Jhulia Gelain

Brown rot, caused by Monilinia fructicola , is the most destructive pre- and postharvest fruit rot of peach worldwide. In 2024, a disease outbreak occurred in a South Carolina orchard despite a rigorous chemical management program consisting of preharvest applications of succinate dehydrogenase inhibitor (SDHI), quinone outside inhibitor, and demethylation inhibitor (DMI) fungicides. Isolates from affected orchards carried the Mona element upstream of the MfCYP51 gene, and propiconazole discriminatory dose assays confirmed reduced sensitivity in vitro, with all 2024 isolates exhibiting relative growth greater than 20%. They displayed either low, moderate, or high levels of resistance to boscalid, isofetamid, fluopyram, and fluxapyroxad based on EC ₅₀ values ranging from 0.30 to 14.74, 0.07 to 1.93, 0.26 to 3.48, and 0.19 to 2.82 µg/ml, respectively. In contrast, EC ₅₀ values obtained for historic isolates were ≤0.05 µg/ml for all SDHI fungicides. Whole-genome sequencing, followed by targeted amplification and sequencing of SDH subunits across the isolate collection, identified five SDH genotypes: wild type (no amino acid substitutions), G1 (N226H in SDHB), G2 (P80H in SDHC), G3 (N226H in SDHB and V26I in SDHD), and G4 (N226H in SDHB, P80H in SDHC, and T24A in SDHD). Isolates harboring amino acid changes in multiple SDH subunits had the highest EC ₅₀ values and were resistant to all SDHIs tested. Detached fruit assays confirmed that label rates of SDHI fungicides failed to control isolates carrying multiple amino acid changes. To the best of our knowledge, this study provides the first report worldwide of SDHI resistance in Monilinia species field isolates linked to amino acid changes in the SDH subunits and combined SDHI and DMI resistance in peach orchards, highlighting the need for resistance monitoring and management.

Venturia effusa, the causal agent of pecan scab, drives the spray program in Georgia’s pecan production. Miravis Top (difenoconazole, FRAC 3 + pydiflumetofen, FRAC 7) has been widely adopted for pecan scab management in recent years due to the high efficacy of its SDHI component, pydiflumetofen. In late summer 2025, a severe outbreak occurred in a commercial orchard in South Georgia despite a well-maintained spray program that included Miravis Top. While resistance to demethylation inhibitor (DMI) fungicides and other fungicide classes in V. effusa has been well documented, resistance to pydiflumetofen has not been reported. Symptomatic leaves and nuts were collected from cultivars ‘Desirable’ and ‘Pawnee’ located on a commercial farm in Georgia. Thirty-five single-spore isolates were obtained and compared with a historic baseline population (n = 12; 1993–1994) presumed unexposed to SDHIs. In mycelial growth assays, baseline isolates showed a mean EC₅₀ of 0.012 µg/ml pydiflumetofen, whereas values from outbreak isolates ranged from reduced sensitive (0.047–0.091 µg/ml; n = 7) to moderate resistant (0.281–0.895 µg/ml; n = 10) and high resistant (1.17–6.70 µg/ml; n = 18), with resistance factors >500. Sequencing of VesdhB, VesdhC, and VesdhD genes identified six amino acid (aa) substitutions associated with reduced sensitivity: H252R (VeSdhB), K73R; M82I; N84S (VeSdhC), and D150E and D150G (VeSdhD). All moderate to high resistant isolates carried at least one substitution, except for a subset of moderate resistant isolates that exhibited the lowest EC₅₀ values within this group. The most resistant isolate (Ve25-22) carried two mutations, K73R (VeSdhC) and D150G (VeSdhD). Cross-sensitivity assays with fluopyram, isofetamid, and benzovindiflupyr showed incomplete cross-resistance with pydiflumetofen (moderate correlations), but a strong correlation between fluopyram and isofetamid (r = 0.998, P < 0.001). Notably, isolates carrying the M82I substitution in VeSdhC were high resistant to both fluopyram and isofetamid. This study provides the first evidence of reduced sensitivity to pydiflumetofen in V. effusa linked to target-site substitutions in the VeSdhB, VeSdhC, and VeSdhD subunits and highlights the importance of resistance management strategies, including monitoring pathogen populations, rotating fungicide modes of action, using fungicide mixtures, and limiting the number of applications.

