title: Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection ofCylindrocladium scopariumon Eucalyptus.
abstract: Eucalyptus dieback disease, caused byCylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback ofC. scopariumprior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection ofC. scopariumbased on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific toC. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR.C. scopariumcould be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples forC. scopariumdetection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.
authors.affiliation: College of Forestry, Sichuan Agricultural University, Wenjiang, Sichuan 611130, P. R. China.
authors.forename: Tian-Min
authors.initials: TM
authors.lastname: Qiao
authors.affiliation: College of Forestry, Sichuan Agricultural University, Wenjiang, Sichuan 611130, P. R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: College of Forestry, Sichuan Agricultural University, Wenjiang, Sichuan 611130, P. R. China.
authors.forename: Shu-Jiang
authors.initials: SJ
authors.lastname: Li
authors.affiliation: College of Forestry, Sichuan Agricultural University, Wenjiang, Sichuan 611130, P. R. China.
authors.forename: Shan
authors.initials: S
authors.lastname: Han
authors.affiliation: College of Forestry, Sichuan Agricultural University, Wenjiang, Sichuan 611130, P. R. China.
authors.forename: Tian-Hui
authors.initials: TH
authors.lastname: Zhu
keywords: Cylindrocladium scoparium; Eucalyptus; loop-mediated isothermal amplification; multiplex PCR; nested PCR