Discrimination and Detection of <i>Erwinia amylovora</i> and <i>Erwinia pyrifoliae</i> with a Single Primer Set

Hyeonheui Ham; Kyongnim Kim; Suin Yang; Hyun Gi Kong; Mi-Hyun Lee; Yong Ju Jin; Dong Suk Park

doi:10.5423/PPJ.OA.03.2022.0027

Plant Pathol J > Volume 38(3); 2022 > Article

Ham, Kim, Yang, Kong, Lee, Jin, and Park: Discrimination and Detection of Erwinia amylovora and Erwinia pyrifoliae with a Single Primer Set

Research Article

The Plant Pathology Journal 2022;38(3):194-202.

Published online: June 1, 2022

DOI: https://doi.org/10.5423/PPJ.OA.03.2022.0027

Discrimination and Detection of Erwinia amylovora and Erwinia pyrifoliae with a Single Primer Set

Hyeonheui Ham, Kyongnim Kim, Suin Yang, Hyun Gi Kong, Mi-Hyun Lee, Yong Ju Jin, Dong Suk Park^*

Crop Protection Division, Department of Agro-food Safety and Crop Protection, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Korea

^*Corresponding author. Phone) +82-63-238-3310, FAX) +82-63-238-3838, E-mail) dspark@rda.go.kr

Handling Editor: Kihyuck Choi

(Received on March 3, 2022; Revised on April 7, 2022; Accepted on April 13, 2022)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Erwinia amylovora and Erwinia pyrifoliae cause fire blight and black-shoot blight, respectively, in apples and pears. E. pyrifoliae is less pathogenic and has a narrower host range than that of E. amylovora. Fire blight and black-shoot blight exhibit similar symptoms, making it difficult to distinguish one bacterial disease from the other. Molecular tools that differentiate fire blight from black-shoot blight could guide in the implementation of appropriate management strategies to control both diseases. In this study, a primer set was developed to detect and distinguish E. amylovora from E. pyrifoliae by conventional polymerase chain reaction (PCR). The primers produced amplicons of different sizes that were specific to each bacterial species. PCR products from E. amylovora and E. pyrifoliae cells at concentrations of 10⁴ cfu/ml and 10⁷ cfu/ml, respectively, were amplified, which demonstrated sufficient primer detection sensitivity. This primer set provides a simple molecular tool to distinguish between two types of bacterial diseases with similar symptoms.

Keywords: detection, discrimination, Erwinia amylovora, Erwinia pyrifoliae, polymerase chain reaction

Fire blight is a bacterial disease of plants in the Rosaceae family caused by Erwinia amylovora. The disease was first reported in New York in 1793 and has since spread to North America, Europe, Africa, Oceania, and Asia. Fire blight adversely impacts apple and pear production worldwide (Bonn and van der Zwet, 2000; Denning, 1794; Drenova et al., 2012; Fatmi et al., 2008; Park et al., 2016). E. amylovora infects the entire tree and young branches by moving systemically through the xylem after entering wounds and natural openings. Symptoms of fire blight are manifested as blights and necrotic lesions in flowers, leaves, and young stems. E. amylovora overwinters in cankers. When the temperature rises the following year, bacterial ooze forms, and the pathogen moves to other trees by rain, wind, insects, and other dispersal agents (Billing, 2011; Thomson, 2000).

Black-shoot blight is another bacterial disease in apples and pears. The disease is caused by Erwinia pyrifoliae. E. pyrifoliae was first reported in pear trees of South Korea in 1995 (Kim et al., 1999) and was also isolated from strawberries in the Netherlands in 2014 (Wenneker and Bergsma-Vlami, 2015). Virulence of E. pyrifoliae is lower than that of E. amylovora, and its host range is, for the most part, limited to pear and some varieties of apple (Kim et al., 2001). The trees infected by E. pyrifoliae show blackened blight and wilt in leaves and shoots, viscous exudates on the surface, and necrosis of the leaves, flowers, and immature fruits (Lee et al., 2020; Rhim et al., 1999).

Visually distinguishing the disease caused by E. amylovora from that caused by E. pyrifoliae is challenging because of their very similar symptoms of the infected trees (Kim et al., 2001). These two pathogens appeared in the South Korean provinces of Gangwon and Gyeonggi (Ham et al., 2020; Kim et al., 1999; Lee et al., 2020). Therefore, molecular detection methods provide valuable tools for distinguishing fire blight from black-shoot blight.

