seven new species of the botryosphaeriaceae from baobab

Seven new species of the Botryosphaeriaceae from baobab and other native treesin Western Australia

Draginja Pavlic1

Michael J. WingfieldForestry and Agricultural Biotechnology Institute(FABI), Centre of Excellence in Tree HealthBiotechnology, Department of Microbiology and PlantPathology, Faculty of Natural and AgriculturalSciences, University of Pretoria, Pretoria, 0002,South Africa

Paul BarberSchool of Biological Sciences and Biotechnology,Murdoch University, Perth, 6150, Australia

Bernard SlippersForestry and Agricultural Biotechnology Institute(FABI), Centre of Excellence in Tree HealthBiotechnology, Department of Genetics, Faculty ofNatural and Agricultural Sciences, University ofPretoria, Pretoria, 0002, South Africa

Giles E. St. J. HardyTreena I. Burgess

School of Biological Sciences and Biotechnology,Murdoch University, Perth, 6150, Australia

Abstract: In this study seven new species of theBotryosphaeriaceae are described from baobab(Adansonia gibbosa) and surrounding endemic treespecies growing in the Kimberley region of north-western Australia. Members of the Botryosphaeria-ceae were predominantly endophytes isolated fromapparently healthy sapwood and bark of endemictrees; others were isolated from dying branches.Phylogenetic analyses of ITS and EF1-a sequencedata revealed seven new species: Dothiorella longicollis,Fusicoccum ramosum, Lasiodiplodia margaritacea,Neoscytalidium novaehollandiae, Pseudofusicoccumadansoniae, P. ardesiacum and P. kimberleyense.

Key words: Adansonia gibbosa, biodiversity, Botry-osphaeria, Dothiorella, Fusicoccum, Lasiodiplodia, Neo-scytalidium, Pseudofusicoccum, systematics, taxonomy

INTRODUCTION

Only eight species of baobabs (Adansonia spp.) areknown. Adansonia gibbosa is the only baobab speciesendemic to Australia and is restricted to the north-western part of the country (Crisp et al 2004).Adansonia digitata has a wide natural distribution

throughout tropical parts of Africa and the six otherspecies are found on Madagascar (Bowman 1997,Baum et al 1998). A recent biogeographical study onbaobabs presented the intriguing view that thedistribution of these unusual trees between Africaand Australia occurred after the division of Gond-wana (Baum et al 1998). The same study revealed thatA. gibbosa in Australia is more closely related to A.digitata from Africa than it is to species fromMadagascar.

In this first study of fungi associated with A. gibbosaand surrounding endemic tree species in northwest-ern Australia members of the Botryosphaeriaceaewere found as non sporulating endophytes inapparently healthy sapwood and bark of branchescollected from all tree species sampled; they also werefound sporulating and releasing conidia on dyingbranches of baobabs. Numerous studies have com-bined phenotype with DNA sequence analyses indefining genera and species in the Botryosphaeria-ceae ( Jacobs and Rehner 1998, Denman et al 2000,Zhou and Stanosz 2001, Slippers et al 2004, Phillips etal 2005). Crous et al (2006) summarized this work andrepresented several lineages in the Botryosphaeria-ceae that were identified with generic names based onlarge subunit (LSU) sequence data, including Botryo-sphaeria, Dothidotthia, Macrophomina, Neofusicoccum,Neoscytalidium, Pseudofusicoccum, Saccharata andGuignardia. The identity and generic placement ofthe numerous species included in Diplodia andLasiodiplodia were unclear in the study of Crous etal (2006), but they are clearly separated in ITS andEF1-a phylogenies (Burgess et al 2005, Phillips et al2005, Damm et al 2007, Alves et al 2008).

In this study we describe seven new species ofBotryosphaeriaceae associated with A. gibbosa andother native trees in the northwestern Australia. Thenew taxa are characterized and described based onITS and EF1-a sequence data combined with ana-morph morphology.

MATERIALS AND METHODS

Isolates.—Those used in this study were collected from A.gibbosa and surrounding native trees in northwesternAustralia in Jun and Jul 2006 (TABLE I). Asymptomaticand dying twigs of A. gibbosa were collected from 26locations approximately 20 km apart along Gibb RiverRoad. At three locations asymptomatic twigs also werecollected from eight other tree species. The other tree

Accepted for publication 8 August 2008.1 Corresponding author. E-mail: [email protected]

Mycologia, 100(6), 2008, pp. 851–866. DOI: 10.3852/08-020# 2008 by The Mycological Society of America, Lawrence, KS 66044-8897

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PAVLIC ET AL: BOTRYOSPHAERIACEAE FROM AUSTRALIA 853

species were different at the three locations but includedAcacia synchronica, Crotalaria medicaginea, Eucalyptuscamaldulensis, an unidentified Eucalyptus sp., Ficus opposita,Grevillia agrifolia, Lysiphyllum cunninghamii and a Termi-nalia sp. (TABLE I). Isolations were made from visuallyhealthy sapwood and bark collected from branches follow-ing Burgess et al (2006b). Collections also were made frompycnidia formed on dying branches. When pycnidia werefound on dying branches masses of conidia were transferreddirectly to 2% malt extract agar (MEA) (Biolab, S.A.).Single-conidial cultures of all isolates in this study aremaintained in the Culture Collection (CMW) of theForestry and Agricultural Biotechnology Institute (FABI),University of Pretoria, Pretoria, South Africa, and theMurdoch University Culture Collection (MUCC). A repre-sentative set of isolates also has been deposited in thecollection of the Centraalbureau voor Scimmelcultures(CBS), Utrecht, The Netherlands.

DNA sequence comparisons.—DNA was extracted fromfungal mycelium from 7 d old single-conidial cultures asdescribed by Burgess et al (2005). DNA was purified withthe UltrabindH DNA purification kit following the instruc-tions given by the manufacturer (MO BIO Laboratories).Two gene regions were used for phylogenetic analyses. Theinternal transcribed spacer (ITS) region of the ribosomalRNA (rRNA) operon was amplified for all isolates usingprimers ITS-1F (Gardes and Bruns 1993) and ITS-4 (Whiteet al 1990). For selected isolates a part of the elongationfactor 1-a (EF1-a) gene was amplified with primers EF1-728F and EF1-986R (Carbon and Kohn 1999). The PCRreaction mixture, PCR conditions and visualization were asdescribed by Pavlic et al (2004) except that 0.5 U of Taqpolymerase (Biotech International, Needville, Texas) wasused. PCR products were cleaned with the UltrabindH DNApurification kit and sequenced with the BigDye terminatorcycle sequencing kit (PE Applied Biosystems) in bothdirections, with the same primers used for the PCRreactions. Products were separated with an ABI 3730 48capillary sequencer (Applied Biosystems, Foster City, Cali-fornia). Data were collected with ABI data collectionsoftware.

