Academic literature on the topic 'Trunk disease of grapevine'

Arthropod-mediated dispersal of pathogens is known in many cropping systems but has never been demonstrated for grapevine trunk disease pathogens. Arthropods from vineyards were screened for the presence of pathogens associated with Petri disease and esca using cultural and molecular techniques. The ability of the most abundant pathogen-carrying species to inoculate healthy grapevine vascular tissues was also determined. Millipedes and ants were allowed to associate with a DsRed- Express-transformed Phaeomoniella chlamydospora, after which they were exposed to freshly pruned healthy grapevines under controlled conditions and wounds were monitored for subsequent infection. In addition, the possibility of millipede excreta, commonly found on pruning wounds in the field, to act as inoculum source was determined. A diverse arthropod fauna was associated with declining grapevines and many of these carried trunk disease pathogens. However, spiders, the ant Crematogaster peringueyi, and the millipede Ommattoiulus moreleti were the most abundant pathogen carriers. The ant and millipede species fed on pruning wound sap and effectively transmitted trunk disease pathogens. Millipede excreta contained viable spores of Phaeomoniella chlamydospora and may serve as an inoculum source. Numerous arthropods, including beneficial predators, are potential vectors of grapevine trunk disease pathogens. Our results highlight the need for an integrated approach, including targeted management of ants and millipedes at the time of pruning, to limit the spread of grapevine trunk diseases.

Trunk diseases have become among the most important grapevine diseases worldwide. They are caused by fungal pathogens that attack the permanent woody structure of the vines and cause various symptoms in woody and annual organs. This study examined modifications of plant responses in green stem, cordon, and trunk of grapevines expressing Esca proper (E) or apoplexy (A) event, which are the most frequent grapevine trunk disease symptoms observed in Europe. Transcript expression of a set of plant defense- and stress-related genes was monitored by quantitative reverse-transcription polymerase chain reaction while plant phytoalexins and fungal metabolites were quantified by high-performance liquid chromatography-mass spectrometry in order to characterize the interaction between the grapevine and trunk disease agents. Expression of genes encoding enzymes of the phenylpropanoid pathway and trans-resveratrol content were altered in the three organs of diseased plants, especially in the young tissues of A plants. Pathogenesis-related proteins and the antioxidant system were severely modulated in A plants, which indicates a drastic stress effect. In the meantime, fungal polyketides 6-MSA, (R)-mellein, and (3R,4R)-4-hydroxymellein, were accumulated in A plants, which suggests their potential effect on plant metabolism during the appearance of foliar symptoms.

Fomitiporia mediterranea M. Fisch. (Fmed) is a basidiomycete first described in 2002, and was considered up to then as part of Fomitiporia punctata (P. Karst) Murrill. This fungus can degrade lignocellulosic biomass, causing white rot and leaving bleached fibrous host residues. In Europe Fmed is considered the main grapevine wood rot (Esca) agent within the Esca disease complex, which includes some of the most economically important Grapevine Trunk Diseases (GTDs). This review summarises and evaluates published research on Fmed, on white rot elimination by curettage or management by treatments with specific products applied to diseased grapevines, and on the relationship between wood symptoms and Grapevine Leaf Stripe Disease (GLSD) in the Esca disease complex. Information is also reviewed on the fungus biology, mechanisms of pathogenicity, and their possible relationships with external foliar symptoms of the Esca disease complex. Information on Fmed control strategies is also reviewed.

The successful infection of a grapevine vascular system by a plant pathogen and expression of disease symptoms occur only when the pathogen has overcome the wound response and other defences of the vine Even when pathogens do successfully infect the vascular system of grapevines symptom expression is not often observed in the first season Symptoms may be observed in one year but the same vine can have reduced or no symptoms the following season Information is presented on physiological stress in association with trunk diseases as one factor that may contribute to symptom expression in vines A hypothesis of grapevine wound response is proposed as part of the discussion of vine physiological response Information on individual trunk diseases and physiological interactions is also provided

Current management options for grapevine trunk diseases are focused on ameliorating symptoms once they have occurred In grapevines and other crops plant stress has been observed to increase symptom expression for different pathogens depending on timing and type of stress This research aimed to establish whether stress to vines at the time of wounding enhances colonisation by fungi associated with trunk disease Potted plants were wounded and inoculated with two grapevine trunk fungi Eutypa lata and Botryosphaeria lutea Stress treatments (leaf removal and girdling) were applied to the vines immediately prior to inoculation Lesion size at the wound site and infection confirmed by reisolation of the pathogen were measured In these experiments stress did not induce greater infection incidence or larger lesions at the site of wounding Some difficulties in using potted plants to simulate responses of mature vines in the field are discussed

Seimatosporium spp. and closely related “pestalotioid fungi” have been isolated from vineyards worldwide, but their ecological status in grapevine wood is unclear. To determine their involvement in the grapevine trunk-disease complex, we tested the pathogenicity of Californian isolates obtained from vines with general symptoms of Botryosphaeria, Eutypa, and Phomopsis diebacks. Multilocus phylogenetic analyses revealed three species: Seimatosporium vitis and two newly described and typified species, S. luteosporum sp. nov. and S. vitifusiforme sp. nov. Inoculations to woody stems of potted grapevines of both isolates of S. vitis and one isolate of S. vitifusiforme, but not S. luteosporum, were associated with significantly larger lesions than those of noninoculated controls. Coinoculations with trunk pathogens (Cryptovalsa ampelina, Diaporthe ambigua, Diatrypella verruciformis, Diplodia seriata, and Eutypa lata), coisolated from the same wood cankers in the field, brought about increased lesion lengths for S. vitifusiforme paired with D. seriata, and S. luteosporum paired with Diaporthe ambigua. In contrast, there were no differences in lesion lengths of S. vitis and Diatrypella verruciformis or S. vitis and E. lata, inoculated alone or together. Our findings suggest that Seimatosporium spp. are involved in the grapevine trunk-disease complex, and their virulence may depend on or affect that of trunk pathogens.

Grapevine trunk diseases make up a disease complex associated with several vascular fungal pathogenic species. Surveys to characterize the composition of grapevine trunk diseases have been conducted for most major grape growing regions of the world. This study presents a similar survey characterizing the fungi associated with grapevine trunk diseases of cold-hardy interspecific hybrid grape varieties grown nearly exclusively in the atypical harsh winter climate of Northern Midwestern United states vineyards. From the 172 samples collected in 2019, 640 isolates obtained by culturing were identified by ITS sequencing and represent 420 sample-unique taxa. From the 420 representative taxa, opportunistic fungi of the order Diaporthales including species of Cytospora and Diaporthe were most frequently identified. Species of Phaeoacremonium, Paraconiothyrium, and Cadophora were also prevalent. In other milder Mediterranean growing climates, species of Xylariales and Botryosphaeriales are often frequently isolated but in this study they were isolated in small numbers. No Phaeomoniellales taxa were isolated. We discuss the possible compounding effects of winter injury, the pathogens isolated, and management strategies. Additionally, difficulties in researching and understanding the grapevine trunk disease complex are discussed.

ABSTRACTGrapevine trunk fungal pathogens, such asDiplodia seriataandPhaeomoniella chlamydospora, can infect plants through pruning wounds. They cause grapevine trunk diseases and are involved in grapevine decline. Accordingly, the protection of pruning wounds is crucial for the management of grapevine trunk diseases. The efficacy of different natural antifungals in inhibiting the growth of several fungi causing grapevine trunk diseases was evaluatedin vitro. The fungi showing greaterin vitroefficacy were tested on autoclaved grape wood assays againstD. seriataandP. chlamydospora. Based on results from these assays, chitosan oligosaccharide, vanillin, and garlic extract were selected for further evaluation on pruning wounds inoculated withD. seriataandP. chlamydosporain field trials. A significant decrease in plant mortality was observed after 2 years of growth in the plants treated with the different natural antifungals compared to the mortality rate observed in infected plants that were not treated with antifungals. Also, the infection rate for the inoculated pathogens was significantly reduced in plants treated with the selected natural antifungals. Therefore, natural antifungals represent a promising alternative for disease control and could provide significant economic benefits for the grape-growing industry.

