Evaluation of Fungicide Resistance of the Brown Shot-hole Pathogens of Cherry Trees in Korea
Article information
Abstract
Cherry trees (Prunus serrulata var. spontanea, Prunus × yedoensis), a widely cultivated ornamental street trees grown in Korea, are vulnerable to variety of diseases particularly the prevailing brown shot-hole disease, thus needs effective maintenance strategies for optimum plant growth and observation. The major fungal genera causing cherry brown shot-hole disease in Korea were identified as Alternaria, Diaporthe, Epicoccum, and Botryosphaeria, and their resistance to tebuconazole and difenoconazole was investigated. Both fungicides are classified as demethylation inhibitors and the emergence of resistant strains has been reported worldwide. In this study, we found significant differences in fungicide resistance depending on the type of pathogen and regional differences and performed base sequence analysis of the CYP51 gene on some Alternaria isolates with fungicide resistance to confirm the occurrence of point mutations and amino acid substitutions. However, point mutations were not detected in some resistant Alternaria isolates, suggesting that the resistance of some isolates is possibly unrelated to the alterations in the target site or may be due to genetic variations in unidentified genomic sequence CYP51 gene. These results will contribute to understanding the mechanism of antifungal resistance development in fungal plant pathogens and are expected to contribute to the control of brown shot hole disease in cherry trees.
Cherry trees (Prunus serrulata var. spontanea, Prunus × yedoensis) account for the largest proportion of street trees planted in Korea and are used as ornamental tourist attractions in many parts of the country (Chang et al., 2007). Cherry trees are susceptible to variety of diseases, among which brown shot-hole disease predominates, inducing impaired photosynthetic efficiency, suppressed flowering, and premature defoliation (Cho and Kim, 2019). Previous investigations have isolated diverse pathogenic taxa belonging to the genus Alternaria, Diaporthe, Epicoccum, and Botryosphaeria from symptomatic cherry trees exhibiting brown shot-hole (Ko et al., 2025). In Korea, only tebuconazole and difenoconazole are two types of fungicides that are currently registered for brown shot-hole disease management and both are classified as demethylation inhibitors (DMI) fungicides (Rural Development Administration, Tsuda et al., 2004). DMI fungicides predominantly repress ergosterol accumulation via suppression of the CYP51 enzyme reaction necessary for the activation of 14α-demethylation of the fungal ergosterol biosynthetic pathway, thereby disrupting membrane fluidity and permeability (Wang et al., 2021; Wieczorek et al., 2015). DMI fungicide is extensively used as a leading chemical control agent and commercial fungicide globally due to its broad-spectrum efficacy (Subramanian et al., 2025). Consequently, application of DMI fungicides continues to expand, underpinning its pivotal role in crop protection strategies and management of fungal crop diseases worldwide (Jørgensen et al., 2021a; Quevedo-Caraballo et al., 2024). Increasing emergence of resistant strains due to extensive utilization of DMI fungicides has been reported globally (Ma and Michailides, 2005). Several DMI fungicide resistant strains including the cherry leaf spot pathogen Blumeriella jaapii, the apple scab pathogen Venturia inaequalis and Alternaria alternata which causes leaf and fruit spot on numerous plants have been documented in previous investigations (Dry et al., 2004; Ma et al., 2006; Schnabel et al., 2001). The emergence of pathogen resistance to these DMI fungicides makes it difficult to control pathogens using fungicides in actual fields. For example, in trees, there are known cases of reduced control effectiveness in Asia due to DMI fungicide resistance of the pear scab pathogen Venturia nashicola, and cases of failure to control the apple scab pathogen V. inaequalis have been investigated in the United States (Ishii et al., 2021; Villani et al., 2015). Inhibition of the effectiveness of DMI fungicides has also been observed in various gramineous crops. It has been shown that continuous use of DMI fungicides against the Septoria tritici blotch pathogen Zymoseptoria tritici results in a decrease in field pathogen control each year, and a decrease in field control efficacy and spread of resistance in the pathogen population against the rice bakanae disease pathogen Fusarium fujikuroi have been reported (Ge et al., 2024; Jørgensen et al., 2021b). Therefore, DMI disinfectant resistance can occur in a very wide range of pathogen taxa. In Korea, the only fungicide targeting cherry tree brown shot-hole disease is DMI fungicide, and it is possible that the pathogen has acquired resistance to DMI fungicide due to the continuous use of a single series of fungicides. However, little research has been conducted on cherry tree brown shot-hole disease in Korea, and no nationwide survey on fungicide resistance of the pathogen has been conducted at present. This study investigated the fungicide resistance of cherry shot-hole disease pathogens isolated from previous studies nationwide to the DMI fungicides tebuconazole and difenoconazole. The national distribution of resistant isolates was analyzed to determine fungicide susceptibility, and the CYP51 genomic sequences of resistant isolates were sequenced to determine the presence of point mutations.
Materials and Methods
Collection, cultivation, and identification of fungal isolates
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.
Fungicide resistance assay of fungal isolates
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).