E. amylovora is usually detected by specific regions in the plasmid of pEA29 and chromosome by polymerase chain reaction (PCR). The pEA29 is indigenous and related to virulence in E. amylovora (Llop et al., 2006). It has been widely used because of its high sensitivity since it has more than one copy in the genome (Bereswill et al., 1995; Obradovic et al., 2007). But after the discovering the E. amylovora strains which do not possess the pEA29 (Llop et al., 2006), the primer designed from their chromosome has been widely used to detect E. amylovora strain. In particular, the primers targeting ams genes involved in exopolysaccharide (EPS) production were designed (Bereswill et al., 1995; Jones and Geider, 2001; Mohammadi et al., 2009), but the false-positive response was found from other bacteria (Powney et al., 2011).

E. pyrifoliae has minor occurrences except for Korea, so there is little information, and the developed primers are limited. The EP16A/EPIG2C primer amplifying the 16S rRNA and internal transcribed spacer region, and the CPS1/2C primer amplifying the cpsB and cpsC region, which encode EPS, were developed (Kim et al., 2001). But these primers were not verified to detect E. tasmaninasis and other closely related species that discovered later than at that time. In addition, the primers detecting both pathogens at once are not developed yet.

Here, comparative genomics was used to design a primer set from a single-copy gene to amplify specific DNA sequences from E. amylovora. This primer set also amplified E. pyrifoliae DNA but produced an amplicon that had a different size than that of E. amylovora. Sequencing the E. pyrifoliae amplicon confirmed the E. pyrifoliae genome binding site. In addition, testing the primers on genomic DNA (gDNA), bacterial suspension cells, and plant extracts revealed detection specificity and sensitivity of the primers. Therefore, this primer set is a valuable addition to molecular tools for the rapid and economical detection of E. amylovora and E. pyrifoliae. The primer set developed in this study is unique in that it can detect and distinguish one bacterial pathogen from the other with a single PCR.

Materials and Methods

Bacterial strains and cultural condition

Twenty-three bacterial species were collected for primer specificity assays (Table 1). Bacterial species from E. amylovora TS3128 and E. pyrifoliae YKB12327 were isolated from diseased pear or apple lesions in 2015. Other bacterial isolates were obtained from the Korean Agricultural Culture Collection (KACC, South Korea) or the Belgian Coordinated Collections of Microorganisms (BCCM). Each bacterial species was cultured in tryptic soy agar (TSA) at 27°C for 48 h, and single colonies were used for the assays.

Total genomic DNA extraction

To extract gDNA from bacteria, single colonies were inoculated on tryptic soy broth and incubated overnight at 27°C under shaking conditions at 150 rpm. Bacterial cultures were pelleted, and gDNA was extracted with the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). DNA concentration was measured with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) before performing conventional PCR.

Primer and PCR amplification

Specific primers were generated from the downloaded E. amylovora CFBP1430 genome sequence (GenBank accession no. GCA_000091565.1) and other closely related species from the National Center for Biotechnology Information (NCBI) GeneBank. The specific genes and primers were selected and designed from the E. amylovora CFBP1430 genome using the genome-wide comparison pipeline as previously described (Lang et al., 2010) with minor modifications.

For PCR amplification, gDNA template (1 μl), 10 mM of each forward and reverse primer, 10× premix (Cellsafe, Yongin, Korea), 1.25 unit of Taq polymerase, and 0.2 mM of dNTPs were combined to produce a 25 μl reaction mixture. PCR was performed with a C1000 Thermal Cycler PCR system (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: pre-denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. For gel electrophoresis, 5 μl of each PCR product was mixed with 1 μl of DNA loading star (Dyne Bio, Seongnam, Korea) and then loaded onto a 1% agarose gel. The gel was visualized with a UV transmission illuminator after electrophoresis.

Configuration of the primer binding site of E. pyrifoliae

To identify the RS24580-205 primer binding site on the E. pyrifoliae genome, the 705 bp amplicon from the E. pyrifoliae template was cloned to the pGEM-T Easy Vector (Promega) and transformed to Escherichia coli DH5α competent cells. The resulting plasmid DNA was purified with GeneAll Hybrid-Q Plasmid Rapidprep (GeneAll Biotechnology, Seoul, Korea) and sequenced (Bionics, Daejeon, Korea) with T7 and SP6 universal primers. The primer binding site was determined by comparing the insertion sequences of the vector with that of the E. pyrifoliae DSM12163 genome sequence (GenBank accession no. GCA_000026985.1).

