Evaluating treatments for the protection of grapevine pruning wounds from natural infection by trunk disease fungi.

Infection of grapevines by fungal pathogens causing grapevine trunk diseases (GTDs) primarily arises from annual pruning wounds made during the dormant season. While various studies have showcased the efficacy of products in shielding pruning wounds against GTDs infections, most of these investigations hinge on artificial pathogen inoculations, which may not faithfully mirror real field conditions. This study aimed to evaluate and compare the efficacy of various liquid formulation fungicides (pyraclostrobin + boscalid) and paste treatments, as well as biological control agents (BCA: Trichoderma atroviride SC1, T. atroviride I-1237, and T. asperellum ICC012 + T. gamsii ICC080), for their potential to prevent natural infection of grapevine pruning wounds by trunk disease fungi in two field trials located in Samaniego (Northern Spain) and Madiran (Southern France) over three growing seasons. Wound treatments were applied immediately after pruning in February. One year after pruning, canes were harvested from vines and brought to the laboratory for assessment of Trichoderma spp. and fungal trunk pathogens. More than 1,200 fungal isolates associated with five GTDs (esca, Botryophaeria, Diaporthe and Eutypa diebacks, and Cytospora canker) were collected from the two vineyards each growing season. Our findings reveal that none of the products under investigation exhibited complete effectiveness against all the GTDs. The efficacy of these products was particularly influenced by the specific year of study. A notable exception was observed with the biocontrol agent T. atroviride I-1237, which consistently demonstrated effectiveness against Botryosphaeria dieback infections throughout each year of the study, irrespective of the location. The remaining products exhibited efficacy in specific years or locations against particular diseases, with the physical barrier (paste) showing the least overall effectiveness. The recovery rates of Trichoderma spp. in treated plants were highly variable, ranging from 17% to 100%, with both strains of T. atroviride yielding the highest isolation rates. This study underscores the importance of customizing treatments for specific diseases, taking into account the influence of environmental factors for BCA applications.

Phaeoacremonium tuscanicum and Phaeoacremonium indicum sp. nov. associated with subcutaneous phaeohyphomycosis.

Two cases of phaeohyphomycotic infections were caused by Phaeoacremonium tuscanicum, not previously identified in human infections, and one new species, Phaeoacremonium indicum, respectively. Morphological and cultural investigation as well as phylogenetic analysis was constructed based on maximum likelihood analyses using actin and -tubulin sequences to identify the fungal isolates.

Exploring Factors Conditioning the Expression of Botryosphaeria Dieback in Grapevine for Integrated Management of the Disease.

Species from the Botryosphaeriaceae family are the causal agents of Botryosphaeria dieback (BD), a worldwide grapevine trunk disease. Because of their lifestyle and their adaptation to a wide range of temperatures, these fungi constitute a serious threat to vineyards and viticulture, especially in the actual context of climate change. Grapevine plants from both nurseries and vineyards are very susceptible to infections by botryosphaeriaceous fungi due to several cuts and wounds made during their propagation process and their entire life cycle, respectively. When decline becomes chronic or apoplectic, it reduces the longevity of the vineyard and affects the quality of the wine, leading to huge economic losses. Given the environmental impact of fungicides, and their short period of effectiveness in protecting pruning wounds, alternative strategies are being developed to fight BD fungal pathogens and limit their propagation. Among them, biological control has been recognized as a promising and sustainable alternative. However, there is still no effective strategy for combating this complex disease, conditioned by both fungal life traits and host tolerance traits, in relationships with the whole microbiome/microbiota. To provide sound guidance for an effective and sustainable integrated management of BD, by combining the limitation of infection risk, tolerant grapevine cultivars, and biological control, this review explores some of the factors conditioning the expression of BD in grapevine. Among them, the lifestyle of BD-associated pathogens, their pathogenicity factors, the cultivar traits of tolerance or susceptibility, and the biocontrol potential of Bacillus spp. and Trichoderma spp. are discussed.

Evaluation of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to control Botryosphaeria dieback and black-foot pathogens in grapevine propagation material.

