Trunk Disease Pathogens Research Articles

A protocol for selection of Trichoderma spp. to protect grapevine pruning wounds against Lasiodiplodia theobromae.

There is no standardized protocol to select Trichoderma against trunk disease pathogens in grapevine. This study describes a protocol to select Trichoderma isolates for protecting the pruning wounds of grapevine against Lasiodiplodia theobromae. Thus, four assays were performed: (1) in vitro antagonism of Trichoderma isolates, molecularly identified, to control L. theobromae; (2) in vitro effect of grapevine fungicides on the conidia of the antagonists; (3) capability of Trichoderma to survive and endophytically penetrate in grapevine canes; and (4) efficiency of Trichoderma and fungicides to protect the pruning wounds of grapevine shoots against L. theobromae. T. asperelloides (02/03), T. koningiopsis (09/02), and T. asperellum (01/13) were the best antagonists for L. theobromae. The conidia of the three isolates were affected only by tebuconazole, with the first two fungi being the most tolerant to the fungicides. The Trichoderma isolates survived on the cane tip for 15days, but only T. asperelloides (02/03) penetrated endophytically 4cm in the cane and showed preventive and curative capability to control L. theobromae, with similar efficiency as that of the fungicides in both the periods tested post inoculation. These four assays are suggested to select Trichoderma against L. theobromae or other trunk disease pathogens.

High-throughput amplicon sequencing-based analysis of active fungal communities inhabiting grapevine after hot-water treatments reveals unexpectedly high fungal diversity

Abstract The ecology of total fungal communities in grapevine is so far largely derived from studies on culture-dependent methods or cultivation-independent rDNA approaches. Sequencing the ribosomal RNA transcripts (rRNA) would rather reveal the functionally and metabolically active important taxa of the fungal community and provide insights into its activity in the wood. The present study investigated changes in the potentially active fungal communities of internal grapevine wood after Hot-Water Treatment (HWT) in planting material from Czech Republic and Spain at two different times during the propagation process and from two plant zones. We examined fungal communities using both traditional isolation into culture and high-throughput amplicon sequencing (HTAS) of the internal transcribed spacer 2 (ITS2) region in extracted total RNA. HTAS from metatranscriptomic RNA increased the resolution of the fungal community analysis and revealed a highly diverse mycobiota of grapevine wood compared to the traditional method. Fungal diversity differed between grapevine genotypes and showed a temporal variation over the vegetative period. Grapevine planting materials exhibited high fungal diversity after HWT, which demonstrates that the HWT process does not sterilize the internal wood of grapevine. HWT reduced the infection caused by fungal trunk disease pathogens but was not completely effective in eliminating their growth. This study provides important and practically useful insights into the dynamics of active fungal communities in hot-water treated plants, and represents the first study of active fungal communities on grapevine grafted plants by comparing traditional and next-generation sequencing methods.

Eutypa, Eutypella, and Cryptovalsa Species (Diatrypaceae) Associated with Prunus Species in South Africa.

Stone fruit trees (Prunus spp.) are economically important fruit trees cultivated in South Africa. These trees are often grown in close proximity to vineyards and are to a large extent affected by the same trunk disease pathogens as grapevines. The aim of the present study was to determine whether stone fruit trees are inhabited by Diatrypaceae species known from grapevines and whether these trees could act as alternative hosts for these fungal species. Isolations were carried out from symptomatic wood of Prunus species (almond, apricot, cherry, nectarine, peach, and plum) in stone fruit growing areas in South Africa. Identification of isolates was based on phylogenetic analyses of the internal transcribed spacer region and β-tubulin gene. Forty-six Diatrypaceae isolates were obtained from a total of 380 wood samples, from which five species were identified. All five species have also been associated with dieback of grapevine. The highest number of isolates was found on apricot followed by plum. No Diatrypaceae species were isolated from peach and nectarine. Eutypa lata was the dominant species isolated (26 isolates), followed by Cryptovalsa ampelina (7), Eutypa cremea (5), Eutypella citricola (5), and Eutypella microtheca (3). First reports from Prunus spp. are E. cremea, E. citricola, and E. microtheca. Pathogenicity tests conducted on apricot and plum revealed that all these species are pathogenic to these hosts, causing red-brown necrotic lesions like those typical of Eutypa dieback on apricot.

