yield observed with seed inoculation was significantly higher than the yield obtained with uninoculated seed under CONVT and CC. However, under NT inoculation had no effect. Furthermore, under CONVT and CC, inoculation with R. leguminosarum LCS0306 produced even higher yields than those obtained in nitrogen-fertilised or control plots. This is the first attempt to explain the inoculation performance of common bean under different tillage systems in Europe. A gene–based hypothesis has been used to explain the effectiveness of indigenous common bean rhizobia as nitrogen fixers in this crop.

Cajanus cajan L. is a legume whose green seeds are consumed in Caribean countries such as Dominican Republic where is a horticultural plant highly appreciated by the consumers. In this work we isolated several slow-growing rhizobial strains nodulating C. cajan that were found to be closely related to Bradyrhizobium yuanmingense according to the analysis of 16S rRNA gene sequences. These strains were genetically diverse on the basis of their RAPD patterns and showed high variability in nodulation ability and N-fixation on hydroponic conditions. Highly effective strains used as biofertilizers in four different soils from Dominican Republic showed that biofertilization of C. cajan with these strains produced the same or even higher yield than the mineral nitrogen fertilization. Therefore, biofertilization with native Bradyrhizobium strains can be adopted instead of chemical fertilization to improve both the yield and the quality of C. cajan . This is the first report about the nodulation of this legume by strains close to B. yuanmingense in Dominican Republic where the natural nodulation of C. cajan is scarce and then, the biofertilization can be crucial to increase the yield of this horticultural crop very important for human feeding.

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.

5 pages, 1 figure, and 2 statistics tables, A bacterial strain designated RTAE36T was isolated from wheat roots in northern Spain. Phylogenetic analyses based on 16S rRNA gene sequence placed the isolate into the genus Paenibacillus with its closest relative being Paenibacillus borealis DSM 13188T with 97.7% sequence similarity. Cells of the isolate were facultatively anaerobic, Gram-stain-positive, motile and sporulating rods. Catalase and oxidase were positive. Gelatin, casein and starch were not hydrolysed. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the only menaquinone detected, and anteiso-C15 : 0, C16 : 0, iso-C14 : 0 and iso-C16 : 0 were the major fatty acids. The polar lipids profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid, two unidentified phospholipids, three unidentified phosphoaminolipids, one unidentified glycolipid and one unidentified lipid. meso-Diaminopimelic acid was detected in the cell-wall peptidoglycan. Strains RTAE36T and P. borealis DSM 13188 had an mean DNA–DNA relatedness of 39% and differed in several phenotypic and chemotaxonomic characteristics, confirming that strain RTAE36 should be considered as a representative of a novel species of the genus Paenibacillus, for which the name Paenibacillus tritici sp. nov. is proposed. The type strain is RTAE36 (=LMG 29502=CECT 9125)., This work was supported by MINECO (Spanish Central Government) grants AGL2013-48098-P and AGL2013-40758-R to E. V. and M. F. P., respectively. E. M. is supported by a postdoctoral contract associated with the project AGL2013-48098-P. J. D. F. F. is supported by a Fellowship of Salamanca University.

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.

Synthesized iron oxide nanoparticles have been proposed as an alternative to non-dispersed iron oxides for in situ environmental remediation. Their colloidal properties enable their injection into porous media, i.e. soils and aquifers, and offer a higher efficiency in removing contaminants. However, this dispersed state is also the cause of concerns over their environmental fate and toxicity, e.g., by increasing the exposure time to aquatic organisms in groundwater remediation activities. Therefore, the objective of in situ groundwater remediation is to establish local reactive barriers in the subsurface by injection by means of reactive colloids with a controllable mobility under in situ conditions and present as colloids as shortly as possible. In this work, we examined the toxicity of humic acid-coated colloidal goethite nanoparticles in Daphnia magna. The adaptation of the ecotoxicological standard tests for nanomaterials is intensely discussed to increase comparability and reliability of results. In the present study, the effect of different exposure conditions on goethite nanoparticles colloidal behaviour and acute Daphnia immobilization effects was investigated. For this purpose, iron concentration in the water column, aggregation state and acute effects were studied in: i) a standard test, ii) test design with exposure dispersions incubated for a week and iii) water accommodated fraction. Despite the different aggregation and settling of the particles found between the approaches tested, no differences in toxicity were observed. Coated nanoparticles were found clogging up the filtering apparatus, and/or adhered to the exoskeleton, hindering the swimming and molting, and causing the immobilization and death of the organisms at doses of ≥ 943 mg/L (EC50). The data suggests that the toxic potential of these nanoparticles is mainly related to the physical interaction with the daphnids.

