AMF communities associated to Vitis vinifera in an Italian vineyard subjected to integrated pest management at two different phenological stages.

Vitis vinifera L. is an economically important crop that can be influenced by soil microorganisms, including arbuscular mycorrhizal fungi (AMF), that establish symbiotic associations with its roots. AMF have beneficial effects on grapevine performance improving water use efficiency and replant success. Most grapevine varieties are susceptible to various diseases, and integrated pest management (IPM) is one of the emerging approaches to perform pest control. In the present study, we examined the AMF communities present in the soil associated to the roots of V. vinifera cv. Pinot Noir (comparing them to those present in a soil not affected by grapevine roots), in a vineyard subjected to IPM at two different phenological stages, using 454 Roche sequencing technology. We proposed a new approach to analyze sequencing data. Most of the taxa were included in the family Glomeraceae. In particular, Glomus sp. Rhizophagus sp. and Septoglomus viscosum were present. The family Archeosporaceae was represented only by the genus Archeospora sp. Different AMF communities were found in the two soils and the importance of the phenological stage in regulating AMF biodiversity was assessed.

Metaproteomic characterization of theVitis vinifera rhizosphere.

The rhizosphere is a hotspot of microbial activity where the release of root exudates stimulates bacterial density and diversity. The majority of the bacterial cells in soil are viable, unculturable, but active. Proteomic tools could be useful in gaining information about microbial community activity and to better understand the real interactions between roots and soil. The aim of this work was to characterize the bacterial community associated with Vitis vinifera cv. Pinot Noir roots using a metaproteome approach. Our results confirmed the large potential of proteomics in describing the environmental microbial communities and their activities: in particular, we showed that bacteria belonging to Streptomyces, Bacillus, Bradyrhizobium, Burkholderia and Pseudomonas genera are the most active in protein expression. Concerning the biological activity of these genera in the rhizosphere, we observed the exclusive presence of the phosphorus metabolic process and the regulation of primary metabolic processes. To our knowledge, this is the first study reporting the rhizosphere proteome of V. vinifera, describing the bacterial community structure and activity of an important ecosystem for the Italian landscape, agriculture and economy.

Antifungal activity of essential oils against azole-resistant and azole-susceptible vaginal Candida glabrata strains.

Candida glabrata is an opportunistic pathogen, associated with endocarditis, meningitis, and disseminated disease, and also with complicated vaginitis. Essential oils derived from aromatic plants are known in traditional medicine as antimicrobial agents and have antifungal properties. The aim of this work was to evaluate whether 12 tested essential oils (tea tree, laurel, anise, basil, bergamot, lavender, mint, oregano, grapefruit, rosemary, winter savory, and ginger) could have a transverse effect on C. glabrata sensitive strains but above all on strains resistant to the three main azole antifungals used (clotrimazole, fluconazole, itraconazole). For this reason, different strains of C. glabrata, vaginal isolated, were characterized (disk diffusion assay, minimal inhibitory concentration) with respect to their response to such antifungals. Electron microscopy analyses were performed to examine cellular damages in depth. Subsequently, we wanted to evaluate the effect of the oils on human cells to estimate their potential cytotoxicity. Oregano and winter savory were the two most effective essential oils, inducing growth inhibition, cell damage of C. glabrata strains (both sensitive and resistant to azole antifungal drugs), and medium-high level of toxicity against human keratinocytes. The results of this work support the research for new alternatives or complementary therapies against vaginal candidiasis.

Sensitivity of Candida albicans to essential oils: are they an alternative to antifungal agents?

AIMS: Candida albicans is an important opportunistic pathogen, responsible for the majority of yeast infections in humans. Essential oils, extracted from aromatic plants, are well-known antimicrobial agents, characterized by a broad spectrum of activities, including antifungal properties. The aim of this work was to assess the sensitivity of 30 different vaginal isolated strains of C. albicans to 12 essential oils, compared to the three main used drugs (clotrimazole, fluconazole and itraconazole). METHODS AND RESULTS: Thirty strains of C. albicans were isolated from vaginal swab on CHROMagar™ Candida. The agar disc diffusion method was employed to determine the sensitivity to the essential oils. The antifungal activity of the essential oils and antifungal drugs (clotrimazole, itraconazole and fluconazole) were investigated using a microdilution method. Transmission and scanning electron microscopy analyses were performed to get a deep inside on cellular damages. Mint, basil, lavender, tea tree oil, winter savory and oregano essential oils inhibited both the growth and the activity of C. albicans more efficiently than clotrimazole. Damages induced by essential oils at the cellular level were stronger than those caused by clotrimazole. CONCLUSIONS: Candida albicans is more sensitive to different essential oils compared to the main used drugs. Moreover, the essential oil affected mainly the cell wall and the membranes of the yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this work support the research for new alternatives or complementary therapies against vaginal candidiasis.

