Phytophthora-ID.org: A Sequence-Based Phytophthora Identification Tool

Grünwald, N. J., Martin, F. N., Larsen, M. M., Sullivan, C. M., Press, C. M., Coffey, M. D., Hansen, E. M., and Parke, J. L. 2011. PhytophthoraID.org: A sequence-based Phytophthora identification tool. Plant Dis. 95:337-342. Contemporary species identification relies strongly on sequence-based identification, yet resources for identification of many fungal and oomycete pathogens are rare. We developed two web-based, searchable databases for rapid identification of Phytophthora spp. based on sequencing of the internal transcribed spacer (ITS) or the cytochrome oxidase (cox) 1 and 2 spacer region, followed by BLAST searching the databases. Both databases are highly selective. For ITS, only sequences associated with published Phytophthora spp. descriptions or classic Phytophthora phylogenetics references are included. For the cox spacer region, only data obtained by resequencing select isolates reported in published work were included. Novel taxa tentatively named are selectively included in the database and labeled as Phytophthora taxon “X”; as in, for example, P. taxon “asparagi”. The database was validated with 700 Phytophthora isolates collected from nursery environments during 2006 to 2009. This resource, found at www. Phytophthora-ID.org, is a robust and validated tool for molecular identification of Phytophthora spp. and is regularly being updated. Contemporary species identification relies strongly on sequencebased identification, yet resources for identifying many fungal and oomycete pathogens are rare. The genus Phytophthora houses some of the most destructive plant pathogens known, including, for example, the potato late blight pathogen Phytophthora infestans (10,11); the sudden oak death pathogen P. ramorum (17,31); and P. cinnamomi, a pathogen affecting in excess of 3,000 species of plants, including over 2,500 Australian native species and crops such as avocado, pineapple, peach, chestnut, and macadamia (19). Since the discovery of P. ramorum in the late 1990s, renewed interest in Phytophthora biology has led to the discovery of many new Phytophthora spp. In 1996, approximately 60 species of Phytophthora were known to science (7). By 2008, around 90 species of Phytophthora were described or proposed. In fact, Brasier (3) estimated that we might expect two to four times that number of Phytophthora spp. to be described. Given the rapid discovery of novel species, an increase in the number of species of regulatory concern, and the prevalence of encountering Phytophthora spp. in diagnostic labs, a contemporary tool for identification of Phytophthora isolates to a species level is urgently needed. A tool for rapid and easy identification of Phytophthora spp. would have to fulfill several requirements. Ideally, this tool should be sequence based because classical morphology is unable to distinguish many of the species currently recognized. For example P. infestans, P. ipomoeae, and P. mirabilis cannot easily be distinguished based on morphology alone because these species are essentially identical for sporangia (semipapillate and caducous) and oospore morphology (7,8,12). Yet, analysis of several sequences clearly distinguishes these species (8). A contemporary Phytophthora spp. identification tool also should be robust enough so that most laboratories with a thermocycler, basic molecular capability, and web access could make use of it. Traditionally, molecular identification of Phytophthora spp. is accomplished by polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region followed by either restriction analysis (5) or direct sequencing and a BLAST search against GenBank or other databases. However, molecular identification based on the ITS region is not without its pitfalls (23). Although PCR-restricted fragment length polymorphism (RFLP) analysis of the ITS region can identify many species, currently, data for only one-half of the described species are available, and there are examples where ITS PCR-RFLP is not effective for differentiating closely related species (e.g., clade 1c species such as P. infestans and P. mirabilis). Identification can also be a challenge when using BLAST analysis with ITS sequences because there can be minimal or no differences between some species (e.g., clade 1c species) or, in some cases, intraspecific variation can blur the boundaries between species (e.g., P. fragariae var. fragariae and P. fragariae var. rubi have identical ITS sequences). Due to its historical importance for the identification and phylogenetic analysis of the genus and the large number of sequences that have been deposited in GenBank, the ITS region remains an important locus for molecular identification; however, additional regions also have Corresponding author: N. J. Grünwald E-mail: [email protected] Mentions of trade names or commercial products in this manuscript are solely for the purpose of providing specific information and do not imply recommendation or endorsement by the United States Department of Agriculture. *The e-Xtra logo stands for “electronic extra” and indicates that two supplemental tables appear online and that Figures 1, 2, and 3 appear in color in the online edition. Accepted for publication 25 October 2010. doi:10.1094 / PDIS-08-10-0609 This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2011. e-Xtra*