Structural basis for galactose side-chain recognition in isoprimeverose-producing oligoxyloglucan hydrolase from Phaeoacremonium minimum.

Grapevine cultivation in Montenegro has long historical and economic significance, with wine being a key trade product. However, under Montenegrin agroecological conditions, grapevine cultivation is increasingly threatened by plant diseases, particularly the Esca disease complex (EDC) which is the most economically damaging among grapevine trunk disease. A 4 year survey from 2021 to 2024 confirmed presence of EDC in all Montenegrin winegrowing regions, as Grapevine Leaf Stripe Disease (GLSD) and apoplexy. Symptom severity varied by grapevine variety and vineyard age, with the greatest incidence in vineyards aged 30–35 years. The indigenous varieties Vranac and Kratošija were more susceptible than introduced varieties (Syrah, Merlot, Chardonnay, Cabernet Sauvignon). Laboratory analyses of symptomatic trunks yielded numerous isolates of endophytic microorganisms. Preliminary identification of these, based on morphology, and molecular PCR diagnostics targeting the ITS rRNA genomic region, plus analyses of the partial tub2 and tef1-α genes, identified three key fungi responsible for EDC in Montenegro: Phaeoacremonium minimum, Phaeomoniella chlamydospora, and Fomitiporia mediterranea. In the investigated vineyards, numbers of empty places remaining after removal of dead grapevines varied, depending on vineyard age and grape variety. In a 21-year-old vineyard where dead vines were not replaced, vine loss of 32%, and direct yield reduction of 44%, were recorded, highlighting the substantial economic impact of esca disease.

Grapevine trunk diseases (GTDs), such as esca, pose a major threat to viticulture worldwide and are associated with complex biochemical responses in woody tissues. Comprehensive metabolome coverage remains a challenge, as conventional methods often overlook non-polar metabolites critical to plant defense mechanisms. This study aimed to expand metabolome and lipidome coverage of grapevine wood by integrating complementary LC-MS approaches, in order to identify metabolic signatures linked to pathogenic fungi and to a biocontrol agent. Woody tissues of Vitis vinifera cv. Cabernet-Sauvignon were inoculated with Phaeomoniella chlamydospora, Phaeoacremonium minimum, and/or the biocontrol fungus Trichoderma atroviride (Vintec®). A biphasic extraction was coupled with three orthogonal LC-MS methods-reverse-phase (RP), hydrophilic interaction chromatography (HILIC), and lipidomics-focused RP. Data were processed through the MSCleanR workflow and integrated using the DIABLO multi-block statistical framework. Compound classification was performed with NPClassifier. The multiplexed strategy enabled the annotation of 1,425 unique features, representing an 83% increase compared to previous studies. Distinct metabolomic and lipidomic signatures were associated with fungal infection and biocontrol treatments. Lipidomic analysis highlighted oxidized fatty acids (oxylipins) -specifically hydroxy-eicosatetraenoic acids (13-HETE, 16(R)-HETE, and 11(R)-HETE)-as potential signaling molecules in defense responses. NPClassifier revealed diverse biosynthetic classes, including phenylpropanoids, terpenoids, and sphingolipids, underscoring the chemical heterogeneity of grapevine responses. This multiplexed LC-MS workflow provides a versatile analytical pipeline for untargeted metabolomics and lipidomics in plants. By integrating complementary methods, the study uncovered novel biomarkers of grapevine defense, particularly oxylipins, emphasizing the critical role of lipidomics in deciphering plant-pathogen interactions.

