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 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: pathogens, Sphaceloma, system- Godwin R. A. Mchau atics Department of Plant Pathology, University of Stellenbosch, Private Bag XI, Matieland 7602 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 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 ( 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, and in South and spp. in Hawaii (Protea Disease Africa, from , Leucadendron, , 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. The ex- ([email protected]). Alignment of the sequences was con- periment was duplicated once. Colony colors were deter- ducted using the CLUSTAL W software program (Thomp- mined according to Rayner (1970). Specimens from host son et al 1994) and improved manually. Unweighted parsi- plants were examined microscopically at x1000 by prepar- mony analyses were performed on the DNA sequence data 368 MYCOLOGIA

TABLEI. Elsinoe isolates used for molecular comparisons

Geographic Date Species Collection Hosta origin isolated GenBank Elsinoe banksiae DAR 50126 B. serrata Australia Sep 1984 AF227197 STE-U 2678 E. leucospermi VPRI 21189 Lcd. sp. Australia 8 Aug 1996 AF097575 STE-U 1502 STE-U 2033 Lcd. pubibracteolatumX chamelaea South Africa 2 Sep 1997 AF131080 VPRI 21187 Lsp. cordifolium Australia 8 Aug 1996 AF097573 STE-U 1505 STE-U 2041 Lsp. cordifoliumcv. Vlam California 9 Apr 1998 AF131088 STE-U 2042 Lsp. cordifolium California 13 Apr 1998 AF131089 STE-U 1607 Lsp. cordifolium South Africa 6 Aug 1996 AF097576 STE-U 2039 Lsp. glabrum X tottum cv. Scarlet Ribbon Zimbabwe 6 Mar 1998 AF131086 STE-U 2040 Lsp. reflexumvar. luteum Zimbabwe 6 Mar 1998 AF131087 VPRI 21188 S. florida Australia 8 Aug 1996 AF097574 STE-U 1511 STE-U 2043 S. florida South Africa 8 Sep 1998 AF131090 STE-U 2044 S. florida South Africa 8 Sep 1998 AF131091 E. proteae STE-U 1349 P cynaroides South Africa 15 Feb 1996 AF097578 Elsinoe sp. STE-U 1609 Citrus sp. South Africa 1995 AF097577 Sphacelomaprotearum STE-U 2034 P compacta x susannae cv. Pink Ice Zimbabwe 9 Mar 1998 AF131081 STE-U 2035 P compacta X susannae cv. Pink Ice Zimbabwe 5 Mar 1998 AF131082 STE-U 2036 P eximia X susannae cv. Sylvia Zimbabwe 6 Mar 1998 AF131083 STE-U 2037 P eximia X susannae cv. Sylvia Zimbabwe 5 Mar 1998 AF131084 STE-U 2038 P magnifica x susannae cv. Susara Zimbabwe 5 Mar 1998 AF131085 Sphaeloma sp. VPRI 21100 B. prionotes Australia 5 Aug 1996 AF097572 STE-U 1508

a B. = Banksia; Lcd. = Leucadendron;Lsp. = Leucospermum;P = Protea; S. = Serruria. with the branch and bound option in PAUP* (Swofford The tree has been evaluated with 1000 bootstrap 1999). Clade stability was assessed by 1000 parsimony boot- replications and decay indices for clade stability. Con- The indices were also calculated strap replications. decay cordant tree topology and a distance matrix of pair- to further test the ro- using AutoDecay (Eriksson 1998) wise distances of the isolates (data not shown but bustness of the branches of the tree. Other measures in- available from the au- tree index, retention index, re- upon request corresponding cluding length, consistency were also obtained the scaled consistency index and homoplasy index (CI, RI, RC thor) using neighbor-joining method. and HI) were also calculated. Neighbor-joining and maxi- mum-likelihood distance methods were also used to infer The phylogenetic tree (FIG. 1) indicates two major the gene tree. clades. The first clade (Elsinol leucospermi) includes isolates with strong bootstrap support (98) and decay indices (3). The second clade RESULTS (Sphaceloma protear- um) also comprises isolates with strong bootstrap sup- Phylogenetic analysis.-DNA sequences were deter- port (100) and decay indices (7). The other isolates, mined for approx. 530 bp of 5.8S rDNA and ITS re- namely E. proteae, E. banksiae and the Elsinoe spp. gions of the Elsinoe isolates. The initial sequence from Citrus and Banksia separated individually. alignment had a total consensus length of 643 sites. These clades correlated well with morphological and The alignment tips at the ends of both ITS1 and ITS2 cultural similarities. containing 38 and 22 uninformative sites of 18S and associ- 28S rDNA respectively, were excluded from the anal- Description of species.-The disease symptoms yses. The alignment has been deposited in TreeBASE ated with the different taxa were in most cases found (S494 & M719). Of the aligned sites, 519 were con- to be reliable to distinguish the various species. The stant and 32 were parsimony-informative. A single Sphaceloma states of the different species were gen- most parsimonious tree (MPT) was obtained with erally morphologically conserved, and could not be PAUP (Swofford 1999), using the branch and bound used as sole characters to identify the different spe- option and Fusarium proliferatum as outgroup. cies. Although cultural characteristics were informa- SWART ET AL: ELSINOE SPECIES DIFFERENTIATION 369

