In 2024, leaf samples identified as showing symptoms of brown shot-hole disease were collected from cherry trees throughout Republic of Korea (Ko et al., 2025). Briefly, the collected sample was cut with sterilized scissors from the tissue showing symptoms, and the piece was washed once with 70% EtOH, once with 1% NaOCl, and twice with sterilized water, and the pathogen was isolated on water agar medium. Afterwards, the isolates were cultured on potato dextrose agar (PDA) medium (potato dextrose broth, 24 g; agar, 20 g per L) at 27°C for about a week and classified into four types (Type 1, Type 2, Type 3, Type 4) according to colony and spore morphology. Forty isolates per type were extracted for DNA according to the CTAB protocol and PCR targeting the ITS region was performed (Rodrigues et al., 2018). Type 1 was identified as Alternaria spp., Type 2 as Diaporthe spp., Type 3 as Epicoccum spp., and Type 4 as Botryosphaeria spp. Moreover, identification of species was completed by sequencing the tef1 (translation elongation factor 1-alpha) region of and Botryosphaeria spp. and Diaporthe spp., and the LSU (28S nrDNA) region of Epicoccum spp. and Alternaria spp.

A total of 160 isolates, previously identified via DNA sequencing, were evaluated for the resistance to the DMI fungicides difenoconazole (active ingredient (a.i.) 10% wettable powder (WP)) and tebuconazole (a.i. 25% WP). Prior to Resistance assays, isolates were maintained on PDA medium at 27°C for one week. Fungicide resistance of isolates was assessed by quantification of the EC₅₀ (half maximal effective concentration), which is the concentration of fungicide at which fungal growth is suppressed by 50% compared to the untreated group after fungicide treatment., and the fungal growth inhibition was evaluated by comparing mycelial growth measurements before and after fungicide treatments (Lee and Kwak, 2023). The mycelial growth inhibition rate according to the concentration of the fungicide was calculated to determine the value of EC₅₀. Measurement of mycelial growth inhibition was conducted by growing fungi in PDA media with or without fungicides with various concentrations that inhibited mycelial growth (Supplementary Table 1). The final concentrations of fungicides in the PDA media by genus were as follows: Alternaria: 0, 0.5, 5, 10, 50, 125 ppm; Diaporthe: 0, 0.25, 0.5, 1, 2, 5 ppm; Epicoccum: 0, 0.5, 5, 10, 50, 250 ppm; Botryosphaeria: 0, 0.5, 1, 5, 10, 125 ppm for tebuconazole; and Alternaria: 0, 0.5, 5, 10, 50, 500 ppm; Diaporthe: 0, 0.25, 0.5, 1, 2, 10 ppm; Epicoccum: 0, 0.5, 1, 5, 50, 500 ppm; Botryosphaeria: 0, 0.5, 1, 5, 50, 500 ppm for difenoconazole (Supplementary Table 1). Fungi were grown in medium placed in a 6-well plate supplemented with various concentrations of fungicide. Culture was performed using mycelial fragments with a diameter of 4 mm as an inoculum, and was performed at 27°C until the mycelial diameter of the untreated group reached 3.5 cm. The assay was performed in three replicates per isolate. Finally, the average mycelial diameter of the treatment groups obtained from the three replicates was quantified and used to calculate the mycelial inhibition rate. Quantification of the mycelial inhibition rate was conducted by dividing the difference between the mycelial diameter of the untreated samples and the mycelial diameter of the samples treated with fungicides by the mycelial diameter of the untreated control samples then converted into percentage (Wang et al., 2023). EC₅₀ value was determined by using the mycelial inhibition rate to the log-logistic model and by using the three-parameter log-logistic model (LL.3) of the R software “drc” package (Noel et al., 2018). Cross-resistance against difenoconazole and tebuconazole in different genera was examined based on the EC₅₀ value. Additionally, the sites where cherry trees were planted along urban roads or in city parks were designated as urban areas, whereas sites located at the interface with rice paddies, upland crop fields, or orchards were designated as rural areas. Spearmen rank correlation was used to compare the Log EC₅₀ of the two fungicides, and the principal coordinates analysis (PCoA) was used to evaluate differences in EC₅₀ values between rural and urban fungal isolates. The Bray-Curtis dissimilarity formula was employed to reprocess the EC₅₀ values of each fungal species, and the permutational multivariate analysis of variance (PERMANOVA) was utilized to analyze the statistical differences of the data collected between rural and urban locations. Between-group statistics on the PCoA1 and PCoA2 axes were evaluated using the Wilcoxon rank-sum analysis. The analyzed and processed data were visualized using R software’s ggplot2 (version 4.0.0), ggalt (version 0.4.0), and ggpubr (version 0.6.1) packages, and data processing and statistical analysis were performed using packages such as dplyr (version 1.1.4), vegan (version 2.7.1), and pairwiseAdonis (version 0.4.1). For the RF values of Alternaria spp., the median value of the isolates with the lower 25% EC₅₀ for each fungicide was set as the baseline, and each isolate was then divided by the baseline to calculate the value (Lim et al., 2025). Fungicide resistance was evaluated by classifying RF values less than 3 as sensitive (S), 3 to 10 as reduced sensitivity (RS), and 10 or more as resistance (R) (Rallos et al., 2016) (Table 1).