Thesis (PhD)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Grapevine trunk diseases are a cause of decline and loss of productivity in grapevines at all stages of growth. These diseases are caused by a complex of wood-inhabiting fungi that infect mainly through pruning wounds. The management of these diseases relies on wound protection to prevent infection since there are no eradicative control measures to cure infected vines. There are few or no fungicides registered for grapevine pruning wound protection in most countries, while Trichoderma biocontrol agents are often available. This study aimed at improving grapevine wound protection by Trichoderma (T.) spp. and to gain a better understanding of the factors and mechanisms involved in biocontrol. The effect of pruning time (early or late) and five timings of application of the biocontrol agent after pruning on pruning wound colonisation by T. atroviride and T. harzianum were determined. Chenin blanc and Cabernet Sauvignon vineyards were pruned in July (early) and August (late) of 2011 and 2012, and pruning wounds were treated with suspensions of the Trichoderma spp. at various times (0, 6, 24, 48 and 96 hours) after pruning. Wound colonisation was depended on the physiological state of the vine at pruning for both cultivars. However, for the 2012 season in Chenin blanc, wound colonisation was similarly high for both pruning times, which was attributed to high rainfall and humidity. Application of the biocontrol agents 6 hours after pruning consistently resulted in high wound colonisation by the Trichoderma spp. in both cultivars and pruning times. In both cultivars, pruning wound infection due to natural inoculum was higher in wounds made in late winter than those made earlier. The effect of conidial formulation in nutritional (glucose, yeast extract and urea) and bio-enhancing (chitin and cell free culture filtrates) additives, on pruning wound colonisation by T. atroviride was also investigated. Nutritional additives increased the extent of pruning wound colonisation by T. atroviride compared to the un-amended conidial suspensions in a glass house study. The additives as well as Garrison, a fungicide containing pruning wound paint, and Eco77®, a registered T. harzianum biocontrol product, were tested in field trials for wound protection from infection by Phaeomoniella (Pa.) chlamydospora. In 2011, the pathogen was inoculated a day after pruning and all the Trichoderma spp. treatments similarly reduced Pa. chlamydospora infection by 75% to 90% in Thompson Seedless, while control was less in Chenin blanc and ranged from 40% to 74%. In 2012, the trial was carried out on Chenin blanc only and the pathogen was inoculated at intervals of 1, 3 and 7 days after pruning. Wound protection by the Trichoderma treatments was highest when wounds were inoculated with Pa. chlamydospora seven days after pruning. Two conidial formulations, a culture filtrate made from a chitin based medium and a combination of yeast extract, urea and glucose, consistently enhanced biocontrol efficacy. These formulations reduced Pa. chlamydospora infection to levels similar to those of Garrison. The integration of chemical and biological wound protection could provide both immediate and long term wound protection, but is limited by the sensitivity of the biocontrol agent to fungicides. Benzimidazole resistant Trichoderma strains were generated by gamma irradiation from the wild type isolates of T. atroviride (UST1 and UST2) and T. harzianum (T77). Mutants from UST1 and UST2 were of similar biological fitness as the wild type isolates and retained their in vitro antagonistic activity against grapevine trunk pathogens, while the mutant from T77 had reduced fitness and was not antagonistic to the pathogens. The wild type, UST1, and its mutant were tested alone and in combination with thiophanate methyl and carbendazim, respectively, for their ability to prevent pruning wound infection by Pa. chlamydospora. The combination of the UST1 mutant and carbendazim was the most effective treatment and gave the highest reduction in Pa. chlamydospora infection (70% to 93% control). Grapevine cell cultures were used to compare the response of grapevines to T. atroviride and Eutypa (E.) lata as a first step to determining the importance of Trichoderma-grapevine interactions in pruning wound bio-protection. The expression of genes coding for enzymes of the phenylpropanoid pathway and pathogenesis related (PR) proteins was profiled over a 48-hour period using quantitative reverse transcriptase PCR. The cell cultures responded to fungal elicitors in a hypersensitive-like response that lead to a decrease in cell viability. Fungal elicitors from both fungi triggered the same genes and caused up-regulation of phenylalanine ammonia-lyase (PAL), 4 coumaroyl Co-A ligase (CCo-A), stilbene synthase (STS), chitinase class IV (CHIT IV), PR 3 and PR 4, and a down regulation of chalcone synthase (CHS) genes. Higher expression of PAL and CHIT IV in cell cultures treated with the T. atroviride elicitor led to a significantly higher (P < 0.05) total phenolic content and chitinolytic enzyme activity of the cell cultures compared to cell cultures treated with the E. lata elicitor. The response of the cell cultures to the T. atroviride elicitor signifies that the induction of grapevine resistance may be involved in wound bio-protection. The role of secondary metabolites produced by Trichoderma spp. used in pruning wound protection was also investigated. A volatile antimicrobial compound, 6-pentyl α-pyrone (6PP), was isolated and found to be the major secondary metabolite from the T. atroviride (UST1 and UST2) and T. harzianum (T77) isolates. This metabolite was found to inhibit mycelial growth, spore and conidia germination of E. lata, Neofussicocum (N.) australe, N. parvum and Pa. chlamydospora. The production of 6PP was induced when the T. atroviride isolates were grown in a grapevine wood extract medium while for UST1, the 6PP concentration was further doubled when it was co-cultured with N. parvum. Results therefore, indicate that 6PP is involved in the Trichoderma-pathogen interactions on pruning wounds. The results of this study have provided new information in regards to the application of Trichoderma-based pruning wound products. The best time of application proved to be 6 hours post pruning. The formulation of conidial suspensions of Trichoderma spp. with nutritional additives and in protein extracts of the biocontrol agent showed potential in reducing variability of wound bio-protection. However, further research would be necessary to develop commercial products. The application of a fungicide together with Trichoderma spp. in the field holds promise to improve control, but would require further trials for possible commercialisation. This study is the first to report on grapevine host defence genes that are activated by the Trichoderma spp. used in pruning wound protection. Together with the characterisation of the major secondary metabolite produced by these Trichoderma spp., this information aids in understanding the mechanisms involved in the complex interaction between the biocontrol agent, the host and the pathogen.
AFRIKAANSE OPSOMMING: Wingerdstamsiektes veroorsaak terugsterwing en verlies aan produktiwiteit in wingerdstokke gedurende alle groeifases. Hierdie siektes word veroorsaak deur „n verskeidenheid van hout-koloniserende swamme wat die wingerdstok meestal deur snoeiwonde infekteer. Die bestuur van hierdie siektes is afhanklik van wondbeskerming om infeksie te verhoed, omdat daar geen uitwissende beheermetodes na infeksie bestaan nie. In meeste lande is daar min of geen swamdoders geregistreer vir snoeiwond beskerming, terwyl Trichoderma biobeheer agente gereëld beskikbaar is. Hierdie studie poog om wingerd wondbeskerming deur Trichoderma (T.) spp. te verbeter en „n meer volledige begrip van die faktore en meganismes betrokke by biologiese beheer te ontwikkel. Die effek van die tydsberekening van snoei (vroeg of laat) en vyf behandelingstye van die biobeheer agent na snoei op die kolonisering van snoeiwonde deur T. atroviride en T. harzianum is bepaal. Chenin blanc en Cabernet Sauvignon wingerde is gesnoei gedurende Julie (vroeg) en Augustus (laat) in 2011 en 2012, en snoeiwonde is behandel met Trichoderma spp. suspensies op verskillende tydspunte (0, 6, 24, 48 en 96 ure) na snoei. Wond-kolonisering was afhanklik van die fisiologiese toestand van die wingerdstok gedurende snoei vir albei kultivars. Gedurende die 2012 seisoen was wond-kolonisering ewe hoog vir albei snoeitye op Chenin blanc. Dit is verklaar deur hoë reënval en humiditeit gedurende daardie seisoen. Die aanwending van biobeheer agente 6 ure na snoei het konsekwent hoë kolonisering deur Trichoderma spp. tot gevolg gehad op albei kultivars en albei snoeitye. In albei kultivars is wondinfeksie as gevolg van natuurlike inokulum hoër gewees in wonde gemaak gedurende laat winter as in wonde wat vroeër in die seisoen gemaak is. Die effek van konidia formulasie in voeding (glukose, gisekstrak en urea) en bioverbetering (chitien en sel-vrye kultuurfiltraat) toevoegings op snoeiwond-kolonisering deur T. atroviride is ook ondersoek. Voeding toevoegings het die omvangs van snoeiwond-kolonisering deur T. atroviride vergroot in vergelyking met ongewysigde konidia suspensies gedurende „n glashuis studie. Die toevoegings, sowel as Garrison, „n snoeiwond verf wat „n swamdoder bevat, en Eco77®, „n geregistreerde T. harzianum biobeheer produk, is getoets in veldproewe vir wondbeskerming teen infeksie deur Phaeomoniella (Pa.) chlamydospora. In 2011 is die patogeen geïnokuleer „n dag na snoei en al die Trichoderma spp. behandelings het infeksie verminder met 75% tot 90% op Thompson Seedless. Beheer was minder suksesvol op Chenin blanc, waar slegs 40% tot 74% beheer behaal is. In 2012 is die proef uitgevoer slegs op Chenin blanc en die patogeen is geïnokuleer teen intervalle van 1, 3 en 7 dae na snoei. Wondbeskerming by die Trichoderma behandelinge was die hoogste wanneer wonde sewe dae na snoei geïnokuleer is met Pa. chlamydospora. Twee konidia formulasies, „n kultuurfiltraat wat bestaan het uit „n chitien-gebaseerde medium en „n kombinasie van gisekstrak, urea en glukose het deurlopend die effektiwiteit van biobeheer verbeter. Hierdie formulasies het Pa. chlamydospora infeksie verminder tot soortgelyke vlakke behaal deur Garrison. Die integrasie van chemiese- en biobeheer in wondbeskerming kan onmiddelike en langtermyn wondbeskerming bied, maar is beperk deur die sensitiwiteit van die biobeheer agent teen swamdoders. Benzimidazole-weerstandbiedende Trichoderma isolate is ontwikkel deur gamma-bestraling van die wilde-tipe isolate van T. atroviride (UST1 en UST2) en T. harzianum (T77). Mutante van UST1 en UST2 het soortgelyke biologiese fiksheid getoon as die wilde-tipe en het hul in vitro antagonistiese aktiwiteit teen wingerd stampatogene behou, terwyl die mutant van T77 verminderde fiksheid getoon het en nie meer antagonisties teen patogene was nie. Die wilde-tipe, UST1, en sy mutant is apart en in kombinasie met thiofanaatmetiel en carbendazim, respektiewelik, getoets vir die vermoë om snoeiwonde te beskerm teen Pa. chlamydospora. Die kombinasie van die UST1 mutant met carbendazim was die mees effektiewe behandeling en het die hoogste vermindering in Pa. chlamydospora infeksie gelewer (70 tot 93% beheer). As „n beginpunt om die belang van Trichoderma-wingerd interaksies in snoiewondbeheer te bepaal, is die invloed van T. atroviride en Eutypa (E.) lata op somatiese selkulture van wingerd vergelyk. Die effek van dié behandelings op ensieme in die fenielpropanoïedweg en patogenese-verwante (PR) proteïene is bepaal deur intydse PKR (real time PCR) van die korresponderende gene oor „n 48 uur tydperk. Die swam-afkomstige ontlokkers het „n hipersensitiewe-tipe reaksie in die selkulture ontlok, wat tot „n afname in sellewensvatbaarheid gelei het. Ontlokkers afkomstig van beide swamme het dieselfde gene aangeskakel en het induksie van fenielalanien ammoniak-liase (PAL), 4 kumaroïel Ko-A ligase (CCo-A), stilbeen sintase (STS), chitienase klas IV (CHIT IV), PR 3 en PR 4 veroorsaak en „n onderdrukking in chalkoon sintase (CHS) gene tot gevolg gehad. Hoër uitdrukking van PAL en CHIT IV in selkulture behandel met die T. atroviride ontlokker het gelei tot „n beduidende hoër (P < 0.05) totale fenoolinhoud en chitienolitiese aktiwiteit in selkulture in vergelyking met selkulture wat behandel is met die E. lata ontlokker. Die reaksie van die selkulture op die T. atroviride ontlokker dui daarop dat die induksie van wingerd weerstandbiedenheid betrokke mag wees in wond biobeheer. Die rol van sekondêre metaboliete geproduseer deur Trichoderma spp. wat gebruik word in snoeiwond beheer is ook ondersoek. „n Vlugtige antimikrobiese verbinding, 6-pentiel α-pyroon (6PP) is geïsoleer en bepaal om die hoof sekondêre metaboliet afkomstig vanuit die T. atroviride (UST1 en UST2) en T. harzianum (T77) isolate te wees. Hierdie metaboliet is betrokke by inhibisie van miselium groei, spoor en konidium ontkieming van E. lata, Neofusicoccum (N.) australe, N. parvum en Pa. chlamydospora. Die produksie van 6PP is geïnduseer deur die T. atroviride in wingerd hout ekstrak te kweek. In die geval van UST1, is die 6PP konsenstrasie verdubbel deur die isolaat met saam met N. parvum te kweek. Hierdie resultaat is „n aanduiding dat 6PP betrokke is in die Trichoderma-patogeen interaksie op snoeiwonde. Die resultate van hierdie studie het nuwe inligting met betrekking tot die aanwending van Trichoderma-gebaseerde snoeiwond produkte verskaf. Die beste tyd vir aanwending van sulke produkte was 6 ure na snoei. Die formulasie van konidia suspensies van Trichoderma spp. met voeding toevoegings en in proteïen ekstrakte van die biobeheer agent het potensiaal getoon in die vermindering van variasie in wondbeskerming deur biobeheer agente. Verdere navorsing sal nodig wees om kommersiële produkte te ontwikkel. Die aanwending van „n swamdoder saam met Trichoderma spp. in die wingerd is belowend om beheer te verbeter, maar het meer proewe nodig voor kommersialisering. Hierdie studie is die eerste om wingerd beskerming gene wat deur Trichoderma spp. geaktiveer word aan te meld. Laasgenoemde, saam met die beskrywing van die hoof sekondêre metaboliete wat deur hierdie Trichoderma spp. geproduseer word, dra by tot „n meer volledige begrip van die meganismes betrokke by die komplekse interaksie tussen die biobeheer agent, die gasheer en die patogeen.

