Validation of a handheld near-infrared spectrophotometer for measurement of chemical intramuscular fat in Australian lamb.

The objective of the study was to independently validate a calibrated commercial handheld near infrared (NIR) spectroscopic device and test its repeatability over time using phenotypically diverse populations of Australian lamb. Validation testing in eight separate data sub-groups (n = 1591 carcasses overall) demonstrated that the NIR device had moderate precision (R2 = 0.4-0.64, RMSEP = 0.70-1.22%) but fluctuated in accuracy between experimental site demonstrated by variable slopes (0.50-0.94) and biases (-0.86-0.02). The repeatability experiment (n = 10 carcasses) showed that time to scan post quartering affected NIR measurement from 0 to 24 h (P < 0.001). On average, NIR IMF% was 0.97% lower (P < 0.001) at 24 h (4.01% ± 0.166), compared to 0 h. There was no difference (P > 0.05) between Time 0 and 1 h or Time 0 and 4 h or between replicate scans within each time point. This study demonstrated the SOMA NIR device could predict lamb chemical IMF% with moderate precision and accuracy, however additional work is required to understand how loin preparation, blooming and surface hydration affect NIR measurement.

Occurrence of fumonisin-producing black aspergilli in Australian wine grapes: effects of temperature and water activity on fumonisin production by A. niger and A. welwitschiae.

Black aspergilli are some of the most common mycotoxigenic fungi in vineyards worldwide. The aims of this research were to assess the occurrence of fumonisin-producing black aspergilli in Australian wine grapes and the effects of environmental factors on fumonisin production by A. niger and A. welwitschiae (syn. A. awamori). Thirty-eight Aspergillus isolates (black aspergilli) were collected from six wine grape varieties grown in Australian vineyards. LC-MS/MS analysis of culture extracts revealed that six isolates produced fumonisins FB2 and FB4. Molecular data revealed that all fumonisin-producing isolates were A. niger and A. welwitschiae. None of the reference isolates, A. carbonarius, A. tubingensis, A. japonicus, and A. foetidus, were positive for fumonisin production. The effects of temperature and water activity on the growth and production of fumonisins were studied using two A. niger and an isolate of A. welwitschiae on synthetic grape juice medium (SGJM) at 20 °C, 25 °C, 30 °C, and 35 °C, and 0.92 aw, 0.95 aw, and 0.98 aw levels. All isolates produced FB2 and FB4 at 0.95 aw and 0.98 aw and 20 °C, 25 °C, and 30 °C. The highest growth rate observed was 14.89 mm/day for A. welwitschiae at 0.98 aw and 35 °C, whereas the highest fumonisin production observed was 25.3 mg/kg at 0.98 aw and 20 °C for A. welwitschiae. None of the isolates produced fumonisins at 35 °C at any water activity levels. To our knowledge, this is the first report on the occurrence of fumonisin-positive isolates of Aspergillus from Australian wine grapes and the impact of the environmental factors on fumonisin production by A. welwitschiae.

Aureobasidium pullulans volatilome identified by a novel, quantitative approach employing SPME-GC-MS, suppressed Botrytis cinerea and Alternaria alternata in vitro.

Volatile organic compounds (VOCs) produced by Aureobasidium pullulans were investigated for antagonistic actions against Alternaria alternata and Botrytis cinerea. Conidia germination and colony growth of these two phytopathogens were suppressed by A. pullulans VOCs. A novel experimental setup was devised to directly extract VOCs using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) from antagonist-pathogen culture headspace. The proposed system is a robust method to quantify microbial VOCs using an internal standard. Multivariate curve resolution-alternating least squares deconvolution of SPME-GC-MS spectra identified fourteen A. pullulans VOCs. 3-Methyl-1-hexanol, acetone, 2-heptanone, ethyl butyrate, 3-methylbutyl acetate and 2-methylpropyl acetate were newly identified in A. pullulans headspace. Partial least squares discriminant analysis models with variable importance in projection and selectivity ratio identified four VOCs (ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol), with high explanatory power for discrimination between A. pullulans and pathogen. The antifungal activity and synergistic interactions of the four VOCs were evaluated using a Box-Behnken design with response surface modelling. Ethanol and 2-phenylethanol are the key inhibitory A. pullulans VOCs against both B. cinerea and A. alternata. Our findings introduce a novel, robust, quantitative approach for microbial VOCs analyses and give insights into the potential use of A. pullulans VOCs to control B. cinerea and A. alternata.

