Search

Viticulturists and winemakers utilize wine grape cultivar clones in order to achieve specific goals related to yield, quality, and wine characteristics. Vitis vinifera cv. Petite Sirah, also known as Durif, is an important wine grape cultivar in California in the production of varietal wines and as a means to improve the color/tannic structure of red wines through blending. Four Petite Sirah clones (FPS01, FPS03, FPS04, and FPS05) were evaluated to compare their agronomic performance and grape and wine characteristics over the course of the 2021 growing season. Eighty vines of each of the four clones were each organized into four biological replicates in a randomized block design at the Robert Mondavi Institute vineyard (Davis, CA). Five vines per biological replicate were evaluated for vine water status, canopy density, ripening parameters, veraison progression, harvest must chemistry, and cluster condition. Clones were harvested at similar ripeness levels, yields determined, and winemaking performed in triplicate. Both the grape and wine phenolic profiles were determined by the Adams-Harbertson Assay and reversed-phase high-performance liquid chromatography (RP-HPLC). Grape and wine aroma profiles were determined by used head space-solid phase micro extraction-gas chromatography-mass spectroscopy (HS-SPME-GC-MS). Wine phenolic extraction during fermentation was monitored daily by spectrophotometric measurements. Results indicate that clones FPS01 and FPS04 may ripen and accumulate sugar quicker than FPS03 and FPS05 depending on the growing season. Grape must from FPS05 contained higher level of malic acid while FPS03 contained the lowest. FPS03 wines had least amount of phenolic compounds in comparison to other clones as they contained the lowest amount of flavan-3-ols, anthocyanins, and flavonols. Wines from FPS01 and FPS04 were higher in flavan-3-ols and flavonols respectively. Grapes and wines from each clone also showed differences in the composition of volatile compounds. Therefore, it can be stated that differences in chemical composition exist among Petite Sirah clones. In this specific year, these chemical differences did not translate to clear, significant differences in sensorial perception based on descriptive analysis.

Xylella fastidiosa was the first bacterial plant pathogen to have its whole genome sequenced. Nonetheless, the scientific community remains uncertain about the mechanism by which this pathogen causes disease. The determinants of its host range are largely unknown. While as a species, X. fastidiosa can infect hundreds of plants, any individual strain’s potential hosts cannot be predicted. This dissertation is an interrogation of host specificity at three vastly different scales. The research is ordered from broad to narrow, beginning with a global analysis. I computationally modeled pathogen host range using 23 plant genera. Through this breadth, I was able to find correlations between hosts and genome content, as well as make estimates for likely ancestral hosts at internal phylogenetic nodes. My second chapter compares strains from Central America with strains introduced into the United States. I show that the introduced pathogen population is poor at infecting an ancestral host. I also found a suite of genes in the introduced population associated with the host jump. Finally, I dive into disease progression in one host species grown in Northern California, Vitis vinifera. Over the course of three years, I documented how disease development can vary among grapevine cultivars. This final chapter provides suggestions for disease management through more accurate detection of X. fastidiosa in an agricultural setting. The overarching goal of these works is to improve our understanding of the determinants of host specificity and disease progression of the economically and environmentally important pathogen species Xylella fastidiosa.

Current climate conditions indicate that California’s viticultural regions will experiencemore frequent heat events, thus leading to greater water stress resulting in lower quality fruit in grapevines. One strategy used to help mitigate the negative effects is irrigating throughout these events to help preserve berry composition. In order to evaluate the usefulness of irrigation throughout heat events, Vitis vinifera cv. Cabernet Sauvignon vines in an established vineyard were exposed to three differential irrigation treatments. In 2020, the baseline treatment was under water deficit (60% ET), while the 2x baseline (120% ET) and 3x baseline (180% ET) treatments had double and triple the irrigation of the baseline, respectively. The irrigation treatments were adjusted in 2021 to see if less water could be used without negatively impacting the phenolics results. The treatments were 60% ET, 90% ET, and 120% ET. Differential irrigation started one to two days prior to a heat wave (HW) and continued until the last day of the HW. In this study, a HW was defined as three consecutive days with maximum temperatures at or above 38°C. Throughout the 2020 and 2021 growing seasons, there were major declines in anthocyanin concentration for the 60% ET treatment, which suggests a suppression of anthocyanin synthesis and promotion of degradation at such high temperatures. A common trend seen throughout both years is that the 180% and 120% treatments from 2020 and 2021 respectively, resulted in lower phenolic concentrations than the 120% and 90% treatments. This is important to note as these results suggest that irrigation prior to heatwaves can be beneficial in maintaining fruit quality, but excessive watering may negate the beneficial aspects of deficit irrigation. Transcriptomic analyses identified the down-regulation of genes involved in the phenylpropanoid and flavonoid pathways, which provides a potential explanation at the molecular level for the lower phenolic concentrations. This study highlights the detrimental effects of too little or excess water application during HWs on grape berry composition and gene expression.

Pythium species are recognised as important soilborne plant pathogens, however, some of these species are aggressive parasites of other fungi and oomycetes, and are referred to as mycoparasitic Pythium species. These have the potential to be developed as biological control agents against plant pathogens. However, there is limited information regarding the population diversity of mycoparasitic Pythium spp. present in New Zealand, especially associated with economically important horticultural crop such as grapevine. The objectives of this study was to determine the best methods to isolate mycoparasitic Pythium species from soil collected from the Lincoln University vineyards, and to use the best methods in a more extensive North Canterbury vineyard survey to identify mycoparasitic Pythium species diversity. Additionally, the in vitro host range and mode of parasitism of mycoparasitic Pythium spp. isolates recovered from North Canterbury vineyard soils were investigated. The first soil sampling was done in summer 2021 from the organically and conventionally managed vineyard sites at Lincoln university. Three isolation methods were tested (1) soil dilution plating, (2) sclerotia baiting, and (3) pre-colonised fungal host baiting. The soil dilution plating was done by plating 1:50 soil dilution on Pythium selective media (CMA-PARP). For the sclerotia baiting method Sclerotinia sclerotiorum sclerotia were placed into soil water suspensions and the sclerotia transferred after 48 h onto selective media. For the pre-colonised fungal host baiting method 1.5 g of each soil sample was placed onto a pre-colonised host fungal culture plate of Botrytis cinerea, Fusarium oxysporum, Ilyonectria liriodendri, Neofusicoccum luteum, and Neofusicoccum parvum. The plates were observed microscopically after 7, 14 and 21 days for the presence of characteristic oospores and isolation was carried out from any positive plates. The sclerotia baiting and F. oxysporum pre-colonised plate methods were the most effective at recovering mycoparasitic Pythium species from the soil samples. Dilution plating method was the least effective method for isolating mycoparasitic Pythium spp. More mycoparasitic Pythium spp. isolates were recovered from the organically managed vineyard. The species identity of the recovered isolates were confirmed as P. acanthicum and P. oligandrum using sequencies of the ITS and cox II gene regions. The six vineyards were surveyed during autumn for soil sampling which included the organic and conventional vineyard sites, Lincoln University, Pegasus Bay Wine (Waipara), Sherwood Estate Wines (Stirling Vineyard, Waipara), Cross Hares Vineyard (Tai Tapu) and Lone Goat Vineyard (Burnham). As for the summer sampling P. acanthicum and P. oligandrum were recovered from the autumn soil sampling. The host fungal range of the P. acanthicum and P. oligandrum isolates recovered was investigated using a pre-colonised host culture assay, with four fungal hosts used B. cinerea, F. oxysporum, I. Iiriodendri and N. parvum. Isolates of mycoparasitic Pythium spp. were inoculated on PDA plates pre-colonised with the fungal host and after 14 days incubation observed microscopically for the presence of characteristic oospores of the mycoparasitic Pythium spp. Presence of oospore on pre-colonised fungal host plate indicated growth of mycoparasite on host colony. Of the four hosts tested N. parvum and F. oxysporum were identified as being susceptible host, I. Iiriodendri was identified at being less susceptible, and B. cinerea was identified as a resistant host. Interactions on I. Iiriodendri pre-colonised host plates indicated that P. acanthicum isolates was less aggressive than P. oligandrum. Four types of parasitic reactions being hyphal lysis, cytoplasmic coagulation, hyphal thickening, and some coiling attempts were observed when interactions between hyphae of P. acanthicum and P. oligandrum and the different host pathogens on coverslip thinly coated with water agar. Pythium acanthicum and P. oligandrum had the same mode of parasitism. The findings of this study showed that both P. acanthicum and P. oligandrum were present in vineyard soils in the North Canterbury region of New Zealand and, with regards to grapevine trunk pathogens, have a similar host range and mode of parasitism. However further glasshouse and field experiments are required to determine their efficacy in reducing infection of grapevines by grapevine trunk pathogens.

