Three new Lasiodiplodia spp. from the tropics, recognized based on DNAsequence comparisons and morphology

Treena I. Burgess1

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

Sari MohaliUniversidad de Los Andes, Facultad de CienciasForestales y Ambientales, Laboratorio de PatologiaForestal, Merida, Venezuela

Geoff PeggDepartment of Primary Industries and Fisheries,Horticulture and Forestry Science, Indooroopilly,Brisbane 4068, Australia

Wilhelm de BeerMichael J. Wingfield

Forestry and Agriculture Biotechnology Institute,University of Pretoria, Pretoria, 0002, Republic ofSouth Africa

Abstract: Botryosphaeria rhodina (anamorph Lasiodi-plodia theobromae) is a common endophyte andopportunistic pathogen on more than 500 treespecies in the tropics and subtropics. During routinedisease surveys of plantations in Australia andVenezuela several isolates differing from L. theobromaewere identified and subsequently characterized basedupon morphology and ITS and EF1-a nucleotidesequences. These isolates grouped into three stronglysupported clades related to but different from theknown taxa, B. rhodina and L. gonubiensis, Thesehave been described here as three new species L.venezuelensis sp. nov., L. crassispora sp. nov. and L.rubropurpurea sp. nov. The three could be distin-guished easily from each other and the two describedspecies of Lasiodiplodia, thus confirming phylogenet-ic separations. Furthermore all five Lasiodiplodia spp.now recognized separated from Diplodia spp. andDothiorella spp. with 100% bootstrap support.

Key words: Botryosphaeria, Diplodia, Dothiorella,Fusicoccum, ITS, molecular phylogenetics, trans-lation elongation factor EF1-a

INTRODUCTION

Botryosphaeria Ces. & de Not. includes pathogenicfungi that cause cankers and dieback on a wide range

of woody hosts (von Arx 1987). The majority ofBotryosphaeria spp. have a cosmopolitan distributionand have been found on most continents and onnumerous hosts. They are rarely primary pathogens,instead causing stress-related diseases or perennialcankers. In temperate climates B. ribis, B. parva andB. dothidea are the most common species isolatedfrom cankers, while in the tropics B. rhodinapredominates (Punithalingam 1980).

Numerous anamorphs have been assigned toBotryosphaeria spp., but recent studies based onDNA sequence comparisons have indicated there isa clear phylogenetic boundary between species withthin-walled, hyaline conidia and those with thick-walled, pigmented spores (Denman et al 2000, Zhouand Stanosz 2001). Those with hyaline conidia havebeen assigned a Fusicoccum anamorph and those withpigmented conidia a Diplodia anamorph (Denman etal 2000). However, among the species with pigmentedconidia, B. rhodina always groups separately from theother species (Denman et al 2000, Zhou and Stanosz2001, Pavlic et al 2004, Slippers et al 2004). Theanamorph of B. rhodina, Lasiodiplodia theobromae, hasconidia much larger than other Diplodia species andcurrently retains the name Lasiodiplodia (Punithalin-gam 1976, Pavlic et al 2004, Phillips et al 2005).

The taxonomic history of B. rhodina/L. theobromaeis confused. During the past 150 y this fungus has hadmany names and has been treated as many differentspecies. This trend ended with the monograph ofPunithalingam (1976) which reduced most species tosynonymy with L. theobromae. Recently Pavlic et al(2004) conducted an extensive review of Lasiodiplodialiterature and searched for herbarium specimensassociated with original descriptions of the genus(Clendinin 1896) and its species (Patouillard and deLagerheim 1892). These could not be found andcultures from the original host and location, Theo-broma cacao in Ecuador, also were not located. It thuswas necessary to rely on descriptions from theliterature and Pavlic et al (2004) found that isolatesfrom USA, South America, South Africa and Asiatypically have conidia that are 18–30 3 10–15 mm.This led to the description of the new species L.gonubiensis, which could be distinguished by bothDNA-based phylogenies and morphological charac-teristics.

