Academic literature on the topic 'Grapevines'

Grapevine Pinot gris virus (GPGV) is considered to be a causal agent of Grapevine Leaf Mottling and Deformation (GLMD) disease that has been reported worldwide through the grapevine-growing regions. Seven grapevines that were collected from a vineyard in the Czech Republic were tested for the presence of GPGV in leaf and phloem tissues. Each of the seven grapevines was infected by GPGV, from which sic symptoms were mostly shown without a typical mottling. The phylogeny based on RNA-dependent RNA polymerase and movement/coat protein sequences indicated the same origin of the GPGV isolates. The GPGV titer was the highest in the grapevines with the highest GLMD-like symptoms; however, some of the grapevines with milder GLMD-like symptoms had a lower GPGV titer than the asymptomatic grapevine. Soil analysis showed uneven boron content in the direct vicinity of the grapevines, while the boron content in the grapevines was more, even showing no boron deficiency. The quantitative analysis of selected gene expressions associated with boron efflux and transport only partially explained the boron content in the soil and grapevines and only in the grapevines growing in soils with the highest or lowest boron contents. The VvBor2 and VvNIP5 genes had a higher expression and VvNIP6 had a lower expression in the grapevine growing in the soil with the lowest boron content, while a low expression of VvBor1 and VvBor2 was observed in the grapevine that was grown in the soil with the highest boron content.

The re-emergence of Grapevine Trunk Diseases (GTDs), mainly Esca, has been observed in most of the world’s vineyards during the last two decades. Development of necrosis in grapevine wood, especially white-rot, is typically associated with Esca-diseased plants. One of the different methods being used in attempts to eradicate GTDs is curettage. This old cultural practice, which consists in surgically removing the necrotic wood, specifically white-rot, retaining only the non-necrotic tissue of Esca-diseased grapevine, is used in some European vineyards (Spain, France, Italy, Portugal), and is being increasingly reintroduced since 10 years ago in France. We, therefore, wanted to study the effect of curettage on vigour, fertility and berry quality, and year after year plant recovery. Our study synthetizes a 3-year experiment on Esca-diseased cv. Sauvignon blanc grapevines curetted in a commercial plot in the Bordeaux region. Asymptomatic control grapevines were compared to Esca-diseased grapevines without curettage (with typical foliar symptoms), and with curetted Esca-diseased grapevines (without foliar symptoms). Even if the curetted grapevines recovered lower vigour and fertility than the control plants, their grape berry quality was comparable, unlike for Esca-diseased grapevines. This cultural practice proved particularly effective in helping Esca-symptomatic grapevines to recover asymptomatic after treatment. Over time, curettage induces the resilience of grapevines, allowing them to recuperate their full physiological functioning, thereby compensating for Esca’s detrimental impact on berry quality.

Grapevine Pinot gris virus (GPGV) is a putative causal agent of grapevine leaf mottling and deformation disease that has been reported worldwide throughout the grapevine-growing regions. Fifty-four grapevines collected from five Algerian grapevine-growing regions were tested for the presence of GPGV in phloem tissues. Eight of the tested grapevines were infected by GPGV. Viromes of two selected Vitis vinifera cv. Sabel grapevines infected by GPGV and showing virus-like symptoms were analyzed by small RNA sequencing. Phylogenetic analyses of the partial coding sequence (cds) of the RNA-dependent RNA polymerase (RdRp) domain showed that all Algerian GPGV isolates were grouped with some already-described asymptomatic isolates. This study provides the first survey of the occurrence of GPGV in Algeria. Moreover, Grapevine fleck virus, Grapevine rupestris stem pitting-associated virus, Grapevine virus B, Grapevine rupestris vein feathering virus, Hop stunt viroid and Grapevine yellow speckle viroid 1 were detected in Algeria for the first time.

Grapevine cultivar (Vitis vinifera) and isolate of Eutypa lata influence wood and foliar symptoms of Eutypa dieback. Foliar symptoms of Eutypa dieback developed within 8 months of inoculating young grapevines (cvs. Grenache, Cabernet Sauvignon, and Merlot) in a shadehouse. Isolates of E. lata from various wine regions in southern Australia varied in their ability to colonize inoculated grapevines and induce wood and foliar symptoms. Grapevine cultivars varied for wood and foliar symptom expression but not for mycelial colonization. However, the severity of foliar symptoms was not related to the rate of spread of the fungus in the grapevine. Furthermore, the staining of wood typically attributed to E. lata did not reflect the presence of the fungus because the fungus was detected up to 80 mm beyond the stain. A field trial with mature grapevines revealed significant differences in the rate of spread of wood staining due to E. lata among eight cultivars, with up to 50 mm/year detected in Cabernet Sauvignon and Shiraz grapevines. In the shadehouse, the maximum growth rate of E. lata was recorded to be 115 mm/year for Grenache rootlings. Information from this study may help to optimize management strategies for maintaining productivity of grapevines with Eutypa dieback, thus reducing the economic impact of the disease.

A severe vein-clearing and vine decline syndrome has emerged on grapevines (Vitis vinifera) and hybrid grape cultivars in the Midwest region of the United States. The typical symptoms are translucent vein-clearing on young leaves, short internodes and decline of vine vigor. Known viral pathogens of grapevines were not closely associated with the syndrome. To obtain a comprehensive profile of viruses in a diseased grapevine, small RNAs were enriched and two cDNA libraries were constructed from a symptomatic grapevine and a symptomless grapevine, respectively. Deep sequencing of the two cDNA libraries showed that the most abundant viral small RNAs align with the genomes of viruses in the genus Badnavirus, the family Caulimoviridae. Amplification of the viral DNA by polymerase chain reaction allowed the assembly of the whole genome sequence of a grapevine DNA virus, which shared the highest homology with the Badnavirus sequences. This is the first report of a DNA virus in grapevines. The new DNA virus is closely associated with the vein-clearing symptom, and thus has been given a provisional name Grapevine vein clearing virus (GVCV). GVCV was detected in six grapevine cultivars showing vein-clearing and vine decline syndrome in Missouri, Illinois, and Indiana, suggesting its wide distribution in the Midwest region of the United States. Discovery of DNA viruses in grapevines merits further studies on their epidemics and economic impact on grape production worldwide.

Virus diseases are major constraints to Vitis vinifera wine grape production worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) was first confirmed in Georgia in 2008. The negative impacts of GLRaV-3, such as decreased fruit yield and quality, were confirmed from samples taken in 2009 and 2010. In light of these findings, studies were initiated to determine the prevalence and types of grapevine viruses present in vineyards in Georgia. Five vineyard blocks were visited during August and September of 2011 and 2012. Leaf samples were collected from a total of 50 grapevines showing symptoms of and suspected as being infected by grapevine leafroll disease (GLD). Samples from individual grapevines were extracted and tested separately for 20 grapevine viruses listed in standard virus indexing programs. Reverse transcription polymerase chain reaction (RT-PCR) or PCR, depending on the nature of the virus genome, was used to detect these viruses with species-specific primers. The results showed the presence of GLRaV-1, -2, -3, and -4 (strains 4, 5, and 6), grapevine virus B, grapevine rupestris stem pitting-associated virus, and grapevine red blotch virus. Many vineyards had multiple viruses detected from individual grapevines showing typical GLD or GLD-like symptoms.