Plant pathology research has long placed a focus on pathogen-derived effector proteins: small, secreted proteins translocated into host cells where they subvert host basal immunity and promote infection. Recent studies suggest that some plant species secrete similar effector-like proteins during mutualistic plant-fungal interactions and affect fungal growth. In this project, a computational tool named SecretePEPPr (Secreted Plant Effector-like Protein Predictor) was written, evaluated, and tested for the purpose of predicting candidate plant effector-like proteins from a set of whole genome annotations. Among other factors, this prediction tool considered classical and non-classical secretion, protein size, and the prevalence and presence of clathrin-mediated endocytic motifs. Analysis on testing data revealed a subcellular localization prediction specificity of 90% on a set of over 500 intracellular plant proteins and sensitivity of 55% on a set of experimentally validated secreted plant proteins. Across four analyzed grape proteomes, several germin-like proteins were identified as potentially haustorially localized through clathrin-mediated endocytic means. Protein length distributions revealed that effector-like candidates containing clathrin-mediated endocytic motifs were mostly in the 150-300 amino acid length range. Follow up in vivo validation was conducted in Erysiphe necator-infected Chardonnay grape leaves. Through this process, the first Erysiphe necator haustorial extraction method was devised using a Percoll Density Gradient followed by fluorescent labeling and fluorescence-activated cell sorting (FACS), resulting in 14 and 18 million purified fungal haustorial cells from 20 grams of heavily infected leaves. Computational streamlining of plant effector-like protein prediction established in this project provides a foundation for the top-down discovery and characterization of this recently discovered class of plant proteins, which have important implications in plant-microbe interactions and may act as targets for breeding and gene editing in plants.

Grapevine trunk diseases (GTDs) are one of the most problematic diseases in grapevines production caused by multiple groups of fungal pathogens that usually invade the vine through pruning wounds. Infection by these pathogens result in a wide range of symptoms negatively affecting vine longevity and productivity in grape-growing regions worldwide including New Zealand. Since chemical options to control GTDs are limited, biological control strategies using plant beneficial microorganisms are of increasing interest. Grapevine tissues harbour a variety of microbes, with endophytic actinobacteria being of particular interest due to their capacity to produce a range of bioactive compounds which ability to inhibit phytopathogens. There have, however, been no studies investigating the diversity of actinobacteria associated with grapevines and their bioactivity against GTD pathogens in New Zealand vineyards. The first aim of this research was to identify the diversity of culturable endophytic actinobacteria associated with different grapevine tissues in New Zealand vineyards and determine the most effective method and media for isolation of endophytic actinobacteria from different grapevine tissues. Grapevine tissues (leaf, stem, and roots) were sampled from Sauvignon blanc vines in both a conventionally and an organically managed vineyard site at Lincoln University. Two isolation methods were tested on surface sterilised tissue: (i) direct plating of tissue pieces, and (ii) tissue maceration, with the tissue homogenized prior plating to dilutions. The tissue samples were plated on four media, International Streptomyces Project 2 agar (ISP2), Starch Casein agar (SC), Tap Water Yeast Extract agar (TWYE) and Actinomycete Isolation agar (AIA). The recovered actinobacteria isolates were identified based on morphology and sequencing of the 16S rRNA gene. A higher frequency of isolates were iii recovered from roots (20 isolates), than leaves (1 isolate) and stems (0 isolates), with more actinobacteria isolated from the organically managed vineyard and using the tissue maceration technique. AIA (8 isolates), ISP2 (5 isolates), SC (5 isolates), and TWYE (3 isolates) were effective at isolating actinobacteria from grapevine tissues. Isolates identified as Streptomyces spp. (20 isolates) and Mycolicibacterium sp. (1 isolate) were recovered. The culturable endophytic actinobacteria species/genera associated with tissues sampled from grapevines of two different ages, young newly planted (2-year-old) and mature (25-year old) Sauvignon blanc vines established in the same vineyard site were identified using the methods developed. Endophytic actinobacteria isolates were more frequently isolated from the mature vines (n=20) than the newly planted vines (n=2) and all isolates were recovered from the root tissues. Sequencing of the 16S rRNA gene revealed that all 22 isolates belonged to the genus Streptomyces. The second aim was to investigate the potential biocontrol activity of actinobacteria isolates to inhibit key pathogens responsible for grapevines trunk diseases (Dactylonectria macrodidyma, Eutypa lata, Ilyonectria liriodendri, Neofusicoccum parvum and N. luteum) and whether these endophytes produce bioactive compounds related to plant growth promotion traits using a range of in vitro bioactivity assays. The inhibitory activity of 40 endophytic actinobacterial isolates was further determined. Thirteen endophytic actinobacterial isolates showed either strong (+++) or moderate (++) antagonistic activity against all five phytopathogenic fungi. In addition to biocontrol activity, four isolates exhibited high siderophore activity on CAS-LB plates, 6 isolates showed moderate or low ability to solubilize phosphate on TCP agar, and 3 isolates were indicated to have high indole acetic acid (IAA) production. Overall, this study revealed the diversity and bioactive potential of culturable endophytic actinobacteria associated with grapevine tissues in New Zealand vineyards. This study identified several isolates belonging to the genus Streptomyces with potential as biocontrol agents against grapevine trunk pathogens. Further study is required to determine their biocontrol activity in planta.

Grapevine trunk diseases (GTDs) are a major economic issue for grapevine production worldwide. Within this group is Botryosphaeria dieback, currently known to be caused by 10 different fungal genera, is an important disease in New Zealand vineyards. Neofusicoccum luteum and N. parvum have been identified as the most common and virulent species both in vineyards and nurseries in New Zealand. Botryosphaeriaceae species can produce spore inoculum throughout the year and can infect grapevines at all crop stages. In addition, these pathogens can be introduced into vineyards through infected propagation material. Hot water treatments (HWT) are carried out by nurseries to reduce the incidence and infection of the planting material by pathogenic bacteria, fungi and nematodes. The overall hypothesis of this study was that HWT can also affect the communities of microbial endophytes colonising the grapevine propagation material as well as causing physiological effects on the grapevine propagation material. Both factors may affect the susceptibility of the grapevine canes to subsequent challenge by the GTD pathogens N. luteum and N. parvum. The first aim of this research was to determine the effect of HWT on the susceptibility of grapevine canes to Botryosphaeriaceae infection. For this, Neofusicoccum luteum isolate CC445 and N. parvum isolate G69a1, both highly pathogenic isolates, were inoculated onto hot water treated Sauvignon blanc and Pinot noir dormant and rooted canes. The susceptibility of the planting material to Neofusicoccum spp. infection was determined by measuring infection progression. Results for dormant canes demonstrated a higher (p=0.025) colonisation by Neofusicoccum spp. in Pinot noir than in Sauvignon blanc. The HWT’s at 48⁰C x 30 min, 50⁰C x 15 min and 50⁰C x 30 min resulted in greater (p

Abstract withheld due to embargo restrictions

Temperature is recognized as a primary influence on grapevine phenology and has been used to classify the climates and regions suited for growing specific grapevine varieties. Many models utilize average temperature to correlate with phenological stages and predict the timing of specific phases. Because average temperature does not account for the hourly temperature or the change in temperature from day to night, it is not known to what degree diurnal temperature range (DTR) influences grapevine phenophases. Utilizing controlled growth conditions (phytotrons) the objective was to determine if DTR impacted the rate of leaf appearance, time of flowering or percent fruit set separate from average temperature (Experiment 1); and to determine if thermal time measured as growing degree hours (GDHs) impacted the rate of leaf appearance, time of flowering and or the degree of fruit set (Experiment 2). For Experiment 1, the time of flowering occurred 3.5 (P

