Fusicoccum arbuti sp. nov. causing cankers on Pacific madrone in western North America with

730

Mycologia, 97(3), 2005, pp. 730–741.q 2005 by The Mycological Society of America, Lawrence, KS 66044-8897

Fusicoccum arbuti sp. nov. causing cankers on Pacific madrone in westernNorth America with notes on Fusicoccum dimidiatum, the correct name for

Scytalidium dimidiatum and Nattrassia mangiferae

David F. FarrSystematic Botany & Mycology Laboratory, USDAAgricultural Research Service, Room 304, B011a,10300 Baltimore Ave., Beltsville, Maryland 20705

Marianne ElliottCollege of Forest Resources, University of Washington,Seattle, Washington 98195

Amy Y. Rossman1

Systematic Botany & Mycology Laboratory, USDAAgricultural Research Service, Room 304, B011a,10300 Baltimore Ave., Beltsville, Maryland 20705

Robert L. EdmondsCollege of Forest Resources, University of Washington,Seattle, Washington 98195

Abstract: Pacific madrone (Arbutus menziesii) is abroadleaf evergreen tree native to western NorthAmerica that has been in decline for the past 30years. A fungus has been isolated and was verified asthe cause of cankers on dying trees. It was deter-mined to belong in the genus Fusicoccum, an asexualstate of Botryosphaeria. This genus in both its sexualand asexual states commonly causes canker diseasesof deciduous woody plants. Using morphological andmolecular data the fungus causing cankers on Pacificmadrone is characterized, described and illustratedas a new species of Fusicoccum, F. arbuti D.F. Farr &M. Elliott sp. nov. No sexual state is known for F.arbuti. Evidence from the literature, cultures andspecimens suggests that F. arbuti, often mistakenlyidentified as Nattrassia mangiferae, has been causingmadrone canker since at least 1968. Authentic iso-lates of Nattrassia mangiferae as the synanamorph Scy-talidium dimidiatum were sequenced and determinedto be different from Fusicoccum arbuti and to belongin Botryosphaeria/Fusicoccum. In addition to molec-ular sequence data, the morphology of the pycnidialand arthric conidial states of Nattrassia mangiferae/Scytalidium dimidiatum resembles that of Fusicoccum.Therefore the correct name for Nattrassia mangiferaeand its numerous synonyms (Dothiorella mangiferae,Torula dimidata, Scytilidium dimidiatum, Fusicoccumeucalypti, Hendersonula toruloidea, H. cypria, Exospor-

Accepted for publication 13 Feb 2005.1 Corresponding author. E-mail: [email protected]

ina fawcetii, H. agathidia, and S. lignicola) is Fusicoc-cum dimidiatum (Penz.) D.F. Farr, comb. nov.

Key words: Arbutus, Botryosphaeria, British Co-lumbia, California, Canada, b-tubulin, forest pathol-ogy, Fusicoccum, Hendersonula, ITS, Nattrassia,Oregon, Scytalidium, systematics, Washington

INTRODUCTION

Pacific madrone (Arbutus menziesii Pursh, Ericaceae)is native to western North America extending fromsouthwestern British Columbia to southern Califor-nia. The tree has distinctive, peeling, reddish bark,shiny, evergreen leaves, clusters of white flowers andbright red berries in the late summer and fall. Forseveral decades Pacific madrone trees have been indecline. Symptoms first were noticed after a severedrought in the late 1960s and have continued up tothe present (Davison 1972, Elliott and Edmonds2003). Although a number of fungi have been isolat-ed from diseased madrone trees, one species hasbeen consistently associated with cankers (Elliott etal 2002, Elliott and Edmonds 2003). This fungus ini-tially was identified as Nattrassia mangiferae (Syd. &P. Syd.) B. Sutton & Dyko.

The objectives of this research were to identify andcharacterize the fungus causing cankers on Pacificmadrone and compare it with N. mangiferae. Molec-ular data confirm that the fungus causing madronecanker is not N. mangiferae. Rather it is a new speciesof Fusicoccum Corda, distinct from known species ofFusicoccum, all of which are the asexual states of Bo-tryosphaeria Ces. & de Not. The fungus causing ma-drone canker is described and illustrated as a newspecies of Fusicoccum. In addition the phylogeneticplacement and accurate scientific name of Nattrassiamangiferae and its synanamorph Scytalidium dimidia-tum were determined.