The domestication and cultivation of the vine required pruning techniques that expose the vines to trunk pathogens, thereby facilitating the spread of Grapevine Trunk Diseases (GTDs). Among these, the Esca Disease Complex (EDC), primarily caused by Phaeomoniella chlamydospora, Phaeoacremonium minimum, and Fomitiporia mediterranea, brings significant challenges to European viticulture. This study examines the effects of curettage and over-grafting as potential methods for containing the expression of the disease in three vineyards located in Tuscany and Veneto (Italy). For several years, symptomatic vines were treated with curettage (removal of necrotic wood) or over-grafting, and the manifestation of symptoms, recurrence, and mortality rates were compared with those of untreated controls. The results revealed that both techniques reduced the incidence of foliar symptoms and mortality from Grapevine Leaf Stripe Disease (GLSD), one of the diseases in the EDC, which was previously simply referred to as “esca”. In the long term, treated vines had a significantly lower probability of resuming symptoms or dying compared to the untreated ones. The effectiveness of over-grafting varied depending on the health status of the rootstock. Overall, both methods offer promising, yet context-dependent, strategies for managing GTDs, highlighting the importance of integrated and site-specific approaches in vineyard disease control.

Phaeoacremonium minimum (formerly P. aleophilum) is one of the causal agents of Petri and Esca disease, which causes disease in numerous woody plants, including stem and branch rot of grape species worldwide (Gramaje et al. 2015; Ye et al. 2020), but has yet to be isolated on American sycamore (Platanus occidentalis). In September of 2022, a large (38 cm diameter at breast height), declining P. occidentalis was reported in Monongalia County, WV. The tree displayed numerous disease symptoms including crown dieback and light brown bark cankers (25-40 cm) down the length of the trunk. Wood plugs were excised from canker margins, surface-disinfected in 10% bleach, plated onto Potato Dextrose Agar (PDA) and incubated at 25°C under dark conditions. After 10-12 days, a pale brown mycelium grew characteristic of Phaeoacremonium (Mostert et al. 2006). DNA sequencing of the ITS1+5.8S+ITS2 of the ribosomal RNA gene repeat (rDNA) and of a fragment of the beta-tubulin gene using primers T1/Bt2b (Glass and Donaldson 1995) matched P. minimum Accession numbers AY179938.1 and MN634159.1. The sequences obtained were deposited in GenBank under the accession numbers PQ135959 and PQ623190. Pathogenicity was confirmed by placing a 6-mm diameter PDA plug from 10-day-old P. minimum cultures into scalpel wounds of twelve sycamore seedlings (mean diam 1.03 cm) at approximately 50 mm from the base of the seedling. Five seedlings were treated with sterile PDA as a control. For all seedlings, the bark was pressed back to cover the wound and closed with parafilm. Inoculated seedings were kept in a growth room at 25°C for 16 weeks and checked regularly for symptom development. By 16 weeks, all P. minimum seedlings displayed necrotic cankers on the stem while controls remained canker-free. A nonparametric Kruskal-Wallis test was used to compare canker ratings between treated and control seedlings. Mean vascular necrosis of P. minimum inoculated isolates were significantly greater (16.18 mm, P = 0.003) than the controls (0.0 mm). Control plants did not show any symptoms. Mean length of streaking for P. minimum inoculated isolates was also significantly longer (47.4 mm, P = 0.001) than controls (0.0 mm). The fungus was reisolated from discolored tissue of all inoculated stems, completing Koch’s postulates and confirming pathogenicity of P. minimum on healthy P. occidentalis. To our knowledge, this is the first documented report of P. minimum infections on American sycamore.

Italy is the leading producer and the main exporting country of table grapes in the European Union. However, table grape production is affected by Grapevine Trunk Diseases (GTDs) which cause serious economic losses to grape growers. Aetiology of GTDs is crucial for application of effective management strategies, particularly regarding the quality of the grapevine propagation material. During 2022˗23, four nurseries in Eastern Sicily, Southern Italy, were surveyed, and high incidence of propagation material with GTDs symptoms was found. Over 100 fungal isolates were collected from 80 symptomatic cuttings of ‘Italia’ and ‘Victoria’ cultivars grafted on rootstock 140RU. Of these isolates, 82 were molecularly analysed, and were found to belong to 22 genera. Isolation results highlighted the presence of well-known GTDs-related pathogens, including species within the Botryosphaeriaceae, and Phaeomoniella chlamydospora, Phaeoacremonium minimum, and Cylindrocarpon-like species. Less common fungi, including Neoscytalydium dimidiatum and Quambalaria cyanescens, were also isolated and characterized by molecular, morphological and phylogenetic analyses, and Koch’s postulates were fulfilled for these two species. This is the first study to associate N. dimidiatum and Q. cyanescens with table grape propagation material in Europe.