Soaking grapevine cuttings in water: a potential source of cross contamination by micro-organisms

Grapevine nurseries soak cuttings in water during propagation to compensate for dehydration and promote root initiation. However, trunk disease pathogens have been isolated from soaking water, indicating cross contamination. Cuttings of Vitis vinifera cv. Sunmuscat and V. berlandieri x V. rupestris rootstock cv. 140 Ruggeri were immersed in sterilized, deionised water for 1, 2, 4, 8 and 16 h. The soaking water was cultured (25°C for 3 days) on non-specific and specific media for fungi and bacteria. The base of each cutting was debarked and trimmed and three 3 mm thick, contiguous, transverse slices of wood cultured at 25°C for 3 days. The soaking water for both cultivars became contaminated with microorganisms within the first hour. Numbers of fungi iso-lated from the wood slices soaked for one hour were significantly greater than those from non-soaked cuttings. The number of bacterial colonies growing from the wood slices increased after soaking for 2‒4 h in Sunmuscat. In a second experiment Shiraz cuttings were soaked for 1, 2, 4, 8 and 24 h. The soaking water became contaminated within the first hour but only the bacterial count increased significantly over time. Microorganisms also established on the container surfaces within the first hour although there were no significant increases over 24 h. These results confirm that soaking cuttings is a potential cause of cross contamination and demonstrate contamination of cuttings occurs after relatively short periods of soaking. Avoiding exposing cuttings to water will reduce the transmission of trunk diseases in propagation.

Protection of Grapevine Pruning Wounds against Eutypa lata by Biological and Chemical Methods

Eutypa dieback, caused by the fungus Eutypa lata, is a serious disease of grapevines that infects mainly through pruning wounds. The aim of this study was to evaluate the in vitro efficacy of fungicides from various chemical groups against E. lata, as well as the in vivo efficacy of the most effective fungicides and selected bacterial and fungal antagonists of E. lata, in grapevine pruning wound protection trials. In vitro studies revealed that flusilazole, tebuconazole, benomyl, fenarimol and myclobutanil were the most effective fungicides to inhibit mycelial growth of E. lata. Two field trials were conducted, one subjected to artificial inoculation and the second to natural infection only. In the first, benomyl, flusilazole and commercially available Trichoderma harzianum-containing products and an experimental Bacillus subtilis strain were applied to fresh pruning wounds. Two Cabernet Sauvignon vineyards were pruned in August 2001 and 2002 and immediately treated and inoculated with a spore suspension of E. lata one day later. Isolations were made from the treated pruning wounds after 12 months to assess the effectiveness of the treatments. The fungicides benomyl and flusilazole were the most effective treatments, although the Trichoderma treatments T77 and Trichoseal spray caused a significant reduction in E. lata infection. In a second trial, pruning wounds of Cabernet Sauvignon, Sauvignon blanc, Red Globe and Bonheur were treated with the Trichoderma products Vinevax (= Trichoseal spray) and Eco77 (= T77) in August 2005 and 2006, subjected to natural infection only and evaluated after seven months. Vinevax and Eco77 not only reduced E. lata, but they also reduced the incidence of other grapevine trunk disease pathogens.

Trunk Disease Fungi Associated With Diospyros kaki in South Africa.

