Rapid In-Field Volatile Sampling for Detection of Botrytis cinerea Infection in Wine Grapes.

Fungal infection of grape berries (Vitis vinifera) by Botrytis cinerea frequently coincides with harvest, impacting both the yield and quality of grape and wine products. A rapid and non-destructive method for identifying B. cinerea infection in grapes at an early stage prior to harvest is critical to manage loss. In this study, zeolitic imidazolate framework-8 (ZIF-8) crystal was applied as an absorbent material for volatile extraction from B. cinerea infected and healthy grapes in a vineyard, followed by thermal desorption gas chromatography-mass spectrometry. The performance of ZIF-8 in regard to absorbing and trapping the targeted volatiles was evaluated with a standard solution of compounds and with a whole bunch of grapes enclosed in a glass container to maintain standard sampling conditions. The results from the sampling methods were then correlated to B. cinerea infection in grapes, as measured and determined by genus-specific antigen quantification. Trace levels of targeted compounds reported as markers of grape B. cinerea infection were successfully detected with in-field sampling. The peak area counts for volatiles 3-octanone, 1-octen-3-one, 3-octanol, and 1-octen-3-ol extracted using ZIF-8 were significantly higher than values achieved using Tenax®-TA from field testing and demonstrated good correlation with B. cinerea infection severities determined by B. cinerea antigen detection.

Detection and prediction of Botrytis cinerea infection levels in wine grapes using volatile analysis.

Infection of grape berries (Vitis vinifera) by the fungus Botrytis cinerea (grey mould) frequently occurs in vineyards, resulting in off-flavours and other odours in wine and potential yield losses. In this study, volatile profiles of four naturally infected grape cultivars, and laboratory-infected grapes were analysed to identify potential markers for B. cinerea infection. Selected volatile organic compounds (VOCs) were highly correlated with two independent measures of B. cinerea infection levels, demonstrating that ergosterol measurements provide accurate quantification of lab-inoculated samples, while B. cinerea antigen detection is more suitable for naturally infected grapes. Excellent predictive models of infection level were confirmed (Q2Y of 0.784-0.959) using selected VOCs. A time course experiment confirmed that selected VOCs 1,5-dimethyltetralin, 1,5-dimethylnaphthalene, phenylethyl alcohol and 3-octanol are good markers for B. cinerea quantification and 2-octen-1-ol could be considered as an early marker of the infection.

Elucidating the interaction of carbon, nitrogen, and temperature on the biosynthesis of Aureobasidium pullulans antifungal volatiles.

The combined biochemical impact of carbon, nitrogen and temperature on the biosynthesis of the antifungal volatile organic compounds (VOCs): ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol produced by Aureobasidium pullulans A1 and A3 was investigated using a Box-Behnken experimental design and response surface methodology (RSM). Normalized peak areas derived from solid phase micro extraction-gas chromatography-mass spectrometry (SPME-GC-MS) analysis, indicated that initial carbon content had a significant influence on the biosynthesis of ethanol and alcohols with greater than three carbon atoms. This result suggests a dominant activity of the A. pullulans anabolic pathway to biosynthesize three higher alcohols via de novo biosynthesis of amino acids from sugar metabolism. Low concentrations of carbon (3-13 g l-1 ) with nitrogen as both ammonium and amino acids in the growth medium resulted in a higher number of significant linear and quadratic relationships. Nitrogen availability and growth temperature had significant negative linear and quadratic correlations with VOCs biosynthesis in most instances. Isolate-dependant metabolic response was evident for all abiotic parameters tested on alcohol production. The findings of this study offer new perspectives to improve the production of key antifungal compounds by antagonists in biological control systems.

Volatile organic compounds produced by Aureobasidium pullulans induce electrolyte loss and oxidative stress in Botrytis cinerea and Alternaria alternata.

Aureobasidium pullulans is a yeast-like fungus that produces volatile organic compounds (VOCs) with antifungal properties. VOCs have the potential to trigger the production of intracellular reactive oxygen species (ROS), lipid peroxidation and electrolyte loss in microorganisms. The relationship among A. pullulans VOCs, induced ROS accumulation and electrolyte leakage was investigated in Botrytis cinerea and Alternaria alternata in vitro. Exposure to a mixture of A. pullulans VOCs: ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol, resulted in electrolyte leakage in both B. cinerea and A. alternata. Fluorescence microscopy using 2',7'-dichlorofluorescein diacetate indicated triggered ROS accumulation in exposed fungal mycelia and the presence of the superoxide radical was evident by intense red fluorescence with dihydroethidium. Partial inhibition of enzymes of the mitochondrial respiratory chain complex I of B. cinerea and A. alternata by pre-treatment with rotenone reduced ROS accumulation in hypha exposed to A. pullulans VOCs and reversed the VOCs inhibition of fungal growth. Scanning electron micrographs revealed that B. cinerea and A. alternata hypha exposed to A. pullulans VOCs had altered cell wall structures. Our findings give insights into the potential mechanisms involved in the antifungal properties of A. pullulans in the suppression of B. cinerea and A. alternata growth in vitro.

