Assessment of chitosan antimicrobial effect on wine microbes.

Chitosan is an active highly charged polysaccharide that has initially been developed in oenology to eliminate the spoilage yeast B. bruxellensis. However, different forms of chitosan exist, some complying with EU regulation for their use in wines, others not. Moreover, with the trend in oenology of limiting SO2, more and more questions arise as to the impact of chitosan on other microorganisms of the grape and wine environment. We investigated the antimicrobial efficiency of chitosan on a large oenological microbial collection, englobing technological as well as spoilage microorganisms. Results show that most species are affected at least transiently. Furthermore, a high variability prevails within most species and sensitive, intermediate and tolerant strains can be observed. This study also highlights different efficiencies depending on the wine parameters or the winemaking stage, giving important indications on which winemaking issues can be solved using chitosan. Chitosan treatment does not seem to be appropriate to limit the musts microbial pressure and Saccharomyces cerevisiae cannot be stopped during alcoholic fermentation, especially in sweet wines. Likewise, acetic acid bacteria are poorly impacted by chitosan. After alcoholic fermentation, chitosan can efficiently limit non-Saccharomyces yeast and lactic acid bacteria but special care should be given as to whether malolactic fermentation is wanted or not. Indeed, O. oeni can be severely impacted by chitosan, even months after treatment. Finally, this study highlights the crucial importance of the chitosan type used in its efficiency towards microbial stabilization. While a high molecular weight chitosan has limited antimicrobial properties, a chitosan with a much lower one, complying with EU and OIV regulation and specifications for its use in wine is much more efficient.

Draft Genome Resources of Two Strains of Xylella fastidiosa XYL1732/17 and XYL2055/17 Isolated from Mallorca Vineyards.

Xylella fastidiosa is a plant-pathogenic bacterium that causes serious diseases in many crops of economic importance and is a quarantine organism in the European Union. This study reports a de novo-assembled draft genome sequence of the first isolates causing Pierce's disease in Europe: X. fastidiosa subsp. fastidiosa strains XYL1732/17 and XYL2055/17. Both strains were isolated from grapevines (Vitis vinifera) showing Pierce's disease symptoms at two different locations in Mallorca, Spain. The XYL1732/17 genome is 2,444,109 bp long, with a G+C content of 51.5%; it contains 2,359 open reading frames and 48 tRNA genes. The XYL2055/17 genome is 2,456,780 bp long, with a G+C content of 51.5%; it contains 2,384 open reading frames and 48 tRNA genes.

Assessment of the impact of PS3-induced resistance to downy mildew on grapevine physiology.

Elicitor-induced resistance against diseases is an attractive strategy that could contribute to reduce the use of fungicides for plant protection. However, activation of defenses has an energetic cost that plants have to fuel by a mobilization of their primary metabolism with possible adverse effect on their physiology. In this context, this study was performed to determine whether elicitor-induced resistance of grapevine leaves against downy mildew impacted its development and metabolism. The elicitor PS3 (sulfated ß-glucan laminarin) was sprayed on grapevine herbaceous cuttings grown in greenhouses once or three times, and its impact was studied on young and older grapevine leaves, prior to, and after Plasmopara viticola inoculation. PS3 did not affect grapevine development during the time course of the experiment. A metabolomic analysis, mainly focused on primary metabolites, highlighted a leaf age dependent effect of PS3 treatment. Nitrogen compounds, and sugars to a lesser extent, were impacted. The results obtained complete the current knowledge of the impact of elicitor-induced resistance on plant physiology. They will be helpful to guide further experiments required to better determine the costs and benefits of elicitor-induced resistance in plants.

Assessment of Erysiphe necator Ascospore Release Models for Use in the Mediterranean Climate of Western Oregon.