Thesis (MScAgric (Plant Pathology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: A survey was undertaken on apple and pear trees in the Western Cape Province to determine the aetiology of trunk diseases with reference to trunk diseases occurring on grapevine. Grapevine trunk diseases cause the gradual decline and dieback of vines resulting in a decrease in the vine’s capability to carry and ripen fruit. In recent years, viticulture has been expanding into several of the well established pome fruit growing areas. The presence of trunk pathogens in pome fruit orchards may affect the health of the pome fruit trees as well as cause a threat to young vineyards planted in close proximity to these potential sources of viable inoculum. Several genera containing species known to be involved in trunk disease on pome fruit and grapevine were found, including Diplodia, Neofusicoccum, Eutypa, Phaeoacremonium and Phomopsis. Diplodia seriata and D. pyricolum, were isolated along with N. australe and N. vitifusiforme. Four Phaeoacremonium species, P. aleophilum, P. iranianum, P. mortoniae and P. viticola, two Phomopsis species linked to clades identified in former studies as Phomopsis sp. 1 and Phomopsis sp. 7, and Eutypa lata were found. In addition, Paraconiothyrium brasiliense and Pa. variabile, and an unidentified Pyrenochaetalike species were found. Of these the Phaeoacremonium species have not been found on pear wood and it is a first report of P. aleophilum occurring on apple. This is also a first report of the Phomopsis species and Eutypa lata found occurring on pome trees in South Africa Two new coelomycetous fungi were also found including a Diplodia species, Diplodia pyricolum sp. nov., and a new genus, Pyrenochaetoides gen. nov. with the type species, Pyrenochaetoides mali sp. nov., were described from necrotic pear and apple wood. The combined ITS and EF1-α phylogeny supported the new Diplodia species, which is closely related to D. mutila and D. africana. The new species is characterised by conidia that become pigmented and 1-septate within the pycnidium, and that are intermediate in size between the latter two Diplodia species. Phylogenetic inference of the SSU of the unknown coelomycete provided bootstrap support (100%) for a monophyletic clade unrelated to known genera, and basal to Phoma and its relatives. Morphologically the new genus is characterised by pycnidial with elongated necks that lack setae, cylindrical conidiophores that are seldomly branched at the base, and Phoma-like conidia. The phylogenetic results combined with its dissimilarity from genera allied to Phoma, lead to the conclusion that this species represents a new genus. A pathogenicity trial was undertaken to examine the role of these species on apple, pear and grapevine shoots. N. australe caused the longest lesions on grapevine shoots, while Pyrenochaetoides mali, Pa. variabile, D. seriata and P. mortoniae caused lesions that were significantly longer than the control inoculations. On pears, D. pyricolum and N. australe caused the longest lesions, followed by D. seriata and E. lata. On apples, the longest lesions were caused by N. australe and P. iranianum. D. seriata, D. pyricolum, E. lata, N. vitifusiforme, Pa. brasiliense, P. aleophilum and P. mortoniae also caused lesions on apple that were significantly longer than the control. The study demonstrated that close cultivation of grapevine to apple and pear orchards may have inherent risks in terms of the free availability of viable inoculum of trunk disease pathogens.
No Afrikaans abstract available.

