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.     

Development of functional symbiotic white clover root hairs and nodules requires tightly regulated production of rhizobial cellulase CelC2

The establishment of rhizobia as nitrogen-fixing endosymbionts within legume root nodules requires the disruption of the plant cell wall to breach the host barrier at strategic infection sites in the root hair tip and at points of bacterial release from infection threads (IT) within the root cortex. We previously found that Rhizobium leguminosarum bv. trifolii uses its chromosomally encoded CelC2 cellulase to erode the noncrystalline wall at the apex of root hairs, thereby creating the primary portal of its entry into white clover roots. Here, we show that a recombinant derivative of R. leguminosarum bv. trifolii ANU843 that constitutively overproduces the CelC2 enzyme has increased competitiveness in occupying aberrant nodule-like root structures on clover that are inefficient in nitrogen fixation. This aberrant symbiotic phenotype involves an extensive uncontrolled degradation of the host cell walls restricted to the expected infection sites at tips of deformed root hairs and significantly enlarged infection droplets at termini of wider IT within the nodule infection zone. Furthermore, signs of elevated plant host defense as indicated by reactive oxygen species production in root tissues were more evident during infection by the recombinant strain than its wild-type parent. Our data further support the role of the rhizobial CelC2 cell wall-degrading enzyme in primary infection, and show evidence of its importance in secondary symbiotic infection and tight regulation of its production to establish an effective nitrogen-fixing root nodule symbiosis.     

Rhizobium promotes non-legumes growth and quality in several production steps: towards a biofertilization of edible raw vegetables healthy for humans

The biofertilization of crops with plant-growth-promoting microorganisms is currently considered as a healthy alternative to chemical fertilization. However, only microorganisms safe for humans can be used as biofertilizers, particularly in vegetables that are raw consumed, in order to avoid sanitary problems derived from the presence of pathogenic bacteria in the final products. In the present work we showed that Rhizobium strains colonize the roots of tomato and pepper plants promoting their growth in different production stages increasing yield and quality of seedlings and fruits. Our results confirmed those obtained in cereals and alimentary oil producing plants extending the number of non-legumes susceptible to be biofertilized with rhizobia to those whose fruits are raw consumed. This is a relevant conclusion since safety of rhizobia for human health has been demonstrated after several decades of legume inoculation ensuring that they are optimal bacteria for biofertilization.     

Cellulase isoenzyme profiles in Frankia strains belonging to different cross-inoculation groups

Carboxymethyl cellulase activities were evaluated in eight strains of Frankia from diverse host specificity groups and geographical origins. Cellulase activity was detected in culture supernatants of all strains in the absence of CMC (Carboxymethylcellulose) as an inducer using both double-layer plate and reducing sugar assays, indicating a constitutive production of CM-cellulases by Frankia. CM-cellulase isoenzyme profiles were visualized using activity gel electrophoresis of concentrated culture supernatants. Different electrophoretic profiles were observed among the eight strains tested, which correlate with the host specificity and taxonomic grouping of Frankia.     

Characterization of Rhizobial Isolates of Phaseolus vulgaris by Staircase Electrophoresis of Low-Molecular-Weight RNA

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli, Rhizobium gallicum, Rhizobium giardinii, Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii.     

Genomic fingerprinting of Frankia strains by PCR-based techniques. Assessment of a primer based on the sequence of 16S rRNA gene of Escherichia coli

Submergence stimulates elongation of the leaves of Rumex palustris and under laboratory conditions the maximum final leaf length (of plants up to 7 weeks old) was obtained within a 9 day period. This elongation response, mainly determined by petiole elongation, depends on the availability of storage compounds and developmental stage of a leaf. A starch accumulating tap root and mature leaves and petioles were found to supply elongating leaves with substrates for polysaccharide synthesis in expanding cell walls. Changes in the composition of cell wall polysaccharides of elongated petioles suggest a substantial cell wall metabolism during cell extension. Reduced starch levels or removal of mature leaves caused a substantial limitation of submerged leaf growth. From the 5th leaf onward enough reserves were available to perform submerged leaf growth from early developmental stages. Very young petioles had a limited capacity to elongate. In slightly older petioles submergence resulted in the longest final leaf lengths and these values gradually decreased when submergence was started at more mature developmental stages. Submerged leaf growth is mainly a matter of petiole elongation in which cell elongation has a concurrent synthesis of xylem elements in the vascular tissue. Mature petioles still elongated (when submerged) by cell and tissue elongation only: the annular tracheary elements stretched enabling up to 70% petiole elongation.     

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

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

Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth,constitute a new genomovar of Acinetobacter calcoaceticus

During a screening of phosphate solubilizing bacteria (PSB) in agricultural soils, two strains, IH9 and OCI1, were isolated from the rhizosphere of grasses in Spain, and they showed a high ability to solubilize phosphate in vitro. Inoculation experiments in chickpea and barley were conducted with both strains and the results demonstrated their ability to promote plant growth. The 16S rRNA gene sequences of these strains were nearly identical to each other and to those of Acinetobacter calcoaceticus DSM 30006^T, as well as the strain CIP 70.29 representing genomospecies 3. Their phenotypic characteristics also coincided with those of strains forming the A. calcoaceticus–baumannii complex. They differed from A. calcoaceticus in the utilization of l-tartrate as a carbon source and from genomospecies 3 in the use of d-asparagine as a carbon source. The 16S–23S intergenic spacer (ITS) sequences of the two isolates showed nearly 98% identities to those of A. calcoaceticus, confirming that they belong to this phylogenetic group. However, the isolates appeared as a separate branch from the A. calcoaceticus sequences, indicating their molecular separation from other A. calcoaceticus strains. The analysis of three housekeeping genes, recA, rpoD and gyrB, confirmed that IH9 and OCI1 form a distinct lineage within A. calcoaceticus. These results were congruent with those from DNA–DNA hybridization, indicating that strains IH9 and OCI1 constitute a new genomovar for which we propose the name A. calcoaceticus genomovar rhizosphaerae.     