X94171 3 Fusariumproliferatum outgroup

2033

2039

2042 Elsinoe leucospermi ,,, 2043

1505

1607

1502

1511

2038

2035 Sphacelomaprotearum

2034

2036

3 Elsinoe proteae

3 Elsinoe sp. (from Citrus) 1508 D Elsinoe sp. (from Banksia) 3 Elsinoe banksiae 5 changes

FIG. 1. Single most parsimonious tree derived from the alignment of the 5.88 and ITS1 and ITS2 rDNA of 20 Elsinoe isolates using parsimony analysis with branch and bound option and 1000 bootstrap replicates. The tree is rooted with the outgroup Fusarium proliferatum (GenBank Accession: X94171). The tree length = 239, CI = 0.913, RI = 0.845, RC = 0.772, HI = 0.087. The bootstrap values and decay indices are indicated above and below the tree branches respectively. tive, and for some species highly specific, this was not quis; constricti ad septum medianum, (15-)17-19(-20) X the case for all taxa involved. The most informative 6-7(-8) p.m. Mycelium internum, compositum ex hyphis vel mediis 3.5-4.5 characters proved to be that of the teleomorph, as ramulosis, septatis, pallidis brunneis, Ijm diam. Conidiomata acervulata, subelevata, coalescentia in ascus and ascospore morphology proved characteris- maturitate, ex textura media brunnea, tic for all taxa studied. Based on these results, four compostia angularis ad 200 pjm lata et 1 mm sub- new are described as follows. usque longa. Conidiophori species cylindrati, pallidi vel medii brunnei, verruculosi, 0-3 septa, 20-30 X 3-5 pm. Conidiogenae cellulae poliphialidibus, Elsinoe banksiae Pascoe et Crous, nov. FIGS. 2-6 cum 1-2 locis integris, pallidis brunneis, verruculosis, doli- sp. 6-13 X 4-6 pm. Conidia Ascomata dispersa, separata, pulvinata, subcuticularia, iformibus, hyalina, aseptata, ellip- cum obtoso cum basi constricta ad locum sub- composita ex textura angularispseudoparenchymatica pal- soidea, apice, truncatum, X pIm. lida vel media brunnea, usque ad 100 pLmlata et alta. Asci (3.5-)4-6(-7) (1.5-)2-3(-3.5) circular to diffusi irregulariterubique in ascoma, ovoidei ad globosos Leaf spots irregular, predominantly epi- cum extremis rotundatis,crasso pariete, octospori,sessiles, phyllous on leaves, extending partially through the hyalini, 20-30 X 20-27 pim.Ascospori hyalini, late ellipso- lamina; initially small gray specks with a brown mar- idei cum extremis rotundatis vel obtusis, cum 2-3 septis gin, surrounded by a chlorotic zone, 0.5-7 mm diam. transversalibuset 1-3 septis verticalibuset 0-2 septis obli- Lesions also develop on stems and midribs of leaves, 370 MYCOLOGIA

-a2 2 3 4 ,7-

FIGS.2-4. Foliar disease symptoms of species of Elsinoe on Banksia. 2. Elsinoe sp. on B. pronotes. Bar = 10 mm. 3, 4. Elsinoe banksiae on B. serrata. Bars: 3 = 3 mm; 4 = 3.5 mm.

