Lee Mi Hyun, Ji Sanghye, Ham Hyeon-Heui, Kong Gi Hyun, Park Dong Suk, Lee Yong Hwan
National Institute of Agricultural Sciences, 230986, Crop Protection Division, Wangju-gun, Jeollabuk-do, Korea (the Republic of);
National Institute of Agricultural Sciences, 230986, Crop Protection Division, Wanju, Jeollabuk-do, KR 55365, Korea (the Republic of);
Plant Dis. 2020 Oct 13. doi: 10.1094/PDIS-09-20-1973-PDN.
During a survey in May 2020, symptoms of blight were observed on apricot ( cv. undetermined) in an orchard (37°06´01.5″N 127°57´44.9″E) in Chungju, South Korea, where fire blight of apple occurred. Three apricot trees in the apple orchard were heavily diseased and showed severe shoot blight and stem canker symptoms. Bacterial isolates were recovered on King's medium B from leaves and twigs that were surface-sterilized with 70% alcohol. Colonies with uniform mucoid, smooth surfaces were collected. DNA from nine isolates did not yield an amplicon in a PCR assay for detection of using primer set CPS1/CPS2c (Kim . 2001). Each isolate was positive in PCR assays for using primer sets A/B (Bereswill . 1992) and AJ75/76 (Llop . 2000) that target pEA29. Sequencing of the PCR products resulted in 99.9% (929 bp out of 930 bp) and 100% (747 bp out of 747 bp) identity with sequences of FB20 (GeneBank: CP050240), respectively. Amplifications of the partial 16S rRNA (GeneBank: LC557153) and hrpN (GeneBank: LC575997) genes were performed, and the products were sequenced. The primers used to amplify 16S rRNA were 518F: 5'-CCAGCAGCCGCGGTAATACG-3' and 800R: 5'-TACCAGGGTATCTAATCC-3', and those for the hrpN genes were HRPN1: 5'-ATGAGTCTGAATACAAG-3' and HRPN3c: 5'-GCTTGCCAAGTGCCATA-3'. BLAST analyses showed 99.8% (1439 bp out of 1442 bp) and 100% (1136 bp out of 1136 bp) identities, respectively, to the sequences of FB20. The ability of the isolates to induce a hypersensitive reaction on tobacco ( cv. Xanthi) leaves was also evaluated. Bacterial suspensions (1.5 ⅹ 10 CFU) of 2 isolates were injected into tobacco leaves, and after 48 h, both isolates caused a hypersensitive response. To confirm pathogenicity of isolates, 3-mm-deep holes in five immature apricot (cv. Goldcot) and five immature apple (cv. Fuji) fruits were inoculated with 10 µl bacterial suspension (1.5 ⅹ 10 CFU/ml). The inoculated fruits were placed in a humid plastic box. After 7 days at 27℃, severe necrosis and bacterial ooze were present at the inoculated sites in three repeated tests. No symptoms were observed on fruits inoculated with sterile water. To complete Koch's postulates, bacteria were reisolated from the inoculated apricot and apple fruits. PCR using the specific primer sets stated above confirmed the identity as . Thus, based on disease symptoms, sequences, and pathogenicity, the bacterium causing blight of apricot was identified as . Natural infections of on apricot trees have been reported in the Czech Republic and Hungary (Korba and Sillerova 2011; Vegh and Palkovics 2013). Fire blight was observed in the Czech Republic on apricot trees near pear seedlings, which are highly susceptible to (Korba and Sillerova 2011). Natural infections of on Japanese plum planted adjacent to an apple orchard with severe fire blight has been reported in the United States (Mohan and Thomson 1996). Moreover, susceptibility to fire blight has been reported for apricot and Japanese plum cultivars (Mohan and Bijman 1999). To our knowledge, this the first report of fire blight of apricot caused by in Korea. This report is important because it provides evidence that apricot may be an overlooked reservoir for , in addition to apple, pear, and other rosaceous plants, in Korea. An intensive survey for additional host plants for the fire blight pathogen will be continued in Korea. This work was supported by a grant from the Agenda program (PJ01530202) of Rural Development Administration, Republic of Korea.
2020年5月的一项调查期间,在韩国忠州市一个果园(北纬37°06´01.5″,东经127°57´44.9″)的杏树(品种未确定)上观察到疫病症状,该果园曾发生过苹果火疫病。苹果园里的三棵杏树严重染病,呈现出严重的嫩梢疫病和茎溃疡症状。用70%酒精对叶片和嫩枝进行表面消毒后,在King氏培养基B上分离出细菌菌株。收集表面均匀、呈黏液状且光滑的菌落。在使用引物对CPS1/CPS2c(Kim等,2001年)进行的检测梨火疫病菌的PCR试验中,9个分离株的DNA未产生扩增子。在针对靶向pEA29的引物对A/B(Bereswill等,1992年)和AJ75/76(Llop等,2000年)的PCR试验中,每个分离株均呈阳性。PCR产物测序结果显示,与梨火疫病菌FB20序列(基因库:CP050240)的同一性分别为99.9%(930 bp中的929 bp)和100%(747 bp中的747 bp)。对部分16S rRNA(基因库:LC557153)和hrpN(基因库:LC575997)基因进行扩增,并对产物进行测序。用于扩增16S rRNA的引物为518F:5'-CCAGCAGCCGCGGTAATACG-3'和800R:5'-TACCAGGGTATCTAATCC-3',用于hrpN基因的引物为HRPN1:5'-ATGAGTCTGAATACAAG-3'和HRPN3c:5'-GCTTGCCAAGTGCCATA-3'。BLAST分析显示,与梨火疫病菌FB20序列的同一性分别为99.8%(1442 bp中的1439 bp)和100%(1136 bp中的1136 bp)。还评估了分离株在烟草(品种Xanthi)叶片上诱导过敏反应的能力。将2个分离株的细菌悬液(1.5×10 CFU)注射到烟草叶片中,48小时后,两个分离株均引发了过敏反应。为确认分离株的致病性,在5个未成熟杏果(品种Goldcot)和5个未成熟苹果(品种富士)上,用10 μl细菌悬液(1.5×10 CFU/ml)接种3毫米深的孔。将接种后的果实置于潮湿的塑料盒中。在27℃下放置7天后,在三次重复试验中,接种部位出现严重坏死和细菌溢出现象。用无菌水接种的果实未观察到症状。为完成柯赫氏法则验证,从接种的杏果和苹果果实中重新分离出细菌。使用上述特异性引物对进行的PCR证实其为梨火疫病菌。因此,基于病害症状、序列和致病性,将引起杏树疫病的细菌鉴定为梨火疫病菌。在捷克共和国和匈牙利已报道过杏树自然感染梨火疫病菌(Korba和Sillerova,2011年;Vegh和Palkovics,2013年)。在捷克共和国,在梨树苗附近的杏树上观察到火疫病,梨树苗对梨火疫病菌高度敏感(Korba和Sillerova,2011年)。在美国已报道过与严重火疫病苹果园相邻种植的日本李自然感染梨火疫病菌(Mohan和Thomson,1996年)。此外,已报道杏和日本李品种对火疫病敏感(Mohan和Bijman,1999年)。据我们所知,这是韩国首次关于梨火疫病菌引起杏树火疫病的报道。本报告很重要,因为它提供了证据,表明在韩国,除苹果、梨和其他蔷薇科植物外,杏可能是梨火疫病菌一个被忽视的宿主库。韩国将继续对火疫病病原菌的其他寄主植物进行深入调查。本研究得到了大韩民国农村发展管理局议程项目(PJ01530202)的资助。