Predictive models have been developed in several major grape-growing regions to correlate environmental conditions to Erysiphe necator ascospore release; however, these models may not be broadly applicable in regions with different climatic conditions. To assess ascospore release in near-coastal regions of western Oregon, chasmothecia (syn. cleistothecia) were collected prior to leaf drop and placed onto natural and artificial grape trunk segments and overwintered outside. Ascospore release was monitored for three overwintering seasons using custom impaction spore traps from leaf drop (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie [BBCH] 97) until the onset of the disease epidemic in the following growing season. Airborne inoculum was concurrently monitored in a naturally infested research vineyard. Weather and ascospore release data were used to assess previously developed models and correlate environmental conditions to ascospore release. Ascospore release was predicted by all models prior to bud break (BBCH 08), and was observed from the first rain event following the start of inoculum monitoring until monitoring ceased. Previously developed models overpredicted ascospore release in the Willamette Valley and predicted exhaustion of inoculum prior to bud break. The magnitude of ascospore release could not be correlated to environmental conditions; thus, a binary ascospore release model was developed where release is a function of the collective occurrence of the following factors within a 24-h period: >6 h of cumulative leaf wetness during temperatures >4°C, precipitation >2.5 mm, and relative humidity >80%. The Oregon model was validated using field-collected ascospore datasets, and predicted ascospore release with 66% accuracy (P = 0.02). Extant methods for estimating ascospore release may not be sufficiently accurate to use as predictive models in wet, temperate climatic regions.

Exposure risk assessment to ochratoxin A through consumption of juice and wine considering the effect of steam extraction time and vinification stages.

The goals of this study were (i) to verify the effect of steam extraction used in juice production and the stages of vinification on the ochratoxin A (OTA) levels found in grapes naturally contaminated, and (ii) evaluate the risk of exposure to this toxin when the daily consumption of juice and wine is followed to prevent cardiovascular disease. OTA-producing fungi were isolated from Cabernet Sauvignon, Moscato Itálico and Concord grapes harvested from the same vineyard and intended to produce red wine, white wine and juice, respectively. The highest levels of this toxin were found in the Concord grapes used for juice production. Although greater reduction in OTA levels occurred during juice production (73%) compared to winemaking (66 and 44%, for red and white, respectively), the estimated OTA exposure through juice was higher than the tolerable intake established for this toxin by JECFA. The risk associated with juice consumption, rather than wine, can be explained by (i) higher OTA levels found in Concord must than those of Cabernet and Moscato, indicating that Concord grapes appear to be more susceptible to OTA production by toxigenic fungi; and (ii) the daily recommended juice consumption is higher than those proposed to red wine.

Evaluation of a decision support strategy for the control of powdery mildew, Erysiphe necator (Schw.) Burr., in grapevine in the central region of Chile.

BACKGROUND: The primary strategy to control powdery mildew in Chilean vineyards involves periodic fungicide spraying, which may lead to many environmental and human health risks. This study aimed to implement and evaluate the effectiveness and economic feasibility of a novel decision support strategy (DSS) to limit the number of treatments against this pathogen. An experiment was conducted between the 2010 and 2013 seasons in two irrigated vine fields, one containing a cultivar of Cabernet Sauvignon (CS) and the other a cultivar of Chardonnay (CH). RESULTS: The results showed that the DSS effectively controlled powdery mildew in CS and CH vine fields, as evidenced by a disease severity lower than 3%, which was lower than that observed in untreated vines (approximately 10 and 40% for CS and CH respectively). The DS strategy required the application of only 2-3 fungicide treatments per season in key vine phenological stages, and the cost fluctuated between $US 322 and 415 ha-1 , which was 40-60% cheaper than the traditional strategy employed by vine growers. CONCLUSION: The decision support strategy evaluated in this trial allows a good control of powdery mildew for various types of epidemic with an early and late initiation. © 2017 Society of Chemical Industry.

Dermal exposure and risk assessment of tebuconazole applicators in vineyards.