MATERIALS AND METHODS

Morphological/cultural characterization.—Fungal isolateswere cultured from symptomatic Pacific madrone branches.A small amount of diseased wood was taken from the cankermargin, surface sterilized in a dilute solution of sodium hy-pochlorite, rinsed in sterile deionized water, then grown on

731FARR ET AL: FUSICOCCUM ON PACIFIC MADRONE

2% malt-extract agar (MEA) at 25 C for 1 wk. Isolates weretransferred to new MEA plates.

For microscopic examination material was rehydratedand mounted in 3% KOH. Conidiomata were sectioned atca. 10 mm thick with a freezing microtome. Sections weremounted in lactic acid with cotton blue. Observations ofmicroscopic features were made with a Zeiss Axioplan 2 mi-croscope with bright field and fluorescence illumination.Calcofluor was used as the fluorescent dye. Photographsand measurements of microscopic features were taken witha Spot 2 digital camera (Diagnostic Instruments Inc., Ster-ling Heights, Michigan) and ImagePro software (Media Cy-bernetics, Silver Spring, Maryland). The description of cul-tural characteristics is based on isolates grown at 25 C inthe dark on Difco potato-dextrose agar (PDA) for 8 d. Theaverage growth rates were determined based on colony di-ameter for five replicates grown on PDA in the dark for 8d at 15, 20, 25, 30 and 35 C. Colors were determined withKornerup and Wanscher (1978). Cultures were stimulatedto sporulate by growing them on oatmeal agar at 25 C inthe dark for 1 wk, and under a 12 h light/12 h dark regimefor 2 wk as well as on prune agar (3 prunes, 5 g lactose, 1g yeast extract, 17 g agar, 1 L H2O). Cultures sporulated onsterilized woody twigs of Pacific madrone and alfalfa stems(Medicago sativa L.) on water agar under the same condi-tions listed above; this technique was used to obtain theholotype specimen and dried culture herbarium specimens.

DNA isolation, sequencing and analyses.—Isolates were cul-tured from madrone or obtained from a culture collection.Isolates were grown 7 d in a basal liquid medium (5 g pep-tone, 0.25 g MgSO4, 0.5 g KH2PO4, 1 L H2O) to preventmelanin formation, which interferes with DNA extraction.Mycelium was dehydrated in acetone (Punekar et al 2003)then ground in microcentrifuge tubes containing the ex-traction buffer (100 mM Tris-HCl, pH 8.0; 1.4 M NaCl; 20mM EDTA; 2% CTAB, w/v; 2% mercaptoethanol; 2% PVP)and a small amount of sterile sand. The mixture was incu-bated 1 h at 65 C. The lysate was extracted with chloroform-isoamyl alcohol (24:1), centrifuged 10 min at 12 000 rpm,then an equal volume of ice-cold isopropanol was added tothe aqueous layer. DNA was precipitated overnight at 25 C(Michiels et al 2003). The precipitate was rinsed with 70%ethanol, dried and resuspended in 100 mL sterile deionizedwater. A total of 10 mg RNAase A was added and the extractswere incubated at 37 C for 30 min. DNA was diluted to aconcentration of 1–2 ng/mL in sterile deionized water.

Six isolates of the madrone fungus and three isolates ofFusicoccum dimidiatum are listed (TABLE I) were sequencedwith the universal fungal primers ITS1F and ITS4 to amplifythe internal transcribed spacer region of the nuclear ribo-somal RNA encoding genes. Part of the b-tubulin gene alsowas sequenced. PCR and sequencing were done as de-scribed for ITS with primers Bt2a and Bt2b for b-tubulin(Slippers et al 2004a). Each reaction was performed in 50mL volumes containing 2.5 U Taq polymerase (FermentasInc., Hanover, Maryland), 13 buffer supplied with the en-zyme, 3 mM MgCl2, 0.2 mM of each dNTP, 0.2 mM of eachprimer, and made up to 50 mL with sterile deionized water.Each reaction mixture was overlaid with mineral oil and

