Marta Marcos-García

Plants Probiotics as a Tool to Produce Highly Functional Fruits: The Case of Phyllobacterium and Vitamin C in Strawberries

The increasing interest in the preservation of the environment and the health of consumers is changing production methods and food consumption habits. Functional foods are increasingly demanded by consumers because they contain bioactive compounds involved in health protection. In this sense biofertilization using plant probiotics is a reliable alternative to the use of chemical fertilizers, but there are few studies about the effects of plant probiotics on the yield of functional fruits and, especially, on the content of bioactive compounds. In the present work we reported that a strain of genus Phyllobacterium able to produce biofilms and to colonize strawberry roots is able to increase the yield and quality of strawberry plants. In addition, the fruits from plants inoculated with this strain have significantly higher content in vitamin C, one of the most interesting bioactive compounds in strawberries. Therefore the use of selected plant probiotics benefits the environment and human health without agronomical losses, allowing the production of highly functional foods.

The high diversity of Lotus corniculatus endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.

Rhizobium as plant probiotic for strawberry production under microcosm conditions

There is increasing interest in the use of plant growth-promoting rhizobacteria (PGPR) as environmental-friendly and healthy biofertilizers. Strawberries (Fragraria x ananassa) are mainly consumed fresh and hence any PGPRs used for biofertilization must be safe for humans, which is the case for members of the genus Rhizobium. In this study, the effects of inoculation of strawberry plants with Rhizobium sp. strain PEPV16, which belongs to the phylogenetic group of R. leguminosarum, and whose plant growth promotion ability has been reported previously for lettuce (Lactuca sativa) and carrots (Daucus carota), was examined. The results demonstrated that PEPV16 promotes strawberry growth through significant increases in the number of stolons, flowers and fruits as compared with uninoculated controls. Compared to uninoculated controls, the fruits of the inoculated plants had higher concentrations of Fe, Zn, Mn and Mo, and they also had higher concentrations of organic acids, such as citric and malic acid, and lower amounts of ascorbic acid than fruits. Although decreases in ascorbic acid have previously been described after the inoculation of strawberry with strains from different PGPR genera, this is the first study to report increases in organic acids after PGPR inoculation.

Mesorhizobium helmanticense sp. nov., isolated from Lotus corniculatus nodules

In this study, three strains belonging to the genus Mesorhizobium, CSLC115NT, CSLC19N and CSLC37N, isolated from Lotus corniculatus nodules in Spain, were characterized. Their 16S rRNA gene sequences were closely related to those of Mesorhizobium metallidurans STM 2683T, Mesorhizobium tianshanense A-1BST, Mesorhizobium tarimense CCBAU 83306T, Mesorhizobium gobiense CCBAU 83330T and Mesorhizobium caraganae CCBAU 11299T with similarity values higher than 99.7 %. The analysis of concatenated recA and glnII genes showed that the most closely related type strains were M. metallidurans STM 2683T, M. tianshanense A-1BST and M. tarimense CCBAU 83306T with 96, 95 and 94 % similarity values in the recA gene and 95, 94 and 94 % in the glnII gene, respectively. M. metallidurans LMG 24485T, M. tianshanense USDA 3592T and M. tarimense LMG 24338T showed means of 44, 41 and 42 % DNA-DNA relatedness, respectively, with respect to strain CSLC115NT. The major fatty acids were those from summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C16 : 0 and C18 : 1ω7c 11-methyl. The results of phenotypic characterization support that the L. corniculatus nodulating strains analysed in this work belong to a novel species of the genus Mesorhizobium for which the name Mesorhizobium helmanticense sp. nov. is proposed, and the type strain is CSLC115NT (= LMG 29734T=CECT 9168T).

Bacterial Probiotics: A Truly Green Revolution

Throughout history, the evolution and progress of all human civilizations have been closely linked to the evolution and development of agriculture, since this is the basis of food production to sustain population and ensure social stability.

Calcofluor white, an Alternative to Propidium Iodide for Plant Tissues Staining in Studies of Root Colonization by Fluorescent-tagged Rhizobia

Aims: To study multiple bacterial colonization In vitro, several limitations are obvious. One of these limitations is the plant autofluorescence generally between green and red fluorescence depending on the plant sections. The most important limitation is the bacterial fluorescence labelling, compromised by different kind of variables. Here we report the use of a secure stain, Calcofluor White, in rhizobial and other kind of beneficial bacteria colonization studies. Study Design: Root colonization assays were designed to confirm the stability of Calcofluor White stain (Sigma) in root cell walls. Place and Duration of Study: Every assay developed in this method article was performed using the technical resources at the Department of Microbiology and Genetics in the University of Salamanca (Spain) in 2013. Methodology: We have labelled rhizobia with two different kinds of fluorescent protein genes (gfp Method Article Flores-Felix et al.; JABB, 2(1): 65-70, 2015; Article no.JABB.2015.009 66 and rfp). We have co-inoculated Lactuca sativa and Daucus carota seedlings with two rhizobia: GFP-tagged Rhizobium sp. PEPV16 and RFP-tagged Mesorhizobium sp. CSLC01. Colonization assays were perfomed in several days post-inoculation, staining inoculated lettuce and carrot roots with Calcofluor White stain (Sigma). Samples were monitorized for several days, using a fluorescence microscope (NIKON Eclipse 80i). Results: Bacterial attachment to plant tissues is observed by fluorescence microscopy after their labelling with fluorescent proteins. Our results show how Calcofluor White staining for plant tissues improves bacterial visualization in contrast with tissues stained with propidium iodide, a carcinogenic agent that cannot be used when bacteria are tagged with red fluorescent proteins such as RFP or mCherry. Conclusion: Calcofluor white is a non-carcinogenic and low toxic compound that has been classically used to stain fungi and plant tissues for different uses. Due to its low wavelength, calcofluor white may be used in combination with several fluorophores. In the present work we showed that this compound is a reliable alternative to propidium iodide for plant tissues staining in multiple rhizobial/bacterial colonization studies.