Thesis (MScAgric (Plant Pathology))--Stellenbosch University, 2008.
In recent years, several studies have conclusively shown that numerous pathogens, including several species in the Botryosphaeriaceae, Phomopsis, Phaeoacremonium, as well as Phaeomoniella chlamydospora and Eutypa lata, contribute to premature decline and dieback of grapevines. These pathogens have the ability to infect grapevines through pruning wounds, which leads to a wide range of symptoms developing that includes stunted growth, cankers and several types of wood necrosis. Pruning wounds stay susceptible for 2 to 16 weeks after pruning and sustained levels of pruning wound protection is therefore required. The aims of this study were to (i) evaluate the ability of several biological agents to protect pruning wounds, (ii) characterise unknown Trichoderma strains and identify their modes of action and (iii) determine the optimal time of season for biological agent application. Several biological agents were initially evaluated in a laboratory for their antagonism against trunk disease pathogens. The best performing control agents were tested in a field trial conducted on Merlot and Chenin blanc vines in the Stellenbosch region. Spurs were pruned to three buds and the fresh pruning wounds were treated with benomyl as a control treatment, Trichoderma-based commercial products, Vinevax® and Eco77®, Bacillus subtilis, and Trichoderma isolates, USPP-T1 and -T2. Seven days after treatment the pruning wounds were spray inoculated with spore suspensions of four Botryosphaeriaceae spp. (Neofusicoccum australe, N. parvum, Diplodia seriata and Lasiodiplodia theobromae), Eutypa lata, Phaeomoniella chlamydospora and Phomopsis viticola. After a period of 8 months the treatments were evaluated by isolations onto potato dextrose agar. Trichodermabased products and isolates in most cases showed equal or better efficacy than benomyl, especially USPP-T1 and -T2. Moreover, these isolates demonstrated a very good ability to colonise the wound tissue. The two uncharacterised Trichoderma isolates (USPP-T1 and USPP-T2), which were shown to be highly antagonistic toward the grapevine trunk disease pathogens, were identified by means of DNA comparison, and their ability to inhibit the mycelium growth of the trunk disease pathogens by means of volatile and non-volatile metabolite production studied. The two gene areas that were used include the internal transcribed spacers (ITS 1 and 2) and the 5.8S ribosomal RNA gene and the translation elongation factor 1 (EF). The ITS and EF sequences were aligned to published Trichoderma sequences and the percentage similarity determined and the two Trichoderma isolates were identified as Trichoderma atroviride. The volatile production of T. atroviride isolates was determined by placing an inverted Petri dish with Trichoderma on top of a dish with a pathogen isolate and then sealed with parafilm. Trichoderma isolates were grown for 2 days on PDA where after they were inverted over PDA plates containing mycelial plugs. The inhibition ranged from 23.6% for L. theobromae to 72.4% for P. viticola. Inhibition by non-volatile products was less than for the volatile inhibition. Inhibition ranged from 7.5% for N. parvum to 20.6% for L. theobromae. In the non-volatile inhibition USPP-T1 caused significantly more mycelial inhibition than USPP-T2. The timing of pruning wound treatment and subsequent penetration and colonisation of the wound site was also determined. One-year-old canes of the Shiraz and Chenin blanc cultivars were grown in a hydroponic system, pruned and spray treated with a spore suspension of Trichoderma atroviride (USPP-T1) as well as a fluorescent pigment. On intervals 1, 3, 5 and 7 days after treatment, the distal nodes were removed and dissected longitudinally. From the one half, isolations were made at various distances from the pruning surface, while the other half was observed under ultra-violet light to determine the depth of fluorescent pigment penetration. Shortly after spray-inoculation of a fresh pruning wound, Trichoderma was isolated only from the wound surface and shallow depths into the wound (2 to 5 mm). One week after inoculation, Trichoderma was isolated at 10 mm depths, and after 2 weeks, at 15 mm depths. Fluorescent pigment particles were observed to a mean depth of 6 mm, which suggests that initial isolation of Trichoderma at these depths was resultant of the physical deposition of conidia deeper into the pruning wound tissue, whereas the isolation of Trichoderma from deeper depths might be attributed to colonisation of grapevine tissue. In a vineyard trial, fluorescent pigment was spray-applied to pruning wounds of Shiraz and Chenin blanc grapevines during July and September at intervals 0, 1, 3, 7 and 14 days after pruning. One week after treatment, the distal nodes were removed and dissected longitudinally. Each half was observed under UV light and the pigment penetration measured. For Chenin blanc and Shiraz, July pruning wounds showed significant deeper penetration of the pigment than pruning wounds treated in September. Moreover, pruning wounds made in September showed pigment particles in longitudinal sections up to 1 day after pruning, whereas wounds made in July showed pigment particles up to 3 days in the xylem vessels. These findings suggest that the best time for application of a biological control agent should be within the first 24 hours after pruning.

Grapevine trunk diseases (GTDs) is one of the most important groups of fungal diseases affecting grapevine plants in all the major growing regions of the world, with more than 130 fungal species associated. All grapevine species are susceptible to these diseases and their complete eradication is not possible for many reasons. In addition, GTDs are influenced by the type of disease and/or pathogens involved, leading management of this diseases complex to focus primarily on disease prevention and mitigation. Focusing on finding alternatives to avoid the spread and higher incidence of the disease, the present work had the aim to identify molecularly the phytopathogenic fungi responsible for GTDs present in vineyards of Alentejo region, and to test the antagonist potential of some endophytes against those pathogens. PCR assays followed sequencing of ITS region were performed to identify fungi and among them, three GTDs fungi were identified at genera level (Diaporthe sp., Pestalotiopsis sp., Neofusicocum sp.) and six at specie level (Hormonema viticola, Stereum armeniaccum, Phialophora fastigiata, Truncatella angustata, Cytospora acaciae, Diplodia pseudoseriata). The most prevalent fungus verified in the samples were Diaporthe sp., Neofusicocum sp. and Hormonema viticola in symptomatic plants. Almost all these pathogens were also verified in asymptomatic plants, highlighting the incidence of Hormonema viticola, what confirms the need of early diagnosis of this diseases complex. All the endophyte used in the direct inhibition antagonism test had the capacity of inhibit the pathogen mycelia growth, showing their potential biocontrol. This study allowed a deeper knowledge of the fungi present in vineyards from Alentejo region associated to GTDs and will contribute to further studies on fungi molecular identification in order to monitor the behaviour of the disease in the vineyards; Resumo: Identificação de espécies de fungos associadas a doenças do lenho em videira na região do Alentejo As doenças do lenho da videira são um dos grupos mais importantes de doenças fúngicas que afetam as plantas da videira em todas as principais regiões produtoras do mundo, com mais de 130 espécies de fungos associadas. Todas as espécies de videira são suscetíveis a essas doenças e sua erradicação completa não é possível por diversos motivos. Além disso, as doenças do lenho são influenciadas pelo tipo de doença e / ou agentes patogénicos envolvidos, levando a gestão deste complexo de doenças a concentrar-se principalmente na prevenção e mitigação de doenças. Com o objetivo de encontrar alternativas para evitar a disseminação e maior incidência da doença, o trabalho aqui apresentado teve como objetivo identificar molecularmente os fungos fitopatogénicos responsáveis pelas doenças do lenho presentes em vinhas da região do Alentejo, e testar o potencial antagonismo de alguns fungos endófitos contra esses agentes patogénicos. Os testes de PCR seguidos de sequenciação da região ITS foram realizados para identificar os fungos, tendo sido identificados três fungos causadores de doenças do lenho ao nível de género (Diaporthe sp., Pestalotiopsis sp., Neofusicocum sp.) e seis ao nível de espécie (Hormonema viticola, Stereum armeniaccum, Phialophora fastigiata, Truncatella angustata, Cytospora acaciae, Diplodia pseudoseriata). Os fungos mais prevalentes verificados nas amostras foram Diaporthe sp., Neofusicocum sp. e Hormonema viticola em plantas sintomáticas. A grande maioria dos agentes patogénicos foram também verificados em plantas assintomáticas, destacando a incidência de Hormonema viticola, o que confirma a necessidade de diagnóstico precoce desse complexo de doenças. Todos os endófitos utilizados no teste de antagonismo de inibição direta tiveram a capacidade de inibir o crescimento mecelial do patogénico, mostrando seu potencial de biocontrole. Este estudo permitiu um conhecimento mais aprofundado dos fungos presentes nas vinhas da região do Alentejo associadas às doenças do lenho, e contribuirá para mais estudos sobre a identificação molecular dos fungos, a fim de monitorizar o comportamento da doença nas vinhas.

