The survey of apple Valsa canker disease occurrence was conducted by the Apple Research Center of the Rural Development Administration and took place over 9 years from 2015 to 2023 in eight major apple-producing regions in Korea: six in Gyeongsangbuk-do, Andong-si, Yeongcheon-si, Yeongju-si, Cheongsong-gun, Gunwi-gun, and Uiseong-gun; Geochang-gun in Gyeongsangnam-do; and Jangsu-gun in Jeollabuk-do. The survey was carried out according to the methods specified in the Guidelines for Crop Disease and Pest Control (Rural Development Administration, 2020) issued by the Rural Development Administration. The disease survey was conducted at 30-day intervals from February to late October each year. At each farm, branches from 100 trees around the survey site were selected to identify infected branches and calculate the infection rate (%). The percentage of infected trees was calculated using the equation disease incidence = (infected trees / 100 trees) × 100. Although there were changes in the surveyed orchards over the lengthy survey period, to the extent possible, efforts were made to conduct the surveys at the same locations. Disease diagnosis was performed by visually inspecting the branches for symptoms and signs, and if necessary, morphological and molecular biological diagnoses were also conducted.

The strains used in this study were isolated from branches showing canker symptoms in the Gyeongsangbuk-do region during the disease survey. The collected samples were cut at the canker site with a scalpel, surface-sterilized with 70% ethanol and 1% NaOCl, rinsed with distilled water, and then plated on potato dextrose agar (PDA) medium. The plates were incubated at 25°C for 1-2 days. The leading edge of any growing mycelium was then transferred to fresh PDA medium to isolate pure cultures, which were used for the experiments. A total of 38 isolates were obtained: three in Andong, one in Yeongcheon, two in Yeongju, two in Cheongsong, 24 in Gunwi, and six in Uiseong (Supplementary Table 1).

For phylogenetic analysis, total genomic DNA was extracted from the isolated strains. Polymerase chain reaction (PCR) was performed using the primer pair ITS1F/ITS4 to amplify the ITS region, a commonly used molecular marker for fungal identification (White et al., 1990). Additionally, PCR was performed using primer pairs for four genes recently shown to improve Cytospora species identification: LROR/LR7 (LSU gene) (Vilgalys and Hester, 1990), ACT512F/ACT783R (act1 gene) (Carbone and Kohn, 1999), RPB2-5F2/fRPB2-7cR (rpb2 gene) (Liu et al., 1999; Sung et al., 2007), and EF1-728F/EF-2 (tef1-α gene) (Carbone and Kohn, 1999; O'Donnell et al., 1998). The amplified PCR products were purified using ExoSAP-IT (GE Healthcare, Buckinghamshire, UK) and sequenced by Macrogen (Daejeon, Korea). The obtained sequences were compared to apple Valsa canker pathogen strains registered in GenBank using BLAST searches of the National Center for Biotechnology Information (NCBI) database. Sequences were aligned using CLUSTAL W (Larkin et al., 2007), and a phylogenetic analysis based on the ITS region alone was performed using the maximum likelihood (ML) method (Felsenstein, 1981) in MEGA 7 (Kumar et al., 2016). Diaporthe eres was used as an outgroup, and branch supports were computed using bootstrap analysis with 1,000 resamples. The sequences of the five molecular markers used to identify the causative agents of apple Valsa canker disease were concatenated in the order presented above, and an ML phylogenetic tree was constructed to analyze the phylogenetic relationships within Cytospora using the same methods. Maximum-likelihood analysis was performed using the nearest-neighbor interchange heuristic search method and Kimura's two-parameter model (Kimura, 1980).

In the survey of apple Valsa canker disease, visually observable symptoms were consistently observed across all regions. When apple Valsa canker disease affected apple trees, the disease typically progressed from the infection site towards the branch ends (Fig. 1A-C), ultimately leading to tree death in most cases. As the disease progressed, becoming more severe, the bark turned brown and peeled off easily and the infected areas swelled slightly (Fig. 1D,E). In cases where the disease occurred on lateral branches, there were often signs of nearby pruning, and the disease progressed from the pruning site towards the branch end or downward (Fig. 1F). In areas infected for longer periods, the size of the black pycnidia increased, and yellow thread-like cirrhi were exuded (Fig. 1G,H). Furthermore, when trees were completely killed by apple Valsa canker disease, Schizophyllum commune was found to develop around the canker area (Melzer and Berton, 1988) (Fig. 1I).

The survey was conducted from 2015 to 2023 to monitor the infection rates of apple Valsa canker disease, and an average annual infection rate of 2.83% was determined. At the beginning of the survey, in 2015, the infection rate was relatively high at 2.20% (Fig. 2). After this period, the infection rates first decreased, reaching as low as around 0.9% in 2016 and 2017, marking the lowest rates observed during the survey period. However, in 2018, the infection rate increased to 3.88%. Higher incidence rates persisted from 2019 to 2022, with the infection rate rising to 4.16% in 2022, and then incidence sharply increased to 6.79% in 2023, nearly three times the average annual infection rate. Thus, the infection rate in 2023 was the highest recorded in the past 9 years.