Persimmon trees with dieback symptoms and cankers were observed in three production areas in Western Cape Province in South Africa. Isolations were made from diseased branches, cankers, and pruning wounds as well as fungal fruiting bodies on dead branches and old pruning wounds. Several trunk disease pathogens were identified based on morphological characteristics and by molecular methods, including Diaporthe eres, D. infecunda, Eutypella citricola, E. microtheca, Phaeoacremonium parasiticum, P. scolyti, P. australiense, P. minimum, Fomitiporia capensis, Fomitiporia sp., Fomitiporella sp., and Inocutis sp., which were isolated from persimmon for the first time in the world. Other first reports from persimmon in South Africa include D. foeniculina, D. ambigua, D. mutila, Diaporthe sp., Neofusicoccum australe, N. parvum, Diplodia seriata, and Eutypa lata. Pathogenicity tests conducted with all species, except the basidiomycetes, confirmed their status as possible persimmon pathogens. This is the first study to determine and identify fungi associated with diseased persimmon in South Africa. The knowledge gained in this study forms the basis for further research to determine the impact of these fungi on persimmon productivity.

Effectiveness of Natural Antifungal Compounds in Controlling Infection by Grapevine Trunk Disease Pathogens through Pruning Wounds.

Grapevine trunk fungal pathogens, such as Diplodia seriata and Phaeomoniella 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 evaluated in vitro. The fungi showing greater in vitro efficacy were tested on autoclaved grape wood assays against D. seriata and P. chlamydospora. Based on results from these assays, chitosan oligosaccharide, vanillin, and garlic extract were selected for further evaluation on pruning wounds inoculated with D. seriata and P. chlamydospora in 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.

Arthropods Vector Grapevine Trunk Disease Pathogens

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.

Phomopsis Dieback: A Grapevine Trunk Disease Caused by Phomopsis viticola in California.

Field surveys recently conducted in California and in other grape-growing regions in the United States showed Phomopsis viticola to be one of the most prevalent fungi isolated from grapevine perennial cankers in declining vines. The current study has not only confirmed the presence of P. viticola from grapevine cankers in California but also has for the first time revealed the occurrence of Diaporthe ambigua, D. eres, and D. neotheicola in symptomatic grapevine wood in California by means of morphological studies and multi-gene sequence analysis. Pathogenicity trials conducted on mature cordons of Vitis vinifera 'Syrah' and 'Red Globe', as well as on lignified Syrah dormant canes, showed P. viticola isolates from California to be capable of causing perennial cankers. Lengths of vascular discoloration caused by P. viticola were similar to those caused by Eutypa lata and several Botryosphaeriaceae spp., which are well-known grapevine trunk disease pathogens. Additionally, a lack of spring growth was commonly observed in dormant canes inoculated with P. viticola spore suspensions in two pathogenicity trials. As part of this study, V. vinifera 'Cabernet Sauvignon' and 'Zinfandel' wood was shown to be more susceptible to infection by P. viticola than 'Barbera', 'Chardonnay', 'Merlot', and 'Thompson Seedless' wood. After more than 40 years overlooking P. viticola as a grapevine wood pathogen, this study provides strong evidence of the role of P. viticola as a canker-causing organism, and suggests its addition to the fungi involved in the grapevine trunk disease complex. Results from this study suggest D. ambigua and D. neotheicola to be saprophytes or weak pathogens on grapevine wood.

Efficacy of paste and liquid fungicide formulations to protect pruning wounds against pathogens associated with grapevine trunk diseases in Chile

Trunk diseases are important on grapevines (Vitis vinifera L.) in most countries where grapevines are grown. In Chile, Diplodia seriata, Inocutis sp. and Phaeomoniella (Pa.) chlamydospora are the most prevalent pathogens associated with this syndrome. It has been demonstrated that fresh pruning wounds are the main infection route for fungal trunk disease pathogens. The objective of this study was to evaluate the efficacy of paste and spray fungicide applications in protecting pruning wounds against D. seriata, Inocutis sp. and Pa. chlamydospora on ‘Cabernet Sauvignon’ grapevines in two important Chilean grapevine production areas. The fungicides used in this study were benomyl, pyraclostrobin, tebuconazole, and thiophanate-methyl, which were applied as paste and liquid fungicide on fresh pruning wounds pre- and post-inoculation with mycelium plugs of the different fungal pathogens used in this study. The efficacy of the fungicides was assessed by measuring the length of the discoloration of the vascular system and percent re-isolation obtained on fresh pruning wounds. The results obtained demonstrate that infections caused by D. seriata, Inocutis sp. and Pa. chlamydospora can be significantly reduced using both paste and spray applications of benomyl, pyraclostrobin, tebuconazole and thiophanate-methyl and that the paste formulations provided a better control than spray applications. Regardless of the fungicide and application method used, the pre-inoculation treatments (performed 24 h before inoculation) resulted in better control than the 24 h post-inoculation treatments. The current study showed that Inocutis sp. can induce vascular discoloration on grapevine spurs. This study supports a broader use of benzimidazole fungicides for the control of the devastating trunk diseases in all grape productions worldwide.