circular to irregular, medium brown, frequently with sparse aerial mycelium; surface pale greenish gray large black acervuli, appearing as longitudinal rup- (35.""f), with a gray olivaceous outer zone (21""b); tures of the epidermis. Ascomata scattered, separate, color underneath same as for surface outer zone. pulvinate, subcuticular, composed of light to medium Colonies reached 8 mm diam on MEA after 1 mo in brown pseudoparenchymatic textura angularis, up to the dark at 15 C. Cardinal temperatures for growth 100 ,Im wide and high. Asci distributed irregularly were min above 5 C, opt 15 C, max below 30 C. throughout the ascoma, ovoid to globose with round- Host. L. f. ed ends, thick-walled, 8-spored, sessile, hyaline, 20- Anamorph. Sphacelomasp. 30 x 20-27 jlm. Ascosporeshyaline, broadly ellipsoid Distribution. Australia. with rounded to obtuse ends, with 2-3 transverse sep- Specimensexamined. AUSTRALIA.NEW SOUTH WALES: ta, 1-3 vertical septa, and 0-2 oblique septa; con- Brisbane Waters National Park, on leaves of Banksia serrata stricted at median septum, (15-)17-19(-20) x 6-7 L. f., 23 Oct 1986, B.C. Sutton, J. Walker& M. Priest (HO- Banksia ser- (-8) jim. Mycelium internal, consisting of branched, LOTYPE,VPRI 14815a);Durras, on leaves of B. on septate, medium to dark brown hyphae, 3.5-4.5 Ixm rata, 23 May 1986, Fleming (VPRI 13976a); Umina, diam. Conidiomata acervular, foliicolous and cauli- leaves of Banksia serrata, Sep 1984, J. Walker(DAR 50126, STE-U Victoria, on leaves and stems of colous, raised, coalescing at maturity, composed of 2678); Longford, Banksia Lindl., 5 1996, D. Tricks& A. Ziehrl medium brown textura angularis, up to 200 jim wide prionotes Aug (VPRI 21100a, VPRI 21100b, STE-U 1508, undescribed El- and 1 mm long. Conidiophoressubcylindrical, light to sinoe sp.). SOUTH AUSTRALIA: Adelaide, on leaves of medium brown, verruculose, 0-3-septate, 20-30 x 3- Banksia sp., 20 Oct 1995, B. Hall (VPRI 20732a). 5 jLm.Conidiogenous cells enteroblastic, polyphialidic, with 1-2 integrated loci, light brown, verruculose, do- liiform, 6-13 x 4-6 jLm. Conidia hyaline, aseptate, Elsinoe leucospermi L. Swart et Crous, sp. nov. ellipsoid, apex obtuse, constricting at base to a sub- FIGS. 7-16 truncate locus, (3.5-)4-6(-7) X (1.5-)2-3(-3.5) uLm Ascomata dispersa, separata, pulvinata, subcuticularia, in vivo. Could not be induced to sporulate in vitro composita ex textura angularis pseudoparenchymatica me- in Fries's medium. Cultures on MEA produced flat dia vel atrobrunnea, usque ad 200 ,xm lata et 60 ,xm alta. colonies with smooth, sinuate margins and with Asci diffusi irregulariter ubique in ascoma, ovoidei ad glo- SWART ET AL: ELSINOE SPECIES DIFFERENTIATION 371

' ' 0 0

FIGS.Elsinoeba 5, 6. 6

FIGS.5, 6. Elsinoe;banksiae. 5. Asci and ascospores. 6. Conidiophores and conidia of the Sphacelomastate. Bar = 10 jLm. bosos, crasso pariete, octospori, sessiles, hyalini, 16-28 X pearing as small, raised, yellow-brown to reddish 13-18 late cum extremis iLm.Ascospori hyalini, ellipsoidei spots, circular to irregular, 1-8 mm diam, enlarging rotundatis vel obtusis, cum 1-4 transversalibus et 1- septis and coalescing to form raised, irregular, medium 2 constricti ad septis verticalibus, septum obliquum raro; brown lesions; borders raised, whitish to brown medianum, X 4-5 plm. light septum (10-)12-14(-19) Mycelium due to the red-brown internum, compositum ex hyphis ramulosis, septatis, hyalin- ruptured epidermis; margins is vel brunneis, 3.5-4.5 pLmdiam. Conidiomata acervulata, frequently present; lesions become roughened and subelevata, coalescentia in maturitate, compostia ex textura corky at maturity, and often have longitudinal cracks, angularis media brunnea, usque ad 200 pLmlata et lmm the smaller of which frequently support acervuli of longa. Conidiophori subcylindrati, pallidi brunnei, verru- the asexual state. Severe infection can also lead to a X culosi, 0-2 septa, 20-30 3-6 pJm.Conidiogenae cellulae twisting and distortion of the stem. Lesions on Ser- cum 1-2 locis brunneis, poliphialidibus, integris, pallidis rura occurring on stems, consisting of small specks verruculosis, ampulliformibus ad doliiformes, 10-15 X 3-4 to raised, circular spots, light brown, 1-3 mm diam, jLm.Conidia hyalina, granulata, aseptata, ellipsoidea, cum surrounded a distinct, wide, red to 2 obtoso apice, cum basi constricta ad locum subtruncatum, by margin, up mm in diam. On leaves from small (2-)5-7(-8) x (1-)2.5-3 Im. symptoms vary necrotic with red to a of the Leaf spots on Leucospermum and Leucadendron cir- spots margins blighting whole cular to irregular, amphigenous, 1-4 mm diam, ex- lamina. Ascomata scattered, separate, pulvi- tending through the lamina, but more prominent on nate, subcuticular, composed of medium to dark the surface initially infected; translucent, becoming brown pseudoparenchymatic textura angularis, up to medium brown, slightly raised, rough and corky with 200 tim wide and 60 ,Im high. Asci distributed irreg- age; border slightly raised, appearing whitish due to ularly throughout the ascoma, ovoid to subglobose, the ruptured epidermis; margin present, thin, red- thick-walled, 8-spored, sessile, hyaline, 16-28 X 13- purple. Lesions also develop on immature shoots, ap- 18 ,im. Ascospores hyaline, broadly ellipsoid with 372 MYCOLOGIA

