PM 7/017 (3) Phyllosticta citricarpa (formerly Guignardia citricarpa)

Abstract

PM 7/017 (3) Phyllosticta citricarpa (formerly Guignardia citricarpa) Specific scope This Standard describes a diagnostic protocol for Phyllosticta citricarpa. 1 This Standard should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols. Specific approval and amendment First approved in 2002-09. First revision 2009-09. Second revision 2020-07. This revision was initially prepared taking into account the IPPC Diagnostic Protocol adopted in 2014 (Appendix 5 to ISPM 27, Phyllosticta citricarpa (McAlpine) van der Aa on fruit). However, the EPPO Diagnostic Standard includes other tests developed after the adoption of the IPPC diagnostic protocol and covers additional matrices than fruits. It also includes new species described since the adoption of the IPPC protocol.

Harnessing the power of comparative genomics to support the distinction of sister species within Phyllosticta and development of highly specific detection of Phyllosticta citricarpa causing citrus black spot by real-time PCR

“…However, according to our internal rules these late Ct values were not considered as positive, since they were later than the mean Ct generated with the VGP assay’s limit of detection control. Considering the VGP qPCR assay as the reference method ( EPPO, 2020 ), the relative sensitivity, specificity and accuracy of the cCBS cPCR and qCBS qPCR assays were all 100%.…”

“…Regions that were clearly distinct from each other in the two Phyllosticta species were identified by comparing the differences in allele frequency in the two populations; the read alignments were visualized using the Integrative Genomics Viewer (IVG) ( Robinson et al, 2017 ). Read alignments of the ITS and tef1 genomic regions in the reference genome were also checked in order to assess the intra- and interspecific polymorphism within these genes recommended as a barcode for identification purposes ( EPPO, 2020 ; Schoch et al, 2012 ). The genomic positions of the ITS and tef1 gene were identified by searching for the target regions of the following PCR primers: V9G ( de Hoog & van den Ende, 1998 ), ITS4 ( White et al, 1990 ), EF1-728F ( Carbone & Kohn, 1999 ), and EF-2 ( O’Donnell et al, 1998 ).…”

“…The description of this new sister species was based on a few fixed nucleotide polymorphisms within one gene from the core genome (translation elongation factor 1-alpha gene, tef1 ) and within a region of the rDNA operon (Large Subunit, LSU ), combined with some morphological differences with P. citricarpa . In general, the morphological features of P. paracitricarpa in pure culture are very similar to P. citricarpa and it is virtually impossible to differentiate the two species based solely on microscopic traits ( EPPO, 2020 ).…”

“…This disease is named after the characteristic symptoms of citrus fruits, i.e ., black spots on the citrus peel, although the symptomatology of this disease can be very variable, ranging from hard spots, virulent spots or false melanose spots to freckle spots. This variability in symptoms is the result of the temperature and maturity of the affected fruits ( EPPO, 2020 ). Phyllosticta citricarpa has a wide host range and is able to infect mostly all Citrus spp.…”

“…However, it has been shown that the ITS is 100% identical in the two sister species P. citricarpa and P. paracitricarpa ( Guarnaccia et al, 2017 ). False positive results can therefore be obtained when testing DNA of the non-target species P. paracitricarpa ( EPPO, 2020 ). Such cross-reactions with P. paracitricarpa DNA were also observed with the assay developed by Ahmed et al (2020) targeting phylogenetic marker MCM7.…”