Disease Focus: Detecting Phytophthora in nursery plants
by Jim Downer
Currently there are over 100 known Phytophthora species with an estimated 200 to 600 unknown members (Brazier, 2009). Phytophthora belongs in the Kingdom Chromalveolata, once considered fungi, these Oomycetes are no longer included in the kingdom fungi. Phytophthora is in a group of pathogens called water molds because they are often isolated from very wet soils, ponds, creeks or marshy/boggy areas in landscapes. The word “Phytophthora” means plant destroyer.
Since Phytophthora species grow into the cells of its host and its reproductive structures are microscopic, it is hard to detect by examination of samples for symptoms and signs. Since many are soil inhabitants and form resting spores in soil, they can remain undetected until plants begin to die. Prior to the advent of modern serological and molecular methods, Phytophthora was isolated form soil tissues or detected in dilutions of soil on selective media. Phytophthora can also be baited from soil with leaf disks or pear and apple fruits (Erwin and Ribiero, 1996) and identified based on morphological features. Microscopic identification of Phytophthora species requires considerable experience and with the advent of many new species, it is more and more challenging. Since the onset of Phytophthora ramorum in California a number of papers have described isolation, bating and identification methods for determination of various species of Phytophthora. Kox and others (2007) compared different Phytophthora detection methods. The most accurate method of detection was with nucleic acid amplification via the polymerase chain reaction (PCR) and sequencing of the resultant amplified DNA. Phythopthora was also positively detected by culturing and subsequent microscopic identification and by use of the Enzyme Linked Immunosorbent assay (ELISA) and Lateral Flow Device (LFD) test kits. Lane and others (2010) reported similar capability of Phytophthora detection with both ELISA and LFD kits.
ELISA serological kits (AgDia Inc. Elkhart In) are simple field test kits that use antibodies to recognize proteins that are unique to specific organisms. ELISA has a low detection limit and can detect Phytophthora at populations below detectability with dilution plating (Graham and Timmer, 1994). A variant of the ELISA serological method is the lateral flow device (LFD) available fromAbingdon Health Products (Foresite Diagnostics Ltc. UK, also known as Pocket Diagnostics), as well as AgDia Inc. Both ELISA and LFD kits detect all known Phytophthora species.
Lateral flow device assays may give a bit of an edge over ELISA test strips as they do not frequently cross react as frequently with other fungi or Ooomycetes. ELISA Phytophthora test kits are known to cross react with Pythium and Peronsopora (Macdonald et al., 1990). The limitation of both ELISA and LFD’s is that they do not identify Phytophthora to species. This may be done the old fashioned way by culturing and identification of microscopic morphologies or more commonly and typically by PCR and sequencing of the amplified DNA.
Both ELISA and LFD kits provide inexpensive (as low as under ten dollars a test) and rapid (under ten minutes) identification of Phytophthora in diseased plant tissues in a field setting and provide a fast and efficient screening method for nurserymen and others who want do detect Phytophthora. If an accurate diagnosis is essential labs can use PCR technology and sequencing to obtain the most accurate identifications for under ten dollars per sample but it requires two days run time.
Brasier, CM. 2009. Phytophthora biodiversity: how many Phytophthora species are there? In: Goheen EM, Frankel SJ, eds. Phytophthoras in Forests and Natural Ecosystems. Albany, CA, USA: USDA Forest
Erwin, D.C. and O.K. Ribeiro. 1996. Phytophthora diseases worldwide. Amer. Phytopathological Soc. Press. St. Paul Minnesota.
Graham, J. H., and Timmer, L. W. 1994. Phytophthora diseases of citrus. Document SL127/CH087. Published online by Soil and Water Science Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida.
Kox, L. F. F., van Brouwershaven, I. R., van de Vossenberg, B. T. L. H., van den Beld, H. E., Bonants, P. J.M., and de Gruyter, J. 2007. Diagnostic values and utility of immunological, morphological, and molecular methods for in planta detection of Phytophthora ramorum. Phytopathology 97:1119-1129rvice: General Technical Report PSW-GTR-221, 101–15.
Lane, C.R., E. Hobden, L.Walker, V.C. Barton, A.J. Inman, K.J.D. Hughes, H. Swan, A. Colyer and I. Barker. 2010. Evaluation of a rapid diagnostic field test kit for identification of Phytophthora species, including P.ramorum and P. kernoviae at the point of inspection.
MacDonald, J.D. Stities, J. and J. Kabashima. 1990. Comparison of serological and culture plate methods of detection species of Phytophthora, Pythium and Rhizoctonia in ornamental plants Plant Disease 74: 665-559.