18 páginas, 2 figuras, 2 tablas. – The definitive versión is available at http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.063560-0 -- MHRB is recipient of a JAE-Doc researcher contract from CSIC cofinanced by ERDF. MJC, JDFF and RM are recipients of contracts supported by this project, A bacterial strain named OHA11T was isolated in the course of a study of phosphate solubilizing bacteria occurring in a forest soil from Salamanca, Spain. The 16S rRNA gene sequence had 99.1% identity with respect to the closest relative Pseudomonas baetica a390T, and the following closest related species with 98.9 % similarity were P. jessenii, P. moorei, P. umsongensis, P. mohnii and P. koreensis, for which OHA11T was classified within genus Pseudomonas. The analysis of housekeeping genes rpoB, rpoD and gyrB confirmed its phylogenetic affiliation and showed identities lower than 95% in almost all cases with respect to the mentioned closest relatives. The strain has two polar flagella. The respiratory quinone is Q9. The major fatty acids are 16:0, 18:1 ω7c and 16:1 ω7c/ 15:0 iso 2OH in summed feature 3. The strain is oxidase, catalase and urease positive, the arginine dihydrolase system is present but nitrate reduction, β–galactosidase production and esculine hydrolysis are negative. It can grow at 31ºC 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 results of genotypic, phenotypic and chemotaxonomic data support the classification of strain OHA11 into a novel species of Pseudomonas, for which the name P. helmanticensis sp. nov. is proposed. The type strain is OHA11T (=LMG 28168T, CECT 8548T)., This research was funded by MINECO (Spanish Central Government) Grant INNPACTO IPT-2011-1283-060000.

Mexico is a center of diversity of Jatropha curcas. Worldwide there are two groups of germplasm, the toxic and the non-toxic, and most of the biofuels programs are based on toxic germplasm. In the mountains of the Totonacapan (Mexico), non-toxic genotypes have been cultivated for human food from ancient times, and now they are in serious risk of being lost because of the "biofuel program'', as well as the ethnobotanical knowledge about this germplasm in the Totonaca culture. The starting hypothesis is that this ethnobotanical knowledge must be related with a high biodiversity of the germplasm of this crop, which is necessary to preserve to avoid genetic erosion. The objective of the work was to assess the biodiversity of the native germplasm using morphological traits, and to analyse socio-economic aspects related with the ethnobotany of this germplasm, to design strategies for the conservation of the germplasm and of the culture linked to it. Other specific objective was to select morphological variables highly discriminant and easy-to-measure, for the characterization of this crop. We selected eight morphological descriptors as the most discriminant, related with the leaves: Length and width of the blade, and length of the petiole; and the fruit: weight, length, diameter, length of the peduncle, and number of fruits per bunch. On the basis of the principal component analysis and canonical discriminant analysis of the morphological data, it was possible to identify as different germplasm pools the six locations sampled, in spite of their proximity, confirming the starting hypothesis that there is high biodiversity of this crop in the region. The pools from Camocuautla and Tuzamapan were the most similar between them, with small fruits and a low number of teeth in the leaves. The germplasm form Jonotla showed also small fruits, but small leaves and a short petiole. Tetelilla's germplasm showed a high number of teeth in the leaves, big fruits and leaves, and long petiole. The accessions from Ecatlan showed big fruits, low number of fruits per bunch and short fruit's peduncle, with small leaves and a short petiole. The differential traits of the accession from Zozocolco were a high number of fruits per bunch and a long fruit's peduncle. Unlike what happened with the germplasm, the socio-economic profile of the farmers did not follow a geographical pattern. Facing the scenario of establishment of the "biofuel program'' in the region, we proposed an on farm conservation strategy consisting in the exclusive use of local nontoxic germplasm, which will need the involvement of all the stakeholders, and in special of the local plant nurseries. As this is difficult to achieve, the ex situ conservation has been proposed as an emergency action. Even if the genetic erosion is stopped, it is necessary to develop other companion measures to avoid the loss of the knowledge about the ancestral uses of this crop, as the farmers could prefer to sell the crop for the industry, instead of use at least a part of it for their own food.

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.

