Black Rot of Orchids Caused by Phytophthora cactorum and Phytophthora palmivora in Florida

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1 2010 Plant Management Network. Accepted for publication 27 April Published. Black Rot of Orchids Caused by Phytophthora cactorum and Phytophthora palmivora in Florida R. A. Cating and A. J. Palmateer, Tropical Research and Education Center, University of Florida, Homestead, FL 33031; C. M. Stiles, Georgia Military College, Valdosta, GA 31605; and P. A. Rayside, Plant Pathology Department, University of Florida, Gainesville, FL Corresponding author: Aaron Palmateer. Cating, R. A., Palmateer, A. J., Stiles, C. M., and Rayside, P. A Black rot of orchids caused by Phytophthora cactorum and Phytophthora palmivora in Florida. Online. Plant Health Progress doi: /php dg. Introduction Orchid sales in the United States have increased steadily since 1997, and sales were estimated to exceed 140 million dollars in 2006 (13). Orchids are the second most economically important flowering plant produced in the United States, and Florida and California are the top producers of orchids in the nation (13). Several species of Phytophthora have been reported to cause economic damage on orchids worldwide (7,11,24). Of these, P. cactorum and P. palmivora have the widest host range across orchid genera, and they are the most common species affecting commercial orchid production in Florida. The objective of this work was to provide a practical diagnostic resource for black rot on orchids in Florida. The Disease Disease caused by Phytophthora cactorum and P. palmivora in orchids can be referred to as black rot, crown rot, and heart rot (10). The Pathogens There are reports of other Phytophthora spp. causing disease on orchids (7,10,19,24), but black rot, in commercial orchid production throughout Florida is most commonly caused by Phytophthora cactorum and P. palmivora. The latter two pathogens can be differentiated by morphological characteristics or by the use of molecular diagnostics (23). A less-common cause of black rot of orchids, Pythium ultimum, can be readily distinguished from Phytophthora based on morphology (7). Taxonomy Phytophthora cactorum and P. palmivora are members of the family Pythiaceae in the kingdom Stramenopila. Recently, Blair et al. (1) produced a comprehensive phylogeny for the genus Phytophthora based on multiple loci. Additional taxonomic information is available in Erwin and Ribeiro (7) and online at (20). Symptoms and Signs Initial symptoms of the disease may include small black lesions on the roots or basal portion of the pseudobulbs. As the lesions age, they enlarge and may engulf the entire pseudobulb and leaf (Fig. 1). White mycelium and sporangia may be seen growing directly on the plant material (Fig. 2). The pathogen can spread through the rhizome to other portions of the plant. Eventually, the entire plant may die.

2 Fig. 1. Symptoms of black rot caused by Phytophthora cactorum on pseudobulbs and lower leaves of a Cattleya orchid hybrid. Fig. 2. White mycelium of Phytophthora palmivora protruding from ends of pseudobulbs of a Cattleya orchid hybrid. Host Range Within the Orchidaceae Phytophthora cactorum and P. palmivora are known to cause disease on many different orchid genera. Black rot is most frequently seen on Cattleya orchids and their hybrids, such as Brassocattleya and Laeliocattleya, but the disease also affects Aerides, Ascocenda, Brassavola, Dendrobium, Gongora, Maxillaria, Miltonia, Oncidium, Paphiopedilum, Phalaenopsis, Rhynchostylis, Schomburgkia, as well as some less commonly grown genera (7,8,19,21). Geographic Distribution Phytophthora cactorum and P. palmivora have wide host ranges and occur on many annual and perennial food and ornamental crops throughout the world (7). Pathogen Isolation Phytophthora cactorum and P. palmivora can be readily isolated from diseased tissue. Wash the symptomatic plant tissue under a gentle stream of tap water for 2 to 3 min. Use a sterile blade to slice multiple sections of diseased tissue from the plant. To increase isolation frequency, one should avoid selecting tissue that has dried and take tissue from margins with actively progressing lesions. Tissue selections should then be cultured in P5ARPH medium (12). Seal the plates with parafilm and incubate them in the dark at 25 to 30 C. Examine for Phytophthora growth after 3 to 5 days. Pathogen Identification Morphological identification is based primarily on the shape of zoosporangia and the presence and shape of oospores and antheridia. Both P. cactorum and P. palmivora produce abundant zoosporangia on orchid host tissue when maintained in a humidity chamber under continuous florescent light at 25 to 28 C (for P. cactorum) or 30 to 33 C (for P. palmivora) (personal observation). Zoosporangia of P. cactorum are ellipsoidal to egg-shaped to spherical, while those of P. palmivora are ellipitical to ovoid (7). Zoosporangia of both species are papillate (i.e., they have a small, swollen translucent tip, and can be detached easily in liquid (7,9). The detached zoosporangia bear a short pedicel (a remnant of the stalk) where the stalk was attached (7,9). Note that the morphological characteristics of sporangia are very similar for isolates of P, cactorum and P. palmivora, so the presence and shape of oospores and characteristics of the antheridia are necessary for species delineation. Phytophthora cactorum is homothallic and produces sex bodies on several common media used in the plant diagnostic laboratory (7,9). Phytophthora 1 palmivora is heterothallic and sex bodies are formed when A and A² mating types are paired in culture. A suitable medium for the formation of sex bodies of P. palmivora (and most other Phytophthora species) is lima bean agar (9). Once the sex bodies are formed, examination of the antheridium and its attachment to the oogonium allows one to distinguish between P. cactorum and