Mesorhizobium bacterial strains isolated from the legume Lotus corniculatus are an alternative source for the production of polyhydroxyalkanoates (PHAs) to obtain bioplastics

Polyhydroxyalkanoic acids (PHAs) are natural polyesters that can be used to produce bioplastics which are biodegradable. Numerous microorganisms accumulate PHAs as energy reserves. Combinations of different PHAs monomers lead to the production of bioplastics with very different properties. In the present work, we show the capability of strains belonging to various phylogenetic lineages within the genus Mesorhizobium, isolated from Lotus corniculatus nodules, to produce different PHA monomers. Among our strains, we found the production of 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxydodecanoate, and 3-hydroxyhexadecanoate. Most of the PHA-positive strains were phylogenetically related to the species M. jarvisii. However, our findings suggest that the ability to produce different monomers forming PHAs is strain-dependent.

Erratum to: The high diversity of Lotus corniculatus endosymbionts in soils of northwest Spain

This paper was unfortunately published with error. The geographical origin of strain MAF 303099 was Japan, not New Zealand as was written in the text and in Figures 2, 3 and 4. The corrected paragraphs as well as the updated figures are shown below. “The strain CSLC22N representing RAPD pattern type V clustered with the type strain of M. erdmanii USDA 3471 (cluster III). This cluster also included strains isolated in Sweden (Ampomah and Huss-Danell 2011), Norway (Gossmann et al. 2012), Uruguay (Sotelo et al. 2011) and two strains nodulating L. corniculatus, R7A and MAFF 303099, isolated in New Zealand and Japan, respectively, and whose genome has been sequenced (Kelly et al. 2014; Kaneko et al. 2000). The strains isolated in Sweden are also misnamed as M. loti because the strain USDA 3471 was considered to be the type strain of this species before its reclassification as M. erdmanii (Martínez-Hidalgo et al. 2015).” “The strain N3 isolated in Uruguay (Sotelo et al. 2011) formed an independent lineage related to the cluster II, which also includes several type strains of Mesorhizobium species nodulating legumes other than Lotus. The cluster III contains the strain CSLC22N isolated in this study and several strains isolated from L. corniculatus nodules in Sweden (Ampomah and Huss-Danell 2011), the strain N33 in Uruguay (Sotelo et al. 2011) and the strains MAFF303099 and R7A isolated in Japan and New Zealand, respectively (Kaneko et al. 2000; Kelly et al. 2014). Moreover, this cluster includes the type strain M. erdmanii USDA 3471 isolated from L. corniculatus nodules.” “The analysis of the atpD gene showed that our strains are divided into four clusters with some differences in their distribution with respect to that found after recA gene analysis (Figure 4). The strains from 16S rRNA gene cluster I, have different atpD gene sequences being particularly divergent in the atpD gene of the strain CSLC14N. This strain formed an independent lineage related to the type strain ofM. caraganae CCBAU 11299with 92.5 % similarity. The remaining strains were phylogenetically related to the type strains of two species nodulating L. corniculatus, M. jarvisii ATCC 33669 and M. erdmanii USDA 3471. The phylogenetic lineage formed by the strain CSLC14N belongs to a cluster that also included two strains, S1302 and S789 isolated from L. corniculatus nodules in Uruguay (Sotelo et al. 2011). The strains CSLC01N, CSLC36N and CSLC28N were equidistant between M. jarvisii ATCC 33669 and M. erdmanii USDA 3471 (similarity higher than 98.5 % in all cases) (Table 1). This makes difficult the identification of these strains as M. jarvisii, as suggested the recA gene analysis. The strain CSLC42N was also related to the type strain of M. jarvisii ATCC 33669 with 98.3 % similarity, but in this case M. erdmanii USDA 3471 was less closely related, with 96.7 % similarity (Table 1). This atpD gene cluster, also contains the strain CSLC22N from 16S rRNA gene cluster III, which present 97.8 % similarity with respect to its closest The online version of the original article can be found at http://dx.doi.org/ 10.1007/s13199-015-0368-5

Rhizobium Symbiotic Enzyme Cellulase CelC2: Properties and Applications

Abstract Rhizobium leguminosarum bv. trifolii CelC2 cellulase is a 1,4-β- d -endoglucanase involved in the hydrolysis and biosynthesis of rhizobial cellulose. These special features make this enzyme essential in the plant infection process and important in root surface colonization and biofilm formation. A loss-of-function of the celC gene causes an increase in the production of external cellulose microfibrils. However, a gain-of-function derivative leads to an enhancement of rhizobial competitiveness, increasing the number of rhizobial cells within the plant. In this chapter, we review the properties and prospective applications of CelC2 cellulase, which reveals itself as a symbiotic enzyme with a high biotechnological potential.