Root collar excavation for control of Armillaria root disease of grapevine was investigated in two California vineyards (vineyard K1 and vineyard N1) from 2002 to 2004. The hypothesis tested was that root collar excavation, when timed in early stages of root collar infection, may cause mycelial fans of the pathogen Armillaria mellea to recede from the root collar before severe disease results from vascular tissue decay. In vineyard N1, excavation significantly increased yield and cluster weight of symptomatic grapevines; symptomatic-excavated grapevines had the same high mean cluster weight as healthy grapevines, and there were no significant effects of excavation on yield or pruning weight of healthy grapevines. In vineyard K1, where excavated root collars frequently refilled with soil, excavation had no significant effects on yield or pruning weight of symptomatic grapevines, and significantly reduced pruning weight and shoot weight of healthy grapevines. Reexamination in March 2004 revealed that mycelial fans had receded from root collars of symptomatic-excavated grapevines, but remained on root collars of symptomatic-nonexcavated grapevines. Root collar excavation appears to be a promising cultural approach for control of Armillaria root disease, as long as excavated root collars are kept clear of soil.

Grapevine Pinot gris virus (GPGV) has been identified in many grape growing countries of the world since 2012. The aim of this work was to investigate the presence of GPGV on some accessions collected from a germplasm collection, in addition to the propagation material and clonal selection samples. During 2019–2020, a total of 199 samples have been analysed by a double antibody sandwich – enzyme-linked immunosorbent assay (DAS-ELISA) for the presence of GPGV, Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus-1+3 (GLRaV-1+3) and Grapevine fleck virus (GFkV). Among them, 107 samples (53.76%) showed a GPGV-infection, associated with or without symptoms on the leaves (deformations, chlorosis, mosaic, wrinkles) or stunting plants. The distribution of infected varieties showed a high rate of infection in old varieties (37.38%), followed by clones (32.71%), rootstocks (11.21%), clonal selections (9.35%) and new varieties (9.35%). The tests revealed the association of GPGV with GFkV (5 cases) and GLRaV-1+3 (2 cases). GPGV should be included in the rules of grapevine certification schemes for the production of virus-free mother plants.

Abstract - The grapevine cultivar Grk, a close relative of Crljenak kaštelanski/Zinfandel, is grown exclusively in southern Croatia. Grapevine yellows-like symptoms were observed on vines in the vineyards in Lumbarda (southern Croatia) and in propagated grapevines near Zadar and Zagreb. The majority of the detected phytoplasma isolates belonged to the 16SrI group. However, RFLP pattern and R16F2n/R2 fragment sequence assigned one isolate to the 16SrIII group. Thus far, on cv. Grk, phytoplasmas belonging to three different groups have been detected: 16SrI, 16SrIII, and 16SrXII, which was confirmed previously. Aside from the 16SrI, 16SrV and 16SrXII phytoplasma groups previously found on grapevines in Croatia, the finding of 16SrIII group, which is not common on grapevines in Europe, adds to the diversity of phytoplasmas in a very small geographic region.

Horseweed is a common pest in vineyards of the San Joaquin Valley (SJV) of California. Interest in controlling this weed has increased with the recent discovery of a glyphosate-resistant (GR) biotype that has been observed to be more vigorous than a glyphosate-susceptible (GS) biotype in the SJV. However, the impact that either biotype may have on grapevine growth has not been assessed. Therefore, two glasshouse experiments were conducted to characterize the competitiveness of GR and GS horseweed biotypes from the SJV with young grapevines. ‘Syrah’ grapevines grafted to Freedom rootstocks were planted in 8-L plastic pots, alone, or with a single GR or GS horseweed. Additional GR and GS horseweeds were also planted separately in individual pots, and all plants were grown for 14 and 16 wk in 2006 and 2007, respectively. Grapevines grown with either biotype of the weed produced fewer leaves and amassed approximately 20% less dry mass (DM) than vines grown alone. The GR biotype reduced grapevine stem DM and length by 30%, but the GS biotype did not. The GR biotype accumulated more than twice the DM as the GS biotype, whether in competition with grapevine or not. Grapevines reduced the total leaf number of both horseweed biotypes by almost 50% and aboveground DM of GR and GS biotypes by 50 and 75%, respectively. These preliminary findings indicate that competition from horseweed can substantially reduce the growth of young grapevines and that the GR biotype may be more competitive than the GS biotype.

Grapevine Leafroll disease (GLD), one of the most destructive diseases of grapevines, has been found in every country where grapevines are grown. Grapevine Leafroll associated virus type 3 (GLRaV-3), one of several viruses associated with GLD globally, is the most prevalent virus in South African grapevines and therefore control of GLRaV-3 takes high priority in any strategy aimed at control of GLD. GLD can be controlled through the use of an integrated strategy which includes using certified plant material, controlling insect vectors through use of systemic insecticides and the removal of infected vines by roguing. Infected individuals are identified each autumn, using either symptom display (in red cultivars, where infected individuals display interveinal reddening and downward rolling of leaves) or ELISA (in symptomless white cultivars). ELISA is laborious, time consuming and relatively insensitivity compared to molecular techniques and a simpler, more rapid and more sensitive means of indentifying GLRaV-3 infected vines is required. A simple RNA extraction procedure combined with a single-tube reverse transcriptase loop-mediated amplification (RT-LAMP) has been developed which allows for the rapid, simple detection of GLRaV-3. Using RT-LAMP, a viral target can be amplified in 2 hours under isothermal conditions. This GLRaV-3 specific RTLAMP uses hydroxy napthol blue (HNB), a colourimetric indicator that changes from violet to sky blue only where a positive RT-LAMP reaction has occurred, making results quick and easy to interpret. The sensitivity of this technique was compared to ELISA and nested PCR by pooling samples at varying ratios of healthy to infected plants. Using nested PCR and RT-LAMP 1 infected sample could be detected amongst 50 healthy individuals while ELISA could only detect 1 amongst 30 infected making RT-LAMP more sensitive than ELISA. Further RT-LAMP could be performed in 2 hours compared to nested PCR and ELISA’s 8 and 48 hours respectively. Based on these results, RT-LAMP is viable alternative for ELISA for the detection of GLRaV-3 in the field. RT-LAMP was also tested for its ability to detect GLRaV-3 in grapevine rootstocks where, due to low viral titres and erratic distribution, it is notoriously difficult to detect. The rootstocks which were used for testing of GLRaV-3 had been tested in a previous study and it was found that only 28% of samples tested positive after 33 months (post inoculation). Using RT-LAMP, 78% of samples tested positive for GLRaV-3. Although further testing must be done, RT-LAMP may also be a viable alternative for testing grapevine rootstocks for GLRaV-3 infection.
Dissertation (MSc)--University of Pretoria, 2013.
gm2014
Microbiology and Plant Pathology
unrestricted