Evaluation of biocontrol agents for grapevine pruning wound protection against trunk pathogen infection.

Trunk diseases of grapevine are caused by numerous pathogens, including Eutypa lata , Phaeomoniella chlamydospora , and species of Botryosphaeriaceae (incl. Botryosphaeria and aggregate genera), Phomopsis and Phaeoacremonium . Since infections occur mainly through pruning wounds, that have been shown by previous research to stay susceptible for up to 16 weeks after pruning, long-term pruning wound protection is required for prevention of infection. This study evaluated several biocontrol agents against a range of trunk disease pathogens in dual plate laboratory trials to determine macroscopic and microscopic interactions. The biocontrol agents had a substantial effect on all the pathogens, with a wide range of macroscopic and microscopic interactions observed. The best performing biocontrol agents were tested in two field trials. Fresh pruning wounds were treated with benomyl, Trichoderma products (Biotricho®, Vinevax® and ECO 77®) and isolates (USPP-T1 and -T2, identified as T. atroviride ) and Bacillus subtilis . Seven days after treatment the pruning wounds were inoculated by spraying with spore suspensions of Neofusicoccum australe , N. parvum , Diplodia seriata , Lasiodiplodia theobromae , Eutypa lata , Phaeomoniella chlamydospora or Phomopsis viticola . Eight months after inoculation, the treatments were evaluated by isolation onto potato dextrose agar. The efficacy of the biocontrol agents was in most cases similar or superior to that observed for benomyl. Isolate USPP-T1, in particular, was very effective, reducing incidence of Ph. viticola , E. lata , Pa. chlamydospora , N. australe , N. parvum , D. seriata and L. theobromae by 69, 76, 77, 78, 80, 85 and 92%, respectively. This is the first report of biological protection of grapevine pruning wounds against this group of grapevine trunk disease pathogens.

Histo-pathology study of the growth of Trichoderma harzianum , Phaeomoniella chlamydospora and Eutypa lata on grapevine pruning wounds

Protecting grapevine pruning wounds by inoculating them with Trichoderma spp. can preventinfection from trunk disease pathogens. The growth and interactions of both, the biological control agent Trichoderma spp. and the vine pathogens, are not well understood. Green fluorescent protein (GFP)-labelled Trichoderma harzianum and red fluorescent protein (DsRed)-labelled T. harzianum , were dual-inoculatedwith Phaeomoniella chlamydospora (DsRed) or Eutypa lata (GFP) on fresh pruning wounds of one-year-oldCabernet Sauvignon and Sauvignon blanc shoots. The inoculated fungi were recovered from varying depthswithin the shoots at 30-day-intervals for 90 days. Trichoderma harzianum suppressed the pathogens and grew deeper in the presence of the pathogens than when it was singly inoculated; possibly an indication of pathogen recognition and competitive response. Eutypa lata was completely eliminated from Sauvignon blancin dual-inoculated canes after 90 days. The mycelium of P. chlamydospora (DsRed) grew extensively in thexylem vessels and possibly contributed to vessel occlusion. Phaeomoniella chlamydospora and E. lata caused blockage of the vessels and thickening of the vessel walls. Grapevine wood produced both tyloses and gums (gels) that blocked xylem vessels as a result of infection. A thickening of the cell walls of xylem fibres occurredonly in E. lata -inoculated shoots, indicative of a different mode of pathogenesis from P. chlamydospora .