Sequence data for isolates of the unknown species weredeposited in GenBank (TABLE I). Sequences of knownspecies were obtained from GenBank, and the isolate code,identity and accession numbers for sequence data used aregiven in TreeBASE (http://www.treebase.org/treebase/index.html, accession number SN3768). Parsimony analysiswas performed on individual datasets (individual trees arenot illustrated) and on the combined dataset after partitionhomogeneity test (PHT) was performed in PAUP (Phylo-genetic Analysis Using Parsimony) version 4.0b10 (Swofford2000) to determine whether sequence data from the ITSand EF-1a gene regions were statistically congruent (Farriset al 1995, Huelsenbeck et al 1996). Noninformativecharacters were removed before analysis and characterswere unweighted and unordered. The most parsimonioustrees were obtained using heuristic searches with randomstepwise addition in 100 replicates, with the tree bisection-reconnection branch-swapping option on and the steepest-

descent option off. MAXTREES were unlimited, branches ofzero length were collapsed and all multiple equallyparsimonious trees were saved. Estimated levels of homo-plasy and phylogenetic signal (retention and consistencyindices) were determined (Hillis and Huelsenbeck 1992).Branch and branch node supports were determined with1000 bootstrap replicates (Felsenstein 1985). The tree wasrooted to a Guignardia sp.

Bayesian analysis was conducted on the same individualand combined datasets as that used in the parsimonyanalysis. First MrModeltest v.2.5 (Nylander 2004) was usedto determine the best nucleotide substitution model.Phylogenetic analyses were performed with MrBayes v. 3.1(Ronquist and Huelsenbeck 2003) applying a general timereversible (GTR) substitution model with gamma (G) andproportion of invariable site (I) parameters to accommo-date variable rates across sites. Two independent runs ofMarkov chain Monte Carlo (MCMC) with four chains wererun 1 000 000 generations. Trees were saved each 1000generations, resulting in 10 001 trees. Burn-in was set at50 001 generations (i.e. 51 trees), well after the likelihoodvalues converged to stationery, leaving 9950 trees fromwhich the consensus trees and posterior probabilities werecalculated.

Morphological characteristics.—To induce sporulation, cul-tures were inoculated onto sterilized pine needles and/oreucalypt twigs placed on the surface of 2% water agar (WA)(Biolab, S.A.) and incubated at 25 C under near-UV light.To obtain single-conidial cultures, releasing conidia frompycnidia formed on pine needles and/or eucalypt twigswere transferred on WA and spread on the medium surfaceby sterilized streaking loop. Plates were incubated at 25 Cunder near-UV light for approximately 12 h and singlegerminating conidia were transferred on the MEA withsterilized needle and incubated under the same conditions.A single pycnidium was placed in a drop of lactoglycerol ona microscope slide and cut in pieces with a sterile medicalneedle before adding the cover slip. Fifty released conidiaand 30 of pycnidia, conidiogenous cells and paraphyseswere measured for each isolate, and the ranges and averageswere computed. Measurements and digital images weremade with an HRc Axiocam digital camera and accompa-nying Axiovision 3.1 software (Carl Zeiss Ltd., Munich,Germany). Drawings were prepared with a drawing tube andfinalized with the method described by Barber and Keane(2007). Colony morphology and color were determinedfrom cultures grown on MEA at 25 C in the dark. Colonycolors (upper surface and reverse) were determined withthe charts of Rayner (1970).

Growth rates at 5–35 C, at 5 C intervals were deter-mined for cultures grown in the dark. To determinegrowth rate, mycelial plugs, 6 mm diam, were takenfrom the actively growing edges of 7 d old single-conidialcultures and transferred to the centers of MEA in 90 mmdiam Petri dishes. Three replicate plates were used for eachisolate at each temperature. Two perpendicular measure-ments were taken of the colony diameter daily until themycelium of the fastest growing isolates had covered theplates.

854 MYCOLOGIA

RESULTS

DNA sequence comparisons.—The partition homoge-neity test comparing the ITS and EF1-a datasets wassignificant (P 5 0.007) indicating that the individualdatasets were not congruent and produced trees withdiffering topology. These differences were not due tothe relationships among species in a genus butinstead were due to the relationship of the generato each other. Thus when the data were combinedsupport for the placement of species within a genuswas high but the support for the deeper branches,indicating relationships between genera, was low.Similar discrepancies were found when comparingthe phylogeny obtained from parsimony and Bayesiananalyses. Thus the analyses for the individual ITS andEF1-a datasets are available from TreeBASE(SN3768), while the results emerging from thecombined dataset are presented here.

The combined dataset consisted of 996 charactersof which 546 were parsimony informative. The datasetcontained significant phylogenetic signal comparedto 1000 random trees (P , 0.01, g1 5 20.43).Heuristic searches resulted in two most parsimonioustrees of 1566 steps (CI 5 0.60, RI 5 0.90) (FIG. 1,TreeBASE SN3768). In the Bayesian analysis thepositions of the genera in relation to each otherdiffered, but within each genus the topology wassimilar to the parsimony tree (TreeBASE SN3768).Eight clades were identified, each corresponding to aseparate genus and each supported with bootstrapvalues of 100% and Bayesian probabilities of 1.00.These were Clade 1 (Lasiodiplodia), Clade 2 (Dip-lodia), Clade 3 (Dothiorella), Clade 4 (Neofusicoccum),Clade 5 (Botryosphaeria), Clade 6 (Macrophomina),Clade 7 (Neoscytalidium) and Clade 8 (Pseudofusicoc-cum). Isolates obtained in this study resided in Clades1, 3, 5, 7 and 8.

