Genetic Diversity and Genotype Distribution of Erwinia amylovora in Korea
Article information
Abstract
Erwinia amylovora, first identified in 1793 in Hudson Valley (New York, USA), has a genome size of 3.7–4.0 Mb. E. amylovora bacterial strains are classified based on the infecting hosts: the Amygdaloideae-infecting (AI) group, targeting apple and pear trees, and the Rubus-infecting group, affecting berry trees. Since the AI-group strains display high genetic similarity (>99.7%), it is challenging to characterize their genotypes. This study investigated the genetic diversity of E. amylovora isolates in Korea and the regional distribution patterns of genotypes using a multilocus variable number of tandem repeat analysis (MLVA). Four specific primers were used to amplify and sequence tandem repeats in the E. amylovora genome, and a distribution map of E. amylovora was created using MLVA genotypes. Thirty-two types of MLVA patterns were identified in Korean strains, and RV19 was the dominant type identified in all South Korean regions. According to the minimal spanning tree, genotypes were differentiated into RV7, RV14, RV20, RV22, and RV27 types, originating from the RV19 type. This finding suggests that the RV19 type, introduced to Korea for the first time, spread to other regions from Anseong-si, Cheonan-si, Chungju-si, and Jecheon-si, depending on the type. We determined the MLVA genotypes of E. amylovora isolates and distribution patterns by region from 2019 to 2023. The distribution of these genotypes by year and region provides basic information for the genetic diversity and spread of E. amylovora in Korea.
Erwinia amylovora, a causal agent of fire blight, is the first bacterium identified to cause disease in plants (Mansfield et al., 2012). The pathogen infects Rosaceae hosts through flowers, shoots, and wounds and systemically infects the whole tree through the xylem (Momol et al., 1998; Norelli et al., 2003). Additionally, E. amylovora imposes a significant loss of apple and pear production worldwide (Norelli et al., 2003; Van Der Zwet et al., 2012). In Korea, fire blight was first discovered in pear and apple tree orchards in Anseong-si and Cheonan-si in 2015 (Myung et al., 2016; Park et al., 2016) and steadily spread to other regions (Ham et al., 2024).
E. amylovora has a relatively small genome size (<4 Mb) compared to other plant pathogenic bacteria such as Xanthomonas spp. and Pseudomonas syringae (Mansfield et al., 2012). E. amylovora has been divided into two subclasses: the Amygdaloideae-infecting (AI) group, usually infecting apple and pear trees, and the Rubus-infecting (RI) group. Most E. amylovora strains belong to the AI group, with a genetic similarity >99.7% (Mann et al., 2013). Since the pathogen displays high sequence homology among strains, high-resolution genotyping methods, such as clustered regularly interspaced short palindromic repeats (CRISPR), variable number of tandem repeat (VNTR), and single-nucleotide polymorphism, have been widely used in E. amylovora (Bühlmann et al., 2014; McGhee and Sundin, 2012; Parcey et al., 2020; Rezzonico et al., 2011; Smits et al., 2017; Tafifet et al., 2020). Recently, large chromosomal inversions and amino acid repeats in RsxC were used for typing, providing new insights into the genetic diversity of E. amylovora (Ham and Park, 2023; Yang et al., 2023). As a result, the AI group has been divided into three clades: the Widely-Prevalent clade, which comprises isolates from various countries; the Eastern N. A. clade; and the Western N. A. clade. The RI group is genetically distinct from the AI group and exhibits distinct protein profiles, and the B-group exhibits limited sequence identity to the AI and RI groups (Albanese et al., 2022; Parcey et al., 2020; Singh and Khan, 2019).
However, no studies compared and analyzed the genotype and distribution patterns of E. amylovora strains isolated from Korea by year and region. All the E. amylovora strains isolated in Korea displayed a 2-22-38 type of CRISPR spacer pattern (Song et al., 2021) and three amino acid repeats in RsxC (Ham and Park, 2023), suggesting that the used genotyping methods are insufficient to discriminate Korean isolates. Therefore, we determined the genotype of E. amylovora isolates using multilocus variable number of tandem repeat analysis (MLVA) to investigate the genetic diversity of E. amylovora in Korea. This method uses VNTRs in the bacterial genome, which efficiently discriminate homogenous species and have been used to determine the population structure of E. amylovora isolates from various countries (Bühlmann et al., 2014). Additionally, the differentiation of each E. amylovora genotype was determined by a minimal spanning tree, and the distribution pattern was confirmed by constructing genotype distribution maps. These results can be used to investigate the genetic diversity and spreading patterns of E. amylovora in Korea.