Formulation development of the deleterious rhizobacterium Pseudomonas trivialis X33d for biocontrol of brome (Bromus diandrus) in durum wheat.

AIM: To develop an appropriate formulation of the deleterious rhizobacterium Pseudomonas trivialis X33d and to evaluate its effectiveness to reduce brome growth. METHODS AND RESULTS: Two formulations of Ps. trivialis X33d, a semolina-kaolin granular formulation (Pesta) and talc-kaolin powder, were prepared and their effectiveness in reducing brome growth was evaluated. Both brome suppression and cell viability of X33d were higher in Pesta granular formulation than in talc-kaolin powder one. The impact of storage temperature and the addition of adjuvants (sucrose and oil) to the granular formulation of X33d were assessed in order to improve the shelf life of the formulation. The longest viability was found in formulated product supplemented with adjuvants and stored at 4°C. The effect of Pesta granules supplemented with adjuvants and stored for 6 months at 4°C on brome and wheat growth under controlled and greenhouse conditions was evaluated. The X33d formulation in Pesta increased the growth of wheat and reduced brome growth. CONCLUSION: Our results indicate that Ps. trivialis X33d formulated in Pesta has potential as a bioherbicide to control brome. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the impracticality of applying bacterial cell suspension on a large scale, the use of Pesta granules of X33d against brome could help in achieving a sustainable agriculture application of a bioherbicide.

Interactions between Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences for the growth of cucumber under salt-stress conditions.

AIMS: After the determination of the toxic but nonlethal concentration of NaCl for cucumber, we examined the interaction between an ACC (1-aminocyclopropane-1-carboxylate) deaminase producing bacterial strain and an arbuscular mycorrhizal fungus (AMF) and their effects on cucumber growth under salinity. METHODS AND RESULTS: In the first experiment, cucumber seedlings were exposed to 0.1, 50, 100 or 200 mmol l(-1) NaCl, and plant biomass and leaf area were measured. While seeds exposed to 200 mmol l(-1) NaCl did not germinate, plant growth and leaf size were reduced by 50 or 100 mmol l(-1) salt. The latter salt cancentration caused plant death in 1 month. In the second experiment, seeds were inoculated with the ACC deaminase-producing strain Pseudomonas putida UW4 (AcdS(+)), its mutant unable to produce the enzyme (AcdS(-)), or the AMF Gigaspora rosea BEG9, individually or in combination and exposed to 75 mmol l(-1) salt. Plant morphometric and root architectural parameters, mycorrhizal and bacterial colonization and the influence of each micro-organism on the photosynthetic efficiency were evaluated. The AcdS(+) strain or the AMF, inoculated alone, increased plant growth, affected root architecture and improved photosynthetic activity. Mycorrhizal colonization was inhibited by each bacterial strain. CONCLUSIONS: Salinity negatively affects cucumber growth and health, but root colonization by ACC deaminase-producing bacteria or arbuscular mycorrhizal fungi can improve plant tolerance to such stressful condition. SIGNIFICANCE AND IMPACT OF THE STUDY: Arbuscular mycorrhizal fungus and bacterial ACC deaminase may ameliorate plant growth under stressful conditions. It was previously shown that, under optimal growth conditions, Ps. putida UW4 AcdS(+) increases root colonization by Gi. rosea resulting in synergistic effects on cucumber growth. These results suggest that while in optimal conditions ACC deaminase is mainly involved in the bacteria/fungus interactions, while under stressful conditions this enzyme plays a role in plant/bacterium interactions. This finding is relevant from an ecological and an applicative point of view.

Microbiological polyphasic approach for soil health evaluation in an Italian polluted site.