The revegetation of land that is no longer cultivated helps reduce soil erosion and runoff. Prior studies have determined the effectiveness of shrub-type cover for this purpose in Spain using small plots sustaining the growth of mature shrubs without considering scaling effects or the establishment and colonisation period, in which the risk of soil erosion is high. In addition, these studies have assessed the effects of high magnitude–low frequency simulated rainfall. The aim of the present study was to measure under natural rainfall conditions the effects of three shrub covers ( Colutea arborescens , Dorycnium pentaphyllum and Medicago strasseri ) on runoff and soil loss. Plots 20.2 m × 4 m were created and runoff discharge and sediment concentrations were measured after each rainfall event for 2.5 years (establishment period) and for the following 4 years when the shrubs were well-developed. The results of this experiment were compared with those of a previous study, in which the same data were recorded under conditions of simulated high-intensity rainfall in small plots (0.25 m 2 ). In the control plot (bare soil), 5 rainfall events were responsible for 85% of the erosion (37.5 Mg ha −1 ) and 59% of the runoff (128.8 mm) produced. When the shrub covers were mature, runoff and erosion were reduced (by 84–95% and 95–99% respectively) compared to the establishment period. The effects of the shrubs at mitigating soil and water losses were more pronounced following high intensity rainfall events. Compared to smaller plots, the 80.8 m 2 plots tested here showed similar soil losses under high intensity thunderstorms but runoff losses were much lower, suggesting that some of the runoff reinfiltrates the slope due to shrub distribution effects.

Legume nodules harbour two types of bacteria, the rhizobia, responsible for their formation, and other endophytic bacteria whose role in the nodule is still poorly known. These bacteria constitute the nodule microbiome from which the rhizobia have been widely studied for decades, whereas the nodule endophytes have been started to be studied in the last years. These studies showed a more complex bacterial composition than previously thought, including bacteria from very different phylogenetic groups. Unlike other plant microbiomes, which have been widely studied by metagenomic techniques, the nodule microbiomes have been basically studied by culture-dependent methods because the main objective of the legume nodule studies is the selection of plant growth-promoting bacteria to be used in agronomic practices in a sustainable agriculture context. In this chapter we revise the groups of bacteria found to date in legume nodules that present in vitro mechanisms of plant growth promotion, with special emphasis in those that are able to promote the plant growth in plant assays.

Phaseolus vulgaris is a legume extensively cultivated in Spain, Leon province being the most important producer. This province produces selected varieties of common bean highly appreciated by their quality that warrants a Protected Geographic Indication (PGI). In this work we analysed the rhizobia present in nodules of the variety “Rinon” in several soils from Leon province in order to select native rhizobial strains to be used as biofertilizers. The analysis of rrs and housekeeping genes of these strains showed that they belong to two phylogenetic groups within Rhizobium leguminosarum (I and II). Although the group II strains were most abundant in nodules, very effective strains were also found in group I. Strains LCS0306 from group I and LBM1123 from group II were the best nitrogen fixers among all strains isolated and were selected for field experiments. The field research showed that the biofertilization of common bean with native and selected rhizobial strains can completely replace the fertilization with chemical N fertilizers. The biofertiliser designed in such way, was valid for the whole agroecological area, regardless the specific properties of each soil and microclimatic conditions. This conclusion can be generalised as a strategy for the development of biofertilisers in different agroecological conditions worldwide.

The genus Crateagus (Rosaceae: Maloideae), is distributed all around the world, but only in the Northern Hemisphere. In Mexico there is a considerable biodiversity with around 15 species, most of them authoctonous and it is called tejocote, a multi-purpose tree with economic importance. However the taxonomy of the Crategus species from Central and Southern Mexico still remains unclear. The gemplasm bank of tejocote of the Universidad Autonoma Chapingo (UACh) (State of Mexico) contains almost 100 accessions of Crataegus from Central and Southern Mexico. Based on previous studies that demonstrated that seeds and inner parts of the fruits are useful for germplasm characterization, the general objective was to investigate the usefulness of seeds and endocarps for the characterization of tejocote genetic pools, using 86 accessions from the tejocote germplasm bank of the UACh, with the specific objectives being: (i) to select from 37 morphological and morphometrical variables, the most informative for the assessment of the biodiversity; (ii) to contribute to the elucidation of the variability of the tejocote genetic pools from Central and Southern Mexico. The traits related with the size and shape of the endocarp, seed and seed cavity, plus the presence or absence of normal vs. abortive seeds, and the number of endocarps per fruit, were the characters that accounted for more variability and in conclusion the most adequate for the characterization of tejocote and some of them have been adopted for the official Crataegus descriptors. The present work confirms prior results with the same germplasm and other characters which postulated that the source from Chiapas is different from the rest of the sources and also that the sources from the State of Mexico and Puebla were not significantly different genetic pools. The accessions from the Chiapas source showed smaller endocarps and seed cavity, lower percentage of endocarps with normal seeds, higher percentage of endocarps without seed, and higher number of endocarps per fruit.