Transposable elements (TEs) are an intrinsic mutagen of eukaryotic genomes and have been proposed to be important in increasing genetic diversity in plants. It has been known that biotic and abiotic stress treatments induce TE transcription, the first stage in TE mobilisation. This research began with an investigation of TE transcription activity in grapevine embryogenic callus subjected to biotic stressors (Botrytis cinerea extracts and live Hanseniaspora uvarum cultures) to determine the location and regulation of autonomous TEs. Short-read RNA sequencing (RNAseq) has been commonly used to determine TE transcription patterns at a family level. This research sought to further these approaches by establishing an analysis pipeline to identify the expression of individual TE loci from Illumina RNAseq data. We efficiently identified that only 1.7%-2.5% of total annotated TE loci were transcribed in our system. This work identified a strong tendency for TE expression candidates to be found within introns of expressed genes. It was also discovered that these pairs of TEs and genes shared the same differential expression patterns in response to applied stressors. Our analysis pipeline was successfully validated using publically available RNAseq datasets from Arabidopsis, wild-type and epigenetic mutant (ibm2 and ddm1) lines, and Drosophila datasets of amyotrophic lateral sclerosis (ALS) models exhibiting a TE transcriptional storm. We successfully identified an Arabidopsis COPIA-93 locus previously proven to mobilise in ddm1 mutant and a subset of Drosophila TE loci that potentially contributed to full-length autonomous TE transcripts in the ALS models that have not been previously reported. Oxford Nanopore Technology (ONT) cDNA sequencing was deployed to determine whether autonomous TEs were being expressed as a precursor of mobilisation. Only low levels of full-length transcription of one Gypsy-V1 locus and three hAT-7 loci was detected in this data, suggesting rare intact transcription from autonomous TE loci despite stress treatments. This finding suggested that TE mobilisation might require inhibition of the epigenetic silencing system. We, therefore, treated embryogenic callus with the histone deacetylase inhibitors (HDACi), trichostatin A (TSA) or 4-phenylbutyric acid (4PBA), to alter the heterochromatic architecture of callus cells. Only the 4PBA treatment showed a noticeable shift in the transcriptional landscape of TE transcription, significantly increasing the proportion of intergenic TE loci in the expression candidate pool and resulting in significant up-regulation of 2,059 TE loci. ONT cDNA sequencing of these samples detected very low levels of intact sequencing reads from different yet a single Gypsy-V1 locus and six hAT-7 loci. Five genes participating in the RNA-dependent DNA methylation (RdDM) pathway (AGO2, AGO4, RDR1, RDR6, and NERD) were upregulated, suggesting that callus exposed to 4PBA responded by an enhancement of RdDM, maintaining effective control of TE transcription and therefore TE mobility. Overall, this thesis contributes to the understanding of the landscape, properties, and determinants of transcriptional activation of endogenous transposable elements, revealing the closely connected transcriptional relationship between TEs and co-localised genes. These findings shed light on the genetic and epigenetic impact of endogenous TE activation on genes in nature.

Agricultural chemicals have been an invaluable tool to fertilize crops and manage diseases, but as consumer demand for sustainable and safer products continues to rise, it’s becoming imperative to find alternative management strategies that are environmentally sustainable and safe to humans. Grapevine (Vitis vinifera L.) is a staple crop to California agriculture and is valued at $6.25 billion. This perennial plant is plagued by Pierce’s Disease (PD), caused by the xylem limited bacterium, Xylella fastidiosa. Under field conditions, grapevine varieties can exhibit a spectrum of disease phenotypes and some vines appear to escape the disease (i.e., no to little symptoms). We hypothesize that the endophytic microbiome, as well as the variety of the host, may impact disease outcome. Harnessing this knowledge can lead to integrated and sustainable approaches for agriculture. We present the first comprehensive culture-independent microbial study of an agricultural crop, including previously unexplored niches: sap and wood. Our results indicate that the majority of endophytes are derived from the rhizosphere. Disease, season, year, and biocompartment all play crucial roles in microbial assemblage. However, a core microbiome shapes the backbone of the grapevine endosphere. Our results showed that two endophytes native to California grapevines, Pseudomonas and Achromobacter negatively correlated with Xylella. We developed novel pathogen-specific qPCR primers to assess X. fastidiosa population in planta. In planta bioassays indicated that‘ Thompson Seedless’ exhibited the highest pathogen titre, disease ratings and lowest stem hydraulic conductivity; suggesting a cultivar’s pedigree and underlining xylem anatomy shape disease susceptibility. In planta, P. viridiflava and A. xylosidans showed protective qualities against X. fastidiosa. In vitro assays were conducted to elucidate potential microbe-pathogen interactions and indicated that the mechanism of protection is not through direct antagonism but possibly via alteration of plant-pathogen interaction. In conclusion, this research elucidates the endophytic grapevine microbiome and deepens our understanding of the grapevine-Xylella-microbe relationship, producing two potential biocontrols and the foundation to new management strategies for Pierce’s Disease.

Amino acids are important primary metabolites and one of the major sources of nitrogen in grapes. Amino acids also occupy a central junction in grapevine biochemistry and are important for vine metabolism and berry homeostasis. The majority of a grapevines photosynthesis and nitrogen assimilation occurs in the leaves, the products of which (sugars and amino acids) are exported to the grape bunches (fruit). In the grape berries themselves, amino acids are the precursors to secondary compounds supporting grapevine growth and physiology, while additionally having an important role in wine quality outcomes. Yet, despite the importance of amino acids in grapevine, their regulation and accumulation in grapes is poorly understood and usually inferred through research in other plant species. Leaf and shoot removal are common practice in commercial vineyards, with any such canopy management interventions having the potential to influence berry biochemical composition. This well-established viticultural intervention was used as an experimental technique to investigate the effect on amino acid biochemistry in Sauvignon blanc grapes. Basal leaf removal had a significant effect on amino acid accumulation in Sauvignon blanc grape berries. This was reflected in reductions of total amino acid concentrations in the berry, differential accumulation at the level of amino acid families and within families, individual amino acids. Individual amino acids also had differential responses to leaf removal. Aside from the quantitative effect of basal leaf removal on amino acid concentrations, leaf removal also had a qualitative effect on some individual amino acids, modifying their proportions of accumulation in the grape berries. The α-ketoglutarate family of amino acids (glutamine, glutamate, arginine and proline) was further studied, being the family that contains the predominant concentrations of amino acids through berry development and having both quantitative and qualitative aspects in their response to basal leaf removal. Glutamine was the dominant amino acid early in development and decreased throughout. Proline and arginine both increased steadily through development to become most abundant amino acids in grape berries at harvest. Glutamate levels stay relatively consistent through development despite being the direct precursor of the two most abundant amino acids at harvest (arginine and proline) and contributing as a substrate / co-factor for other amino acids via other metabolic reactions (pathways). The significant reduction in amino acid concentrations in grape berries due to leaf removal, was hypothesised to be predominantly due to the effect of an altered source/sink balance and partitioning impacts. The accumulation of sugars in the berry was less impacted by the same leaf removal treatments. A model was proposed, whereby the younger leaves further up the shoot can compensate with an increased carbohydrate export to the bunches, but this same compensation is not achieved with respect to amino acids. The mechanisms of amino acid accumulation in the grape berries was further examined, by investigating a number of genes involved in different aspects of α-ketoglutarate amino acid biochemistry. Investigating transcriptional changes of genes involved in these amino acid metabolic pathways, demonstrated differential expression of a range of transcripts involved in metabolism and regulation of glutamine, glutamate, arginine and proline. These experiments also present novel information, regarding the up-regulation of expression of genes involved in arginine and proline metabolism through berry development. In other plant species, proline is induced as a stress response, but in grapevine, the proline metabolic pathways are not well characterised and the accumulation of proline is poorly understood. An increase in expression of proline biosynthetic genes through veraison was demonstrated with a maintenance of transcript counts at postveraison time points, overlapping the period in berry development when proline accumulation in the berry is increasing and the berry is starting to accumulate larger amounts of sugars. An increase in expression of genes related to proline and arginine degradation pathways was also demonstrated through berry development. This implied that even as the berry is accumulating arginine and proline, there appeared to be the potential for interconversion and turnover of these amino acids through the upregulation of enzymes involved in their catabolism. The relative activity of an associated enzyme (ornithine aminotransferase) which was transcriptionally up-regulated, was also studied. Enzyme assays performed on treatment samples indicated that ornithine aminotransferase specific activity increased through Sauvignon blanc berry development and ripening. Additionally, the maintenance of a leaf canopy was important in regulating ornithine aminotransferase enzyme activity, as relative specific activity was increased by basal leaf removal compared to control (maintained leaf canopy) samples. This indicated the potential for allosteric regulation of key amino acid pathway enzymes in grape berries. Overall, the results presented in this thesis substantially improve the understanding of the mechanisms influencing amino acid biochemistry in Vitis vinifera L. var. Sauvignon blanc grapes.