Specificity and sensitivity assays

The gDNA of bacterial strains listed in Table 1 was used to test the specificity of the RS24580-205 primer set. DNA concentration was adjusted to 25 μg/μl, and 1 μl was used as a template. To test primer sensitivity, E. amylovora and E. pyrifoliae gDNA templates were serially diluted by 10-fold (i.e., from 5 ng/μl to 5 fg/μl). Primer sensitivity was also tested on bacterial cell suspension. Overnight cultures E. amylovora and E. pyrifoliae cells were adjusted to an optical density (OD) of 1.0 at 600 nm with a NanoDrop spectrophotometer and serially diluted by 10-fold. Cell suspensions (100 μl) were spread on TSA plates to determine E. amylovora and E. pyrifoliae cell density. Cell density of E. amylovora at an OD of 1.0 was 2.55 × 10⁹ cfu/ml, while that of E. pyrifoliae was 1.25 × 10⁹ cfu/ml. Diluted gDNA (1 μl) and cell suspensions were used for PCR. The detection limits were determined by examining the amplified target bands.

Detection of E. amylovora and E. pyrifoliae on inoculated apple shoots

Shoot segments (15-20 cm long) were collected from branches of 2-year-old apple trees (cultivar Fuji) and surface sterilized with 70% ethanol. Shoots were put into water-soaked floral foam (Oasis, Smithers-Oasis Korea, Cheonan, Korea) and inoculated with 10 μl E. amylovora or E. pyrifoliae cell suspensions adjusted to an OD₆₀₀ of 0.1 (10⁸ cfu/ml) with 200 μl sterilized tips (TipOne UltraPoint Graduated Tip, Starlab Ltd., Hamburg, Germany). Inoculated shoots were incubated in a growth chamber maintaining 27°C and 100% relative humidity, and disease symptoms were observed after 7 days. After sterilizing the surface of the shoots with 70% ethanol, lesions were cut into 5 × 5 mm pieces. Five lesions were ground in 1 ml sterile distilled water, and 200 μl of suspension was used to extract total DNA using the taco plant DNA/RNA extraction kit (GeneReach Biotechnology Corp., Taichung City, Taiwan) with nucleic acid automatic extraction system (taco, GeneReach Biotechnology Corp.).

Results and Discussion

Primer design, PCR detection, and specificity test

From the NCBI GenBank FTP site (ftp://ftp.ncbi.nlm.nih.gov/genomes/all/), we identified unique chromosome loci of E. amylovora against the loci of all available Erwinia genomes using a BLASTN (E = 1e-1) search. By the Primer3 module (Rozen and Skalestsky, 2000), primers producing 95-310 bp of amplicons were designed from the unique E. amylovora loci. Using reverse-ePCR (n = 2, g = 1) (Schuler, 1997), predicted primer pairs were confirmed against all Erwinia genomes. Primers only for targeting the E. amylovora genome were selected. Finally, the forward primer, RS24580-205F (5′-CAC TGC GCC TGT TGT TCA-3′), and reverse primer, 205R (5′-ATG TAT CTG GTA GCC GGG TAA GTT-3′), were selected which encodes a hypothetical protein (WP_162010770.1) from the E. amylovora genome (Fig. 1). We also found that the RS24580-205 primers amplified not only 205 bp of PCR product from E. amylovora genome but also 751 bp from E. pyrifoliae (Table 1, Fig. 2, lanes 1-10). Other closely related bacterial species, such as Pectobacterium, Dickeya, and Pantoea sp., which were previously classified as Erwinia species, and five strains belonging to the Erwinia genus, were used to test the specificity of the RS24580-205 primers (Kado, 2006; Samson et al., 2005) (Table 1). No PCR products were amplified from other bacterial species with the RS24580-205 primer set (Table 1, Fig. 2, lanes 11-23).

In contrast to the RS24580-205 primers developed in this study, it was reported that A/B primers, which were designed to specifically amplify the sequences in the E. amylovora pEA29 plasmid at the amplicon size between 900 bp, produced non-specific bands by PCR. The detection of Erwinia herbicola (Pantoea agglomerans) and Erwinia carotovora sub-species atroseptica (Pectobacterium atrosepticum) by A/B primers did not produce 900 bp of amplicon but shorter than or above the 900 bp (Bereswill et al., 1992).