Introduction . Tejocote (Crataegus spp.) is an edible fruit species that some Mesoamerican cultures have cultivated since pre-Hispanic times in Mexico; however, this fruit crop has not undergone formal breeding. It is unknown whether morphological markers associated with fruit size exist to be used for indirect selection; this could be important, because this species has a large cycle. Our research aimed at studying phenotypic correlations between leaf and stomata characters with fruit weight; such correlations might be used as morphological markers for selection of superior germplasm for fruit weight. Materials and methods . Pearson’s correlation coefficients of 36 leaf variables and three leaf stomata variables with fruit weight were calculated in 94 genotypes of four different species. Results . As expected, the correlations calculated for each species and for the species pool differed. However, the number of veins in leaves of reproductive shoots was positively correlated with fruit weight (P ≤ 0.05) in the two most important horticultural species. On the other hand, C. mexicana was the best option for selecting superior germplasm, and the most significant variables correlated with fruit weight in this species were leaf major axis length of large vegetative shoots, leaf elongation ratio and number of veins of reproductive shoot leaves. Discussion . These variables could be good selection indices or morphological markers that can be used for early screening of germplasm for yield potential.

Tejocote (Crataegus spp.) is a genus of fruit-bearing trees distributed widely throughout Mexico; 13 species are reported for the north and central zones and two or more species may be present in southern Mexico. Accessions of this genus are safeguarded in the Germplasm Bank of Tejocote at the Autonomous University of Chapingo, mainly from three regional sources, i.e. the states of Puebla, Mexico, and Chiapas, including five different species that belong to series Mexicanae and series Crus-galli. They can be morphologically characterized by leaves from different shoot types, flowers and fruits. Multivariate statistical methods were used to elucidate patterns of variation in each of these regional sources. The sources displayed very low intra-source variability. The source from Chiapas showed significant statistical differences in all morphological variables evaluated, as a result, this genetic pool is considered as different from the other two sources. The sources from the states of Puebla and Mexico only differed by 22.79% (with P ≤ 0.05), and thus they could be considered as components of a single genetic pool. The most highly discriminant variables were from the leaf, such as basal angle, petiole length/major axis length ratio, minor axis length/major axis length ratio, and number of veins.

Black sigatoka caused by Mycosphaerella fijiensis (Morelet) is one of the main diseases affecting banana trees worldwide, resulting in a reduced fruit yield . Banana is a major export crop in many tropical and subtropical countries, and many farmers have decided to shift to an organic production system for produce that is destined for the international market. Induced systemic resistance (ISR) protects plants against disease-causing pathogens, and this mechanism can be triggered by some bacteria associated with plant roots. The inoculation of plant roots with bacteria able to induce systemic resistance in plants has been proposed for disease control in organic agriculture as an alternative to chemicals. Fifty-six nonpathogenic and endophytic strains isolated from roots of banana trees cv. Cavendish in organic systems from four regions of the Dominican Republic were the starting point for the present work. We used 26 of them to inoculate the root systems of banana plants, the leaves of which were inoculated with M. fijiensis inoculum, in order to analyse their effect on the control of black sigatoka under growth chamber conditions. The selection of the 26 strains to be tested in plants was based on the following plant growth-promoting (PGP) traits : production of siderophore (17 strains), production of IAA (two strains) and ACC deaminase activity (two strains); the remaining five showed low values for all of the PGP traits. The best controllers of sigatoka were five strains from the genus Bacillus (related to B. licheniformis, B. siamensis and B. subtilis ssp. inaquosorum) and one Rhizobium massiliae. With the six strains, the severity index (SI) of the plants coinoculated with the bacteria and the pathogen did not differ from the noninoculated control. The sigatoka control observed has been tentatively assigned to ISR phenomena, which is discussed in the chapter. The six selected strains could be used in the short term to control black sigatoka in organic banana production systems in the Dominican Republic.