Fungi associated with die-back symptoms of apple and pear trees, a possible inoculum source of grapevine trunk disease pathogens

A survey was undertaken on apple and pear trees in the main pome fruit growing areas ofthe Western Cape of South Africa to determine the aetiology of trunk diseases with specific reference to pathogens known to occur on grapevine, which are frequently cultivated in close proximity of these orchards. Several fungal genera containing known trunk disease pathogens were found. Two Diplodia species, D. seriata and Diplodia sp., were isolated along with Neofusicoccum australe and N. vitifusiforme . Four Phaeoacremonium species, Phaeoacremonium aleophilum , Pm. iranianum , Pm. mortoniae and Pm. viticola , two Phomopsis species linked to clades identified in former studies as Phomopsis theicola and Phomopsis sp. 7, and Eutypa lata were found. In addition, Paraconiothyrium brasiliense , Pr. variabile and a Pyrenochaeta like species were also isolated. Diplodia seriata (56% of total isolates) and Pm. aleophilum (22%) were most frequently isolate. First reports from pear wood include the Phaeoacremonium spp. and Paraconiothyrium brasiliense , while new reports from apple include Pm. aleophilum , Ph. theicola , Phomopsis sp. 7, Pr. variabile and E. lata . A pathogenicity trial was undertaken to determine the role of these species on apple, pear and grapevine shoots. Neofusicoccum australe caused the longest lesions on grapevine shoots, while Pr. variabile , D. seriata , Pm. mortoniae and the Pyrenochaeta -like sp. caused lesions that were longer than non-inoculatedand non-pathogen experimental controls. On pear shoots, Diplodia sp. and N. australe caused the longest lesions,followed by D. seriata and E. lata . On apple shoots, the longest lesions were caused by N. australe and Pm. iranianum . These results demonstrate that apple and pear trees in Western Cape orchards are hosts to many known trunk pathogens along with potential new trunk disease-causing fungi.

The distribution and symptomatology of grapevine trunk disease pathogens are influenced by climate

Grapevine trunk diseases, caused by a range of phytopathogenic fungi, represent a serious impediment to wine and table grape production wherever these crops are cultivated. Previous studies have shown that the distribution of these pathogens is influenced by climate and that they are associated with a variety of internal wood decay symptoms. Little was known, however, about the influence of climate on the disease symptomatology of the different pathogens in a specific area. To address this, a survey was conductedin 30 wine and table grape vineyards in summer, marginal and winter rainfall regions of South Africa. Apart from Eutypa lata , which occurred only in the winter rainfall region, Phaeomoniella chlamydospora , species of Phaeoacremonium , Botryosphaeriaceae and Phomopsis occurred in all regions surveyed. The incidence of the fungal genera and species associated with trunk disease varied between regions, with overlapping symptom profiles that differed based on the climatic region. These findings suggest that symptom-based disease diagnosis alone is unreliable and that distribution and symptomatology of grapevine trunk pathogens are strongly influenced by climatic conditions in a specific production region.

Temporal spore dispersal patterns of grapevine trunk pathogens in South Africa

Trunk disease pathogens of grapevines, viz. Phaeomoniella chlamydospora, Eutypa lata and several species in Botryosphaeriaceae, Phaeoacremonium and Phomopsis are known to infect fresh pruning wounds by means of air-borne inoculum released after rainfall or prolonged periods of high relative humidity. Recent surveys have demonstrated that most or all of these pathogens are present in climatically diverse grape growing regions of South Africa. However, the factors controlling spore dispersal of these pathogens in vineyards were largely unknown. To address this question, spore trapping was done in a Chenin Blanc vineyard in the Stellenbosch area, South Africa, for 14 weeks during the grapevine pruning period from June to mid-September of 2004 and 2005. Hourly recordings of weather data were done by a weather station in the row adjacent to the spore trap. Spores of E. lata and Phomopsis and species in Botryosphaeriaceae were trapped throughout the trapping periods of 2004 and 2005, with higher levels of trapped spores recorded in 2005. The spores of all three pathogens were trapped during or after periods of rainfall and/or high relative humidity. In neither of the 2 years were spores of Pa. chlamydospora or Phaeoacremonium spp. trapped. Results indicated that spore event incidence, as well as the amount of spores released during a spore event of above-mentioned pathogens, were governed by rainfall, relative humidity, temperature and wind speed prior to and during the spore events.