BACKGROUND: Grapevine trunk diseases (GTDs) are a complex group of diseases that lead to major economic losses in all wine-producing countries. The investigation of biocontrol agents (BCAs) capable of forestalling or at least minimizing the development of GTDs has, recently, become a priority. Nursery experiments were set up to (i) assess the biocontrol effect of Trichoderma atroviride (Ta) SC1 and Bacillus subtilis (Bs) PTA-271, alone and in simultaneous application, against Botryosphaeria dieback (BOT)- and black-foot (BF)- associated pathogens during the grapevine propagation process and (ii) evaluate the success of the BCA inoculation during the grapevine propagation process, using quantitative reverse-transcription polymerase chain reaction techniques. RESULTS: The results demonstrated a significant reduction in the percentage of potentially infected plants and the percentage of fungal isolation from wood fragments of BOT and BF pathogens in nursery material treated with Ta SC1 and Bs PTA-271, respectively. In one of the experiments, simultaneous treatments with Bs PTA-271 and Ta SC1 caused a reduction in percentages of potentially infected plants and fungal isolation, from wood fragments containing BOT and BF pathogens. CONCLUSION: These biological treatments may be relevant components of an integrated approach, using complementary management strategies to limit infection by GTD pathogens, but further research is still needed to elucidate the effectiveness of Bs PTA-271 and the benefits of simultaneous application with Ta SC1 for the control of GTD pathogens in nurseries. © 2022 Society of Chemical Industry.

Esca grapevine disease involves leaf hydraulic failure and represents a unique premature senescence process.

Xylem anatomy may change in response to environmental or biotic stresses. Vascular occlusion, an anatomical modification of mature xylem, contributes to plant resistance and susceptibility to different stresses. In woody organs, xylem occlusions have been examined as part of the senescence process, but their presence and function in leaves remain obscure. In grapevine, many stresses are associated with premature leaf senescence inducing discolorations and scorched tissue in leaves. However, we still do not know whether the leaf senescence process follows the same sequence of physiological events and whether leaf xylem anatomy is affected in similar ways. In this study, we quantified vascular occlusions in midribs from leaves with symptoms of the grapevine disease esca, magnesium deficiency and autumn senescence. We found higher amounts of vascular occlusions in leaves with esca symptoms (in 27% of xylem vessels on average), whereas the leaves with other symptoms (as well as the asymptomatic controls) had far fewer occlusions (in 3% of vessels). Therefore, we assessed the relationship between xylem occlusions and esca leaf symptoms in four different countries (California in the USA, France, Italy and Spain) and eight different cultivars. We monitored the plants over the course of the growing season, confirming that vascular occlusions do not evolve with symptom age. Finally, we investigated the hydraulic integrity of leaf xylem vessels by optical visualization of embolism propagation during dehydration. We found that the occlusions lead to hydraulic dysfunction mainly in the peripheral veins compared with the midribs in esca symptomatic leaves. These results open new perspectives on the role of vascular occlusions during the leaf senescence process, highlighting the uniqueness of esca leaf symptoms and its consequence on leaf physiology.

The road to molecular identification and detection of fungal grapevine trunk diseases.

Grapevine is regarded as a highly profitable culture, being well spread worldwide and mostly directed to the wine-producing industry. Practices to maintain the vineyard in healthy conditions are tenuous and are exacerbated due to abiotic and biotic stresses, where fungal grapevine trunk diseases (GTDs) play a major role. The abolishment of chemical treatments and the intensification of several management practices led to an uprise in GTD outbreaks. Symptomatology of GTDs is very similar among diseases, leading to underdevelopment of the vines and death in extreme scenarios. Disease progression is widely affected by biotic and abiotic factors, and the prevalence of the pathogens varies with country and region. In this review, the state-of-the-art regarding identification and detection of GTDs is vastly analyzed. Methods and protocols used for the identification of GTDs, which are currently rather limited, are highlighted. The main conclusion is the utter need for the development of new technologies to easily and precisely detect the presence of the pathogens related to GTDs, allowing to readily take phytosanitary measures and/or proceed to plant removal in order to establish better vineyard management practices. Moreover, new practices and methods of detection, identification, and quantification of infectious material would allow imposing greater control on nurseries and plant exportation, limiting the movement of infected vines and thus avoiding the propagation of fungal inoculum throughout wine regions.