In conclusion, these results showed that in addition to the high specificity of RS24580-205 primers for E. amylovora and E. pyrifoliae, the generation of a different amplicon size specific for each bacterial species made it possible to distinguish one pathogen from the other using a single primer set (Fig. 2).

Amplification regions by RS24580-205F/R primer in E. pyrifoliae

The forward primer, RS24580-205F, bound to a ‘metal-dependent hydrolase (WP_012667018.1)’ sequence of the E. pyrifoliae DSM12163 genome, while the reverse primer, RS24580-205R, bound to a gene encoding a ‘patatin-like phospholipase family protein (WP_012667017.1)’ (Fig. 3). Patatin-like proteins are the storage proteins and have lipolytic enzyme activity that related to the plant-pathogen interactions (Banerji and Flieger, 2004). For the 18-mer RS24580-205F primer, 14-mer at the 3′ end was complementary to the target template, while 3-mer at the 5′ end was mismatched. In the 24-mer RS24580-205R primer, 12-mer at the 3′ end was complementary to the target template, while 4-mer at the 5′ end was mismatched. The binding features of the RS24580-205 primer set to the E. pyrifoliae templates resulted in a 751 bp amplicon. Although each primer did not bind perfectly to the E. pyrifoliae genome sequence, more than 12-mer at the 3′ end was complementary, which was sufficient to generate a PCR product.

In general, mismatched bps at the 3′ end of the primer have a more adverse effect on PCR amplification than those at the 5′ end. If there are several bp mismatches at the 5′ end or the middle of the primer, target amplification can still occur, but efficiency is reduced (Ye et al., 2012). Also, 3-bp mismatches at the 5′ end of the forward primer cannot amplify the target template when its concentration is low (Sipos et al., 2007). The efficiency of RS24580-205F and RS24580-205R primers was reduced because of 4-mer and 8-mer bp mismatches, respectively, at the 5′ end. However, the 12-mer match at the 3′ end of the RS24580-205 primer set still allowed template amplification. The binding regions of the primers to E. pyrifoliae templates were compared to those of E. amylovora, E. billingiae, E. rhapontici, E. tasmaniensis, P. agglomerans, and P. dispersa (Supplementary Fig. 1). The binding sequence of the metal-dependent hydrolase gene to the RS24580-205F primer was the same for E. pyrifoliae, E. amylovora, and E. tasmaniensis. However, the binding sequence of the patatin-like phospholipase family protein gene to the RS24580-205R primer was unique to the E. pyrifoliae template, which led to the 751 bp amplicon. The ability of the RS24580-205 primer set to simultaneously detect E. amylovora and E. pyrifoliae was tested by mixing the gDNA or bacterial cells for a 1:1 ratio from both pathogens. Only the 205 bp amplicon from E. amylovora was identified, which indicated partial target detection of E. pyrifoliae when both pathogens were infected together.

Sensitivity tests of the designed primers

Sensitivity tests of RS24580-205 primers showed that in E. amylovora, gDNA was detected at concentrations as low as 500 fg, while that of E. pyrifoliae was 50 pg (Fig. 4A and B). The detection limit for E. amylovora gDNA was 100 times higher than that for E. pyrifoliae. In bacterial suspensions, the RS24580-205 primer detected E. amylovora template from cells at a concentration of 10⁴ cfu/ml, which corresponded to 10 cfu/reaction or higher. On the other hand, the primer set only detected E. pyrifoliae template from cells at a concentration of 10⁷ cfu/ml, which corresponded to 10⁴ cfu/reaction (Fig. 4C and D).