Copper induces transcription of BcLCC2 laccase gene in phytopathogenic fungus, Botrytis cinerea.

Laccases are one of many groups of inducible enzymes produced by the filamentous fungus, Botrytis cinerea during colonisation of host plant tissues. While the processes involved in laccase induction are not fully understood, Cupric ions (e.g. CuSO4) and gallic acid (GA) have been reported as laccase inducers. This study investigates laccases activities and the expression of three laccase genes (BcLCC1, BcLCC2, BcLCC3) in three B. cinerea isolates grown in laccase-inducing medium (LIM) supplemented with CuSO4 and GA. Laccase activity in culture filtrates with CuSO4 increased after 48 h of growth in LIM at 24°C. The induction of BcLCC2 transcription was greatest at a concentration of 0.6 mM CuSO4, concentrations greater than 0.6 mM inhibited fungal growth. In contrast, no laccase induction was observed in the presence of GA. Liquid chromatography-mass spectroscopy (NanoLC ESI MS/MS) analysis confirmed the presence of a 63.4 kDa protein, the BcLCC2 isoform in the culture filtrate with 0.6 mM CuSO4. Analysis of mRNA transcripts further showed BcLCC3 was also inducible and the expression of BcLCC2 and BcLCC3 was isolate-dependent. In conclusion, CuSO4 induces a 63.4 kDa laccase in B. cinerea by induced transcription of the BcLCC2 gene.

The characterization and diversity of bacterial endophytes of grapevine.

The diversity of culturable and nonculturable bacterial endophytes of grapevine (Vitis vinifera L.) was examined using a combination of cultivation and molecular methods. Entire grapevines were sampled to characterize bacterial diversity from different locations throughout the vine. Gas chromatography of fatty acid methyl esters (FAMEs) was used to identify culturable isolates prior to subsequent further microbiological characterization, whilst denaturing gradient gel electrophoresis (DGGE) was used to profile the ribosomal DNA of the bacterial endophyte community extracted from grapevines. Gas chromatography of FAMEs identified 75% of culturable bacterial endophytes to genus level (similarity index >0.3). Many isolates were identified as Bacillus spp., Pseudomonas spp., and Curtobacterium spp. Additionally, actinomycetes are reported for the first time as endophytes of grapevines, with a number of isolates identified as Streptomyces spp. DGGE was successfully used to identify major bands present in samples and indicated a degree of homogeneity of bacterial endophyte community profiles within the grapevines sampled. The major bacterial bands were sequenced and used in identification. Comparison with bacterial markers produced from cultured bacterial endophytes suggested that bacteria in the DGGE profiles were not the species most commonly cultured. Additional research demonstrated similarities between epiphytic and endophytic populations and examined potential entry vectors. Endophyte entry was demonstrated in both field-grown and potted grapevines ('Chardonnay') using a rifampicin-resistant Bacillus cereus mutant. The possibility of grapevine epiphytes becoming endophytes, if the opportunity arises, was supported by comparison of gas chromatography of FAMEs from epiphytic and endophytic populations. This research adds grapevine bacterial endophyte communities to those that have been characterized by a multifaceted approach.

Influence of UV-B irradiation on the carotenoid content of Vitis vinifera tissues.

The carotenoid content was examined in leaf and berry tissues of grapevines (cv. Cabernet Sauvignon) grown either under ambient conditions or under a polyester film to reduce UV light by 98%. Total carotenoids in leaves were less in vines grown under the UV screen. Levels of beta-carotene decreased with berry development around veraison. This effect was more pronounced in vines grown under reduced UV light. The lutein content of berries appeared to remain relatively constant with berry development, but levels were decreased under the UV screen. These observations are important for the wine industry because of the biosynthetic link between carotenoids and wine flavour and aroma compounds.

Radio-detection high-performance liquid chromatographic enzyme assay for inhibitors of fungal sterol delta 14-reductase.

An enzyme assay for inhibitors of fungal sterol delta 14-reductase employing isocratic reversed-phase high-performance liquid chromatography is described. A Hypersil 5-microns octadecylsilyl (ODS) column (250 mm x 4.6 mm I.D.) was used and a mobile phase consisting of methanol-water-ethanol (86:4:10, v/v) was pumped at a flow-rate of 1.5 ml/min. Typical analysis times were 15 min. Using [4-14C]ignosterol as a substrate and an enzyme preparation from Saccharomyces cerevisiae, this method was used to compare the inhibition of sterol delta 14-reductase by the fungicides fenpropidin and fenpropimorph with three N-substituted 8-azadecaline compounds.