Nanosecond Pulsed Dielectric Barrier Discharge Ionization Mass Spectrometry.

Dielectric barrier discharge ionization (DBDI) is an emerging technique for ionizing volatile molecules directly from complex mixtures for sensitive detection by mass spectrometry (MS). In conventional DBDI, a high frequency and high voltage waveform with pulse widths of ∼50 µs (and ∼50 µs between pulses) is applied across a dielectric barrier and a gas to generate "low temperature plasma." Although such a source has the advantages of being compact, economical, robust, and sensitive, background ions from the ambient environment can be formed in high abundances, which limits performance. Here, we demonstrate that high voltage pulse widths as narrow as 100 ns with a pulse-to-pulse delay of ∼900 µs can significantly reduce background chemical noise and increase ion signal. Compared to microsecond pulses, ∼800 ns pulses can be used to increase the signal-to-noise and signal-to-background chemical noise ratios in DBDI-MS by up to 172% and 1300% for six analytes, including dimethyl methylphosphonate (DMMP), 3-octanone, and perfluorooctanoic acid. Using nanosecond pulses, the detection limit for DMMP and PFOA in human blood plasma can be lowered by more than a factor of 2 in comparison to microsecond pulses. In "nanopulsed" plasma ionization, the extent of internal energy deposition is as low as or lower than in electrospray ionization and micropulsed plasma ionization based on thermometer ion measurements. Overall, nanosecond high-voltage pulsing can be used to significantly improve the performance of DBDI-MS and potentially other ion sources involving high voltage waveforms.

Syringopeptin Contributes to the Virulence of Pseudomonas fuscovaginae, Based on sypA Biosynthesis Mutant Analysis.

Pseudomonas fuscovaginae, first reported from Japan in 1976, is now present in many agroecological regions around the world; it causes sheath brown rot of rice and is reported as a pathogen of a broad range of hosts. The pathogen can infect rice plants at all stages of growth and is known to cause significant losses due to grain discoloration, poor spike emergence and panicle sterility. Limited information is available on the virulence and mechanisms of pathogenicity for P. fuscovaginae. To address this, an analysis of genomes was conducted, which identified the presence of a gene showing homology to one of the genes contributing to syringopeptin synthetase (sypA) of P. syringae pv. syringae. To study the potential role of this gene in the virulence and pathogenicity of P. fuscovaginae, a site-specific mutation was created. Following inoculation of seeds and plantlets of rice and wheat with P. fuscovaginae wild types and their respective mutants, we demonstrated that the mutation significantly reduced virulence. This was evident on rice and wheat inoculated with mutants causing a significantly higher number of roots, length of roots and seedling height compared with their respective wild types. Characteristic disease symptoms of necrotic lesions were significantly less in rice seedlings infected with bacterial suspensions of mutants indicating a reduction in virulence. Chromatography analysis of bacterial exudates showed suppression of synthesis of metabolites analogous to syringopeptin in the mutants. These data demonstrate that the protein encoded by this sypA homolog gene is a major virulence determinant of P. fuscovaginae.

A GC-MS untargeted metabolomics approach for the classification of chemical differences in grape juices based on fungal pathogen.

Fungal bunch rot of grapes leads to production of detrimental flavour compounds, some of which are well characterised but others remain unidentified. The current study uses an untargeted metabolomics approach to classify volatile profiles of grape juices based on the presence of different fungal pathogens. Individual grape berries were inoculated with Botrytis cinerea, Penicillium expansum, Aspergillus niger or A. carbonarius. Grape bunches were inoculated and blended with healthy fruit, to provide 10% (w/w) infected juice. Juices from the above sample batches were analysed by GC/MS. PLS-DA of the normalised summed mass ions indicated sample classification according to pathogen. Compounds identified from those mass ion matrices that had high discriminative value for classification included 1,5-dimethylnaphthalene and several unidentified sesquiterpenes that were relatively higher in B. cinerea infected samples. A. niger and A. carbonarius samples were relatively higher in 2-(4-hexyl-2,5-dioxo-2,5-dihydrofuran-3-yl)acetic acid, while P. expansum samples were higher in γ-nonalactone and m-cresol.

Gas chromatography-mass spectrometry method optimized using response surface modeling for the quantitation of fungal off-flavors in grapes and wine.