There are several primers developed to detect E. amylovora DNA sequences. For example, the A/B primer alluded to earlier, which targets the E. amylovora pEA29 plasmid, can detect 1 pg of E. amylovora gDNA or 1.9 × 10³ cfu/ml of bacterial cells (Bereswill et al., 1992; Ivanović et al., 2019). The G1/G2 primers (also called pEA71 primers), which target an E. amylovora chromosomal sequence, have a detection limit of 10 cfu/reaction (Taylor et al., 2001). Another E. amylovora chromosome-targeted primer set, FER1/R, reproducibly shows a detection limit of 3 cfu/reaction (Obradovic et al., 2007). However, another report shows that the detection limit of FER1/R for bacterial cells is as low as 1.9 × 10⁷ cfu/ml, which corresponds to 1.9 × 10⁵ cfu/reaction (Ivanović et al., 2019). The RS24580-205 primer set developed in this study showed similar or higher sensitivity for E. amylovora detection than that of other primers, except for the A/B primer set. Nonetheless, A/B primers cannot detect E. amylovora strains lacking pEA29 (Llop et al., 2006). Also, there are several copies of pEA29 in the E. amylovora genome, which increases the sensitivity of the A/B primer set (Bereswill et al., 1995; Obradovic et al., 2007). By contrast, the RS24580-205 primer was designed to target a single-copy gene. Primers that target single-copy genes are beneficial for measuring PCR efficiency. When we use 10-fold serial dilution of target DNA to draw a calibration curve from the real-time PCR, single-copy gene makes the slope of −3.322 of regression equation at 100% of PCR efficiency. This means polymerase reaction of target DNA is in the exponential rate, which enables reliable quantification results (Ginzinger, 2002; Kralik and Ricchi, 2017). Furthermore, the PCR amplicon of E. amylovora from the primer is 205 bp, suitable for applying real-time PCR. Therefore, the RS24580-205 primer set can provide a new tool for quantitative studies of E. amylovora.

There are also primers for detecting E. pyrifoliae cells. For instance, the EP16A/EPIG2C primer set detects DNA from E. pyrifoliae bacterial cells at 20 cfu/reaction, which corresponds to 2 × 10⁴ cfu/ml, while CPS1C/2R detects 200 cfu/reaction, which is about 2 × 10⁵ cfu/ml (Kim et al., 2001). Moreover, the EpERF/R primer set, which targets enterobacterial repetitive intergenic consensus sequences, has a detection limit of 50 pg for gDNA and 10⁴ cfu/ml for bacterial cells (Park et al., 2010). The sensitivity of these primers is higher than that of the RS24580-205 primer. Because they are only targeted for E. pyrifoliae, which enables more sensitive detection. Nonetheless, the fact that RS24580-205 primer set detects and discriminates E. amylovora and E. pyrifoliae at once is the greatest advantage of this primer.

Detection of pathogens from inoculated apple shoots

DNA from the lesions of apple shoots inoculated with bacterial suspensions (Fig. 5A) was amplified with the RS24580-205 primer set. The detection limit of the primers for plant extracts was 150 pg of total gDNA, which corresponded to 3.7 × 10⁵ cfu/ml in E. amylovora (Fig. 5B), and 15 ng of total g DNA, which corresponded to 1.1 × 10⁷ cfu/ml in E. pyrifoliae (Fig. 5C). The detection sensitivity of the RS24580-205 primers for DNA of plant extracts was 100 times higher in E. amylovora than in E. pyrifoliae. Also, the detection sensitivity of the primers for bacterial gDNA was 300 times higher than that for DNA from plant extracts. By contrast, A/B primers detect E. amylovora from DNA of plant extracts at 10⁵-10⁶ cfu/ml. G1/G2 and FER1-F/rgER2R detect plant DNA at 10³-10⁵ cfu/ml, while that of PEANT1/2 is 10⁴-10⁶ cfu/ml (European and Mediterranean Plant Protection Organization, 2013). On the other hand, EP16A/EPIG2c and CPS1/CPS2c primers, which target E. pyrifoliae, detect 10³ cfu/ml of bacterial cells from extracts of diseased plant materials (Kim et al., 2001). The ERIC1R/ERIC2 primer set was reported to detect E. pyrifoliae from the total DNA of plant materials, inoculated with 10⁶ cfu/ml of E. pyrifoliae (Park et al., 2010). The detection sensitivity of the RS24580-205 primer set from the E. amylovora infected plant materials showed similar levels with the previously developed primers. However, the detection of the primer set from the plant materials infected by E. pyrifoliae was less sensitive than the former studies.

In summary, the RS24580-205 primer set described here detected templates of E. amylovora and E. pyrifoliae by conventional PCR. By binding to different regions of the E. amylovora and E. pyrifoliae genome, this primer set produced amplicons of 205 bp and 751 bp for E. amylovora and E. pyrifoliae, respectively, making it possible to distinguish one pathogen from the other with a single PCR reaction. The RS24580-205 primer set detected E. amylovora with high sensitivity. However, sensitivity for E. pyrifoliae was somewhat low because of primer mismatches at the 5′ end. Despite limitations in sensitivity, the ability of the RS24580-205 primer set to distinguish E. amylovora from E. pyrifoliae based on amplicon size is a valuable addition to the suite of molecular tools to combat these bacterial diseases through early detection.