Evaluation of the grapevine nursery propagation process as a source of Phaeoacremonium spp. and Phaeomoniella chlamydospora and occurrence of trunk disease pathogens in rootstock mother vines in Spain

Five commercial nurseries were sampled in 2007 to evaluate the grapevine nursery propagation process as a source of Petri disease pathogens (Phaeoacremonium spp. and Phaeomoniella chlamydospora). Samples were taken at four stages of the propagation process: pre-grafting hydration tanks, scissors used for cutting buds, grafting machines and peat used to promote root development. All samples were analysed using two different techniques: nested PCR using specific primers for Phaeoacremonium spp. (Pm1/Pm2) and Pa. chlamydospora (Pch1/Pch2); and fungal isolation by culturing on semi-selective medium. Either Phaeoacremonium spp. or Pa. chlamydospora were detected at any of these stages, and more importantly they were viable since they were detected by isolating on culturing medium. Additionally, the importance of grapevine rootstock mother fields as sources of inoculum in the nurseries was studied. Fourteen grapevine rootstock mother fields were surveyed in 2006 and 2007 for the occurrence of fungal trunk pathogens. A total of 16.4% and 30% of the plants sampled in 2006 and 2007, respectively were infected. Petri disease pathogens (Pa. chlamydospora, Phaeoacremonium aleophilum, Pm. parasiticum) and several Botryosphaeriaceae species (Neofusicoccum parvum, Botryosphaeria dothidea, Lasiodiplodia theobromae, N. australe, N. mediterraneum and N. vitifusiforme) and Phomopsis viticola were isolated. This is the first time N. mediterraneum has been isolated from grapevines and the first report of N. australe, N. mediterraneum and N. vitifusiforme in Spain. This work shows that grapevine rootstock mother plants and the propagation process of grapevine plants should be considered as important sources of inoculum for fungal trunk pathogens, and especially of Petri disease pathogens.

Reporter gene transformation of the trunk disease pathogenPhaeomoniella chlamydosporaand biological control agentTrichoderma harzianum

The economically important trunk disease pathogen Phaeomoniella chlamydospora causes Petri disease in Vitis vinifera and is also associated with the Esca trunk disease complex. Not much is known about the pathogen’s epidemiology and interactions with the grapevine host, other trunk disease pathogens and biological control agents such as Trichoderma harzianum. Reporter gene labelling of plant pathogens and biocontrol agents can facilitate host-pathogen-biocontrol interaction studies. Therefore, the first aim of this study was to stably transform T. harzianum strain T77 and a South African isolate of P. chlamydospora with the Green fluorescent protein (GFP) and the Red fluorescent protein (DsRed-Express) reporter genes. Several stably transformed reporter gene labelled transformants were obtained for both fungi. The second aim was to evaluate these transformants and their respective wild type isolates for several in vitro biological characteristics, virulence (P. chlamydospora) and host colonisation (T. harzianum) on hydroponically grown grapevine cuttings. Additionally, a previously described New Zealand P. chlamydospora GFP transformant (pCT74-P7) and its wild type isolate were also biologically characterised. In vitro biological and virulence characteristics of P. chlamydospora transformants did not vary much from their respective wild type isolates. In vitro biological characterisation of T. harzianum transformants showed that one of the transformants exhibited a faster growth rate at lower temperatures. Nonetheless, characterisation of its host colonisation ability on grapevine cuttings showed no difference when compared with another transformant and the wild type isolate. All Trichoderma isolates showed differential colonisation of grapevine cultivars Chenin Blanc and Merlot.