In a study using SSR markers to examine hostrelationships and geographic isolation among isolates

Accepted for publication 4 Apr 2006.1 Corresponding author. E-mail: tburgess@murdoch.edu.au

Mycologia, 98(3), 2006, pp. 423–435.# 2006 by The Mycological Society of America, Lawrence, KS 66044-8897

423

of L. theobromae, Mohali et al (2005) identified severalisolates that shared no common alleles with 177 otherisolates at any of the eight SSR loci. This is consideredto be indicative of the presence of related butdifferent species. In addition disease surveys intropical Australia have led to the discovery of severalLasiodiplodia isolates with conidia morphologicallydifferent from those of L. theobromae. These observa-tions prompted the current study aimed at character-izing isolates of Lasiodiplodia based on morphologyand multiple gene genealogies.

MATERIALS AND METHODS

Isolates.—During routine sampling in 2002/2003 andsubsequent studies using SSR markers, a number of fungiresembling Lasiodiplodia theobromae were collected. Closerexamination of some showed cultural characteristics thatdistinguished them from L. theobromae sensu stricto. Thesecollections included two isolates from cankered sandal-wood (Santalum album) in Kununnara, Western Australia,one isolate from Eucalyptus urophylla in Venezuela, threeisolates from Acacia mangium in Venezuela and fourisolates collected from cankered E. grandis near Tully,North Queensland (TABLE I).

DNA sequence comparisons.—For each isolate approxi-mately 50 mg of fungal mycelium was scraped from thesurface of 7 d old cultures, ground with a glass rod,suspended in 200 mL of DNA extraction buffer (200 mMTris-HCL pH 8.0, 150 mM NaCl, 25 mM EDTA, 0.5%

SDS) and incubated 1 h at 70 C. DNA was purified withthe UltrabindH DNA purification kit following themanufacturer’s instructions (MO BIO Laboratories, So-lana Beach, California).

Two gene regions were used for phylogenetic compar-isons. A part of the internal transcribed spacer (ITS) regionof the ribosomal DNA operon was amplified with theprimers ITS-1F (59 CTT GGT CAT TTA GAG GAA GTA A)(Gardes and Bruns 1993) and ITS4 (59 TCC TCC GCT TATTGA TAT GC) (White et al 1990). In addition a part of theelongation factor 1-a was amplified with primers EF1-728F(59 CAT CGA GAA GTT CGA GAA GG) and EF1-986R (59

TAC TTG AAG GAA CCC TTA CC) (Carbone and Kohn1999). The PCR reaction mixture (25 mL), PCR conditionsand visualization of products were as described by Pavlic etal (2004) except that 0.5 U of Taq polymerase (BiotechInternational, Needville, Texas) were used in each reaction.PCR products were cleaned with UltrabindH DNA purifica-tion kit (MO BIO Laboratories). Products were sequencedwith the BigDye terminator cycle sequencing kit (PEApplied Biosystems) with the same primers used in theinitial amplification. The products were separated with anABI 3730 48 capillary sequencer (Applied Biosystems, FosterCity, California) and a BioRad Biofocus 2000 capillary gelelectrophoresis system. Data were collected with ABI datacollection software.

To compare the Botryosphaeria isolates used in this studywith other Botryosphaeria spp., 30 ITS rDNA and 15 EF-1a

sequences obtained from GenBank were included in thephylogenetic analysis (TABLE I). Sequence data were ana-lyzed with Sequence Navigator version 1.0.1TM (PerkinElmer Corp., Foster City, California) and manually alignedby inserting gaps. PCR products of approximately 500 bpand 300 bp were amplified for the ITS and EF-1a regionsrespectively. Ambiguous sequences at the 59 and 39 endswere deleted in the aligned dataset. Lasiodiplodia spp. havea large deletion (35–38 bp), compared with other Botryo-sphaeria spp. in this study, in the ITS1 region, which wasexcluded and coded. Two regions in EF-1a sequences werealso excluded and liberally coded. The first region wasa 9 bp insertion found only in B. rhodina and the seconda 13 bp insertion found only in B. ribis. Gaps were treated asa fifth character, all ambiguous characters and parsimonyuninformative characters were excluded before analysis.The initial analysis was performed on an ITS dataset thatincluded 33 isolates of B. rhodina (TABLE I). Subsequentanalyses, including 11 isolates of B. rhodina, were per-formed on individual datasets as well as combined datasetsafter partition homogeneity tests (PHT) were performed inPAUP version 4.0b10 (Swofford 2000) to determinestatistical congruence (Farris et al 1995, Huelsenbeck et al1996).