Electronic Supplementary Material

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).

PPJ-OA-03-2022-0027-suppl.pdf

Acknowledgments

This work was supported by the “Cooperative Research Program for Agriculture Science and Technology Development” (Project No. PJ01421904), Rural Development Administration, Republic of Korea.

Notes

Conflicts of interest

No potential conflict of interest relevant to this article was reported.

Fig. 1

Comparative analysis of Erwinia amylovora strains of specific binding sitez in hypothetical protein (WP_162010770.1) gene. (A) The BLASTN search of hypothetical protein gene from E. amylovora CFBP1430. (B) Comparison and alignment of primer binding sites among E. amylovora strains.

Fig. 2

Polymerase chain reaction amplification of Erwinia amylovora and Erwinia pyrifoliae with the RS24580-205 primer set. M, 1 kb size marker; lanes 1-4, 25 ng E. amylovora gDNA; lanes 5-10, E. pyrifoliae gDNA; lanes 11-23, other Erwinia, Pectobacterium, Dickeya, and Pantoea species listed in Table 1; lane −, negative control.

Fig. 3

Binding site of the RS24580-205F/R primer to the Erwinia pyrifoliae genomic DNA template.

Fig. 4

Sensitivity of the RS24580-205 primer set using serially diluted bacterial genomic DNA and suspension cells. Genomic DNA (gDNA) of Erwinia amylovora TS3128 (A) and E. pyrifoliae CP12327 (B). M, 1 kb size marker; lane 1, 5 ng gDNA; lane 2, 500 pg gDNA; lane 3, 50 pg gDNA; lane 4, 5 pg gDNA; lane 5, 500 fg gDNA; lane 6, 50 fg gDNA; lane 7, 5 fg gDNA; −, negative control. Bacterial cells of E. amylovora TS3128 (C) and E. pyrifoliae CP12327 (D). M, 1 kb size marker; lane 1, 10⁹ cfu/ml; lane 2, 10⁸ cfu/ml; lane 3, 10⁷ cfu/ml; lane 4, 10⁶ cfu/ml; lane 5, 10⁵ cfu/ml; lane 6, 10⁴ cfu/ml; lane 7, 10³ cfu/ml; lane 8, 10² cfu/ml; −, negative control.

Fig. 5

Polymerase chain reaction amplification of bacterial isolates from diseased apple leaves using the RS24580-205 primer set. (A) Pathogen-inoculated apple shoots. SDW, inoculated with sterilized distilled water; EA, Erwinia amylovora TS3128; EP, Erwinia pyrifoliae CP12327. Red arrows point to inoculated sites. (B) Genomic DNA (gDNA) of E. amylovora TS3128-inoculated diseased apple shoots. M, 1 kb size marker; lane 1, 15 ng gDNA; lane 2, 1.5 ng gDNA; lane 3, 150 pg gDNA; lane 4, 15 pg gDNA; lane 5, 1.5 pg gDNA; lane 6, 150 fg gDNA; lane 7, 15 fg gDNA. (C) gDNA of E. pyrifoliae CP12327 inoculated diseased apple shoots. M, 1 kb size marker; lane 1, 15 ng gDNA; lane 2, 10 ng gDNA; lane 3, 5 ng gDNA; lane 4, 1 ng gDNA; lane 5, 500 pg gDNA; lane 6, 50 pg gDNA.

