Mycologia, 106(3), 2014, pp. 415–423. DOI: 10.3852/13-202 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

Exobasidium maculosum, a new species causing leaf and fruit spots on blueberry in the southeastern USA and its relationship with other Exobasidium spp. parasitic to blueberry and cranberry Marin Talbot Brewer1 Ashley N. Turner Phillip M. Brannen

phylogenetically from other described species and is described herein as Exobasidium maculosum. Within the southeastern USA, clustering based on host species, host tissue type (leaf or fruit ) or geographic region was not detected; however, leaf spot isolates from lowbush blueberry were genetically different and likely represent a unique species. Key words: Basidiomycota, Ericaceae, Exobasidiomycetes, plant pathogen, SEM

Department of Plant Pathology, University of Georgia, 120 Carlton Street, Athens, Georgia 30602

William O. Cline Department of Plant Pathology, North Carolina State University, Horticultural Crops Research Station, 3800 Castle Hayne Road, Castle Hayne, North Carolina 28429

Elizabeth A. Richardson

INTRODUCTION

Department of Plant Biology, University of Georgia, 120 Carlton Street, Athens, Georgia 30602

Exobasidium Woronin is a diverse genus of biotrophic plant pathogens in the Ustilaginomycotina that are parasites of the Ericaceae and related families. The genus Vaccinium, which includes wild and commercial blueberry and cranberry species, is susceptible to multiple different species of Exobasidium, many of which are host-specific (Nannfeldt 1981, Begerow et al. 2002, Pia˛tek et al. 2012). Exobasidium species cause plant deformities including leaf spots, witches’ broom, shoot infections characterized by reddened leaves, or galls on leaves, stems, flowers, shoots and buds (Burt 1915; Nannfeldt 1981; Chandramouli 2003; Nagao et al. 2003, 2004, 2006; Sinclair and Lyon 2005). Infections can be annual or perennial and local or systemic and often are characterized by a white, felt-like growth and reddish discoloration. The felt-like layer that forms on the underside of leaves or on the entire surface of plant organs is an exposed hymenium where the basidia and basidiospores are produced. Asexual reproduction occurs when basidiospores bud to produce conidia (Mims and Richardson 1987). Members of the genus are dimorphic with a yeast-like growth form in culture. Host ranges and species limits for Exobasidium are not clearly defined, especially outside Europe (Nannfeldt 1981, Nickerson and Vander Kloet 1997, Pia˛tek et al. 2012). Although Burt (1915) and Savile (1959) proposed broad morphological species concepts within the genus and considered most species to be confined to E. vaccinii (Fuckel) Woronin, Nannfeldt (1981) postulated that species of Exobasidium are symptom specific and specialized to one or a few closely related hosts and, therefore, defined E. vaccinii as restricted to V. vitis-idaea L. (lingonberry). Molecular phylogenetic studies have supported

Abstract: Exobasidium leaf and fruit spot of blueberry (Vaccinium section Cyanococcus) is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA. To determine whether this disease is caused by a new species of Exobasidium, we studied the morphology and phylogenetic relationship of the causal fungus compared with other members of the genus, including the type species E. vaccinii and other species that parasitize blueberry and cranberry (V. macrocarpon). Both scanning electron microscopy and light microscopy were used for morphological characterization. For phylogenetic analyses, we sequenced the large subunit of the rDNA (LSU) from 10 isolates collected from leaf or fruit spots of rabbiteye blueberry (V. virgatum), highbush blueberry (V. corymbosum) and southern highbush blueberry (Vaccinium interspecific hybrid) from Georgia and North Carolina and six isolates from leaf spots of lowbush blueberry (V. angustifolium) from Maine and Nova Scotia, Canada. LSU was sequenced from isolates causing red leaf disease of lowbush blueberry and red leaf spot (E. rostrupii) and red shoot (E. perenne) of cranberry. In addition, LSU sequences from GenBank, including sequences with high similarity to the emerging parasite and from Exobasidium spp. parasitizing other Vaccinium spp. and related hosts, were obtained. All sequences were aligned and subjected to phylogenetic analyses. Results indicated that the emerging parasite in the southeastern USA differs morphologically and Submitted 27 Jun 2013; accepted for publication 27 Nov 2013. 1 Corresponding author. E-mail: [email protected]