INTRODUCTION: Models used in the pre-marketing evaluation do not cover all work scenarios and may over- or underestimate exposure. OBJECTIVES: Uncertainties present in the extrapolation from pre-marketing to the post-marketing warrant exposure and risk assessment in real-life working conditions. METHODS: Seven vineyard pesticide applicators were followed for a total of 12 work-days. A data collection sheet was developed specifically for this study. Workers' body exposure, hands, and head exposure were measured. Tebuconazole was analyzed using LC-MS/MS. RESULTS: Median potential and actual body exposures were 22.41 mg/kg and 0.49 mg/kg of active substance applied, respectively. The median protection factor provided by the coverall was 98% (range: 90-99%). Hand exposure was responsible for 61% of total actual exposure, and was reduced by more than 50% in workers using gloves. The German Model underestimated the exposure in one work-day, and grossly overestimated it in 3 work-days. CONCLUSIONS: High levels of potential body exposure were efficiently controlled by the cotton coverall. Use of personal protective devices, especially chemically-resistant gloves and head cover is the main determinant of skin protection. Field studies on pesticide exposure in real-life conditions and development of methods and tools for easier risk assessment are necessary to complement and confirm the risk assessment done in the authorization process.

Efficiency of Artemisia nilagirica (Clarke) Pamp. essential oil as a mycotoxicant against postharvest mycobiota of table grapes.

BACKGROUND: In order to get a potent botanical fungicide for the management of fungal decay of table grapes, an experiment was conducted in which 20 essential oils of higher plants were screened at 0.33 µL mL(-1) against dominant fungi causing decay of table grapes, including Aspergillus flavus, A. niger and A. ochraceus. Furthermore, the minimum inhibitory/fungicidal concentration, fungitoxic spectrum and mycotoxin inhibition activity of the most potent oil were determined. The efficacy of the most potent oil in preservation of table grapes, along with organoleptic evaluation, was also carried out by storing 1 kg of grapes in the oil vapour. RESULTS: Artemisia nilagirica oil was found to be most toxic, exhibiting 100% mycelia inhibition of all test fungi. Moreover, 0.29 µL mL(-1) A. nilagirica oil was fungistatic and 0.58 µL mL(-1) was fungicidal for all tested species of Aspergillus. The oil exhibited a broad range of fungitoxicity against other grape berry-rotting fungi. Artemisia nilagirica oil completely suppressed the growth and mycotoxin (AFB1 and OTA) secretion of aflatoxigenic and ochratoxigenic strains of Aspergillus at 1.6 µL mL(-1) . During the in vivo experiment, fumigation of 1 kg of table grapes with 200 and 300 µL dosage of A. nilagirica oil enhanced the shelf life for up to 9 days. The oil did not show any phytotoxic effect. Besides, oil application did not substantively change the sensory properties of the fruits. CONCLUSION: Artemisia nilagirica oil can be used as an alternative botanical fungicide for the control of fruit-rotting fungi of stored grapes.

Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis vinifera.

Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies.

Penicillium strains isolated from Slovak grape berries taxonomy assessment by secondary metabolite profile.

The secondary metabolite profiles of microfungi of the genus Penicillium isolated from samples of grape berries collected in two different phases during two vegetative seasons in Slovakia is described to assess the taxonomy. Three Slovak vine regions have been selected for this study, based on their climatic differences and national economic importance. Cultures of microfungi isolated from berries were incubated on different selective media for macro and micromorphology identification. The species Penicillium brevicompactum, Penicillium crustosum, Penicillium chrysogenum, Penicillium expansum, Penicillium palitans and Penicillium polonicum were identified according to growth and morphology. The related strains were found to produce a broad spectrum of fungal metabolites, including roquefortine C, chaetoglobosin A, penitrem A, cyclopeptin, cyclopenin, viridicatin, methylviridicatin, verrucofortine, secalonic acid D, cyclopiazonic acid, fumigaclavine and mycophenolic acid. Chemotaxonomy was performed using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Dried grape berries were also analyzed allowing to assess the presence of patulin, roquefortine C and penicillic acid; this last one has been identified in dried berries but not in vitro.

