Early Brown Rot Infections in Sweet Cherry Fruit Are Detected by Monilinia-Specific DNA Primers

Abstract

ABSTRACT Visible and nonvisible quiescent infections of immature and mature fruit are an integral component of the disease cycle of brown rot of sweet cherry in California. Detection of these infections is critical for developing efficient and efficacious fungicide management programs. The previously published DNA amplification primers mfs3 and NS5 for the identification of Monilinia fructicola were very specific in amplifying DNA of M. fructicola only and not M. laxa. This primer set, however, only detected DNA from some of the California isolates of M. fructicola. This genetic diversity was supported by random amplified polymorphic DNA (RAPD) analysis. Using eight 10-mer primers, seven M. fructicola isolates from California were all identified as genetically distinct. Using the same primers, only one polymorphism was detected among seven isolates of M. laxa. The multiple genotypes identified within the small population sample of M. fructicola, but not of M. laxa, using RAPD analysis could be indicative of genetic recombination within M. fructicola but not within M. laxa. To detect early brown rot infections in fruit, two primer sets that were developed from DNA sequences of either ribosomal DNA (MF5/ITS4/ITS3) or a RAPD fragment (X-09intF3/X-09R) specifically amplified DNA from isolates of M. fructicola and Monilinia species, respectively. No amplification products were present when using DNA from Botrytis cinerea or from other fungi commonly found on sweet cherry fruit. Primers X-09intF3 and X-09R were more sensitive and reliable for detecting small amounts of target DNA either extracted from conidia or from laboratory-inoculated cherry fruit with early brown rot infections that showed no visual symptoms or with visible quiescent infections. Furthermore, these primers also were effective for detecting visible quiescent infections in cherry fruit that were collected in the field.