Xylella fastidiosa is a xylem-limited bacterium that causes Pierce’s Disease (PD) in grapevine. PD symptom progression is correlated with systemic colonization by X. fastidiosa, which occurs via pit membrane degradation. Previous studies determined that a polygalacturonase, one of several cell wall degrading enzymes (CWDEs) secreted by X. fastidiosa, is necessary for pit membrane degradation. Here, it is demonstrated that the loss of two X. fastidiosa β-1,4-endoglucanases (EGases) in tandem causes significantly slower PD symptom development and progression, and reduces X. fastidiosa virulence and population size. Furthermore, differences in the PD symptom progression rate between two cultivars appear to be caused by variations in pit membrane carbohydrate composition, which influence the rate of enzymatic degradation, and subsequently, the rate of systemic colonization. How these CWDEs and other virulence factors are secreted has not been fully elucidated. However, several are predicted to be secreted via the Type II secretion system (T2SS). T2SS-deficient X. fastidiosa mutants did not induce PD symptoms, and the mutant bacteria were not recovered from infected vines, indicating that the T2SS and its secreted substrates are necessary for virulence and survival. Analysis of wild type X. fastidiosa and T2SS-deficient mutant secretomes found six potential Type II secreted proteins for further characterization, including three lipases, a β-1,4-cellobiohydrolase, a protease, and a hypothetical protein. However, none of the CWDEs were present in the secretomes, but their high transcript abundance indicates that these enzymes could be post-transcriptionally regulated. Several PD symptoms appear similar to drought stress symptoms. Under drought conditions, decreased photosynthesis, cessation of plant growth, and carbon starvation occur. Tylose formation in response to X. fastidiosa infection occludes vessels to prevent the spread of pathogens and air embolisms. Interestingly, genes involved in photosynthesis and plant growth are down-regulated during PD symptom development, and tylose formation increases significantly. This increased vessel occlusion in the absence of new vessel formation over time could cause prolonged water stress. Furthermore, starch in the ray parenchyma depletes as PD symptoms progress, indicating that carbon starvation occurs. These results indicate that, along with X. fastidiosa-mediated virulence factors, host responses also contribute to PD.

(1) Background: Vitis vinifera L. cv. Tempranillo is cultivated over the world for its wine of high quality. The association of Tempranillo with arbuscular mycorrhizal fungi (AMF) induced the accumulation of phenolics and carotenoids in leaves, affected the metabolism of abscisic acid (ABA) during berry ripening, and modulated some characteristics and quality aspects of grapes. The objective of this study was to elucidate if AMF influenced the profiles and the content of primary and secondary metabolites determinants for berry quality in Tempranillo. (2) Methods: Fruit-bearing cuttings inoculated with AMF or uninoculated were cultivated under controlled conditions. (3) Results: Mycorrhizal symbiosis modified the profile of metabolites in Tempranillo berries, especially those of the primary compounds. The levels of glucose and amino acids clearly increased in berries of mycorrhized Tempranillo grapevines, including those of the aromatic precursor amino acids. However, mycorrhizal inoculation barely influenced the total amount and the profiles of anthocyanins and flavonols in berries. (4) Conclusions: Mycorrhizal inoculation of Tempranillo grapevines may be an alternative to the exogenous application of nitrogen compounds in order to enhance the contents of amino acids in grapes, which may affect the aromatic characteristics of wines.

Climate change and their resulting impacts are becoming a concern for winegrowers due to the high socioeconomic relevance of the winemaking sector worldwide. In fact, the projected climate change is expected to have detrimental impacts on the yield of grapevines, as well as on the quality and properties of grapes and wine. It is well known that arbuscular mycorrhizal fungi (AMF) can improve the nutritional quality of edible parts of crops and play essential roles in the maintenance of host plant fitness under stressed environments, including grapevines. The future scenarios of climate change may also modify the diversity and the growth of AMF in soils as well as the functionality of the mycorrhizal symbiosis. In this review, we summarize recent research progress on the effects of climate change on grapevine metabolism, paying special attention to the secondary compounds involved in the organoleptic properties of grapes and wines and to the levels of the phytohormones implied in the control of berry development and fruit ripening. In this context, the potential role of AMF for maintaining fruit quality in future climate change scenarios is discussed.

Vineyards in the Mid-Atlantic often have large, vigorous vines that can be costly to manage and produce inadequate fruit for wine production. Dense canopies increase the incidence of fungal disease, require greater allocation of resources to manage, and inhibit fruit development. The primary objective of these studies was to determine effective vine-size modification treatments that would optimize fruit quality, while reducing labor and chemical control. Research factors included root manipulation, under-trellis ground cover, and rootstock. Treatment levels were root bag (RBG) or no root manipulation (NRM); under-trellis cover crop (CC) or herbicide strip (HERB); and one of three rootstocks: 101-14, Riparia Gloire, or 420-A. Effects of these treatments were measured in two experiments: Experiment I compared combinations of all three treatments, while Experiment II explored the individual effects of root restriction using root bags of varying volumes. Root restriction consistently demonstrated the ability to reduce vegetative growth and vine water status. In the first experiment fruit-zone photosynthetic photon flux density (PPFD) was increased by 234% in RBG vines. Timed canopy management tasks indicated that RBG canopies required about half the labor time of NRM canopies. Anthocyanin concentration and total phenolic content were increased by 20% and 19% respectively in RBG fruit. CC increased fruit-zone PPFD by 62%, and increased soluble solids and color compounds. The 420-A rootstock reduced potassium uptake, resulting in lower must potassium concentration. Results demonstrated that these treatments significantly reduce vegetative growth in a humid climate, decrease management labor, and produce higher quality fruit.

To combat excessive vine vigor, many vintners have employed intensive cover cropping techniques. While cover crops provide a multitude of benefits to the farming system, they can compete for nutrients and water. The seemingly ubiquitous adoption of cover crops in the Eastern United States has led to vines and grape musts which are deficient in nitrogen (N). A must that is deficient in yeast assimilable nitrogen (YAN) can lead to the production of off aromas and stuck or sluggish fermentations. It has also been suggested that musts with limited amino nitrogen sources can result in wines with less fruity aromas than those with a higher starting amino acid content. Varying rates of calcium nitrate were applied to the soil at bloom and foliar urea was sprayed at a Sauvignon blanc and Petit Manseng (Vitis vinifera L.) vineyard. Perennial White and Crimson clover as well as foliar urea applications at véraison were utilized at a Vidal blanc (Vitis spp.) site. Foliar urea was effective at significantly increasing YANs in all experiments with some year to year variation in efficacy. Foliar urea applications slightly favored the production of ammonia over primary amino nitrogen. While most of the measured amino acids in fruit increased in concentration with the application of either soil or foliar N, foliar applications were more effective at increasing fruit amino acids. Of the amino acids measured, arginine and glutamine were the most increased by foliar urea applications, whereas proline was relatively unaffected. The use of clover as a perennial under-vine cover crop did not increase berry YAN. The application of foliar urea sprays may present an effective means by which vintners can easily increase must YANs and amino acid contents.

Vineyards in the Eastern United States are often prone to excessive vegetative growth. In order to suppress excessive vine vigor, many viticulturists have employed cover cropping strategies. Cover crops provide a myriad of agronomic benefits, however they are known to compete with the vine for water and nutrients. Due to the widespread use of cover crops in Eastern vineyards, many vineyards experience nitrogen (N) deficiencies in both the vegetative vine tissue and yeast assimilable nitrogen (YAN) in the juice. Soil applications of calcium nitrate and foliar applications of urea were assessed as a means of vineyard N amelioration at cover cropped sites comprised of Petit Manseng and Sauvignon blanc (Vitis vinifera L.). Perennial White and Crimson clover cover crops and foliar urea applications were also used in a Vidal blanc (Vitis spp.) vineyard. Treatments were imposed in the Sauvignon blanc vineyard for five years. The Petit Manseng and Vidal blanc vineyards were subjected to treatments for two years. Soil-applied N at bloom was most effective at increasing leaf petiole N at véraison, season-long chlorophyll content index (CCI), vine capacity and fruit yield. Fruit yield was increased due to more berries per cluster and greater berry weights. Increased rates of soil-applied N decreased the fruit weight:pruning weight ratio. Clover cover crops offered little to no benefit as a N source in the two-year period of evaluation. None of the N management schemes negatively impacted canopy density, fruit zone light interception, or botrytis bunch rot incidence. The combination of both a soil-applied and foliar-applied N fertilizer may be the most effective means to increase both vine capacity and YAN in vineyards where vineyard floor cover crops are compromising vine N status.