Thesis (MScAgric)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Grapevine trunk diseases are responsible for reduced wine and table grape production world-wide. Trunk disease infections are caused by xylem-inhabiting pathogens which include species of Botryosphaeriaceae, Diatrypaceae, Hymenochaetales and Diaporthales, as well as Phaeomoniella chlamydospora and Phaeoacremonium spp. Winter pruning wounds are regarded as the main infection-sites for trunk disease pathogens. However, the role of sucker wounds as portals of trunk disease infections has been minimally investigated. Knowledge of the potential role of grapevine trunk pathogen infections that occur through sucker wounds is important for better wound protection strategies. The aim of this study was to determine the role of grapevine sucker wounds as portals of entry for trunk disease pathogens and to assess the use of Trichoderma spp. for sucker wound protection. The susceptibility of sucker wounds to different trunk disease pathogens was assessed from natural as well as artificial infections. In addition the duration of sucker wound susceptibility in the field was also ascertained. Sucker wounds were sampled from three wine and two table grape vineyards during 2011 and 2012 in the Western Cape province of South Africa. Thereafter, fungal isolations were made from 161 sucker wounds and the cultures were identified based on cultural and morphological characteristics as well as the internal transcribed spacer regions and 5.8S ribosomal RNA gene. Sixty-two percent of the wounds were naturally infected by at least one of the trunk pathogens. Phomopsis (Po.) viticola (46%; 18%), Diplodia (D.) seriata (30%; 9%) and Phaeomoniella (Ph.) chlamydospora (27%; 5%) were the most predominant trunk disease pathogens isolated from sucker wounds of field wine and table grape cultivars, respectively. Lower incidences of Phaeoacremonium aleophilum (18%), Eutypella sp. (3%), Cryptovalsa ampelina (2%), Diplodia sp. (1%) and Neofusicoccum australe (1%) were obtained, however, only from wine grapes. Sucker wounds on 1-year-old potted grapevine plants of Chardonnay cultivar were inoculated with spore suspensions of Eutypa lata, N. parvum, Pa. aleophilum, Ph. chlamydospora and Po. viticola in the glasshouse. After 4 months all the inoculated pathogens could be re-isolated at the following incidences: N. parvum (85%), Ph. chlamydospora (75%), Po. viticola (65%), Pa. aleophilum (55%) and E. lata (45%). Sucker wound susceptibility was further ascertained under field conditions on 12-year-old Cabernet Sauvignon vines by artificial inoculation of the same pathogen species. After 5 months three pathogens could be re-isolated at the following incidences: Po. viticola (65%), N. parvum (32.5%) and Ph. chlamydospora (7.5%). The duration of susceptibility of field sucker wounds to Ph. chlamydospora was assessed for a period of 4 weeks. The wounds remained susceptible for 4 weeks with a decline in susceptibility after one week. This study showed that sucker wounds are susceptible to the major trunk disease pathogens and thus could play an important role in grapevine trunk disease epidemiology. In the second part of this thesis a possible management strategy to prevent infections of sucker wounds was investigated. The use of Trichoderma (T.) harzianum against two trunk pathogens on sucker wounds was tested in the field. Additionally the sensitivity of T. harzianum and T. atroviride was tested in vitro against 16 fungicides that are used to control powdery mildew, downy mildew, Botrytis rot and Phomopsis cane and leaf spot. In October 2012, sucker wounds were made on 1-year-old wood of Cabernet Sauvignon and spray-treated with Eco-77® immediately after desuckering, and then inoculated with spore suspensions of either Ph. chlamydospora or Po. viticola after 24 hours. After 5 months, isolations were made from the sucker wounds to evaluate the efficacy of the Trichoderma treatment. Trichoderma harzianum reduced the incidence of Ph. chlamydospora by 66.65%. Although the incidence of Po. viticola was reduced by 15.37%, it was not significantly different from the control treatment. The inhibition of mycelial growth and conidial germination of T. harzianum and T. atroviride were screened against 16 fungicides. The fungicides were applied at 0, 0.25, 0.5, 1 and 2 times the recommended dosages. Systemic fungicides boscalid, metrafenone and trifloxystrobin, as well as contact fungicides quinoxyfen and meptyldinocap were least toxic to Trichoderma spp. isolates. For the conidial germination assay, boscalid, trifloxystrobin, penconazole and metrafenone (systemic) plus quinoxyfen and folpet (contact) were compatible with Trichoderma spp. These fungicides were regarded as being compatible with Trichoderma spp. isolates because they gave mean percentage inhibitions of less than 50% at all the tested dosages. Spiroxamine and pyrimethanil gave the highest mean percentage inhibitions for both mycelial inhibition and conidial germination. The findings of this study showed that T. harzianum can protect sucker wounds against Ph. chlamydospora in the field. Furthermore, some fungicides applied for the control of powdery mildew and Phomopsis cane and leaf spot can be alternatively or simultaneously applied with T. harzianum and T. atroviride, however, this will have to be verified with field trials.
AFRIKAANSE OPSOMMING: Wingerd stamsiektes is wêreldwyd verantwoordelik vir verminderde wyn- en tafeldruif produksie. Stamsiektes word veroorsaak deur patogene wat in die xileem voorkom, insluitend verskeie spesies in die Botryosphaeriaceae, Diatrypaceae, Hymenochaetales en Diaporthales, asook Phaeomoniella chlamydospora en Phaeoacremonium spp. Winter snoeiwonde word beskou as die hoof bron van infeksies vir stamsiekte patogene. Die rol van suierwonde as poorte van infeksie vir stamsiektes is nog nie goed bestudeer nie. Kennis van die potensiële rol van wingerd stamsiekte patogeen infeksies wat deur suierwonde plaasvind is belangrik vir die formulasie van beter wondbeskerming strategieë. Die mikpunt van hierdie studie was om die rol van suierwonde as ingangsportale vir wingerd stamsiekte patogene te bepaal en om die gebruik van Trichoderma spp. vir suierwond beskerming te evalueer. Die vatbaarheid van suierwonde vir verskillende stamsiekte patogene is geëvalueer vanuit natuurlike, sowel as kunsmatige infeksies. Die duur van suierwond vatbaarheid in die veld is ook bepaal. Suierwonde is versamel vanuit drie wyn- en twee tafeldruif wingerde gedurende 2011 en 2012 in die Wes Kaap provinsie van Suid Afrika. Hierna is swam isolasies gemaak vanuit 161 suierwonde en die kulture is geïdentifiseer volgens kultuur en morfologiese kenmerke, sowel as die interne transkribeerde spasieerders en 5.8S ribosomale RNA geen. Twee-en-sestig persent van die wonde was geïnfekteer deur ten minste een van die stamsiekte patogene. Phomopsis (Po.) viticola (46%; 18%), Diplodia (D.) seriata (30%; 9%) en Phaeomoniella (Ph.) chlamydospora (27%; 5%) was die mees algemene stamsiekte patogene wat, respektiewelik, vanuit die wyn- en tafeldruif kultivars verky is. Laer hoeveelhede Phaeoacremonium aleophilum (18%), Eutypella sp. (3%), Cryptovalsa ampelina (2%), Diplodia sp. (1%) en Neofusicoccum australe (1%) is verkry, en slegs vanaf wyndruiwe. Suierwonde op 1-jaar oue Chardonnay wingerdplante in potte is in die glashuis geïnokuleer met spoorsuspensies van Eutypa lata, N. parvum, Pa. aleophilum, Ph. chlamydospora en Po. viticola. Na 4 maande kon al die geïnokuleerde patogene her-isoleer word teen die volgende hoeveelhede: N. parvum (85%), Ph. chlamydospora (75%), Po. viticola (65%), Pa. aleophilum (55%) en E. lata (45%). Suierwond vatbaarheid is verder geëvalueer onder veld kondisies op 12-jaar oue Cabernet Sauvignon plante deur kunsmatige inokulasie van die selfde patogeen spesies. Na 5 maande kon drie patogene her-isoleer word teen die volgende hoeveelhede: Po. viticola (65%), N. parvum (32.5%) en Ph. chlamydospora (7.5%). Die duur van vatbaarheid van suierwonde teen Ph. chlamydospora in die veld is geevalueer oor ‘n periode van 4 weke. Die wonde het vatbaar gebly vir 4 weke met ‘n afname in vatbaarheid na ‘n week. Hierdie studie demonstreer dat suierwonde vatbaar is vir die hoof wingerd stamsiektes en dus ‘n belangrike rol in die epidemiologie van wingerd stamsiektes kan speel. In die tweede deel van hierdie tesis is ‘n moontlike bestuurs-strategie ondersoek om infeksie van suierwonde te verhoed. Die gebruik van Trichoderma (T.) harzianum teen twee stampatogene op suierwonde is getoets in die veld. Verder is die in vitro sensitiwiteit van T. harzianum en T. atroviride getoets teen 16 fungisiedes wat gebruik word in die beheer van poeieragtige meeldou, donsskimmel, Botrytis vrot en Phomopsis streepvlek. Gedurende Oktober 2012 is suierwonde gemaak op 1-jaar oue hout van Cabernet Sauvignon en onmiddelik behandel met Eco-77® na suiering. Wonde is dan geïnokuleer met spoorsuspensies van óf Ph. chlamydospora óf Po. viticola na 24 uur. Na 5 maande is isolasies gemaak vanaf suierwonde om die doeltreffendheid van van die Trichoderma behandeling te evalueer. Trichoderma harzianum het die voorkoms van Ph. chlamydospora met 66.65% verminder. Alhoewel die voorkoms van Po. viticola verminder is met 15.37%, was dit nie ‘n beduidende verskil in vergelyking met die kontrole behandeling nie. Die inhibisie van miselium groei en konidia ontkieming van T. harzianum en T. atroviride is getoets teen 16 fungisiedes. Die fungisiedes is aangewend teen 0, 0.25, 0.5, 1 en 2 keer die aanbevole dosisse. Sistemiese fungisiedes boscalid, metrafenone en trifloxystrobin, sowel as kontak fungisiedes quinoxyfen en meptyldinocap was die minste toksies teen Trichoderma spp. Gedurende die konidia ontkiemingstoets was boscalid, trifloxystrobin, penconazole en metrafenone (sistemies) en quinoxyfen en folpet (kontak) versoenbaar met Trichoderma spp. Die fungisiedes is beskou as bruikbaar met Trichoderma spp. isolate omdat hulle gemiddelde persentasie inhibisies van minder as 50% teen al die getoetste dosisse gelewer het. Spiroxamine en pyrimethanil het die hoogste gemiddelde persentasie inhibisie gelewer vir beide die miselium inhibisie en konidia ontkieming. Die bevindings van hierdie studie het gewys dat T. harzianum suierwonde kan beskerm teen Ph. chlamydospora in die veld. Verder sou sommige fungisiedes wat aangewend word vir die bestuur van poeieragtige meeldou en streepvlek moontlik alternatiewelik of gelyktydig met T. harzianum en T. atroviride aangewend word, alhowel dit met veldproewe bevestig moet word.