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Nannfeldt’s species concept (Begerow et al. 2002, Kennedy et al. 2012, Pia˛tek et al. 2012). Exobasidium leaf and fruit spot is an emerging disease of blueberry in the southeastern USA that has been reported on rabbiteye blueberry (V. virgatum Aiton), highbush blueberry (V. corymbosum L.) and southern highbush blueberry (Vaccinium interspecific hybrid) in Georgia, North Carolina and Mississippi. The fruit stage of this disease, which can cause significant economic losses due to unmarketable fruit, was first described on highbush blueberry in North Carolina (Cline 1998). The cause of Exobasidium leaf and fruit spot of blueberry has not been identified previously to species and it is unknown how it relates to other Exobasidium species. An Exobasidium leaf spot occurring sporadically on lowbush blueberry (V. angustifolium Aiton) in Canada also has been described (Nickerson and Vander Kloet 1997), but the causal fungus was not identified to species. The symptoms are similar to the disease in the southeastern USA, but fruit are rarely affected. Lowbush blueberry is also susceptible to red leaf, a systemic and perennial shoot disease caused by Exobasidium (Hilborn and Hyland 1956, Lockhart 1958, Mims and Nickerson 1986, Caruso and Ramsdell 1995). The causal fungus of red leaf disease is reported in the literature as E. vaccinii; however, it is likely a different species according to the host and symptom-specific species concept presented by Nannfeldt (1981). The objectives of this study were to describe both morphologically and phylogenetically the fungus causing Exobasidium leaf and fruit spot of blueberry in the southeastern USA to determine: (i) whether it is a new species of Exobasidium; (ii) how it is related to other Exobasidium spp., particularly those that cause leaf spots or are parasitic to blueberry and cranberry; and (iii) whether it is differentiated in the southeastern USA by Vaccinium host species, tissue type (leaf or fruit) or geographic region of origin. MATERIALS AND METHODS Sample collection and preservation.—Isolates of Exobasidium from blueberry leaf and fruit spots were obtained from cultivars of rabbiteye blueberry, highbush blueberry and southern highbush blueberry from commercial fields in Georgia and North Carolina (TABLE I). Isolates of Exobasidium from leaf spots on cranberry and lowbush blueberry were obtained from New Jersey and Maine, respectively. Isolations were made by cutting the leaf spot or fruit spot from the plant tissue with a sterile scalpel, affixing the cut tissue with petroleum jelly to the lid of a Petri dish of potatodextrose agar (PDA) and incubating 8–24 h in the dark at 23 C. Five single basidiospores that fell or were ejected onto the agar surface were identified by examination with a dissecting microscope and transferred to a new dish of PDA.

