Development of Molecular Detection Technology and Quarantine Applications of Cherry Blossom Tree Witch''s Broom Disease

Witch’s broom disease of cherry blossom tree, commonly known as Tengu nest disease in Japan, is caused by the ascomycetous fungus Taphrina wiesneri (Rathay) Mix. With typical symptoms including swollen and hyperplasic branches, stubby and deformed leaves, and reduced buds, the disease poses big threat to tourism industry. Recently, the disease infection in Japan has increased alarmingly. Trying to prevent the spread of disease, Komatsu et al (2010) designed a specific primer pair TwITSF/TwITSR (amplicon 123 bp) for PCR detection of T. wiesneri in host plants. In recent years, the symptoms of witch’s broom disease of cherry blossom tree had been observed in some mountain areas in Taiwan. In this study, we first detected the existence of T. wiesneri in symptomatic cherry trees using the TwITSF/TwITSR primers. We successfully isolated and pure-cultured the pathogen, which then confirmed to be T. wiesneri based on its morphological characteristics and ITS sequences. To test the pathogenicity, we inoculated the plants by spraying with sporidial suspension, stabbing with sporidia, and grafting with diseased scions. Combined with our newly-developed detection techniques, we provided evidence of the proliferation and migration of our T. wiesneri isolates in host branches. The detection techniques developed in this study include the conventional PCR (primer pair Tw-F/Tw-R, amplicon 326 bp), the quantitative real-time PCR (qPCR, primer pair TWrt-F/TWrt-R, amplicon 80 bp), and the loop-mediated isothermal amplification (LAMP, primer set 2) techniques. The sensitivity tests showed that the detection limits for PCR, qPCR, and LAMP can reach 1.5 pg/ul, 15 fg/ul, and 15 pg/ul, respectively. The specificity tests using Botryosphearia dothidea, a common parasite in cherry blossom trees, indicated that conventional PCR with Tw-F/Tw-R has the highest specificity. False-positive signals can appear during the final cycles of the LAMP and qPCR reactions, suggesting that the reaction time should not be set for too long. In addition, we applied the PCR and qPCR techniques for cherry witch’s broom disease detection in field investigation. It was found that in Alishan of southern Taiwan, T. wiesneri was detected in 50% adult trees and 33.3% seedlings in the samples collected in May 2012. In May 2013, Taiwan Forestry Research Institute randomly tested 50 cherry blossom trees and found that 100% of them were infected. However, in Oct 2013, the infection rate has decreased to 14%, which might be due to the effective disease control by applying fungicides in June 2013. In CingJing mountain area of central Taiwan, T. wiesneri was clearly detected in all the symptomatic host tissues; minor infection was also found in some symptomless parts nearby the symptomatic tissues. The cherry blossom trees in Yangmingshan of northern Taiwan did not show any symptoms, and the detection results were all negative. The growth of T. wiesneri may have been inhibited under the relatively higher temperatures in Yanmingshan. With regard to the research and management of witch’s broom disease of cherry blossom tree, the highly-sensitive qPCR technique can be used for quarantine detection as well as the quantification of T. wiesneri in host during the latent infection period; the LAMP technique can be applied for rapid on-site screening in the field; the PCR will be suitable for annual sampling and disease monitoring. Since T. wiesneri multiplies and migrates slowly in the host, early detection and removal of infected branches would help prevent the occurrence and spread of the disease.