The use of microorganisms as bioindicators of soil health is quite a new feature, rarely considered for the soil health evaluation in chronically-polluted industrial sites, and still suffering of the bias related to the technique applied. In this work we applied a microbiological polyphasic approach, relying on soil indigenous microorganisms as bioindicators and combining culture-dependent and -independent methods, in order to evaluate soil health of four sites (1a, 1b, 2a and 2b) inside a chemical factory with a centenary activity. Functional as well as structural aspects were comprehensively considered. Results were related to the kind of pollutants found in each site. Heavy metal pollution was recorded in sites 1b and 2b, while both organic and inorganic substances were detected in sites 1a and 2a. Based on the chemical and physical properties of the four soils, site 1b and 2b grouped together, while 1a and 2a were separated from the others. The density of the culturable bacteria was very low in site 2a, where only gram-positive were found. According to the identification of culturable bacteria, site 2a showed the lowest similarity with the other sites. Microbial activity was detected only in sites 1b and 2b. PCR-DGGE (Denaturing Gradient Gel Electrophoresis), was performed on the culturable, total and active microbial communities. Consistently with the identification of culturable bacterial strains, the molecular profile of the culturable fraction of site 2a, was clearly separated from the molecular profiles of other sites in cluster analysis. Molecular fingerprintings of the whole and active bacterial communities differed among the sites, but clustered according to the pollutants present in each site. The presence of possible key species in each site has been discussed according to the whole and active species. Since the results obtained by microbiological analysis are consistent with the chemical data, we suggest that the use of this microbiological polyphasic approach and of microorganisms as intrinsic bioindicators, can be suitable for the evaluation of soil health.

Identification of bacterial groups preferentially associated with mycorrhizal roots of Medicago truncatula.

The genetic structures of bacterial communities associated with Medicago truncatula Gaertn. cv. Jemalong line J5 (Myc+ Nod+) and its symbiosis-defective mutants TRV48 (Myc+ Nod-) and TRV25 (Myc- Nod-) were compared. Plants were cultivated in a fertile soil (Châteaurenard, France) and in soil from the Mediterranean basin showing a low fertility (Mas d'Imbert, France). Plant growth, root architecture, and the efficiency of root symbiosis of the three plant genotypes were characterized in the two soils. Structures of the bacterial communities were assessed by automated-ribosomal intergenic spacer analysis (A-RISA) fingerprinting from DNA extracted from the rhizosphere soil and root tissues. As expected, the TRV25 mutant did not develop endomycorrhizal symbiosis in any of the soils, whereas mycorrhization of line J5 and the TRV48 mutant occurred in both soils but at a higher intensity in the Mas d'Imbert (low fertility) than in the Châteaurenard soil. However, modifications of plant growth and root architecture, between mycorrhizal (J5 and TRV48) and nonmycorrhizal (TRV25) plants, were recorded only when cultivated in the Mas d'Imbert soil. Similarly, the genetic structures of bacterial communities associated with mycorrhizal and nonmycorrhizal plants differed significantly in the Mas d'Imbert soil but not in the Châteaurenard soil. Multivariate analysis of the patterns allowed the identification of molecular markers, explaining these differences, and markers were further sequenced. Molecular marker analysis allowed the delineation of 211 operational taxonomic units. Some of those belonging to the Comamonadaceae and Oxalobacteraceae (beta-Proteobacteria) families were found to be significantly more represented within bacterial communities associated with the J5 line and the TRV48 mutant than within those associated with the TRV25 mutant, indicating that these bacterial genera were preferentially associated with mycorrhizal roots in the Mas d'Imbert soil.

Dynamic of the genetic structure of bacterial and fungal communities at different developmental stages of Medicago truncatula Gaertn. cv. Jemalong line J5.

The genetic structure of bacterial and fungal communities was characterized in the rhizosphere of Medicago truncatula Gaertn. cv. Jemalong line J5 at five developmental stages (three vegetative and two reproductive stages), and in three compartments (bulk soil, rhizosphere soil and root tissues). The genetic structure of microbial communities was determined by cultivation-independent methods using directly extracted DNA that was characterized by automated ribosomal intergenic spacer analysis (ARISA). Principal component analyses (PCA) indicate that, for all developmental stages, the genetic structure of microbial communities differed significantly by compartment, with a major shift in the community in root tissues corresponding to the most intimate compartment with the plant. Differences were also recorded during plant development, the most significant being observed during the transition between vegetative and reproductive stages. Throughout this period, plants were shown to establish the highest level of symbiotic association (mycorrhization, nodulation) with arbuscular mycorrhizal fungi and Rhizobia. During the reproductive stages, the dynamics of the genetic structure differed between bacterial and fungal communities. At the last reproductive stage, the genetic structure of bacterial communities became close to that recorded during the first vegetative stages, suggesting a resilience phenomenon, whereas the genetic structure of fungal communities remained different from the vegetative stages and also from the early reproductive stages, suggesting a persistence of the rhizosphere effect.