A single developing colony was selected and maintained. Single basidiospore isolates of Exobasidium from leaf spots and red leaf disease of lowbush blueberry and red leaf spot (E. rostrupii Nannf.) and red shoot disease (E. perenne N.L. Nick.) of cranberry from Nova Scotia, Canada, were obtained from the collection of N. Nickerson (Atlantic Food and Horticulture Research Centre, Kentville, Nova Scotia, Canada). All isolates were stored in 30% glycerol at 280 C. Morphological observations.—Fresh material from leaf and fruit spots from rabbiteye blueberry and southern highbush blueberry from Georgia was used for morphological observations. Descriptions of color include hexadecimal RGB codes for clarity. For observations with scanning electron microscopy (SEM), infected leaves of V. virgatum cv. Premier were cut into 7 mm segments with a razorblade and fixed in 2.5% v/v glutaraldehyde in 0.05 M potassium phosphate buffer (pH 6.8) overnight at 4 C. Samples were washed with buffer and post-fixed 2 h at 4 C in similarly buffered 1% osmium tetroxide. Samples were rinsed in distilled water, dehydrated in an ethanol series (25%, 50%, 75%, 95%) to 100% and critical-point dried with a Samdri model 780-A Critical Point Dryer (Tousimis, Rockville, Maryland). Leaf pieces were mounted on aluminum stubs with sticky carbon tabs and sputter-coated (SPI Module Sputter Coater, Structure Probe Inc., West Chester, Pennsylvania) with gold-palladium and viewed with a Zeiss 1450EP variable pressure SEM (Carl Zeiss MicroImaging Inc., Thornwood, New York) operating at 15 kV. Measurements of leaf spots and fruit spots from rabbiteye blueberry and southern highbush blueberry were made by hand. Observations by differential interference contrast (DIC) microscopy of germinating basidiospores and conidia were made at 20 and 48 h. Slides were prepared by pipetting a thin layer of PDA onto a glass slide, placing the slide inside a Petri dish and affixing a leaf spot from V. virgatum cv. Premier to the lid as described above. All measurements of microscopic features were made with ImageJ (http://rsb. info.nih.gov/ij). Specimens were deposited at the Fungal Herbarium at the University of Florida (FLAS) and the American Type Culture Collection (ATCC). DNA sequencing and phylogenetic analyses.— DNA was purified from colonies derived from single basidiospores incubated in the dark at 23 C for 2–3 wk. Approximately 50 mg fresh weight of cells was harvested and added to 1 mL lysis buffer (50 mM EDTA pH 8, 100 mM Tris pH 8, 3.5% SDS, 250 mg/mL proteinase K, 1% sodium bisulfite), vortexed 1 min and incubated at 65 C for 15 min. Samples were vortexed briefly and centrifuged at 14 000 g for 5 min. The supernatant was removed and added to 200 mL 7.5 M sodium acetate, vortexed 10 s, placed on ice for 15 min and centrifuged at 14 000 g for 3 min. The supernatant was added to 700 mL isopropanol, gently mixed to precipitate the DNA and centrifuged at 14 000 g for 5 min. The supernatant was discarded and the pellet was rinsed twice with 70% ethanol, dried and resuspended in 100 mL sterile H2O. The D1–D2 region of LSU rDNA was amplified with the primers NL1 and NL4 (O’Donnell 1993). PCR was carried

macrocarpon (CB) macrocarpon (CB) angustifolium (LB) angustifolium (LB)

red red red red

shoot disease shoot disease leaf disease leaf disease

Bacon County, Georgia Sampson County, North Carolina Georgia Bacon County, Georgia Bacon County, Georgia Bladen County, North Carolina Bacon County, Georgia Sampson County, North Carolina Sampson County, North Carolina Sampson County, North Carolina Waldo County, Maine Kings County, Nova Scotia Kings County, Nova Scotia Shelburne County, Nova Scotia Kings County, Nova Scotia Lunenburg County, Nova Scotia Kings County, Nova Scotia Burlington County, New Jersey Burlington County, New Jersey Kings County, Nova Scotia Halifax County, Nova Scotia Kings County, Nova Scotia Hants County, Nova Scotia

Location

Jul 1981 (N. Nickerson) Jul 1982 (N. Nickerson) Jun 1991 (N. Nickerson) Jun 1991 (N. Nickerson)

Jun 2012 (J. Polashock)

Aug 1980 (N. Nickerson) Jun 2012 (J. Polashock)

Jul 1992 (N. Nickerson) Jul 1992 (N. Nickerson)

Aug 2012 (S. Annis) Jul 1988 (N. Nickerson) Jul 1986 (N. Nickerson) Jul 1989 (N. Nickerson)

May 2012 (M. Brewer)

May 2012 (M. Brewer)

May 2012 (M. Brewer) May 2012 (M. Brewer)

2011 (H. Scherm) May 2012 (M. Brewer) Apr 2012 (M. Brewer) May 2012 (M. Brewer)

Apr 2012 (M. Brewer) May 2012 (M. Brewer)

Collection date (collector)

E81-3 E82-1 E91-3 E91-5

E12CNJ1-2

E80-1 E12CNJ1-1

E92-26 E92-50

E12ME1-13 E88-9 E86-1 E89-5

E12NCLC1-45

E12NCLC1-44

E12FS2-1 E12NCLC1-15

E11-1 E12A7-4 E12B2 E12FS1-10

E12A1-1 E12NCLC1-22

Isolate name

KF134418 KF134419 KF134423 KF134424

KF134409

KF134417 KF134408

KF134426 KF134425

KF134412 KF134421 KF134420 KF134422

KF134416

KF134415

KF134411 KF134413

KF134404 KF134406 KF134407 KF134410

KF134405 KF134414

Accession no.