Materials and Methods
E. amylovora isolates and DNA extraction
We collected and selected 391 E. amylovora isolates from 2019 to 2023 and sorted them by year and region to study E. amylovora genetic diversity (Supplementary Table 1). Selected isolates were streaked onto tryptic soy agar (Difco, Sparks, MD, USA) and incubated at 27°C for 48 h before DNA extraction. Genomic DNA (gDNA) was extracted using the Wizard gDNA purification kit (Promega, Madison, WI, USA), following the manufacturer’s instructions. The concentration of the extracted gDNA was quantified by spectrophotometer (Hidex F1/Sense, Turku, Finland) and adjusted at 25 ng/μl.
Multiplex PCR for VNTR amplification
Multiplex PCR amplification and fragment analysis were conducted using the methods of Bühlmann et al. (2014) with slight modifications to analyze MLVA patterns. Tandem repeats from loci C, D, F, and H, located in the E. amylovora chromosome, were used for the analysis. Each primer was tagged with FAM, NED, VIC, and PET at the 5′ site (Supplementary Table 2). The reaction was conducted with 25 ng of template DNA and 10 mM of each primer, for a final volume of 20 μl, using the AccuPower Multiplex PCR PreMix (Bioneer, Daejeon, Korea). The multiplex PCR conditions included a pre-denaturation at 95°C for 15 min, 30 cycles at 95°C for 30 s, 56°C for 90 s, and 72°C for 4 min, and a final extension at 72°C for 30 min. The amplicons’ sequencing and capillary electrophoresis were conducted by Macrogen Corporation (Seoul, Korea).
Analysis of VNTR locus patterns
For the VNTR pattern analysis of E. amylovora, the MLVA information on the pathogens from the USA was acquired from Table S5 of Bühlmann et al. (2014). The MLVA patterns of 391 Korean isolates were estimated by the number of tandem repeats in each locus according to the amplicons’ size acquired by fragment analysis. Additionally, the sequence information for the MLVA of six strains (TS3128, TS3238, FB20, F207, FB86, and FB307) isolated in 2015 was acquired from NCBI GenBank. The allele frequency analysis of E. amylovora by nation and Korean regions was conducted using GenAlEx (v6.503) (Peakall and Smouse, 2006, 2012). The haploid diversity was calculated using the formula, 1 − ∑pi2 (p = allele frequency). After organizing the number of repetitions for each locus, 32 repetition patterns were identified from the 397 Korean isolates and named RV1–RV32 (Supplementary Table 3).
Production of a genotype distribution map
The GPS locations of each E. amylovora isolate’s isolation site were collected. Each isolate was sorted by MLVA genotype and isolation year. The distribution maps of E. amylovora isolates by genotype were produced using QGIS (v3.8). Vector maps of the Korean regions were downloaded from the National Geographic Information Service (https://map.ngii.go.kr/).
Phylogenetic analysis by minimal spanning tree
The sequences of VNTR C, D, F, and H loci of each E. amylovora strain were aligned and concatenated using Clustal W of the Mega-X program (v10.0.5). The goBURST distance was used to create a minimal spanning tree (Feil et al., 2004) by entering the alignment file and isolate information for each genotype into Phyloviz (v2.0).
Results
VNTR patterns of E. amylovora isolated from Korea
The VNTR patterns of Korean E. amylovora isolates in loci C, D, F, and H were analyzed. On locus C, the repeat motif “AACAAT” exhibited nine different repeat types, ranging from 8 to 17 repeats, depending on the strain. The repeat motif “TGCCAA” on the locus D had ten different repeat types, ranging from 6 to 16, and the repeat motif “RGCAGCGTARGYGYYMGT” on the locus F had two different repeat types with five or six repeats among the E. amylovora strains. The repeat motif “ATATCACGC” on locus H had seven different repeat types ranging from 5 to 12 (Table 1). The dominant repeat type comprised 12 repetitions on locus C, 13 on locus D, 6 on locus F, and 9 on locus H. The haploid diversity was 0.337, 0.149, 0.020, and 0.404 for loci C, D, F, and H, respectively, suggesting a high diversity of the repeat pattern of loci C and H depending on the E. amylovora isolate. In contrast, E. amylovora from the USA displayed different patterns: locus C had nine different repeat types ranging from 3 to 11, locus D had nine ranging from 4 to 15, locus F had four ranging from 5 to 8, and locus H had four ranging from 4 to 7 repetitions. Among them, the dominant type comprised six repetitions on locus C and seven repetitions on loci D, F, and H. Additionally, Korean strains accounted for most of the specific patterns, whereas American strains had multiple patterns evenly distributed with high diversity.