Pseudomonas syringae pv. syringae (Pss) is the causal agent of bacterial canker, a disease that can result in yield losses, aerial tissue damage, and tree mortality in stone fruits worldwide. Peach, one of the major stone fruit crops, experiences significant yield losses and tree mortality attributed to bacterial canker in the United States. As the second-largest peach-producing state, South Carolina faces direct and significant impacts due to Pss. Early evaluations of peach scion responses to Pss infection have relied primarily on circumstantial field observations in rootstock trials. Although laboratory evaluations in peach have been reported, these studies primarily focused on pathogen virulence testing or small accession sets and did not establish a standardized, scalable detached twig protocol for systematic germplasm phenotyping. The absence of a clearly described laboratory assay has limited reproducible and large-scale evaluation of bacterial canker tolerance in peach. To address this gap, a detached dormant twig assay, previously developed for cherry, was adapted and optimized for peach. Dormant shoots from nine peach accessions were cut into 10 cm segments, surface-sterilized, and inoculated with a Pss suspension prepared in 10 mM MgCl₂ buffer or with the buffer alone. After six weeks of incubation, inner bark lesion size was evaluated visually and quantified using ImageJ. A newly developed visual rating scale was established and compared with quantitative lesion measurements. Spearman correlation analysis showed strong positive correlations between visual disease scores and ImageJ-based lesion measurements across two independent replicates (ρ = 0.80–1.00, p < 0.01), while shoot segment diameter showed weak-to-moderate negative correlations with disease severity. This adapted and consolidated dormant twig assay provides a practical, reproducible, and scalable method for phenotyping bacterial canker tolerance in peach and supports future germplasm screening and breeding efforts.

Resistance to propiconazole in Geotrichum candidum was reported previously in isolates collected from peaches after cold storage, but the origin of resistance was unclear. If resistance had been generated and selected in the packinghouse with postharvest propiconazole drenches, we would expect to find resistance in the sour rot pathogen only in orchards that had received cull fruit returned to the orchard floor from the packinghouse. In this study, 70 G. candidum isolates were collected from seven orchards that had received cull fruit in the past from a packinghouse with documented resistance to propiconazole in G. candidum in stored fruit and six orchards that had never received cull fruit. Species identification was confirmed through species-specific PCR. Results showed a similar prevalence of sensitive, reduced-sensitive, and resistant isolates in cull and non-cull orchards. Sequencing of the GcCYP51B gene identified previously reported genotypes leading to E126K, Y143F, and G460S mutations and a newly described genotype consisting of a set of combined mutations, Y143F and L389V. Further investigation into alternate genetic determinants of resistance revealed that overexpression of the GcCYP51A or GcCYP51B genes was not involved in resistance to propiconazole. Our findings suggest that propiconazole resistance in G. candidum, first found in a peach packinghouse, originated in the field, likely from decades of demethylation inhibitor (DMI) fungicide applications to control preharvest diseases of peaches.

Geotrichum candidum Link (1809) is a yeast-like fungus that causes sour rot of peach (Prunus persica). Outbreaks of the disease have occurred since 2021 in peach fruit kept in cold storage despite postharvest treatments with propiconazole at a commercial farm in South Carolina (SC). A total of 58 isolates, 40 from symptomatic fruit from cold storage in Saluda County (SC packing house isolates), 11 from three SC orchards in Saluda County, Spartanburg County, and Pickens County (SC non–packing house isolates), and 7 California (CA) isolates (at least 3 from packing houses) were evaluated for propiconazole sensitivity. Mycelial growth assays revealed that six of seven CA isolates had the lowest fungicide concentration causing inhibition of mycelial growth by 50% (EC₅₀) values and were considered sensitive (S) to propiconazole with an average EC₅₀ value of 0.02 μg/ml and minimum inhibitory concentration (MIC) values of >1 to 10 to 300 μg/ml. Two CYP51 genes, GcCYP51A and GcCYP51B, encoding two demethylase inhibitor (DMI) target enzyme 14α-demethylases were identified, sequenced, and characterized. Two GcCYP51A and three GcCYP51B variants were found. Although both GcCYP51A variants were linked to S isolates, the GcCYP51B2 variant possessing the mutation Y143F was found in RS, and the GcCYP51B3 variant possessing Y143F, E126K, and G460S mutations was identified in R isolates. The Y143F and G460S mutations had been associated with DMI fungicide resistance in other plant pathogens. No increased constitutive expression of GcCYP51A or GcCYP51B was observed in RS or R isolates. Detached fruit assays revealed that label rates of propiconazole controlled sour rot caused by S and RS but not R isolates. Our results suggest that sour rot outbreaks in an SC packing house were linked to target gene–induced propiconazole resistance in G. candidum.