Fungi of the genus Trichoderma show strong potential as biological control agents (BCAs) against grapevine trunk diseases (GTDs) through mechanisms like antibiotic metabolite production and lytic enzymes. This study evaluated the biocontrol activity of four native Trichoderma strains—T. gamsii T065 and T071, T. carraovejensis T154, and T. harzianum T214—against Phaeoacremonium minimum, Phaeomoniella chlamydospora, and Diplodia seriata. Culture filtrates obtained at 8, 16, and 24 days post-incubation were tested using antibiogram and mycelial inhibition assays. Strains T071, T154, and T214 effectively inhibited D. seriata, while T154 and T214 also suppressed P. chlamydospora. Nevertheless, the limited effectiveness of all filtrates against P. minimum suggests that antibiosis is not the predominant mechanism involved in its control. These findings highlight the potential of specific Trichoderma strains and incubation times to directly control GTD pathogens and support the development of scalable biocontrol solutions.

Grapevine Trunk Diseases (GTD) reduce global grape production and requires vineyards to be replanted every 10-15 years. One possible management option is to deploy biocontrol agents such as those based on the fungal genus Trichoderma. Therefore, the objective of this study was to identify and evaluate two Californian strains of Trichoderma, T. saturnisporopsis strain RSI and T. asperellum strain TLI, for potential to combat common fungal pathogens associated with GTD. The ability of these Trichoderma strains to reduce growth of four GTD pathogens, Diplodia seriata, Neofusicoccum parvum, Eutypa lata, and Phaeoacremonium minimum, was initially examined in co-plating assays that observed both RSI and TLI significantly inhibited pathogen growth. Subsequently, greenhouse-grown Cabernet Sauvignon vines previously non-treated or inoculated with one of Trichoderma strains were later inoculated with Diplodia seriata, Neofusicoccum parvum, or Eutypa lata, and had pathogen lesion sizes measured as an assessment of relative susceptibility. Vines previously inoculated with RSI or TLI had reduced pathogen lesion growth compared to vines that were not inoculated with Trichoderma. Therefore, both Trichoderma strains demonstrated potential as biological control agents to reduce GTD severity. Further research is warranted to observe how RSI and TLI may be utilized to facilitate GTD management in vineyards.

Grapevine trunk diseases (GTDs) cause significant losses in viticulture. To enhance the phytosanitary quality of planting material, a method was developed to control the common GTD pathogens Phaeomoniella chlamydospora, Phaeoacremonium minimum, and Diplodia seriata using hot water treatment (HWT) and inoculation with the biocontrol agent Trichoderma atroviride SC1 (Vintec®, Certis Belchim B. V.) (Ta SC1). Sensitivity of P. minimum and P. chlamydospora isolates to HWT was tested in vitro and in autoclaved wood at conidial and mycelium stages and at various time/temperature combinations (30 and 40 min at 40 °C, 45 °C, 50 °C and 55 °C). The results showed that P. minimum had greater tolerance to HWT compared to P. chlamydospora. Spore germination of all P. chlamydospora isolates was completely inhibited at 45 °C, while P. minimum isolate 117607 tolerated 50 °C. Mycelium growth of both pathogens was completely inhibited at 55 °C. In autoclaved wood, P. chlamydospora growth was inhibited after 30 min at 50 °C, while treatments for 45 min were necessary to inhibit the growth of P. minimum. In nursery experiments, cuttings were artificially inoculated and incubated with the pathogens and treated with HWT at 50 °C for 45 min under conditions of common viticultural practice. In these conditions, no recovery was recorded for P. chlamydospora and D. seriata seven days post-inoculation (dpi), but the survival rate of P. minimum was 5 %. In 2021, P. minimum recovered in 40 % of the samples six months post-inoculation, P. chlamydospora in 7 % twelve months post-inoculation, and D. seriata was eliminated. In 2022, D. seriata was detected in 10 % of samples six months post-inoculation and P. chlamydospora in 25 % of the samples twelve months post-inoculation. P. minimum recovered at all sampling time points during the assessment period. Combined applications of HWT and Ta SC1 provided protection of the planting material over the twelve months assessment period, with molecular analysis confirming 90 % recovery of Ta SC1. Field experiments carried out with naturally infected scions showed that HWT at 50 °C for 45 min significantly reduced the incidence of Diplodia spp. Overall, HWT effectively reduced GTD pathogens in grapevine planting material, and the combined treatment with Ta SC1 enhanced long-lasting protection in the nursery.