RB 5 rabbiteye blueberry, SH 5 southern highbush blueberry, HB 5 highbush blueberry, LB 5 lowbush blueberry, CB 5 cranberry; cultivar (cv.), when known, is listed. b GenBank accession number for the large subunit of the rDNA. c Two distinct species of Exobasidium are not formally named that are designated as either Exobasidium sp. A or B for clarity.

a

V. V. V. V.

red leaf spot

V. macrocarpon (CB)

E. rostrupii

E. perenne E. perenne Exobasidium sp. B Exobasidium sp. B

red leaf spot red leaf spot

V. macrocarpon (CB) V. macrocarpon (CB)

E. rostrupii E. rostrupii

spot spot spot spot

leaf spot leaf spot

leaf leaf leaf leaf

V. angustifolium (LB) V. angustifolium (LB)

sp. sp. sp. sp.

Exobasidium sp. A Exobasidium sp. A

Exobasidium Exobasidium Exobasidium Exobasidium

(LB) (LB) (LB) (LB)

leaf spot

V. corymbosum cv. Duke (HB)

E. maculosum

angustifolium angustifolium angustifolium angustifolium

leaf spot

V. V. V. V.

fruit spot leaf spot

Vaccinium hybrid cv. Star (SH) Vaccinium hybrid cv. Legacy (SH) V. corymbosum cv. Duke (HB)

E. maculosum E. maculosum

Ac A A A

fruit spot leaf spot leaf spot fruit spot

V. virgatum (RB) V. virgatum cv. Climax (RB) V. virgatum cv. Tifblue (RB) Vaccinium hybrid cv. Star (SH)

maculosum maculosum maculosum maculosum

E. E. E. E.

E. maculosum

leaf spot leaf spot

Symptom

V. virgatum cv. Premier (RB) V. virgatum cv. Columbus (RB)

Host species of origina

E. maculosum E. maculosum

Exobasidium species

TABLE I. Origin, collection date and GenBank accession number for the large subunit of the rDNA (LSU) for Exobasidium isolates from blueberry and cranberry (Vaccinium spp.)

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out in 50 mL reaction volume with 5 mL 103 ExTaq buffer (Takara Bio Inc., Otsu, Shiga, Japan), 5 mL 2.5 mM dNTPs, 2.5 mL 10 mM primer, 1 U ExTaq (Takara Bio Inc.), and 1 mL DNA template (10–200 ng/mL). Thermal-cycling conditions were 94 C for 2 min, 40 cycles of 94 C for 30 s, 55 C for 30 s, 72 C for 30 s followed by 72 C for 5 min. Amplification of single PCR products within the expected size range was confirmed by electrophoresis on a 1% (w/v) agarose gel. PCR products were purified with QIAquick spin columns (QIAGEN, Valencia, California). All DNA fragments were sequenced at the Georgia Genomics Facility (Athens, Georgia) with the Applied Biosystems Automated 3730xl DNA Analyzer with BigDye 3 Terminator chemistry. All gene regions were sequenced in both directions with NL1 and NL4 primers. Sequences were aligned and manually edited in Geneious 6 (Biomatters Ltd., Auckland, New Zealand). New sequences were deposited in GenBank under accession numbers KF134404–KF134426. In addition to sequences obtained in this study (TABLE I), published sequences of other species of Exobasidium, including ones with high similarity based on basic local alignment search tool (BLASTN; Altshul et al. 1990) to those causing leaf and fruit spots from southeastern USA and the type species of E. vaccinii from V. vitis-idaea, were obtained from the sequence database NCBI GenBank. The total dataset included 23 LSU sequences of Exobasidium obtained in this study and 18 LSU sequences from GenBank including Graphiola cylindrica, which was used as outgroup. Sequences were assembled in Geneious 6, and manual adjustments were made as needed. The alignment was deposited in TreeBASE under accession number 14780. Phylogenies were reconstructed by Bayesian inference (BI) in MrBayes 3.2 (Huelsenbeck and Ronquist 2001, Ronquist and Huelsenbeck 2003) and by maximum likelihood (ML) in MEGA 5 (Tamura et al. 2011). For both methods the Kimura 2-parameter model of evolution including estimation of invariable sites and assuming a gamma distribution (K2+I+G) as determined most appropriate in MEGA 5 was implemented. For BI, the analysis was based on parallel runs of 5 000 000 generations with four chains, sampling every 1000 generations. The 50% majority consensus tree was visualized with FigTree 1.4 (http://tree. bio.ed.ac.uk/software/figtree). Support for internal branches of the ML tree was determined by 1000 bootstrap replicates. Only clades with 95% posterior probability for BI or 70% bootstrap for ML analyses were recognized and reported.