Fludioxonil and propiconazole are frequently used fungicides for managing fungal diseases in specialty crops. They are often used in mixture to manage postharvest brown rot caused by Monilinia fructicola and sour rot caused by the yeast-like fungus Geotrichum candidum on stone fruits. Previous research indicated, however, that fludioxonil can increase the expression of the ATP-binding cassette transporters CDR1 and CDR2 in the yeast Candida albicans, leading to increased resistance to demethylation inhibitor (DMI) fungicides. In this study, mycelial growth assays and detached fruit assays were conducted with G. candidum isolates of varying sensitivity to propiconazole to investigate potential antagonism between the two active ingredients. Isolates sensitive, reduced-sensitive, and resistant to propiconazole were able to grow with or without 20 μg/ml of fludioxonil on potato dextrose agar containing up to 0, 3, and 30 μg/ml of propiconazole and on water agar containing up to 7.5, 7.5, and 30 μg/ml of propiconazole, respectively, and no antagonistic interactions were detected. Propiconazole controlled disease with or without 20 μg/ml of fludioxonil equally on detached cherry fruit caused by isolates with different DMI sensitivity phenotypes. Our results indicated no detectable antagonism between fludioxonil and propiconazole that would interfere with the mixture’s ability to manage sour rot of cherry.

Emerging fungal pathogens have always been an issue of concern in southeastern U.S. strawberry production. In 2023, an unusual outbreak of Gnomonia leaf blotch occurred at one North Carolina and multiple South Carolina (SC) strawberry farms and marked the first reports of its occurrence in SC. Molecular identification and phylogenetic analysis of isolates from multiple locations identified the fungus Gnomoniopsis fructicola as the causal agent. In vitro germination of G. fructicola progressed slowly and remained less than 40% even after 24 h of incubation. Similarly, germ tube growth was slow compared with other pathogens. Slow symptom development on strawberry leaves of young strawberry plants grown in the greenhouse started 5 weeks after inoculation. Once the pathogen established on greenhouse plants, leaf necrosis forming blotches was observed. The baseline sensitivity of G. fructicola isolates to commonly used chemical classes of fungicides was assessed. Propiconazole, cyprodinil, pyraclostrobin, and fludioxonil were highly effective in mycelial growth assays with effective concentration of fungicide required for 50% growth inhibition (EC₅₀) values ≤0.01 μg/ml. Iprodione and thiophanate-methyl were also effective, with EC₅₀ values ranging from 0.05 to 1.38 and 2.01 to 23.96 μg/ml, respectively. Fluopyram and fenhexamid were ineffective. with EC₅₀ values >100 μg/ml. Based on conversations with the producers, the disease outbreak was linked to transplants from the same nursery source. This study reports for the first time the presence of Gnomonia leaf blotch in SC and provides valuable insights into chemical management options for G. fructicola.