Fusarium oxysporum f.sp. phaseoli and Phytophthora capsici are soil pathogens which cause diseases to common bean (Phaseolus vulgaris) and pepper ( Capsicum annuum) , respectively. Some microorganisms associated with plants show different biocontrol mechanisms against pathogens. The agricultural use of these mechanisms is a chief strategy as an alternative to chemicals. In a previous work, we isolated and taxonomically identified 68 endophytic bacteria from common bean and 54 from pepper. The isolations were carried out in farms located in regions protected by a PGI: “Alubia de La Baneza—Leon” in the case of common bean, and “Pimiento de Fresno-Benavente” in the case of pepper. The aim of the present work was the evaluation of such bacteria and AM fungus, as potential biocontrollers of F. oxysporum f.sp. phaseoli and P. capsici. In total 18 out of the 122 isolates controlled in vitro the growth either of F. oxysporum or P. capsici, or both pathogens. Such controller bacteria were tested in the plants in which they were isolated (common bean or pepper), which were grown in hydroponic conditions and inoculated with F. oxysporum f.sp. phaseoli and P. capsici, respectively. Two isolates belonging to the species Pseudomonas brassicacearum and Bacillus siamensis , respectively, were the best controllers of Fusarium root rot in common bean. Moreover, two isolates belonging to Bacillus pumilus were the best controllers of P. capsici in pepper plants . In trials carried out in microcosm conditions, common bean plants inoculated with the pathogen F. oxysporum, plus an autochthonous Rhizobium leguminosarum strain and the B. siamensis isolate, showed a very weak pathogen attack. Regarding the pepper microcosm assay, the co-inoculation with one of the B. pumilus isolates plus an autochthonous mycorrhiza controlled P. capsici in the plants inoculated with the pathogen, even at the same level than the chemical fungicide. This opens the door for the development of biocontrollers, alternative to chemicals, in these two crops.

In the present study, we isolated 14 bacterial strains from the roots of maize plants growing in a soil located at Leon (Spain). All these strains presented some of the following in vitro plant growth promotion mechanisms: (i) phosphate solubilization, (ii) ACC deaminase activity, (iii) siderophore and/or (iv) IAA production. These strains displayed 11 different RAPD profiles, showing they were genetically diverse. Representative strains from each RAPD type were identified on the basis of the 16S rRNA gene sequencing analysis, showing that most of them belonged to gamma-Proteobacteria and, specifically, to the families Enterobacteriaceae and Pseudomonadaceae (genus Pseudomonas). Most of the isolated strains belong to species that can be pathogenic for humans, such as Pantoea agglomerans, Pantoea ananatis, Lelliottia amnigena (formerly Enterobacter amnigenus), Kosakonia cowanii (formerly Enterobacter cowanii), Enterobacter ludwigii and Rahnella aquatilis, or for plants, such as Pseudomonas corrugata, Pseudomonas brassicacearum and Agrobacterium larrymoorei. These results showed the need of a correct identification of bacterial endophytes before selecting strains for the design of biofertilizers, which must be pathogen-free.

All the accessions (35) of white lupin (Lupinus albus L.) landraces collected from northwest Iberian plateau and maintained at the Spanish germplasm collection (CRF of INIA), were studied with the general objective of assessing the biodiversity of these landraces and to ascertain their value as genetic resources for the development of germplasm adapted to the areas where they were collected with long and chilly winters. The characterization study comprised 51 qualitative and 50 quantitative characters. Quantitative parameters were analyzed by Principal Components Analysis (PCA). The 2-dimensional plot (49.3% of cumulative variability) formed a main group of accessions and 4 outliers (#1, #9, #27 and #28) separated in the first PC. The characters with a higher contribution to the first PC were inflorescence length, leaf petiole length and leaf central foliole area. The presence of alkaloids, the percentage of plants killed by soil borne fungal diseases, the phenology, the yield per plant and yield components were also assessed. Twenty six accessions (the main group in the PCA plot) showed high homogeneity, with the following highlights: bitterness, indeterminate growth habit, early ripeness and spring sowing with the exception of #18, in which seeding date is unknown and it had a later phenology. By contrast accessions #9 and #27 were bitter of autumn sowing with determinate growing habit and they had the longest values of primary inflorescence length and a later phenology when they were sown in spring. Average values for yield and yield components showed a broad range of variation between accessions. The main yield component was the number of pods per plant. Accession #22 had the highest mean seed mass, although this had not significant influence in yield. Accession #17 showed the highest yield and #1 the lowest. This last also had the lowest values of inflorescence length, leaf petiole length and leaf central foliole area. No significant differences of resistance to soil-borne fungi were found between accessions. The year had a significant effect in the phenology, yield and yield components except for number of seeds per pod. The studied material might be of interest for the development of spring sowing germplasm adapted to north Iberian peninsula.