Exploring the Temporal Dynamics of the Fungal Microbiome in Rootstocks, the Lesser-Known Half of the Grapevine Crop.

Rootstocks are the link between the soil and scion in grapevines, can provide tolerance to abiotic and biotic stresses, and regulate yield and grape quality. The vascular system of grapevine rootstocks in nurseries is still an underexplored niche for research, despite its potential for hosting beneficial and pathogenic microorganisms. The purpose of this study was to investigate the changes in the composition of fungal communities in 110 Richter and 41 Berlandieri rootstocks at four stages of the grapevine propagation process. Taxonomic analysis revealed that the fungal community predominantly consisted of phylum Ascomycota in all stages of the propagation process. The alpha-diversity of fungal communities differed among sampling times for both rootstocks, with richness and fungal diversity in the vascular system decreasing through the propagation process. The core microbiome was composed of the genera Cadophora, Cladosporium, Penicillium and Alternaria in both rootstocks, while the pathogenic genus Neofusicoccum was identified as a persistent taxon throughout the propagation process. FUNguild analysis showed that the relative abundance of plant pathogens associated with trunk diseases increased towards the last stage in nurseries. Fungal communities in the vascular system of grapevine rootstocks differed between the different stages of the propagation process in nurseries. Numerous genera associated with potential biocontrol activity and grapevine trunk diseases were identified. Understanding the large diversity of fungi in the rootstock vascular tissue and the interactions between fungal microbiota and grapevine will help to develop sustainable strategies for grapevine protection.

Cultivar Contributes to the Beneficial Effects of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to Protect Grapevine Against Neofusicoccum parvum.

Grapevine trunk diseases (GTDs) are a big threat for global viticulture. Without effective chemicals, biocontrol strategies are developed as alternatives to better cope with environmental concerns. A combination of biological control agents (BCAs) could even improve sustainable disease management through complementary ways of protection. In this study, we evaluated the combination of Bacillus subtilis (Bs) PTA-271 and Trichoderma atroviride (Ta) SC1 for the protection of Chardonnay and Tempranillo rootlings against Neofusicoccum parvum Bt67, an aggressive pathogen associated to Botryosphaeria dieback (BD). Indirect benefits offered by each BCA and their combination were then characterized in planta, as well as their direct benefits in vitro. Results provide evidence that (1) the cultivar contributes to the beneficial effects of Bs PTA-271 and Ta SC1 against N. parvum, and that (2) the in vitro BCA mutual antagonism switches to the strongest fungistatic effect toward Np-Bt67 in a three-way confrontation test. We also report for the first time the beneficial potential of a combination of BCA against Np-Bt67 especially in Tempranillo. Our findings highlight a common feature for both cultivars: salicylic acid (SA)-dependent defenses were strongly decreased in plants protected by the BCA, in contrast with symptomatic ones. We thus suggest that (1) the high basal expression of SA-dependent defenses in Tempranillo explains its highest susceptibility to N. parvum, and that (2) the cultivar-specific responses to the beneficial Bs PTA-271 and Ta SC1 remain to be further investigated.

Relationship Between the Xylem Anatomy of Grapevine Rootstocks and Their Susceptibility to Phaeoacremonium minimum and Phaeomoniella chlamydospora.

Fungal grapevine trunk diseases (GTDs) are some of the most pressing threats to grape production worldwide. While these diseases are associated with several fungal pathogens, Phaeomoniella chlamydospora and Phaeoacremonium minimum are important contributors to esca and Petri diseases. Recent research has linked grapevine xylem diameter with tolerance to Pa. chlamydospora in commercial rootstocks. In this study, we screen over 25 rootstocks for xylem characteristics and tolerance to both Pa. chlamydospora and Pm. minimum. Tolerance was measured by fungal incidence and DNA concentration (quantified via qPCR), while histological analyses were used to measure xylem characteristics, including xylem vessels diameter, density, and the proportion of the stem surface area covered by xylem vessels. Rootstocks were grouped into different classes based on xylem characteristics to assess the potential association between vasculature traits and pathogen tolerance. Our results revealed significant differences in all the analyzed xylem traits, and also in DNA concentration for both pathogens among the tested rootstocks. They corroborate the link between xylem vessels diameter and tolerance to Pa. chlamydospora. In Pm. minimum, the rootstocks with the widest xylem diameter proved the most susceptible. This relationship between vasculature development and pathogen tolerance has the potential to inform both cultivar choice and future rootstock breeding to reduce the detrimental impact of GTDs worldwide.