Thesis (MSc)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: The latest strategies in the protection of crops against microbial pathogens are rooted in harnessing the natural, highly complex defense mechanisms of plants through genetic engineering to ultimately reduce the application of chemical pesticides. This approach relies on an in-depth understanding of plant-pathogen interactions to develop reasonable strategies for plant improvement. Among the highly specialized defense mechanisms in the plant’s arsenal against pathogen attack, is the de novo production of proteinaceous antimicrobial peptides (AMPs) as part of the plant’s innate immunity. These AMPs are small, cysteine-rich peptides such as plant defensins that are known for their broad-spectrum of antifungal activity. These plant defensin peptides have been found to be present in most, if not all plant species and the defensin encoding genes are over-represented in plant genomes. Most of these defensins are generally the products of single genes, allowing the plant to deliver these molecules relatively rapidly and with minimal energetic expense to the plant. These factors contribute to establishing AMPs as excellent candidates for genetic engineering strategies in the pursuit of alternative crop protection mechanisms. The first antimicrobial peptide identified and isolated from grapevine, Vv-AMP1, was found to be developmentally regulated and exclusively expressed in berries from the onset of ripening. Recombinantly produced Vv-AMP1 showed strong antifungal activity against a wide range of plant pathogenic fungi at remarkably low peptide concentrations in vitro, however, no in planta defense phenotype could thus far be linked to this peptide. In this study, the antifungal activity of Vv-AMP1 constitutively overexpressed in its native host (Vitis vinifera) was evaluated against grapevine-specific necrotrophic and biotrophic fungi. Firstly, a hardened-off genetically characterised transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was generated and morphologically characterized. In order to evaluate the in planta functionality of Vv-AMP1 overexpressed in grapevine, this confirmed transgenic population was subjected to antifungal assays with the necrotrophic fungus, B. cinerea and the biotrophic powdery mildew fungus, Erysiphe necator. For the purpose of infection assays with a biotrophic fungus, a method for the cultivation and infection with E. necator was optimized to generate a reproducible pathosystem for this fungus on grapevine. Detached leaf assays according to the optimized method with E. necator revealed programmed cell death (PCD) associated resistance linked to overexpression of Vv-AMP1 that can be compared to that of the highly resistant grapevine species, Muscadinia rotundifolia. Contrastingly, whole-plant infection assays with B. cinerea revealed that Vv-AMP1 overexpression does not confer V. vinifera with elevated resistance against this necrotrophic fungus. An in silico analysis of the transcription of defensin-like (DEFL) genes previously identified in grapevine was included in this study. This analysis revealed putative co-expression of these DEFL genes and other genes in the grapevine genome driven by either tissue- or cultivar specific regulation or the plant’s response to biotic and abiotic stress stimuli. In conclusion, this study contributed to our knowledge regarding Vv-AMP1 and revealed an in planta defense phenotype for this defensin in grapevine. In silico analysis of the DEFL genes in grapevine further revealed conditions driving expression of these genes allowing for inferences to be made regarding the possible biological functions of DEFL peptides in grapevine.
AFRIKAANSE OPSOMMING: Die nuutste strategieë wat deel vorm van die beskerming van plant gewasse teen mikrobiese patogene het hul oorsprong in die inspanning van die natuurlike, hoogs gekompliseerde verdedigingsmeganismes van die plant deur middel van genetiese enginieurswese ten einde die gebruik van chemiese plaagdoders te verlaag. Hierdie benadering maak staat op ‘n in-diepte begrip van plant-patogeen interaksies om verstandige strategieë vir plantverbetering te kan ontwikkel. Van hierdie hoogs-gespesialiseerde verdedigingsmeganismses in die plant se arsenaal teen patogeen aanvalle sluit die de novo produksie van proteinagtige antimikrobiese peptiede (AMPs) in as deel van die plant se ingebore immuunstelsel. Hierdie AMPs is klein, sisteïen-ryke peptiede soos die plant “defensins” en is bekend vir hul breë-spektrum antifungiese aktiwiteit. Hierdie plant defensinpeptiede word aangetref in meeste, indien nie alle plant spesies nie en die defensin koderende gene word oor-verteenwoordig in plant genome. Meeste van hierdie defensins is gewoonlik die produkte van enkele gene wat die plant in staat stel om hierdie molekules relatief spoedig en met minimale energie verbruik in die plant te vorm. Hierdie faktore dra by tot die vestiging van AMPs as uitstekende kandidate vir genetiese ingenieursstrategieë as deel van die strewe na alternatiewe gewasbeskermingsmeganismes. Die eerste antimikrobiese peptied wat geïdentifiseer en geïsoleer is uit wingerd, Vv-AMP1, word beheer deur die ontwikkelingsstadium en word eksklusief uitgedruk in korrels vanaf die aanvang van rypwording. Rekombinant-geproduseerde Vv-AMP1 het sterk antifungiese aktiwiteit getoon teen ‘n wye reeks plantpatogeniese swamme teen merkwaardige lae peptied konsentrasies in vitro, alhoewel geen in planta verdedigingsfenotipe tot dusver gekoppel kon word aan hierdie peptied nie. In hierdie studie was die antifungiese aktiwiteit van Vv-AMP1 wat ooruitgedruk is in sy natuurlike gasheerplant (Vitis vinifera) ge-evalueer teen wingerd-spesifieke nekrotrofiese- en biotrofiese swamme. Eerstens is ‘n afgeharde geneties-gekarakteriseerde transgeniese V. vinifera (cv. Sultana) populasie wat Vv-AMP1 ooruitdruk gegenereer en morfologies gekarakteriseer. Om die in planta funksionaliteit van Vv-AMP1 ooruitgedruk in wingerd te evalueer is hierdie bevestigde transgeniese populasie blootgestel aan antifungiese toetse met die nekrotrofiese swam, B. cinerea en die biotrofiese swam, Erysiphe necator. Vir die doel om infeksiestudies uit te voer met ‘n biotrofiese swam is ‘n metode geoptimiseer vir die kweek en infeksies met E. necator wat gelei het tot ‘n herhaalbare patosisteem vir hierdie swam op wingerd. Blaarstudies, volgens die pas-verbeterde metode vir E. necator infeksies het ‘n geprogrammeerde seldood-geassosieërde weerstand, gekoppel aan die ooruitdrukking van Vv-AMP1 onthul, wat vergelyk kan word met dié van die hoogs-weerstandige wingerdspesie, Muscadinia rotundifolia. Hierteenoor het heel-plant infeksie studies met B. cinerea onthul dat Vv-AMP1 ooruitdrukking geen verhoogde weerstand teen dié nekrotrofiese swam aan V. vinifera bied nie. ‘n In silico analise van die transkripsie van defensin-agtige (DEFL) gene wat vroeër in wingerd geïdentifiseer is, is by hierdie studie ingesluit. Hierdie analise het vermeende gesamentlike uitdrukking van hierdie DEFL gene en ander gene in die wingerd genoom onthul wat aangedryf word deur weefsel- of kultivar-spesifieke regulering of die plant se reaksie tot biotiese en abiotiese stress stimuli. Ten slotte, hierdie resultate het bygedra tot ons kennis in verband met Vv-AMP1 en het ‘n in planta verdedigingsfenotipe vir hierdie defensin in wingerd onthul. In silico analiese van die DEFL gene in wingerd het verder toestande onthul wat die uitdrukking van hierdie gene aandryf wat ons toelaat om aannames te maak ten opsigte van die moontlike biologiese funksies van DEFL peptiede in wingerd en ondersteun die opstel en toets van hipoteses vir die rol en megansimes van aksie van die wingerd defensin familie.

Grapevine leafroll-associated virus-2 (GLRaV-2), GLRaV-3, and grapevine fleck virus (GFkV) are widespread in grapes around the world. These viruses can cause significant crop loss and affect wine quality by reducing sugar accumulation and compromising skin color. Mealybugs are vectors of grapevine leafroll-associated viruses (GLRaVs). A statewide survey of commercial and wild grapevines in Virginia was conducted during 2009 through 2011. Also, vector management options were tested in two field studies. GLRaV-2, GLRaV-3, and GFkV were detected in 8%, 25%, and 1%, respectively, of over 1,200 vine samples (41 wine grape varieties) from 77 locations, and 64% of vineyards were positive for at least one of the tested viruses. All 100 wild grapevines tested were free of these three viruses, indicating that they are not alternative hosts. The majority of infected vines from commercial vineyards were planted prior to the 1990\'s; however, some new plantings were also found to be positive, indicating movement of the viruses among vineyards and also potential infection prior to planting. The high frequency of virus-infected vines emphasizes the importance of clean plant materials, as well as management of vector insects. The insecticide trials resulted in promising vector control with dinotefuran and spirotetramat; however, acetamiprid and pryrethroid resulted in an increase in mealybug population. This study is the first to examine multiple grape viruses in VA. It will aid in developing better strategies aimed at controlling mealybugs to restrict the movement of viral diseases.
Master of Science in Life Sciences