Thesis (MScAgric)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, and Botryosphaeria spp. are important trunk disease pathogens that cause premature decline and dieback of grapevine. Previous research has focused primarily on wine grapes and the incidence and symptomatology of these pathogens on table grapes were largely unknown. A survey was therefore conducted to determine the status and distribution of these pathogens and associated symptoms in climatically diverse table grape growing regions. Fifteen farms were identified in the winter rainfall (De Doorns, Paarl and Trawal) and summer rainfall (Upington and Groblersdal) areas. Samples were taken in July and August 2004 from Dan-ben-Hannah vineyards that were 8 years and older. Distal ends of arms were removed from 20 randomly selected plants in each vineyard. These sections were dissected and isolations were made from each of the various symptom types observed: brown or black vascular streaking, brown internal necrosis, wedge-shaped necrosis, watery necrosis, esca-like brown and yellow soft wood rot, as well as asymptomatic wood. Fungal isolates were identified using molecular and morphological techniques. Pa. chlamydospora was most frequently isolated (46.0%), followed by Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea and an undescribed Diplodia sp. (0.2% each), while E. lata was not found. Most of these pathogens were isolated from a variety of symptom types, indicating that disease diagnosis can not be based on symptomatology alone. Pa. chlamydospora was isolated from all areas sampled, although most frequently from the winter rainfall region. Pm. aleophilum was found predominantly in Paarl, while P. viticola only occurred in this area. Although B. obtusa was not isolated from samples taken in De Doorns and Groblersdal, it was the most commonly isolated Botryosphaeria sp., being isolated from Upington, Paarl and Trawal. B. rhodina occurred only in Groblersdal and B. parva in Paarl, Trawal and Groblersdal, while B. australis was isolated from Paarl only. The rest of the isolates (33%) consisted of sterile cultures, Exochalara, Cephalosporium, Wangiella, Scytalidium, Penicillium spp. and two unidentified basidiomycetes, which were isolated from five samples with yellow esca-like symptoms from the Paarl area. These findings clearly illustrate that grapevine trunk diseases are caused by a complex of fungal pathogens, which has serious implications for disease diagnosis and management. Protection of wounds against infection by any of these trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. However, previous research on the effectiveness of chemical pruning wound protectants has mostly focused on the control of Eutypa dieback only. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and Eutypa lata, but no such studies have been conducted for the pathogenic Botryosphaeria species from grapevine in South Africa. Ten fungicides were therefore tested in vitro for their efficacy on mycelial inhibition of the four most common and/or pathogenic Botryosphaeria species in South Africa, B. australis, B. obtusa, B. parva and B. rhodina. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid were ineffective in inhibiting the mycelial growth at the highest concentration tested (5 μg/ml; 20 μg/ml for copper ammonium acetate). Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole were the most effective fungicides with EC50 values for the different species ranging from 0.36-0.55, 0.07-0.17, 0.07-1.15 and 0.04-0.36 μg/ml, respectively. These fungicides, except prochloraz manganese chloride, are registered on grapes in South Africa and were also reported to be effective against Pa. chlamydospora, P. viticola and E. lata. Results from bioassays on 1-year-old Chenin Blanc grapevine shoots indicated that benomyl, tebuconazole and prochloraz manganese chloride were most effective in limiting lesion length in pruning wounds that were inoculated with the Botryosphaeria spp after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation data of the inoculated lesions. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can nonetheless be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against Botryosphaeria spp. as well as the other grapevine trunk disease pathogens.
AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, en Botryosphaeria spesies is die mees belangrikste stamsiekte patogene wat agteruitgang en vroeë terugsterwing van wingerd veroorsaak. Voorafgaande navorsing het hoofsaaklik gefokus op wyndruiwe en die voorkoms en simptomatologie van hierdie patogene op tafeldruiwe is dus grootliks onbekend. ‘n Opname is gevolglik gedoen in verskillende klimaaatsareas waar tafeldruiwe verbou word om die voorkoms en verspreiding, asook die simptome geassosieer met hierdie patogene, te bepaal. Vyftien plase is geïdentifiseer in die winter- (De Doorns, Paarl en Trawal) en somer-reënval (Upington en Groblersdal) streke. Wingerde (8 jaar en ouer) met die kultivar Dan-ben-Hannah is gekies vir opname en monsters is gedurende Julie en Augustus 2004 geneem. Die distale deel van ‘n arm is verwyder vanaf 20 lukraak gekose plante in elke wingerd. Hierdie dele is ontleed en isolasies is gemaak vanuit elke simptoomtipe wat beskryf is, naamlik bruin en swart vaskulêre verkleuring, bruin interne nekrose, wig-vormige nekrose, waterige nekrose, esca-geassosieerde bruin en geel sagte houtverrotting en asimptomatiese hout. Identifikasie van die swamagtige isolate is gedoen op grond van morfologiese eienskappe en molekulêre tegnieke. Pa. chlamydospora is die meeste geïsoleer (46.0%), gevolg deur Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea en ‘n onbeskryfde Diplodia sp. (0.2% elk), terwyl E. lata nie geïsoleer is nie. Hierdie patogene is elk geïsoleer vanuit ‘n verskeidenheid simptoomtipes, wat daarop dui dat siektediagnose nie alleenlik op simptomatologie gebaseer kan word nie. Pa. chlamydospora is geïsoleer vanuit al die gebiede, alhoewel die patogeen opmerklik meer voorgekom het in die winter-reënval area. Pm. aleophilum het hoofsaaklik voorgekom in Paarl, terwyl P. viticola slegs in hierdie area voorgekom het. Alhoewel B. obtusa nie voorgekom het in die De Doorns en Groblersdal areas nie, was dit die mees algemeen geïsoleerde Botryosphaeria sp. en het in Upington, Paarl en Trawal voorgekom. B. rhodina het slegs in Groblersdal voorgekom, B. parva in Paarl, Groblersdal en Trawal en B. australis het slegs in Paarl voorgekom. Die res van die isolate (33%) het bestaan uit steriele kulture, Exochalara, Cephalosporium, Wangiella, Scytalidium, en Penicillium spesies asook twee onbekende basidiomycete isolate, geïsoleer vanuit vyf monsters met geel eska-geassosieerde simptome vanuit die Paarl area. Hierdie resultate illustreer dus die feit dat wingerdstamsiektes deur ‘n kompleks van swampatogene veroorsaak word, wat belangrike implikasies het vir die bestuur en diagnose van hierdie siektes. Wondbeskerming teen infeksie van enige van hierdie stamsiekte patogene is die mees doeltreffende en koste-effektiewe manier om wingerdstamsiektes te voorkom. Vorige navorsing aangaande die effektiwiteit van chemiese wondbeskermingsmiddels het egter slegs gefokus op die beheer van Eutypa terugsterwing. In vitro swamdoder sensitiwiteitstoetse is gedoen vir Pa. chlamydospora, P. viticola en Eutypa lata, maar geen studies is al gedoen ten opsigte van die patogeniese Botryosphaeria spesies op wingerd in Suid-Afrika nie. Tien swamdoders is dus getoets vir inhibisie van in vitro miseliumgroei van die vier mees algemene en/of patogeniese Botryosphaeria spesies wat in Suid-Afrika voorkom, naamlik B. australis, B. obtusa, B. parva en B. rhodina. Iprodione, pyrimethanil, koper ammonium asetaat, kresoxim-metiel en boscalid was oneffektief by die hoogste konsentrasies getoets (5 μg/ml; 20 μg/ml vir koper ammonium asetaat). Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool was die mees effektiewe swamdoders met EC50 waardes tussen 0.36-0.55, 0.07-0.17, 0.07-1.15 en 0.04-0.36 μg/ml, onderskeidelik vir die verskillende spesies. Hierdie fungisiedes, behalwe prochloraz mangaan chloried, is geregistreer op druiwe in Suid-Afrika en is ook effektief gevind teenoor Pa. chlamydospora, P. viticola en E. lata. Resultate van biotoetse op 1-jaar-oue Chenin Blanc wingerd lote het getoon dat benomyl, tebuconasool en prochloraz mangaan chloried die effektiefste was om die lengte van letsels in snoeiwonde, geinokuleer met Botryosphaeria spesies na die aanwending van swamdoder behandelings, te verminder. Die bevindinge was egter onbeslis as gevolg van die lae en variërende her-isolerings data. Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool kan egter geïdentifiseer word as swamdoders wat verder geevalueer kan word as snoeiwond beskermingsmiddels teen Botryosphaeria spesies asook ander wingerd stamsiekte patogene in verdere biotoetse en wingerdproewe.

