First Report of Canker Caused by Pectobacterium actinidiae on Asian Pear Trees (Pyrus pyrifolia) in South Korea.

In July 2020, pear trees (Pyrus pyrifolia cv. Niitaka) with cankers displaying dark-red bacterial ooze on the trunk and branches were found in two pear orchards located in Naju, Jeollanam-do, South Korea (34°57'50″ N, 126°43'52″ E and 34°56'14″ N, 126°33'42″ E). The incidence was 1.5% (3 out of 200 trees) and 0.83% (1 out of 120 trees), respectively. The symptoms were similar to those of the bleeding canker caused by Dickeya fangzhongdai (Choi et al. 2021), which is typically observed in October. The bacterial ooze was suspended in sterile water and streaked in Luria-Bertani (LB) medium to isolate single bacterial colonies. Two isolates (PRI-B16 and PRI-B17) from representative diseased trees were selected for investigation. Physiological and biochemical characteristics of the isolates analyzed using the BIOLOG GEN III MicroPlate™ system (Biolog, Hayward, CA, USA) were similar to the characteristics of Pectobacterium actinidiae (Portier et al. 2019). These isolates were positively utilized stachyose, L-galactonic acid-g-lactone, guanidine hydrochloride and weakly utilized (-)-D-arabitol (Portier et al. 2019). Bacterial genomic DNA was extracted from cell cultured in 5 ml LB at 28C for 2 days using G-spin DNA extraction kit (iNtRON Biotechnology, Korea) according to the manufacturer's protocol. PCR amplification was amplified as Portier et al. (2019). The generated their sequences of the small subunit ribosomal RNA (16S rRNA) using primers 27f and 1492r (Heuer et al. 1997) (Genebank accession numbers: ON951863 and ON951864) were 99.86% and 99.76% identical, respectively, to that of P. actinidiae isolate SCPJ-1 (KY307837.1) by a BLAST search against gene bank databases. The dnaX (Genebank accession nos: ON960281 and ON960282), leuS (Genebank accession nos: ON960283 and ON960284), and recA (Genebank accession nos: ON960285 and ON960286) genes of these isolates were also amplified and sequenced by previously described Stawiak et al. (2009) for dnaX and leuS, and Waleron et al. (2002) for recA. A neighbor-joining phylogenetic analysis based on the concatenated dnaX, leuS, and recA sequences placed the two isolates in a clade containing previously identified P. actinidiae isolates. A pathogenicity test was conducted using two-year-old pear (P. pyrifolia cv. Nittaka) trees grown in a greenhouse. Wounded and unwounded pear tree branches were inoculated with 10 µL of the bacterial suspension (108 CFU/ml) or sterile water as a control. The inoculated plants were maintained at 30°C without light for 2 days under 85-90% humidity. At 7 days post-inoculation, bacterial ooze was observed on the branches inoculated with a bacterial suspension, whereas branches subjected to unwounded inoculation and water inoculation exhibited no symptoms. This assay was performed three times. We reisolated two colonies from each sample showing typical bleeding symptoms and confirmed their identity by sequencing the dnaX locus. Pectobacterium actinidiae has been reported to cause canker in pear trees in Brazil (Araujo et al. 2021) as well as kiwifruit in South Korea (Koh et al. 2012). This is the first report of P. actinidiae causing canker on pear trees in South Korea and is, therefore, pathologically significant.

First Report of Bleeding Canker of Pear Tree Trunks Caused by Dickeya fangzhongdai in Korea.