3 P. palmivora (7). Antheridia of P. cactorum attach to the side of the oogonium, whereas antheridia of P. palmivora surround the oogonium stalk (Fig. 3). Light is inhibitory to oospore formation but stimulatory to oospore germination. Mature oospores can be induced to germinate by treatment with 0.25% KMnO 4 for 20 min and incubation under light during germination. Fig. 3. Paragynous antheridium and oogonium of Phytophthora cactorum (top of figure) and amphigynous antheridium and oogonium of Phytophthora palmivora (bottom of figure). Molecular Diagnostics Sequencing of the internal transcribed spacer (ITS) regions of rdna can be used to discriminate between P. cactorum, P. palmivora, and among many other species of Phytophthora (2,3,4). However, DNA sequence variation in the ITS region is not sufficient to discriminate among closely related Phytophthora species (17), so multiple criteria should be used to make species identifications, especially when the identifications have regulatory significance. Recently, we sequenced the ITS1, 5.8S rrna gene, and ITS2 regions from a P. palmivora isolate affecting Cattleya orchids in Florida (GenBank accession # GQ131800). Species-specific primers for the ITS region are available for both P. cactorum and P. palmivora (16,23). In addition to DNA sequencing, other molecular techniques have been used to identify Phytophthora species. These include single-strand-conformation polymorphism (SSCP) of ribosomal DNA (9,15), isozyme analysis (18), and restriction fragment analysis (5). A useful tool for the identification of Phytophthora species using the ITS region and restriction fragment analysis is available online at phytophthora-id.org. Pathogen Storage Storage of Phytophthora isolates can be difficult, and frequent transfers may be necessary to maintain viability; however, transfers can cause mutations or other changes (7). Ko (14) demonstrated that isolates of P. palmivora can be stored in sterile water at room temperature for as long as 23 years. To store isolates in this manner, remove plugs from isolates actively growing on V-8 juice agar and place 4 plugs in 7 ml of sterile distilled water in a sterile test tube and tighten the cap. Store the tubes in a test tube rack on a laboratory shelf. Another method for storage of isolates involves the use of liquid nitrogen (7,22). In this method, plugs of agar containing the isolate are removed and stored in polypropylene vials containing 10% glycerol or DMSO and placed in liquid nitrogen (22). However, a pretreatment of -80 C may be required to successfully recover the isolate (22). For detailed information on cryopreservation, see Dahmen et al. (6) and Tooley (22). Pathogenicity Tests Hine (10) tested the pathogenicity of P. palmivora on several different orchid genera by using inocula of zoospore suspensions or mycelium-covered V- 8 agar blocks. More recently, Orlikowski and Szkuta (19) examined the pathogenicity of P. palmivora on Phalaenopsis, Dendrobium, Cymbidium, and Epidendrum orchids using mycelia disks of P. palmivora cultures on potato

