Mycol Progress DOI 10.1007/s11557-009-0649-6 ORIGINAL ARTICLE Phylogenetic relationships of sugarcane rust fungi Linley J. Dixon & Lisa A. Castlebury & M. Catherine Aime & Neil C. Glynn & Jack C. Comstock Received: 29 September 2009 /Revised: 2 December 2009 /Accepted: 7 December 2009 # US Government 2010 Abstract The phylogenetic positions of Puccinia spp. in- within the Pucciniales. Phylogenetic analyses place P. fecting sugarcane (a complex hybrid of Saccharum spp.) melanocephala most closely to P. miscanthi, P. nakanishi- were determined using 38 newly generated rust sequences kii, and P. rufipes infecting Miscanthus sinensis, Cymbo- and 26 sequences from GenBank. Rust specimens on pogon citratus,andImperata cylindrica, respectively. sugarcane were collected from 164 locations in 23 countries Puccinia kuehnii is basal to a clade of Poaceae-infecting and identified based on light microscopy. The morphology rusts including P. agrophila, P. polysora, P. substriata, and for all samples matched that of Puccinia kuehnii or P. Uromyces setariae-italicae infecting Schizachyrium spp., melanocephala, the orange and brown rust pathogens of Zea mays, Digitaria spp., and Urochloa mosambicensis, sugarcane, respectively. Nuclear ribosomal DNA sequences respectively. Light and scanning electron microscopy (rDNA) including portions of the 5.8S rDNA, the complete images highlight morphological differences distinguishing internal transcribed spacer 2 (ITS2) and 5′ region of the the two sugarcane-infecting species. This study confirms large subunit (nLSU) rDNA were obtained for each species the separation of rust species infecting Poaceae from along with 36 additional rust taxa. Despite a shared host, Cyperaceae- and Juncaceae-infecting rusts and also the two Puccinia spp. on sugarcane are not closely related provides support for the presence of an additional group that includes P. kuehnii and other grass-infecting relatives. Product names and trademarks are mentioned to report factually on available data; however, the USDA-ARS neither guarantees nor Keywords Molecular systematics . Puccinia kuehnii . warrants the standard of the product, and the use of the name by Puccinia melanocephala . Orange and brown rusts . USDA-ARS does not imply approval of the product to the exclusion of others that may also be suitable. The experiments reported comply Plant pathogenic fungi with current US laws. L. J. Dixon : L. A. Castlebury USDA-ARS Systematic Mycology and Microbiology Laboratory, Introduction Beltsville, MD 20705, USA Two species of fungi are known to cause rust diseases on M. C. Aime sugarcane (Saccharum officinarum L.). Puccinia kuehnii Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, (W. Krüger) E.J. Butler, which causes orange rust, is more Baton Rouge, LA 70803, USA prevalent in high humidity, whereas P. melanocephala : Syd. & P. Syd., which causes brown rust, occurs in cool, N. C. Glynn J. C. Comstock dry conditions (Braithwaite et al. 2009). Brown rust is USDA-ARS Sugarcane Field Station, Canal Point, FL 33438, USA found almost everywhere sugarcane is grown. After its first appearance in the Western Hemisphere in 1978, L. J. Dixon (*) brown rust spread throughout the Americas within a year, USDA ARS Systematic Mycology Laboratory, and yield losses have been reported as high as 50% in Rm 304, Bldg 011A, BARC-West, 10300 Baltimore Ave, Beltsville, MD 20705-2350, USA Mexico (Purdy et al. 1983). In contrast, orange rust, once e-mail: [email protected] restricted to scattered outbreaks in Australia and Asia Mycol Progress (Egan 1980), was only recently reported for the first time Though differences in morphology of the sugarcane rusts in the western hemisphere, in Florida in 2007 (Comstock have been clarified previously, their phylogenetic relation- et al. 2008). Since then, orange rust has spread to several ships to each other and other rust fungi remain unknown. In countries in Central America and appears to be moving response to the threat of rusts on sugarcane production, a throughout the Americas (Chavarria et al. 2009;Floreset morphological assessment of rust fungi causing diseases on al. 2009;Ovalleetal.2008). Economic losses from orange sugarcane was conducted using globally distributed sam- rust during a single season have been estimated at US$40 ples to determine the current distribution of the two million in Florida and US$177 million in Australia (Raid sugarcane-infecting species. In addition, DNA sequence and Comstock 2006; Braithwaite et al. 2009). As a result data were obtained to clarify the phylogenetic relationships of the recent movement of orange rust, there is great con- of sugarcane infecting rust fungi to each other and to other cern that the disease will spread to the