'/ 7'

6': ;"

; ,,-'"' ..i . V.0j

,~

1.?.~'.:~ . . . ~'.;.,? .... b ~..;. . . , , 10mm; 9 = 4 mm. 10, 11. cospermum?~"r hybrid Bars 10 75:-:'...~,...," mm 11 = 2.5 mm. ,i

FIGS~~~~~~~~~~~~~~~~~~~~~~~~. ?-11 10 ram~;Ci.1,1.L?oe~hbi.Bas 4 0 9 . a;1 : -25mn

FIGS. 7-11. Foliar and stem disease symptoms of Elsinoe leucospermzon different hosts. 7-9. Sernrura florzda. Bars: 7, 8 = I

SWART ET AL: ELSINOE SPECIES DIFFERENTIATION 373

I

- 121 S FIGS.12-14. Foliar and stem disease symptoms of Elsinoe leucospermion Leucadendron spp. 12. Leucadendron cv. Silvan Red. Bar = 8 mm. 13. Lcd. thymifolium.Bar = 6 mm. 14. Lcd. laureolum. Bar = 7 mm. rounded to obtuse ends, 1-4 transversely septate, and leavesof Lsp. cordifolium(Salisb. ex Knight)Fourc., 11 May 1-2 vertical septa; oblique septa rare; constricted at 1996, L. Swart (HOLOTYPE,PREM 54980), ex-type cul- tures median septum, (10-)12-14(-19) X 4-5 p,m. Myce- (STE-U1378-1380); Kleinmond,on stems and leaves of L. Benic lium internal, consisting of branched, septate, hya- Leucospermumsp., 1983, (STE-U 1605-1606); On stems and leaves of 8 L. line to brown hyphae, 3.5-4.5 ,pm diam. Conidiomata Lsp. cordifolium, May 1996, Swart (STE-U1610); Stellenbosch,on stems and leaves of acervular, foliicolous but primarily caulicolous, Lsp. 6 1996, L. Swart raised, at of medium cordifolium, Aug (STE-U 1607-1608); coalescing maturity, composed Constantia,on stems and leaves of 17 brown textura to 200 ,umwide and 1 Lsp. cordifolium, Sep angularis, up 1996, L. Swart (STE-U 1604). McGregor,on stems and mm brown, long. Conidiophoressubcylindrical, light leaves of , 8 Sep 1998, L. Swart (STE-U 20-30 x 3-6 ,xm. Conidi- verruculose, 0-2-septate, 2043-2044). EASTERNCAPE: Humansdorp, on stems and cells ogenous enteroblastic, polyphialidic, with 1-2 in- leaves of Lcd. pubibracteolatumI. Williams x Lcd. chamelaea tegrated loci, light brown, verruculose, ampulliform (Lam.) I. Williams, 2 Sep 1997, L. Swart (STE-U 2033). to doliiform, 10-15 x 3-4 ,um. Conidia hyaline, gran- AUSTRALIA.Victoria, Red Hill, on stemsand leavesof Lcd. ular, aseptate, ellipsoid, apex obtuse, constricting at thymifolium (Salisb. ex Knight) I. Williams, 8 Aug 1994, I. base to a subtruncate locus, (2-)5-7(-8) x (1-)2.5- Pascoe & A. Ziehrl (VPRI 20330a); Gembrook, on stems and 3 ,im in vivo, 5-7 X 2-3 ,xm in vitro in Fries's me- leaves of Leucospermumsp., 8 Mar 1995, I. Pascoe & A. Ziehrl dium. Cultures on MEA produced irregular, erum- (VPRI20521a); on stems and leaves of Leucadendronsp., 8 Mar I. Pascoe & A. on stems pent, folded colonies, with smooth, sinuate margins, 1995, Ziehrl, (VPRI 20522a); and leaves of 8 A. Ziehrl without aerial mycelium; surface red (71i) to blood Lsp. cordifolium, Aug 1996, (VPRI on stems and leaves of Leucadendron 8 red (71k), sienna (13i) or coral (3'b). Colonies 21187a); sp., Aug A. Ziehrl Merricks on stems reached 7-12 mm in diam on MEA after 1 mo in the 1996, (VPRI 