Thesis (MScAgric)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Petri grapevme decline, also known as black goo, slow die-back and Phaeoacremonium grapevine decline, causes significant losses of young vines worldwide. Species of Phaeoacremonium, Phaeomoniella chlamydospora and related genera are associated with this grapevine disease. This study investigates the Phaeoacremonium-complex and Phaeomoniella chlamydospora, focussing on the species isolated from grapevines. Fungicide sensitivity of Pa. chlamydospora and the possibility of employing molecular techniques for the detection of Pa. chlamydospora in grapevines were also investigated. In an overview of the literature on Petri grapevine decline the disease history and the relatedness of Petri grapevine decline to esca is discussed. Petri grapvine decline occurs in propagation material or young vines. Infected material can appear asymptomatic and therefore the possibilities of molecular techniques for identification were also investigated in the literature. In South Africa Pa. chlamydospora is the dominant organism causing Petri grapevine decline and therefore different fungicides were evaluated to control this fungus. Six isolates of Pa. chlamydospora, from Stellenbosch, Wellington, Somerset West and Malmesbury of Western Cape province, South Africa, were screened against twelve fungicides testing their effect on mycelial inhibition in vitro. These fungicides included benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole and thiram. Results provided the base-line sensitivity of South African isolates of Pa. chlamydospora. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride and tebuconazole were the most effective (with EC50 values ranging from 0.01 to 0.05 ug/ml) for inhibiting mycelial growth of Pa. chlamydospora in vitro. This in vitro test gave a good indication of which fungicides could be selected for further studies in glasshouses and nurseries. The molecular phylogeny of Phaeoacremonium and Phaeomoniella isolates from grapevines of South Africa, or isolates obtained from the Centraalbureau voor Schimmelcultures (CBS) in the Netherland, were investigated. Sequence data were created from the rONA region and partial B-tubulin gene of 33 of these isolates using the PCR technique. This sequence data were analysed with PAUP* version 4.Ob2a. An analysis of the sequence data confirmed the genus Phaeomoniella to be distinct from Phaeoacremonium (Pm.) based on DNA phylogeny. Although morphologically similar, the species status of Pm. aleophi/um and Pm. angustius was confirmed with DNA phylogeny and cultural characteristics. Pm. aleophilum has an optimum growth rate at 30°C and the ability to grow at 35°C, where as Pm. angustius has an optimum growth rate at 25°C and cannot grow at 35°C_ Pm. viticola was shown to be synonymous with Pm. angustius, and a new species, Pm. mortoniae, was newly described from grapevine occurring in California. Futhermore, Pm. aleophilum was newly reported from South Africa and grapevine isolates thought to be Pm. inflatipes were all re-identified as Pm. aleophilum. These findings therefore also shed some doubt on the possible role of Pm. inflatipes in Petri grapevine decline. It was confirmed that Pa. chlamydospora, Pm. aleophilum and Pm. angustius are the species involved in Petri grapevine decline. Pm. mortoniae was isolated from grapevines, but its pathogenicity should still be confirmed and the role of Pm. injlatipes in Petri grapevine decline remains unclear. Pa. chlamydospora has been routinely isolated from symptomless propagation and nursery material. Because the disease can take years to develop, it is crucial that healthy propagation material is used at planting. Pa. chlamydospora is a slowgrowing fungus, and positive identification from symptomless grapevine tissue can take up to 4 wks. The possibility of employing molecular techniques for the detection of Pa. chlamydospora in apparently healthy grapevines was investigated. Speciesspecific primers (PCLI and PCL2) based on the regions ITSI and ITS2 were designed for Pa. chlamydospora. These primers were highly sensitive and amplification was achieved from genomic DNA of Pa. chlamydospora from as low as 16 pg. Phaeoacremonium spp., related genera and common fungal taxa from grapevines were tested with these primers, but positive amplification was achieved for Pa. chlamydospora only. The presence of Pa. chlamydospora in symptomless grapevine tissue culture plants was confirmed by PCR within 24 hours. These primers therefore allow rapid and accurate identification of Pa. c~lamydospora. Testing on a larger scale with nursery material should be conducted to determine the feasibility of using these species-specific primers in the grapevine industry.
AFRIKAANSE OPSOMMING: Petri-terugsterwing van jong wingerde, ook algemeen bekend as "black goo" en Phaeoacremonium-terugsterwing, veroorsaak wêreldwyd groot geldelike verliese in die wingerdbedryf. Spesies van Phaeoacremonium, Phaeomoniella chlamydospora en verwante genera word met hierdie wingerdsiekte geassosieer. In die tesis word In oorsig gegee van die geskiedenis van hierdie siekte, die verwantskap tussen Petriterugsterwing en esca, en moontlike maniere van siektebestuur. Swamme wat by die siektekompleks betrokke is, kan in simptoomlose plantweefsel voorkom en daarom is die moontlikhede van die gebruik van molekulêre tegnieke vir swamidentifikasie in oënskou geneem. In Suid-Afrika is Pa. chlamydospora die dominante swam wat met Petriterugsterwing geassosieerword, gevolglik is verskillende fungisiedes vir die chemiese beheer van Pa. chlamydospora geëvalueer. Ses isolate van Pa. chlamydospora, versamel vanaf verskillende areas in die Wes-Kaap provinsie, is in dié studie gebruik. Benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole en thiram se effek op miselium inhibisie van Pa. chlamydospora is in vitro geëvalueer. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole was die mees effektiewe middels. Die effektiewe konsentrasie waarby 50% van die miselium groei geïnhibeer is (EKso),was tussen 0.01 en 0.05 ug/ml vir die mees effektiewe groep middels. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole het in vitro goeie potensiaal getoon, en verder toetse moet in vivo uitgevoer word. 'n Molekulêre studie is van Phaeoacremonium en Phaeomoniella isolate; verkry uit Suid-Afrikaanse wingerde, of vanaf die "Centraalbureau voor Schimmelcultures" (CBS) van Nederland; gedoen. Deur van die PKR tegniek gebruik te maak, is die basispaaropeenvolgingsdata van 33 isolate, van die ITSl, 5.8S, ITS2 rDNA area en die gedeeltelike B-tubullen geen verkry. Gekombineerde molekulêre data het die teorie ondersteun dat Phaeomoniella (Herpotrichiellaceae) gedistansieerd is van Phaeoacremonium (Magnaporthaceae). Pm. aleophilum en Pm. angustius was morfologies moeilik onderskeibaar, maar kon op grond van molekulêre data en kulturele eienskappe onderskei word. Pm. aleophilum se optimum groeitemperatuur was by 30°C en die swam besit die vermoë om by 35°C te groei. Pm. angus/ius se optimum groeitemperatuur was by 25°C, maar het nie by 35°C gegroei nie. 'n Studie van molekulêre en kulturele eienskappe het getoon dat Pm. angus/ius en Pm. viticola sinoniem is. 'n Nuwe spesie, Pm. mortoniae, wat uit wingerde van Kalifornie geïsoleer is, is beskrywe. Verder is Pm. aleophilum die eerste keer in Suid-Afrikaanse wingerde aangetref en Pm. tnflatipes isolate, wat vanuit wingerde geïsoleer is, is almal met molekulêre data gewys om Pm. aleophilum te wees. Hierdie bevindinge trek die rol van Pm. inflatipes in Petri-terugsterwing van wingerde in twyfel. Phaeomoniella chlamydospora IS m voortplantingsmateriaal en kwekerystokkies opgespoor. Omdat dit jare kan duur voordat siektesimptome ontwikkel, is dit belangrik om vroegtydig te weet of jong stokkies met Pa. chlamydospora geïnfekteer is. Pa. chlamydospora groei baie stadig en positiewe identifikasie van simptoomlose infeksies duur tot vier weke. Die toepassing van molekulêre tegnieke vir die vinnige identifikasie van Pa. chlamydospora in wingerde is dus ondersoek. Spesie-spesifieke oligonukleotiedes (PCU en PCL2) is vir Pa. chlamydospora ontwerp. Hierdie oligonukleotiedes is uiters sensitief en genomiese DNA van Pa. chlamydospora is van so laag as 16 pg geamplifiseer. Phaeoacremonium spp., verwante genera en algemene swamme vanuit wingerdmateriaal is met die oligonukleotiedes getoets, maar positiewe amplifikasie was slegs met Pa. chlamydospora moontlik. Die teenwoordigheid van Pa. chlamydospora is binne 24 uur in asimptomatiese wingerd weefselkultuurplantjies bevestig. Hierdie oligonukleotiedes identifiseer Pa. chlamydospora vinnig en akkuraat en toetsing op 'n groter skaal moet vervolgens met kwekerymateriaal onderneem word.