PCR was carried out in a MJ PTC-100 thermal cycler (MJResearch Inc., Watertown, Massachusetts) according to thethis program: denaturation at 95 C for 85 s, followed bydenaturation (95 C for 35 s), annealing (55 C for 55 s) andelongation (72 C for 1 min) with increased elongation timesof 1 min every 10 cycles, for a total of 32 cycles. DNA waspurified with the QIAGEN QIAquick PCR Purification kit(QIAGEN Inc., Chatsworth, California), labeled with ABIPrism BigDye Terminator Cycle Sequencing Kit (PE Biosys-tems, Foster City, California) and sequenced in the Univer-sity of Washington Biochemistry DNA Sequencing Facilityon an ABI 3700 high-throughput capillary DNA analyzer.

GenBank sequence numbers for newly sequenced isolatesare listed (TABLE I) along with numbers for sequences ob-tained from GenBank. DNA sequences (partial 18S, ITS-1,5.8S, ITS-2, partial 28S) were aligned with Clustal X Multi-ple Sequence Alignment Program (version 1.81, Mar 2000)with gap opening penalty 5 15.00 and gap extension pen-alty 5 6.66. Sequences were truncated at the 59 and 39 endsand manually aligned when necessary with BioEdit v. 5.0.9(Hall 1999). Phylogenetic analysis was carried out withPAUP* version 4.0b10 (Swofford 2003). Alignment gapswere treated as a fifth character, and all characters wereunordered and of equal weight. The heuristic search formaximum parsimonious trees was done using only infor-mative characters in random (stepwise) addition 100 timesper replicate. The branch swapping algorithm was tree bi-section and reconstruction (TBR). Maxtrees were unlimitedand branches of zero length were collapsed. The ITS treewas rooted to the outgroup containing two species of Cer-cospora (Genbank AY343371 and AY342272) (van Niekerket al 2004). Most parsimonious trees were saved and branchsupports using 1000 bootstrap replicates were calculated.Goodness of fit for these trees was calculated as well as es-timated level of homoplasy (consistency, retention and ho-moplasy indices). A 50% majority rule consensus tree wascalculated in PAUP*. Trees were observed with TreeView(Page 1996).

To determine the placement of F. arbuti within Botryos-phaeria-Fusicoccum, ITS and b-tubulin sequences from B.australis, B. dothidea, B eucalyptorum, B. luteum, B. parvaand B. ribis were subjected to the same analysis as describedabove for the full ITS dataset. Trees were rooted with themidpoint method. The ITS and b-tubulin datasets werecombined and a partition homogeneity test was performedin PAUP*. All alignments have been submitted to TreeBaseas No. SN2105.

TAXONOMY

Fusicoccum arbuti D.F. Farr & M. Elliott sp. nov.FIGS. 1–10

Conidiomata nigra, uni- bis multiloculata, 0.5–1.5 3 1.5–3 mm. Conidiophora deminuta conidiogenis cellulis. Con-idiogenae cellulae 9.0–16.5 3 2.5–3.5 mm, cylindraceae-su-bobpyriformes, holoblastinae, discretae, determinatae velinterdum indeterminatae et proliferantes percurrentes inincrassates annulos. Conidia 20.0–29.2 3 4.8–7.4 mm, obo-voidea, fusiformes, basi tuncata, apice obtuse vel subobtusa,

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734 MYCOLOGIA

FIGS. 1–4. Conidiomata of Fusicoccum arbuti. 1. Conidiomata on alfalfa stem. 2. Conidiomata on prune agar with conidialcirrhus. 3. Section of conidioma on alfalfa stem. 4. Section of conidiomata on prune agar. All of holotype culture CBS 116131.Bar 5 100 mm.

hyalina, 2–septata ubi vesta. Microconidia 3.4–6.3 3 1–1.6mm, cylindracea vel allantoidea, flexuosa vel aliquantum inmedio angustata, hyalina, laevigata, non septata.