Within the Lasiodiplodia clade two isolates werefound to be distinct from the known species in thisgenus (FIG. 1). Three isolates, two from Lysiphyllumcunninghamii and one from a Terminalia sp., formeda well supported lineage in the Dothidotthia/Dothior-ella clade (FIG. 1). Within the Botryosphaeria clade asingle isolate from Eucalyptus camaldulensis wasphylogenetically distinct from the two previouslysequenced (for ITS and EF1-a) species, B. dothideaand B. corticis (FIG. 1). Four isolates obtained in thepresent study from Acacia synchronica, Adansoniagibbosa, Crotalaria medicaginea and Grevillia agrifoliaformed a separate subclade within the Neoscytalidiumclade (FIG. 1). Although support for this subclade waslow, these isolates produce Dichomera-like synana-morphs that distinguish them from known Neoscyta-lidium species and are described in this study as a new

Neoscytalidium species. Pseudofusicoccum is currentlymonotypic for P. stromaticum. In this study three newspecies were found that phylogenetically reside in thisgenus (FIG. 1).

Morphology.—With exception of one isolate ofPseudofusicoccum ardesiacum (CMW 26160) all iso-lates of the Botryosphaeriaceae obtained from A.gibbosa and other native trees in northwesternAustralia produced pycnidia on the pine needlesand eucalypt twigs on WA within 2–3 wk. No ascomatawere observed. Based on culture and conidialmorphology, isolates were separated into sevenspecies: three in Pseudofusicoccum and one specieseach in Dothiorella, Fusicoccum, Lasiodiplodia andNeoscytalidium. These species are described as follows.

TAXONOMY

Pseudofusicoccum adansoniae Pavlic, Burgess, M.J.Wingfield, sp. nov. MB512048 FIGS. 2, 3Pycnidia subimmersa solitaria globosa papillata castanea,

mycelio tecta, usque ad 500 mm diam. Cellulae conidio-genae holoblasticae laeves cylindricae hyalinae, conidioprimo holoblastico, posteriora enteroblastica. Conidiamediocriter 22.5 3 5.2 mm, 4.3 plo longiora quam latiora,hyalinae, parietibus tenuibus, viscida strato persistenti mucitecta, laeves contentu tenue granulari, raro subflexa velirregularia, apicibus rotundatis, unicellularia, ante germi-nationem 1–2 septa formantia.

Pycnidia semi-immersed, solitary, globose, papil-late, chestnut, covered by hyphal hairs, up to 500 mmdiam. Conidiogenous cells holoblastic, smooth, cylin-drical, hyaline, the first conidium produced holoblas-tically and subsequent conidia enteroblastically, (9–)10–15(–16) 3 (1.5–)2–3(–3.5) mm (av. 12.7 3

2.4 mm). Conidia ellipsoid, occasionally slightly bentor irregularly shaped, (19–)21–24(–26) 3 (3.5–)4.5–6(–6.5) mm (av. 22.5 3 5.2 mm, l/w 4.3), apicesrounded, smooth with fine granular content, hyaline,thin-walled, covered with a persistent mucus layer,unicellular, forming 1 or 2 septa before germination.Cultural characteristics. Colonies initially white withmoderately dense, appressed mycelial mat. Sub-merged mycelium turning gray olivaceous (219999b)to olivaceous black (279999m) from the middle ofcolony after 3–5 d and becoming dark slate-blue(399999k) with age. Aerial mycelium slightly fluffy,becoming dense, cottony with age, sometimes re-maining white to smoke gray (219999f), usually turningpale olivaceous gray (2199999d) within 7 d andbecoming olivaceous gray (2199999i) to iron gray2399999k) with age. Colonies slightly irregular, occa-sionally radially striated with lobate edges and/orforming concentric, irregular circles. Conidiomatareadily formed from the middle of colony within 7–


FIG. 1. One of two most parsimonious trees of 1566 steps resulting from the analysis of the combined ITS-EF1a sequencedata. Bootstrap values of the branch nodes are given in italics and the posterior probabilities resulting from Bayesian analysisare indicated in bold. Isolates from this study are in bold. Tree is rooted to a Guignardia sp. The strongly supported clades thatrepresent different genera within the Botryosphaeriacea according to Crous et al. (2006) are indicated by circles at the nodes.

856 MYCOLOGIA

10 d, covering the entire surface of the colony andimmersed in the medium (seen as a round blackstructures on the reverse side of Petri dishes) 14 dafter incubation. Optimum growth at 30 C, coveringthe 90 mm diam Petri dish after 4 d in the dark.

Teleomorph. Not known.Etymology. Refers to the host from which the type

specimen was isolated.Habitat. Dying branches of Adansonia gibbosa and

asymptomatic branches of Acacia synchronica, Eucalyp-tus sp. and Ficus opposita.

Known distribution. Western Australia.HOLOTYPE. AUSTRALIA. WESTERN AUSTRALIA: Derby

(17u21903.150S, 123u40907.578E), on Adansonia gibbosa, Jul2006, T.I. Burgess (PREM 59841, a dry culture ex CMW26147 on pine needles; ex-type culture CMW 26147 5 CBS122055).

Additional specimens examined. See TABLE I.

Pseudofusicoccum kimberleyense Pavlic, Burgess,M.J. Wingfield, sp. nov. MB512049 FIGS. 4, 5Pycnidia subimmersa solitaria globosa papillata castanea,

mycelio tecta, usque ad 500 mm diam. Cellulae conidio-genae holoblasticae laeves cylindricae vel subcylindricaehyalinae, conidio primo holoblastico, posteriora enteroblas-tica. Conidia mediocriter 30.7 3 7.4 mm, 4.1 plo longioraquam latiora, hyalinae, parietibus tenuibus, viscida stratopersistenti muci tecta, laeves contentu tenue granulari,ellipsoidea, recta vel subfalcata, apicibus rotundatis, uni-cellularia, ante germinationem 1–4 septa formantia.

