Colorimetric test by loop-mediated isothermal amplification for rapid identification of Fusarium circinatum in the field

Fusarium circinatum the causal agent of pine pitch canker (PPC), is one of the most important disease that affects conifer species in nurseries and plantations worldwide. In Chile the fungus is subject to official control regulations, enforced by the Agriculture and Livestock Service (Servicio Agrícola y Ganadero, SAG) and the fungus is found mainly in nurseries or in one-year-old plantations due to nursery infection. Identification methods for F. circinatum are time consuming, labor intensive and require, qualified personnel, laboratory equipment and special facilities. There is no user-friendly diagnostic method that is applicable in the field and that delivers results quickly and efficiently. In this study, a rapid and efficient colorimetric detection method based on loop-mediated isothermal amplification (LAMP) was developed to identify F. circinatum using a portable and easy-to-use method in the field. The results of the field identification can be obtained in about 60 min, by visual observation of color change from yellow (negative samples) to pink (positive samples). The assays successfully detected the pathogen in infected plants collected from inoculated plants in laboratory condition and symptomatic plants from Arauco nurseries, whereas no cross-reactions were observed with healthy plants and other Fusarium species. This method was also able to identify F. circinatum DNA extracted from mycelium and from symptomatic plants tissues which allowing to reduce time in the sample preparation process. Therefore, the colorimetric LAMP protocol developed is simple, cost-effective, fast, specific and sensitive tool for the visual identification of F. circinatum and will contribute to improve the disease management.