Pears (Pyrus pylifolia L.) are cultivated nationwide as one of the most economically important fruit trees in Korea. At the end of October 2019, bleeding canker was observed in a pear orchard located in Naju, Jeonnam Province (34°53'50.54″ N, 126°39'00.32″ E). The canker was observed on trunks and branches of two 25-year-old trees, and the diseased trunks and branches displayed partial die-back or complete death. When the bark was peeled off from the diseased trunks or branches, brown spots or red streaks were found in the trees. Bacterial ooze showed a rusty color and the lesion was sap-filled with a yeasty smell. Trunks displaying bleeding symptoms were collected from two trees. Infected bark tissues (3 × 3 mm) from the samples were immersed in 70% ethanol for 1 minute, rinsed three times in sterilized water, ground to fine powder using a mortar and pestle, and suspended in sterilized water. After streaking each suspension on Luria-Bertani (LB) agar, the plates were incubated at 25°C without light for 2 days. Small yellow-white bacterial colonies with irregular margins were predominantly obtained from all the samples. Three representative isolates (ECM-1, ECM-2 and ECM-3) were subjected to further characterization. These isolates were cultivated at 39 C, and utilized (-)-D-arabinose, (+) melibiose, (+)raffinose, mannitol and myo-inositol but not 5-keto-D-gluconate, -gentiobiose, or casein. These isolates were identified as Dickeya sp. based on the sequence of 16S rRNA (MT820458-820460) gene amplified using primers 27f and 1492r (Heuer et al. 2000). The 16S rRNA sequences matched with D. fangzhongdai strain ND14b (99.93%; CP009460.1) and D. fangzhongdai strain PA1(99.86%; CP020872.1). The recA, fusA, gapA, purA, rplB, and dnaX genes and the intergenic spacer (IGS) regions were also sequenced as described in Van der wolf et al. (2014). The recA (MT820437-820439), fusA (MT820440-820442), gapA (MT820443-820445), purA (MT820446-820448), rplB (MT820449-820451), dnaX (MT820452-820454) and IGS (MT820455-820457) sequences matched with D. fangzhongdai strains JS5, LN1 and QZH3 (KT992693-992695, KT992697-992699, KT992701-992703, KT992705-992707, KT992709-992711, KT992713-992715, and KT992717-992719, respectively). A neighbor-joining phylogenetic analysis based on the concatenated recA, fusA, gapA, purA, rplB, dnaX and IGS sequences placed the representative isolates within a clade comprising D. fangzhongdai. ECM-1 to 3 were grouped into a clade with one strain isolated from waterfall, D. fangzhongdai ND14b from Malaysia. Pathogenicity test was performed using isolate ECM-1. Three two-year-old branches and flower buds on 10-year-old pear tree (cv. Nittaka), grown at the National Institute of Horticultural and Herbal Science Pear Research Institute (Naju, Jeonnam Province in Korea), were inoculated with 10 µl and 2 µl of a bacterial suspension (108 cfu/ml), respectively, after wounding inoculation site with a sterile scalpel (for branch) or injecting with syringe (for flower bud). Control plants were inoculated with water. Inoculated branches and buds in a plastic bag were placed in a 30℃ incubator without light for 2 days (Chen et al. 2020). Both colorless and transparent bacterial ooze and typical bleeding canker were observed on both branches and buds at 3 and 2 weeks post inoculation, respectively. No symptoms were observed on control branches and buds. This pathogenicity assay was conducted three times. We reisolated three colonies from samples displaying the typical symptoms and checked the identity of one by sequencing the dnaX locus. Dickeya fangzhongdai has been reported to cause bleeding canker on pears in China (Tian et al. 2016; Chen et al. 2020). This study will contribute to facilitate identification and control strategies of this disease in Korea. This is the first report of D. fangzhongdai causing bleeding canker on pears in Korea.

Development of Carrot Medium Suitable for Conidia Production of Venturia nashicola.

The causal fungus of pear scab, Venturia nashicola, grows slowly and rarely produces conidia on artificial media in the laboratory, but it produced conidia on the Cheongah medium containing Cheongah powder. V. nashicola grew too slow to produce conidia until 15 days after cultivation but produced conidia with 4 × 104 conidia/plate 30 days after cultivation on the Cheongah medium containing 1% Cheongah powder. V. nashicola showed a peak production of conidia with 4.5 × 105 conidia/plate 60 days after cultivation on the carrot medium containing 2% carrot powder, one of the constituents of Cheongah powder. The carrot medium is considered to be the best medium to obtain conidia of V. nashicola in the laboratory until now. This is the first report on the development of a suitable medium for conidia production of V. nashicola, as far as we know.

Specific and Sensitive Detection of Venturia nashicola, the Scab Fungus of Asian Pears, by Nested PCR.

The fungus Venturia nashicola is the causal agent of scab on Asian pears. For the rapid and reliable identification as well as sensitive detection of V. nashicola, a PCR-based technique was developed. DNA fingerprints of three closely related species, V. nashicola, V. pirina, and V. inaequalis, were obtained by random amplified polymorphic DNA (RAPD) analysis. Two RAPD markers specific to V. nashicola were identified by PCR, after which two pairs of sequence characterized amplified region (SCAR) primers were designed from the nucleotide sequences of the markers. The SCAR primer pairs, designated as D12F/D12R and E11F/E11R, amplified 535-bp and 525-bp DNA fragments, respectively, only from genomic DNA of V. nashicola. The specificity of the primer sets was tested on strains representing three species of Venturia and 20 fungal plant pathogens. The nested PCR primer pair specific to V. nashicola was developed based on the sequence of the species-specific 525-bp DNA fragment amplified by primer set E11F/E11R. The internal primer pair Na11F/Na11R amplified a 235-bp fragment from V. nashicola, but not from any other fungal species tested. The nested PCR assay was sensitive enough to detect the specific fragment in 50 fg of V. nashicola DNA.