Evaluation of fungicides as potential grapevine pruning wound protectants againstBotryosphaeriaspecies

Protection of wounds against infection by trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. Studies done to determine the effectiveness of chemical pruning wound protectants have mostly focused on the control of Eutypa lata. However, other important wound pathogens, such as Phaeomoniella chlamydospora, Phaeoacremonium spp., Phomopsis spp. and species of Botryosphaeriaceae (including Botryosphaeria and aggregate genera), pose just as significant a threat to sustainable grape production. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and E. lata. However, no such studies have been conducted for the pathogenic species of Botryosphaeriaceae from grapevines in South Africa. Ten fungicides were, therefore, tested in vitro for their efficacy on mycelial inhibition of the four most common or pathogenic species of Botryosphaeriaceae in South Africa, ‘B.’ obtusa, Neofusicoccum australe, N. parvum and Lasiodiplodia theobromae. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalidwere ineffective in inhibiting themycelial growth at the highest concentrations tested (20 μg/mL for copper ammonium acetate, 5 μg/mL for other agents tested). 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 for use 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 species of Botryosphaeriaceae after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation incidences. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against species of Botryosphaeriaceae as well as the other grapevine trunk disease pathogens.

Chemical and biological protection of grapevine propagation material from trunk disease pathogens

Wound protection during all stages of grapevine propagation is of utmost importance to prevent infection of propagation material by decline and dieback pathogens. In semi-commercial nursery trials, grapevine rootstock and scion cuttings were soaked in water (control), chemical or biological sanitation products prior to cold storage, prior to grafting (machine- or hand-grafting) and prior to planting in field nurseries. Natural infection levels in basal ends and graft unions of uprooted nursery grapevines were evaluated 8 months after planting. Total pathogen incidences in the water-treated control plants ranged from 30% in basal ends to 13.5% in graft unions. Phaeomoniella chlamydospora was the most commonly isolated pathogen, followed by Phaeoacremonium, Cylindrocarpon + Campylocarpon, Botryosphaeria and Phomopsis spp. Machine-grafted unions generally had lower pathogen incidences compared with hand-grafted graft unions. In general, repeated soak-treatments of propagation material in the tested products resulted in reduced pathogen incidences in nursery grapevines. However, products containing T. harzianum (Trichoflow-T), hydrogen peroxide (Bio-sterilizer) and 8-hydroxyquinoline sulphate (Chinosol) gave inconsistent results, whereas Bronocide (a blend of halogenated alcohols and water) proved to be a good sterilising agent, but reduced certifiable plant yield significantly. Benomyl (at 100 g/100 l), Sporekill (a patented didecyldimethylammonium chloride formulation at 150 ml/100 l) and captan (at 1000 ml/100 l) were consistently the best treatments as growth parameters were not negatively influenced and pathogen incidences in basal ends and graft unions of uprooted plants were reduced.

Occurrence of Fungal Pathogens Associated with Grapevine Nurseries and the Decline of Young Vines in Spain

AbstractThe occurrence of fungal grapevine trunk pathogens associated with grapevine nurseries and the decline of young vines in Spain was determined in extensive surveys conducted in nurseries and vineyards with young plants. The presence of Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, Cylindrocarpon spp., Botryosphaeria obtusa and Botryosphaeria spp. was detected in all the surveys that were carried out. This study provides evidence for the presence of these pathogens in Spanish grapevine nurseries. Cylindrocarpon spp., B. obtusa and Botryosphaeria spp., were isolated very early in the planting material production process, nevertheless, P. aleophilum and P. chlamydospora were not detected until the rootstock–scion combinations were planted in the field to develop shoots and roots during summer. The occurrence of trunk disease pathogens in symptomatic young vineyards was also high, suggesting that infected plant material might have been used. Phaeomoniella chlamydospora and P. aleophilum were the main species associated with the decline of young vines, followed by Botryosphaeria spp., Cylindrocarpon spp. and B. obtusa. This research confirmed the importance of fungal grapevine trunk pathogens associated with the decline of young vineyards in Spain and suggested the connection between the presence of these fungi in the nurseries and the disease in the fields.