La Vite ultracentenaria del Fantini, situata ai piedi della collina del podere Terzanello di Sotto a Pianoro, è una delle viti più vecchie dell’Appenino Tosco-Emiliano, scampata dall’epidemia di filossera dell’800. La Vite, fotografata per la prima volta nel 1956 da Luigi Fantini, fu ritrovata nel 1990 da Stefano Galli, ma in condizioni decisamente diverse rispetto a quelle di quarant’anni prima. La Vite mostrava una forte perdita di vigoria, con il tronco in buona parte marcito e disseccamenti fogliari e dei tralci. Galli cercò negli anni di smuovere interesse nei confronti della vite su vari fronti e nel 2019 decise di contattare i laboratori di Patologia vegetale dell’Università di Bologna per un’analisi più approfondita sulle possibili patologie presenti sulla Vite. A tale scopo, il seguente progetto di tesi si è incentrato sull’analisi morfologica e molecolare dei diversi patogeni isolati dal materiale prelevato dalla Vite in seguito a diversi isolamenti effettuati nel 2019/2020 per comprendere l’eziologia delle diverse malattie presenti e trovare possibili soluzioni per il miglioramento della salute della stessa. Si è proceduto all’identificazione morfologica e molecolare delle colonie fungine e dei virus presenti nei tessuti vegetali analizzati; si è valutata l’efficacia di diversi prodotti a basso contenuto cuprico e con estratti vegetali in vitro al fine di poter scegliere il prodotto da utilizzare per il successivo trattamento in endoterapia. I funghi d’interesse, isolati su PDA e sequenziati (gene ITS), sono risultati essere Diplodia mutila, Diplodia seriata, Fomitiporia mediterranea e Kalmusia variispora, tutte specie responsabili delle malattie del legno e del Mal dell’esca della vite, mentre non si è riscontrata la presenza di alcun virus. Durante la ripresa vegetativa della Vite, in seguito del trattamento in endoterapia effettuato con due diversi prodotti (RESOLV 5 e ES PLUS), sarà possibile comprendere meglio il decorso della patologia.

As doenças do lenho são das mais importantes doenças associadas à videira. Este trabalho teve como objetivo principal estudar as diferenças da diversidade dos fungos endofíticos e patogénicos e a suscetibilidade de duas cultivares distintas: Trincadeira e Aragonez, em dois locais (A e B). Situadas na Região do Alentejo todas testadas em material lenhoso. Os fungos endofíticos mais encontrados nas amostras de planta foi o pertencente ao género Alternaria, tanto para os dois locais como para as duas cultivares. Nos fungos patogénicos, os mais encontrados nas amostras das plantas foram os fungos das espécies Diplodia seriata, Botrytis cinerea e Rhizopus spp. Verificou-se que a cultivar Aragonez, no local A é menos suscetível às doenças do lenho, ao contrário do que acontece no local B, neste local a cultivar menos suscetível às doenças do lenho é a Trincadeira. As espécies Botryosphaeria dothidea e Diplodia seriata são causadores das doenças do lenho; Evaluation of endophytic fungal communities in two grapevine cultivars from Alentejo Region showing different susceptibility to trunk diseases Abstract: Trunk diseases is one of the most important diseases of the grapevine. The main objective of this work was to study the differences in the diversity of endophytic and pathogenic fungi and the susceptibility of two cultivars: Trincadeira and Aragonez in two sites (A and B) located in Alentejo Region and all tested in woody material. The most endophytic fungi found in the plant samples was, the one belonging to the genus Alternaria, for both sites and cultivars. For pathogenic fungi, the most found in the plant samples were the species Diplodia seriata, Botrytis cinerea and Rhizopus spp. It was also found, that the cultivar Aragonez in the organic production mode is less susceptible to trunk diseases, unlike what happens in the conventional production mode. In this mode the cultivar Trincadeira is less susceptible to these diseases. Wood diseases are caused by the species: Botryosphaeria dothidea and Diplodia seriata.

O trabalho teve como objetivo testar o potencial antagonista de fungos presentes em videiras do Alentejo, contra fungos patogénicos responsáveis por doenças do lenho. Foram identificados 14 isolados provenientes de ramos de videiras aparentemente saudáveis, 12 dos quais pertencentes aos géneros Alternaria, Epicoccum e Cladosporium, sendo considerados fungos endofíticos, dos quais foi testada a capacidade antagonista, e dois identificados como Botryosphaeria dothidea e Phomopsis viticola, fungos patogénicos responsáveis por doenças do lenho. Todos os isolados apresentaram capacidade antagonista contra B. dothidea nos três testes realizados. No caso de P. viticola todos os fungos apresentaram capacidade antagonista no teste de antagonismo direto e compostos voláteis, enquanto que no teste de compostos não voláteis apenas um não apresentou capacidade antagonista. Os resultados apresentados mostram o potencial antagonista dos fungos endofíticos como agentes de luta biológica; ABSTRACT: Antagonistic activity of endophytic fungi of Vitis vinifera L. against the most important grapevine trunk diseases in Alentejo The objective of this work was to test the potential antagonism of fungi present in Alentejo vines against pathogenic fungi responsible for trunk diseases. 14 isolates from apparently healthy grapevines were identified, 12 were considered endophytic fungi belonging to the genera Alternaria, Epicoccum and Cladosporium, and two were identified as Botryosphaeria dothidea and Phomopsis viticola, pathogenic fungi responsible for trunk diseases. All the isolates presented antagonistic capacity against to B. dothidea, in the direct antagonism test, the volatile compounds test and the non-volatile compounds test. In the case of P. viticola all fungi in the test of direct antagonism and volatile compounds presented antagonistic capacity, whereas in the test of non-volatile compounds only one did not present antagonistic capacity. The results presented show the potential antagonism of endophytic fungi as biological control agents.