La sequera és una de les majors limitacions per a l’agricultura en general, factor que pot incrementar d’acord amb les prediccions del canvi climàtic. Per això, reduir l’ús de l’aigua en el reg i augmentar l’eficiència en l’ús de l’aigua (EUA) constitueix una de les majors prioritats per aconseguir una agricultura sostenible. L’EUA és un balanç entre guanys de biomassa i les despeses d’aigua. Aquesta Tesi s’ha centrat en l’estudi de 3 dels processos fisiològics que afecten a l’ús de l’aigua i a la producció de la planta en el cas de la vinya, i que per tant poden ser considerats factors potencials per millorar l’EUA: (1) conductància del mesòfil, (2) transpiració nocturna (3) respiració. Els resultats d’aquesta Tesi revelen que la conductància del mesòfil i la respiració són els principals components per millorar l’EUA permetent millorar l’assimilació de carboni o minimitzant les pèrdues del carboni fixat per la fotosíntesis

Thesis (MScAgric )--Stellenbosch University, 2004.
ENGLISH ABSTRACT: In an attempt to combat some of the pathogens that are associated with trunk diseases and disorders of grapevines, research in this thesis focused on the taxonomy and pathological aspects of Coniellai/Pilidiella, Botryosphaeria and Phomopsis spp. Previously, conidial pigmentation was used to separate Pilidiella from Coniella. Recently, however, the two genera have been regarded as synonymous, with the older name, Coniella, having priority. The most important species in the Coniellai/Pilidiella complex of grapevines is C. diplodiella (Speg.) Petr. & Syd., the causal organism of white rot of grapevines. Previous studies found it difficult to distinguish between C. diplodiella and C. fragariae (Oudem.) B. Sutton, which is known to occur in soil and caused leaf diseases of Fragaria and Eucalyptus. Both these species have previously been reported from South Africa. None of the reports on C. diplodiella could be scientifically substantiated; therefore it is still a quarantine organism. However, this status has been questioned. Based on sequence analyses of the internal transcribed spacer region (ITS 1, ITS 2), 5.8S gene, large subunit (LSU) and elongation factor 1- α gene (EF l- α) from the type species of Pilidiella and Coniella, Coniella was separated from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by species with hyaline to pale brown conidia (avg. length: width >1.5), with Coniella having dark brown conidia (avg. length: width ≤1.5). Pilidiella diplodiella, previously C. diplodiella, causal organism of white rot of grapevines, was shown to be an older name for C. petrakii. This fungus is present in South Africa and is therefore no longer of quarantine importance. Based on analyses of the histone (H3) gene sequences of isolates in the P. diplodiella species complex, P. diplodiella was separated from a newly described species, P. diplodiopsis. A new species, P. eucalyptorum, is proposed for isolates formerly treated as C. fragariae, associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens was newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. The genus Botryosphaeria Ces. & De Not. are known to be cosmopolitan, with broad host ranges and geographical distributions. Several saprotrophic species have been reported from grapevines, while others are severe pathogens of this host. These species include B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.J.L. Phillips and B. ribis Grossenb. & Duggar. Species reported from South Africa as grapevine pathogens are B. obtusa, B. dothidea, B. ribis and B. vitis (Schulzer) Sacco. In the present study, morphological, DNA sequence data (ITS 1, 5.8S, ITS 2 and EFI-α) and pathological data were used to distinguish 11 Botryosphaeria spp. associated with grapevines from South Africa and other parts of the world. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. were confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme were described as new species. Although isolates of B. dothidea and B. stevensii were confirmed from grapevines in Portugal, neither of these species, nor B. ribis, were isolated in this study. All grapevine isolates from Portugal, formerly presumed to be B. rib is, are identified as B. parva based on EF1-α sequence data. Artificial inoculations on grapevine shoots showed that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes was identified as morphologically similar, but phylogenetically distinct from D. sarmentorum, while D. sarmentorum was confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria, and is thus regarded as a probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs. The genus Phomopsis (Sacc.) Bubak contains many species that are plant pathogenic or saprotrophic. Ten species are known from grapevines. However, only two have been confirmed as being pathogenic, namely P. viticola (Sacc.) Sacc., causal organism of Phomopsis cane and leaf spot and P. vitimegaspora Kuo & Leu (teleomorph Diaporthe kyushuensis Kajitani & Kanem.), causal organism of swelling arm disease of grapevines. P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, a known pathogen from Prunus sp., was shown to be a possible pathogen of grapevines in a previous study. D. perjuncta Niessl. causes bleaching of dormant canes only and is therefore of little importance as a grapevine pathogen. Recently a number of Phomopsis isolates were obtained from grapevines in the Western Cape province of South Africa. Isolations were made from Phomopsis-like symptoms, pruning wounds and asymptomatic nursery plants. These isolates showed great variation in morphology and cultural characteristics. Earlier taxonomic treatments of Phomopsis, based species identification on host specificity, cultural characteristics and morphology. Recent studies have indicated that these characteristics can no longer be used to distinguish species of Phomopsis due to wide host ranges and morphological plasticity of some species. The use of anamorph/teleomorph relationships in species identification is also untenable, since Diaporthe teleomorphs have only been described for approximately 20% of the known Phomopsis species. In this study morphological data, DNA sequences (ITS-I, 5.8S, ITS-2) and pathogenicity data were combined to distinguish Phomopsis spp. from grapevines. Fifteen species of Phomopsis were delineated by phylogenetic analysis of ITS sequence data. Diaporthe helianthi, a sunflower pathogen, was reported from grapevines for the first time, with a further six, unknown species also distinguished. Three different clades contained isolates previously identified as D. perjuncta. Based on type studies, it appeared that the name D. viticola was available for collections from Portugal and Germany, a new species, D. australafricana, was proposed for South African and Australian isolates, formerly treated as D. perjuncta or D. viticola. An epitype specimen and culture were designated for D. perjuncta. This species was distinguished from D. viticola and D. australafricana based on morphology and DNA phylogeny. Artificial inoculations of green grapevine shoots indicated that, of the species tested, P. amygdali, a known pathogen of peaches in the USA, and P. viticola were the most virulent.
