Identification of Monilinia fructigena, M. fructicola, M. laxa, and Monilia polystroma on Inoculated and Naturally Infected Fruit Using Multiplex PCR

Abstract

Monilinia fructigena, M. fructicola, M. laxa, and Monilia polystroma each have a different regulatory status. To monitor imported and exported fruit for the presence of quarantined Monilinia or Monilia species, a timely identification method is required. Random amplified polymorphic DNA analysis was used to generate an M. fructigena-specific band that was characterized by sequencing. Using the sequence obtained, primers were designed to amplify bands in the same genomic region of M. fructicola and M. laxa. These bands were also characterized by sequencing. From all three sequences, a multiplex polymerase chain reaction (PCR) method based on a common reverse primer (MO368-5) and three species-specific forward primers (MO368-8R, MO368-10R, and Laxa-R2) was established for the differentiation of the three Monilinia species. The multiplex PCR was tested with additional isolates and consistently produced a 402-bp PCR product for M. fructigena, a 535-bp product for M. fructicola, and a 351-bp product for M. laxa. The method was also used with isolates of the recently characterized Monilia polystroma, and all isolates amplified a 425-bp PCR product. The identification method was shown to amplify a PCR product directly from inoculated apples, and the PCR band produced was specific to the inoculated Monilinia or Monilia species. Furthermore, the multiplex PCR was used to identify Monilinia species on naturally infected stone fruits. The method correctly identified infections by both M. laxa and M. fructicola by successful amplification of corresponding PCR products for each species.