The most parsimonious trees were obtained by usingheuristic searches with random stepwise addition in 100replicates, with the tree bisection-reconnection branch-swapping option on and the steepest-descent option off.MAXTREES were unlimited, branches of zero length werecollapsed and all multiple equally parsimonious trees weresaved. Estimated levels of homoplasy and phylogeneticsignal (retention and consistency indices) also were de-termined with PAUP (Hillis and Huelsenbeck 1992). In theinitial analysis all characters were unweighted and un-ordered; for the ITS analysis, characters were reweightedaccording to the consistency index. Branch and branchnode supports were determined with 1000 bootstrapreplicates (Felsenstein 1985) and characters were sampledwith equal probability but weights were applied. ITS treeswere rooted with Saccharata protea (Wakefield) Denman &Crous. This study focussed on Botryosphaeria spp. withLasiodiplodia anamorphs, and to avoid long-branch attrac-tion associated with phylogenetically distant outgroupstrees from the combined dataset were rooted with B. ribis,a species with hyaline conidia and a Fusicoccum anamorph.

Morphological characteristics.—A total of 32 single co-nidium isolates representing the different culturalmorphologies were used in this study (TABLE I).Sporulation was induced by transferring isolates to tapwater agar overlaid with pine needles and/or eucalypttwigs as a substrate and exposing these to near UV lighton a 24 h light cycle at 22 C for 2–4 wk. Cultures weremaintained on one-half strength potato-dextrose agar(one-half PDA; Becton, Dickinson & Co., Sparks, Mary-land) at 25 C and stored on this medium at 4 C.Cultures also were stored at room temperature in sterilewater.

Colony morphology, color (Rayner 1970) and growthrates at 5–35 C of representative isolates were determined

424 MYCOLOGIA

on one-half strength PDA. Fruiting structures weremounted in lactoglycerol. Observations and measurementsof conidial characteristics (30–40 per isolate) were madewith a light microscope and an Axiocam digital camera(Carl Zeiss, Germany) and drawings prepared with a drawingtube. Approximately 30 conidia were measured for eachisolate. All isolates in this study are maintained in theculture collection (CMW) of the Forestry and AgricultureBiotechnology Institute, University of Pretoria, SouthAfrica, and the Department of Agriculture, Perth, WesternAustralia (WAC). Herbarium material is held at theMurdoch University Herbarium (MURU).

RESULTS

DNA sequence comparisons.—Initially 44 Lasiodiplodiaisolates, including 33 isolates of B. rhodina fromdifferent hosts and locations, were compared basedon ITS sequence alone (TABLE I). The aligneddataset consisted of 541 characters, of which a 38 bpindel was coded and excluded, resulting in 133parsimony informative characters. The datasetcontained significant phylogenetic signal com-pared to 1000 random trees (P , 0.01, g1 5

21.02). Heuristic searches of unweighted charac-ters in PAUP resulted in 1224 most parsimonioustrees of 257 steps (CI 5 0.74, RI 5 0.92). The largenumber of trees was due to the small (1–5 bp)differences among isolates of L. theobromae. Of the133 informative characters, 47 characters hada weight of less than 1, indicating homoplasy.Reweighting of characters based on the consistencyindex resulted in only 18 trees of 190 steps (CI 5

0.83, RI 5 0.95). Sequence alignments are availablefrom TreeBASE (SN2399-9015). In the analysisLasiodiplodia isolates grouped in five stronglysupported clades (FIG. 1). One large clade con-tained isolates of L. theobromae from a wide range ofhosts and locations, another clade of two isolatesrepresented L. gonubiensis and three further cladeswere thought to represent undescribed taxa.