Fabry disease is a rare X-linked lysosomal storage disorder in which deficiency of alpha-galactosidase A leads to accumulation of substrate, mostly globotriaosylceramide, which causes a progressive, multiorgan disease affecting predominantly the kidneys, skin, heart, gut, and nervous system. Painful peripheral (‘acral’) neuropathy is characteristic. The disease is commonly diagnosed in children and young men often after some years of usually neuropathic symptoms, with exacerbations (Fabry crises), that commonly elude diagnosis for a long time. These usually occur years in advance of overt involvement of other organs. Diagnosis may also be suspected from renal biopsy, echocardiographic evidence of cardiomyopathy commonly beginning as left ventricular hypertrophy, or characteristic angiokeratomas typically in ‘bathing trunk’ distribution on skin. Renal manifestations are of proteinuria leading to progressive chronic kidney disease associated with deposits in podocytes. Diarrhoea is common. Disordered sweating is typical. Corneal lesions are also typical and there may be tortuosity of retinal vessels. Strokes are increased in frequency, and sensorineural deafness may occur. Women have fewer and later overt manifestations but some develop severe disease.

For almost 40 years, Australian researchers have been part of an international group of scientists who have studied graft-transmitted disorders of the grapevine. The Australian wine and grape industries are undergoing significant expansion as is the case in some other countries. Preventing the spread of pathogens, by producing clean propagation material, and minimising the disease load on new vines, is essential for the continuing success of the industry. This book covers the characteristics of each class of graft-transmitted pathogen, their effect on vines, how they spread and strategies for their control. Eleven of the most important diseases are illustrated and described comprehensively, including information about occurrence, symptoms, detection, transmission and effect on yield and quality. Finally there is a discussion of quarantine issues and disease management. This book will be an invaluable teaching tool and is intended for vineyard managers, grape growers, consultants, extension offers and students. While it provides a basic understanding of the nature of pathogens, it will aid in field assessment and identification of the often confusing disease symptoms.

Basal Cell Nevus Syndrome – Bathing Trunk Nevus – Cowden Disease – Cylindromatosis – Dysplastic Nevus Syndrome – Epidermal Nevus – Gardner Syndrome – Hereditary Keratoacanthomas – Infantile Myofibromatosis – Multiple Endocrine Neoplasia Types 1, 2A, and 2B/3 – Multiple Leiomyomatosis – Pilomatricoma – Proteus Syndrome – Sebaceous Nevus Syndrome – Tumoral Calcinosis

Tremors are characterized by rhythmic oscillations of one or more body parts. Although typically seen in the upper limbs, almost any area may be involved, including the trunk, head, facial muscles, and legs. Sometimes, tremor is not visible at all but may be heard or palpated, for example, in vocal or orthostatic tremor, respectively. In neurological practice, the diagnosis and treatment of tremor is an everyday problem. A common scenario is the distinction between essential tremor and Parkinson’s disease. In this chapter, the wide range of tremors are discussed, with their aetiolology, pathophysiology, diagnosis and management described.Ataxia is a term used to describe a wide range of neurological disorders affecting muscle coordination, speech and balance that reflect dysfunction of a part of the central nervous system involved in motor function. Many of ataxias have a cerebellar pathology as root cause, although it must be remembered that ataxia, clumsiness, disordered ocular motility, dysarthria, and even kinetic or intention tremor are not always caused by cerebellar disease. This chapter describes the wide range of cerebellar disorders and ataxias, as are non-cerebellar ataxias such as Friedreich’s ataxia.

Context: Pisa Syndrome (PS) is a rare postural disorder, characterized by dystonia of the trunk muscles, lateral deviation as well as rotation of the axial axis. There is a strong association with Parkinson’s disease (PD) due to the possible imbalance between neurotransmitters. It happens either due to a decrease in dopaminergic stimuli, either because of an excess of cholinergic stimuli or drugs (an example of antidopaminergics). The diagnosis is clinical, showing at least a 10-degree trunk flexion with improvement of pharmacological and non- pharmacological measures. Case report: A 60-year-old man was diagnosed with PD five years ago due to tipical clinical complaints and physical examination. The treatment recquired an increase of Pramipexole as well as the use of Levodopa and Benserazide. After two years, he complained about neck pain, low back pain, hip pain and a slight trunk twisting. After six months, his pain was worse and he reported right hemidystonia. Thus, he was diagnosed with PS associated with PD. It was decided to optimize the therapy with Pregabalin, muscle relaxants and rehabilitation. However, it did not show any good result. In 2020, the application of botulinum toxin (BTX) evidenced excellent results, improving both the pain and the spasticity of the patient. Conclusions: Early recognition is necessary to introduce the right treatment as soon as possible, especially BTX and rehabilitation, ensuring functionality and avoiding negative outcomes.

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.

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.

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.

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.

Vineyards are found on six of seven continents, producing a crop of high economic value with much historic and cultural significance. Because of the wide range of conditions under which grapes are grown, management approaches are highly varied and must be adapted to local climatic constraints. Research has been conducted in the traditionally prominent grape growing regions of Europe, Australia, and the western USA, but far less information is available to guide production under more extreme growing conditions. The overarching goal of this project was to improve understanding of vineyard water management related to the critical inter-row zone. Experiments were conducted in moist temperate (North Carolina, USA) and arid (Negev, Israel) regions in order to address inter-row water use under high and low water availability conditions. Specific objectives were to: i) calibrate and verify a modeling technique to identify components of evapotranspiration (ET) in temperate and semiarid vineyard systems, ii) evaluate and refine strategies for excess water removal in vineyards for moist temperate regions of the Southeastern USA, and iii) evaluate and refine strategies for water conservation in vineyards for semi-arid regions of Israel. Several new measurement and modeling techniques were adapted and assessed in order to partition ET between favorable transpiration by the grapes and potentially detrimental water use within the vineyard inter-row. A micro Bowen ratio measurement system was developed to quantify ET from inter-rows. The approach was successful at the NC site, providing strong correlation with standard measurement approaches and adding capability for continuous, non-destructive measurement within a relatively small footprint. The environmental conditions in the Negev site were found to limit the applicability of the technique. Technical issues are yet to be solved to make this technique sufficiently robust. The HYDRUS 2D/3D modeling package was also adapted using data obtained in a series of intense field campaigns at the Negev site. The adapted model was able to account for spatial variation in surface boundary conditions, created by diurnal canopy shading, in order to accurately calculate the contribution of interrow evaporation (E) as a component of system ET. Experiments evaluated common practices in the southeastern USA: inter-row cover crops purported to reduce water availability and thereby favorably reduce grapevine vegetative growth; and southern Israel: drip irrigation applied to produce a high value crop with maximum water use efficiency. Results from the NC site indicated that water use by the cover crop contributed a significant portion of vineyard ET (up to 93% in May), but that with ample rainfall typical to the region, cover crop water use did little to limit water availability for the grape vines. A potential consequence, however, was elevated below canopy humidity owing to the increased inter-row evapotranspiration associated with the cover crops. This creates increased potential for fungal disease occurrence, which is a common problem in the region. Analysis from the Negev site reveals that, on average, E accounts for about10% of the total vineyard ET in an isolated dripirrigated vineyard. The proportion of ET contributed by E increased from May until just before harvest in July, which could be explained primarily by changes in weather conditions. While non-productive water loss as E is relatively small, experiments indicate that further improvements in irrigation efficiency may be possible by considering diurnal shading effects on below canopy potential ET. Overall, research provided both scientific and practical outcomes including new measurement and modeling techniques, and new insights for humid and arid vineyard systems. Research techniques developed through the project will be useful for other agricultural systems, and the successful synergistic cooperation amongst the research team offers opportunity for future collaboration.

For Plant Protection and Quarantine (PPQ) and our partners, 2016 was a year of remarkable successes. Not only did we eradicate 10 fruit fly outbreaks, but we also achieved 4 years with zero detections of pink bollworm, moving us one step closer to eradicating this pest from all commercial cotton-growing areas of the continental United States. And when the U.S. corn industry faced the first-ever detection of bacterial leaf streak (Xanthomonas vasicular pv vasculorum), we devised a practical and scientific approach to manage the disease and protect valuable export markets. Our most significant domestic accomplishment this year, however, was achieving one of our agency’s top 10 goals: eliminating the European grapevine moth (EGVM) from the United States. On the world stage, PPQ helped U.S. agriculture thrive in the global market-place. We worked closely with our international trading partners to develop and promote science-based standards, helping to create a safe, fair, and predictable agricultural trade system that minimizes the spread of invasive plant pests and diseases. We reached critical plant health agreements and resolved plant health barriers to trade, which sustained and expanded U.S. export markets valued at more than $4 billion. And, we helped U.S. producers meet foreign market access requirements and certified the health of more than 650,000 exports, securing economic opportunities for U.S. products abroad. These successes underscore how PPQ is working every day to keep U.S. agriculture healthy and profitable.