AFRIKAANSE OPSOMMING: In 'n poging om sommige patogene geassosieer met stamsiektes en syndrome, te beveg, het die navorsing in die tesis gefokus op die taksonomie en patologiese aspekte van ConiellaiPilidiella, Botryosphaeria en Phomopsis spp Voorheen is konidium pigmentasie gebruik om Pilidiella (hialien tot ligbruin konidia) van Coniella (donkerbruin konidia) te skei. Onlangs is hierdie twee genera egter as sinoniem beskou met die ouer naam, Coniella, wat voorkeur gekry het. Die belangrikste spesies in die ConiellaiPilidiella kompleks van wingerd is C. diplodiella (Speg.) Petr. & Syd., die veroorsakende organisme van witvrot van wingerd. Vorige studies het dit moeilik gevind om te onderskei tussen C. diplodiella en C. fragariae (Oudem.) B. Sutton, wat bekend is dat dit in grond voorkom en ook blaarsiektes van Fragaria en Eucalyptus veroorsaak. Beide hierdie spesies is tevore in Suid-Afrika aangemeld. Geen van die aanmeldings van C. diplodiella is egter wetenskaplik bewys nie en daarom is dit steeds 'n kwarantyn organisme. Hierdie kwarantyn status is egter bevraagteken. Op grond van DNS volgordes van die interne getranskribeerde spasieerder area ("ITS 1", "ITS2"), die 5.8S rRNS geen, die groot ribosomale subeenheid ("LSU") en die verlengingsfaktor 1-α geen ("EF-lα") van die tipe spesies van Pilidiella en Coniella, is Coniella van Pilidiella geskei, met die meerderheid van die taxa wat binne Pilidiella resorteer. Pilidiella word gekarakteriseer deur spesies met hialien tot ligbruin konidia (gem. lengte: breedte > 1.5), in teenstelling met die donkerbruin konidia van Coniella (gem. lengte: breedte ≤ 1.5). Daar is verder bewys dat Pilidiella diplodiella, voorheen C. diplodiella, veroorsakende organisme van witvrot van wingerd, die ouer naam van C. petrakii is. Hierdie swam is teenwoordig in Suid-Afrika en P. diplodiella is dus nie meer van kwarantyn belang nie. Op grond van analises van die histoon (H3) volgordes van spesies in die P. diplodiella spesies kompleks, is P. diplodiella geskei van 'n nuut beskryfde spesie, P. diplodiopsis. 'n Nuwe spesie, P. eucalyptorum, is ook voorgestel vir isolate voorheen beskou as C. fragariae, geassosieer met blaarvlek van Eucalyptus spp. Hierdie spesie het basaal van Pilidiella gegroepeer en mag moontlik nog 'n derde genus binne hierdie kompleks verteenwoordig. Pilidiella destruens is nuut as anamorf van Schizoparme destruens beskryf, wat geassosieer word met loot terugsterwing van Eucalyptus spp. in Hawaii. Die genus Botryosphaeria Ces. & De Not. is bekend as kosmopolitaans met 'n wye gasheerreeks en geografiese verspreiding. Verskeie saprofitiese spesies is aangemeld vanaf wingerd, terwyl ander ernstige patogene van hierdie gasheer is. Laasgenoemde spesies sluit in B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.1.L. Phillips en B. ribis Grossenb. & Duggar. Spesies aangemeld in Suid-Afrika as wingerdpatogene, is B. obtusa, B. dothidea, B. ribis en B. vitis (Schulzer) Sacco In hierdie studie is morfologiese, DNS volgorde data ("ITSl", "ITS2", 5.8S en "EF-Iα") en plantpatologiese data gebruik om II Botryosphaeria spesies, geassosieer met wingerde in Suid-Afrika en verskeie ander werelddele, te onderskei. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina en 'n Diplodia sp. is bevestig van wingerde in Suid-Afrika, terwyl Diplodia porosum, Fusicoccum viticlavatum en F. vitifusiforme as nuwe spesies beskryf is. AIhoewel isolate van B. dothidea en B. stevensii bevestig is van wingerde in Portugal, is geen van hierdie spesies en ook nie B. ribis geïsoleer nie. AIle isolate vanaf wingerd in Portugal, voorheen beskou as B. rib is, is as B. parva op grond van hul "EF-lα" volgordes geïdentifiseer. Uit kunsmatige isolasies gemaak op wingerdlote is die gevolgtrekking gemaak dat B. australis, B. parva, B. ribis en B. stevensii meer virulent is as die ander spesies wat bestudeer is. Die Diplodia sp. versamel vanaf wingerdlote is geïdentifiseer as morfologies eenders, maar filogeneties verskillend van D. sarmentorum, terwyl D. sarmentorum bevestig is as die anamorf van Otthia spiraeae, die tipe spesie van die genus Otthia (Botryosphaeriaceae). 'n Kultuur wat as 0. spiraeae geïdentifiseer is, het binne Botryosphaeria gegroepeer, en word dus as 'n moontlike sinoniem beskou. Hierdie bevindinge bevestig vroeëre voorstelle dat die generiese konsep van Botryosphaeria uitgebrei behoort te word om genera met gesepteerde askospore en Diplodia anamorwe in te sluit. Die genus Phomopsis (Sacc.) Bubak bevat verskeie spesies wat as of plantpatogenies, of saprofities, beskryf is. Tien spesies is bekend op wingerd. Slegs twee is as patogenies bevestig, naamlik P. viticola (Sacc.) Sacc., veroorsakende organisme van loot-en-blaarvlek ("streepvlek") en P. vitimegaspora Kuo & Leu (teleomorf Diaporthe kyushuensis Kajitani & Kanem.), veroorsakende organisme van geswelde arm van wingerd. In 'n vroeëre studie is bevind dat P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, 'n bekende patogeen van Prunus sp., moontlik ook 'n patogeen van wingerd mag wees. D. perjuncta Niessl. veroorsaak egter net verbleiking van dormante lote en is dus van min belang as 'n wingerd patogeen. Gedurende die afgelope twee jaar is verskeie Phomopsis isolate van wingerde in die Wes-Kaap provinsie van Suid-Afrika verkry. Isolasies is gemaak van Phomopsis-agtige simptome, snoeiwonde en asimptomatiese kwekeryplante. Die isolate verkry uit hierdie materiaal het groot variasie ten opsigte van morfologie en kultuureienskappe getoon. Vroeëre taksonomiese verhandelings van Phomopsis het spesies-identifikasie op gasheerspesifisiteit, kultuureienskappe en morfologie gebasseer. Onlangse studies het egter getoon dat, weens wye gasheerreekse en morfologiese plastisiteit van somnuge spesies, hierdie eienskappe me meer gebruik kan word om Phomopsis spesies te identifiseer nie. Die gebruik van anamorflteleomorf verwantskappe in die identifikasie van Phomopsis spesies ook onbruikbaar omdat Diaporthe teleomorwe vir slegs ongeveer 20% van die bekende Phomopsis spesies beskryf is. Die huidige studie het dus morfologiese data, DNS volgordes ("ITS 1", 5.8S, "ITS2") en patogenisiteitsdata gekombineer ten einde Phomopsis spp. vanaf wingerd te identifiseer. Vyftien Phomopsis spesies is deur die filogenetiese analise van die interne getranskribeerde spasieerder area ("ITS") volgordes geskei. Diaporthe helianthi, 'n bekende patogeen van sonneblomme, is vir die eerste maal op wingerd aangeteken, terwyl 'n verdere ses, tans onbekende spesies van Phomopsis ook geidentifiseer is. Drie verskillende groepe het isolate bevat wat voorheen as D. perjuncta geidentifiseer is. Gebasseer op studies van tipes, het dit voorgekom dat die naam D. viticola beskikbaar is vir isolate uit Portugal en Duitsland. 'n Nuwe spesie, D. australafricana, is voorgestel vir Suid-Afrikaanse en Australiese isolate wat voorheen behandel is as D. perjuncta of D. viticola. 'n Epitipe monster en kultuur is vir D. perjuncta benoem. Hierdie spesie is van D. viticola en D. australafricana onderskei op grond van morfologie en DNS filogenie. Kunsmatige inokulasies van groen wingerdlote het getoon dat P. amygdali, bekende perske patogeen, en P. viticola die mees virulent was.