The aligned dataset of the combined ITS and EF-1asequences consisted of 877 characters of which threeindels of 57 bp were coded and excluded leaving 276parsimony informative characters that were includedin the analysis. A partition homogeneity test showedno significant difference (P 5 0.77) between the datafrom the different gene regions (sum of lengths oforiginal partition was 549, range for 1000 randomiza-tions was 543–552) and these therefore could becombined. The combined dataset contained signifi-cant phylogenetic signal compared to 1000 randomtrees (P , 0.01, g1 5 20.75). Initial heuristic searchesof unweighted characters in PAUP resulted in eightmost parsimonious trees of 552 steps (CI 5 0.75, RI 5

0.93). Sequence alignments are available from Tree-

BASE (SN2399-9016). The resulting tree (FIG. 2)clearly separated isolates (with 100% bootstrapsupport) with Lasiodiplodia anamorphs from thosewith Diplodia, Dothiorella and Fusicoccum anamorphs.The Lasiodiplodia isolates were grouped further intofive clades corresponding to L. theobromae, L.gonubiensis and three undescribed taxa labeled CladeIII, Clade IV and Clade V (FIG. 2). All clades weresupported strongly by high bootstrap value and nofurther subdivision was seen for clades II–V. Withinthe B. rhodina clade (I), there was some substructurebut this had low bootstrap support.

Morphology.—Anamorph structures were producedby isolates representing the three unique phylo-genetic groups, on both pine needles and eucalypttwigs after 3 wk. No sexual structures wereobserved, either on the original wood from whichisolations were made or on the needles or twigs inculture. The conidia produced by the cultureswere similar to those described for L. theobromae,although in all cases they were slightly larger,although not as large, as those observed for L.gonubiensis (TABLE II). Isolates in clades III and IVhad septate paraphyses, whereas those observedfor L. theobromae and L. gonubiensis were aseptate.Pycnidia of isolates in Clade V were reddish/purple and covered with mycelium. Pycnidia of L.theobromae, and those for isolates in clades III andIV are smooth.

TAXONOMY

DNA sequence comparisons have shown that isolatescollected from Western Australia and Venezuela,forming the basis of this study, represent threedistinct and strongly supported phylogenetic groups.These groups separated clearly from L. theobromaeand L. gonubiensis. The isolates in these three cladesalso could be separated from L. theobromae and L.gonubiensis as well as from each other, based onmorphological characteristics. They consequently aredescribed here as three new species:

Lasiodiplodia crassispora MB500235 Burgess, Barber,sp. nov. FIGS. 3A–C, 4Pycnidia superficialia, pro parte maxima solitaria,

conica laevia, chalybea, 0.5–1 mm diam. Paraphysescylindricae, septatae, hyalinae. Cellulae conidiogenaeholoblasticae, subcylindricae, cylindricae vel ampulli-formes, percurrenter proliferantes. Conidia in culturafacta (Statura media 28.8 3 16.0 mm, longitudo/latitudo 1.8), primo hyalina, unicellularia, ellipsoideavel obovoidea, parietibus crassis cum contento gran-ulari, apice rotundata, interdum basin truncata, cum

BURGESS ET AL: THREE NEW LASIODIPLODIA SPP. 425

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BURGESS ET AL: THREE NEW LASIODIPLODIA SPP. 427

maturitate vel ante germinationem colorascentia unocum septo, in maturis strias verticales visas.

Pycnidia superficial, mostly solitary, conical,smooth, iron gray (21–23’’’’’k), 0.5–1 mm diam.Paraphyses cylindrical, septate, hyaline (21)30–62(66) 3 2–3.5(4) mm (average of 50 paraphyses

45.7 3 2.7 mm). Conidiogenous cells holoblastic,hyaline, subcylindrical to cylindrical to ampulliform,(6)8–16(19) 3 3–7 mm (average of 50 conidiogenouscells 11.8 3 5.0 mm), proliferating percurrently.Conidia produced in culture initially hyaline, unicel-lular, ellipsoid to obovoid, thick-walled (2–3 mm,

FIG. 1. Bootstrap consensus tree from analysis of ITS sequence data. Bootstrap support (based on 1000 replication) is givenabove the branches. The sequence of Botryosphaeria rhodina from GenBank is compared with isolates sequenced in this study.The locality and host of each isolate is provided next to the culture number or GenBank accession number.

