Biological and chemical pruning wound protectants reduce infection of grapevine trunk disease pathogens

Grapevine trunk diseases (GTDs) are currently considered some of the most important challenges for viticulture, curtailing vineyard longevity and productivity in nearly every raisin, table and wine grape production region in California and worldwide. Pruning wounds provide the main entry point for fungal pathogens responsible for these diseases; pathogens enter the wounds following precipitation events. The aim of this study was to evaluate the efficacy of selected chemical and experimental biological fungicides for protection of pruning wounds against two of the most common and virulent fungal pathogens causing GTDs: Eutypa lata and Neofusicoccum parvum. This study was conducted on sauvignon blanc at the UC Davis Department of Plant Pathology Field Station. Results showed that several chemical and biological fungicides, notably the chemical fungicide Luna Sensation, the biofungicide Vintec and a combination of the biofungicides Bio-Tam and CrabLife Powder, provided significant protection against at least one of the two canker pathogens used in this study. However, the majority of products tested did not provide simultaneous control of both E. lata and N. parvum pathogens, highlighting the continuing challenge of controlling GTDs.

Grapevine wood microbiome analysis identifies key fungal pathogens and potential interactions with the bacterial community implicated in grapevine trunk disease appearance

Background Grapevine trunk diseases (GTDs) is a disease complex caused by wood pathogenic fungi belonging to genera like Phaeomoniella, Phaeoacremonium, Fomitiporia, Eutypa and members of the family Botryosphaeriaceae. However, the co-occurrence of these fungi in symptomatic and asymptomatic vines at equivalent abundances has questioned their role in GTDs. Hence, we still lack a good understanding of the fungi involved in GTDs, their interactions and the factors controlling their assemblage in vines. We determined the fungal and bacterial microbiome in wood tissues of asymptomatic and symptomatic vines of three main Greek cultivars (Agiorgitiko, Xinomavro, Vidiano), each cultivated in geographically distinct viticultural zones, using amplicon sequencing. Results We noted that cultivar/biogeography (lumped factor) was the strongest determinant of the wood fungal microbiome (p < 0.001, 22.7%), while GTD symptoms condition had a weaker but still significant effect (p < 0.001, 3.5%), being prominent only in the cultivar Xinomavro. Several fungal Amplicon Sequence Variants (ASVs), reported as GTD-associated pathogens like Kalmusia variispora, Fomitiporia spp., and Phaemoniella chlamydosporα (most dominant in our study), were positively correlated with symptomatic vines in a cultivar/viticultural zone dependent manner. Random Forest analysis pointed to P. chlamydosporα, K. variispora, A. alternata and Cladosporium sp., as highly accurate predictors of symptomatic vines (0% error rate). The wood bacterial microbiome showed similar patterns, with biogeography/cultivar being the main determinant (p < 0.001, 25.5%) of its composition, followed by the GTD status of vines (p < 0.001, 5.2%). Differential abundance analysis revealed a universal positive correlation (p < 0.001) of Bacillus and Streptomyces ASVs with asymptomatic vines. Network analysis identified a significant negative co-occurrence network between these bacterial genera and Phaemoniella, Phaeoacrominum and Seimatosporium. These results point to a plant beneficial interaction between Bacillus/Streptomyces and GTD pathogens. Conclusions Our study (a) provides evidence that GTD symptomatic plants support a wood fungal microbiome, showing cultivar and biogeography-dependent patterns, that could be used as a proxy to distinguish between healthy and diseased vines, (b) points to strong interactions between the bacterial and fungal wood microbiome in asymptomatic vines that should be further pursued in the quest for discovery of novel biocontrol agents.

Phytotoxins Produced by Two Biscogniauxia rosacearum Strains, Causal Agents of Grapevine Trunk Diseases, and Charcoal Canker of Oak Trees in Iran