Assessment of Cymbopogon citratus (DC.) stapf essential oil as herbal preservatives based on antifungal, antiaflatoxin, and antiochratoxin activities and in vivo efficacy during storage.

Thirty-five randomly collected samples of stored table grapes (Vitis vinifera L.) from different markets of Gorakhpur city, Uttar Pradesh, India, revealed occurrence of 11 types of fungi. Of which, Aspergillus flavus, Aspergillus niger, and Aspergillus ochraceus were dominant causing severe decay of grapes with 58%, 52%, and 67% incidence, respectively. On screening of 15 essential oils at 0.33 µL/mL, Cymbopogon citratus oil caused 100% mycelial inhibition against aforesaid dominant fungi. Oil was fungistatic at 0.29 µL/mL and exhibited broad fungitoxicity against other fruit rotting fungi associated with collected samples. C. citratus oil completely inhibited the growth and mycotoxin (AFB1 and OTA) secretion of the aflatoxigenic and ochratoxigenic strains of A. flavus, A. niger, and A. ochraceus at 0.8 µL/mL. E-Citral (52.9%) and Z-Citral (39.38%) were the major components of C. citratus oil during gas chromatography and gas chromatography-mass spectrometry analysis. Application of 200 and 300 µL of C. citratus oil on 1 kg of stored grapes showed enhancement of shelf life up to 10 d. The oil did not exhibit any phytotoxic effect on fruits. These results confirm that C. citratus oil could be a natural alternative to commercial fungicide for control of fruit rotting fungi of stored grapes.

A novel low-cost sensor prototype for monitoring temperature during wine fermentation in tanks.

This paper presents a multipurpose and low cost sensor for temperature control over the wine fermentation process, in order to steadily communicate data through wireless modules in real time to a viticulturist's mobile or fixed device. The advantage of our prototype is due to the fact that it will be used by small winemakers in the "Ribera del Duero" area, and as it is a cheaper sensor and easy to use for the control and monitoring of the grape fermentation process, it will probably be used by other business men with the same necessities in the region. The microcontroller MSP430G2553 is among the components that make up the sensor, that are integrated onto a motherboard. It communicates with the RN-42 Bluetooth module through an UART interface. After verifying that all elements are working correctly, the parts are assembled to form the final prototype. This device has been tested in a winery in the region, fulfilling the initial project specifications.

Stilbene levels and antioxidant activity of Vranec and Merlot wines from Macedonia: effect of variety and enological practices.

The content of the stilbenes trans-resveratrol and piceid as well as the antioxidant activity of Macedonian red wines from the two main grape varieties Vranec and Merlot have been evaluated. Тhe effects of time of maceration, type of yeast and the level of sulphur dioxide applied on stilbene content and antioxidant activity have been studied. The most important factor in winemaking technology is the maceration time since the highest concentrations of trans-resveratrol, piceid and highest antioxidant activity were found following 6 and 10 days of maceration. Concerning the yeast type, higher concentrations of trans-resveratrol and piceid have been obtained with French yeast "Levuline CHP" in comparison to Macedonian yeast "Vinalco". In contrast, the higher antioxidant activity of wines from both varieties of grapes was observed by application of Macedonian yeast "Vinalco".

Strong resistance to the fungicide fenhexamid entails a fitness cost in Botrytis cinerea, as shown by comparisons of isogenic strains.