Three commercially available cherimoya cultivars (Campas, Burtons and White), one cultivar recently obtained and registered in Mexico (Cortes-II-31) and seven Mexican germplasm selections (S-196, S-256, S-260, S-266, S-9651, S-Selene and S-Carapan) were characterized by using the morphometric traits of various organs of the adult plant, together with several agronomical and chemical characteristics. The objectives were: (i) to seek an alternative approach to the definition of cherimoya cultivars through multivariate analysis, using commercial varieties and Mexican germplasm selections and (ii) to elucidate the grouping of cultivars and selections obtained by multivariate analysis on the basis of their origin and geographical distribution. Plant material was collected in 2002 from adult plants 3 years after grafting. Twenty-one morphometric characteristics (seven of leaves, nine of flowers, two of fruits and three of seeds), plus five fruit characteristics of agronomical importance and three chemical parameters of the fruit were selected for characterizing accessions. The intra-accession variability recorded for the traits selected made them suitable for identifying cultivars. All of the traits but one were capable of showing up differences between accessions at a significance level of 0.001. Principal Component Analysis (PCA) showed that the traits which yielded the maximum separation between accessions were: leaf blade form factor, angle of the fifth vein of leaves, upper angle of leaves, area of the cross-section of petals, sepal maximum projected area, weight of fruits, total soluble solids in fruits, resistance of the skin of fruits to a penetrometer and width-to-length ratio of the maximum projected area of seeds. Consequently, all of these characteristics may be of interest as descriptors for cherimoya varieties. Furthermore, four consistent groups of accessions were defined by the three-dimensional plot obtained through projecting the accessions onto the first three principal components and the tree-diagram, or dendrogram, obtained through Cluster Analysis (CA). Two of these groups were made up of accessions with a well-defined common origin. The first consisted of selections S-196, S-256, S-260 and S-266 obtained from Coatepec Harinas in Mexico State, while the second comprised selections S-Selene and S-Carapan from Purepecha native Indian communities in Michoacan State. For the other two groups the origin of the accessions forming them is not fully known. Hence, further studies, based on molecular markers, might be carried out in order to ascertain if these accessions are genetically related.

Medicago strasseri, M. citrina, Colutea arborescens, and Dorycnium pentaphyllum are legume shrubs potentially useful for the revegetation of semiarid Mediterranean ecosystems and for animal grazing. As nodulation with specific rhizobia and/or mycorrhizal fungi, may become essential for the settlement and growth of legumes, the objective was to ascertain the specificity/promiscuity of the plant–rhizobia interaction, by detecting the occurrence of infective rhizobia in the soils to be revegetated and by cross inoculation tests. Native rhizobia for the target species, were isolated from nondisturbed native areas. The four species were inoculated with four semiarid soils located in central Spain, that we intend to revegetate. Specific rhizobia for M. strasseri and M. citrina were detected in three of the four soils. The soil that did not show infective rhizobia had received sewage-sludge from a waste-water treatment plant, and levels of heavy metals were high. None of the 4 soils had specific rhizobia for C. ...

Galega officinalis L. is a leguminous perennial herb that presents a demonstrated lac‐togenic effect. However, it is toxic to the ewe if it is eaten in substantial amounts, due to the presence of some alkaloids. The objective of this work was i) to optimise its management on Mediterranean‐type climate regions, and ii) to determine if controlled amounts of G. officinalis could increase milk yield in “Churra” ewes, without toxicity problems. The high level of specificity of the infective rhizobia makes it necessary to inoculate plants with selected strains, prior to the cultivation of G. officinalis. The optimum stand was 160 000 plants ha−1 obtained after broadcast autumn sowing, which in Mediterranean‐type climatic conditions increased yield in the first cutting year. Optimum cutting height was between 6 and 10 cm above ground level. The maximum yield was 13 t dry matter ha−1 in the first year, and 10 in the second year which allowed five cuttings per year before flowering. This avoids an increas...

Seven Pinus species (P. canariensis, P. halepensis, P. pinaster, P. pinea, P. nigra, P. uncinata, P. sylvestris) present on the Iberian Peninsula and the Canary Islands were characterized using polyacrylamide gel electrophoresis (PAGE). Acid phosphatases (ACP), esterases (EST), glutamate oxalacetate transaminase (GOT) and superoxide dismutase (SOD) isoenzyme systems showed a clear expression and were used for the study. Pinus canariensis, Pinus halepensis, Pinus pinaster, Pinus pinea and the group formed by Pinus nigra, Pinus uncinata and Pinus sylvestris were clearly distinguished by means of ACP or both GOT and SOD. On the other hand Pinus nigra, Pinus uncinata and Pinus sylvestris were distinguished using the EST system. Consequently the technique allows for the characterization of the studied taxa. Further work is being carried out in order to analyze the possibility of discriminating different populations of a given species.