Biscogniauxia rosacearum, recognized for the first time as a pathogen involved in grapevine trunk diseases in Paveh (west of Iran) vineyards, produced meso-2,3-butanediol (1) as the only phytotoxin. Nectriapyrone (2), (3R)-5-methylmellein (3), (3R)-5-methyl-6-methoxymellein (4), and tyrosol (5) were instead produced as phytotoxins from a strain of the same fungus isolated from oak trees in Zagros forests of Gilan-e Gharb, Kermanshah Province. They were identified comparing their 1H and 13C NMR, ESIMS, and specific optical rotation data with those already reported in the literature. The phytotoxicity of metabolites (1–5) was estimated by leaf puncture assay on Quercus ilex L. and Hedera helix L., and by leaf absorption assay on grapevine (Vitis vinifera L.) at a concentration of 5 × 10−3 and 10−3 M. Tested on grapevine, meso-2,3-butanediol (1) and (3R)-5-methyl-6-methoxymellein (4) resulted to be the most phytotoxic compounds. On Q. ilex, nectriapyrone (2) and tyrosol (5) showed severe necrosis at the highest concentration while none of the compounds (1–5) was active on H. helix. Furthermore, the phytotoxicity of compounds 3 and 4 was also compared with that of some related natural melleins to perform a structure-activity relationship (SAR) study. The results of this study were also discussed.

The compositional turnover of grapevine-associated plant pathogenic fungal communities are greater among intraindividual microhabitats and terroirs than among healthy and Esca-diseased plants

Grapevine trunk diseases (GTD) are a major threat to the wine industry, causing yield loss and dieback of grapevines. While the increasing damage caused by GTDs in recent decades have spurred several studies on grapevine-associated pathogenic fungi, key questions about the emergence and severity of GTDs remain unanswered, including possible differences in plant pathogenic fungal communities in asymptomatic and symptomatic grapevines. We generated fungal DNA metabarcoding data from soil, bark, and perennial wood samples from asymptomatic and symptomatic grapevines sampled in three terroirs. We observed larger compositional differences in plant pathogenic fungi among different plants parts within grapevine plants than among individual grapevines. This is driven by the dominance of GTD-associated fungi in perennial wood and non-GTD pathogens in soil, as well as by the lack of significant differences among asymptomatic and Esca symptomatic grapevines. These results suggest that fungi generally associated with Esca disease belong to the core grapevine microbiome and likely are commensal endophytes and/or latent saprotrophs, some of which can act as opportunistic pathogens on stressed plants. In addition, we found significant compositional differences among sampling sites, particularly in soil, which suggest a certain influence of local edaphic and mesclimatic factors on plant pathogenic fungal communities. Furthermore, the observed differences among terroirs in plant pathogenic fungal communities in grapevine woody parts indicate that environmental factors likely are important for the development of Esca disease and further studies are needed to investigate the abiotic conditions on fungal compositional dynamics in Esca-affected plants.

Combining an HA + Cu (II) Site-Targeted Copper-Based Product with a Pruning Wound Protection Program to Prevent Infection with Lasiodiplodia spp. in Grapevine

The genus Lasiodiplodia has been reported from several grape growing regions and is considered as one of the fastest wood colonizers, causing Botryosphaeria dieback. The aim of this study was to (i) evaluate the efficacy of Esquive®, a biocontrol agent, on vineyard pruning wound protection, applied single or, in a combined protection strategy with a new site-targeted copper-based treatment (LC2017), and (ii) compare their efficacy with chemical protection provided by the commercially available product, Tessior®. For two seasons, protectants were applied onto pruning wounds, while LC2017 was applied throughout the season according to the manufacturer’s instructions. Pruning wounds of two different cultivars were inoculated with three isolates of Lasiodiplodia spp. Efficacy of the wound protectants, varied between both years of the assay and according to the cultivar studied but were able to control the pathogen to some extent. The application of LC2017 did not show clear evidence of improving the control obtained by the sole application of the other products tested. Nevertheless, LC2017 showed a fungistatic effect against Lasiodiplodia spp., in vitro, and has previously shown an elicitor effect against grapevine trunk diseases. Therefore, this combination of two protection strategies may constitute a promising long-term approach to mitigate the impact of Botryosphaeria dieback.

Hunting the plant surrender signal activating apoplexy in grapevines after Neofusicoccum parvum infection

Apoplectic breakdown from Grapevines Trunk Diseases (GTDs) has become a serious challenge to viticulture in consequence to drought stress. We hypothesise that fungal aggressiveness is controlled by a chemical communication between host and colonising fungus. We introduce the new concept of a "plant surrender signal" accumulating in host plants under stress and triggering aggressive behaviour of the strain Neofusicoccum parvum (Bt-67) causing Botryosphaeriaceae-related dieback in grapevines. Using a cell-based experimental system (Vitis cells) and bioactivity-guided fractionation, we identify trans-ferulic acid, a monolignol precursor, as "surrender signal". We show that this signal specifically activates secretion of the fungal phytotoxin Fusicoccin A. We show further that this phytotoxin, mediated by 14-3-3 proteins, activates programmed cell death in Vitis cells. We arrive at a model pinpointing the chemical communication driving apoplexy in Botryosphaeriaceae-Vitis interaction and define the channelling of phenylpropanoid pathway from the lignin precursor, trans-ferulic acid to the phytoalexin trans-resveratrol as target for future therapy.