Table 1

Bacterial strains used in this study

No.	Bacterial strains	Source	Host	Origin
1	Erwinia amylovora	TS3128	Pear	Korea
2	Erwinia amylovora	CP200930	Apple	Korea
3	Erwinia amylovora	CP201324	Apple	Korea
4	Erwinia amylovora	CP20102102	Apple	Korea
5	Erwinia pyrifoliae	YKB12327	Apple	Korea
6	Erwinia pyrifoliae	KACC 13945	N/A	Korea
7	Erwinia pyrifoliae	KACC 13946	N/A	Korea
8	Erwinia pyrifoliae	KACC 13947	N/A	Korea
9	Erwinia pyrifoliae	KACC 13948	N/A	Korea
10	Erwinia pyrifoliae	KACC 13949	N/A	Korea
11	Erwinia rhapontici	KACC 13973	N/A	Korea
12	Erwinia rhapontici	KACC 14080	N/A	Korea
13	Erwinia sp.	LMG 2466	N/A	Belgium
14	Pectobacterium carotovorum subsp. carotovorum	LMG 2404	N/A	Belgium
15	Pectobacterium carotovorum subsp. carotovorum	KACC 10225	N/A	Korea
16	Pectobacterium carotovorum subsp. odoriferum	KACC 13954	N/A	Korea
17	Pectobacterium carotovorum subsp. brasiliense	KACC 17662	Paprika	Korea
18	Pectobacterium wasabiae	KACC 10406	N/A	Korea
19	Pectobacterium atrosepticum	KACC 10463	N/A	Korea
20	Dickeya chrysanthemi	KACC 10165	Orchid (Cymbidum sp.)	Korea
21	Pantoea agglomerans	KACC 15275	Knee laceration	Korea
22	Pantoea stewartii	KACC 17663	Dracaena sanderiana	Korea
23	Pantoea ananatis	KACC 19138	Rice	Korea

KACC, Korean Agricultural Culture Collection, Republic of Korea; LMG, Belgian Coordinated Collections of Microorganisms (BCCM); N/A, unknown.

References

Banerji, S and Flieger, A 2004. Patatin-like proteins: a new family of lipolytic enzymes present in bacteria? Microbiology. 150:522-525.

Bereswill, S, Bugert, P, Bruchmüller, I and Geider, K 1995. Identification of the fire blight pathogen, Erwinia amylovora, by PCR assays with chromosomal DNA. Appl Environ Microbiol. 61:2636-2642.

Bereswill, S, Pahl, A, Bellemann, P, Zeller, W and Geider, K 1992. Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis. Appl Environ Microbiol. 58:3522-3526.

Billing, E 2011. Fire blight. Why do views on host invasion by Erwinia amylovora differ? Plant Pathol. 60:178-189.

Bonn, WG and van der Zwet, T 2000. Distribution and economic importance of fire blight. In: Fire blight: the disease and its causative agent, Erwinia amylovora, eds. by JL Vanneste, pp. 37-53. CABI Publishing, Wallingford, UK.

Denning, W 1794. On the decay of apple trees. Trans Soc Promotion Agric Arts Manufact Inst State N Y. 2:219-222.

Drenova, NV, Isin, MM, Dzhaimurzina, AA, Zharmukhamedova, GA and Aitkulov, AK 2012. Bacterial fire blight in the Republic of Kazakhstan. Plant Health Res Pract. 1:44-48.

European and Mediterranean Plant Protection Organization 2013. PM 7/20 (2) Erwinia amylovora . Bull OEPP/EPPO Bull. 43:21-45.

Fatmi, M, Bougsiba, M and Saoud, H 2008. First report of fire blight caused by Erwinia amylovora on pear, apple, and quince in Morocco. Plant Dis. 92:314.

Ginzinger, DG 2002. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol. 30:503-512.

Ham, H, Lee, Y-K, Kong, HG, Hong, SJ, Lee, KJ, Oh, G-R, Lee, M-H and Lee, YH 2020 Outbreak of fire blight of apple and Asian pear in 2015-2019 in Korea. Res Plant Dis. 26:222-228. (in Korean).

Ivanović, M, Kuzmanović, N, Gašić, K, Prokić, A, Zlatković, N and Obradović, A 2019. Specificity and sensitivity of three PCR-based methods for detection of Erwinia amylovora in pure culture and plant material. Genetika. 51:1039-1052.

Jones, AL and Geider, K 2001. Gram negative bacteria, Erwinia amylovora group. In: Laboratory guide for identification of plant pathogenic bacteria, eds. by NW Schaad, JB Jones and W Chun, pp. 40-55. APS Press, St. Paul, MN, USA.

Kado, CI 2006. Erwinia and related genera. In: The prokaryotes A handbook on the biology bacteria: proteobacteria: gamma subclass, eds. by M Dworkin, S Falkow, E Rosenberg, K-H Schleifer and E Stackebrandt, 3rd ed. pp. 443-450. Springer, New York, NY, USA.