Assessment of resistance factor (RF) values and fungicide resistance levels according to the pathogen collection regions
Qualitative fungicide resistance assay of fungi
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 ExpinTM 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.
Results
Screening for DMI fungicide resistant pathogens
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.
Inhibition rate of mycelial growth induced by tebuconazole and difenoconazole on (A) Alternaria, (B) Diaporthe, (C) Epicoccum, and (D) Botryosphaeria. Fungicide inhibition rates were quantified using the agar dilution method, where pathogens were cultured on PDA medium supplemented with four varying fungicide concentrations. The percent inhibition rate was calculated by dividing the difference in mycelial diameter between the fungicide-treated and untreated groups by the mycelial diameter of the control group, then converting to a percentage.
EC50 values for tebuconazole and difenoconazole in (A) Alternaria, (B) Diaporthe, (C) Epicoccum, and (D) Botryosphaeria calculated based on the log-logistic model. The EC50 value was calculated by integrating the mycelial growth inhibition and fungicide treatment concentration into the three-parameter log-logistic model (LL.3) of the R software “drc” package. Measurements of EC50 values for the two fungicides were quantified for each fungal genera, with the blue dotted line depicting the mean EC50 value and the red dotted line representing the twice the mean EC50 value.
Fungicide cross-resistance assay
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).
Scatter plot of cross-resistance tests for (A) Alternaria, (B) Diaporthe, (C) Epicoccum, and (D) Botryosphaeria against tebuconazole and difenoconazole. Cross-resistance was assessed by Spearman’s rank correlation (ρ) between the LogEC50 values for the two fungicides, and the 95% confidence interval was estimated by paired bootstrapping of the isolates. The Benjamini–Hochberg procedure was applied to control the false discovery rate (FDR) at the 0.05 level for multiple comparisons, quantifying the q-value. The linear regression is illustrated by a solid black line, with the 95% confidence interval shaded in black, and the Spearman’s rank correlation (ρ), degrees of freedom (df), and two-tailed P-value (P) are displayed in the upper left corner of the scatter plot.
EC50 variation of pathogens in each region classified into urban and rural sites
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.
Principal coordinates analysis (PCoA) plot depicting the variations in fungicide resistance among pathogens collected nationwide from urban and rural locations in South Korea. PCoA was independently conducted for (A) tebuconazole and (B) difenoconazole datasets. The EC50 values of pathogens for tebuconazole and difenoconazole were recalculated using the Bray-Curtis dissimilarity formula and plotted on a coordinate plane. Group data distributions were expressed with 70% data-concentration ellipse, and the distribution of data by group on each axis (PCoA1, PCoA2) was visualized as a marginal boxplot. Permutational multivariate analysis of variance (PERMANOVA) was used for statistical analysis of PCoA results on the coordinate plane, and the Wilcoxon rank-sum test evaluated group differences on each axis (ns: P ≥ 0.05, *: 0.01 ≤ P < 0.05).
Distribution of fungicide-resistant isolates of Alternaria spp. in Korea
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).
Distribution of fungicide resistant isolates against (A) tebuconazole and (B) difenoconazole among Alternaria spp. isolates collected nationwide. Alternaria isolates were categorized into three phenotypes (sensitive, reduced sensitivity, resistance) based on the RF values, and the distribution ratios of categories were converted to a percentage for each geographic region.
Investigation of qualitative resistance occurrence in fungicide-resistant isolates
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).
Confirmation of CYP51 gene point mutations in Alternaria spp. isolates exhibiting elevated resistance to fungicides. (A) Mycelial growth status was compared between isolates with elevated (AJA3, AGM1, ACU1) and reduced (ANW2, AHD1) EC50 values for tebuconazole and difenoconazole. (B) Confirmation of amino acid substitution due to SNP in CYP51 gene, which is a critical site for DMI fungicide resistance. Point mutations were confirmed through sequencing of the CYP51 genes in resistant and sensitive isolates, and DNA sequence alignments were conducted using the MEGA 11 software to verify mutations.
Discussion
Brown shot-hole disease, which occurs on cherry trees in Korea, is a disease that exists frequently every year, and its control relies on chemical control using fungicides (Han et al., 2019). In Korea, only two fungicides are registered as pesticides for cherry trees: tebuconazole and difenoconazole, and both are DMI fungicides Previous studies showed tthat the continuous use of single-acting fungicides for plant disease control is the most important cause of the progression of fungicide resistance in pathogens (Corkley et al., 2022). Previous research has reported that brown shot-hole disease, which occurs in domestic cherry trees, can be caused by various fungal taxa (Ko et al., 2025). However, no studies have investigated the resistance of different causative agents of brown shot-hole disease to DMI fungicides in Korea. In this study, we investigated the resistance of pathogens of the genus Botryosphaeria, Diaporthe, Alternaria and Epicoccum isolated from brown shot-hole disease symptoms in Korea to tebuconazole and difenoconazole.
