Utilizing Disease Prediction Models to Time Fungicide Applications for Controlling Ripe Rot, Caused by Colletotrichum spp., in Maryland Vineyards.

Two previously published ripe rot prediction models, DF2-NN and GH2-DT, were evaluated for fungicide application timing efficacy in Maryland vineyards. Both models utilize leaf wetness duration (LWD), temperature, and grape cluster phenological stages as model parameters. These three parameters were tracked throughout the 2021 to 2023 seasons in three vineyards. The fungicide efficacy trials started at the veraison phenological stage and included a nontreated control, a 12-day interval treatment, and two model-triggered treatments when risk predicted by the models crossed a threshold. The severity of ripe rot on the clusters in each treatment was assessed when the fruit were mature. Ripe rot severity in the nontreated controls was higher during seasons with more LWD and more precipitation. Days in which the models were triggered by the environmental conditions primarily coincided with precipitation events and lengthy LWDs. The model-triggered treatments never had significantly higher ripe rot severity than the 12-day interval treatment but had significantly lower severities than the nontreated control in most trials which had high ripe rot pressure. Furthermore, the model-triggered treatments resulted in fewer fungicide applications than the 12-day interval treatment on average. The DF2-NN model appeared to be more accurate and useful for ripe rot prediction and treatment than the GH2-DT model because it triggered fewer fungicide applications while reducing ripe rot. This model may be useful for improving or maintaining ripe rot control with fewer fungicide inputs, which may be beneficial for the environment and the reduction of fungicide resistance selection.

Identification and Pathogenicity of Cladosporium, Fusarium, and Diaporthe spp. Associated with Late-Season Bunch Rots of Grape.

Fungal pathogens continue to pose a significant threat to grape production. Previous studies of pathogens associated with late-season bunch rots in Mid-Atlantic vineyards had elucidated the primary causal agents of these diseases, but the significance and identity of the less commonly isolated genera was unclear. Therefore, to more fully understand the identity and pathogenicity of Cladosporium, Fusarium, and Diaporthe spp. associated with late-season bunch rots of wine grapes in the Mid-Atlantic, phylogenic analyses and pathogenicity assays were conducted. Isolates were characterized to the species level by sequencing the TEF1 and Actin, TEF1 and TUB2, and TEF1 genes for 10, 7, and 9 isolates of Cladosporium, Diaporthe, and Fusarium, respectively. Four Cladosporium, three Fusarium, and three Diaporthe species were identified, and C. allicinum, C. perangustum, C. pseudocladosporioides, F. graminearum, and D. guangxiensis had not yet been isolated from grape in North America. The pathogenicity of each species was evaluated on detached table and wine grapes, and D. eres, D. ampelina, D. guangxiensis, and F. fujikuroi were found to be the most aggressive on both table and wine grapes. Further investigations through more extensive isolate collection and of myotoxicity testing may be warranted due to the prevalence and pathogenicity of D. eres and F. fujikuroi.

Ontogenic Susceptibility of Grapevine Clusters to Ripe Rot, Caused by the Colletotrichum acutatum and C. gloeosporioides Species Complexes.

Natural infection by Colletotrichum spp. and the subsequent development of ripe rot were observed in susceptible grape (Vitis vinifera) clusters either protected or exposed to environmental conditions and naturally occurring inoculum by the application or removal of paper bags at various phenological stages at two Mid-Atlantic vineyards. During each of the three experimental seasons, most treatments with grape clusters that were exposed between veraison and harvest developed significantly more severe ripe rot than clusters that were exposed during only the early season or protected throughout the entire season. Spore traps were placed in one vineyard over two seasons and were analyzed via quantitative PCR. DNA of the Colletotrichum acutatum and C. gloeosporioides species complexes was detected from the bloom to the harvest stage, with a higher quantity of C. acutatum DNA than C. gloeosporioides DNA. From ripe rot symptomatic clusters, 417 isolates were collected, and a multilocus phylogenetic analysis of 51 representative isolates identified six Colletotrichum spp., with C. fioriniae (C. acutatum complex) being the most frequently isolated. Weather data were also monitored, and ripe rot-conducive conditions were observed at multiple times throughout each season. In summary, only clusters that were exposed to inoculum and environmental conditions in the late season developed severe ripe rot. The data collected in this study suggest that grape clusters have ontogenic susceptibility to ripe rot, becoming more susceptible as they mature, in contrast to the susceptible bloom stage for other fruit rotting diseases of grapevine.

Off-target selection of resistance to azoxystrobin in Aspergillus species associated with grape late season rots.