Kralik, P and Ricchi, M 2017. A basic guide to real time PCR in microbial diagnostics: definitions, parameters, and everything. Front Microbiol. 8:108.

Kim, W-S, Gardan, L, Rhim, S-L and Geider, K 1999. Erwinia pyrifoliae sp. nov., a novel pathogen that affects Asian pear trees (Pyrus pyrifolia Nakai). Int J Syst Bacteriol. 49:899-906.

Kim, WS, Jock, S, Paulin, J-P, Rhim, S-L and Geider, K 2001. Molecular detection and differentiation of Erwinia pyrifoliae and host range analysis of the Asian pear pathogen. Plant Dis. 85:1183-1188.

Lang, JM, Hamilton, JP, Diaz, MGQ, Van Sluys, MA, Burgos, MRG, Vera Cruz, CM, Buell, CR, Tisserat, NA and Leach, JE 2010. Genomics-based diagnostic marker development for Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola . Plant Dis. 94:311-319.

Lee, GM, Ko, S, Oh, E-J, Song, Y-R, Kim, D and Oh, C-S 2020. Comparative genome analysis reveals natural variations in the genomes of Erwinia pyrifoliae, a black shoot blight pathogen in apple and pear. Plant Pathol J. 36:428-439.

Llop, P, Donat, V, Rodríguez, M, Cabrefiga, J, Ruz, L, Palomo, JL, Montesinos, E and López, MM 2006. An indigenous virulent strain of Erwinia amylovora lacking the ubiquitous plasmid pEA29. Phytopathology. 96:900-907.

Mohammadi, M, Moltmann, E, Zeller, W and Geider, K 2009. Characterisation of naturally occurring Erwinia amylovora strains lacking the common plasmid pEA29 and their detection with real-time PCR. Eur J Plant Pathol. 124:293-302.

Obradovic, D, Balaz, J and Kevresan, S 2007. Detection of Erwinia amylovora by novel chromosomal polymerase chain reaction primers. Microbiology. 76:748-756.

Park, DH, Thapa, SP, Kim, W-S, Hur, JH and Lim, CK 2010. Species-specific detection of Erwinia pyrifoliae by PCR assay using enterobacterial repetivive intergenic consensus (ERIC) primers. Plant Pathol J. 26:267-270.

Park, DH, Yea, MC, Lee, SJ, Myung, I-S, Shim, HS and Oh, C-S 2016. First report of fire blight disease on Asian pear caused by Erwinia amylovora in Korea. Plant Dis. 100:1946.

Powney, R, Beer, SV, Plummer, K, Luck, J and Rodoni, B 2011. The specificity of PCR-based protocols for detection of Erwinia amylovora . Australas Plant Pathol. 40:87-97.

Rhim, SL, Völksch, B, Gardan, L, Paulin, JP, Langlotz, C, Kim, WS and Geider, K 1999. Erwinia pyrifoliae, an Erwinia species different from Erwinia amylovora, causes a necrotic disease of Asian pear trees. Plant Pathol. 48:514-520.

Rozen, S and Skaletsky, H 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 132:365-386.

Samson, R, Legendre, JB, Christen, R, Saux, MF-L, Achouak, W and Gardan, L 2005. Transfer of Pectobacterium chrysanthemi (Burkholder et al., 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. Int J Syst Evol Microbiol. 55:1415-1427.

Schuler, GD 1997. Sequence mapping by electronic PCR. Genome Res. 7:541-550.

Sipos, R, Székely, AJ, Palatinszky, M, Révész, S, Márialigeti, K and Nikolausz, M 2007. Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis. FEMS Microbiol Ecol. 60:341-350.

Taylor, RK, Guilford, PJ, Clark, RG, Hale, CN and Forster, RLS 2001. Detection of Erwinia amylovora in plant material using novel polymerase chain reaction (PCR) primers. N Z J Crop Hortic Sci. 29:35-43.

Thomson, SV 2000. Epidemiology of fire blight. In: Fire blight: the disease and its causative agent, Erwinia amylovora , eds. by JL Vanneste, pp. 9-36. CABI Publishing, Wallingford, UK.

Wenneker, M and Bergsma-Vlami, M 2015. Erwinia pyrifoliae, a new pathogen on strawberry in the Netherlands. J Berry Res. 5:17-22.

Ye, J, Coulouris, G, Zaretskaya, I, Cutcutache, I, Rozen, S and Madden, TL 2012. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 13:134.