Pycnidia semi-immersed, solitary, globose, papil-late, chestnut, covered by hyphal hairs, up to 500 mmdiam. Conidiogenous cells holoblastic, smooth, cylin-drical to subcylindrical, hyaline, the first conidiumproduced holoblastically and subsequent conidiaenteroblastically, (7–)8.5–11(–14) 3 (2.5–)3–3.5(–4)mm (av. 9.8 3 3.3 mm). Conidia ellipsoid, straight orslightly curved, (24–)28–33(–34) 3 (6.5–)7–8(–8.5)mm (av. 30.7 3 7.4 mm, l/w 4.1), apices rounded,smooth with fine granular content, hyaline, thin-walled, covered with a persistent mucus layer,unicellular, forming 1–4 septa before germination.Cultural characteristics. Colonies initially white, hy-phae forming a moderately dense, appressed mycelialmat. Submerged mycelium citrine (21 k) to grayolivaceous (219999b) from the middle of colony after3–5 d, becoming olivaceous black (279999m) to blackwith age. Aerial mycelium slightly fluffy, becomingdense, cottony with age, smoke gray (219999f) to paleolivaceous gray (2199999d). Colonies slightly irregularwith sinuate edges. Optimum growth at 30 C,covering the 90 mm diam Petri dish after 4 d in thedark.

FIG. 2. Pseudofusicoccum adansoniae. a. Conidiogenouscells (CBS122056). b. Conidia (CBS122054, CBS122055,CBS122056). c. Germinating conidia (CBS122056). Bar 5

10 mm.

FIG. 3. Pseudofusicoccum adansoniae. a. Pycnidia formedin culture on pine needles (CBS122054). b. Aseptateconidia (CBS122055). c, d. Conidiogenous cells(CBS122053). Bars: a 5 500 mm, b–d 5 10 mm.


Teleomorph. Not known.Etymology. Refers to Kimberley region, Western

Australia, where the substratum was collected fromwhich the fungus was isolated.

Habitat. Dying branches of Adansonia gibbosa andasymptomatic branches of Acacia synchronica, Eucalyp-tus sp. and Ficus opposita.

Known distribution. Western Australia.HOLOTYPE. AUSTRALIA. WESTERN AUSTRALIA: Tunnel

Creek National Park (17u54933.342S, 125u17901.686E), onAcacia synchronica, Jul 2006, T.I. Burgess (PREM 59842, adry culture on pine needles ex CMW 26156; ex-type cultureCMW 26156 5 CBS 122058).


Pseudofusicoccum ardesiacum Pavlic, Burgess, M.J.Wingfield, sp. nov. MB512051 FIGS. 6, 7Pycnidia subimmersa solitaria globosa papillata castanea,

mycelio tecta, usque ad 510 mm diam. Cellulae conidio-genae holoblasticae laeves cylindricae vel subcylindricaehyalinae, conidio primo holoblastico, posteriora enteroblas-tica. Conidia mediocriter 25 3 7.5 mm, 3.3 plo longioraquam latiora, hyalinae, parietibus tenuibus, viscida stratopersistenti muci tecta, laeves contentu tenue granulari,

ellipsoidea vel vergata, recta vel subflea, apicibus rotundatis,unicellularia, ante germinationem 1–3 septa formantia.

Pycnidia semi-immersed, solitary, globose, papil-late, chestnut, covered by hyphal hairs, up to 510 mmdiam. Conidiogenous cells holoblastic, smooth, cylin-drical, hyaline, the first conidium produced holoblas-tically and subsequent conidia enteroblastically, (6–)7.5–10(–11) 3 (2.7–)3–4(–4.3) mm (av. 8.6 3 3.5 mm).Conidia ellipsoid to rod-shape, straight or slightlybent, (17.5–)21–29(–32) 3 (6.3–)7–8(–9) mm (av. 253 7.5 mm, l/w 3.3), apices rounded, smooth with finegranular content hyaline, thin-walled, covered with apersistent mucus layer, unicellular, forming 1–3 septabefore germination. Cultural characteristics. Coloniesinitially white with sparse to moderately denseappressed mycelial mat. Submerged mycelium darkviolet (59 m) to dark blue (47 m) (middle of thecolony) and smoke gray (219999f) to gray olivaceous(219999b) toward edges within 3–5 d, becomingviolaceous gray (599999i) to slate blue (47999k) withage. Aerial mycelium slightly fluffy, becoming dense,cottony with age, turning smoke gray (219999f) to palepurplish gray (719999d) in the middle of colony andsmoke gray (219999f) to gray olivaceous (219999b)toward edges after 5–7 d, becoming lavender gray(4599999f) with age; occasional columns of aerialmycelium in the middle of colony, reaching the lid.Colonies slightly irregular with sinuate edges. Con-idiomata readily formed and immersed in aerialmycelia on the entire colony surface within 7–10 d.

FIG. 4. Pseudofusicoccum kimberleyense. a. Conidiogenouscells (CBS122059). b. Aseptate conidia (CBS122058,CBS122060, CBS122061). c. 1–4-septate conidia(CBS122059, CBS122060, CBS122061). Bar 5 10 mm.

FIG. 5. Pseudofusicoccum kimberleyense. a. Pycnidiaformed in culture on pine needles (CBS122058). b, c.Aseptate conidia (CBS122058). d, e. Conidiogenous cells(CBS122058). f, g. Aseptate and 2–4-septate conidia(CBS122060). h. Aseptate conidia (CBS122061). Bars: a 5

500 mm, b–h 5 10 mm.

858 MYCOLOGIA

Optimum growth at 30 C, covering the 90 mm diamPetri dish after 4 d in the dark.

Teleomorph. Not known.Etymology. Refers to the slate blue-violet pigment

found in cultures.Habitat. Dying branches of Adansonia gibbosa and

asymptomatic branches of Eucalyptus sp.Known distribution. Western Australia.HOLOTYPE. AUSTRALIA. WESTERN AUSTRALIA: Mount

Hardman, Great Northern Highway (17u16905.952S,123u45926.930E), on Adansonia gibbosa, Jul 2006, T.I.Burgess (PREM 59843, a dry culture ex CMW 26159 onpine needles; ex-type culture CMW 26159 5 CBS 122062).