Vineyards in the Eastern United States are often prone to excessive vegetative growth. In order to suppress excessive vine vigor, many viticulturists have employed cover cropping strategies. Cover crops provide a myriad of agronomic benefits, however they are known to compete with the vine for water and nutrients. Due to the widespread use of cover crops in Eastern vineyards, many vineyards experience nitrogen (N) deficiencies in both the vegetative vine tissue and yeast assimilable nitrogen (YAN) in the juice. Soil applications of calcium nitrate and foliar applications of urea were assessed as a means of vineyard N amelioration at cover cropped sites comprised of Petit Manseng and Sauvignon blanc (Vitis vinifera L.). Perennial White and Crimson clover cover crops and foliar urea applications were also used in a Vidal blanc (Vitis spp.) vineyard. Treatments were imposed in the Sauvignon blanc vineyard for five years. The Petit Manseng and Vidal blanc vineyards were subjected to treatments for two years. Soil-applied N at bloom was most effective at increasing leaf petiole N at véraison, season-long chlorophyll content index (CCI), vine capacity and fruit yield. Fruit yield was increased due to more berries per cluster and greater berry weights. Increased rates of soil-applied N decreased the fruit weight:pruning weight ratio. Foliar-applied N after fruit set was most effective at increasing berry YAN. While most of the measured amino acids in fruit increased in concentration with the application of either soil or foliar N, foliar applications were more effective at increasing fruit amino acids. Clover cover crops offered little to no benefit as a N source in the two-year period of evaluation. None of the N management schemes negatively impacted canopy density, fruit zone light interception, or botrytis bunch rot incidence. The combination of both a soil-applied and foliar-applied N fertilizer may be the most effective means to increase both vine capacity and YAN in vineyards where vineyard floor cover crops are compromising vine N status.

Grape seed extract (GSE) and peanut skin extract (PSE) are waste products in the wine and peanut industries. Both extracts have high concentrations of polyphenols, known to possess antioxidant and antimicrobial properties. A subcategory of polyphenol is procyanidin, which can be divided into two types, type A and type B. Type A (PSE), contains two single bonds connecting the phenolic groups while type B (GSE), contains one single bond connecting the phenolic groups. The minimum inhibitory concentration (MIC) of the two extracts was evaluated for their antimicrobial effect on Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella Typhimurium using the pour plate method. GSE was found to have a significantly lower MIC (p ≤ 0.05) than PSE for L. monocytogenes (GSE=60.60ppm, PSE=not found), S. aureus (GSE=38.63ppm, PSE=51.36ppm), and S. Typhimurium (GSE=45.73ppm, PSE=60.60ppm). There was no significant difference in inhibition of E. coli O157:H7 (GSE=47.44ppm, PSE=51.13ppm). Since GSE, contributed to greater pathogen inhibition, its extract was fractionated into monomer and oligomers components. Growth curves of all four pathogens inoculated in the monomer and oligomer fractions were compared using the BioScreen method. Oligomers inhibited growth of L. monocytogenes, S. aureus, and E. coli O157:H7 while monomers inhibited growth of S. Typhimurium. These results indicate that an extract with type B procyanidins that are high in oligomers may be more effective as antimicrobials. Type B procyanidins have also been shown to prevent bacterial adhesion, as is the case with urinary tract infections, and may aid in the prevention of biofilms.

Grape root borer, Vitacea polistiformis (Harris), is an oligophagous pest of grapevines in the eastern USA. Neonates must burrow into the soil to find grape roots. In Virginia, larvae feed on roots for ~2 years, then pupate just beneath the soil surface. Emerging adults leave an empty pupal exuviae at the soil surface around the vine base. There was no relationship between weekly captures in pheromone traps and pupal exuviae counts, indicating that exuviae sampling is most appropriate to assess infestations. Exuviae sampling in Virginia vineyards revealed infestations that ranged from light to very heavy. Eighteen biotic and abiotic variables were measured and used in analyses that assessed their relative contributions to differences in exuviae density. Water holding capacity and clay/sand ratio were most strongly associated with pupal exuviae density; these variables were used to develop a model for predicting the extent of infestation of individual vineyards. The spatial distribution of pupal exuviae was characterized using non-spatial and geospatial techniques. Although the non-spatial method (Taylor's Power Law) indicated that exuviae showed an aggregated distribution in all blocks, spatial methods (variograms, SADIE) revealed aggregated distributions only in blocks with ≥ 0.5 pupal exuviae per vine. Independent pupal exuviae samples for population assessment in vineyards can be achieved using sampling points separated by >8.8 m. Combined results from geospatial analyses and the temporal distribution of pupal exuviae within years enabled the development of a practical and quantitative sampling protocol. Bioassays used to measure the behavioral response of larvae to host stimuli revealed that neonates were attracted to grape root volatiles. In soil column bioassays, larvae moved vertically and horizontally over distances of up to 120 cm and apparently perceived the presence of grape roots from a distance of 5 cm in soil. Results are discussed in relation to their potential implications for monitoring and managing grape root borer.

Under-trellis cover crops have become more prevalent in East Coast grape growing regions through either intentional planting or adoption of native vegetation, to minimize the potential for erosion and to help regulate grapevine size and vigor. These companion crops, however, have sometimes resulted in increased competition for soil nitrogen, leading to decreased vine nitrogen status and berry yeast assimilable nitrogen (YAN). The aim of this study was to determine the effects of different nitrogen fertilization methods applied at varying doses and different times, on vine and berry nitrogen parameters of cover cropped grapevines. The research described herein involved Sauvignon blanc, Merlot, and Petit Manseng grapevines (Vitis vinifera L.) subjected to different sets of nitrogen treatments, and was primarily conducted over two years. There were very few differences in pruning weights, canopy architecture, components of yield, and primary fruit chemistry amongst nitrogen treatments. Sauvignon blanc petiole nitrogen concentration, season-long chlorophyll content index (CCI) values, and berry YAN were most affected by the highest rate of soil nitrogen treatment (60 kg N/ha total split between two calcium nitrate applications at bloom and six weeks post bloom) and foliar fertilization (40 kg N/ha split over seven to nine urea applications); however, the foliar fertilization was most effective at increasing the concentration of certain individual amino acids. Petit Manseng berry YAN at harvest was increased in response to post-véraison foliar applications (10 kg N/ha split between two urea applications), corresponding to an increased concentration of nine amino acids. Merlot berry YAN, petiole nitrogen concentration, and season-long CCI values were most affected by a high rate of soil nitrogen treatment (60 kg N/ha total split between two calcium nitrate applications at bloom and six weeks post bloom) and establishing clover as the under-trellis cover crop. This study identified nitrogen treatments that improved berry nitrogen concentration and content in cover cropped sites.

Leaf removal is a normal commercial practice in viticulture to lower disease pressure through reduction in humidity and increased light exposure. However, New Zealand has 30-40% higher levels of UV radiation compared to similar latitudes in the Northern hemisphere, an increase of light exposure may therefore have a profound influence on berry composition. The aim of this research was to determine the effects of leaf removal and UV-B on flavonoid biosynthesis and regulation of gene activity in Vitis vinifera L. var. Sauvignon blanc grapes, particularly those genes associated with the low and high fluence UV-B responses. During the 2010 and 2011 seasons, three north-south oriented rows of Sauvignon blanc grapevines in the Lincoln University research vineyard were chosen to study the specific effects of UV-B on flavonoid accumulation and gene expression. This was achieved by using a combination of leaf removal and plastic screening techniques, which alter the light environment that the fruits are exposed to. UV-B exposure had a dramatic effect on the physical appearance of the berries causing specific pigmentation post-veraison. However, the accumulation of the total soluble solids and berry tissue development were not affected either by leaf removal around the fruiting zone or increased exposure to UV-B. In contrast, flavonols, particularly quercetin and kaempferol glycosides, increased substantially on fruit exposure due to UV-B. Spatial analysis of these flavonols located the changes to the berry skin. To understand the role of UV-B in regulating flavonoid biosynthesis, the transcript abundance for genes and transcription factors that are involved in the flavonoid biosynthetic pathway were analysed by qRT-PCR. These genes include five flavonol synthase genes (VvFLS1-5), three chalcone synthase genes (VvCHS1-3), and genes for the MYB-bHLH-WD40 transcription factor complex (VvMYB12, VvMYCA1 and VvWDRs). Of the five identified VvFLS, only two (VvFLS4 and VvFLS5) were found to be transcriptionally active in Sauvignon blanc berries and responsive to UV-B exposure. Of three VvCHS, only VvCHS1 and VvCHS2 showed a significant UV-B induction. Of the transcription factor genes, only VvMYB12 was found to be responsive to UV-B radiation and its expression pattern was consistent with VvFLS. The relative involvement of genes in the high and low fluence UV-B transduction pathways (VvUVR8, VvHY5 and VvCOP1; PR genes and VvMAPK3; respectively) were also studied. VvUVR8 did not response to UV-B but showed a developmental regulation. A similar lack of a UV-B response was found for VvCOP1. In contrast, VvHY5 showed a UV-B induction and higher levels of transcript abundance took place at harvest. All of PR genes showed a significant developmental regulation, with very low or no detectable transcript abundance pre-veraison and high transcript abundance post-veraison. These PR genes were not responsive to the natural fluence UV-B in the vineyard environment, but some showed a significant UV-B induction to a relatively high fluence UV-B under the controlled environment. Overall, this study clearly demonstrates the effect of UV-B exposure on the flavonoid biosynthesis in Sauvignon blanc grapes is achieved through a complex regulation of genes and transcription factors involved in the flavonoid biosynthetic pathway. This research makes a substantial contribution, not only in improvement of our scientific understanding of UV-B responses in important commercial species, but also provides valuable information for vineyard management to ultimately improve wine quality.