21189a); North, and leaves of Lcd. laureolum(Lam.) Fourc.,9 Mar 1995, I. dark at 25 C. Cardinal for were temperatures growth Pascoe & A. Ziehrl (VPRI 20515a). on stems and min above 5 20-25 max above 35 C. Bunyip, C, opt C, leaves of Leucadendron 9 Mar 1995, I. Pascoe & A. Ziehrl Hosts. Leucadendron sp., spp., Leucospermumspp., Ser- (VPRI 20526a). Victoria, on stems and leaves of Serruria ruria florida (Thunb.) Salisb. ex Knight. florida 12 May 1995, W Tregea (VPRI 20591a). On stems Anamorph. Sphacelomasp. and leaves of Serruria florida, 8 Aug 1996, D. Tricks & A. Distribution. Australia, South Africa, USA (Califor- Zierhl (VPRI 21188a). USA. CALIFORNIA:San Diego, on nia), Zimbabwe. stems and leaves of Lsp. cordifolium cv. Vlam, 9 Apr 1998, Specimensexamined. SOUTH AFRICA. : L. Swart (STE-U 2041); on stems and leaves of Lsp. cordi- Harold PorterBotanical Gardens, Betty's bay, on stems and folium, 13 Apr 1998, L. Swart (STE-U 2042). ZIMBABWE. 374 MYCOLOGIA

i -~~-4 0 G

*111 15 r

/ C00

FIGS.15, 16. Elsinoe leucospermi.15. Asci and ascospores. 16. Conidiophores and conidia of the Sphacelomastate. Bar = 10 Mxm.

Rusape, on stems and leaves of Lsp. glabrum E. Phillips x hospite. Conidiophori reducti ad cinidiogenas cellulas. Lsp. tottum (L.) R. Br. cv. Scarlet Ribbon, 6 Mar 1998, L. Conidiogenae cellulae poliphialidibus, cum 1-3 locis inte- Swart (STE-U 2039). On stems and leaves of Lsp. reflexum gris, hyalinae, leves, subcylindratae ad doliiformes, 6-12 X H. Buek ex Meisn. var. luteum, 6 Mar 1998, L. Swart (STE- 4-6 jm. Conidia hyalina, granulata, aseptata, ellipsoidea, U 2040). cum obtoso apice, cum basi rotundata ad subtruncatam, (5-) 6-7(-8) X 2-3(-4) ,Im in vivo. Lesions on leaves and petioles, separate, becoming Elsinoe Crous et Swart, nov. FIGS. 17-20 proteae sp. confluent, circular, 2-12 mm diam, amphigenous, Ascomata dispersa, separata, pulvinata, subcuticularia, not extending through the leaf lamina, raised with a composita ex textura angularis pseudoparenchymatica me- discrete border, white-gray, with black ascomata visi- dia vel atrobrunnea, usque ad 250(pm lata et 80 prm alta. ble to the naked eye. Ascomata scattered, separate, Asci diffusi irregulariter ubique in ascoma, ovoidei ad glo- subcuticular, of medium to dark bosos, crasso pariete, octospori, sessiles, hyalini, 24-45 x pulvinate, composed brown textura to 19-24 jim. Ascospori hyalini ad olivaceosi, late ellipsoidei pseudoparenchymatic angularis, up wide and 80 ptm Asci distributed cum extremis rotundatis, cum 3-4 septis transversalibus et 250 ILm high. irreg- 1-3 septis verticalibus, septum obliquum interdum; pler- ularly throughout the ascoma, ovoid to subglobose, umque leviter constricti ad septum medianum, (14-)16- thick-walled, 8-spored, sessile, hyaline, 28-45 x 19- 17(-20) X (5-)6-7 fIm. Sphaceloma stata non observata in 24 Lm. Ascospores hyaline to olivaceous, broadly ellip- SWART ET AL: ELSINOE SPECIES DIFFERENTIATION 375