The VNTR patterns of Korean strains by region (n = 10 or more) indicated that various repetition patterns for each locus were found in E. amylovora isolated from Anseong-si, Cheonan-si, and Chungju-si, the main regions of fire blight emergence (Table 2). The dominant VNTR type of each region was identical in loci C, D, and F, with 12, 13, and 6 repetitions, respectively. However, regarding locus H, Chungju-si, Jecheon-si, and Andong-si mostly had ten repeats, whereas other regions had nine. Each allele frequency from isolated regions displayed high diversity, with 16 types in Anseong-si and Cheonan-si and 12 types in Chungju-si.
Diversity of E. amylovora isolated from Korea according to the MLVA genotype
The repetition number of each repeat motif at loci C, D, F, and H was compared among the 397 E. amylovora isolates, including six strains from 2015 and 391 isolates from 2019 to 2023 in Korea to characterize their MLVA patterns. Finally, the MLVA patterns were divided into 32 genotypes, and the isolates were classified from RV1 to RV32 (Table 3, Supplementary Table 3). RV19 accounted for the largest proportion with 210 isolates, followed by RV20 with 77 isolates, RV7 with 25 isolates, RV8 with 13 isolates, RV27 with 11 isolates, and RV2 with 10 isolates. RV19 had 12 repetitions on locus C, 13 on locus D, 6 on locus F, and 9 on locus H. The repetition type of RV20 was identical to RV19 except that locus H had 10 repetitions. In the case of RV7, it was identical to RV19 except that locus C had 11 repetitions. Therefore, RV19 and RV20 were dominant, accounting for more than 70%, and other RV types represented fewer than 25 strains. By host, more than 98% of the pathogens were isolated from apple and pear trees, three were isolated from hawthorn trees, one from mountain ash trees, and two from quince trees. Five out of six isolates from hawthorn, mountain ash, and quince trees showed the RV19 type, and one isolated from quince showed RV7 type, suggesting that RV19 was also dominant in these hosts.
The minimal spanning tree constructed by each RV type indicated that Korean isolates were differentiated into RV7, RV10, RV14, RV20, RV22, and RV27, all originating from RV19 (Fig. 1). Among them, RV20 was differentiated into five types: RV8, RV15, RV21, RV23, and RV28. Additionally, RV7 was differentiated into RV4 and RV6, RV22 was differentiated into RV24 and RV1, and RV27 was differentiated into RV29 and RV30. Among the RV types that originated from RV7, RV10, RV14, RV20, RV22 and RV27, only RV23, RV26, and RV32 showed differences in repeat numbers at two loci, while other RV types differed at only one locus from their original group. We assumed that RV7, RV10, RV14, RV20, RV22 and RV27, which are differentiated RV groups originated from RV19, formed a clonal complex that shares identical repeat numbers at more than two loci. Therefore, we focused on monitoring these clonal complexes which could provide insights into the overall differentiation patterns and regional distribution of E. amylovora in Korea.

Minimal spanning tree of Erwinia amylovora isolates sorted by RV types. The tree was constructed using Phyloviz (v2.0) with eBURST analysis defined by Feil et al. (2004). Isolate names and their RV type were linked as underbar, like K19-1_RV19. The isolates in green were divided into two or more RV types. The number on the line indicates the distance between the linked isolates.
E. amylovora MLVA genotype distribution
The regional distribution of RV types indicated that RV19 appeared from all the fire blight-occurring regions (Fig. 2). RV20 was discovered in Chungju-si and Jecheon-si in 2019-2020, Wonju-si, Dangjin-si, and Andong-si in 2021, Cheonan-si in 2022, and Jeongseon-gun, Cheonan-si, Anseong-si, Asan-si, Andong-si, Bonghwa-gun, and Muju-gun in 2023 (Fig. 3). Additionally, RV20 was estimated to be subdivided into RV21 (Paju-si), RV8 (Chungju-si, Wonju,-si Andong-si, and Muju-gun), RV15 (Chungju-si, Jecheon-si, and Andong-si), RV23 (Chungju-si), and RV28 (Chungju-si, Danyang-si, and Cheonan-si) and spread to each region.

Distribution of the prevalent genotype RV19 among Erwinia amylovora isolates collected from Korea. The map was generated using QGIS (v.3.8). The symbol shape represents the isolation year: triangle, 2019; circle, 2020; square, 2021; diamond, 2022; pentagon, 2023.