Dothiorella longicollis Pavlic, Burgess, M.J. Wingfield,sp. nov. MB512053 FIGS. 8, 9Pycnidia subimmersa plerumque solitaria, basi globosa

usque ad 550 mm diam, collis longis, interdum ramosis,usque ad 1.5 mm longis, e substrato orientia. Cellulaeconidiogenae holoblasticae cylindricae vel subcylindricaehyalinae, conidio primo holoblastico, posteriora enteroblas-tica. Conidia mediocriter 20.4 3 8.7 mm, 2.3 plo longioraquam latiora, primo hyalina unicellularia, dum etiam adcellulas conidiogenas affixa cinnamomeo- vel sepiaceo-brunnescentia, uniseptata, ovalia vel ovoidea apice rotun-data basi truncata.

Pycnidia semi-immersed, mostly solitary, with glo-bose base (,550 mm diam) and long neck (some-times branching), up to 1.5 mm long, arising fromthe substrate. Conidiogenous cells holoblastic, cylindri-cal to subcylindrical, hyaline, the first conidiumproduced holoblastically and subsequent conidiaenteroblastically, (5–)6–8(–10) 3 (2.5–)3–4(–4.5)mm (av. 7.3 3 3.4 mm). Conidia oval to ovoid, (17–)19–22(–23) 3 (7–)8–9.5(–10.5) mm (av. 20.4 3

8.7 mm, l/w 2.3), apices rounded and truncate base,initially hyaline, unicellular, becoming cinnamon(1399) to sepia (1399k) and 1-septate while stillattached to conidiogenous cells. Cultural characteris-tics. Colonies initially white to olivaceous-buff(21999d), becoming greenish-olivaceous (23999) tocitrine (21 k) from the middle of colonies within7 d, iron gray (2399999) (surface) and black (beneath)with age, with suppressed, moderately fluffy myceli-um, edges smooth appearing sinuate as the colonydarkens with age. Conidiomata readily formed fromthe middle of colony within 7–10 d, covering theentire surface of the colony and immersed in themedium (seen as round black structures on thereverse side of Petri dishes) 14 d after incubation.Optimum growth at 25 C, covering 90 mm diam Petridish after 4 d in the dark.

Teleomorph. Not known.Etymology. Refers to the fact that the pycnidia have

long necks.Habitat. Asymptomatic branches of Lysiphyllum

cunninghamii (Caesalpiniaceae) and Terminalia sp.(Combretaceae).

Known distribution. Western Australia.HOLOTYPE. AUSTRALIA. WESTERN AUSTRALIA: Tunnel

FIG. 7. Pseudofusicoccum ardesiacum. a. Pycnidia formedin culture on eucalypt twig (CBS122063). b, c. Conidiogen-ous cells (CBS122063). d. Conidium attached to conidiogen-ous cell (CBS122062). e. Aseptate conidia (CBS122062). f.Two-septate conidium (CBS122062). g. Aseptate conidiacovered with mucus layer (indicated by arrow) (CBS122063).Bars: a 5 500 mm, b–g 5 10 mm.

FIG. 6. Pseudofusicoccum ardesiacum. a. Conidiogenouscells (CBS122062). b. Conidia (CBS122062, CBS122063).Bar 5 10 mm.


Creek National Park (17u54933.342S, 125u17901.686E), onLysiphyllum cunninghamii, Jul 2006, T.I. Burgess (PREM59845, a dry culture ex CMW 26166 on pine needles; ex-typeculture CMW 26166 5 CBS 122068).

Additional specimens examined. See TABLE I.Note. Cultures transferred onto WA with pine needles

formed numerous pycnidia on the surface and immersed inthe medium. Dothiorella longicollis conforms well tomorphological concept of the genus proposed by Phillipset al (2005).

Lasiodiplodia margaritacea Pavlic, Burgess, M.J. Wing-field, sp. nov. MB512052 FIGS. 10, 11Pycnidia subimmersa solitaria globosa papillata nigra

mycelio tecta, usque ad 520 mm diam. Paraphyses cylindri-cae, 1–2-septatae, hyalinae. Cellulae conidiogenae holoblas-ticae, cylindricae vel subcylindricae, hyalinae, conidio primoholoblastico, posteriora enteroblastica. Conidia mediocriter15.3 3 11.4 mm, 1.3 plo longiora quam latiora primounicellularia, hyalina globosa subglobosa vel obovoidea,parietibus crassis, contentu granuloso, cinnamomeo- velsepiaceo-brunnescentia, cum maturitate 1-septata longitu-dinaliter striata.

Pycnidia semi-immersed, solitary, globose, papil-late, black, covered by hyphal hairs, up to 520 mmdiam. Paraphyses cylindrical, 1–2-septate, hyaline,

(19–)28–46(–54) 3 (1.5–)2–2.5(–3) mm (av. 37.1 3

2.2 mm), formed among conidiogenous cells. Con-idiogenous cells holoblastic, cylindrical to subcylindri-cal, hyaline, the first conidium produced holoblasti-cally and subsequent conidia enteroblastically, (6–)10–11(–19.5) 3 (2–) 3–4 (–4.5) mm (av. 10.3 3

3.3 mm). Conidia globose to subglobose to obovoid,(12–)14–17(–19) 3 (10–)11–12(–12.5) mm (av. 15.33 11.4 mm, l/w 1.3), with granular content, thick-walled (1–2 mm), initially unicellular, hyaline, becom-ing cinnamon (1399) to sepia (1399k), forming oneseptum and longitudinal striations with maturation.Cultural characteristics. Colonies initially white tosmoke gray (219999f) with woolly aerial mycelium,becoming pale olivaceous gray (2199999d) within 5–7 d, olivaceous gray (2199999i) to iron gray (2399999k)with age, margins regular. Submerged myceliumdense, reverse gray olivaceous (219999b) to olivaceousblack (279999m) after 7 d, becoming black with age.Optimum growth at 30 C, covering the 90 mm diamPetri dish after 3 d in the dark.

Teleomorph. Not known.Etymology. The name refers to the conidia that have

a pearl-like appearanceHabitat. Asymptomatic branches of Adansonia gib-

bosa.Known distribution. Western Australia.HOLOTYPE. AUSTRALIA. WESTERN AUSTRALIA: Tunnel

Creek Gorge (17u36922.884S, 125u108946.056E), on Adan-

FIG. 9. Dothiorella longicollis. a. Pycnidia formed inculture on water agar (CBS122068). b. Pycnidium formedin culture releasing dark 1-septate conidia (CBS122068). c.Cross section through a pycnidium showing outer layers ofdark brown cells and inner layers of hyaline cells withconidiogeneous cells arising from the pycnidial wall(CBS122067). d. Dark 1-septate conidia (CBS122067). Bars:a 5 500 mm, b–d 5 10 mm.

