Recently Fire blight was reported and eradication program has been executed in Korea. Fast diagnosis makes possible to shorten time to make decision on eradication, which is important for prevention of the disease spread. In this study, 2 diagnostic tools, antibody based EB (Erwinia blight) strip an...
Recently Fire blight was reported and eradication program has been executed in Korea. Fast diagnosis makes possible to shorten time to make decision on eradication, which is important for prevention of the disease spread. In this study, 2 diagnostic tools, antibody based EB (Erwinia blight) strip and DNA sequence based DS-LAMP, for on-site diagnosis of fire blight were developed and evaluated their specificity to Erwinia amylovora (Ea) and detection sensitivity. Also their on-site diagnosis ability were validated on the fire-blight occurred orchards. Ea AgriStrip, a commercial fire blight diagnostic kit, was used as control for EB strip. EB strip and Ea AgriStrip reacted positively to the all tested Korean Ea strains and negatively to all Pusedomonas syringae pv. syringae (Pss) strains that cause shoot blight on apple. Their specificity to Erwinia pyrifoliae (Ep) causing black shoot blight on apple and pear was difference depending on strains of Ep. Detection sensitivity was similar between the 2 strips. For on-site diagnosis, the two strips reacted positively only to the extractions of the different fire-blighted samples on all fire blight occurred orchards except one orchard at which on-site diagnosis was carried out at winter time. In addition, they also reacted positively to the black-shoot blighted extractions from the black shoot blight occurred apple orchard. LAMP (Loop-mediated isothermal amplification) is the best tool for on-site disease diagnosis because of faster, more specific, and simpler than any other tools for detection of target pathogens. Erwinia amylovora (Ea) specific LAMP (DS-LAMP) was developed in this study. DS-LAMP is consisted of the 5 primers, F3, B3, FIP, BIP, LP designed from Ea gene, Genie III (OptiGene Limited, West Sussex, United Kingdom) master mix, and bacterial DNA or different sample extracts of host trees. DS-LAMP amplified DNA from all 8 Ea strains tested, whereas it didn’t amplify from 11 Ep strains causing black shoot blight and 8 Pss strains causing shoot blight on apple. Validation of the DS-LAMP for on-site diagnosis was carried out on the 5 fire blight-occurred orchards and 1 black shoot blight-occurred orchard. For each tested sample, Ea detection was confirmed by isolation and identification of Ea from each sample extract. DS-LAMP detected Ea from the all samples showing fire blight symptoms or bacterial ooze, while it didn’t from healthy samples and negative control on all of the 5 fire blight occurred orchards. It also detected Ea from the pathogen-inactive samples at winter season, January, 2017. On black shoot blight occurred apple orchard, it didn’t amplify DNA from various sample extracts from which Ep was isolated in the laboratory. LAMP developed by Bühlmann et al. in 2013 amplified DNA from 3 out of 11 Ep strains tested and, also, showed false positive during on-site diagnosis occasionally. These results indicate that EB strip is useful for on-site diagnosis of lesions showing fire blight and black shoot blight symptoms, and DS-LAMP is very sensitive and specific to detect Ea, and suitable for on-site diagnosis of fire blight. Using these 2 tools together on orchards, not only fire blight and black shoot blight, both are eradication diseases in Korea, can be diagnosed fast and accurate.
Recently Fire blight was reported and eradication program has been executed in Korea. Fast diagnosis makes possible to shorten time to make decision on eradication, which is important for prevention of the disease spread. In this study, 2 diagnostic tools, antibody based EB (Erwinia blight) strip and DNA sequence based DS-LAMP, for on-site diagnosis of fire blight were developed and evaluated their specificity to Erwinia amylovora (Ea) and detection sensitivity. Also their on-site diagnosis ability were validated on the fire-blight occurred orchards. Ea AgriStrip, a commercial fire blight diagnostic kit, was used as control for EB strip. EB strip and Ea AgriStrip reacted positively to the all tested Korean Ea strains and negatively to all Pusedomonas syringae pv. syringae (Pss) strains that cause shoot blight on apple. Their specificity to Erwinia pyrifoliae (Ep) causing black shoot blight on apple and pear was difference depending on strains of Ep. Detection sensitivity was similar between the 2 strips. For on-site diagnosis, the two strips reacted positively only to the extractions of the different fire-blighted samples on all fire blight occurred orchards except one orchard at which on-site diagnosis was carried out at winter time. In addition, they also reacted positively to the black-shoot blighted extractions from the black shoot blight occurred apple orchard. LAMP (Loop-mediated isothermal amplification) is the best tool for on-site disease diagnosis because of faster, more specific, and simpler than any other tools for detection of target pathogens. Erwinia amylovora (Ea) specific LAMP (DS-LAMP) was developed in this study. DS-LAMP is consisted of the 5 primers, F3, B3, FIP, BIP, LP designed from Ea gene, Genie III (OptiGene Limited, West Sussex, United Kingdom) master mix, and bacterial DNA or different sample extracts of host trees. DS-LAMP amplified DNA from all 8 Ea strains tested, whereas it didn’t amplify from 11 Ep strains causing black shoot blight and 8 Pss strains causing shoot blight on apple. Validation of the DS-LAMP for on-site diagnosis was carried out on the 5 fire blight-occurred orchards and 1 black shoot blight-occurred orchard. For each tested sample, Ea detection was confirmed by isolation and identification of Ea from each sample extract. DS-LAMP detected Ea from the all samples showing fire blight symptoms or bacterial ooze, while it didn’t from healthy samples and negative control on all of the 5 fire blight occurred orchards. It also detected Ea from the pathogen-inactive samples at winter season, January, 2017. On black shoot blight occurred apple orchard, it didn’t amplify DNA from various sample extracts from which Ep was isolated in the laboratory. LAMP developed by Bühlmann et al. in 2013 amplified DNA from 3 out of 11 Ep strains tested and, also, showed false positive during on-site diagnosis occasionally. These results indicate that EB strip is useful for on-site diagnosis of lesions showing fire blight and black shoot blight symptoms, and DS-LAMP is very sensitive and specific to detect Ea, and suitable for on-site diagnosis of fire blight. Using these 2 tools together on orchards, not only fire blight and black shoot blight, both are eradication diseases in Korea, can be diagnosed fast and accurate.
주제어
#현장진단 LAMP 스트립 Erwinia amylovora
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