Cadophora luteo-olivacea, Phaeoacremonium minimum, and Pseudophaeomoniella oleicola have been associated with branch dieback, shoot blight, and vascular wilt in super-high-density olive plantations in Spain. In this study, the effects of the interaction between these three fungal species on the disease progression were evaluated through multi-infections inoculated by spore suspensions or mycelial plugs in olive plants of cultivar Arbequina. The susceptibility of six olive cultivars was also evaluated against these three pathogens by single inoculations with conidial suspensions. The detection and inoculum quantification of each fungal species from coinfected plant tissues or from inoculated plants of different cultivars was conducted by qPCR, using species-specific primers. In addition, a species-specific primer to identify Ps. oleicola was developed in this study. In the multi-infections experiment with conidial suspensions, lesions on olive infected simultaneously with all three species were larger than those on plants coinfected by two species, which were, in turn, larger than those inoculated by a single species. However, when inoculations were conducted with mycelial plugs, C. luteo-olivacea caused larger lesions than those in coinfected plants. The quantification of these fungi through qPCR suggests that their interaction during coinfections significantly alters their relative abundance, potentially impacting their aggressiveness. Regarding the cultivar resistance experiment, the most susceptible cultivar to the disease was 'Koroneiki', followed by 'Arbequina' and 'Picual'. In all the samples, the detection of C. luteo-olivacea was an order of magnitude lower than that of the other fungal species. Nevertheless, the relative quantification of fungi by qPCR, despite being useful to detect the presence of the pathogen, does not correlate with the lesions observed.

Diversity and Pathogenicity of <i>Phaeoacremonium</i> Species Infecting <i>Haloxylon</i> and <i>Tamarix</i> Species in Iran

ABSTRACT Fungal trunk diseases are of major concern for tree fruit, nut, and grape growers throughout the world. These diseases include Eutypa dieback of grape, caused by Eutypa lata, band canker of almond, caused by Neofusicoccum mediterraneum and Neofusicoccum parvum, and twig and branch dieback of walnut, caused by N. mediterraneum, Botryosphaeria dieback of grape, caused by Diplodia mutila, Diplodia seriata, N. mediterraneum, and N. parvum, and esca of grape, caused by Phaeomoniella chlamydospora and Phaeoacremonium minimum. Given the common occurrence of mixed infections, and the similar wood symptoms at the macroscopic level, species-specific detection tools are needed. Fatty acid methyl ester (FAME) profiling can be an effective and inexpensive diagnostic tool. FAME analyses were conducted on pure cultures of multiple isolates per species to characterize profiles and assess whether this technique could result in consistent identification. FAME profiles were dominated by oleic acid (18:1 ω9c) and palmitic acid (16:0), with less abundant FAMEs in different ratios for each species and isolates within species. Canonical discriminant analyses revealed which minor FAMEs were most variable, with a total of 20 different FAMEs that can explain 69.01% of profile variance in the first two canonicals. Using these analyses, samples were self-tested and correctly sorted 97.18% of the time. Within species, canonical discriminant analyses were able to separate isolates further, often by original geographic location or by host plant species. These results further suggest that potential novel species, subspecies, or races may be present among the isolates analyzed, demonstrating the capacity of FAME profiling to have a role in discovering cryptic species and accurately identifying fungal pathogens in conjunction with other molecular techniques and genomic analyses.