sugarcane-growing rust fungi. areas of Hawaii, Louisiana and Texas in the United States and to South America. Important commercial sugarcane clones are susceptible to both orange and brown rust, and Materials and methods even resistant clones demonstrate increased susceptibility over time due to genetic variability within pathogen Origins of fungal strains and microscopy populations (Braithwaite et al. 2009; Magarey et al. 2001). Puccinia kuehnii and P. melanocephala are the only Rust specimens on sugarcane were sent from 164 different species known to cause rust disease on commercial sugar- field locations in 23 countries to the Systematic Mycology cane and are commonly misidentified (Chona and Munjal and Microbiology Laboratory in Beltsville, MD, between 1950; Egan 1980; Roy et al. 1998; Virtudazo et al. 2001a). 2007 and 2008, and specimens were deposited in the Virtudazo et al. (2001b) reviewed the taxonomy of these United States National Fungus Collection (Table 1). Mi- two species using freshly collected samples from Japan and croscopic features of pustules, urediniospores, and paraphy- the Philippines and dried specimens from seven herbaria ses were examined for all specimens. Representative worldwide. All specimens were classified as either P. isolates of P. kuehnii and P. melanocephala were observed melanocephala or P. kuehnii, the former distinguished by under the scanning electron microscope (BPI 878930 and having paraphyses, dark brown urediniospores with uni- 878929, respectively). To increase taxon sampling, rust formly thick cell walls, and 2-celled teliospores with upper specimens from grasses and other hosts were obtained from cells darker than the lower cells (Virtudazo et al. 2001b). In specimens intercepted by USDA, APHIS, Plant Health, contrast, P. kuehnii lacks paraphyses and has orange to Plant Protection and Quarantine mycologists or collected in cinnamon urediniospores with apically thickened walls, and the field, pressed and dried until further processing in the 2- to 3-celled hyaline to whitish teliospores. Reported hosts laboratory. Additional DNA sequences from rusts were for P. melanocephala include the following: Aquilegia spp. obtained from GenBank (http://www.ncbi.nlm.nih.gov/) L., Bambusa spp. Schreb., Eulalia fastigiata (Nees ex (Table 2). Steud.) Haines, Miscanthus floridulus (Labill.) Warb. Ex K. Schum. & Lauterb., M. sacchariflorus (Maxim.) Benth., M. DNA isolation, amplification, and sequencing sinensis Andersson, Phyllostachys aurea Rivière & C. Rivière, P. bambusoides Siebold & Zucc., P. glauca Portions of the ITS2 and nLSU locus were sequenced from McClure, P. nigra (Lodd. Ex Lindl.) Munro, Saccharum a representative specimen of P. kuenhii (BPI 878930) and P. barberi Jeswiet, S. narenga (Nees ex Steud.) Wall. Ex Hack., melanocephala (BPI 878929). For each sampled individual, S. officinarum,S. robustum E. W. Brandes & Jeswiet ex 5–6 sori were excised from dried symptomatic leaf material Grassl, S. ravennae (L.) L., S. rufipilum Steud., and S. and placed in 2-ml bead solution tubes of the UltraClean spontaneum L.; and for P. kuehnii include the following Plant DNA Isolation Kit (MoBio Laboratories, Solana grasses: Saccharum arundinaceum Retz., S. barberi, S. edule Beach, CA, USA). Tubes were processed twice for 30 s at Hassk., S. narenga, S. officinarum, S. robustum, S. sinense 5.0 m/s in a FastPrep™ (Bio101) instrument with 10-min Roxb., S. spontaneum,andSclerostachya fusca (Roxb.) A. room-temperature incubations between runs (BIO101, Vista, Camus (Farr and Rossman 2009; Virtudazo et al. 2001b). In CA, USA). Tubes were incubated at 55°C overnight (15– some cases, the above-listed host names were changed 17 h) with solution P1 followed by the remaining DNA according to USDA, ARS, National Genetic Resources extraction steps according to the manufacturer’s instructions. Program, Germplasm Resources Information Network PCR amplifications were performed with forward primer (GRIN) Online Database (National Germplasm Resources Rust2inv and reverse primer LR6 according to the proce- Laboratory, Beltsville, MD. URL: http://www.ars-grin.gov/ dures outlined in Aime (2006). PCR products were cleaned cgi-bin/npgs/html/index.pl (27 August 2009)). by adding 2 μl ExoSAP-IT (Affymetrics/USB, Santa Clara, Mycol Progress Table 1 Sugarcane rust samples identified in this study Country Puccinia sp. Variety and specimen voucher number Australia melanocephala Q117 (BPI879669), Q124 (BPI879693), Q190 (BPI879692) kuehnii Q124 (BPI879696) Barbados melanocephala DB9627 (BPI879727), HQ3097 (BPI879726) Brazil melanocephala B8008 (BPI879657), RB835486 (BPI879651, BPI879654, BPI879656), RB918639 (BPI879653), RB92579 (BPI879655) China kuehnii RB72-454 (BPI879703, BPI879704, BPI879705)