The formation of inflorescence primordia (IP) marks an important step in the reproductive cycle of grapevines. The development of an IP to a grape bunch occurs over the course of two growing seasons. During season one, an IP initiates from a group of uncommitted cells, termed an anlage, where initiation is thought to occur around the time of flowering. Further branching and development of IP continues until the onset of winter dormancy. During spring of the following season, branching of IP resumes and each branch terminates in a floral identity, where each floral identity has the potential to form a berry. Inflorescence primordia initiation and development is sensitive to carbohydrate (CHO) availability and temperature. This thesis explores the influence of CHO availability and temperature during IP initiation and their continued development the following season. An inflorescence or bunch has two main components: the inner and outer arm. For the purpose of this thesis, inflorescence or bunch architecture is defined by the type of structure occurring at the outer arm position: and outer arm with floral identities; a tendril; or no structure. While the inner arm is required for an inflorescence structure to occur, development of the outer arm to a floral bearing structure does not always occur. Although initiation of the inner and outer arm likely occurs at the same time, the development of the outer arm into a floral bearing structure is frequently delayed compared to its inner arm structure. The causes and consequences of delayed outer arm formation on yield and grape composition at harvest are also addressed in this thesis. The effect of restricting CHO availability to IP during their second season of development was achieved by pre-budbreak (BB) cane girdling. First, it was established that pre-BB cane girdling alters the CHO availability to developing shoots and IP structures (Chapter 3.1). Canes were girdled 5, 10 or 20 cm from the terminal bud of the cane and shoot growth of the terminal bud was monitored over a single growing season. A linear relationship was found between the initial rate of shoot growth and the amount of cane isolated by the girdle. A decrease in available CHOs during initial shoot growth appeared to inhibit the shoot’s ability to produce new vegetative nodes past the point of discontinuity. This resulted in a decrease in total leaf area due to incomplete leaf expansion. The transition of the vine’s dependence on reserve CHOs to a net positive state appeared to occur when shoot growth reached a steady state. In the case of severe CHO restriction, no lateral growth occurred, suggesting the CHO status of the vine may play a role in lateral bud growth. The cross sectional area of canes or shoots were shown to have a linear relationship to their CHO content, which allowed for an estimation of the amount of CHOs required to obtain growth similar to the Control treatment. Additionally, main shoot leaf area can be used to predict total CHO content in the shoot at harvest. In the same experiment, the dates of flowering, flower number, berry number and grape berry soluble solids (SS) were measured for the inner and outer arm components of the basal and apical inflorescence and bunches separately (Chapter 3.2). Restricting pre-BB CHOs resulted in the abortion of some pre-formed inflorescences and reduced branching of the inflorescences that did develop. In general, berry SS were greatest for the basal inner arm, followed by those of the apical bunch inner arm, then those of the basal bunch outer arm, then lastly by those of the apical bunch outer arm. However, this was influenced by the relative berry numbers between the inner and outer arm. Bunches with more similar berry numbers on the inner and outer arms had more synchronous flowering and uniform SS. The differences in SS were largely a reflection of the timing of flowering of the various inflorescence components and may be an important source of variation in SS within a vine at harvest. The effects of girdling shoots and / or periodic leaf removal post fruit set on the initiation and development of IP was studied (Chapter 4). Dormant latent buds from treatment shoots (shoot node positions one to ten) were grown as single node cuttings (SNCs). Inflorescence number per SNC and their architecture were scored for every SNC. Girdling increased the proportion of SNC basal bunches with an outer arm, but had no effect on the number if inflorescence structures per bud (fruitfulness). However, there was a decrease in fruitfulness per bud and in the proportion of SNC basal inflorescences with an outer arm when girdled shoots had their leaves removed at zero and four weeks post fruit set respectively. This chapter presents novel information regarding the timing of IP initiation, including the initiation and development of the outer arm. As well, for the first time, the formation of an outer arm is shown to be sensitive to girdling and / or leaf removal. The influence of temperature during IP initiation on the resulting fruitfulness, distribution and architecture over the course of two consecutive growing seasons, at a single vineyard (2-Cane, 4-Cane and Spur pruning) was studied (Chapter 5). The pruning system had no effect on the fruitfulness per bud, or on the resulting bunch architecture. Warmer temperatures during IP initiation were correlated to: an increase in fruitfulness; an increase in the occurrence of an outer arm; and a decrease in the basal bunch insertion point on a shoot. As well, an increase in cane cross sectional area correlated to an increase in the average fruitfulness per shoot along a cane, where the influence of cane cross sectional area on fruitfulness was consistent between seasons. Additionally, an increase in cane cross sectional area correlated to an increase in the average occurrence of an outer arm per shoot along a cane, where the influence of cane cross sectional area was greater when temperatures during IP initiation were cooler. Temperatures during winter dormancy and BB were altered to determine its influence on the timing of BB and the branching of IP structures (Chapter 6). Winter dormant buds were passively heated using plastic heating chambers for different periods during dormancy to BB. Heating buds from either July or August to BB advanced the date of BB and the start date of flowering for the basal inner arm component by 14 and 16 days respectively compared to the Control treatment. Heating buds during winter dormancy had no effect on the fruitfulness of buds, the distribution of inflorescence structures on shoots or on the identity of the structure occurring at the outer arm position. However, elevated bud temperatures 12 days pre-BB caused a statistically significant decrease in the flower number per shoot (P < 0.05). Additionally, it was found that any delay in the start of flowering (which can be influenced by the date of BB, the position of the inflorescence on the shoot and / or the inflorescence component in question) is reflected in the accumulation of SS at harvest. The influence of temperature during IP initiation and CHO availability during BB were combined in a final experiment to improve the ability to predict when IP is occurring (Chapter 7). In this experiment, the influence of temperatures during IP initiation on fruitfulness and inflorescence architecture was studied between vineyards from separate growing regions throughout New Zealand. Winter dormant canes were harvested from each vineyard, and nodes three and ten were grown as SNCs. The volume of the SNC, inflorescence number, the position of the bunch on the SNC shoot and the occurrence of an outer arm were recorded. Inflorescence number and the occurrence of an outer arm increased as the volume of the SNC increased. The timing of IP initiation was predicted using stepwise regressions from 80 days pre to 90 days post 50% flowering with a variable window of time. Regressions of average daily temperature during IP initiation versus bunch number and versus the occurrence of an outer arm resulted in a linear continuum between node three and node ten for both bunch number (R²=0.73) and the occurrence of an outer arm (R²=0.47). The results also indicate that the use of 50% flowering as a reference point to calculate IP initiation is a valid alternative to destructively sampling buds to determine bud fruitfulness and IP architecture. This thesis provides an understanding of two of the major factors affecting bunch architecture, specifically outer arm development, which are temperature and CHO availability. The results indicate that temperature is likely the major factor influencing bunch architecture. Whereas CHO availability is a modifying factor, and is likely not to be an issue in New Zealand where grapevines are rarely CHO stressed.