The results of this study indicate that apple Valsa canker disease incidence was low, around 0.9%, in 2016 and 2017, but has been increasing sharply since 2018. Several factors likely contributed to this rising rate of infection. One major factor is the restriction on the supply of the pesticide Neoasozin imposed by the Rural Development Administration due to concerns about misuse and potential hazards. Neoasozin was an important fungicide for controlling apple Valsa canker disease (Uhm and Sohn, 1995), and its reduced availability may have led to insufficient disease management. Additionally, other fungicides, such as thiophanate-methyl and iminoctadine triacetate, while effective, are costly and less convenient to use, limiting their adoption by farmers. The high cost of these fungicides may deter farmers from using them regularly or in sufficient quantities, reducing the effectiveness of disease control. Furthermore, these fungicides may require specific application techniques or equipment that are not readily available or feasible for all farmers, further limiting their use. Although this study did not specifically investigate the potential factors behind the increasing incidence of apple Valsa canker, it is possible that agronomic practices, such as leaving infected branches in orchards after pruning, improper sanitation of tools, and changing environmental conditions, may have contributed to the rise in infections, as suggested by previous research (Cheon et al., 2018). Overall, our findings underscore the need for continuous monitoring and effective disease management strategies to mitigate the economic impact of apple Valsa canker disease in Korea.

To identify the species and determine the taxonomic positions of the apple canker pathogens occurring in domestic apple trees, 38 isolates collected during the survey period were analyzed. Sequence analysis of the ITS region, the most fundamental marker for fungal classification, revealed that the nucleotide sequences of all isolates were identical. The GenBank database search confirmed that these isolates had the highest match with C. mali. Therefore, out of the 38 isolates, one collected in 2015, the first year of the survey period, and one collected in 2023, the last year of the survey period, were selected for further analysis and named ARI-15-US and ARI-23-GW, respectively. Additionally, to accurately determine these isolate's taxonomic positions, a phylogenetic analysis was performed using the five molecular markers currently used to accurately distinguish species within the genus Cytospora.

A phylogenetic re-identification of the two isolates from this study based on the ITS region nucleotide sequences of the 12 species known to cause apple Valsa canker disease in apple trees in East Asia (Wang et al., 2020) was conducted (Table 1). The resulting phylogenetic tree revealed that ARI-15-US and ARI-23-GW formed a clade with the two C. mali sequences. However, phylogenetic trees based solely on the ITS region have limitations, for example, in our analysis, C. mali-sylvestris MFLUCC 16-0638 and C. melostoma A 846 formed a cluster, making it impossible to distinguish between the two strains (Fig. 3). Therefore, to more accurately determine phylogenetic relationships in Cytospora, partial sequences of the LSU, act1, rpb2, and tef1-α genes, which have, along with the ITS sequence, recently been shown to collectively differentiate species within the genus Cytospora, were utilized. No differences were observed in the ITS region sequences among the 38 isolates, and a search of the LSU, act1, rpb2, and tef1-α gene sequences in the NCBI database showed nearly 100% homology with Cytospora mali. However, small variations in the LSU, act1, rpb2, and tef1-α gene sequences were detected among the 38 isolates, and two strains with the most noticeable differences, ARI-15-US and ARI-23-GW, were selected for further analysis. To determine whether these sequence variations were significant enough to distinguish between the two strains at the species level and to ensure the accurate identification of ARI-15-US and ARI-23-GW, a phylogenetic analysis was conducted using Cytospora species for which sequences of all five molecular markers were available in the NCBI database (Table 2). This analysis used concatenated sequences of the ITS region and LSU, act1, rpb2, and tef1-α genes, resulting in a combined dataset of 44 sequences, each 2,167 bp long. Diaporthe eres (CBS 145040) was used as the outgroup. The phylogenetic tree revealed that ARI-15-US and ARI-23-GW formed a distinct clade with other C. mali strains (Fig. 4).

In Korea, since the discovery of the pathogen C. mali causing apple Valsa canker disease in apple trees in 1919 (Kim et al., 1970), no other species of the genus Cytospora have been reported to our knowledge. However, a total of 21 species in the genus Cytospora that cause disease in apples have been reported in East Asia. Given the sharp increase in the incidence of apple Valsa canker disease in 2023 and, thus, a growing need for effective control measures, it is important to investigate the potential presence of Cytospora species other than C. mali in domestic orchards. Therefore, this study aimed to identify all 38 isolates collected during the survey period and determine the specific species involved. As all 38 isolates possessed identical ITS regions, a more detailed phylogenetic analysis was conducted using four additional genetic markers (LSU, act1, rpb2, tef1-α), which has recently been deemed essential for accurate Cytospora taxonomy. The phylogenetic tree constructed using all five markers showed that the isolates formed a single clade with C. mali, confirming that C. mali is the predominant species causing apple Valsa canker disease in Korea.

In the survey conducted from 2015 to 2023 in major apple-producing regions in Korea, the highest incidence of apple Valsa canker disease was recorded in 2023, indicating an increasing trend in disease occurrence. This trend suggests that the incidence may continue to rise in the future. While 21 species are known to cause Valsa canker in apples in East Asia, this study identified only one species, Cytospora mali, in Korea. Although it cannot be definitively concluded that other fungal species do not contribute to the disease, it is clear that C. mali is the dominant species. This finding provides essential baseline data for establishing effective control strategies against apple Valsa canker disease.