Drought Influences Fungal Community Dynamics in the Grapevine Rhizosphere and Root Microbiome.

Plant roots support complex microbial communities that can influence nutrition, plant growth, and health. In grapevine, little is known about the impact of abiotic stresses on the belowground microbiome. In this study, we examined the drought-induced shifts in fungal composition in the root endosphere, the rhizosphere and bulk soil by internal transcribed spacer (ITS) high-throughput amplicon sequencing (HTAS). We imposed three irrigation regimes (100%, 50%, and 25% of the field capacity) to one-year old grapevine rootstock plants cv. SO4 when plants had developed 2-3 roots. Root endosphere, rhizosphere, and bulk soil samples were collected 6- and 12-months post-plantation. Drought significantly modified the overall fungal composition of all three compartments, with the root endosphere compartment showing the greatest divergence from well-watered control (100%). The overall response of the fungal microbiota associated with black-foot disease (Dactylonectria and "Cylindrocarpon" genera) and the potential biocontrol agent Trichoderma to drought stress was consistent across compartments, namely that their relative abundances were significantly higher at 50-100% than at 25% irrigation regime. We identified a significant enrichment in several fungal genera such as the arbuscular mycorrhizal fungus Funneliformis during drought at 25% watering regime within the roots. Our results reveal that drought stress, in addition to its well-characterized effects on plant physiology, also results in the restructuring of grapevine root microbial communities, and suggest the possibility that members of the altered grapevine microbiota might contribute to plant survival under extreme environmental conditions.

Cultivar Susceptibility to Natural Infections Caused by Fungal Grapevine Trunk Pathogens in La Mancha Designation of Origin (Spain).

Grapevine trunk diseases (GTDs) are one of the main biotic stress factors affecting this crop. The use of tolerant grapevine cultivars would be an interesting and sustainable alternative strategy to control GTDs. To date, most studies about cultivar susceptibility have been conducted under controlled conditions, and little information is available about tolerance to natural infections caused by GTD fungi. The objectives of this study were: (i) to identify tolerant cultivars to GTD fungi within a Spanish germplasm collection, based on external symptoms observed in the vineyard; and (ii) to characterize the pathogenic mycoflora associated with symptomatic vines. For this purpose, a grapevine germplasm collection including 22 white and 25 red cultivars was monitored along three growing seasons, and their susceptibility for esca foliar symptoms was assessed. Fungi were identified by using morphological and molecular methods. Cultivars such as, 'Monastrell', 'Graciano', 'Cabernet Franc', 'Cabernet Sauvignon', 'Syrah', 'Moscatel de Alejandría', 'Sauvignon Blanc', and 'Airén' displayed high susceptibility to GTDs, whereas others such as 'Petit Verdot', 'Pinot Noir', 'Chardonnay', and 'Riesling' were considered as tolerant. The prevalent fungal species isolated from symptomatic vines were Phaeomoniella chlamydospora (27.9% of the fungal isolates), Cryptovalsa ampelina (24.6%), and Dothiorella sarmentorum (21.3%).

Cadophora sabaouae sp. nov. and Phaeoacremonium Species Associated with Petri Disease on Grapevine Propagation Material and Young Grapevines in Algeria.

A field survey conducted on asymptomatic grapevine propagation material from nurseries and symptomatic young grapevines throughout different regions of Algeria yielded a collection of 70 Phaeoacremonium-like isolates and three Cadophora-like isolates. Based on morphology and DNA sequence data of ß-tubulin (tub2) and actin, five Phaeoacremonium species were identified including Phaeoacremonium minimum (22 isolates), Phaeoacremonium venezuelense (19 isolates), Phaeoacremonium parasiticum (17 isolates), Phaeoacremonium australiense (8 isolates), and Phaeoacremonium iranianum (4 isolates). The latter two species (P. australiense and P. iranianum) were reported for the first time in Algeria. Multilocus phylogenetic analyses (internal transcribed spacer, tub2, and translation elongation factor 1-α) and morphological features, allowed the description of the three isolates belonging to the genus Cadophora (WAMC34, WAMC117, and WAMC118) as a novel species, named Cadophora sabaouae sp. nov. Pathogenicity tests were conducted on grapevine cuttings cultivar Cardinal. All the identified species were pathogenic on grapevine cuttings.