FIG. 8. Dothiorella longicollis. a. Conidiogenous cells(CBS122067, CBS122068). b. Conidia (CBS122067,CBS122068). Bar 5 10 mm.

860 MYCOLOGIA

sonia gibbosa, Jul 2006, T.I. Burgess (PREM 59844, a dryculture ex CMW 26162 on pine needles; ex-type cultureCMW 26162 5 CBS 122519).

Additional specimens examined. See TABLE I.Notes. Isolates of L. margaritacea clustered with other

Lasiodiplodia species with high bootstrap support (100%).The septate conidia with striations that darken with age, aswell as paraphyses, are typical of the genus (Punithalingham1976, Pavlic et al 2004, Burgess et al 2006a). However thesmaller, subglobose conidia clearly distinguish this speciesfrom previously described species (Punithalingham 1976,Pavlic et al 2004, Burgess et al 2006a, Damm et al 2007, Alveset al 2008).

Fusicoccum ramosum Pavlic, Burgess, M.J. Wingfield,sp. nov. MB512054 FIGS. 12, 13Pycnidia subimmersa solitaria globosa papillata castanea,

mycelio tecta, usque ad 510 mm diam, interdum collis ad1.7 mm longis, e substrato orientia. Cellulae conidiogenaeholoblasticae, cylindricae vel subcylindricae, hyalinae, con-idio primo holoblastico, posteriora enteroblastica. Conidio-phorae laeves cylindricae septatae usque ad 2 mm latae50 mm longae, simplices vel ramosae. Conidia mediocriter13.4 3 5.7 mm, 2.3 plo longiora quam latiora, hyalinae,parietibus tenuibus vel subcrassis, laeves contentu tenuegranulari, fusiformia ellipsoidea vel ovalia apicibus rotun-datis basibus truncatis, unicellularia vel 1-septata.

Pycnidia semi-immersed, solitary, globose, papil-late, chestnut, covered by hyphal hairs, up to 510 mmdiam, sometimes with a neck to 1.7 mm long, arisingfrom the substrate. Conidiogenous cells smooth,cylindrical to subcylindrical, hyaline, the first conid-ium produced holoblastically and subsequent conidiaenteroblastically, (6–)7.5–10(–11) 3 (2–)2–3(–3.5)mm (av. 8.7 3 2.5 mm). Conidiophores smooth,cylindrical, septate, up to 2 mm wide and 50 mm long,simple or branching. Conidia fusiform to ellipsoid tooval, (11–)12–15(–16) 3 (4.7–)5–6(–7) mm (av. 13.43 5.7 mm, l/w 2.3), apices rounded or round at apexand truncate at base, smooth with fine granularcontents, hyaline, wall thin to slightly thickened,unicellular or 1-septate. Cultural characteristics. Colo-nies initially white turning gray olivaceous (219999b)from the middle of colonies within 5–7 d, withappressed mycelial mat and white moderately dense,cottony aerial mycelium toward the edge of colony,becoming smoke gray (219999f) to olivaceous gray

FIG. 11. Lasiodiplodia margaritacea. a. Pycnidia emerg-ing through the eucalypt bark in culture. b. Conidiumattached to conidiogenous cell. c. Germinating conidium.d. Paraphyses. e. Conidia in various stages of development,including young, hyaline, aseptate conidia, unicellularconidia with developing pigmentation with and withoutseptation, mature striate conidia. (a–e CBS122519). Bars: a5 500 mm, b–e 5 10 mm.

FIG. 10. Lasiodiplodia margaritacea. a. Conidiogenouscells and paraphyses. b. Immature conidia. c. Matureconidia. (a–c CBS122519). Bar 5 10 mm.


(2199999i) (surface) and iron gray (2399999k) (beneath)within 10–14 d. Optimum growth at 25 C, coveringthe 90 mm diam Petri dish after 4 d in the dark.

Teleomorph. Not known.Etymology. Name refers to the branched conidio-

phores of this species.Habitat. Asymptomatic branches of Eucalyptus ca-

maldulensis.Known distribution. Western Australia.HOLOTYPE. AUSTRALIA. WESTERN AUSTRALIA: Bell

Gorge (17u00958.584S, 125u13947.866E), on Eucalyptuscamaldulensis, Jul 2006, T.I. Burgess (PREM 59846, a dryculture ex CMW 26167 on pine needles; ex-type cultureCMW 26167 5 CBS 12206).

Notes. The only known culture of ‘‘Botryosphaeria’’,anamorph Fusicoccum ramosum, is distinguished from otherspecies in the genus by its long, simple or branchingconidiophores. Its conidia develop a single septum beforegerminating, as is typical of Botryosphaeria (Slippers et al2004). It did not produce a Dichomera synanamorph, whichis reported for type species Botryosphaeria dothidea (Barberet al 2005). The conidia of Fusicoccum ramosum aresignificantly shorter then those of known species in thisgenus.

Neoscytalidium novaehollandiae Pavlic, Burgess, M.J.Wingfield, sp. nov. MB512103 FIGS. 14, 15

Pycnidia ad dimidium immersa vel superficiales, solitariavel in stromata multilocularia, nigra, cum basim globosa,diametrus usque ad 300 mm, collis usque ad 600 mm longis.Cellulae conidiogenae holoblasticae, cylindricae vel sub-cylindricae, hyalinae, conidio primo holoblastico, poster-iora enteroblastica. Conidia (1) mediocriter 11.5 3 4.4 mm,2.6 plo longiora quam latiora, apices rotundati, primohyalina, evadentes cinnamomeo- vel sepiaceo-brunnescentiacum maturitate, sive ellipsoidea vel ovoidea et 0–2-septatacum maturitate; (2) mediocriter 10.6 3 6.9 mm, 1.5 plolongiora quam latiora, primo hyaline, evadentes cinnamo-meo- vel sepiaceo-brunnescentia cum maturitate, sive informa variabilia, irregularia, globosa, subglobosa vel obpyr-iformia, cum septis muriformibus, Arthroconidia catenulatain mycelio aerio, mediocriter 6.5 3 4 mm, 1.6 plo longioraquam latiora, pulveriformia, disarticulantia, cylindrica,oblonga, obtusa vel doliiformia, crasse tunicata, primohyalina et unicellularia, cinnamomeo- vel sepiaceo-brun-nescentia et 0–1-septata cum maturitate.

