Mycologia, 93(2), 2001, pp. 366-379. ? 2001 by The Mycological Society of America, Lawrence, KS 66044-8897 Differentiation of species of Elsinoe associated with scab disease of Proteaceae based on morphology, symptomatology, and ITS sequence phylogeny Lizeth Swart' from Leucospermumin California and Zimbabwe are Pedro W. Crous2 representative of the same species. Ji-Chuan Kang Key Words: plant pathogens, Sphaceloma, system- Godwin R. A. Mchau atics Department of Plant Pathology, University of Stellenbosch, Private Bag XI, Matieland 7602 South Africa INTRODUCTION Ian Pascoe Species of Elsinoe usually are associated with leaf, Institute for Horticultural Development,Private Bag stem and disease of various 15, South-EasternMail Centre, Victoria 3176 pod symptoms plants Australia (Pan 1994, Phillips 1994, Gottwald 1995). These dis- eases, commonly referred to as scab, have been re- Mary E. Palm corded worldwide on numerous crops important to USDA-APHIS-PPQ SystematicBotany and Mycology agriculture as well as to forestry (Farr et al 1989). Laboratory,Beltsville, Maryland, USA 20705 A scab disease of Proteaceae Benth. & Hook. f. was first observed in South Africa in 1981. The disease, locally known as corky bark or scab, is associated with Abstract: Scab disease of Proteaceae, which was ini- severe losses of commercial pincushion (Leucosper- tially observed on Leucospermumin South Africa in mum R. Br.) (Lsp.) plantings. The causal agent was 1981, has subsequently been reported on this host identified as a species of Elsinoe but was never for- from Australia and Hawaii. The disease, commonly mally described (Benic and Knox-Davies 1983). Sim- known as corky bark or scab, is associated with severe ilar disease symptoms have been observed on cone- losses of commercial plantings of Leucospermumin bushes (Leucadendron R. Br.) (Lcd.) in South Africa South Africa, and has also been collected from spe- (Benic and Knox-Davies 1983, Von Broembsen 1989) cies of Leucadendron, Protea and Serruria in South and Leucospermum spp. in Hawaii (Protea Disease Africa, from Banksia, Leucadendron, Mimetes, Protea Letter Management Group, University of Hawaii un- and Serruria in Australia, and from Leucospermum publ) and Australia (Ziehrl et al 1995). Similarities and Protea in California and Zimbabwe. The causal in disease symptoms and the appearance of the caus- agent was determined to be a species of Elsinoe, al organism in South Africa and Australia have led to which has not been formally described. The aim of speculation that the causal organism could possibly the present study was to elucidate the taxonomy of be the same species. In Australia, scab disease symp- the species of Elsinoe associated with scab disease of toms have been observed on species of Banksia L. f., Proteaceae in these countries. Morphology, symptom- Leucadendron, Mimetes Salisb., Protea L. and Serruria atology and DNA sequence analysis of the 5.8S rDNA Salisb. (Forsberg 1993, Pascoe et al 1995). In the pre- gene and its flanking ITS1 and ITS2 regions were sent study, scab diseases are recorded on species of used. Anamorph and teleomorph characteristics of Leucadendron, Leucospermum,Protea, and Serruria in isolates from Leucospermum,Protea and Banksia sug- South Africa and Australia, on Banksia in Australia, gest that there are at least four distinct species in- and on species of Leucospermumand Protea in Cali- volved. These findings are strongly supported by the fornia, USA, and Zimbabwe. phylogenetic tree inferred from DNA sequence data. Characterization of species of Elsinoe is difficult Furthermore, these results also show that the Elsinoe since their teleomorph states are rarely observed and isolates from Leucadendron, Leucospermum and Ser- their Sphaceloma De Bary anamorphs generally are ruria in South Africa and Australia, and the isolates morphologically conserved. Molecular tools have be- come increasingly important in confirming the inter- of variation. Random am- for 28, 2000. pretation morphological Accepted publication August DNAs with Current address: ARC, Private Bag X1, Elsenburg 7607, South Af- plified polymorphic (RAPDs) arbitrary rica. primers (Welsh and McClelland 1990, Williams et al 2Corresponding author, Email: [email protected] 1990) have proven useful at taxonomic levels ranging 366 SWART ET AL: ELSINOE SPECIES DIFFERENTIATION 367 from cultivars to species (Demeke and Adams 1994). ing mounts in lactophenol. Averages were derived from at least 30 and the in For example, RAPDs have been used to distinguish observations, range given parentheses. of Elsinoe isolates as- between species and pathotypes DNA extraction.