Due to recent evidence of Aspergillus uvarum pathogenicity on wine grapes and variable fungicide sensitivity to quinone outside inhibitor (QoI) fungicides, the identity and QoI sensitivity of Aspergillus isolates from the Mid-Atlantic United States was investigated. Phylogenic analysis of 31 isolates revealed 26 as A. uvarum and 5 as A. japonicus, both of which have been previously isolated from grape. The A. uvarum isolates had variable sensitivities to the QoI azoxystrobin, and the genomic region that codes for the target of QoIs, cytochrome b, was sequenced. Translation of the cytochrome b coding sequence revealed that the most resistant isolates (termed cytb3) contained three mutations, S108A, F129L, and A194V, and the moderately sensitive isolates (termed cytb2) contained two mutations S108A and A194V. This is the first report of an amino acid variation in cytochrome b at position 108. Cytb3 isolates were significantly less inhibited than the cytb2 and wild-type isolates (cytbWT) in vitro, and were significantly less inhibited than the cytbWT isolates on detached fruit. Molecular docking analysis revealed similar differences, with azoxystrobin binding most securely in the cytbWT variant of cytochrome b than cytb2 and cytb3. As Aspergillus rot has not been a target disease of fungicide sprays in the U.S., the selection of resistant phenotypes is likely resultant from sprays for other diseases. Resistance is of concern due to the pathogenicity of A. uvarum and A. japonicus on wine grapes, and the ability of these species to be mycotoxigenic or pathogenic for humans.

Diversity, Pathogenicity, and Fungicide Sensitivity of Fungal Species Associated with Late-Season Rots of Wine Grape in the Mid-Atlantic United States.

Late-season bunch rots cause major losses in grape production every year in the Mid-Atlantic United States, but the causal agents are not well characterized. In this study, 265 fungal isolates were collected from rotten grapes from 2014 to 2020 and identified to the genus level according to internal transcribed spacer sequences. The most prevalent of the 15 genera were Botrytis, Colletotrichum, Aspergillus, Alternaria, Pestalotiopsis, and Neopestalotiopsis. Of these, isolates within three prevalent, yet understudied, genera were identified to be Aspergillus uvarum, Alternaria alternata, and Neopestalotiopsis rosae. The pathogenicity of these three fungal species was evaluated in two field trials by artificially inoculating wounded and nonwounded grapes (Vitis vinifera) of four cultivars at the phenological stages of bloom, véraison, and preharvest. Upon ripening, fruit were weighed and assessed for severity of multiple diseases. On nonwounded fruit, A. uvarum caused significantly higher disease severity than the control in both seasons. On wounded fruit, each inocula caused significantly higher disease than the respective controls in the first season, but only A. uvarum and Botrytis cinerea caused this in the second season. Also, wounding was found to have a detrimental effect on cluster weight, which was significantly influenced by inoculation timing and cultivar. Lastly, A. uvarum and N. rosae were tested for sensitivity to azoxystrobin, boscalid, and difenoconazole. The A. uvarum isolates were found to be more sensitive to boscalid and difenoconazole in general, with varying sensitivity to azoxystrobin. N. rosae isolates were resistant to boscalid and azoxystrobin but displayed much higher sensitivity to difenoconazole. Evidence from the isolate collection and field trials demonstrates that A. uvarum could be a significant pathogen of wine grapes in the Mid-Atlantic United States. Results from this study will be useful for the identification and management of the understudied Alternaria, Aspergillus, and Neopestalotiopsis fruit rots of wine grapes.

Validation of the Strawberry Advisory System in the Mid-Atlantic Region.

Anthracnose fruit rot (AFR) and Botrytis fruit rot (BFR) are primary diseases affecting strawberry (Fragaria × ananassa), which typically drive fungicide applications throughout the growing season. The Strawberry Advisory System (StAS), a disease forecasting tool, was originally developed in Florida to better time the fungicide sprays by monitoring AFR and BFR infection risk based on leaf wetness and temperature input in real-time. Thirteen field trials were conducted in Maryland and Virginia between 2017 and 2019 to evaluate the StAS performance in the Mid-Atlantic region. As a result, 55, 18, and 31% fewer sprays were recorded on average in the model-based StAS treatment compared with the grower standard treatment in 2017, 2018, and 2019, respectively. Marketable yield, as well as AFR and BFR incidence, were largely comparable between the two treatments. However, poor disease control occurred during the StAS treatment in four trials in 2017, presumably because of a missed fungicide spray during a high-risk infection event and attributable to heavy rainfall that led to impassable fields. The implementation of the StAS may be further challenged by the employment of floating row covers that are essential for growing strawberries in plasticulture systems in open fields in the Mid-Atlantic region. Preliminary results indicated that row covers can alter canopy-level microclimatic conditions, possibly increasing the risk for disease occurrence. Overall, the StAS can be a valuable tool for Mid-Atlantic growers to control AFR and BFR, but sprays may need to be promptly applied when consecutive or heavy rainfalls are predicted, especially for highly susceptible cultivars. Complications in disease forecasting and management arising from the use of row covers need to be further addressed in this region because of its highly diverse climate.

Competitive ability of multi-fungicide resistant Botrytis cinerea in a blackberry planting over three years.