Harold Porter BotanicalGardens, Betty's bay, on leaves of (L.) L., 15 Feb 1996, P.W Crous (HOLO- TYPE,PREM 54979), ex-typecultures (STE-U1348-1350). Sir Lowry'sPass Village, on stem of Protearepens (L.) L., 21 Feb 1978,J.E. Taylor(PREM 56547).

Sphaceloma protearum L. Swart et Crous, sp. nov. FIGS.21-23 Myceliuminternum, compositum ex hyphis ramulosis, septatis,hyalinis vel brunneis, 3-4 Ixmdiam. Conidiomata acervulatain caulibus,sporodochia in foliis, compostiaex texturaangularis media brunnea, usque ad 100 Ixmlata et 1mm longa. Conidiophora subcylindrataad doliiformia, pallidavel nedia brunnea,verruculosa, 0-2 septa, 12-20 X 5-6 ixm.Conidiogenae cellulae poliphialidibus,cum 1-2 lo- cis integris, pallidae brunneae, verruculosae,doliiformes, 5-12 X 4-6 I,m. Conidia hyalina,granulata, aseptata, ellip- soidea, cum obtoso apice, cum basi constrictaad locum sub- truncatum,(3.5-)5-6(-7) X (1.5-)2-2.5 jim. Leaf spots circular, reddish, 5-15 mm diam, covered with erumpent conidiomata, appearing as reddish sporodochia on the necrotic tissue; commonly asso- ciated with Shepherd's crook symptoms on young shoots, leading to a blackening, withering and death of shoot tips. Mycelium internal, consisting of branched, septate, hyaline to brown hyphae, 3-4 ,xm diam. Conidiomata acervular on stems, or sporodo- 18 chial on leaves, composed of medium brown textura angularis, up to 100 uim wide and 1 mm long. Conid- to to medium FIGS.17, 18. Foliar disease symptoms of Elsinoe proteaeon iophoressubcylindrical doliiform, light X 5-6 Protea cynaroides. Bars: 17 = 1.5 mm; 18 = 2 mm. brown, verruculose, 0-2-septate, 12-20 jxm. Conidiogenous cells enteroblastic, polyphialidic, with 1-2 integrated loci, light brown, verruculose, doli- soid with rounded ends, with 3-5-transverse septa, iform, 5-12 X 4-6 ,xm. Conidia hyaline, aseptate, el- and 1-3 vertical septa; oblique septa sometimes pre- lipsoid, apex obtuse, constricting at base to a sub- sent; mostly slightly constricted at the median sep- truncate locus, (3.5-)5-6(-7) X (1.5-)2-2.5 jLm in tum, (14-)16-17(-20) X (5-)6-7 jxm. Sphaceloma vivo, not in vitro in Fries's medium. Cul- state not observed on host, but induced in Fries's sporulating tures on MEA producing irregular, erumpent, folded medium. Conidiophores reduced to conidiogenous colonies with sinuate, smooth margins, without aerial cells. Conidiogenous cells enteroblastic, polyphialidic, surface blood red (71k-71m), with a with 1-3 loci, smooth, mycelium; slight integrated hyaline, subcylindri- diffuse red visible in the with cal to doliiform, 6-12 X 4-6 jxm. Conidia pigment becoming agar hyaline, Colonies 11 mm diam on MEA after 1 obtuse, base round- age. reaching granular, aseptate, ellipsoid, apex mo in the dark at 25 C. Cardinal for ed to subtruncate, (5-)6-7(-8) X 2-3(-4) jim in temperatures were min above 5 max vivo. Cultures on MEA growth C, opt 20-25 C, below produce irregular, erumpent, 30 C. folded colonies with sinuate, smooth aerial margins; Hosts. Protea whitish, surface rose to red (71b- spp. mycelium sparse; Distribution. Zimbabwe. 71i); older colonies surrounded a diffuse red by pig- examined. ZIMBABWE. MUTARE: Gomo Re- ment. Colonies reached 12 mm in diam on MEA af- Specimens miti Farm, on leaves and stems of X susannae ter 1 mo in the dark at 20 C. Cardinal temperatures cv. Sylvia, 5 Mar 1998, L. Swart (HOLOTYPE,PREM for were min above 5 15-20 max growth C, opt C, 56301), ex-type culture (STE-U 2037). Mutare, on stems below 30 C. and leaves of R. Br. X Protea susannae E. Hosts. P cynaroides,P repens. Phillips cv. Pink Ice, 5 Mar 1998, L. Swart (STE-U2035). Anamorph. Sphacelomasp. On stems and leaves of Proteaeximia (Salisb. ex Knight) Distribution. South Africa. Fourc. X Protea susannae cv. Sylvia, 5 Mar 1998, L. Swart Specimensexamined. SOUTH AFRICA. WESTERN CAPE: (STE-U2037). On stems and leavesof Proteamagnifica Link 376 MYCOLOGIA

