Browsing by Author "Morgan, Horatio Herbert"

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    Investigating the effect of leaf removal on the grape-associated microbiome through culture-dependent and –independent approaches
    (Stellenbosch : Stellenbosch University, 2016-12) Morgan, Horatio Herbert; Setati, Mathabatha Evodia ; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Leaf removal is a common practice, which is often performed at either, veraison to increase the air circulation, light exposure, penetration of fungicides and decrease disease incidence; or at fruit-set to enhance wine colour, flavour and aroma. While there is greater understanding of how leaf removal influences the grape chemical composition and grapevine associated pathogenic fungi, most notably Botrytis cinerea, there is limited information on how the complete microbial community responds to these leaf removal procedures. The current study focused on a phylogenetic survey of both the bacterial and fungal communities present in Sauvignon blanc grape must prepared from shaded (SH) and sun-exposed (EX) grapes. A combination of culture-based methods, ARISA community fingerprinting as well as direct amplicon sequencing was employed to analyse the community. The yeast community was analysed using culture-based methods for four consecutive vintages (2012 – 2015), while the bacteria were only isolated and identified for the 2015 samples. Furthermore, the two culture-independent approaches were used to profile and identify the bacterial and fungal species present in the 2014 & 2015. A comparison of the cultivable yeast communities in the shaded and exposed grape derived must using ANOSIM revealed that there was more variation in the yeast community structures between vintages than between the treatments, thus suggesting the leaf removal only had a small effect on the yeast community (R = -0.04, p = 0.65). In contrast, ARISA data revealed a more diverse community which was different between the treatment (R = 0.5, p = 0.63) albeit with significant overlap. Our ARISA data suggested that leaf removal might have a significant influence on the filamentous fungal community and to a lesser extent on the yeast community. A more in-depth analysis of the microbiome was performed using Illumina target amplicon sequencing of the ITS-1 region, and the fungal population in the SH and EX musts exhibited significant overlap (R = -0.5, p =1) within treatments, while significant overlap between years was observed (R = 0.5, p =0.331) and the difference between the two populations was not significant. However, minor reductions in both Botrytis and Penicillium were observed in the sun-exposed derived grape must. The cultivable bacterial community derived from the 2015 must samples demonstrated complete variation between the samples, with the absence of AAB in the exposed derived must. However, this was only based on a single vintage. Contrastingly, the B-ARISA data demonstrated that leaf removal had no influence of on the bacterial community, with the variation greatest within replicate samples (treatment) (R = -1, p = 1). Our ARISA data suggested that vintage might have a greater influence in driving bacterial communities. The in-depth analysis, was achieved by targeting the protein encoding region rpoB as it has been demonstrated to provide greater resolution and overcome biases associated with the 16S rRNA region. The results were similar to that obtained in the fungal analysis (R = -0.5, p =1), with significant overlap within treatments with, some overlap between years (R = 0.25, p =0.64). However, potential plant pathogens Stenotrophomonas and Xanthomonas were reduced in the exposed treatment, together with Achromobacter and Bordetella. Interestingly concordant with the culture-dependent analysis, Gluconobacter was not identified in the 2015 exposed sample, but only in the shaded derived must. Overall our data show similar trends with regards to the microbial community composition in the shaded and exposed must and whether these are largely influenced by defoliation or not. As a whole the data set suggest that the differences in the microbial community can largely be ascribed to the absence and presence of minor species and relative abundance of a few major species, that dominate the berry surface. On the surfaces leaf removal appears to have no impact on the microbial community, however on closer inspection it seems as if a few groups are impacted, ever so slightly. Therefore, future work should focus on selectedmicrobial communities, which has been shown to be influenced by leaf removal or have the potential for disease. Moreover, the study did however only focus on a single variety, for two consecutive vintages, a greater sample set is required over several vintages and maybe two cultivars.