428 MYCOLOGIA

average of 50 conidia 2.6 mm) with granular content,round at apex, occasionally truncate at base, becom-ing pigmented with one septa when mature or beforegermination, vertical striations observed at matura-tion, 27–30(–33) 314–17 mm (average of 75 conidia,28.8 3 16.0, l/w 1.8). Cultural characteristics. Moder-

ately dense, appressed mycelial mat. Aerial mycelia/colonies initially white to buff turning pale olivaceousgray (21’’’’’d) within 7 d and becoming olivaceousgray (21’’’’’i) with age. At 7 d the submerged myceliaare olivaceous gray (21’’’’’i), becoming iron gray (21–23’’’’’k) to black with age. Optimum temperature for

FIG. 2. A phylogram of one of the eight most parsimonious trees obtained from the combined ITS and EF-1a sequencedata of Botryosphaeria isolates. The three new Lasiodiplodia spp. are in bold. Branch support (bootstrap values) is given abovethe branches based on 1000 bootstrap replicates. The tree is rooted to Botryosphaeria ribis.

BURGESS ET AL: THREE NEW LASIODIPLODIA SPP. 429

TABLE II. Conidial dimensions of Lasiodiplodia species examined in the present study and described previously inthe literature

Identity Conidial size (mm) L/W paraphyses Source of data

L. theobromae 15–35 3 10–15 Cited in Pavlic et al (2004)17–33 3 10–15 [Av. 22.6 3 12.2] 1.9 aseptate This study

L. gonubiensis (28–)32–36(–39) 3 (14–)16–18.5(–21)[Av. 33.8 3 17.3]

1.9 aseptate Pavlic et al (2004)

L. venezuelensis 26–33 3 12–15 [Av. 28.4 3 13.5] 2.1 septate This studyL. crassispora 27–30(–33) 3 14–17 [Av. 28.8 3 16.0] 1.8 septate This studyL. rubropurpurea 24–33 3 13–17 [Av. 28.2 3 14.6] 1.9 aseptate This study

FIG. 3. Micrographs of fruiting structures of (a) Lasiodiplodia crassispora, conical pycnidia formed in culture on pineneedles; (b) Lasiodiplodia crassispora, immature conidia with thick walls; (c) Lasiodiplodia crassispora, mature conidia withmelanized banding; (d) Lasiodiplodia rubropurpurea, pycnidia formed in culture on pine needles covered with mycelium; (e)Lasiodiplodia rubropurpurea, immature conidia; (f ) Lasiodiplodia rubropurpurea, mature conidia; (g) Lasiodiplodiavenezuelensis, pycnidia formed in culture on pine needles oozing immature conidia; (h) Lasiodiplodia venezuelensis immatureconidia; (i) Lasiodiplodia venezuelensis mature conidia with apparent vertical striations. Bar 5 0.5 mm (a, d, g) or 10 mm (b–c,e–f, h–i).

430 MYCOLOGIA

growth 30 C, reaching 74 mm diam on PDA after 3 dat 30 C in the dark.

Teleomorph. Botryosphaeria sp. (based on phylo-genetic inferences, but unknown)

Etymology. Having thick-walled sporesSpecimens examined. AUSTRALIA, Western Australia:

Kununurra from canker of Santalum album, Dec 2003,T.I. Burgess (HOLOTYPE MURU 407) (cultureWAC12533); Kununurra, S. album, T.I. Burgess (MURU408) (culture WAC12534): VENEZUELA, Portuguesa State:Acarigua from wood of living Eucalyptus urophylla, Oct2003, S. Mohali (culture CMW13448)

Lasiodiplodia rubropurpurea MB500236 Burgess,Barber, Pegg, sp.nov. FIGS. 3D–F, 5Pycnidia superficialia, globosa, atrocarminea vel

atrovinosa, pro parte maxima solitaria, 0.5–1 mmdiam, mycelio tecta. Paraphyses cylindricae, aseptatae,hyalinae. Cellulae conidiogenae holoblasticae, hyali-nae, subcylindricae vel ampuliformes, percurrenterproliferantes cum una annelatione. Conidia (staturamedia 28.2 3 14.6 mm, longitudo/latitudo 1.90)primo hyalina, unicellularia, ellipsoidea vel obovoi-dea, parietibus crassis cum contento granulari, cum

maturitate vel ante germinationem colorascentia unocum septo, in maturis strias verticales visas.