7 I

I,

FIGS.19, 20. Elsinoeproteae. 19. Asci and ascospores. 20. Vertical section through an ascoma on leaf tissue. Bars = 10 jm. SWART ET AL: ELSINOESPECIES DIFFERENTIATION 377

t I

FIG. 23. Conidiophores and conidia of Sphacelomapro- tearum. Bar = 10 ,xm.

DISCUSSION

The present study provides the first evidence that sev- eral distinct species of Elsinoe are associated with scab diseases of Proteaceae. This is not totally unexpected, as the general symptomatology of the disease on Pro- tea and Banksia is totally different from that observed on Leucospermum, Leucadendron and Serruria. More- over, the symptoms on Protea in South Africa differ significantly from the symptoms on Protea in Zimbab- we. The ascus and ascospore morphology of the El- FIGS.21, 22. Foliarand stem disease symptomsof Spha- sinoe teleomorph obtained on three host genera, celoma protearum on a Protea sp. Bars: 21 = 2 mm; 22 = 9 namely Leucospermum, Protea, and Banksia suggest mm. that they represent three distinct species. These observations are corroborated by the se- quence data of the 5.8S and ITS rDNA, which also x Protea susannae cv. Susara, 5 Mar 1998, L. Swart (STE-U suggest that these isolates represent three distinct 2038). Harare,on stemsand leavesof Proteaeximia X Protea species. Based on the DNA sequences for the Elsinoe susannaecv Sylvia,6 Mar1998, L. Swart(STE-U 2036). Ban- isolates from Leucadendron, Leucospermum, and Ser- ket, on stems and leaves of Protea compacta X Protea susan- ruria in South Africa and Australia, and isolates from nae cv Pink 9 Mar L. Swart STE- Ice, 1998, (PREM56302, Leucospermumin California and Zimbabwe, we con- U 2034). AUSTRALIA.Victoria, Red Hill, on leaves of Pro- clude that these isolates are representative of the tea sp., 8 Aug 1994, I. Pascoe & I. Porter (VPRI 20320a, same species. These data further suggest that some 20320b, undeterminedElsinoe sp.). On leaves of Proteacy- Elsinoe are less than others, and naroides,8 Aug 1994, I. Pascoe (VPRI 20321a, undeter- species host-specific can occur on more than one in the same fam- mined Elsinoe sp.). Bunyip, on leaves of Protea compacta, 8 genus Mar 1995, I. Pascoe & A. Ziehrl (VPRI 20525a, undeter- ily. mined Elsinoe sp.); on leaves of Protea compacta, 9 Mar The present study demonstrates that in South Af- 1995, I. Pascoe & A. Ziehrl (VPRI 20549a, undetermined rica there are at least two species of Elsinoe causing Elsinoe sp.). diseases of Proteaceae. The scab disease induced by 378 MYCOLOGIA