Target gene sequencing was performed on Alternaria spp. isolates that had higher EC₅₀ values and RF values of 10 or higher compared to other genera, and a search for isolates that expressed qualitative resistance through point mutations was conducted. Target gene sequencing was conducted on isolates AJA3, AGM1, and ACU1 with RF values higher than 20, and ANW2 and AHD1 with low RF values. Afterwards, PCR was performed using primers AaCYP51-1-F (5′-TAT TCT TGA AGC AGG TGA C-3′)/AaCYP51-1-R (5′-GCT ATC ATA CAA CGG ACA T-3′) and AaCYP51-2-F (5′-CCC AGC CTG CAT TGA TA-3′)/AaCYP51-2-R (5′-CCG TTC TCC GCT CGT TA-3′) targeting the CYP51 gene, respectively, with specific parameters such as initial denaturation (1 cycle) at 95°C for 5 min; denaturation (35 cycles) at 95°C for 30 s, annealing at 55°C for 40 s, and elongation at 72°C for 50 s; and 1 cycle of final elongation at 72°C for 7 min (Sun et al., 2021). PCR product purification was performed using a Expin^TM Gel SV kit (GeneAll, Seoul, Korea). The purified PCR product was analyzed for DNA sequence by Cosmogenetech (Seoul, Korea), and the two sequences were merged and extended. The base sequences of the CYP51 gene of five isolates were aligned using MEGA 11 software, and the presence or absence of SNPs was confirmed to evaluate whether there was a point mutation.

In this study, a fungicide resistance test was conducted on four genera of pathogens isolated from cherry tree brown shot hole disease. It was first verified that the R² value of the mycelial inhibition rate of most isolates was 0.9 or higher, which means that the data can be used for EC₅₀ measurement (Supplementary Table 2). The results of the mycelial inhibition rate for tebuconazole and difenoconazole of fungi showed that in the case of Alternaria and Epicoccum, mycelial growth was inhibited when relatively high concentrations of fungicides were treated (Fig. 1A, 1C). On the other hand, in the case of Diaporthe and Botryosphaeria, mycelial growth was inhibited at relatively low concentrations of fungicide (Fig. 1B, 1D). The variation in mycelial inhibition rate by isolate in each genus was different. In the case of Alternaria and Diaporthe, the variation by isolate was relatively large, but in the case of Epicoccum and Botryosphaeria, the variation by isolate was relatively small (Fig. 1). The EC₅₀ for each genus, measured based on mycelial inhibition rate, showed relatively high values for both fungicides for Alternaria and Epicoccum (Fig. 2A, 2C). The mean EC₅₀ of Alternaria was 10.11 ppm for tebuconazole and 6.33 ppm for difenoconazole, showing more sensitivity to difenoconazole (Fig. 2A). Additionally, there were 5 isolates (AJA3, AGM1, ASA3, ACU1, ASU4) for tebuconazole and 2 isolates (ACU1, ASU4) for difenoconazole with an EC₅₀ more than twice the mean (Fig. 2A). For Epicoccum, the mean EC₅₀ was 35.26 ppm for tebuconazole and 10.48 ppm for difenoconazole, showing more sensitivity to difenoconazole, similar to Alternaria (Fig. 2C). And isolates with EC₅₀ more than twice the mean was identified as 1 isolate (BGR5) from tebuconazole and 1 isolate (BNW3) from difenoconazole (Fig. 2C). In contrast, Diaporthe and Botryosphaeria showed relatively low EC₅₀ for the two fungicides (Fig. 2B, 2D). The mean EC₅₀ of Diaporthe was 1.01 ppm for tebuconazole and 0.83 ppm for difenoconazole, showing more sensitivity to difenoconazole (Fig. 2B). Additionally, isolates with EC₅₀ more than twice the mean was identified as 3 isolates (DHD2, DJJ1, DGH1) from tebuconazole and 4 isolates (DJJ1, DHD2, DGS4, DGS3) from difenoconazole (Fig. 2B). Botryosphaeria was susceptible to tebuconazole, with EC₅₀ of 0.67 ppm for tebuconazole and 2.08 ppm for difenoconazole (Fig. 2D). For isolates with an EC₅₀ more than twice the average EC₅₀ of Botryosphaeria, no isolates were found for tebuconazole, and 1 isolate (CNW3) was identified for difenoconazole (Fig. 2D). As a result, among the four genera, Alternaria was found to have a high EC₅₀ and at the same time, a large variation among isolates.