Holotypus: UNITED STATES: WASHINGTON,King County, Seattle, Magnolia Bluffs, isolated fromcankers of Arbutus menziesii, Oct 2003, collected byMarianne Elliott, isolated by Amy Rossman AR 40365 CBS 116131. The culture sporulated on sterilewood of A. menziesii on water agar, which was driedand deposited as BPI 843970 herein designated HO-LOTY PE.

Mycelium immersed, of branched, septate, smooth,hyaline hyphae, becoming brown, constricted withage, forming sparse, brown, thick-walled, intercalary,serial chlamydospores. Conidiomata black, scattered,uniloculate to multiloculate, 0.5–1.5 3 1.5–3 mm, be-coming clumped and irregular in shape, papillate.Stromata in longitudinal section of dark brown tex-tura intricata, locule walls of several layers of thick-walled, dark-brown textura angularis, becoming hya-

line toward conidiogenous region. Conidiophores re-duced to conidiogenous cells. Conidiogenous cells9.0–16.5 3 2.5–3.5 mm, cylindrical to subobpyriform,hyaline, holoblastic, discrete, determinate, occasion-ally indeterminate, proliferating percurrently result-ing in periclinal thickenings or rarely indistinct an-nellations, lining inner wall of pycnidium. Conidiaobovoid, fusiform, base truncate, apex obtuse to su-bobtuse, hyaline, guttulate. Older conidia may be-come brownish and 2-septate before germination.Conidia on sterile wood 18.5–27.8 3 5.5–7.8 mm, av-erage 22.8 3 6.4 mm, L/W ratio 3.6 (n 5 235); onoatmeal agar 21.4–30.4 3 4.6–7.6 mm, average 25.73 6.4 mm, L/W ratio 4.0 (n 5 244); on prune agar16.8–28.7 3 4.7–7.8 mm, average 22.3 3 6.2 mm, L/W ratio 3.6 (n 5 263). Microconidia cylindric to al-lantoid, flexuous or somewhat dumbbell-shaped, hy-aline, smooth, nonseptate 3.4–6.3 3 1–1.6 mm, aver-age 4.3 3 1.2 mm (n 5 37).

Colonies on PDA at 25 C in the dark for 8 d, light


FIGS. 5–10. Microscopic characteristics of Fusicoccum arbuti. 5. Conidiogenous cells. 6. Conidiogenous cells as seen byfluorescence microscopy. 7. Conidia occasionally becoming 2-septate, on oat agar. 8. Conidia on prune agar. 9. Microconidia.10. Chlamydospores. 5–9 Holotype culture CBS 116131. Bar 5 10 mm.

yellow (3A5) to olive grey (3E4) or olive brown(4F4), darkest around plug, pigmentation extendingabout two-thirds of colony width, outer area white,reverse same, surface mycelium cottony exceptaround plug where mycelium is appressed, obscurelyzonate, margin irregular. Growth: 25 mm at 15 C, 63mm at 20 C, 70 mm at 25 C, 37 mm at 30 C, nogrowth at 35 C. Colonies not producing yellow pig-ment diffusing into agar.

Specimens/cultures examined. CANADA. BRITISH CO-LUMBIA: Nanoose Bay, isolated from canker of A. menziesii,8 Mar 1972, collected by A. Funk (UAMH 6800); VancouverIsland, Northwest Bay, isolated from canker of Arbutus men-ziesii, 6 Jan 1972, collected by G. Helem (UAMH 6799).UNITED STATES. CALIFORNIA: Del Norte County, Gas-quet, isolated from cankers of A. menziesii, 20 Oct 1998(UW 52 5 CBS 116574, dried culture BPI 863594); Nevada

County, Nevada City, isolated from cankers of A. menziesii,20 Oct 1998 (UW 32 5 CBS 116573, dried culture BPI863593); San Luis Obispo County, isolated from cankers ofA. menziesii, Feb 1997 (UW13 5 CBS 117090, dried cultureBPI 863936); Santa Cruz County, Santa Cruz, isolated fromcankers of A. menziesii, 20 Oct 1998 (culture ME 1-4 5 CBS116575, dried culture BPI 863595); Sonoma County, isolat-ed from cankers of A. menziesii, Oct 1998 (UW22 5 CBS117089, dried culture BPI 863937). WASHINGTON: Jeffer-son County, Port Townsend, isolated from cankers of A.menziesii, 18 Jun 2003, collected by Marianne Elliott, isolat-ed by Amy Rossman (culture ME 7324 5 CBS 116576, driedculture BPI 863596).