Eutypa dieback of grapevines, caused by Eutypa lata, is a major cause of reduced longevity in vineyards worldwide. The fungus grows in the woody tissue of infected vines, producing translocatable toxins that cause foliar symptoms of the disease. By the time foliar symptoms are evident the pathogen may have become well established in the vine. One aim of this study was to develop DNA markers to allow rapid reliable identification of E. lata and to detect the pathogen in infected wood. The second aim was to analyse secondary metabolite production by E. lata in order to gain information on the compounds responsible for the foliar symptoms of the disease and to identify metabolites which could be used as markers to detect the early stages of the disease prior to the expression of foliar symptoms. In addition, genetic variation of the pathogen was assessed using RFLP and RAPD analysis. Two techniques were used to develop DNA markers; first, SCAR markers derived from RAPD fragments were developed and, second, an E. lata genomic DNA library was constructed, from which DNA fragments specific to E. lata were identified. These markers were used in either PCR- or Southern hybridisation-based assays to detect the pathogen in infected wood. PCR-based detection of the pathogen in infected wood was prone to inhibition by phenolic compounds, however, Southern hybridisation techniques were capable of detecting E. lata in wood. Genetic variation among 38 isolates of E. lata was assessed using six randomly selected clones from the genomic DNA library. A subset of 11 isolates was subjected to RAPD analysis using 10 random primers. Considerable genetic diversity, in terms of RFLP and RAPD profiles, was observed among isolates. There was no apparent correlation between grouping of isolates following neighbour joining analysis and either host species or geographic origin of isolates. The RAPD and RFLP profiles of two isolates differed significantly from the majority of the other isolates. These isolates, which were morphologically similar to all other isolates, were subsequently found not to be E. lata. Secondary metabolite production of 11 isolates was analysed by HPLC following growth on a range of media. A wider range of secondary metabolites was detected in E. lata than has previously been reported. Two of the secondary metabolites, eutypine and an unidentified compound with a retention time of 19.6 min, were produced by eight of nine isolates of E. lata. Neither of the non-E. lata isolates produced these compounds. It was concluded that the remaining isolate of E. lata may have lost the ability to produce these compounds following storage. Whilst a wider range of isolates needs to be screened before a candidate marker can be selected, these results suggest that certain compounds are present in the majority of E. lata isolates and, hence, may prove suitable markers for the detection of the pathogen prior to the expression of foliar symptoms. The molecular probes developed in this study will allow the rapid and reliable identification and detection of E. lata in grapevine cane or wood. These probes also have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata. Suitable control measures could then be applied to vines which have been shown by the use of chemical markers to have latent infection. Used in combination, therefore, the DNA and biochemical markers could facilitate improved management of eutypa dieback.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2003.

The possibility of grapevines infecting with F. proliferatum and F. oxysporum at the flowering stage is proven. The methods of infection penetration can be different, both through injuring the conductive tissue of inflorescence (axes, ridges), and through flowers (in case of violation of the integrity of delicate tissues by the wind, agricultural tools, etc.). However, the damage caused to the plant by the disease in all these cases is almost the same.

The purpose of this work was to study the development of the eyelets and shoots of white table grapes variety ‘Huseyne’ in different ecological zones of Northern Tajikistan depending on the load left (60, 80, 100 eyelets), planting pattern and height of the stem (3 × 2 m, height of the stem 80 cm; 3 × 2 m, height of the stem 120 cm; 4 × 3.7 + 0.6 m, paired, height of the stem 140 cm). On high-stem grapevines, the fruit-bearing capacity (productivity) of shoots increases. This is especially noticeable in paired plantings, where the amount of manual works reduces and becomes more automated. Also, due to good aeration, the resistance of plants to diseases like mildew and gray mold increases.

Grapevine yellows diseases characterized by similar symptoms have been reported in several countries including Israel, the United States, France, Italy, Spain, Germany and Australia. These diseases are among the most serious known in grapevine, but precise knowledge of the pathogens' identities and modes of their spread is needed to devise effective control stratgegies. The overall goals of this project were to develop improved molecular diagnostic procedures for detection and identification of the presumed mycoplasmalike organism (MLO) pathogens, now termed phytoplasmas, and to apply these procedures to investigate impact and spread and potential for controlling grapevine yellows diseases. In the course of this research project, increased incidence of grapevine yellows was found in Israel and the United States; the major grapevine yellows phytoplasma in Israel was identified and tis 16S rRNA gene characterized; leafhopper vectors of this grapevine yellows phytoplasma in Israel were identified; a second phytoplasma was discovered in diseased grapevines in Israel; the grapevine yellows disease in the U.S. was found to be distinct from that in Israel; grapevine yellows in Virginia, USA, was found to be caused by two different phytoplasmas; both phytoplasmas in Virginia grapevines were molecularly characterized and classified; commercial grapevines in Europe were discovered to host a phytoplasma associated with aster yellow disease in the USA, but this phytoplasma has not been found in grapevine in the USA; the Australian grapevine yellows phytoplasma was found to be distinct from the grapevine phytoplasmas in Israel, the United States and Europe and was described and named "Candidatus phytoplasma australiense", and weed host plants acting as potential reservoirs of the grapevine phytoplasmas were discovered. These and other findings from the project should aid in the design and development of strategies for managing the grapevine yellows disease problem.

The original objectives are listed below: 1. Design vectors for constitutive expression of endochitinase from Trichoderma harzianum strain P1. Design vectors with signal peptides to target gene expression. 2. Extend transformation/regeneration technology to other cultivars of importance in the U.S. and Israel. 3. Transform cultivars with the endochitinase constructs developed as part of objective 1. A. Characterize foliar powdery mildew resistance in transgenic plants. Background of the topic Conventional breeding of grapevines is a slow and imprecise process. The long generation cycle, large space requirements and poor understanding of grapevine genetics prevent rapid progress. There remains great need to improve existing important cultivars without the loss of identity that follows from hybridization. Powdery mildew (Uncinula necator) is the most important fungal pathogen of grapevines, causing economic losses around the world. Genetic control of powdery mildew would reduce the requirement for chemical or cultural control of the disease. Yet, since the trait is under polygenic control, it is difficult to manipulate through hybridization and breeding. Also, because grapevines are heterozygous and vegetatively propagated cultivar identity is lost in the breeding process. Therefore, there is great need for techniques to produce transgenic versions of established cultivars with heterologous genes conferring disease resistance. Such a gene is now available for control of powdery mildew of grapevines. The protein coded by the Endochitinase gene, derived from Trichoderma harzianum, is very effective in suppressing U. necator growth. The goal of this proposal is to develop transgenic grapevines with this antifungal gene, and to test the effect of this gene on resistance to powdery mildew. Conclusions, achievements and implications Gene transfer technology for grape was developed using commercial cultivars for both wine and table grapes. It paved the way for a new tool in grapevine genetic studies enabling the alteration of specific important traits while maintaining the essential features of existing elite cultivars. Regeneration and transformation technologies were developed and are currently at an advanced stage for USA wine and Israeli seedless cultivars, representing the cutting edge of grape genetic engineering studies worldwide. Transgenic plants produced are tested for powdery mildew resistance in greenhouse and field experiments at both locations. It is our ultimate goal to develop transgenic grapes which will be more efficient and economical for growers to produce, while also providing consumers with familiar products grown with reduced chemical inputs.

Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.