Field evaluation of biocontrol agents against black-foot and Petri diseases of grapevine.

BACKGROUND: Black-foot and Petri diseases are the main fungal diseases associated with young grapevine decline. Two field experiments were established to evaluate the preventive effect of two potential biocontrol agents (BCAs), that is Streptomyces sp. E1 + R4 and Pythium oligandrum Po37, and three BCA-commercial products containing Trichoderma atroviride SC1, Trichoderma koningii TK7 and Pseudomonas fluorescens + Bacillus atrophaeus on fungal infection in grafted plants and plant growth parameters. RESULTS: The effectiveness of some BCA in reducing the incidence and severity of both diseases was dependent on the plant part analyzed and the plant age. No single BCA application was able to control both diseases. Streptomyces sp. E1 + R4 were able to reduce significantly the infection of the most prevalent black-foot disease fungi while P. oligandrum Po37 and Trichoderma spp. were able to reduce significantly Phaeomoniella chlamydospora and Phaeoacremonium minimum (Petri disease) infection. BCA treatments had no effect on the shoot weight, and root weight was significantly lower in all BCA treatments with respect to the control. CONCLUSIONS: The combination of the disease-suppressive activity of two or more beneficial microbes in a biocontrol preparation is required to prevent infection by black-foot and Petri disease fungi in vineyards.

Comparative Genomic Analysis of Dactylonectria torresensis Strains from Grapevine, Soil and Weed Highlights Potential Mechanisms in Pathogenicity and Endophytic Lifestyle.

The soil-borne fungus Dactylonectria torresensis is the most common causal agent of black-foot disease in Europe. However, there is a lack of understanding on how this fungus can provoke plant symptoms. In this study, we sequenced, annotated and analyzed the genomes of three isolates of D. torresensis collected from asymptomatic vine, weed and soil. Sequenced genomes were further compared to those of 27 fungal species including root and aerial pathogens, white rot degraders, indoor biodeterioration agents, saprotrophs, dark septate endophytes and mycorrhiza. Strains of D. torresensis present genomes with between 64 and 65 Mbp and with up to 18,548 predicted genes for each strain. Average Nucleotide Identity (ANI) shows that strains are different according to genome contents. Clusters of orthologous groups were compared, and clusters of genes related to necroses were particularly detected in all strains of D. torresensis (necrosis inducing peptides and proteins, and ethylene inducing peptides) as well as several genes involved in resistance against fungicides frequently used in viticulture such as copper. Interestingly, an expanded high number of genes related to carbohydrate-active enzymes were detected in each Dactylonectria strain, especially those related to glycoside hydrolases that could be involved in penetration of plant tissues or pathogenicity. An increased number of candidate genes for CAZyme classes AA9 and AA3-1 supports the ability of strains to efficiently degrade plant material. High numbers of genes of D. torresensis related to secretome and small secreted proteins were further characterized. Moreover, the presence of several gene clusters such as fujikurin-like genes was detected and were normally found in Fusariumfujikuroi, that have been linked to fungal pathogenicity. The phenotypes of the three strains investigated showed further difference in light response. We found that Dactylonectria strains have an increased number of photoreceptor encoding genes and we showed sequence alterations. Altogether, the results highlight several gene clusters present in D. torresensis strains that could be linked to endophytic lifestyle, pathogenicity, plant maceration and degradation of plant tissues as well as adaptation to soil contaminated with metals and metalloids and light response.

A Survey of Trunk Disease Pathogens within Citrus Trees in Iran.