Host and distribution. Known only from Arbutusmenziesii Pursh (Pacific madrone) in the westernUnited States and Canada from British Columbia toCalifornia.

736 MYCOLOGIA

RESULTS

Phylogenetic sequence analyses.—Sequence analyses ofITS alone and combined with b-tubulin suggest thatFusicoccum arbuti is distinct from all other species ofFusicoccum (FIGS. 11, 12). The aligned ITS datasetcontained 484 characters of which 151 were parsi-mony informative and 333 were constant (FIG. 11).Heuristic searches in PAUP* resulted in 266 mostparsimonious trees with length of 322 steps (CI 50.7640, RI 5 0.9345, RC 5 0.7140 and HI 5 0.2360).In this dataset isolates of Fusicoccum arbuti group to-gether with 81% bootstrap support. Isolates of Fusi-coccum dimidiatum group together in a separate cladein Botryosphaeria-Fusicoccum with 100% bootstrap val-ue.

The ITS dataset was combined with b-tubulin anda partition homogeneity test was performed inPAUP* (P 5 0.04). Although this P-value is signifi-cant, the datasets can be combined because the datapartitions are only weakly incongruent. Combininggenes has been shown to improve phylogenetic ac-curacy even though incongruence tests are signifi-cant (Cunningham 1997). Adding more genes to theanalysis would provide better statistical support forthe final tree, as would eliminating third codon po-sitions in coding regions. The combined dataset con-tained 870 characters with 97 parsimony informativecharacters and 773 constant characters (FIG. 12). Atotal of 104 most parsimonious trees of 132 stepswere obtained (CI 5 0.8258, RI 5 0.9555, RC 50.7890 and HI 5 0.1742). In the two gene dataset,isolates of F. arbuti grouped with bootstrap values of99% in a well supported clade that is sister of the B.ribis/B. parva clade (FIG. 12).

Morphological analysis.—In reviewing the two mostrecent keys to Fusicoccum, namely Slippers et al(2004a) and Phillips (2004 http://www.crem.fct.unl.pt/botryosphaeriapsite/key.htm), Fusicoccum arbuti isreadily differentiated from its closest relatives by co-nidial size. The conidia in culture of F. arbuti (FIGS.7–8) are longer than those of F. ribis Slippers et aland F. parvum Pennycook & Samuels but are not aslong as those of F. aesculi Corda. In addition F. arbutidoes not produce a diffuse yellow pigment into theculture medium as in F. luteum Pennycook & Samu-els. Fusicoccum arbuti is similar morphologically to F.parvum, anamorph of Botryosphaeria parva Penny-cook & Samuels (1985). As in F. arbuti, the conidiaof F. parvum occasionally become 2-septate with thecentral cell becoming noticeably olivaceous to brownbefore germination as illustrated in Slippers et al(2004a, FIG. 15). Although similar in shape and sep-tation, the conidia of F. arbuti generally are largerthan those of F. parvum, which vary from (11–)14–

18(–23) 3 5–7(–10) mm, average 16.0 3 5.9 mm(Pennycook and Samuels 1985, Slippers et al 2004a).Small microconidia termed spermatia by Pennycookand Samuels (1985) as described for F. parvum wereobserved in older cultures of F. arbuti (FIG. 9). Thesealso are produced in cultures of F. ribis (Morgan-Jones and White 1987 as Fusicoccum anamorph of B.ribis Grossenb. & Duggar).

DISCUSSION

Madrone canker caused by Fusicoccum arbuti.—Atpresent Fusicoccum arbuti is known only from Arbutusmenziesii along the western coast of North America.The fungus invades heat-injured bark when branchesare exposed to strong sunlight and also may infectthrough wounds made by pruning or other mechan-ical devices (Elliott and Edmonds 2003). Fusicoccumarbuti can be isolated from the expanding cankerson madrone. If inoculated with this fungus, pycnidiaof F. arbuti are observed on the substratum in nature(Elliott pers comm). Although a sexual state neverhas been seen in nature or in culture, F. arbuti un-doubtedly is derived from within Botryosphaeria. Spe-cies of Botryosphaeria, primarily in their asexual statesof Fusicoccum or related pycnidial genera, are knowncommonly to cause twig, stem and basal cankers ofwoody plants (Swart and Blodgett 1998, Besoain et al2000, Sanchez et al 2003, van Niekerk et al 2004).They also have been reported to cause twig and stemblights (Ko et al 1999) and commonly are isolated asleaf endophytes ( Johnston 1998).

