Culturable bacterial diversity from the chestnut (Castanea sativa Mill.) phyllosphere and antagonism against the fungi causing the chestnut blight and ink diseases.

The phyllosphere supports a large and complex bacterial community that varies both across plant species and geographical locations. Phyllosphere bacteria can have important effects on plant health. The sweet chestnut (Castanea sativa Mill.) is an economically important tree species affected worldwide by the fungal pathogens Cryphonectria parasitica and Phytophthora cinnamomi. We examined the culturable phyllosphere bacterial community of the sweet chestnut at two nearby locations in Central Spain in order to know its geographical variability and to explore its potential as source of biological control agents against these two pathogenic fungi. The bacterial diversity at strain level was high but it varied significantly between locations; however, phylotype richness and diversity were more comparable. The isolates were affiliated with the phyla Actinobacteria, Firmicutes and Proteobacteria. Most of them were members of recognized bacterial species, with a notable proportion of representative of the genera Dietzia and Lonsdalea, but a small fraction of the strains revealed the existence of several potential novel species or even genera. Antagonism tests showed the occurrence in the chestnut phyllosphere of bacterial strains potentially useful as biological control agents against the two pathogenic fungi, some of which belong to species never before described as fungal antagonists. Chestnut phyllosphere, therefore, contains a great diversity of culturable bacteria and may represent an untapped source of potential biocontrol agents against the fungi causing blight and ink diseases of this tree species.

Herbaspirillum canariense sp. nov., Herbaspirillum aurantiacum sp. nov. and Herbaspirillum soli sp. nov., isolated from volcanic mountain soil, and emended description of the genus Herbaspirillum.

Three Gram-negative, motile and slightly curved rod-shaped bacteria, strains SUEMI03(T), SUEMI08(T) and SUEMI10(T), were isolated from an old volcanic mountain soil on Tenerife (Canary Islands). The three strains were related phylogenetically to Herbaspirillum seropedicae. 16S rRNA gene sequence similarity was 99.2-99.6 % among strains SUEMI03(T), SUEMI08(T) and SUEMI10(T), which presented 97.5, 97.8 and 97.7 % identity, respectively, with respect to H. seropedicae DSM 6445(T). The three strains grew optimally in TSB at 28 °C and contained summed features 3 (C(16:1)ω6c and/or C(16:1)ω7c) and 8 (C(18:1)ω6c and/or C(18:1)ω7c) and C(16:0) as major cellular fatty acids. The DNA G+C contents of strains SUEMI03(T), SUEMI08(T) and SUEMI10(T) were 61.6, 60.4 and 61.9 mol%, respectively. Strains SUEMI03(T), SUEMI08(T) and SUEMI10(T) presented less than 60 % interstrain DNA relatedness and less than 30 % relatedness with respect to H. seropedicae DSM 6445(T). In spite of their common geographical origin, the three strains isolated in this study presented several phenotypic differences, presenting phenotypic profiles highly divergent from that of H. seropedicae. Therefore, we propose that the strains isolated in this study represent three novel species of the genus Herbaspirillum, named Herbaspirillum canariense sp. nov. (type strain SUEMI03(T) = LMG 26151(T) = CECT 7838(T)), Herbaspirillum aurantiacum sp. nov. (type strain SUEMI08(T) = LMG 26150(T) = CECT 7839(T)) and Herbaspirillum soli sp. nov. (type strain SUEMI10(T) = LMG 26149(T) = CECT 7840(T)).

Diversity and community structure of culturable arsenic-resistant bacteria across a soil arsenic gradient at an abandoned tungsten-tin mining area.

We studied the bacterial diversity at a single location (the Terrubias mine; Salamanca province, Spain) with a gradient of soil As contamination to test if increasing levels of As would (1) change the preponderant groups of arsenic-resistant bacteria and (2) increase the tolerance thresholds to arsenite [As(III)] and arsenate [As(V)] of such bacteria. We studied the genetic and taxonomic diversity of culturable arsenic-resistant bacteria by PCR fingerprinting techniques and 16S rRNA gene sequencing. Then, the tolerance thresholds to As(III) and As(V) were determined for representative strains and mathematically analyzed to determine relationships between tolerances to As(III) and As(V), as well as these tolerances with the soil contamination level. The diversity of the bacterial community was, as expected, inversely related to the soil As content. The overall preponderant arsenic-resistant bacteria were Firmicutes (mainly Bacillus spp.) followed by γ-Proteobacteria (mainly Pseudomonas spp.), with increasing relative frequencies of the former as the soil arsenic concentration increased. Moreover, a strain of the species Rahnella aquatilis (γ-Proteobacteria class) exhibited strong endurance to arsenic, being described for the first time in literature such a phenotype within this bacterial species. Tolerances of the isolates to As(III) and As(V) were correlated but not with their origin (soil contamination level). Most of the strains (64%) showed relatively low tolerances to As(III) and As(V), but the second most numerous group of isolates (19%) showed increased tolerance to As(III) rather than to As(V), even though the As(V) anion is the prevalent arsenic species in soil solution at this location. To our knowledge, this is the first study to report a shift towards preponderance of Gram-positive bacteria (Firmicutes) related to high concentrations of soil arsenic. It was also shown that, under aerobic conditions, strains with relatively enhanced tolerance to As(III) predominated over the most As(V)-tolerant ones.

Over-expression of GTP-binding proteins and GTPase activity in mouse astrocyte membranes in response to Theiler's murine encephalomyelitis virus infection.

Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease that resembles human multiple sclerosis. In order to delineate the early events in this virus-induced neuroinflammatory disease, we have analyzed global GTPases gene activation following TMEV infection of murine brain astrocytes. DNA hybridization microchip analysis demonstrated that 10 sequences described as GTPbinding proteins and GTPases in different protein databases were over-expressed, in response to this infectious agent in astroglial cells. We have first characterized both the GTP-binding and GTPase activities in uninfected astrocyte membranes from a biochemical point of view. The increase in such activities was further validated in TMEV-infected astrocytes, peaking 2-4 h after infection. Over-expression is also induced by the inflammation-related chemokines interleukin-6 and interferon-gamma but not by interleukin-1alpha or tumor necrosis factor-alpha. From the many GTPases that could be over-expressed we have studied two, because of its biological significance; Ras p21 and the subunit alphai2 of G proteins. Western blots revealed increases in both proteins after infection with TMEV, in accordance with the previous enzymologic results. An increase in the active form of Ras (the GTP bound form) in cell lysates was also confirmed by affinity binding to a glutathione-S-transferase-fusion protein, following TMEV infection. A final demonstration of physiological up-regulation is provided by UV cross-linking of membrane proteins with the hydrolysis-resistant GTP agonist GTP [gamma-(35)S]. This technique allow us to detect, after SDS-PAGE, the increase of two further majoritary GTPbinding proteins with MW of 62 and 49 KDa. A quantitative analysis of four selected genes coding for p21 ras, Galphai2 subunit of protein G, Munc-18 and protein interacting with C kinase 1, was performed by real-time RT-PCR to verify the microarray results. The study of GTPase activity and of the above genes by RT-PCR in brains of sick mice, demonstrated a significative increase in mRNA coding for p21ras and protein interacting with C kinase 1 in vivo. Here we demonstrate that one of the mechanisms triggered by TMEV infection of astrocytes is the up-regulation of proteins related to GTP metabolism, one important signal transduction system in mammalian cells.