Citrus trees with cankers and dieback symptoms were observed in Bushehr (Bushehr province, Iran). Isolations were made from diseased cankers and branches. Recovered fungal isolates were identified using cultural and morphological characteristics, as well as comparisons of DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, translation elongation factor 1α, ß-tubulin, and actin gene regions. Dothiorellaviticola, Lasiodiplodia theobromae, Neoscytalidiumhyalinum, Phaeoacremonium (P.) parasiticum, P. italicum, P. iranianum, P. rubrigenum, P. minimum, P. croatiense, P. fraxinopensylvanicum, Phaeoacremonium sp., Cadophora luteo-olivacea, Biscogniauxia (B.) mediterranea, Colletotrichum gloeosporioides, C. boninense, Peyronellaea (Pa.) pinodella, Stilbocrea (S.) walteri, and several isolates of Phoma, Pestalotiopsis, and Fusarium species were obtained from diseased trees. The pathogenicity tests were conducted by artificial inoculation of excised shoots of healthy acid lime trees (Citrus aurantifolia) under controlled conditions. Lasiodiplodia theobromae was the most virulent and caused the longest lesions within 40 days of inoculation. According to literature reviews, this is the first report of L. theobromae and N. hyalinum on citrus in Iran. Additionally, we report several Phaeoacremonium species, S. walteri, Pa. pinodella and C. luteo-olivacea on citrus trees for the first time in the world.

Quantification of Cadophora luteo-olivacea From Grapevine Nursery Stock and Vineyard Soil Using Droplet Digital PCR.

Cadophora luteo-olivacea is the most prevalent Cadophora species associated with Petri disease and esca of grapevine. Accurate, early, and specific detection and quantification of C. luteo-olivacea are essential to alert growers and nurseries to the presence of the pathogens in soil and to prevent the spread of this pathogen through grapevine planting material. The aim of this study was to develop molecular tools to detect and quantify C. luteo-olivacea inoculum from environmental samples. Species specific primers based on the ß-tubulin gene and a TaqMan probe for droplet digital PCR (ddPCR) and quantitative PCR (qPCR) were first developed to detect and quantify purified DNA of the target fungus. Specificity tests showed that the primers were able to amplify the C. luteo-olivacea DNA (20 isolates) while none of the 29 nontarget fungal species (58 isolates) tested were amplified. The ddPCR was shown to be more sensitive compared with qPCR in the detection and quantification of C. luteo-olivacea at very low concentrations and was further selected to accurately detect and quantify the fungus from environmental samples. Twenty-five of the 94 grafting plants (26.6%) analyzed by ddPCR tested positive to C. luteo-olivacea DNA (>3 copies/µl). C. luteo-olivacea was barely detected from vineyard soils. The procedure employed in this study revealed the presence of the pathogen in symptomless vines, which makes implementation of this technique suitable for certification schemes of C. luteo-olivacea-free grapevine planting material.

Temporal Dispersal Patterns of Phaeomoniella chlamydospora, Causal Agent of Petri Disease and Esca, in Vineyards.

Although the fungus Phaeomoniella chlamydospora is the most commonly detected causal agent of Petri disease and esca, two important fungal grapevine trunk diseases, little is known about the dispersal patterns of P. chlamydospora inoculum. In this work, we studied the dispersal of P. chlamydospora airborne inoculum from 2016 to 2018 in two viticultural areas of eastern (Ontinyent) and northern (Logroño) Spain. The vineyards were monitored weekly from November to April using microscope slide traps, and P. chlamydospora was detected and quantified by a specific real-time quantitative (qPCR) method set up in this work. The method was found to be sensitive, and a good correlation was observed between numbers of P. chlamydospora conidia (counted by microscope) and DNA copy numbers (quantified by qPCR). We consistently detected DNA of P. chlamydospora at both locations and in all seasons but in different quantities. In most cases, DNA was first detected in the last half of November, and most of the DNA was detected from December to early April. When rain was used as a predictor of P. chlamydospora DNA detection in traps, false-negative detections were observed, but these involved only 4% of the total. The dispersal pattern of P. chlamydospora DNA over time was best described (R2 = 0.765 and concordance correlation coefficient = 0.870) by a Gompertz equation, with time expressed as hydrothermal time (a physiological time accounting for the effects of temperature and rain). This equation could be used to predict periods with a high risk of dispersal of P. chlamydospora.