In this study, isolates of Phaeoacremonium minimum and Phaeomoniella chlamydospora, fungal pathogens associated with Petri and esca diseases of grapevine, were used to determine the effect of temperature on the development of their fruiting bodies in vitro. Perithecia of Pm. minimum and pycnidia of Pa. chlamydospora were induced at 5, 10, 15, 20, 25, and 30°C on pieces of 1-year-old grapevine cuttings of 110 Richter rootstock, which were incubated for 45 days under continuous white light. Both species were able to produce abundant fruiting bodies at temperatures ranging from 15 to 25°C, but Pm. minimum produced more perithecia at 25°C and Pm. chlamydospora produced more pycnidia at 20°C. At 30°C, only very few reproductive structures were observed. Calculated optimal temperatures ranged from 23.3 to 25.6°C, and equations providing a proper description of temperature effect on Pm. minimum and Pa. chlamydospora fruiting body development were obtained. Moreover, the development of fruiting bodies and the survival of both pathogens on artificially inoculated grapevine cuttings were investigated in two vineyards. No fruiting bodies were observed during the vineyard experiments, but both fungal species were systematically recovered by fungal isolation from the cuttings. Differences in pathogen survival based on incidence data were observed relative to the species, location, and time of exposure, and generalized linear mixed-models analysis showed a progressive reduction of inoculum viability with time. The present research increases our knowledge about the biology and epidemiology of Pm. minimum and Pa. chlamydospora, being particularly useful to improve epidemiological models that could be developed for the prediction of Petri and esca diseases.

Esca is one of the main grapevine trunk diseases affecting vineyards worldwide. Phaeoacremonium minimum and Phaeomoniella chlamydospora are thought to be two of the main causal agents of this disease. However, the molecular mechanisms underlying plant defense responses in the grapevine trunk against esca-associated pathogens are poorly understood. To provide a first glimpse at the trunk responses to P. minimum and P. chlamydospora, transcriptomic and metabolomic analyses were performed to compare and contrast host responses to these pathogens. Transcriptomic analysis revealed different gene expression reprogramming in the trunk in response to each fungus. The main significant differences were found among genes associated with secondary metabolism, signaling, and hormone signaling. An untargeted liquid chromatography–high resolution mass spectrometry metabolomic approach performed 3 weeks after inoculation was used, and dereplication mainly highlighted flavonoids and stilbenes as plant defense metabolites in the infected trunk. Some metabolites were overproduced with both fungi, but specific responses were also observed. Particularly, a lipophilic flavonoid cluster was emphasized after P. minimum inoculation. The assessment of fungal infection 6 weeks postinfection showed more copies of P. minimum than P. chlamydospora. This dissimilarity in the level of colonization could be linked to the metabolomic responses observed. Our results reveal both different gene expression reprogramming and metabolomic-specific signatures depending on the wood pathogen. Altogether, these observations suggest that grapevine trunks can differently perceive and respond to P. minimum and P. chlamydospora. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Vine training and pruning are cultural strategies that can be deployed to manage grapevine trunk diseases (GTDs). Forty-year-old commercial vineyards in the Cognac region, France, trained to either Guyot-Arcure (severe pruning) or Guyot-Poussard (minimal pruning), were studied to determine how the two systems affected trunk disease symptomatology. Effects of pruning practices on the pathobiome and mycobiome of asymptomatic grapevines were also assessed, using culture- and amplicon-based Illumina sequencing approaches. The hypothesis examined was that severe pruning of Guyot-Arcure increases trunk diseases incidence and severity, and causes higher pathogen load and microbial diversity, compared to Guyot-Poussard. Numbers of symptomatic and asymptomatic vines for the two training systems were recorded over 3 years, including numbers of vines with esca foliar symptoms, and partially unproductive and dead vines. Six asymptomatic vines from each pruning method were selected, and culturing and sequencing data were obtained from 27 samples per vine. Fungi in the Phaeomoniellaceae, Togniniaceae, and Botryosphaeriaceae were the most frequently identified. The data indicated that severe pruning increased risk of pathogen infections, with Phaeomoniella chlamydospora, Phaeoacremonium minimum and Diplodia sp. being the most commonly identified fungi. Greater numbers of dead or dying vines were recorded in the severely pruned vineyard, indicating that this strategy shortens vine longevity. Results also showed that severe pruning increased endophytic microbial diversity, and that the pruning methods influenced mycobiome community composition. This knowledge will improve recommendations to growers for practical and cost-effective ways to manage GTDs.