An optimized method for the quantitation of volatile compounds responsible for off-aromas, such as earthy odors, found in wine and grapes was developed. The method involved a fast and simple headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) for simultaneous determination of 2-isopropyl-3-methoxypyrazine, 2-isobutyl-3-methoxypyrazine, 3-octanone, fenchone, 1-octen-3-one, trans-2-octen-1-ol, fenchol, 1-octen-3-ol, 2-methylisoborneol, 2,4,6-trichloroanisole, geosmin, 2,4,6-tribromoanisole, and pentachloroanisole. The extraction of the temperature and time were optimized using response surface methodology in both wine base (WB) and grape base (GB). Low limits of detection (0.1-5 ng/L in WB and 0.05-1.6 in GB) and quantitation (0.3-17 in WB and 0.2-6.2 in GB) with good recoveries (83-131%) and repeatability [4.3-9.8% coefficient of variation (CV) in WB and 5.1-11.1% CV in GB] and reproducibility (3.6-10.2 in WB and 1.9-10.9 in GB) indicate that the method has excellent sensitivity and is suitable for the analysis of these off-flavor compounds in wine and grape juice samples.

Grapevine bunch rots: impacts on wine composition, quality, and potential procedures for the removal of wine faults.

Bunch rot of grape berries causes economic loss to grape and wine production worldwide. The organisms responsible are largely filamentous fungi, the most common of these being Botrytis cinerea (gray mold); however, there are a range of other fungi responsible for the rotting of grapes such as Aspergillus spp., Penicillium spp., and fungi found in subtropical climates (e.g., Colletotrichum spp. (ripe rot) and Greeneria uvicola (bitter rot)). A further group more commonly associated with diseases of the vegetative tissues of the vine can also infect grape berries (e.g., Botryosphaeriaceae, Phomopsis viticola ). The impact these fungi have on wine quality is poorly understood as are remedial practices in the winery to minimize wine faults. Compounds found in bunch rot affected grapes and wine are typically described as having mushroom, earthy odors and include geosmin, 2-methylisoborneol, 1-octen-3-ol, 2-octen-1-ol, fenchol, and fenchone. This review examines the current state of knowledge about bunch rot of grapes and how this plant disease complex affects wine chemistry. Current wine industry practices to minimize wine faults and gaps in our understanding of how grape bunch rot diseases affect wine production and quality are also identified.

Evaluation of Fungicides for the Management of Botryosphaeria Canker of Grapevines.

The family Botryosphaeriaceae comprises a number of species that are associated with the dieback disease of grapevine (Vitis vinifera), referred to as Botryosphaeria canker. To date, there are few effective agents available for the management of this disease. In this study, fungicides were evaluated for controlling the disease using a combination of in vitro tests and field trials. Twenty fungicides registered for use on other diseases in Australian viticulture were tested in vitro for their effect on mycelial growth of four species within the Botryosphaeriaceae. The concentrations of fungicide at which 50% of mycelial growth is inhibited (EC50 values) were significantly affected both by fungicide and isolate (P < 0.001). Differences in sensitivities of the four species to the fungicides were negligible (0.41 to 0.59 mg/liter). The most effective fungicides were fludioxonil, carbendazim, fluazinam, tebuconazole, flusilazole, penconazole, procymidone, iprodione, myclobutanil, and pyraclostrobin, for which EC50 values were <1.0 mg/liter. These fungicides were evaluated under field conditions, in addition to the pruning wound protectants Bacseal Super, Garrison, and ATCS tree wound dressing, as well as the biological control agent Vinevax. In field trials, carbendazim (Bavistin), fluazinam (Shirlan), tebuconazole (Folicur), Garrison, and ATCS tree wound dressing applied to freshly cut pruning wounds were the most effective and reduced infection by Diplodia seriata and D. mutila by 41 to 65%. These results suggest that the occurrence of Botryosphaeria canker on grapevines may be reduced via treatment of pruning wounds with selected fungicides as soon as possible after pruning.

Detection and Monitoring of Greeneria uvicola and Colletotrichum acutatum Development on Grapevines by Real-Time PCR.

Bitter rot (Greeneria uvicola) and ripe rot (Colletotrichum acutatum, syn. C. simmondsii) occur frequently in subtropical grape-growing regions of Australia, where they cause yield loss and bitter taints in wine. To further advance the epidemiological studies of G. uvicola and C. acutatum and contribute toward their effective management and control, a rapid and reliable species-specific real-time polymerase chain reaction (PCR) method was developed based on the polymorphic portion of the internal transcribed spacer region of the two fungi. It was found that, within 6 to 8 h postinoculation, the assay could detect as little as 20 fg of genomic DNA and 10 conidia for both species. Artificially and naturally infected grape inflorescences and mature berries were analyzed by both conventional plating methods and real-time PCR. Fungal presence was demonstrated on all plant material but development was observed only on mature berries. The results demonstrate that the real-time PCR technique is a highly specific, rapid, and sensitive method that can be used to detect and study the dynamics of G. uvicola and C. acutatum during different stages of infection and on different grape tissues.