Distribution of RV20, RV21, RV8, RV15, RV23, and RV28 genotypes. The map was generated using QGIS (v.3.8). The symbol shape represents the isolation year: triangle, 2019; circle, 2020; square, 2021; diamond, 2022; pentagon, 2023. The symbol color represents the RV type: gray, RV8; green, RV15; blue, RV20; light green, RV21; orange, RV23; deep red, RV28.
RV27 first appeared in Jecheon-si in 2015, and then in Jecheon-si in 2019, Eumseong-gun and Yongin-si in 2021, Anseong-si and Cheonan-si in 2022, and Yanggu-gun, Pyeongtaek-si, Dangjin-si, Jeungpyeong-gun, and Chungju-si in 2023 (Fig. 4). This RV type was estimated to have differentiated and spread into RV29 (Chungju-si), RV30 (Cheonan-si), RV31, and RV32 (Chungju-si).

Distribution of RV27, RV29, RV30, and RV31 genotypes. The map was generated using QGIS (v.3.8). The symbol shape represents the isolation year: triangle, 2019; circle, 2020; square, 2021; diamond, 2022; pentagon. The symbol color represents the RV type: purple, RV27; yellow, RV29; light gray, RV30; orange, RV31.
RV7 was discovered in Anseong-si in 2019 and then appeared in Cheonan-si in 2020, Anseong-si, Dangjin-si, and Andong-si in 2021, Icheon-si, Pyeongtaek-si, Gwangju-si, Goesan-gun, Jincheon-gun, Chungju-si, and Wonju-si in 2022, and Anseong-si, Icheon-si, Asan-si, Eumseong-gun, Jincheon-gun, and Chungju-si in 2023 (Fig. 5). RV7 was estimated to have differentiated and spread into RV4 (Anseong-si, Yesan-gun), RV3 (Cheonan-si), RV2 (Yeoju-si, Icheon-si, Anseong-si, and Cheonan-si), RV6 (Pyeongtaek-si), and RV5 (Cheonan-si).

Distribution of RV7, RV4, RV3, RV2, RV6, and RV5 genotypes. The map was generated using QGIS (v.3.8). The symbol shape represents the isolation year: triangle, 2019; circle, 2020; square, 2021; diamond, 2022; pentagon, 2023. The symbol color represents the RV type: light pink, RV2; gray, RV3; green, RV4; deep purple, RV5; black, RV6; light green, RV7.
RV22 was first discovered in Cheonan-si and Jecheon-si in 2019 and then appeared in Cheonan-si and Icheon-si in 2021 and Anseong-si and Eumseong-gun in 2022 (Fig. 6). This RV type was estimated to have differentiated into RV24 and RV25 (Anseong-si) and RV1 and RV13 (Pyeongtaek-si).

Distribution of RV22, RV24, RV25, RV1, and RV13 genotypes. The map was generated using QGIS (v.3.8). The symbol shape represents the isolation year: triangle, 2019; circle, 2020; square, 2021; diamond, 2022; pentagon, 2023. The symbol color represents the RV type: light gray, RV1; gray, RV13; pink, RV22; light orange, RV24; light green, RV25.
RV14 was first discovered in Anseong-si and Cheonan-si in 2022, then in Anseong-si again in 2023 (Fig. 7). This genotype was estimated to have differentiated into RV12 (Icheon-si, Anseong-si, Pyeongtaek-si), RV11 (Cheonan-si), RV9 (Pyeongchang-gun), and RV26 (Cheonan-si).

Distribution of RV14, RV12, RV11, RV9, and RV26 genotypes. The map was generated using QGIS (v.3.8). The symbol shape represents the isolation year: triangle, 2019; circle, 2020; square, 2021; diamond, 2022; pentagon, 2023. The symbol color represents the RV type: deep purple, RV9; light pink, RV11; red, RV12; green, RV14; yellow, RV26.
The E. amylovora genotypes from Korea indicated that RV19 was first discovered in Anseong-si and spread to various parts of the country. RV20 differentiated from RV19 and into RV8, RV15, and RV23 in Chungju-si and Jecheon-si and spread to Gangwon, Gyeongbuk, Chungnam, Gyeonggi, and Jeonbuk provinces. Additionally, RV7 differentiated from RV19, spread from Anseong-si and Cheonan-si to the adjacent regions in Gyeonggi, Chungnam, Gangwon, and Chungbuk provinces, and differentiated into RV2, RV3, RV4, RV5, and RV6.