Transposable elements (TEs) are recognised as a significant and ubiquitous component of eukaryotic genomes. This thesis contributes to the current knowledge of these elements by describing the stimulated mobilisation of multiple class I and class II elements from eight TE superfamilies in grapevine somatic embryo cultures following stress treatments and tissue culture, leading to the production of new vegetative material. An in silico analysis of class I TEs in the grapevine genome revealed that although the majority of the 137 defined retrotransposon families exist mainly as eroded fragments, several families show evidence of recent mobility or appear in transcript databases. Based on these results, a high-resolution S-SAP technique was used to identify insertion polymorphisms of three Ty1-Copia TE families (Edel, Noble and Cremant) and one Ty3-Gypsy family (Gret1) across 32 grapevine genotypes, demonstrating the contribution of these elements to genetic diversity in Vitis. By supplementing bacterial suspensions with an organosilicone surfactant, the efficiency of Agrobacterium-mediated transient transformation of grapevine leaf tissue was improved by an average of 72-fold. This protocol was used to show that of the above four TE families, only the long-terminal repeat (LTR) sequence of Edel is able to drive expression of reporter genes in grapevine leaf tissue, but all four are capable of stimulating expression in the model plant N. tabaccum. After two generations, the LTR sequences of Gret1 and Edel no longer induced reporter gene expression in stable N. tabaccum transgenic plants, but the LTR sequences of Cremant and Noble retained a wound-responsive expression pattern. Due to their sessile lifestyle, plants are forced to endure and adapt to environmental challenges. Biotic (e.g. pathogen attack) and abiotic (e.g. wounding / drought) stress events have previously been shown to stimulate the activity of certain TEs in plants. In this study, transcripts all four TE families (Gret1, Edel, Cremant and Noble) were found to increase when Pinot noir embryogenic callus (EC) cultures were co-cultivated with live yeast species endemic to New Zealand vineyards. Abiotic stresses and fungal extracts did not elicit the same response. A total of 24 new TE polymorphisms, relating to all four of the TE families analysed, were detected by S-SAP in a population of 183 vines regenerated from EC cultures. The majority (14) of these polymorphisms were found in vines regenerated from yeast-stressed EC tissue. The regenerated vines also displayed a variety of phenotypic abnormalities. Finally, whole-genome sequence data from twenty of the regenerated vines revealed that vines passaged through somatic embryogenesis experienced a general activation of the mobilome, resulting in an average of 64 new TE insertions per plant from both TE classes. Yeast stress at the embryogenic callus stage increased the number of new TE insertions identified by 63%. Despite a strong general bias against coding DNA sequence (CDS) insertions, approximately 2 insertions were found in this context per plant. These data are discussed with regards to the biological implications of endogenous TE mobilisation and the potential use of these elements for saturation mutagenesis in grapevine.

Grapevine powdery mildew, caused by Erysiphe necator, is a cosmopolitan pathogen that threatens grape production worldwide. Accurate disease forecasts would enhance disease management, particularly where severity varies substantially between years, such as in New York State. Ascospore release occurred before local bud break of Vitis vinifera in NY, WA, NC and NJ, and was quantitatively related to accumulation of degree days and rain events during overwintering. Acute cold temperature events (

Wine vineyards in humid environments like the mid-Atlantic United States are characterized by vines that develop too much vegetative growth for optimum quality wine production. Cover crops, rootstocks and rootzone restriction were evaluated for their effect on vegetative and reproductive growth on Cabernet Sauvignon. Treatments were arranged in a strip-split-split plot arrangement with under-trellis cover crops (UTCC) compared to row-middle only cover crop combined with 1-m weed-free strips in the vine row as main plots. Rootstocks riparia Gloire, 420-A, and 101-14 were sub-plots, while sub-sub-plots comprised two treatments: vines were either planted in root-restrictive (RR), fabric bags (0.016 m3) at vineyard establishment (2006), or were planted without root restriction. All three factors were effective in suppressing vegetative development as measured by rate and extent of shoot growth, lateral shoot development, trunk circumference, and dormant pruning weights. Canopies of vines with UTCC and RR had reduced leaf layer values by approximately 21% and 23% compared to conventional controls. The principal effect of the UTCC and the RR treatments was a sustained reduction in stem (xylem) water potential. UTCC and RR caused significant 7 and 10% reductions in berry weight, compared to their conventional controls. Berry weights of vines grafted to riparia were greater than those of vines grafted to other rootstocks. Wine made from UTCC and RR treatments increased red wine color compared to herbicide UTGC and NRR, respectively. This study identified treatments that improve vine balance while simultaneously improving grape composition and potential wine quality.

Green leaf volatiles (GLVs) are short-chain acylic aldehydes, alcohols and esters produced by plants via enzymatic metabolism of polyunsaturated fatty acids (PUFAs). GLVs are known to affect flavour and aroma of fruits and vegetables, including grapes. It has also been suggested that C6 and C5 GLVs are the precursors of volatile thiols, the aroma compounds that are important in Sauvignon blanc wine. GLVs are produced during grape berry development and released in high quantities upon berry crush. GLV aldehydes are formed from PUFAs by the action of two enzymes, lipoxygenase (LOX) and hydroperoxide lyase (HPL). This biochemical pathway is well characterised in other plant species and is known as the GLV pathway. To date, the GLV pathway has not been characterised in grapes. This thesis focuses on identification and initial characterisation of LOX and HPL genes and enzymes involved in the GLV-pathway. LOXs are a group of non-haem iron-containing dioxygenases that catalyse oxygenation of PUFAs producing PUFA-hydroperoxides. The most common PUFA substrates in plants are linoleic acid (LA, 18:2) and α-linolenic acid (LnA, 18:3). Depending on the position of oxygenation of the 18-carbon chain PUFAs, all plant LOXs are classified as 13(S) - and 9(S)-LOXs. 13(S)-LOXs are further regarded as type II and type I enzymes, depending on the presence or absence of transit peptides in the amino acid sequences of these enzymes respectively. HPLs belong to the CYP74 enzyme family, which is represented by atypical members of cytochrome P450 oxidases superfamily. HPLs cleave PUFA-hydroperoxides, producing aldehydes and oxoacids. Depending on the substrate specificity, HPLs are classified as 13-HPLs, 9/13-HPLs or 9-HPLs. The research reveals the complexity of the genetic makeup of the GLV metabolic pathway in grapes. Eighteen putative LOX genes and six putative HPL genes were identified in the Pinot noir grape genome. Phylogenetic analysis of the identified grape LOXs classified them as members of two groups, type II 13-LOXs and 9-LOXs, whereas all identified grape HPLs were classified as 13-HPLs (CYP-74B) and 9/13-HPLs (CYP-74C). Several LOX and HPL genes were expressed at different levels in Sauvignon blanc berry. Study of selected LOX and HPL gene expression revealed different levels of expression and differential tissue distribution of individual LOX and HPL genes within the berry. The studied genes also displayed different patterns of expression across different stages of berry development, upon wounding and in berries infected with Botrytis cinerea. Amongst the four LOX gene studied, transcripts of VvLOXA were the most abundant at all stages during berry development. VvLOXO was induced transiently upon berry damage and was a clear candidate involved in berry response to wounding. Expression levels of VvLOXC and VvLOXO were significantly increased in berries infected with Botrytis cinerea compared to the uninfected berries. In vitro biochemical analysis of the reaction products of recombinant VvLOXA (LOXA-TP) and VvLOXO (LOXO-TP) confirmed that these two enzymes are 13-LOXs. Both enzymes preferred LnA as a substrate. Both enzymes had the same temperature optima of 25°C, but preferred different pH conditions. Recombinant LOXA-TP preferred acidic environment and had pH optimum of pH 5.5, while LOXO-TP preferred neutral-to-basic conditions and had pH optimum of pH 7.5. Preliminary experiments with recombinant VvHPLA showed its ability to metabolise 13(S)-hydroperoxides, releasing C6 volatile aldehydes. Recombinant VvHPLA exhibited maximum activity with 13(S)-hydroperoxides of LnA as substrate at pH 5.0.

Phomopsis cane and leaf spot is a disease of grape ( Vitis spp.) caused by Phomopsis viticola(Sacc.). The fungus survives winter in grape cane tissues that were infected in previous years. In the spring, conidia are splashed by rain onto new growth where they infect the plant tissues. The fungus can infect many parts of the grape, including shoots, rachises, leaves, and fruits, and infection typically takes place when the tissues are immature. The disease is considered to be monocyclic. The control of the disease has usually been done by either selective pruning of diseased canes (reducing inoculum) or preventative spraying of protectant fungicides onto new tissues. To extend our understanding of the epidemiology and control the disease, studies were conducted to: 1) evaluate a disease warning system by applying fungicides and fungicide-adjuvant combinations based on predicted infection periods utilizing measured weather conditions; 2) determine efficacy of a dormant fungicide spray program for controlling the disease in its early stage of development (spring); 3) correlate commercial control practices and environmental conditions with disease incidence based on a state-wide survey of commercial vineyards; and 4) determine spatial pattern of the disease in small (within a vine) and large (among vines) spatial scales using a range of spatial analyses. With the warning system, control was often equal to that obtained with a 7-day calendar-based protectant program, but with fewer fungicide applications. However, fungicides and fungicide-adjuvant combinations used with the warning system did not show curative activity in a controlled-environment study. A dormant fungicide application provided consistent, yet only moderate, control of the disease; however, growers who applied a dormant application or spring protectants tended to have less disease incidence based on the results of the commercial survey. Analyses of spatial patterns revealed that the disease tended to aggregate within a vine, but clustering of the disease among vines was not common. Based on the control achieved with the warning system, the moderate control by dormant applications, and management-practice information from the survey, early season application of protective fungicides was shown to be a key factor for successful management of P. viticola.