4 dextrose agar (PDA); the inoculum was placed on orchid leaf blades and roots. The inoculated plant material was placed on sterile, moist blotting paper in polystyrene boxes and incubated at 22 to 25 C in the dark. Small black lesions were initially observed at the point of inoculation, and after 3 days the disease progressed causing affected tissue to appear water soaked and black in color. Literature Cited 1. Blair, J. E., Coffey, M. D., Park, S.-K., Geiser, D. M., and Kang, S A multilocus phylogeny for Phytophthora utilizing markers derived from complete genome sequences. Fungal Genet. Biol. 45: Brasier, C. M., Cooke, D. E. L., and Duncan, J. M Origin of a new Phytophthora pathogen through interspecific hybridization. Proc. Natl. Acad. Sci. USA 96: Cooke, D. E. L., and Duncan, J. M Phylogenetic analysis of Phytophthora species based on ITS1 and ITS2 sequences of the ribosomal RNA gene repeat. Mycol. Res. 101: Cooke, D. E. L., Drenth, A., Duncan, J. M., Wagels, G., and Brasier, C. M A molecular phylogeny of Phytophthora and related oomycetes. Fungal Genet. Biol. 30: Cooke, D. E. L., Duncan, J. M., Williams, N. A., Hagenaar-de Weerdt, M., and Bonants, P. J. M Identification of Phytophthora species on the basis of restriction enzyme fragment analysis of the internal transcribed spacer regions of ribosomal RNA. EPPO Bulletin 30: Dahmen, H., Staub, T., and Schwinn, F. J Technique for long-term preservation of phytopathogenic fungi in liquid nitrogen. Phyopathology 73: Erwin, D. C., and Ribeiro, O. K Phytophthora Diseases Worldwide. American Phytopathological Society, St. Paul, MN. 8. Farr, D. F., Bills, G. F., Chamuris, G. P., and Rossman, A. Y Fungi on Plants and Plant Products in the United States. American Phytopathological Society, St. Paul, MN. 9. Gallegly, M. E., and Hong, C Phytophthora: Identifying Species by Morphology and DNA Fingerprints. American Phytopathological Society, St. Paul, MN. 10. Hine, R. B Pathogenicity of Phytophthora palmivora in the Orchidaceae. Plant Dis. Rep. 46: Ilieva, E., Man in t Veld, W. A., Veenbaas-Rijks, W., and Pieters, R Phytophthora multivesiculata, a new species causing rot in Cymbidium. Eur. J. Plant Pathol. 104: Jeffers, S. N., and Martin, S. B Comparison of two media selective for Phytophthora and Pythium species. Plant Dis. 70: Jerardo, A Floriculture and Nursery Crops Outlook, FLO-05. Economic Research Service, USDA, Washington, DC. 14. Ko, W.-H Long-term storage and survival structure of three species of Phytophthora in water. J. Gen. Plant Pathol. 69: Kong, P., Hong, C., Richardson, P. A., and Gallegly, M. E Single-strandconformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytopthora. Fungal Genet. Biol. 39: Lacourt, I., Bonants, P. J. M., Van Gent-Pelzer, M. P., Cooke, D. E. L., Hagenaar-De Weerdt, M., Surplus, L., and Duncan, J. M The use of nested primers in the polymerase chain reaction for the detection of Phytophthora fragariae and P. cactorum in strawberry. Acta Hort. (ISHS) 439: Martin, F. N., and Tooley, P. W Identification of Phytophthora isolates to species level using restriction fragment length polymorphism analysis of a polymerase chain reaction-amplified region of mitochondrial DNA. Phytopathology 94: Mchau, G. R. A., and Coffey, M. D Isozyme diversity in Phytophthora palmivora: Evidence for a southeast Asia center of origin. Mycol. Res. 98: Orlikowski, L. B., and Szkuta, G Phytophthora rot of some orchids-new disease in Poland. Phytopathol. Pol. 40:57-61.

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