The permanent organs of grapevines (Vitis vinifera L.), like those of other woody perennials, are colonized by various unrelated pathogenic ascomycete fungi secreting cell wall-degrading enzymes and phytotoxic secondary metabolites that contribute to host damage and disease symptoms. Trunk pathogens differ in the symptoms they induce and the extent and speed of damage. Isolates of the same species often display a wide virulence range, even within the same vineyard. This study focuses on Eutypa lata, Neofusicoccum parvum, and Phaeoacremonium minimum, causal agents of Eutypa dieback, Botryosphaeria dieback, and Esca, respectively. We sequenced 50 isolates from viticulture regions worldwide and built nucleotide-level, reference-free pangenomes for each species. Through examination of genomic diversity and pangenome structure, we analyzed intraspecific conservation and variability of putative virulence factors, focusing on functions under positive selection and recent gene family dynamics of contraction and expansion. Our findings reveal contrasting distributions of putative virulence factors in the core, dispensable, and private genomes of each pangenome. For example, carbohydrate active enzymes (CAZymes) were prevalent in the core genomes of each pangenome, whereas biosynthetic gene clusters were prevalent in the dispensable genomes of E. lata and P. minimum. The dispensable fractions were also enriched in Gypsy transposable elements and virulence factors under positive selection (polyketide synthase genes in E. lata and P. minimum, glycosyltransferases in N. parvum). Our findings underscore the complexity of the genomic architecture in each species and provide insights into their adaptive strategies, enhancing our understanding of the underlying mechanisms of virulence. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Field surveys were conducted in British Columbia fruit tree growing regions to determine the incidence of dieback and to identify the main fungal species causing cankers. Fruit trees showing cankers and dieback symptoms were recorded from 94% of orchards and 5.5% of trees surveyed. Overall, higher dieback incidence was observed in cherry than apple with 33% of cherry blocks showing between 5% and 26% of trees affected. Morphological studies along with DNA sequencing of the internal transcribed spacer (ITS) including the 5.8S rDNA, and parts of the translation elongation factor 1-α (TEF1), beta-tubulin (TUB2), and actin (ACT1) genes, identified seven fungi for the first time in fruit trees in Canada, including Calosphaeria pulchella, Cytospora parasitica, Cytospora populicola, Cytospora sorbicola, Ilyonectria robusta, Nectria dematiosa, and Phaeoacremonium minimum. In addition, this study reports for the first time Diplodia mutila and Diplodia seriata from cankers in sweet cherry in Canada. The already known fungal pathogens Neofabraea perennans and Neonectria ditissima were also identified. Pathogenicity studies showed N. ditissima and C. sorbicola to cause the largest vascular lesions in apple and cherry, respectively. This study identified the main fungal pathogens causing tree fruit cankers and dieback in British Columbia providing important information for the development of effective control strategies.