Bitter rot, caused by the fungus Greeneria uvicola (Berk. & Curtis) Punith., is one of the most important fruit rot diseases that threaten the burgeoning winegrape (Vitis vinifera L.) industry in the southeastern United States. Epidemiological studies were conducted to examine the variation in aggressiveness among isolates, period of fruit susceptibility in V. vinifera, relative susceptibility of cultivars to bitter rot, and influence of temperature and duration of wetness on infection. Detached V. vinifera 'Chardonnay' fruit were inoculated with 10 isolates of G. uvicola obtained from fruit of V. vinifera, V. rotundifolia (muscadine grape), and a French-American hybrid. Isolates HCMD1 and HCMD5, obtained from V. vinifera grapes from a Maryland vineyard, were the most aggressive. Severity of disease on fruit inoculated with isolates collected from V. vinifera was significantly higher than with isolates collected from V. rotundifolia. The period of fruit susceptibility was distinguished by inoculating intact clusters of grapes in vineyards in Alamance Co. and Rockingham Co., NC, every 2 weeks from bloom until 2 weeks before harvest. Susceptibility of V. vinifera 'Merlot,' 'Chardonnay,' and 'Cabernet Franc' fruit increased from bloom until véraison in 2003 and from bloom until 2 weeks before véraison in 2004. The relative susceptibility of 38 cultivars and selections, including 23 V. vinifera cultivars and 5 French-American hybrids, was determined by inoculating and incubating detached fruit at 26°C. Fruit of V. vinifera were significantly more susceptible to infection by G. uvicola than French-American hybrids. V. vinifera 'Petite Sirah,' 'JB97-8-0-7,' 'MissBlanc,' 'Roussanne,' 'Mourvédre,' and 'Petit Verdot' were among the most susceptible to the bitter rot pathogen. V. aestivalis 'Cynthiana Norton,' V. vinifera 'Arkansas 1271' and 'Riesling,' and French-American hybrid 'Traminette' and 'Chardonel' were among the most resistant. Growth chamber studies also were conducted to examine the influence of temperature and duration of wetness on infection. Detached fruit of V. vinifera 'Cabernet Sauvignon,' 'Cabernet Franc,' and 'Chardonnay' were inoculated and incubated at 14, 18, 22, 26, or 30°C for 6, 12, 18, or 24 hours. The optimal conditions for infection of fruit by G. uvicola were a temperature of 23.7°C and 9 hours of wetness.

In field experiments conducted in northern Virginia during 1998, oils reduced photosynthesis, fruit maturity, and crop yield. Three applications of a 1.5% (v/v) oil/water emulsion were made to Chardonnay and Cabernet Sauvignon vine canopies at 6200 L/ha (Chardonnay) and 2440 L/ha (Cabernet Sauvignon). Net assimilation rate (NAR), soluble solids concentration (SSC), and berry size were reduced by oil treatments when compared to an untreated control. Also in 1998, a 1.5% (v/v) oil/water emulsion at 5550 L/ha and 4680 L/ha was applied to 23 wine grape cultivars. Eleven cultivars had significant foliar injury but injury was not related to reductions in fruit maturity. Experiments conducted in 1999 determined if reduced spray volumes or applications to only the fruit zone minimize reductions in NAR and SSC. Chardonnay and Cabernet Sauvignon were treated with JMS Stylet oil (1.5%) using 5600 L/ha or 1870 L/ha applied to the whole canopy or 930 L/ha applied to the fruit zone. The NAR of 5600 L/ha treated Cabernet Sauvignon was significantly lower than the NAR of control and other oil treatments on three measurement dates. The NAR of Chardonnay in the 5600 L/ha treatment was significantly lower than the NAR of control treatments in three measurements. Cabernet Sauvignon SSC was reduced by the 5600 L/ha and 1870 L/ha treatments, relative to the water treated control, on three sample dates but not at harvest. The SSC of Chardonnay in the 5600 L/ha treatment was reduced at harvest as compared to all other treatments. The SSC of Chardonnay in the 1870 and 930 L/ha treatments was reduced relative to the water control. Low volumes of oil caused less reduction in NAR and SSC; however, there are concerns about the efficacy of oils used at low volumes due to poor coverage.

Over the past decade, the face of Canterbury viticulture has changed substantially. This dissertation documents those changes and ascertains why certain cultivars were planted in Canterbury. A systems study of the Canterbury wine grape industry is presented incorporating production, economic and marketing aspects. A literature review incorporated current world and New Zealand statistics on vineyard areas and wine production and trade. Survey results from all known wine grape growers in Canterbury are presented. There are 215 ha planted, 141 ha of white and 74 ha of red cultivars. Pinot Noir and Riesling represent 47 ha each. Chardonnay (38 ha) has rapidly emerged as a promising cultivar and there is increasing interest in Sauvignon Blanc. An economic analysis revealed that annual gross returns are insufficient to cover direct costs. Estimated gross returns per hectare were low; Chardonnay $5700, Pinot Noir $5466, Riesling $4900, Sauvignon Blanc $4900, Merlot $5600 and Cabernet Franc $5600 are the cultivars recommended with most potential, but yields and prices for these are still too low to make the industry economically viable. Key problems identified include: • limited knowledge on, and certified availability of reliable clones • low yields resulting from original planting of poor clones • bird damage • seasonal climatic variation • spacing and training factors • lack of a suitable database for reliable management decisions Suggestions are made on the future direction for the Canterbury industry with emphasis on coordinated husbandry / economic / marketing research.

The effect of pre-fermentation enzyme maceration on anthocyanins, phenolics, colour and colour parameters was investigated in Pinot noir (Vitis vinifera L.) wines. Wines were made from Pinot noir musts that were macerated for 12 hours at 20°C with a commercial enzyme preparation, Rohapect VR-C (Rohm, Darmstadt, Germany), with and without additions of SO₂. Anthocyanins and phenolics were determined by spectrophotomeric and HPLC methods. Extraction of anthocyanins was not increased by enzyme treatment. However, wines produced by enzyme maceration had increased visible colour intensity which corresponded with increased colour density and polymeric pigment formation. By the end of fermentation, enzyme treated wines had more polymeric pigment and less monomeric anthocyanins than non-enzyme treated wines. The promotion of the formation of polymeric pigments in enzyme treated wines continued after the wines were bottled, and the greater content of polymeric pigment led to greater colour density in the enzyme treated wines. The promotion of polymeric pigment formation in wines by macerating enzyme treatment of the must may be due to the breakdown of protective polysaccharides-protein colloids by hemicellulase and protease activities in the enzyme preparation.

Early bunch-stem necrosis (EBSN) is a physiological disorder which can severely affect fruit set in grapevines. Poor set due to EBSN has been responsible for up to 50% crop losses in many New Zealand and Californian vineyards. The specific cause(s) of EBSN are unclear. Involved researchers presently acknowledge EBSN's correlation with site specific stress(es), with most maintaining that EBSN is the grapevines acclimation response to a reduced carbon status, with damage symptoms mediated through elevated rachis NH₄⁺ levels. The experiments reported here investigated the influence of NH₄NO₃, (NH₄)₂SO₄, CaCl₂, ethephon, and α-ketoglutarate treatments on the leaf and opposing inflorescence of single node Cabernet Sauvignon (Vitis vinifera L.) cuttings. Results from these investigations concluded that: • Incubation of cuttings in ethephon concentrations of 0.1 and 1 mM induced significant levels of EBSN. • Incubation of cutting in 10, 20, 30 mM NH₄NO₃, 5, 10, 15 mM (NH₄)₂SO₄, and 10, 20, 30 mM CaCl₂ induced only 'EBSN-like symptoms'. • Incubation of cuttings in 0.1, 1, and 10 mM α-ketoglutarate induced no significant reduction in EBSN or EBSN-like symptoms. Secondly ethylene production levels by excised grape inflorescences following NH₄NO₃, (NH₄)₂SO₄, CaCl₂, ethephon, and α-ketoglutarate treatments were measured. Conclusions drawn from investigations were that: • Incubation of cuttings in 0.1, 1, and 10 mM ethephon significantly increased ethylene production by excised grape inflorescences. • Incubation of cuttings in NH₄NO₃ or (NH₄)₂SO₄ treatments did not significantly influence ethylene production by the excised inflorescence. • Incubation of cuttings in CaCl₂ treatments did not significantly influence ethylene production by the excised inflorescence. • Incubation of cuttings in 0.1, 1, and 10 mM α-ketoglutarate treatments significantly increased ethylene production by excised grape inflorescences. The above conclusions clearly support the involvement of ethylene in the development of, and manifestations associated with, EBSN. The involvement of NH₄⁺, Ca²⁺ and α-ketoglutarate however remains unclear.