-Total DNA of single conidial isolates was sociated with scab disease of Citrus L. (Tan et al extracted from 1-mo-old colonies grown on potato dextrose 1996) and Phaseolus (Tourn.) L. (Mchau et al 1998). agar (PDA; Biolab, Johannesburg, South Africa) at room In preliminary studies, this technique has also proven temperature. Fungal colonies were scraped clean of agar, frozen in and to a fine useful in separating several species occurring on Pro- liquid nitrogen, ground powder. DNA was extracted the method of Della- teaceae (Viljoen et al 1998). The phylogenetic value subsequently by porta et al (1983) and quantified by ethidium bromide fluo- of rDNA sequences has been discussed in previous rescence on an UV transilluminator with known quantities reviews (Bruns et al 1991, Hibbett 1992, Kohn 1992, of lambda DNA (Sambrook et al 1989). Kurtzman 1992, Samuels and Seifert 1995). Ribosom- PCR were carried out in al DNA sequences, in particular the 5.8S rDNA and amplification.-Reactions 25-[LL volumes in thin walled tubes in an Idaho Tech- flanking internal transcribed spacers (ITS1 and Eppendorf nology Air Thermo Cycler, model 1605 (Idaho Technology, ITS2) have been used to study the phylogeny of a Idaho Falls, Idaho, USA). Reaction mixtures contained 1-5 number of plant pathogens (Lee and Taylor 1992, ng template DNA, 100 pIMeach of dATP, dCTP, dGTP and Morales et al 1993, Zambino and Szabo 1993, Schoch dTTP, 2.5 FL 10x ammonium sulphate buffer [670 mM et al 1999, Stewart et al 1999). Tris-HCl, pH 8.8, 40 mM MgCl2, 160 mM (NH4)2SO4,0.01% Our aim was to delineate the Elsinoi species among Tween 20], 0.2 pIMoligonucleotide primers, and 0.5 units isolates from Proteaceae collected in Australia, Cali- of Taq DNA polemerase (Boehringer, Mannheim Ltd., fornia, South Africa, and Zimbabwe, using morphol- Mannheim, Germany). The 5.8S nuclear ribosomal RNA ogy, symptomatology and phylogenetic analysis of the gene and the two flanking internal transcribed spacers and were with ITS1 5.8S, ITS1 and ITS2 rDNA operon. (ITS1 ITS2) amplified primers (5'-d- TCCGTAGGTGAACCTGCGG)and ITS4 (5' dTCCTCCG- CTTATTGATATGC) (White et al 1990). Thermal cycling MATERIALSAND METHODS conditions for amplification included an initial hold at 94 C for 2 min; followed by 30 cycles of 94 C for 15 s, 55 C for Isolates and cultures.-Single spore isolations were made 30 s and 72 C for 35 s; and finally followed by a 4-min from acervuli and ascomata that occurred on the stems and incubation at 72 C. leaves of the various hosts. Conidia and ascospores were reactions.-PCR of iso- cultured on 2% malt extract agar (MEA, Oxoid), and in- Sequencing products representative lates from different hosts were used for se- cubated at 25 C under near-ultraviolet light. Sub-cultures proteaceous (TABLEI). Prior to the PCR are maintained in the Culture Collection of the Depart- quencing sequencing, products were from nucleotides and ment of Plant Pathology, University of Stellenbosch (STE- purified unincorporated prim- ers using WizardTM PCR DNA Purification U). Accession numbers and other data pertaining to the Preps System various Elsinoe isolates examined are listed in TABLEI. Fu- (Promega Corporation, Madison, Wisconsin, USA). Both the forward and reverse strands of each fragment were se- sarium proliferatum (Matsushima) Nirenberg (GenBank quenced to achieve greater fidelity of the sequence data, X94171) was used as outgroup. using an automated sequencer ABI Prism 377 DNA Se- Morphological and cultural studies.-Sporulation in culture quencer (PE Biosystems, Inc., Foster City, California, USA). was induced by using Whiteside's (1975) method, as isolates The primers used for sequencing were the same as those used for PCR. A did not sporulate on MEA. The modified Fries's medium Dye Terminator Cycle Sequencing Ready Reaction Kit DNA [5 g (NH4)2C4H406, 1 g NH4NO3, 1 g K2HPO4, 0.5 g containing AmpliTaq Polymerase (Per- was used for the reactions. The re- MgSO4-7H20, 0.1 g CaCl2, 0.1 g NaCl, and 20 g sucrose in kin-Elmer) sequencing actions were carried out with a concentration of 1 L of distilled water] was poured into 90-mm-diam Petri 20-40 ng of DNA and 3.2 in a total volume of dishes (10 mL per dish). Mycelial fragments were taken template pmol primer from colonies on MEA and distributed in Petri dishes con- 10 .IL. The cycle sequencing reaction was done by PCR under conditions of 96 C for 30 50 C for 15 and 60 C taining Fries's medium. After 4 d at 20 C the small colonies s, s, for 4 min. This was for 25 DNA was (1 mm diam) that developed on the bottom of the Petri repeated cycles. finally columns dishes were flushed with sterile distilled water, scraped from purified using Centri-Sep Spin (Princeton Sepa- rations, New USA) and loaded onto the the dish with a scalpel and transferred to drops of lacto- Adelphia, Jersey, phenol on glass slides to determine the dimensions of mor- sequencing gel. phological structures formed in culture. Phylogenetic analysis.-The DNA sequences of the isolates Isolates were characterized in culture by growing the fun- in TABLEI (GenBank: AF097572-AF097578; AF131080- gus on MEA in the dark at 5-35 C in 5 C intervals for 1 AF131091) were edited with the Tex-Edit Plus program mo, with three replicate plates per temperature.