Pycnidia semi-immersed or superficial, solitary or inmultilocular stromata, black, with globose base, up to300 mm diam and long neck, up to 600 mm long.Conidiogenous cells holoblastic, cylindrical to subcy-

FIG. 13. Fusicoccum ramosum. a. Pycnidia emergingthrough the eucalypt bark in culture releasing white massesof conidia. b. Conidiophores with attached conidia. c.Germinating 1-septate conidium. d. Conidium attached toconidiogenous cell. e. Conidiophores arising from thepycnidial wall. f, g. Conidiophore with attached conidium,at two different focuses. h. Aseptate conidia. (a–hCBS122069). Bars: a 5 500 mm, b–h 5 10 mm.

FIG. 12. Fusicoccum ramosum. a. Conidiogenous cellsand conidiophores. b. Branching conidiophores. c. Conid-ia. (a–c CBS122069). Bar 5 10 mm.

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lindrical, hyaline, the first conidium produced holo-blastically and subsequent conidia enteroblastically,(6–)7–10(–11) 3 (2–)2–3(–4) mm (av. 8.6 3 2.5 mm).Conidia of two distinct types: (i) ellipsoidal to oval,(8–)10.5–12.5(–14) 3 (3–)4–5(–5) mm (av. 11.5 3

4.4 mm, l/w 2.6), apices rounded, initially hyaline,unicellular, becoming cinnamon (1399) to sepia(1399k), and 0–1-septate or 2-septate with darkercentral cell; (ii) variable in shape, globose, subgloboseto obpyriform with muriform septa, (8–)8.5–12.5(–15.5) 3 (5–)5.5–7.5(–8) mm (av. 10.6 3 6.9 mm, l/w 1.5), initially hyaline becoming cinnamon (1399) tosepia (1399k). Aerial mycelium forms chains ofarthroconidia, (5–)5.5–7.5(–9.5) 3 (3–)3.5–4.5(–5)mm (av. 6.5 3 4 mm, l/w 1.6), unicellular, powdery tothe touch, disarticulating, cylindrical, oblong toobtuse to doliiform, thick-walled, initially hyalinebecoming becoming cinnamon (1399) to sepia(1399k) and 0–1-septate. Cultural characteristics. Col-onies initially white to olivaceous-buff (21999d),becoming greenish-olivaceous (23999) to citrine(21 k) from the middle of colonies within 7 d, andblack (surface and beneath) with age, with sup-pressed, moderately fluffy mycelium, edges smooth.

Optimum growth at 35 C, covering the 90 mm diamPetri dish after 3 d in the dark.

Teleomorph. Not known.Etymology. Name refers to original Dutch name for

Western Australia, where the substratum was collectedfrom which the fungus was isolated.

Habitat. Asymptomatic branches (sapwood) of Aca-cia synchronica, Adansonia gibbosa, Crotalaria medica-ginea and Grevillia agrifolia.

Known distribution. Western Australia.HOLOTYPE. AUSTRALIA. WESTERN AUSTRALIA: Bell

Gorge (17u00958.584S, 125u13947.866E), on Crotalariamedicaginea, Jul 2006, T.I. Burgess (PREM 60069, a dryculture ex CMW 26170 on pine needles; ex-type cultureCMW 26170 5 CBS 122071).

Additional specimens examined. See TABLE I.Note: Isolates of Neoscytalidium novaehollandiae

are similar in morphological characteristics to those ofthe type specimen N. dimidiatum (Punithalingam andWaterston 1970, Crous et al 2006). However isolatesobtained in this study produce muriform, Dichomera-likeconidia that distinguish this species from known Neoscyta-lidium spp.

KEY TO PSEUDOFUSICOCCUM SPECIES

1. Blue-violet pigment in cultures visible after 3–5 d;conidia averaging 25 mm long, l/w 3.3, aseptate,forming 1–3 septa before germination . . P. ardesiacum

1. Blue-violet pigment absent in cultures . . . . . . . . . . 22. Conidia on average .30 mm long. . . P. kimberleyense

2. Conidia on average ,25 mm long. . . . . . . . . . . . . . 33. Conidia aseptate, l/w 4 . . . . . . . . . . . P. stromaticum

3. Conidia aseptate, forming 1or 2 septa beforegermination, l/w 4.3 . . . . . . . . . . . . . . P. adansoniae

FIG. 14. Neoscytalidium novaehollandiae. a. Conidiogen-ous cells (CBS122610). b. Conidia. c. Muriform conidia. d.Chains of arthroconidia. (b–d CBS122071). Bar 5 10 mm.

FIG. 15. Neoscytalidium novaehollandiae. a. Pycnidiaemerging from a pine needle in culture. b. Conidiogenouscells (CBS122610). c. Hyaline aseptate conidia. d. Two-septate dark conidia. e–g. Muriform conidia. h, i. Chains ofarthroconidia. (a, c–i CBS122071). Bars: a 5 500 mm, b–i 5

10 mm.


DISCUSSION

Seven new species of Botryosphaeriaceae were isolat-ed from endemic trees in Western Australia. Com-bined ITS and EF1-a sequence data distributed theseisolates among genera Botryosphaeria, Dothiorella,Lasiodiplodia, Neoscytalidium and Pseudofusicoccum.Teleomorphs were not observed for any of the speciesidentified in this study.

Three of the seven new fungi are species ofPseudofusicoccum, a genus previously monotypic forP. stromaticum (Crous et al 2006, Mohali et al 2006).Pseudofusicoccum is separated from Fusicoccum by thepresence of persistent mucous sheaths surroundingthe conidia (Crous et al 2006). Pseudofusicoccumstromaticum was described on non-native Acacia andEucalyptus spp. in Venezuela (Mohali et al 2006).Strains of P. adansoniae and P. kimberleyense de-scribed in this study were obtained from fourunrelated hosts (Acacia sp., Eucalyptus sp., Ficus sp.and A. gibbosa) residing in four families all native toWestern Australia. Isolates of P. ardesiacum wereobtained from two of these native hosts, Eucalyptusand A. gibbosa. The fact that all Pseudofusiccum spp.occured on native hosts in a relatively undisturbedarea of Australia or in the case of P. stromaticum onAustralian plants suggests that the species are mostlikely native to the country.