Pycnidia superficial, globose, livid red (69’’i) todark vinaceous (69’’m), mostly solitary, 0.5–1.5 mmdiam and covered with mycelium. Paraphyses cylin-drical, aseptate, hyaline (30)32–52(58) 3 1.5–3.5 mm(average of 50 paraphyses 42.4 3 2.6 mm). Conidio-genous cells holoblastic, hyaline, subcylindrical toampulliform, 7–13(15) 3 3–5 mm (average of 50conidiogenous cells 10.2 3 4.0 mm), proliferatingpercurrently with up to 1 annellation. Conidia initiallyhyaline, unicellular, ellipsoid to obovoid, thick-walled(1 mm, average of 50 conidia 5 1 mm) with granularcontent, round at apex, occasionally truncate at base,initially hyaline and unicellular, becoming pigmentedwith one septa when mature or before germination,vertical striations observed at maturation, 24–33 3

13–17 mm (average of 100 conidia 28.2 3 14.6, l/w1.9). Cultural characteristics. Moderately dense, ap-pressed mycelial mat. Aerial mycelia/colonies initially

FIG. 4. Lasiodiplodia crassispora, (a) conidiogenous cellsand paraphyses, (b) immature conidia, (c) mature conidia.Bar 5 10 mm.

FIG. 5. Lasiodiplodia rubropurpurea, (a) conidiogenouscells and paraphyses, (b) immature conidia (c) matureconidia. Bar 5 10 mm.

BURGESS ET AL: THREE NEW LASIODIPLODIA SPP. 431

white to buff turning smoke gray (21’’’’d) to paleolivaceous gray (21’’’’’d) within 7 d and becominggray olivaceous (21’’’’b) to olivaceous gray (21’’’’’i)with age. At 7 d the submerged mycelia are grayolivaceous (21’’’’b) to olivaceous gray (21’’’’’i),becoming iron gray (23’’’’’k) to black with age.Optimum temperature for growth 25–30 C, reaches76 mm diam on PDA after 3 d at both 25 C and 30 Cin the dark.

Teleomorph. Botryosphaeria sp. (based on phylo-genetic inferences, but unknown)

Etymology. Refers to the reddish-purple pycnidiaSpecimens examined. AUSTRALIA. Queensland: Tully,

from canker of Eucalyptus grandis, May 2003, T.I. Burgess(HOLOTYPE MURU 409) (culture WAC12535); Tully, E.grandis, T.I. Burgess (MURU 410) (culture WAC12536);Tully, E. grandis, T.I. Burgess (MURU 411) (cultureWAC12537); Tully, E. grandis, T.I. Burgess (MURU 412)(culture WAC12538)

Lasiodiplodia venezuelensis MB500237 Burgess, Bar-ber, Mohali, sp. nov. FIGS. 3H–J, 6Pycnidia superficialia, chalybea, laeves, cylindrica,

pro parte maxima solitaria, 0.5–1 mm diam, saepeconidia immatura stillantia. Paraphyses cylindricae,septatae, hyalinae. Cellulae conidiogenae holoblasti-cae, hyalinae, subcylindricae vel cylindricae velampulliformes, percurrenter proliferantes. Conidia(statura media 28.4 3 13.5 mm, longitudo/latitudo2.1) primo hyalina, unicellularia, ellipsoidea velobovoidea, parietibus crassis cum contento granulari,apice rotundata, interdum basin truncata, cummaturitate vel ante germinationem colorascentiauno cum septo, in maturis strias verticales visas.

Pycnidia. superficial, iron gray (21–23’’’’’k),smooth, cylindrical, mostly solitary, 0.5–1 mm diamoften oozing immature conidia. Paraphyses cylindri-cal, septate, hyaline (12)16–41(45) 3 (1.5)2–5 (aver-age of 50 paraphyses 28.3 3 3.5 mm). Conidiogenouscells holoblastic, hyaline, subcylindrical to cylindricalto ampulliform, (5)7–14 (15) 3 3–4.5(5) mm (averageof 50 conidiogenous cells 10.4 3 3.7 mm), proliferat-ing percurrently. Conidia initially hyaline, unicellular,ellipsoid to obovoid, thick-walled (1.5–2.5(3) mm,average of 50 conidia 5 1.96 mm) with granularcontent, round at apex, occasionally truncate at base,becoming pigmented with one septa when mature orbefore germination, vertical striations observed atmaturation, 26–33 3 12–15 mm (average of 75conidia 28.4 3 13.5, l/w 2.1),. Cultural characteristics.Moderately dense, appressed mycelial mat. Aerialmycelia/colonies initially white to buff turning paleolivaceous gray (21’’’’’d) within 7 d and becomingolivaceous gray (21’’’’’i) with age. At 7 d the sub-merged mycelia are olivaceous gray (21’’’’’i), becom-ing iron gray (23’’’’’k) to black with age. Optimumtemperature for growth 25 C, reaching 75 mm diamon PDA after 3 d at 25 C in the dark.