Sea-level dynamics and palaeoecological factors affecting trace fossil distribution in Eocene turbiditic deposits (Gorrondatxe section,N Spain)

Ichnological analysis of the upper Ypresian–lower Lutetian interval at the Gorrondatxe section (W Pyrenees, N Spain), reveals a relationship between sea-level dynamics and the eco-sedimentary factors influencing trace fossil assemblages. The 600 m thick section of deep-sea turbiditic deposits contains 41 ichnospecies belonging to 28 ichnogenera, which are typical of the Nereites ichnofacies, and mostly of the Paleodictyon ichnosubfacies, suggesting deposition in a basin plain to fan-fringe setting. The trace fossil diversity and abundance fluctuate, irrespective of turbidite frequency. These ichnological features are strongly affected by trophic level changes related partly to sea-level dynamics according to the sequence stratigraphic interpretations for the studied section. Temperature, oxygenation and substrate changes are also considered as relevant factors. Increased ichnodiversity, particularly among graphoglyptids, coincides with moderate oligotrophy and stable ecological conditions. Eutrophisation, lowered oxygenation and drop of temperature, typical of low sea level, can reduce ichnodiversity.     

Rhizobia from Lanzarote,the Canary Islands,That Nodulate Phaseolus vulgaris Have Characteristics in Common with Sinorhizobium meliloti Isolates from Mainland Spain

The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and the group II LMW RNA S. meliloti isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of S. meliloti. The isolates from P. vulgaris nodulated bean but not Medicago sativa, while those recovered from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The bean isolates also were distinguished from those of Medicago, Melilotus, and Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean isolates were most similar to those reported for S. meliloti bv. mediterranense and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"DQ333891","term_id":"84872507","term_text":"DQ333891"}}DQ333891 and {"type":"entrez-nucleotide","attrs":{"text":"AF217267","term_id":"13810627","term_text":"AF217267"}}AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne transmission of Rhizobium etli from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.A remarkable attribute of Phaseolus vulgaris (common bean) is its ability to nodulate with rhizobia from at least 20 different legume genera (summarized in reference 1). Of particular relevance is the report by Ishizawa (16), who described P. vulgaris nodulation ranging from doubtful to good by 14 strains recovered from Medicago sativa, Medicago denticulata, and Melilotus alba, while nodulation of the latter three legumes by four bean strains was negative.At the time of the host range experiments, such as those described by Ishizawa (16), rhizobial nomenclature depended on the legume host of origin; the taxonomy of the strains was based on cross-inoculation groups. Consequently, no information was available about the genetic relationships among the rhizobial strains that originated from the different host legume genera and formed nodules on P. vulgaris. Eventually, rhizobial nomenclature based on the cross-inoculation groups was abandoned because of the many unexplainable and incongruous nodulation data (44). The cross-inoculation groups consisted of different rhizobial species within the single genus Rhizobium. Eventually, rhizobial taxonomy was expanded to several different genera based on estimates of their phylogeny (38). Phylogenies of bean-nodulating rhizobia were estimated from variations in the 16S rRNA gene sequence (39), even though subsequently it became clear that this method is significantly limited by histories of genetic exchange and recombination (6, 40). Most reported phylogenies of rhizobia nodulating P. vulgaris have placed them in the genus Rhizobium (3, 39), but several surveys with isolates from North Africa and Spain have demonstrated that rhizobia in the genus Sinorhizobium also nodulate this legume species (12, 23, 24, 25, 41), supporting the nodulation data originally published by Ishizawa (16). The number of isolates described as originating from nodules of P. vulgaris in the genus Sinorhizobium is small, and for the most part, from the published evidence, it has been suggested that they are affiliated with Sinorhizobium fredii. However, nodules of P. vulgaris growing in a single Tunisian soil where beans are cultivated yielded four isolates that, according to the data, appeared to support an affiliation with Sinorhizobium meliloti rather than S. fredii (25). Whether these four cultures were of the same rhizobial genotype constituting a single example of S. meliloti isolated from P. vulgaris is unknown.P. vulgaris was introduced into Europe as a crop plant as early as the 16th century (31) but never became a very important part of agriculture in Lanzarote, one of the Canary Islands that lie in the Atlantic Ocean to the west of the North African coast. Since there is no record of any nodulation studies with P. vulgaris cultivated on Lanzarote Island, the first objective of this study was to examine bean plants that had grown in Lanzarote soil for nodulation. Considering that the diversity of rhizobia able to nodulate bean plants is extremely wide, the second objective was to characterize the isolates originating from the nodules of plants grown in Lanzarote soil.(Part of this work was presented as a poster at the First International Meeting on Microbial Phosphate Solubilization, Salamanca, Spain, July 2002.)