For both tebuconazole and difenoconazole, Alternaria and Epicoccum showed relatively high EC50, while Diaporthe and Botryosphaeria showed low EC50. The fungicide resistance clearly differed depending on the taxon, suggesting that the control effect may differ depending on the taxon of the pathogen causing cherry brown shot-hole disease. In addition, Alternaria and Diaporthe were confirmed to have a high variation in fungicide resistance compared to other genera, and it is predicted that intensive monitoring is needed for fungicide-resistant strains of the genus Alternaria, which have a high average EC50. A notable point is the difference in fungicide efficacy between tebuconazole and difenoconazole, which are both DMI fungicides. Alternaria, Diaporthe, and Epicoccum were more susceptible to difenoconazole, while Botryosphaeria was more susceptible to tebuconazole. Results suggest that difenoconazole, known as a potent fungicide against wide range of pathogens, is more efficient in the management of brown shot hole disease in cherry trees. Variation in the expression of cross-resistance in each genus exists due to the difference between antifungal capacity of the two fungicides. Significant cross-resistance to two fungicides was observed in Diaporthe and Alternaria implicating that efficient fungal management is possible through alternate application of several fungicides with various modes of action (Ballu et al., 2023). Diverse fungal efficacy is attributed to the structure variations of the binding site of each fungicide and the different concentrations of fungicide located inside the cellular components, since the two DMI fungicides possess distinct molecular structures (Zhang et al., 2020). Unique differences in the mode of action between fungicides affect differences in the progression of fungicide resistance (Chen et al., 2022). The difference in fungicide resistance between pathogens isolated from urban and rural areas is likely due to variation in the amount or frequency fungicide application via spray in the two regions. The increase in fungicide resistance of the pathogen population due to a decrease in the density of susceptible strains and an increase in the density of resistant strains caused by repeated use of fungicides will make it difficult to control cherry tree brown shot-hole disease in the future (Massi et al., 2021). The distribution of fungicide-resistant isolates among domestic administrative districts of Alternaria isolates with high EC50 on average did not show significant regional differences. Sensitive isolates were more than half in all regions, and resistant, reduced sensitivity isolates were approximately 20–30%, indicating that regional differences in fungicide resistance were not significant for both fungicides. However, in the present study, quantitative data on fungicide application intensity such as frequency of application, total amounts applied, and potential spray drift, were not incorporated into the analysis for either urban or rural sites. Consequently, the observed differences in fungicide sensitivity between isolates from the two regions cannot be conclusively attributed to differences in fungicide exposure. To clearly elucidate regional variation in fungicide sensitivity, further studies are required that integrate detailed information on application records, and management practices at each sampling site.
Next, we selected isolates of the genus Alternaria with high resistance and measured the occurrence of point modifications in the CYP51 gene. As a result, we confirmed the occurrence of point mutations in one of the three resistant isolates. A. alternata AJA3, in which a point mutation was confirmed, had an amino acid substitution of G462S, and this mutation has already been reported in China (Feng et al., 2025). Studies showed that the resistance to DMI fungicide increased in dose-dependent manner, and the substitutions in specific sequence of CYP51 gene induce fungicide resistance such as I300S, A303T, and A303V (Li et al., 2023). Therefore, AJA3, which shows significant increase in resistance to tebuconazole, possibly contains amino acid substitutions in specific regions of the CYP51 gene sequence aside from the specified G462S mutation. Furthermore, in AGM1 and ACU1, which are resistant isolates with unidentified point mutations in this study exhibited the possibility of amino acid substitutions present in CYP51 genetic region that was not sequenced. Moreover, previous reports characterized the mechanism of various pathogens in the development of resistance to DMI fungicides includes several factors related to genetic modifications such as increased transcriptional expression of CYP51, overexpression of ABC transporter proteins, alteration in the sterol biosynthesis pathway, and the modification of the cell membrane structure of the pathogen (Karaoglanidis et al., 2003; Leroux and Walker, 2011; Price et al., 2015; Spanner et al., 2021). Therefore, the resistance progression exhibited by AJA3, AGM1, and ACU1 was possibly induced by the modification of gene expression levels, thus further research including the transcriptional expression analysis of related genes is necessary (He et al., 2019).
In this study, the fungicide resistance of cherry tree brown shot-hole pathogens to two types of DMI fungicides was investigated and results indicated that the EC50 for the fungicides was significantly different and is found dependent on the type of pathogens Moreover, a point mutation in one Alternaria isolate strain was identified and exhibited fungicide resistance confirming that this was a similar type of mutation as previously reported. These findings provide valuable insights to the development of effective strategies against cherry brown shot-hole disease and for the advancement of domestic strategies for disease control methods.
Notes
Conflicts of Interest
No potential conflict of interest relevant to this article were reported.
Acknowledgments
This work was supported by R&D Program for Forest Science Technology (Project No. RS-2024-00405244) provided by the Korea Forest Service (Korea Forestry Promotion Institute) and Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by Ministry of Education (grant No. 2022R1A6C101B724).
Electronic Supplementary Material
Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).