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Title:   Author  item
                                            SIKDAR, P - Washington State Universityitem
                                            Okubara, Patriciaitem
                                            Mazzola, Markitem
                                            Xiao, Chang-Lin
  Submitted to: Plant Disease
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/3/2013
                                    Publication  Date: 2/1/2014
                                    Citation: Sikdar, P., Okubara, P.A., Mazzola, M., Xiao, C. 2014. Development of PCR assays for diagnosis of the pathogens Phacidiopycnis washingtonensis and Sphaeropsis pyriputrescens in apple fruit. Plant Disease. 98:241-246.Interpretive Summary: Apples may rot during storage or in the market if they are infected by rot-causing pathogens. Two new fruit rot diseases have recently been reported to occur on apples grown in Washington State, which produces over 60% of the nation's apples with the production value exceeding one billion dollars. The two diseases have now been listed as quarantine diseases in certain export markets. Apples are subject to inspection for rots prior to shipping. It is very challenging for inspectors to distinguish the quarantined diseases from other common rots because of the similarity of rot symptoms. In this study, we developed a quick and reliable method using DNA fingerprinting technology to assist with diagnosis of the two quarantine diseases. The new method can also assess the risk of apple fruit infection by these two diseases before harvest and thus help growers implement relevant measures to control them.
                                Technical Abstract: Speck rot caused by Phacidiopycnis washingtonensis and Sphaeropsis rot caused by Sphaeropsis pyriputrescens are two recently reported postharvest diseases of apple.  Fruit infection by the pathogens occurs in the orchard but remains latent before harvest. Symptoms develop after harvest and are similar to that of gray mold caused by Botrytis cinerea. Accurate diagnosis of these diseases is important to the fruit inspection process, particularly in the instance of fruit destined for export. Early near-harvest detection of latent infections in apple fruit is important to implementation of relevant pre- and postharvest measures for control of these diseases. The aim of this study was to develop PCR assays for disease diagnosis and early detection of latent infection of apple by P. washingtonensis and S. pyriputrescens. Species-specific primers based on sequences of the rDNA internal transcribed spacer region were designed for use in PCR assays. Conventional and real-time PCR assays were developed and validated using fruit artificially inoculated with P. washingtonensis, S. pyriputrescens or B. cinerea in comparison with identifications as determined using traditional isolation-based assays. For wound-inoculated fruit, the PCR assays consistently provided the correct identification of the pathogen used as the inoculant in 6 hours of processing time, compared to 5-6 days using culture-based methods.  Real-time PCR assays effectively detected latent infections in symptomless stem and calyx tissues of fruit that were inoculated with the pathogens in the orchard during the growing season. The PCR assays provide a rapid, accurate method for diagnosis and early detection of these diseases.


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