BACKGROUND: Fenhexamid, a sterol biosynthesis inhibitor effective against Botrytis, inhibits the 3-ketoreductase (Erg27) involved in C-4 demethylation. Several fenhexamid-resistant phenotypes have been detected in Botrytis cinerea populations from French vineyards. The field isolates with the highest resistance levels display amino acid changes in Erg27 (F412S, F412I or F412V). RESULTS: Fenhexamid-resistant mutants were generated by site-directed mutagenesis of the erg27 gene in a sensitive recipient strain to overcome the impact of different genetic backgrounds. The wild-type erg27 allele was replaced by the three mutated alleles (erg27(F412S/I/V)) by homologous recombination. These isogenic strains were shown to be fenhexamid-resistant and were used to quantify the impact of F412 mutations on fungal fitness. Several parameters, including radial growth, the production of sclerotia and conidia, freezing resistance and aggressiveness, were quantified in laboratory conditions. Analysis of variance demonstrated significant differences between the mutant and parental strains for some characters. In particular, the mutants grew more slowly than the wild-type strain and displayed variations in the production of sclerotia and conidia with temperature and susceptibility to freezing. CONCLUSIONS: The results highlight a moderate but significant impact of F412 mutations on the survival capacity of B. cinerea strains displaying high levels of resistance to fenhexamid in laboratory conditions, potentially limiting their dispersal and persistence, particularly in terms of overwintering, in field conditions.

Production of the phytoalexins trans-resveratrol and delta-viniferin in two economy-relevant grape cultivars upon infection with Botrytis cinerea in field conditions.

Leaves, shoots and flowers from two different economy-relevant grape cultivars, Merlot and Cabernet Sauvignon, were examined to assess the distribution of phytoalexins upon inoculation with Botrytis cinerea at pre-bloom, bloom, and post-bloom stages. Mass spectrometric analysis evidenced considerable levels of trans-resveratrol (3,5,4'-trihydroxystilbene), albeit higher in Cabernet Sauvignon, in leaves from both grape cultivars following fungal infection at all the examined stages of development. Although both these cultivars are reported to be sensitive against fungal infections, in Cabernet Sauvignon leaves and flowers, we were also able to measure relevant quantities of the resveratrol dehydrodimer delta-viniferin. While infection by B. cinerea occurs at bloom stage, high-sensitivity of the HPLC-mass spectrometric analytic method allowed detecting measurable levels of viniferins even in early pre-bloom stages in Cabernet Sauvignon flowers and to evidence even slight resveratrol differences between the cultivars. Concordingly, Cabernet Sauvignon better responded to fungal infection. This analysis allowed us to conclude that, even when analyzing fungal infection-sensitive cultivars, the HPLC-MS method holds the sensitivity to highlight the slightest differences in the concentrations of the two phytoalexins and correlate them to different anti-fungal response potential.

Assessment of fungicide resistance and pathogen diversity in Erysiphe necator using quantitative real-time PCR assays.

BACKGROUND: Management of grapevine powdery mildew Erysiphe necator Schw. requires fungicide treatments such as sterol demethylation inhibitors (DMIs) or mitochondrial inhibitors (QoIs). Recently, reduction in the efficacy of DMIs or QoIs was reported in Europe and the United States. The aim of the present study was to develop real-time qPCR tools to detect and quantify several CYP51 gene variants of E. necator: (i) A versus B groups (G37A) and (ii) sensitive versus resistant to sterol demethylase inhibitor fungicides (Y136F). RESULTS: The efficacy of the qPCR tools developed was better than the CAPS method, with a limit of 2 pg for E necator DNA, 0.06 ng for genetic group A and 1.4 ng for the DMI-resistant allele. The detection limits of qPCR protocols (LOD) ranged from 0.72 to 0.85%, and the quantification limits (LOQ) ranged from 2.4 to 2.85% for the two alleles G47A and Y136F respectively. The application of qPCR to field isolates from French vineyards showed the presence of DMI-resistant and/or QoI-resistant alleles in French pathogen populations, linked to genetic group B. CONCLUSION: The real-time PCR assay developed in this study provides a potentially useful tool for efficient quantification of different alleles of interest for fungicide monitoring and for population structure of E. necator.

Assessment of the process of movement of Xylella fastidiosa within susceptible and resistant grape cultivars.

