Loop-Mediated Isothermal Amplification (LAMP) for the Rapid and Sensitive Detection of Alternaria alternata (Fr.) Keissl in Apple Alternaria Blotch Disease with Aapg-1 Encoding the Endopolygalacturonase

Abstract

Apple Alternaria blotch disease, caused by <i>Alternaria alternata</i> (Fr.) Keissl, is one of the most famous leaf diseases. When the disease is prevalent, it causes leaf abscission and influences the formation of flower buds and photosynthesis. Therefore, a simple, rapid, high-specificity and sensitivity method for monitoring infected leaves at early developmental stages is urgently needed, so that the occurrence and expansion of <i>A. alternata</i> can be controlled in time. In our research, a rapid, specific and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect <i>A. alternata</i> within 60 min. Six primers of LAMP detection can only specifically amplify the <i>aapg-1</i> gene in <i>A. alternata</i> but not in four other important fungi in apples. The <i>aapg-1</i> gene encodes endopolygalacturonase in <i>A. alternata</i>, and there are significant differences among different species. Thus, it was applied as the target for LAMP primers. Compared to conventional PCR detection, our LAMP method had the same sensitivity as that of detecting as little as 1 fg of pure genomic DNA of <i>A. alternata</i>. When leaves were inoculated with <i>A. alternata</i> conidia, LAMP detected 1 × 10² conidia/mL as the minimum concentration. However, the traditional tissue isolation and identification method only isolated <i>A. alternata</i> from leaves inoculated with 1 × 10⁵ and 1 × 10⁶ conidia/mL, indicating that the LAMP method was more sensitive than the traditional tissue isolation and identification method for <i>A. alternata</i> before symptoms. Further tests also indicated that LAMP detection was more accurate and sensitive than the traditional tissue isolation and identification method for <i>A. alternata</i> in leaves with the Alternaria blotch symptom collected from the field. Our results showed that the LAMP-targeting the <i>aapg-1</i> gene has the advantages of high sensitivity, specificity and simplicity and can be used for rapid detection and early monitoring of <i>A. alternata</i> in the field. LAMP is instructive for us to effectively prevent and control apple Alternaria blotch disease.