Sterol demethylation inhibitor (DMI) fungicides continue to be essential components for the control of brown rot of peach caused by Monilinia fructicola in the United States and worldwide. In the southeastern United States, resistance to DMIs had been associated with overexpression of the cytochrome P450 14α-demethylase gene MfCYP51 as well as the genetic element Mona, a 65 bp in length nucleotide sequence located upstream of MfCYP51 in resistant isolates. About 20 years after the first survey, we reevaluated sensitivity of M. fructicola from South Carolina and Georgia to propiconazole and also evaluated isolates from Alabama for the first time. A total of 238 M. fructicola isolates were collected from various commercial and two experimental orchards, and sensitivity to propiconazole was determined based on a discriminatory dose of 0.3 μg/ml. Results indicated 16.2, 89.2, and 72.4% of isolates from Alabama, Georgia, and South Carolina, respectively, were resistant to propiconazole. The detection of resistance in Alabama is the first report for the state. All resistant isolates contained Mona, but it was absent from most sensitive isolates. It was unclear if the resistance frequency had increased in South Carolina and Georgia. However, the resistance levels (as assessed by the isolate frequency in discriminatory dose-based relative growth categories) did not change notably, and no evidence of other resistance genotypes was found. Analysis of the upstream MfCYP51 gene region in the resistant isolate CF010 revealed an insertion sequence described for the first time in this report. Our study suggests that current fungicide spray programs have been effective against increasing resistance levels in populations of M. fructicola and suppressing development of new resistant genotypes of the pathogen.

Fungal diseases, including downy mildew (caused by Plasmopara viticola) and gray mold (caused by Botrytis cinerea), significantly impact the marketable yield of grapes produced worldwide. Cytochrome b of the mitochondrial respiratory chain of these two fungi is a key target for Quinone outside inhibitor (QoI)-based fungicide development. Since the mode of action (MOA) of QoI fungicides is restricted to a single site, the extensive usage of these fungicides has resulted in fungicide resistance. The use of fungicide combinations with multiple targets is an effective way to counter and slow down the development of fungicide resistance. Due to the high cost of in planta trials, in silico techniques can be used for the rapid screening of potential fungicides. In this study, a combination of in silico simulations that include Schrödinger Glide docking, molecular dynamics, and Molecular Mechanism-Generalized Born Surface Area calculation were used to screen the most potent QoI and non-QoI-based fungicide combinations to wild-type, G143A-mutated, F129L-mutated, and double-mutated versions that had both G143A and F129L mutations of fungal cytochrome b. In silico docking studies indicated that mandestrobin, famoxadone, captan, and thiram have a high affinity toward WT cytochrome b of Botrytis cinerea. Although the QoIs mandestrobin and famoxadone were effective for WT based on in vitro results, they were not broadly effective against G143A-mutated isolates. Famoxadone was only effective against one isolate with G143A-mutated cytochrome b. The non-QoI fungicides thiram and captan were effective against both WT and isolates with G143A-mutated cytochrome b. Follow-up in silico docking and molecular dynamics studies suggested that fungicide combinations consisting of famoxadone, mandestrobin, fenamidone, and thiram should be considered in field testing targeting Plasmopara viticola and Botrytis cinerea fungicide resistance.

A new Neopestalotiopsis sp. was recently reported causing outbreaks of leaf spot and fruit rot on strawberry in Florida, Georgia, and South Carolina. In contrast to other Pestalotiopsis pathogens, the new species appears more aggressive and destructive on strawberry. Current chemical options for management are disease suppressive at best, and affected growers have been experiencing major yield losses. In this study, we developed a molecular method based on polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) for identification of the new Neopestalotiopsis sp. from strawberry. Isolates of the new Neopestalotiopsis sp. collected in Florida; isolates of N. rosae, N. honoluluana, N. ellipsopora, N. saprophytica, N. samarangensis, and P. rhododendri; and isolates from South Carolina suspected to be the new Neopestalotiopsis sp. were included in this study. This method is based on PCR amplification of a β-tubulin gene fragment using a previously published set of primers (Bt2a and Bt2b), followed by use of the restriction enzyme BsaWI. The enzyme cuts the PCR product from the new Neopestalotiopsis sp. twice, yielding fragments of 290 base pairs (bp) and 130 and 20 bp in size, whereas fragments from other species are only cut once, yielding fragments of 420 and 20 bp. This method will aid research labs and diagnostic clinics in the accurate and fast identification of the aggressive Neopestalotiopsis sp. variant from strawberry.