A cross-resistance test was conducted against the DMI fungicides tebuconazole and difenoconazole (Fig. 3). As a result, in the case of Alternaria, Spearman’s rank correlation analysis showed a moderate correlation of ρ = 0.485 (Fig. 3A), and in the case of Diaporthe, a strong correlation of ρ = 0.732 (Fig. 3B). Next, Epicoccum had a weak correlation with ρ = 0.259 (Fig. 3C), and Botryosphaeria also had a weak correlation with ρ = 0.19 (Fig. 3D). Therefore, the four genera showed different cross-resistance to the same DMI fungicides, with Diaporthe and Alternaria showing significantly higher cross-resistance (Fig. 3A, 3B). In contrast, Epicoccum and Botryosphaeria did not show cross-resistance to the two fungicides (Fig. 3C, 3D).

Each isolate for which the EC₅₀ for tebuconazole and difenoconazole was measured was analyzed to see if there was a difference in EC₅₀ according to the collection sites (Fig. 4). PCoA based on EC₅₀ data reprocessed employing the Bray-Curtis dissimilarity formula, revealed significant differences in fungicide resistance between urban and rural isolates for both fungicides (Fig. 4). In the case of tebuconazole, a significant difference was found in the Wilcoxon rank-sum test statistical analysis between PCoA1 values (P = 0.024), and a significant numerical difference was found in the PERMANOVA statistical analysis between the two sites (P = 0.001), (Fig. 4A). Difenoconazole showed a significant difference in the Wilcoxon rank-sum test analysis between PCoA2 values (P = 0.0068), and a statistically significant difference in the PERMANOVA analysis between the two sites (P = 0.001), (Fig. 4B). Therefore, it was predicted that differences in fungicide resistance between urban and rural isolates were likely to occur.

The RF value was measured for Alternaria, which has high EC₅₀ values on mean and isolates with large variations, to determine the proportion of fungicide-resistant isolates. As a result, one resistant (R) isolates with an RF value of 10 or higher against tebuconazole was identified in Gyeongsangbuk-do and Jeollabuk-do, and one resistant isolate against difenoconazole was identified in Jeollanam-do and Chungcheongbuk-do, respectively (Table 1 and Fig. 5). Reduced sensitivity (RS) isolates with RF values of 3 or more and less than 10 were found to have the highest rate in Gyeongsangnam-do for tebuconazole, and the highest rate in Gyeongsangbuk-do for difenoconazole (Table 1 and Fig. 5). For both tebuconazole and difenoconazole, the majority of sensitive (S) isolates with RF values less than 3 were present in all regions, and in South Chungcheongnam-do, all isolates were sensitive to tebuconazole (Table 1 and Fig. 5).

Three resistant isolates (AJA3, AGM1, ACU1) and two sensitive isolates (ANW2, AHD1) with high RF values of 20 or higher for the DMI fungicides tebuconazole and difenoconazole were selected, and the occurrence of qualitative resistance in the fungicide-resistant isolates was investigated (Fig. 6). As a result of base sequence analysis of the CYP51 gene, which is the location where genetic point mutations in previously known DMI fungicide-resistant strains occur, the base sequence of the 462nd codon of the sensitive isolates ANW2 and AHD1 and the resistant isolates AGM1 and ACU1 matched as GGC (Fig. 6B). On the other hand, the resistant isolate AJA3 was confirmed to have a base sequence of AGC at the 462nd codon of CYP51 (Fig. 6B). The 462nd codon of CYP51 in AJA3, where the SNP occurred encodes serine, and the 462nd codon of the remaining isolates, where the SNP did not occur, encodes glycine (Fig. 6B). Therefore, we verified that the occurrence of a SNP at 462nd codon of CYP51 of the resistant isolate AJA3 caused the G462S amino acid substitution (Fig. 6B).