Lim Yeon-Jeong, Ham Hyeonheui, Lee Mi Hyun, Park Dong Suk, Roh Eunjung, Park Duck Hwan, Lee Yong Hwan
National Institute of Agricultural Sciences, Crop Protection Division, Wanju-gun, Jeollabuk-do, Korea (the Republic of).
Kangwon National University, 34962, Interdisciplinary Program in Smart Agriculture, Chuncheon, Gangwon-do, Korea (the Republic of);
Plant Dis. 2023 Jul 10. doi: 10.1094/PDIS-04-23-0703-PDN.
Fire blight is one of the destructive plant diseases caused by and causes enormous economic losses worldwide. Fire blight was initially reported in apples, pears, and Chinese quince (Park et al. 2016; Myung et al. 2016a, 2016b) in Korea, but recent studies have reported new hosts such as apricot (Lee et al. 2021) and mountain ash (Lim et al, 2023). These reports indicate that fire blight is likely to disperse to new hosts in Korea. During the nationwide survey in June 2021, we observed typical symptoms of blossom blight and shoot blight on a Chinese hawthorn ( Bunge) just near an orchard (37°09'21.7"N, 127°35'02.6"E) in Icheon, Gyeonggi Province, where fire blight of Asian pear occurred. For identifying its causal agent, bacterial isolates were recovered after incubating at 28 ℃ for 24 hours on tryptic soy agar (TSA) medium (BD Difco, USA) from blighted leaves and shoots that were surface sterilized with 70% alcohol for 30 sec and homogenized in 500 µl of 10mM MgCl. Pure cultures of white to mucoid colonies were grown on mannitol glutamate yeast extract (MGY) medium, a semi-selective medium for (Shrestha et al, 2003). Two isolates produced 1.5 kb amplicon through colony PCR using amsB primers (Bereswill et al. 1995). Two strains (CPFB26 and CPFB27) from the Chinese hawthorn produced amplicons identical to that from the TS3128 strain of , isolated from the pear tree and identified in 2016 (Park et al. 2016). For the partial 16s rRNA sequences, the total DNA of these two strains was extracted using the Wizard DNA prep kit (Promega, USA), and PCR was performed using fD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and Rp2 (5'-ACGGCTACCTTGTTACGACTT-3') primer sets and further sequenced (Weisburg et al. 1991). These sequences belonged to the clade and were identified as in phylogenetic analysis (GenBank accession no. OP753569 and OP753570). Based on BLASTN analysis, CPFB26 and CPFB27 showed 99.78% similarity to the sequences of the strains TS3128, CFBP 1430, and ATCC 49946. To confirm pathogenicity of the isolates, 10 ㎕ bacterial suspensions (1.5 ⅹ 10 CFU/ml) was injected through the veins of the upper 2nd leaf of 3-month-old clone of apple rootstock ( cv. M29) and incubated for six days at 28 ℃ in a chamber with 12 hours of light per day. Petioles and stems turned red hue, and the shoots finally blighted. To complete Koch's postulates, colonies were recovered on TSA medium from the inoculated apple rootstocks and verified through colony PCR for the amsB and A/B primer set (Powney et al. 2011). Hawthorn has been reported as an epidemiologically important alternate host plant of fire blight (van der Zwet et al. 2012). This study is the first to report fire blight caused by in Chinese hawthorn in Korea. Because Chinese hawthorn is natively distributed in Korea and is widely used as a landscaping tree (Jang et al. 2006), the findings of this study suggest that early monitoring could prevent the spread of fire blight through natural hosts.
火疫病是由[病原体名称未明确给出]引起的毁灭性植物病害之一,在全球范围内造成巨大经济损失。火疫病最初在韩国的苹果、梨和中国榅桲中被报道(Park等人,2016年;Myung等人,2016a,2016b),但最近的研究报告了新的寄主,如杏(Lee等人,2021年)和花楸(Lim等人,2023年)。这些报告表明火疫病在韩国可能会传播到新的寄主上。在2021年6月的全国性调查中,我们在京畿道利川市一个果园(北纬37°09'21.7",东经127°35'02.6")附近的一棵中国山楂(Crataegus pinnatifida Bunge)上观察到了典型的花腐和枝枯症状,该果园曾发生过亚洲梨火疫病。为了鉴定其病原菌,从经70%酒精表面消毒30秒并在500微升10mM氯化镁中匀浆的枯萎叶片和嫩枝上,在胰蛋白胨大豆琼脂(TSA)培养基(美国BD Difco公司)上于28℃培养24小时后分离得到细菌分离物。白色至黏液状菌落的纯培养物在甘露醇谷氨酸酵母提取物(MGY)培养基上生长,MGY培养基是[病原菌名称未明确给出]的半选择性培养基(Shrestha等人,2003年)。两个分离物通过使用amsB引物(Bereswill等人,1995年)的菌落PCR产生了1.5kb的扩增子。来自中国山楂的两个菌株(CPFB26和CPFB27)产生的扩增子与2016年从梨树分离并鉴定的[病原菌名称未明确给出]的TS3128菌株相同(Park等人,2016年)。对于部分16s rRNA序列,使用Wizard DNA制备试剂盒(美国Promega公司)提取这两个菌株的总DNA,并使用fD1(5'-AGAGTTTGATCCTGGCTCAG-3')和Rp2(5'-ACGGCTACCTTGTTACGACTT-3')引物对进行PCR并进一步测序(Weisburg等人,1991年)。这些序列属于[病原菌名称未明确给出]进化枝,在系统发育分析中被鉴定为[病原菌名称未明确给出](GenBank登录号OP753569和OP753570)。基于BLASTN分析,CPFB26和CPFB27与[病原菌名称未明确给出]菌株TS3128、CFBP 1430和ATCC 49946的序列显示出99.78%的相似性。为了确认分离物的致病性,将10微升细菌悬液(1.5×10[CFU/ml具体数值未明确])通过3个月大的苹果砧木(M29品种)克隆苗上部第二片叶的叶脉注射,并在每天光照12小时的培养箱中于28℃培养6天。叶柄和茎变成红色,嫩枝最终枯萎。为了完成科赫法则,从接种的苹果砧木上在TSA培养基上回收菌落,并通过针对amsB和A/B引物对的菌落PCR进行验证(Powney等人,2011年)。山楂已被报道为火疫病在流行病学上重要的替代寄主植物(van der Zwet等人,2012年)。本研究首次报道了韩国中国山楂上由[病原菌名称未明确给出]引起的火疫病。由于中国山楂原产于韩国且被广泛用作园林树木(Jang等人,2006年),本研究结果表明早期监测可以防止火疫病通过自然寄主传播。