Isolates of P. adansoniae came from different hostsbut were morphologically uniform. This is in contrastto isolates of P. kimberleyense, which displayeddifferences in conidial morphology and variation inDNA sequences in both gene regions analyzed. Thesedifferences could indicate that P. kimberleyense com-prises more than one species. Pseudofusicoccumardesiacum was easily distinguished from other speciesin the genus by its smaller conidia and the distinctslate blue-violet pigment that it produces in culture.

Two species with dark conidia were identified inthis study. Based on phylogenetic analyses andphenotype, they have been placed in Lasiodiplodiaand Dothiorella. Lasiodiplodia margaritacea was iden-tified only from dying branches of A. gibbosa. This wasinteresting because high numbers of dead and dyingbaobabs (A. digitata) have been reported in southernAfrica, particularly in Zimbabwe (Anonymous 1991,Piearce et al 1994). The symptoms identified on thetrees in Zimbabwe originally were reported as ‘‘sootybark disease’’ caused by species of sooty mold fungi(Calvert 1989, Anonymous 1991). However Piearce etal (1994) reported that the ‘‘sooty’’ baobabs weredying due to drought, related to climatic change,rather than being caused by fungal pathogens. Arecent study on diseases of baobabs in South Africa,showing symptoms of die-back and death of branches

followed by sap exudation, revealed that Lasiodiplodiatheobromae was the most abundant fungus present(Roux 2002). This fungus is a well known latent,stress-associated pathogen on more then 500 hostsworldwide (Punithalingham 1976) and as such couldbe involved in the decline of baobab trees in Africancountries (Roux 2002). Because Lasiodiplodia margar-itacea was found only on A. gibbosa that shows die-back symptoms this fungus could be pathogenic tothis tree.

Dothiorella longicollis is another species with darkconidia described in this study. This species ismorphologically similar to the other species withDothiorella anamorphs, D. iberica, D. sarmentorum andD. viticola (Luque et al 2005, Phillips et al 2005).Other than the pycnidia with long necks, which are adistinct feature of D. longicollis, other morphologicalcharacteristics, such as conidial shape and size,overlap among these species and cannot be used toseparate them with confidence. However their dis-tinction is well supported in the ITS and EF1-aphylogenies. Dothiorella longicollis occured as anendophyte in asymptomatic branches of two unrelat-ed hosts, Lysiphyllum cunninghamii (Caesalpiniaceae)and a Terminalia sp. (Combretaceae) endemic toWestern Australia and nothing is known regarding itsecology.

A number of isolates obtained from asymptomaticbranches on different hosts, including Acacia, Adan-sonia, Crotalaria and Grevillia, were identified asNeoscytalidium novaehollandiae. Neoscytalidium, withN. dimidiatum as a type, accommodates species withScytalidium-like synanamorphs (Crous et al 2006).These are characterized by conidia held in arthricchains in the aerial mycelium. In addition toarthroconidia the cultures produce Fusicoccum-likeconidia in pycnidia. Isolates of N. novaehollandiaeidentified in this study produce a Dichomera-likesynanamorph, which is not known for other speciesin this genus. Dichomera-like synanamorphs recentlywere described for Botryosphaeria dothidea, Neofusi-coccum parvum, N. ribis and N. australe (Barber et al2005), however this is the first time that a Dichomera-like synanamorph has been found in Neoscytalidium.Neoscytalidium dimidiatum has been isolated fromdifferent substrates including plant tissues, soil,human skin and nails, and is known as a plantpathogen (Punithalingam and Waterston 1970, Crouset al 2006). The isolates examined in this study werecollected as endophytes from plant tissues, and theyseem unlikely to be important pathogens. Fusicoccumramosum was isolated as endophyte from asymptom-atic twigs of Eucalyptus camaldulensis.

Numerous species of Botrosphaeraceae with ‘‘Fusi-Fusicoccum’’ anamorphs identified from Eucalyptus

864 MYCOLOGIA

now have been placed in a new genus Neofusicoccum(Crous et al 2006). Neofusicoccum spp. are the mostcommon endophytes and latent pathogens of Euca-lyptus (Burgess et al 2006b, Slippers and Wingfield2007), however no Neofusicoccum spp. were isolatedfrom Euclyptus in this study.

Species of Botryosphaeriaceae are well known asendophytes and latent, opportunistic canker and die-back pathogens on numerous woody hosts worldwide(von Arx 1987, Slippers and Wingfield 2007, de Wet etal 2008). However this is the first detailed study toconsider these fungi on Adansonia gibbosa and otherendemic trees in Western Australia, including Acaciasynchronica, Crotalaria medicaginea, Eucalyptus camal-dulensis, Eucalyptus sp., Ficus opposita, Grevilliaagrifolia, Lysiphyllum cunninghamii and Terminaliasp. The seven new species emerging from this study,of which five were recorded on A. gibbosa, reflect alack of knowledge regarding the fungi on A. gibbosaand of the Botryosphaeriaceae on native plants in thisregion. The role of these fungi in the ecology of thetrees from which they were collected will be consid-ered in future studies.

ACKNOWLEDGMENTS

We thank Dr H.F. Glen (Natal Herbarium. P.O. Box 52099,Berea Road, Durban, 4007, South Africa) for providingLatin descriptions and Monique Sakalidis for assisting us inmaking some of the primary isolations. In Australia thisproject was financially support in part by the ARC Discoveryproject DP0664334 for a sabbatical research project ofMichael J. Wingfield. Portions of the research conducted inSouth Africa were supported financially by the NationalResearch Foundation (NRF), members of the Tree Protec-tion Co-operative Programme, the THRIP initiative of theDepartment of Trade and Industry as well as the Depart-ment of Science and Technology/ National ResearchFoundation Centre of Excellence in Tree Health Biotech-nology (CTHB).

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seven new species of the botryosphaeriaceae from baobab

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