Madrone canker probably has been present inwestern North America for at least the past three de-cades. A canker disease on madrone was reported inWashington in the summers of 1968 and 1969 afteran ‘‘unseasonably hot and dry’’ summer of 1967 and‘‘an unusually cold, severe period in Jan 1969’’ (Dav-ison 1972). The causal fungus was reported as Hen-dersonula toruloidea Nattrass, a synonym of Fusicoc-cum dimidiatum (see below), although it is likely thatthis canker was caused by Fusicoccum arbuti. Novoucher specimens or cultures exist to verify the caus-al agent, however this is probably the first report ofmadrone canker caused by F. arbuti. Later reports ofthe madrone canker caused by F. arbuti were listedmistakenly as Nattrassia mangiferae, also a synonymof Fusicoccum dimidiatum (Elliott et al 2002, Elliottand Edmonds 2003). Specimens and cultures docu-ment the presence of F. arbuti causing madrone can-ker since 1972 throughout the range of the host(Hunt and Funk 1992).

Placement of Fusicoccum arbuti.—The genus Fusicoc-cum is one of several anamorphs connected with spe-


FIG. 11. Most parsimonious tree showing phylogenetic relationships among Botryosphaeria species based on the ITS rDNAsequence data. The tree is rooted to the outgroup Cercospora beticola and C. penzigii. Isolates of Fusicoccum arbuti clearly aredifferent from F. dimidiatum. Heuristic searches in PAUP* resulted in 266 most parsimonious trees with length of 322 steps(CI 5 0.7640, RI 5 0.9345, RC 5 0.7140 and HI 5 0.2360). Numbers at nodes are percentage of trees from bootstrapanalysis that support the topography of the consensus tree. The aligned ITS dataset contains 484 characters of which 151are parsimony informative and 333 are constant. Isolates are listed with their hosts and country of origin (Aus 5 Australia,Neth 5 Netherlands, NZ 5 New Zealand, SA 5 South Africa, Swit 5 Switzerland).

738 MYCOLOGIA

FIG. 12. Most parsimonious tree showing phylogenetic relationships among Botryosphaeria species with Fusicoccum ana-morphs based on the combined dataset of ITS rDNA and b-tubulin DNA sequences. Numbers at the nodes are percentageof trees from bootstrap analysis that support the topography of the consensus tree. The tree is rooted to the clade containingBotryosphaeria dothidea. The combined dataset contains 870 characters with 97 parsimony informative characters and 773constant characters. A total of 104 most parsimonious trees of 132 steps were obtained (CI 5 0.8258, RI 5 0.9555, RC 5


←

0.7890 and HI 5 0.1742). Isolates are listed with their hosts and country of origin (Aus 5 Australia, Neth 5 Netherlands,NZ 5 New Zealand, SA 5 South Africa, Swit 5 Switzerland).

cies of Botryosphaeria. In a paper by Crous and Palm(1999) the concept of Fusicoccum was re-evaluatedand the genus was typified based on F. aesculi forwhich a neotype was designated. Fusicoccum had beendefined by Sutton (1980) to include ‘‘coelomyceteswith fusiform, hyaline, non-septate conidia producedholoblastically in stromatic conidiomata’’ indicatingthat this genus best could accommodate the ana-morphs of B. dothidea and B. ribis. Although the co-nidia are produced primarily holoblastically, the con-cept of Fusicoccum had been expanded by Pennycookand Samuels (1985) to include the older conidiogen-ous cells of F. aesculi that are enteroblastic and pro-liferate percurrently. Both authors noted that the co-nidia of Fusicoccum are initially hyaline but may be-come olivaceous with age and are primarily aseptate,sometimes becoming septate before germination. Al-though many species have been described as Fusicoc-cum, the genus now includes only species that haveteleomorphs in Botryosphaeria, if known, and are re-lated genetically to the type species, Fusicoccum aes-culi (Slippers et al 2004a). For many species of Fusi-coccum including F. arbuti, a Botryosphaeria sexualstate has not been seen or reported. The sexual statemight be formed only rarely or might have been lost,as is the case for many asexually reproducing fungi(Taylor et al 1999).