E. leucospermiis an important disease of Leucosper- been supported by the molecular data. As the Elsinoe mum spp., especially Lsp. cordifoliumin South Africa. state is seldom collected for these fungi, molecular This disease, however, has been found to vary in se- studies are required to determine the genetic varia- verity on different cultivars and seedlings. Of the var- tion between isolates of Elsinoe from various hosts in ious Leucospermum cultivars, Lsp. cordifolium cv. the family Proteaceae. Vlam, Red Sunset and cv. Gold Dust have been found Although scab disease has also been reported from to be extremely susceptible (Knox-Davies et al 1986). Proteaceae occurring in Hawaii (Protea Disease Let- Because the same pathogen occurs on Leucospermum ter Management Group, University of Hawaii, un- and Leucadendronspp. in Australia, California, South publ), no cultures were available for inclusion in the Africa and Zimbabwe, communal programs for the present study. More detailed collections therefore are selection of resistant hybrids and the implementation required from Australia, California, Hawaii, South Af- of different fungicide regimes for disease control can rica, and Zimbabwe to suitably determine the differ- now be initiated in these countries. ences that exist between the isolates, as well as the The occurrence of an Elsinoe sp. on Protea cv. Pink host ranges and distribution of the various species of Ice in Australia was the first record of a scab disease Elsinoe associated with scab diseases of Proteaceae in on this host (Ziehrl et al 1995). It also has been ob- these countries. served on P cynaroides, P compacta and on a Protea sp. in Australia, and on P mundii in California, USA. KEY TO SPECIES OF ELSINOEoccurring on proteaceae The leaf symptoms on P cynaroides from Australia 1. Occurringon Banksia...... 2 correlate with those observed on this host in South 1. On other Proteaceae...... 3 Africa. no cultures or ma- 2. Leaf spots on B. serrata;colonies gray olivaceous, Unfortunately, teleomorph at 15 C ...... E. terial from P. in Australia were available for optimalgrowth banksiae cynaroides 2. Lesions on leaves and veins of B. colo- with the two known prionotes; comparison species presently nies blood red, optimal growth at 20-25 C .... from Protea in South Africa and Zimbabwe, respec- ...... Elsinoesp. tively. Symptoms similar to those on P cynaroideswere 3. Occurringon Leucadendron,Leucospermum and Ser- observed on leaves of P repens (PREM 56547) in ruria;ascospores with 1-4 transverse,1-2 verticaland South Africa, with ascospores (12-17 X 4.5-7 IJm) rarelyany oblique septa, (10-)12-14(-19) X 4-5 jim; resembling those of E. proteae.However more isolates colonies blood red, optimal growth at 20-25 C; co- X from Protea spp. in South Africa will be required to nidia (2-)5-7(-8) (1-)2.5-3 lm .... E. leucospermi 3. on Protea...... 4 determine the variation in this species. Occurring 4. Leaf spots on mature leaves;ascospores with 3-5 The type material of Elsinoe banksiae was obtained transverse,1-3 verticaland rarelyany oblique sep- from leaf of Banksia serrata (VPRI 13976) spots ta (14-)16-17(-20) X (5-)6-7 Vm; colonies rose 3, A culture obtained from another collec- (FIGS. 4). to red, with diffuse red pigment, optimal growth tion from this host was used in the (STE-U 2678) at 15-20 C; Sphacelomastate not observedon host molecular studies. Elsinoe banksiae is regarded as dis- ...... E. proteae tinct from the Sphacelomaspecies occurring on Bank- 4. Shepherd'scrook and leaf spot symptomson ju- sia prionotes (VPRI 21 00b, STE-U 1508), that is more venile growthflushes, leading to shoot blight;col- onies blood red with diffuse red commonly associated with prominent stem lesions slight pigment, at 20-25 C; Elsinoestate not ob- than leaf spots (FIG. 2). Furthermore, on MEA cul- optimal growth served on host ...... Sphacelomaprotearum tures of E. banksiae are greenish gray, while those from B. prionotes are red. Cultures from these hosts also differ in temperature requirements for growth, with E. banksiae growing best at 15 C, and the Spha- ACKNOWLEDGMENTS celoma sp. from B. prionotes reaching optimal growth The authors gratefullyacknowledge J. Walkerfor making at 20-25 C, and also growing at 30 C, thus appearing his Elsinoecollections from Banksiaavailable for inclusion to be tolerant of higher temperatures than E. bank- in this study.We are also thankfulfor the USDA-FAS-ICD- RSED for siae. Although we believe the Sphaceloma sp. on B. grant 98IARA6578,as well as financial support from the National Research the prionotes to be new, we presently lack sufficient ma- Foundation, European terial to describe it as such. Union and the South African Protea Producersand Ex- portersAssociation (SAPPEX). A comprehensive classification of the Elsinoi spe- cies occurring on Proteaceae is attainable through a molecular phylogenetic approach. ITS sequence data LITERATURE CITED Banksia and isolates separate Protea, Leucospermum Benic LM,Knox-Davies PS. 1983. Scabof Leucospermumcor- from each other and correlate with morphology. The difoliumand other Proteaceaecaused by an Elsinoesp. value of these properties as taxonomic characters has Phytophylactica 15:95-107. SWART ET AL: ELSINOE SPECIES DIFFERENTIATION 379

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