Teleomorph. Botryosphaeria sp. (based on phylo-genetic inferences, but unknown)

Etymology. Country of origin, VenezuelaSpecimens examined. VENEZUELA, Estado Portuguesa:

Acarigua from wood of living Acacia mangium, Oct 2003, S.Mohali (HOLOTYPE MURU 413) (culture WAC12539);Acarigua, A. mangium, S. Mohali (MURU 414) (cultureWAC12540); Acarigua, A. mangium, S. Mohali (cultureCMW13513)

KEY TO LASIODIPLODIA SPP.

1. Paraphyses septate . . . . . . . . . . . . . . . . . . . . . 21. Paraphyses aseptate . . . . . . . . . . . . . . . . . . . . 32. spore wall of immature conidia thin (,2 mm),

striations in mature conidia narrow . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . L. venezuelensis

2. spore wall of immature conidia thick (.2 mm),striations in mature conidia wide . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . L. crassispora

FIG. 6. Lasiodiplodia venezuelensis, (a) conidiogenouscells and paraphyses, (b) immature conidia, (c) matureconidia. Bar 5 10 mm.

432 MYCOLOGIA

3. conidia on average ,30 um long . . . . . . . . . . 43. conidia on average .30 um long . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . L. gonubiensis4. conidia on average ,25 um long, pycnidia

smooth and dark with oozing spores. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . L. theobromae

4. conidia on average .25 um long, pycnidia fluffyand reddish-purple. . . . . . . . . . L. rubropurpurea

DISCUSSION

Recognition of new Lasiodiplodia species in this studyhighlights both the underestimation of fungal speciesnumbers in well studied groups and the importanceof combining DNA techniques with classical taxono-my. Discovery of new Lasiodiplodia spp. is perhaps notsurprising because more than 10 species wereassigned to this genus before the monograph ofPunithalingam (1976), which reduced them tosynonymy. This study supports the views of Pavlic etal (2004), who suggested that undescribed species ofLasiodiplodia are likely to emerge through phyloge-netic studies based on DNA sequence comparisons.

Consideration must be given to the possibility thatsome of the Lasiodiplodia spp. described before thestudy of Punithalingam (1976) are the same as thoseemerging from DNA sequence comparisons. We donot believe this to be the case because Punithalingam(1976) was able to merge all species based on a lack ofmorphological differences. Pavlic et al (2004) soughtherbarium specimens for L. theobromae to comparewith L. gonubiensis, but neither cultures nor herbar-ium specimens pertaining to the original host andlocation (Theobroma cacao L. in Ecuador) could belocated. Thus, until original material can be locatedor an epitype specimen assigned, it is necessary to relyon descriptions from the literature.

The species described in this study are morpholog-ically and phylogenetically distinct from both L.theobromae and L. gonubiensis. It is not possible totest the genetic relatedness of the new species toother previously described species due to the absenceof cultures. In recent studies we have examined morethan 200 isolates from pines, eucalypts, acacia, andsandalwood in Australia, South Africa, Venezuela andMexico, and among all these isolates only 10 (lessthan 5%) were found to be distinct from L.theobromae. In our opinion L. theobromae is a commonspecies in tropical parts of the world but other lesscommon species of this genus are yet to be described.