First Report of Diaporthe foeniculina Associated with Grapevine Trunk Diseases on Vitis vinifera in Cyprus

In June 2017, three vineyards were surveyed in the regions of Droushia (30-year-old, cv Mavro), Ineia (50-year-old, cv Xynisteri), and Lemona (15-year-old, cv Carignan) at the province of Paphos, Cyprus, with dieback incidence of 22%, 32%, and 14%, respectively. More specifically, affected grapevines exhibited severe dieback symptoms in spur and cordon positions, related to perennial cankers and internal brown discoloration. Thirty symptomatic samples, were surface-sterilized (95% ethanol) and wood chips were plated on potato dextrose agar (PDA), amended with streptomycin (500 μg/ml) at 25 °C for 3-5 days. Based on colony morphology (white to creamy color, with sparse aerial mycelium) and conidia production, nine Diaporthe-like isolates were obtained. For species identification, the internal transcribed spacer (ITS) region and β-tubulin (BT) genes were amplified using the primer pairs ITS1/ITS4 and Bt2a/Bt2b, respectively (Úrbez-Torres et al. 2008). Sequences of the isolates P101b, P114c, and P289a revealed >99.8% homology to NCBI voucher specimens of Diaporthe foeniculina (Sacc.) Udayanga & Castl. (ITS: CBS111553, MH050434; ΒΤ: KY511368, KF778966), and were deposited in the GeneBank (ITS: MT735646, MT737289, MT737287; BT: MT903969, MT903970, MT903971). Thus, 8.3% of the collected isolates (3 of 36) were identified as D. foeniculina, while the rest Diaporthe-like isolates were identified as D. ampelina. D. foeniculina isolates were also transferred on 2% water agar with sterile pine needles under a 12h/12h near-ultraviolet, light/darkness regime, at 25 °C, to induce sporulation (Guarnaccia and Crous 2017). Two weeks later, microscopic observations revealed dark brown to black, globose to sub-globose, ostiolate pycnidia (n = 30) 291 to 897 μm (595 ± 173) x 192 to 655 μm (364 ± 113) containing hyaline, unbranched conidiophores, bearing alpha‐ and beta‐conidia in the form of yellowish cirri. Alpha-conidia were aseptate, hyaline, ovate to ellipsoidal, ranging (n=100) from 5.6 to 9.9 μm (7.5 ± 0.8) x 1.9 to 3.3 μm (2.7 ± 0.3). Beta-conidia were abundant, aseptate, hyaline, filiform, slightly curved (n = 100) from 22.4 to 35.3 μm (28.1 ± 2.5) x 1.2 to 2.3 μm (1.6 ± 0.2) (Udayanga et al. 2014). Pathogenicity tests were conducted with isolates P101b and P289a under greenhouse conditions (24-32 ⁰C, 70% RH). Ten 1-year-old rooted canes cv Mavro were inoculated with 4 mm mycelium plugs from actively growing cultures into wounds made by drilling between two internodes at the middle of the trunk. The same number of cuttings were inoculated with sterile PDA plugs, sealed with Vaseline, and wrapped with parafilm, serving as controls. Seven months later, all inoculated cuttings developed brownish wood discolorations (average 39 ± 13 mm), similar to naturally infected plants. No symptoms were observed in the controls. Successful re-isolations were made only from the inoculated cuttings and confirmed by colony morphology. Previously, D. foeniculina (as D. neotheicola) has been reported as grapevine wood saprophyte (Úrbez-Torres et al. 2014). It has also been reported to cause shoot canker and dieback in numerous hosts, including almond, avocado, citrus, and sweet chestnut (Annesi et al. 2016; Guarnaccia and Crous 2017; Diogo et al. 2010; Mathioudakis et al. 2020). This is the first record of D. foeniculina associated with grapevine trunk diseases (GTDs) in Cyprus. However, its relative importance as the causal agent of GTDs remains to be further investigated.