The three species belonging to the sect. Dendrotelis of the genus Medicago: M. arborea L., M. citrina (Font Quer) Greuter, and M. strasseri Greuter, Matthas & Risse, were characterized using 12 morphometrical characters of seeds and 20 of seedlings. These species have interest for the regeneration of degraded lands in semi-arid climates. M. arborea presents the largest phenotypic variability and is dispersed throughout the Mediterranean basin, while the other two species are endemic to a very reduced area, M. citrina, in Columbretes Islands and Cabrera Islands (Spain) and M. strasseri in Crete. Several populations of M. arborea were measured, in order to compare the intraspecific variability within M. arborea with the differences among all three species. The multivariate analysis of the data used characters that were independent of the size of the studied organs. The three species were clearly differentiated on the basis of seed characters. On the other hand, seedling characters distinguished M. citrina from the other two species, but M. strasseri and the populations of M. arborea were intermingled. In spite of this, we have described relevant morphological differences among M. arborea, M. strasseri and M. citrina, which are consistent with other ecological or bromatological differences. The study supports that the three accessions merit to be considered as three different species.

Genista monspessulana and Genista linifolia are woody legumes with a high potential value for the rehabilitation of degraded soils , although field establishment of the plantlets in nonnative soils is very difficult because of the lack of specific rhizobial strains . In this work the rhizobia that nodulate the two species were tenta tively classified as a Bradyrhizobium sp . Only those soils with naturally occurring G. monspessulana or G. linifolia, now or in the past , had specific bradyrhizobia . Out of the six rhizobial strains analyzed , only Bradyrhizobium sp . ISLU21 , iso lated from Lupinus luteus, nodulated with the species studied . Out of the 11 legumes inoculated with the strains isolated from G. monspessulana and G. linifolia, only L. luteus produced infective nodules . In a field experiment , plants inocu lated with the specific bradyrhizobia isolates showed higher survival , growth rates , and N content than the noninoculated controls .

Nineteen strains isolated from nodules of the legume Genista monspessulana (Group M) and 19 strains isolated from nodules of G. linifolia (Group L) were studied on the basis of their random amplified polymorphic DNA (RAPD) patterns, to ascertain whether the rhizobia isolated from them are species‐specific. Another objective was to determine whether the random primers used could distinguish our strains from other rhizobial species or biovars. The variance among the patterns obtained for the strains of Group M versus the strains of Group L was estimated using analysis of molecular variance (AMOVA). Variance within every group was much higher than variance among groups. Consequently, the dendrogram obtained after cluster analysis of the presence‐absence data matrix for the RAPD profiles, did not show independent branches for the strains of Group M and Group L. A complementary physiological study showed that there were no significant differences between the efficiency of strains from Groups M and L. ...

The chemical composition of herbage produced by legume shrubs was determined to appraise their potential value for forage. Six species were assessed for seasonal variation in the proportion of leaves, and ash content, neutral detergent fibre (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), and crude protein (CP) concentrations. Hippocrepis balearica, Medicago citrina, Medicago strasseri, and Medicago arborea showed an average ADF content 18%. The chemical composition of Medicago strasseri was very close to that of Medicago arborea throughout the year. In winter and summer, Medicago citrina showed lower ADF and ADL content than Medicago arborea. Therefore, Medicago citrina could be useful as a summer or a winter forage. Hippocrepis balearica showed higher crude protein content in summer and lower ADF content in winter than Medicago arborea, but significantly higher ADL contents (9.7% to 12.5% versus 7.1% to 9.4%). Therefore, Hippocrepis bal...

The phenology of nineteeen accessions belonging to fourteen shrubby species of the genus Cytisus and allied taxa (Genisteae: Leguminosae) has been studied. The species are potentially interesting for introduction in agroforestry systems, or non-irrigated gardens, in extreme-climated dry mediterranean areas. The methodology used for the description of the phenology was based on nine key phenophases. This system can be generally applicable to most of the shrubby species interesting for the above-mentioned purposes. Cytisophyllum sessilifolium started the growth season significantly later than the rest of the species. Bloom extended from mid-March for some of the species to the beginning of June for others, and maturation of pods from the end of March to the beginning of August. The dendrogram obtained on the mean dates for every phenophase contributes to support Chamaecytisus hirsutus and Chamaecytisus supinas as consistently different species, as well as the close relation between Cytisus villosus and the genus Chamaecytisus.

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 β-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).

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.