In addition to the clear phylogenetic differencesbetween the newly described species, these fungidiffer from each other and existing species based onseptation of the paraphyses, the size of the spores,thickness of spore walls and the color of pycnidia. All

species described as new in this study have largerconidia than those of L. theobromae but smaller thanL. gonubiensis. Lasiodiplodia venezuelensis and L.crassispora have septate paraphyses, while they areaseptate in other species. Lasiodiplodia crassispora hasnotably thicker cell walls in the immature spores andthe striations appear to be wider and the cytoplasmwart-like in appearance, which is different from allother species. Lasiodiplodia rubropurpurea is uniquein having red-purple pycnidia.

The three new species, L. crassispora, L. rubropur-purea and L. venezuelensis, have been recognized asresiding in Lasiodiplodia, based on size and shape ofconidia and the presence of vertical striations, whichare characteristic of this genus. No teleomorphstructures were found for these new species butphylogenetic inference leads us to conclude that theyare species of the teleomorph genus Botryosphaeria.Botryosphaeria is a large genus with clear mono-phyletic groups emerging from phylogenetic studiesbased on DNA sequence data. Thus clear subdivisionshave been recognized between those species that haveDiplodia anamorphs with primarily dark-colored co-nidia and those that have hyaline conidia andFusicoccum anamorphs (Denman et al 2000, Zhouand Stanosz 2001). These might be assigned to newgenera, and if that is the case the generic placementof Lasiodiplodia will come into question.

In this study we have chosen to retain the nameLasiodiplodia and not reduce it to synonymy withDiplodia as had been considered (Denman et al 2000,Zhou and Stanosz 2001). This decision was madebecause all species of Lasiodiplodia group together ina highly supported (100% bootstrap) clade related tobut distinct from the clade encompassing Diplodiaand Dothiorella species. Through the addition of threenew species the cohesiveness and separate nature ofthis clade is enhanced. Phillips et al (2005) resur-rected Dothiorella to encompass two new Botryo-sphaeria spp. with dark conidia. Based on a combinedITS and EF-1a phylogeny, these new species formeda clade, which like the Lasiodiplodia clade is close tobut distinct from the Diplodia clade (Phillips et al2005).

Species in Diplodia, Dothiorella and Lasiodiplodiaare clearly separated from those in Fusicoccum bythick-walled conidia with a much smaller length towidth ratio (Luque et al 2005, Phillips et al 2005).When mature the conidia of most Diplodia, Dothiorel-la and Lasiodiplodia species are dark and septate,however conidia of B. stevensii and B. corticola aremostly hyaline and often aseptate and can germinatebefore darkening (Denman et al 2000, Zhou andStanosz 2001, Alves et al 2004). Conidia of D. pineaand B. obtusa while dark are often aseptate (de Wet et

BURGESS ET AL: THREE NEW LASIODIPLODIA SPP. 433

al 2003, Alves et al 2004). Conidia of the newlydescribed B. iberica, B. sarmentorum and B. viticolabecome brown and 1-septate early in their develop-ment (Luque et al 2005, Phillips et al 2005). Diplodia,Dothiorella and Lasiodiplodia spp. conidia sizes over-lap, although in general those of Lasiodiplodia arewider and more obovoid. The other distinguishingfeature of Lasiodiplodia spp. is the obvious verticalstriations in mature conidia (von Arx 1974). Webelieve there is a reasonable argument to retainLasiodiplodia as distinct from Diplodia and Dothiorellaand expect that if the genus Botryosphaeria is sub-divided those species with Lasiodiplodia anamorphswill be retained in a discrete teleomorph genus.

All new species described in this study were isolatedfrom cankers on various tree species. In each casethey were associated with L. theobromae and otheropportunistic pathogens such as Cytospora eucalypti-cola sensu lato and B. ribis. Their distribution and hostrange currently is limited, especially compared withL. theobromae which has more than 500 host speciesand a global distribution (Punithalingam 1976, 1980).Pathogenicity studies were not conducted, but itwould be interesting to compare the pathogenicityof the new species with that of L. theobromae.Although the new species were isolated from cankers,it is likely they are also endophytes and latentpathogens and not primary pathogens as is the casefor many Botryosphaeria spp. (von Arx 1987, Smith etal 1996, Burgess et al 2001, Pavlic et al 2004).

ACKNOWLEDGMENTS

We thank Hugh Glen for Latin descriptions and DianneWhite for technical support. This work was financed in partby the Australian Research Council DP0343600.

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