RESULTS Phylogenetic analyses.—The total dataset consisted of 41 sequences and 573 characters of which 118 were variable and 60 were phylogenetically informative. Phylogenetic reconstruction with BI and ML produced congruent topologies except a single node separating E. pachysporum was not inferred by ML. The leaf and fruit spot isolates from blueberry, labeled Exobasidium maculosum and Exobasidium sp. A (FIG. 1), form a distinct clade. Within this clade the

isolates from the southeastern USA cluster separately from isolates that cause leaf spots on lowbush blueberry in Maine and Nova Scotia. Within the southeastern USA there is no apparent clustering based on Vaccinium host species, geographic region or host tissue (leaf or fruit) of origin. The cause of red leaf disease on lowbush blueberry, labeled Exobasidium sp. B (FIG. 1), is distinct from E. vaccinii from V. vitis-idaea and from the clade of isolates that cause leaf and fruit spot diseases on lowbush blueberry and other blueberry hosts. Both E. perenne and E. rotrupii, which cause different symptoms on cranberry, are distinct from each other and from other Exobasidium species that affect blueberry. TAXONOMY Exobasidium maculosum M.T. Brewer, sp. nov. FIGS. 2, 3 MycoBank MB804774 Hymenial layer on surface of fruit spots or abaxial surface of leaf spots; leaf spots circular, chlorotic, 2– 14 mm diam, pale yellow (EEE8AA) to pale green (98FB98) turning crimson (DC143C) to maroon (800000) with age, adaxial surface concave, abaxial surface white to pale yellow, raised and felt-like. Fruit spots pale green and leathery, turning powdery and pale yellow to crimson or maroon with age. The number of leaves with spots increased toward the interior and lower portions of affected bushes. Host symptoms aside from leaf and fruit spots were not observed. Basidia 8.5–11.5 3 5–6.5 mm, cylindrical, emerging from lower epidermis, straight or slightly curved at base, arranged in clusters of varying stages, 5–6-sterigmate. Sterigmata 2–3 3 1–2 mm (at base). Basidiospores 8–10 3 3–3.5 mm, hyaline, smooth, ellipsoid to banana-shaped, hooked at base, distinct knob at hila appendix, often transversely one-septate after detachment from basidium. Germination occurs at both ends of basidiospores, giving rise to conidia or hyphae. On PDA, most basidiospores produce conidia that give rise to more conidia; however, some germinate to form up to 30 mm hyphal filaments that do not branch. Conidia 7–9 3 1–1.5 mm, ellipsoid, hyaline, smooth, aseptate, arise from basidiospores or other conidia. Hyphae 1–2.5 mm diam, abundant within leaf spongy mesophyll, some hyphae on abaxial surface, thickened tissue layer throughout leaf epidermis. Colonies derived from single basidiospores on PDA incubated in the dark at 23 C showed irregular, radial growth that is moist, wrinkled and superficial with no penetration of the agar, beige (F5F5DC) to peach (FFDAB9) front and reverse with no pigmentation in the media, growth is slow with colonies reaching 0.5–1 cm diam in 1 month; 1-mo