The chemical composition of the herbage produced by shrubs belonging to the genus Cytisus and allied genera (Cytisophyllum, Chamaecytisus, and Genista sect. Teline) was determined to appraise its potential value for forage. In their third year, eight accessions were assessed in a semi‐arid environment (400 mm annual rainfall) for seasonal variation of leaf/stem ratio, and contents of ashes, neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), and crude protein (CP). Fibre and crude protein concentration were similar to those of best‐quality shrubby legumes like Medicago sp. pi. Colutea sp. pi., or Chamaecytisus palmensis (ADF, 18–35%; CP, 14–24%). Genista monspessulana had low fibre and high protein contents, which were the most constant during the year. From the results obtained in this and the previous agronomic study, Part 1 of this series (Gonzalez‐Andres & Ortiz 1996), Genista monspessulana, Chamaecytisus podolicus, and C. supinus are recommended as potenti...

The chemical composition of the herbage produced by shrubs belonging to the genus Cytisus and allied genera (Cytisophyllum, Chamaecytisus, and Genista sect. Teline) was determined to appraise its potential value for forage. In their third year, eight accessions were assessed in a semi‐arid environment (400 mm annual rainfall) for seasonal variation of leaf/stem ratio, and contents of ashes, neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), and crude protein (CP). Fibre and crude protein concentration were similar to those of best‐quality shrubby legumes like Medicago sp. pi. Colutea sp. pi., or Chamaecytisus palmensis (ADF, 18–35%; CP, 14–24%). Genista monspessulana had low fibre and high protein contents, which were the most constant during the year. From the results obtained in this and the previous agronomic study, Part 1 of this series (Gonzalez‐Andres & Ortiz 1996), Genista monspessulana, Chamaecytisus podolicus, and C. supinus are recommended as potenti...

Twenty-four accessions belonging to the genus Cytisus and allied taxa were characterized by adult plant morphometry. Twenty-six characters were measured in flowers, 9 in leaves, and 5 in fruits. Two data sets were prepared, the first including only floral parameters and the second with all the parameters. Two different multivariate analyses were carried out for every data set: cluster analysis and principal components analysis. All these studies produced a similar grouping of the operational taxonomic units. Four clear groups were defined: (i) Cytisophyllum sessilifolium; (ii) Cytisus baeticus, C. reverchonii, C. scoparius., (iii) Chamaecytisus species; (iv) Genista species. On the other hand, Cytisus villosus showed an intermediate position between Cytisus and Chamaecytisus, and Cytisus heterochrous and C. purgans an intermediate position between Cytisus and Genista. This grouping agrees with that obtained by other recent seed morphometry and biochemical studies, and supports the generic arrangement presented by Bisby (1981).

SummaryThe number of recognized taxa within the genus Echinocloa Beauv. varies from one author to another. As the morphological variation is quite continuous, it is difficult to establish different taxa. In this work, caryopsis morphometry and isoenzymes were used as an aid for the discrimination of the eastern Spain species. The multivariate analysis of isoenzymes clearly defines three species: E. colonum, E. crus-galli and E. oryzicola and a group formed by E. hispidula and E. oryzoides in which the variability between populations of E. hispidula is higher than the variability between both taxa.

Twenty-five accessions belonging to the genus Cytisus and allies (Genisteae: Leguminosae), potentially useful for agrosylvopastoral systems in the Mediterranean basin, were analyzed using isoenzymes. The aim was to assess the taxonomic usefulness of these characters at the specific and generic levels, and its value as an aid for the taxonomy. A high level of infrageneric and infraspecific variability was detected. Numerical analysis showed that isoenzymes are useful for the characterization both at the specific and generic levels. The obtained dendrogram supports the generic arrangement proposed by Bisby [(1981) Advances in Legume Systematics , Vol. 1, pp. 409–425], therefore helping to elucidate the difficult taxonomy of the studied species.

Twenty-five accessions belonging to the genus Cytisus and allied taxa (Genisteae: Fabaceae) were analyzed using RAPD (Random Amplified Polymorphic DNA) techniques in order to study the usefulness of this technique for characterization of species and genera and for estimation of its potential value for further populational studies and prebreeding programs. A high level of intrapopulational variability was detected. Nevertheless, when only common bands to all the studied individuals per accession were considered for numerical analysis, the technique was useful for characterization at the specific and generic levels. The dendrogram obtained supports the generic arrangement proposed by Bisby (1981), helping to elucidate the difficult taxonomy of the studied taxa. The high intrapopulational variability indicates that RAPD could be useful for populational studies and also as molecular markers.