Young vine decline (YVD), caused by several taxonomically different fungi, results in the decline and death of grapevines within a few years after planting. Infection can occur in nursery mother blocks and/or at several stages in the nursery propagation process, but the final plant material may remain asymptomatic. Four nurseries that sell ready-to-plant grapevines in Canada were sampled to evaluate the health status with regard to YVD fungi, including Botryosphaeriaceae spp., Cadophora luteo-olivacea, Dactylonectria macrodidyma, Dactylonectria torresensis, Phaeoacremonium minimum, and Phaeomoniella chlamydospora. Plants representing three cultivars, 'Chardonnay', 'Merlot', and 'Pinot noir', either grafted onto '3309C' rootstock or self-rooted, were provided by the nurseries. Samples from the roots, base of the rootstock or self-rooted cultivar, graft-union, and scion were collected from each plant. DNA was extracted, and the total abundance of each fungus was quantified using Droplet Digital PCR. Results revealed that 99% of plants harbored at least one of the fungi studied, with a mean of three different fungal species that were present per grapevine. Droplet Digital PCR results showed that the abundance of the different fungi significantly varied between different sections of each plant, individual plants for each cultivar, and cultivars from the same nursery. Necrosis measurements were recorded from the base of the rootstock or self-rooted cultivars and did not correlate with fungal abundance recorded in that section for each grapevine, but necrosis was consistent across cultivars within nurseries. Five different rootstocks were compared from one nursery, and results showed no differences between rootstocks and their health status. Among all nurseries, C. luteo-olivacea was the most prevalent fungus (97% of the plants), while D. macrodidyma was the least commonly found (13% of the plants). This study shows that ready-to-plant nursery material sold in Canada is likely to be infected with several YVD fungi and that presence and abundance of fungi vary significantly among individual grapevines and nurseries.

Abstract Esca is a grapevine trunk disease (GTD) that is caused by filamentous fungi. It is responsible for considerable economic losses in viniculture on a global scale. Despite many unknown factors contributing to the development of symptoms in affected plants, Phaeoacremonium minimum (PMI), Phaeomoniella chlamydospora (PCH) and Fomitiporia mediterranea (FMED) are generally considered as the main causative fungal species. Early detection and specific identification of these pathogens therefore play an important role in disease control and evaluation of suitable countermeasures. In this study, loop‐mediated isothermal amplification (LAMP) assays were developed for each of the three pathogens. A genome‐based approach was applied for detection and selection of unique target DNA sequences. The designed primer sets showed overall good specificities, with some observed cross‐reactions towards closely related Phaeoacremonium species for the PMI primer set. The developed assays had detection limits of 100 pg (FMED, PMI) and 1 pg (PCH) per reaction (corresponding to 1460 [FMED]; 1950 [PMI]; 342 [PCH] genome copies per reaction). The application of the assays to field samples was demonstrated by testing individual infected grapevine trunks from two European viticultural regions using crude DNA obtained in a rapid sample preparation step. LAMP assay results matched those of PCR following a conventional DNA extraction protocol. The study showed that LAMP‐based rapid molecular detection of major Esca agents can serve as a useful tool for further research and surveillance of a highly devastating grapevine disease. The application of computer‐based whole genome comparison between target and non‐target species for the identification of unique target sequences as the basis for LAMP (or PCR) primer design was demonstrated to be a useful approach in species for which scarce sequence information is available. Moreover, the developed method for rapid DNA preparation from grapevine trunks may potentially be adapted to the DNA‐based detection also of other fungal species that cause grapevine trunk diseases.

Grapevine trunk disease fungi infect vines through openings, primarily pruning wounds. The main objective of this study was to understand the role of sucker wounds and wounds made by the removal of green shoots from the stems of potted grapevines as potential points of infection for grapevine trunk disease pathogens. Six wine and four table grape vineyards of different ages were sampled in different production areas in the Western Cape grape region of South Africa. Isolations were made from 161 sucker wounds, and fungal pathogens were identified using morphology and DNA sequence analysis of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S ribosomal RNA gene, the translation elongation factor 1alpha or the partial β-tubulin gene. The results show that 62% of the sucker wounds were infected by trunk disease pathogens, including Diaporthe ampelina, Diplodia seriata, Phaeomoniella chlamydospora, Phaeoacremonium minimum, Eutypella microtheca, Cryptovalsa ampelina and Neofusicoccum australe. Diaporthe ampelina was the most common, followed by D. seriata and P. chlamydospora, in both the wine and table grape sucker wounds. Under glasshouse conditions, wounds made by the removal of young green shoots on one-year-old potted grapevine plants were inoculated with spore suspensions of D. ampelina, E. lata, N. parvum, P. minimum and P. chlamydospora. After four months, all the inoculated pathogens could be re-isolated. This study shows that grapevine sucker and green shoot wounds are susceptible to different grapevine trunk disease pathogens and may therefore play a role in the epidemiology of trunk diseases.