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FIG. 1. Bayesian inference 50% majority rule consensus tree based on LSU rDNA of Exobasidium maculosum and related species rooted with Graphiola cylindrica. Posterior probabilities $ 95% are above branches, while ML bootstrap values $ 70% are below branches. The maximum likelihood (ML) tree has the same topology except the branch separating E. pachysporum was not inferred. Host species are in parentheses and the symptom from which the isolates were derived are to the right: LS 5 leaf spot; FS 5 fruit spot. The locations of origin are indicated for blueberry spot isolates: GA 5 Georgia, NC 5 North Carolina, ME 5 Maine, CAN 5 Nova Scotia, Canada. Sequences generated in this study are indicated by boldface. GenBank accession numbers are given for sequences obtained in previous studies.

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FIG. 2. Morphological features of Exobasidium maculosum. A. Exobasidium leaf spot of blueberry caused by E. maculosum in a commercial planting of rabbiteye blueberry (Vaccinium virgatum) in Bacon County, Georgia. B. Adaxial (left) and abaxial (right) surface of E. maculosum on leaves of V. virgatum. C. Exobasidium fruit spot on southern highbush blueberry (Vaccinium interspecific hybrid cv. Star). D. Colonies of E. maculosum derived from single basidiospores incubated on PDA in the dark at 23 C for 25 d. E, F. Differential interference contrast (DIC) micrographs of basidiospores of E. maculosum ejected from leaf spots onto PDA and incubated in the dark at 23 C. E. Conidial production after 20 h incubation. F. Conidial production after 48 h incubation. White bars 5 1 cm, black bars 5 10 mm.

old colonies consist solely of single-celled conidia with no hyphae or pseudohyphae present. Type: USA: Georgia, Bacon County, commercial planting of blueberry in Alma, leaf spot on Vaccinium virgatum cv. Premier, collected by M.T. Brewer Apr 2012 (HOLOTYPE: FLAS-F-58748). The ex-type strain (E12A1-1) is deposited with the American Type Culture Collection (ATCC MYA-4957). Host range and distribution: Southeastern USA, including North Carolina, Georgia and Mississippi, on commercial Vaccinium virgatum (rabbiteye blueberry), V. corymbosum (highbush blueberry), and Vaccinium interspecific hybrid (southern highbush blueberry). Etymology: refers to spotted growth on host tissue.

DISCUSSION The fungus causing Exobasidium leaf and fruit spot of blueberry in southeastern USA is a novel fungus, Exobasidium maculosum, that is morphologically and phylogenetically distinct from previously described species. This pathogen is economically important because affected fruit are unmarketable (Cline 1998), thus it is useful for it to be taxonomically described so it can be identified and referred to properly and without confusion. Members of the genus Exobasidium that form galls and spots cause localized, annual and ephemeral infections (Nannfeldt 1981). We found no evidence that E. maculosum is any different; however, additional studies on the life cycle and biology of this parasite are ongoing. Among isolates

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FIG. 3. Scanning electron micrographs of Exobasidium maculosum causing leaf spots on rabbiteye blueberry (Vaccinium virgatum). A, B. Cross section of a leaf spot with abaxial leaf surface and hymenium toward top. B. Hyphae in spongy mesophyll and lower epidermal layers. C–E. Basidia of E. maculosum at various developmental stages. C. Young basidia emerging from lower epidermis with one at right developing five sterigmata. D. Developing, six-sterigmate basidium with basidiospore initials. E. Developing and mature basidia, some with developing or mature basidiospores. F. Hyphae and germinating basidiospores on abaxial leaf surface.