In the past 10 years molecular sequence data havebeen applied to the Botryosphaeria complex resultingin a more accurate circumscription of species withinthis genus (Phillips et al 2002, 2004, Slippers et al2004a, b, van Niekerk 2004, Zhou and Stanosz 2001).Major groups within Botryosphaeria recently havebeen recognized that correlate with anamorph gen-era including Diplodia, Dothiorella, Fusicoccum andLasiodiplodia (Phillips et al 2004). Fusicoccum arbutigroups with the type species of Fusicoccum, F. aesculi,the anamorph of Botryosphaeria dothidea, the typespecies of Botryosphaeria (Slippers et al 2004a). With-in Botryosphaeria-Fusicoccum several new species havebeen recognized, most recently, B. protearum Den-man & Crous on Protea (Denman et al 2003), Fusi-coccum viticlavatum Niekerk & Crous and F. vitifusi-forme Niekerk & Crous on Vitis (van Niekerk et al2004) and B. australis Slippers & Crous on Acaciaand Sequoiadendron in Australia (Slippers et al2004b), Robinia in Portugal and Vitis in South Africa(van Nierkerk et al 2004).

Fusicoccum arbuti belongs in Botryosphaeria-Fusicoc-cum and is differentiated readily from all other de-

scribed species based on morphological and molec-ular data. In both the ITS tree and the combinedITS-b-tubulin trees the six isolates of F. arbuti form aunique group with 81% and 99% bootstrap valuesrespectively. Based on molecular data, F. arbuti ismost closely related to F. parvum and F. ribis. F. arbutiis distinguished morphologically by the conidia (FIGS.7–8) that are longer than either F. parvum or F. ribis.Fusicoccum ribis also is similar to F. arbuti in produc-ing chains of brown, thick-walled chlamydospores(FIG. 10) (Morgan-Jones and White 1987, Rayachhe-try et al 1996).

Fusicoccum dimidiatum, the correct name for Scytali-dium dimidiatum and Nattrassia mangiferae.—Fusi-coccum arbuti initially was identified as Nattrassiamangiferae based on the 2-septate conidia with a dark-ened central cell and presence of chlamydospores(Davison 1972, Elliott et al 2002, Elliott and Edmonds2003). The monotypic genus Nattrassia B. Sutton &Dyko based on the type species N. mangiferae wasconsidered distinct because of the 2-septate conidiathat become brown in the middle cell (Sutton andDyko 1989). A conspecific synanamorph of N. man-giferae was recognized as S. dimidiatum. Three au-thentic cultures of N. mangiferae as S. dimidiatum(Penz.) B. Sutton & Dyko were examined and in-cluded in the molecular analyses. One of these iso-lates represents the causal organism of a branch wiltdisease on Persian walnut trees (Juglans regia L.) inCalifornia (Wilson 1947, 1949). These moleculardata reveal that N. mangiferae belongs in Botryos-phaeria/Fusicoccum, most closely related to B. ma-mane (Gardner 1997) (FIG. 11). No sexual state isknown for N. mangiferae or S. dimidiatum. The char-acteristics of the pycnidial state of N. mangiferae re-semble those of Fusicoccum aesculi and other speciesof Fusicoccum. Unlike most species in the genus Fu-sicoccum, the conidia of N. mangiferae often become2-septate with a brown central cell. In addition to aFusicoccum pycnidial state, Nattrassia mangiferae pro-duces a synanamorph that consists of chains ofbrown, disarticulating, arthric conidia. This synana-morph has been referred to as Scytalidium dimidia-tum (5 S. lignicola Pesante) based on Torula dimi-diata Penz. (Ellis 1971, Sutton and Dyko 1989). Thesynonymous name Scytalidium lignicola is the typespecies of the genus Scytalidium. This arthric synan-amorph of Nattrassia mangiferae can be regarded asa similar but more extensive development of the

740 MYCOLOGIA

brown, thick-walled chlamydospores produced byboth F. arbuti and F. ribis.