To better understand the processes contributing to symptoms and resistance to Pierce's disease of grape, we examined the movement and multiplication of a green fluorescent protein-marked strain of Xylella fastidiosa in the stems and petioles of Cabernet Sauvignon, Chenin Blanc, Roucaneuf, and Tampa grape cultivars that differ in their susceptibility to this disease. X. fastidiosa achieved much lower population sizes and colonized fewer xylem vessels in the stem of resistant cultivars compared with more susceptible cultivars. In contrast, X. fastidiosa achieved similarly high population sizes and colonized a similar proportion of the vessels in petioles of susceptible and resistant cultivars, suggesting that, compared with the stem, X. fastidiosa is relatively unrestricted in its movement and growth within the petiole. There was not a direct relationship between the population size of X. fastidiosa in the stem and the proportion of vessels colonized; a much higher population size of the pathogen was observed in susceptible cultivars than expected based on the proportion of vessels colonized. The high population sizes of X. fastidiosa in stems of susceptible genotypes were associated with both a high number of infected vessels and a much higher extent of colonization of those vessels that become infested than in more resistant cultivars. The formation of large cellular aggregates in vessels is not required for X. fastidiosa to move laterally in the stem to adjacent vessels because most vessels harbored only small assemblages, especially in resistant cultivars such as Roucaneuf, in which some intervessel movement was detected. Resistance to Pierce's disease is apparently not due to inhibitory compounds that circulate in the xylem because they might be expected to operate similarly in all tissues.

A low-cost procedure for production of fresh autochthonous wine yeast.

A low-cost procedure was designed for easy and rapid response-on-demand production of fresh wine yeast for local wine-making. The pilot plant produced fresh yeast culture concentrate with good microbial quality and excellent oenological properties from four selected wine yeasts. The best production yields were obtained using 2% sugar beet molasses and a working culture volume of less than 60% of the fermenter capacity. The yeast yield using 2% sugar grape juice was low and had poor cell viability after freeze storage, although the resulting yeast would be directly available for use in the winery. The performance of these yeasts in commercial wineries was excellent; they dominated must fermentation and improved its kinetics, as well as improving the physicochemical parameters and the organoleptic quality of red and white wines.

Natural products alone or with copper vs. grape downy mildew: efficacy, costs, Cu impact.

In organic farming copper is one of the few fungicides that can be used and the only effective against downy mildew. Due to the problems of environmental impact associated with the use of this heavy metal, the European Union decided to restrict its use (Annex II Regulation EC n. 889/2008). In order to assess the possibility of replacing the copper or reduce the quantities used, we tested natural products and low rate copper formulations in field trials. The anti-downy mildew activity of these formulations has been studied in an organic vineyard. During the two years of activity we also estimated the economic cost of use of the products to assess their possible use in agricultural practice.

Predictive assessment of ochratoxin A accumulation in grape juice based-medium by Aspergillus carbonarius using neural networks.

AIMS: To study the ability of multi-layer perceptron artificial neural networks (MLP-ANN) and radial-basis function networks (RBFNs) to predict ochratoxin A (OTA) concentration over time in grape-based cultures of Aspergillus carbonarius under different conditions of temperature, water activity (a(w)) and sub-inhibitory doses of the fungicide carbendazim. METHODS AND RESULTS: A strain of A. carbonarius was cultured in a red grape juice-based medium. The input variables to the network were temperature (20-28 degrees C), a(w) (0.94-0.98), carbendazim level (0-450 ng ml(-1)) and time (3-15 days after the lag phase). The output of the ANNs was OTA level determined by liquid chromatography. Three algorithms were comparatively tested for MLP. The lowest error was obtained by MLP without validation. Performance decreased when hold-out validation was accomplished but the risk of over-fitting is also lower. The best MLP architecture was determined. RBFNs provided similar performances but a substantially higher number of hidden nodes were needed. CONCLUSIONS: ANNs are useful to predict OTA level in grape juice cultures of A. carbonarius over a range of a(w), temperature and carbendazim doses. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a pioneering study on the application of ANNs to forecast OTA accumulation in food based substrates. These models can be similarly applied to other mycotoxins and fungal species.