of E. maculosum from southeastern USA there was no detectable genetic differentiation, indicating that the same species is infecting leaves and fruit, is occurring throughout the region and is causing disease on diverse cultivars of rabbiteye blueberry, highbush blueberry and southern highbush blueberry. Future studies based on analyses of additional regions of the genome may reveal subtle differences in population structure of E. maculosum due to host specialization or geographic subdivision. Isolates of Exobasidium causing a leaf spot on lowbush blueberry in Maine and Nova Scotia, Canada, (referred to as Exobasidium sp. A in FIG. 1) are most similar to, yet are distinct from, E.

maculosum in our phylogenetic analyses. In addition, leaf spot isolates from lowbush blueberry have slight morphological differences with isolates from blueberry in southeastern USA. Based on observations by Nickerson and Vander Kloet (1997), isolates from lowbush blueberry usually produce four sterigmata per basidium, whereas E. maculosum usually produces five (80% of observations) and sometimes six (20% of observations) sterigmata per basidium. Basidiospores were morphologically similar, but those on lowbush blueberry were reported as slightly longer (8–12 mm) and narrower (2–2.5 mm) than what was observed for E. maculosum (8–10 3 3– 3.5 mm). Moreover, Nickerson and Vander Kloet

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(1997) found that leaf spot isolates from lowbush blueberry were pathogenic on few Vaccinium species other than lowbush blueberry but that they were not pathogenic on highbush blueberry, which is a host of E. maculosum; pathogenicity of lowbush isolates on rabbiteye blueberry and southern highbush blueberry was not determined in their study. These genetic and phenotypic differences suggest that the Exobasidium on lowbush blueberry is a different species than E. maculosum, but this needs to be confirmed by analysis of additional loci. The fungus that causes the systemic and perennial red leaf disease of lowbush blueberry (referred to as Exobasidium sp. B in FIG. 1) is clearly different from E. maculosum and the Exobasidium sp. that causes Exobasidium leaf spot on lowbush blueberry. In addition, we determined that it is phylogenetically distinct from E. vaccinii, the name used for the cause of red leaf disease in most studies (Hilborn and Hyland 1956, Lockhart 1958, Mims and Nickerson 1986, Caruso and Ramsdell 1995) based on the broad species concept of E. vaccinii proposed by Burt (1915) and Savile (1959). Sundstro¨m (1964) determined that many members of E. vaccinii from different hosts had narrow host ranges and consequently were later classified as belonging to different species (Nannfeldt 1981). According to Nannfeldt (1981), E. vaccinii refers to the species that causes concave leaf galls, or less often, spots or galls of shoot tips or flower buds, on V. vitis-idaea. Thus, the cause of red leaf disease on lowbush blueberry needs revision. Highbush blueberry is reportedly susceptible to red leaf disease as well, but in contrast to the disease on lowbush blueberry it is rare (Caruso and Ramsdell 1995). Studies to determine whether it too is caused by a different, but closely related species of Exobasidium may be warranted. The Exobasidium isolates from cranberry, E. rostrupii and E. perenne, were clearly different from isolates from blueberry. Of note, the different Exobasidium spp. from blueberry and cranberry clustered based on disease symptom rather than original host species (FIG. 1). For example, E. rostrupii that causes a leaf spot on cranberry and the Exobasidium spp. that cause leaf spots on blueberry are more closely related to each other and to other leaf-spot isolates from different hosts than to E. perenne that causes red shoot on cranberry or the Exobasidium sp. that causes red leaf disease on lowbush blueberry. Similarly the species causing red leaf or red shoot are more closely related to each other and to another red shoot disease caused by E. myrtilli than to the spot isolates on the same host. Our results support an earlier hypothesis that there is independent evolution of Exobasidium lineages based

on infection mode, such as local galls, or systemic shoot infections, in addition to cospeciation of pathogen and host within lineages (Begerow et al. 2002). ACKNOWLEDGMENTS We thank Amy Savelle and Harald Scherm for assistance in collecting samples and Seanna Annis, Paul Hildebrand (N. Nickerson collection) and James Polashock for sending samples or isolates of Exobasidium. We thank Harald Scherm and two anonymous reviewers for helpful comments on earlier drafts of the manuscript. This work was supported by grants from the Georgia Blueberry Commission and Southern Region Small Fruit Consortium.

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