Rugose wood is a complex disease of grapevines, which occurs in all growing areas. The disease is spread in the field by vector transmission (mealybugs). At least five elongated-phloem- limited viruses are implicated in the various rugose wood disorders. The most fully characterized of these are Grapevine virus A (GV A) and GVB, members of a newly established genus, the vitivirus. GVC, a putative vitivirus, is much less well characterized than GV A or GVB. The information regarding the role of GVC in the etiology and epidemiology of rugose wood is fragmentary and no sequence data for GVC are available. The proposed research is aimed to study the etiology and epidemiology of rugose wood disease, and to construct genetically engineered virus-resistant grapevines. The objectives of our proposed research were to construct transgenic plants with coat protein gene sequences designed to induce post-transcriptional gene silencing (pTGS); to study the epidemiology and etiology of rugose wood disease by cloning and sequencing of GVC; and surveying of rugose wood- associated viruses in Californian and Israeli vineyards. In an attempt to experimentally define the role of the various genes of GV A, we utilized the infectious clone, inserted mutations in every ORF, and studied the effect on viral replication, gene expression, symptoms and viral movement. We explored the production of viral RNAs in a GV A-infected Nicotiana benthamiana herbaceous host, and characterized one nested set of three 5'-terminal sgRNAs of 5.1, 5.5 and 6.0 kb, and another, of three 3'-terminal sgRNAs of 2.2, 1.8 and 1.0 kb that could serve for expression of ORFs 2-3, respectively. Several GV A constructs have been assembled into pCAMBIA 230 I, a binary vector which is used for Angrobacterium mediated transformation: GV A CP gene; two copies of the GV A CP gene arranged in the same antisense orientation; two copies of the GV A CP gene in which the downstream copy is in an antigens orientation; GV A replicase gene; GV A replicase gene plus the 3' UTR sequence; and the full genome of GV A. Experiments for transformation of N. benthamiana and grapevine cell suspension with these constructs have been initiated. Transgenic N. benthamiana plants that contained the CP gene, the replicase gene and the entire genome of GV A were obtained. For grapevine transformation, we have developed efficient protocols for transformation and successfully grapevine plantlets that contained the CP gene and the replicase genes of GV A were obtained. These plants are still under examination for expression of the trans genes. The construction of transgenic plants with GV A sequences will provide, in the long run, a means to control one of the most prevalent viruses associated with grapevines. Our many attempts to produce a cDNA library from the genome of GVC failed. For surveying of rugose wood associated viruses in California vineyards, samples were collected from different grape growing areas and tested by RT-PCR for GV A, GVB and GVD. The results indicated that some of the samples were infected with multiple viruses, but overall, we found higher incidence of GVB and GV A infection in California vineyards and new introduction varieties, respectively. In this research we also conducted studies to increase our understanding of virus - induced rootstock decline and its importance in vineyard productivity. Our results provided supporting evidence that the rootstock response to virus infection depends on the rootstock genotype and the virus type. In general, rootstocks are differ widely in virus susceptibility. Our data indicated that a virus type or its combination with other viruses was responsible in virus-induced rootstock decline. As the results showed, the growth of the rootstocks were severely affected when the combination of more than one virus was present.

Original objectives . 1. Test anti-fungal gene products for activity against Uncinula necator, Aspergillus niger, Rhizopus stolonifer and Botrytis cinerea. 2. For Agrobacterium transformation, design appropriate vectors with gene combinations. 3. Use biolistic bombardment and Agrobacterium for transformation of important cultivars. 4. Characterize gene expression in transformants, as well as level of powdery mildew and Botrytis resistance in foliage of transformed plants. Background The production of new grape cultivars by conventional breeding is a complex and time-consuming process. Transferring individual traits via single genes into elite cultivars was proposed as a viable strategy, especially for vegetatively propagated crops such as grapevines. The availability of effective genetic transformation procedures, the existence of genes able to reduce pathogen stress, and improved in vitro culture methods for grapes, were combined to serve the objective of this proposal. Effective deployment of resistance genes would reduce production costs and increase crop quality, and several such genes and combinations were used in this project. Progress The efficacy of two-way combinations of Trichoderma endochitinase (CHIT42), synthetic peptide ESF12 and resveratrol upon the control of growth of Botrytis cinerea and Penicillium digitatum were evaluated in vitro. All pairwise interactions were additive but not synergistic. Per objective 2, suitable vectors with important gene combinations for Agrobacterium transformation were designed. In addition, multiple gene co-transformation by particle bombardment was also tested successfully. In New York, transformation work focused on cultivars Chardonnay and Merlot, while the technology in Israel was extended to 41B, R. 110, Prime, Italia, Gamay, Chardonnay and Velika. Transgenic plant production is summarized in the appendix. Among plants developed in Israel, endochitinase expression was assayed via the MuchT assay using material just 1-5 days after co-cultivation. Plants of cv. Sugraone carrying the gene coding for ESF12, a short anti-fungal lytic peptide under the control of the double 358 promoter, were produced. Leaf extracts of two plants showed inhibition zones that developed within 48 h indicating the inhibitory effect of the leaf extracts on the six species of bacteria. X fastidiosa, the causal organism of Pierce's disease, was very sensitive to leaf extracts from ESF12 transformed plants. Further work is needed to verify the agricultural utility of ESF12 transformants. In New York, some transformants were resistant to powdery mildew and Botrytis fruit rot. Major conclusions, solutions, achievements and implications The following scientific achievements resulted from this cooperative BARD project: 1. Development and improvement of embryogenesis and tissue culture manipulation in grape, while extending these procedures to several agriculturally important cultivars both in Israel and USA. 2. Development and improvement of novel transformation procedures while developing transformation techniques for grape and other recalcitrant species. 3. Production of transgenic grapevines, characterization of transformed vines while studying the expression patterns of a marker gene under the control of different promoter as the 35S CaMV in different part of the plants including flowers and fruits. 4. Expression of anti-fungal genes in grape: establishment of transgenic plants and evaluation of gene expression. Development of techniques to insert multiple genes. 5. Isolation of novel grape specific promoter to control the expression of future antimicrobial genes. It is of great importance to report that significant progress was made in not only the development of transgenic grapevines, but also in the evaluation of their potential for increased resistance to disease as compared with the non engineered cultivar. In several cases, increased disease resistance was observed. More research and development is still needed before a product can be commercialized, yet our project lays a framework for further investigations.

RNA silencing is a defense mechanism that functions against virus infection and involves sequence-specific degradation of viral RNA. Diverse RNA and DNA viruses of plants encode RNA silencing suppressors (RSSs), which, in addition to their role in viral counterdefense, were implicated in the efficient accumulation of viral RNAs, virus transport, pathogenesis, and determination of the virus host range. Despite rapidly growing understanding of the mechanisms of RNA silencing suppression, systematic analysis of the roles played by diverse RSSs in virus biology and pathology is yet to be completed. Our research was aimed at conducting such analysis for two grapevine viruses, Grapevine virus A (GVA) and Grapevine leafroll-associated virus-2 (GLRaV- 2). Our major achievements on the previous cycle of BARD funding are as follows. 1. GVA and GLRaV-2 were engineered into efficient gene expression and silencing vectors for grapevine. The efficient techniques for grapevine infection resulting in systemic expression or silencing of the recombinant genes were developed. Therefore, GVA and GLRaV-2 were rendered into powerful tools of grapevine virology and functional genomics. 2. The GVA and GLRaV-2 RSSs, p10 and p24, respectively, were identified, and their roles in viral pathogenesis were determined. In particular, we found that p10 functions in suppression and pathogenesis are genetically separable. 3. We revealed that p10 is a self-interactive protein that is targeted to the nucleus. In contrast, p24 mechanism involves binding small interfering RNAs in the cytoplasm. We have also demonstrated that p10 is relatively weak, whereas p24 is extremely strong enhancer of the viral agroinfection. 4. We found that, in addition to the dedicated RSSs, GVA and GLRaV-2 counterdefenses involve ORF1 product and leader proteases, respectively. 5. We have teamed up with Dr. Koonin and Dr. Falnes groups to study the evolution and function of the AlkB domain presents in GVA and many other plant viruses. It was demonstrated that viral AlkBs are RNA-specific demethylases thus providing critical support for the biological relevance of the novel process of AlkB-mediated RNA repair.