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 Alternaria alternata (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 A. alternata can be controlled in time. In our research, a rapid, specific and efficient loop-mediated isothermal amplification (LAMP) method was developed to detect A. alternata within 60 min. Six primers of LAMP detection can only specifically amplify the aapg-1 gene in A. alternata but not in four other important fungi in apples. The aapg-1 gene encodes endopolygalacturonase in A. alternata, 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 A. alternata. When leaves were inoculated with A. alternata conidia, LAMP detected 1 × 102 conidia/mL as the minimum concentration. However, the traditional tissue isolation and identification method only isolated A. alternata 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 A. alternata before symptoms. Further tests also indicated that LAMP detection was more accurate and sensitive than the traditional tissue isolation and identification method for A. alternata in leaves with the Alternaria blotch symptom collected from the field. Our results showed that the LAMP-targeting the aapg-1 gene has the advantages of high sensitivity, specificity and simplicity and can be used for rapid detection and early monitoring of A. alternata in the field. LAMP is instructive for us to effectively prevent and control apple Alternaria blotch disease.