Based on the molecular sequence data that placesNattrassia mangiferae/Scytalidium dimidiatum in Bo-tryosphaeria/Fusicoccum and the morphological simi-larities of the pycnidial and arthric conidial states toFusicoccum, this species is recognized in the genusFusicoccum as follows:

Fusicoccum dimidiatum (Penz.) D.F. Farr, comb. nov.[ Torula dimidiata Penz., Michelia 2:466. 1882. (Bas-

ionym)[ Scytalidium dimidiatum (Penz.) B. Sutton & Dyko,

Mycol Res 93:484. 1989.5 Dothiorella mangiferae Syd. & P. Syd. in Syd. et al, Ann

mycol 14:192. 1916.[ Nattrassia mangiferae (Syd. & P. Syd.) B. Sutton &

Dyko, Mycol Res 93:484. 1989.5 Fusicoccum eucalypti Da Camara, An Inst Super Agron

3:32. 1929.5 Hendersonula toruloidea Nattrass, Trans Brit Mycol Soc

18:97. 1933.5 Hendersonula cypria Nattrass, Cyprus Fungi, Nicosia. p

43. 1937.5 Exosporina fawcettii E.E. Wilson, Hilgardia 17:427.

1947.5 Hendersonula agathidis H.E. Young, Queensland J

Agric Sci 5:12. 1948.5 Scytalidium lignicola Pesante, Ann Sper Agron N.S. 11:

supplement p. cclxv. 1957.All these fungal names are synonyms (i.e. they referto the same species). Because the respective type spe-cies of these genera are synonyms of F. dimidiatum,Nattrassia and Scytalidium are synonyms of the genusFusicoccum.

Fusicoccum dimidiatum has been reported on di-verse woody plants (Punithalingam and Waterston1970, Sutton and Dyko 1989, Farr et al 2004) andoccasionally is isolated from human skin and nails(Moore 1988, de Hoog et al 2000). Although report-ed to be cosmopolitan, the diseases caused by thisfungus tend to occur in tropical countries as well asCalifornia. Symptoms include gummosis and diebackof stone fruit trees in Egypt (Nattrass 1933), branchwilt and drying of grape vines in India and Iraq (Na-tour and Ahmed 1969, Wangikar et al 1969), branchwilt, decline and death on citrus in Iraq (Alizadeh etal 2000), leaf spot diseases in India (Chandra 1974),leaf spot and dieback of mango in India and Niger(Pandey et al 1981, Reckhaus and Adamous 1987),and tip rot of bananas in Jamaica and Hawaii (Mer-edith 1963, 1969). In North America F. dimidiatumhas been reported in California to cause branch wiltand canker of walnut (Wilson 1947, 1949), diebackand canker of citrus (Calavan and Wallace 1954), sec-ondary canker infection of almond (English et al

1975) and a canker and dieback of Eucalyptus in Ar-izona (Matheron and Sigler 1993). The temperatureminimum (15 C), optimum (30–35 C) and maxi-mum (38–40 C) for growth of F. dimidiatum (Nattrass1933, Wilson 1947) ranges about 5 C higher than forF. arbuti, which is 10, 25 and 30–35 C respectively(Davison 1972, Elliott 1999).

A recent report of Hendersonula toruloidea causinga foliar disease of strawberry tree (Arbutus unedo) inGreece (Tsahouridou and Thanassoulopoulos 2000)was not documented with a voucher specimen or cul-ture, thus it cannot be determined if the causal agentwas actually Fusicoccum arbuti or F. dimidiatum.

ACKNOWLEDGMENTS

This project was financed by Save Magnolia’s Madrones.The authors thank Joe Ammirati, Erica Cline, Suzanne Jo-neson, Brandon Matheny and Amy Ramsey for their help.

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Documents

Fusicoccum arbuti sp. nov. causing cankers on Pacific madrone in western North America with