Discussion
Fire blight is a very destructive plant disease that can kill the entire tree by systemic infection (Billing, 2011). Therefore, many countries growing apple or pear trees in temperate climates are making efforts to prevent the outbreak of fire blight. Particularly, the European and Mediterranean Plant Protection Organization placed E. amylovora on the A2 list and recommended managing it as a quarantine pathogen (European and Mediterranean Plant Protection Organization, 2022). In Korea, all the trees infected by E. amylovora must be eradicated (Park et al., 2017). Therefore, identifying the distribution of E. amylovora and tracking its movement routes is crucial to block its spread and prevent the disease outbreak.
MLVA is promising for exploring geographic diversity and understanding the dynamic evolution of E. amylovora as this method has sufficient discriminatory power for isolates from identical regional backgrounds (Alnaasan et al., 2017; Bühlmann et al., 2014; Puławska and Sobiczewski, 2012). Other techniques, such as ribotyping and repetitive element PCR, random amplified polymorphic DNA fragment analysis, and amplified fragment length polymorphism analysis, failed to differentiate the AI group of E. amylovora isolates (Alnaasan et al., 2017). In addtition, pulsed field gel electrophoresis and CRISPR had insufficient discriminatory power to distinguish the North American isolates or European strains. MLVA allowed the discrimination of isolates from worldwide origins, Mediterranean countries, and even single orchard outbreaks (Alnaasan et al., 2017; Bühlmann et al., 2014).
In this study, we determined that MLVA also differentiates Korean isolates. Four VNTRs located on the chromosome were used to analyze the genotype of E. amylovora isolated in Korea. Two more VNTR markers (loci A and B) are present on the plasmid pEA29 in E. amylovora (Bühlmann et al., 2014); however, it was challenging to compare them under the same conditions because some isolates do not possess this plasmid (Llop et al., 2006). Additionally, two tandem repeats were found on locus B in all the strains isolated from Korea (data not shown). Interestingly, when we compare the repeat number of each locus, the strains from Korea had a higher repetition number than the strains from the USA. During DNA replication, expansion or contraction can emerge in tandem repeats through slipped-strand mispairing or uneven cross-over when bacteria undergo extensive genetic variation in response to various environmental conditions (Bayliss and Palmer, 2012; Levinson and Gutman, 1987). The pathogen may have evolved to increase the number of tandem repeats as it adapted to the Korean environment.
The allele frequencies of loci C, D, F, and H sorted by isolated regions, MLVA genotype frequencies, and the minimal spanning tree of Korean E. amylovora isolates described in this study indicated that bacterial isolates from Anseong-si and Cheonan-si have high genetic diversity. These regions are where fire blight first emerged in Korea and exhibit high frequencies of fire blight emergence (Ham et al., 2024; Myung et al., 2016; Park et al., 2016). Additionally, most E. amylovora strains, such as TS3128 (Kang et al., 2021), TS3238, FB-20, FB-86, and FB-207 (Song et al., 2021) isolated in 2015, were classified as RV19. Therefore, RV19 types originating from Anseong-si and Cheonan-si would differentiate into other genotypes, including RV7, RV20, RV22, and RV27, as described in the minimal spanning tree in this study. Therefore, Korean strains were divided into genotypes originating from Anseong-si and Cheonan-si and genotypes originating from Chungju-si and Jecheon-si, the main emerging regions; they subsequently dispersed to other regions.
E. amylovora strains that emerged in Korea exhibited 32 RV types and six clonal complexes based on MLVA analyzed by four tandem repeats. The RV19 was a dominant type found in all areas where fire blight occurred, and it was subdivided into genotypes derived from Anseong/Cheonan-si and Chungju/Jecheon-si, with its differentiation type. The regional distribution of genotypes is thought to be decisive in identifying the differentiation pattern and predicting the spread route of E. amylovora. For example, MLVA showed a potential correlation with the virulence of Pseudomonas syringae pv. actinidiae and discriminated hypervirulent and low virulent strains isolated from different geographic origin into different groups (Ciarroni et al., 2015). Likewise, by linking the genotype of E. amylovora with biological characteristics, the reason for the dominance and distribution of genotypes would be elucidated. Moreover, we believe these results could contribute to establishing regional control strategies tailored to the pathogens’ characteristics, preventing the movement and spread of E. amylovora.
Notes
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
This research was supported by a Cooperative Research Program (Project No. RS-2020-RD009337) from the Rural Development Administration, Republic of Korea.
Electronic Supplementary Material
Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).