Botrytis cinerea isolates with multi-fungicide resistance have frequently been isolated from small fruit fields such as strawberries and blackberries. Individual B. cinerea isolates have been found resistant to up to seven chemical classes of fungicides. Fitness costs and less competitiveness have been observed in multi-fungicide resistant isolates, but this has not been examined under field conditions. In the spring of 2016, flowers of field-grown blackberries were either not inoculated or inoculated with B. cinerea isolates sensitive (0CCR), resistant to five or six chemical classes excluding phenylpyrroles (5CCR), or resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The experimental field was left unsprayed for the duration of this study and isolates of B. cinerea were collected from flowers and/or fruit in each of the three experimental years. Isolates collected in summer of 2016 revealed resistance phenotypes in each plot closely matching those of the respective inoculum, with 95% 0CCR, 55% 5CCR, and 91% 6CCR/MDR1h isolates recovered from 0CCR, 5CCR, and 6CCR/MDR1h inoculation plots, respectively. In the 2017 and 2018 isolate collections, 6CCR/MDR1h resistance phenotypes were found in plots inoculated and non-inoculated with this phenotype, indicating their persistence and movement between plots. Resistance phenotypes different from the inoculum were also recovered each year, indicating that the inoculum was successfully competing with a native Botrytis population. Despite the competition, 6CCR/MDR1h isolates were recovered in high frequency from all inoculated plots in 2018. G3pdh and mrr1 sequences of 6CCR/MDR1h isolates collected in 2018 were identical to the sequences of the inoculum, indicating that these isolates likely descended from the inoculum. This study demonstrates that isolates carrying multi-fungicide resistant phenotypes, specifically 6CCR/MDR1h, are competitive in the absence of fungicide selection pressure.

Within-Season Shift in Fungicide Resistance Profiles of Botrytis cinerea in California Strawberry Fields.

Sensitivity of Botrytis cinerea to seven fungicide chemical classes was determined for 888 isolates collected in 2016 from 47 California strawberry fields. Isolates were collected early season (minimum fungicide exposure) and late season (maximum fungicide exposure) from the same planting block in each field. Resistance was determined using a mycelial growth assay, and variable frequencies of resistance were observed to each fungicide at both sampling times (early season %, late season %): boscalid (12, 35), cyprodinil (12, 46), fenhexamid (53, 91), fludioxonil (1, 4), fluopyram (2, 7), iprodione (25, 8), isofetamid (0, 1), penthiopyrad (8, 25), pyraclostrobin (77, 98), and thiophanate-methyl (81, 96). Analysis of number of chemical class resistances (CCRs) revealed an increasing shift in CCR from the early to late season. Phenotypes of 40 isolates that were resistant or sensitive to different chemical classes were associated with presence or absence of mutations in target genes. Fungicide-resistance phenotypes determined in the mycelial growth assay closely matched (93.8%) the genotype observed. Previously described resistance-conferring mutations were found for each gene. A survey of fungicide use from 32 of the sampled fields revealed an average of 15 applications of gray mold-labeled fungicides per season at an average interval of 12 days. The most frequently applied fungicides (average number of applications during the 2016 season) were captan (7.3), pyraclostrobin (2.5), cyprodinil (2.3), fludioxonil (2.3), boscalid (1.8), and fenhexamid (1.4). Multifungicide resistance is widespread in California. Resistance management tactics that reduce selection pressure by limiting fungicide use, rotating among Fungicide Resistance Action Committee codes, and mixing/rotating site-specific fungicides with multisite fungicides need to be improved and implemented.

CRISPR-enabled investigation of fitness costs associated with the E198A mutation in ß-tubulin of Colletotrichum siamense.

Introduction: Understanding fitness costs associated with fungicide resistance is critical to improve resistance management strategies. E198A in b-tubulin confers resistance to the fungicide thiophanate-methyl and has been widely reported in several plant pathogens including Colletotrichum siamense. Method: To better understand potential fitness costs associated with the resistance, a ribonucleoprotein (RNP) complex mediated CRISPR/Cas9 system was used to create a point mutation (E198A) through homology directed repair (HDR) in each of the sensitive (E198) C. siamense isolates collected from strawberries, raspberries, and peaches. The RNP complex was delivered into fungal protoplasts using polyethylene glycol-mediated (PEG) transfection. Results: The transformation efficiency, the proportion of transformants of sensitive parental isolates containing the E198A mutation, averaged 72%. No off-target mutations were observed when sequences similar to the b-tubulin target region with a maximum of four mismatch sites were analyzed, suggesting that the CRISPR/Cas9 system used in this study was highly specific for genome editing in C. siamense. Of the 41 comparisons of fitness between mutant and wild type isolates through in vitro and detached fruit assays, mutant isolates appeared to be as fit (24 of 41 comparisons), if not more fit than wild-type isolates (10 of 41 comparisons). Discussion: The use of CRISPR/Cas9 to evaluate fitness costs associated with point mutations in this study represents a novel and useful method, since wild-type and mutant isolates were genetically identical except for the target mutation.