Phylogeny and taxonomy of Botryosphaeria and Neofusicoccum speciesin Iran, with description of Botryosphaeria scharifii sp. nov.

Jafar Abdollahzadeh1

Department of Plant Protection, Faculty of Agriculture,University of Kurdistan, PO Box 416, Sanandaj, Iran

Rasoul ZareDepartment of Botany, Iranian Research Institute ofPlant Protection, PO Box 1454, Tehran 19395, Iran

Alan J.L. PhillipsCentro de Recursos Microbiologicos, Departamento deCiencias da Vida, Faculdade de Ciencias e Tecnologia,Universidade Nova de Lisboa, 2829-516 Caparica,Portugal

Abstract: Species of Botryosphaeriaceae are impor-tant pathogens and endophytes associated with woodyplants. Botryosphaeria and Neofusicoccum are two wellknown genera of the family. In this study 125 isolatesmorphologically resembling members of this familywere collected from about 20 different fruit and foresttrees in Iran. Based on morphology, MSP-PCR profileand DNA sequence data (ITS and tef1-a), four specieswere identified. Of these, Botryosphaeria dothidea,Neofusicoccum mediterraneum and N. parvum areknown while Botryosphaeria scharifii is described hereas new. N. mediterraneum is a new record for Iran andis reported here for the first time on mango trees.High diversity within Iranian population of N. parvumsuggests the need to revise and reassess the morpho-logical species description of N. parvum and closelyrelated species.

Key words: Botryosphaeria, Botryosphaeriaceae,Fusicoccum, ITS, Neofusicoccum, Phylogeny, tef1-a

INTRODUCTION

In Iran forest and horticultural crops cover an areaestimated respectively at 14.2 and 2.6 million ha. Themost common forest trees are oak, beech, spruce,elm, alder, maple, juniper, wild pistachio and cypress.Among the horticultural crops pistachio, grape, date,apple, walnut, orange and almond are the mainwoody plants (http://www.maj.ir). All of these treeshave great value in forest and horticultural industries ofthe country and are affected by a variety of biotic andabiotic stresses. Dieback, canker and fruit rot arefrequently observed. Members of the Botryosphaeriaceae

are well known fungi associated with these symptomsworldwide.

Based on extensive phylogenetic studies 18different genera including Botryosphaeria (anam.Fusicoccum) and Neofusicoccum have been describedin this family (Phillips et al. 2008; Rojas et al. 2008;Phillips and Alves 2009). The genus Botryosphaeriawas described 150 y ago (Cesati and de Notaris1863). Phylogenetic analysis based on LSU sequencedata together with morphological characteristics byCrous et al. (2006) divided Botryosphaeria into 10lineages representing 10 distinct genera includingNeofusicoccum and Pseudofusicoccum. Most of thespecies were transferred to Neofusicoccum. Fusicoc-cum stromaticum was transferred to Pseudofusicoc-cum, and only Botryosphaeria dothidea, B. mamaneand B. corticis were retained in Botryosphaeria. Morerecently F. ramosum (Pavlic et al. 2008), F. atrovirens(Mehl et al. 2011) and F. fabicercianum (Chen et al.2011) were described in the Botryosphaeria s.s.clade.

The genus Neofusicoccum was established by Crouset al. (2006) to accommodate Botryosphaeria-likespecies with Fusicoccum-like anamorphs and Dicho-mera-like synanamorphs. Botryosphaeria and Neofusi-coccum are similar morphologically, but they differ inproducing a Dichomera synanamorph that occurs onlyin some Neofusicoccum species. However, a Dichomeraanamorph is not produced by all Neofusicoccumspecies or by all isolates of a species, and furthermoresome B. dothidea isolates occasionally may form aDichomera-like state (Phillips et al. 2005). Neverthe-less, the two genera are phylogenetically distinct(Crous et al. 2006; Phillips et al. 2008).

Despite intensive mycological studies in the pasttwo decades, little is known about the ascomycetousfungi, including Botryosphaeriaceae, associated withcankers, dieback, gummosis and fruit rots on trees inIran. Before 2009, studies on this family were limitedto Abdollahzadeh et al. (2007a, b) and listed reportsin Ershad (2009). Phylogenetic and morphologicalstudies on the family Botryosphaeriaceae in Iranhave resulted in the description of Barriopsisiraniana and Phaeobotryon cupressi (Abdollahzadehet al. 2009) and four Lasiodiplodia species (Abdol-lahzadeh et al. 2010). In this paper new findingsfrom morphological and molecular studies onBotryosphaeria and Neofusicoccum species are present-ed and discussed.

Submitted 2 Apr 2012; accepted for publication 20 Jun 2012.1 Corresponding author. E-mail: J.abdollahzadeh@yahoo.com

Mycologia, 105(1), 2013, pp. 210–220. DOI: 10.3852/12-107# 2013 by The Mycological Society of America, Lawrence, KS 66044-8897

210

MATERIALS AND METHODS

Fungal isolation.—Isolates were derived from conidiaproduced in pycnidia on rotting fruits or branches withcanker or dieback symptoms. Some isolates were obtainedby transferring pieces of disinfested symptomatic tissues tohalf-strength potato dextrose agar (1/2 PDA, Difco Labo-ratories) supplemented with chloramphenicol (100 mg/L).Single conidium cultures were prepared for all isolates.Representative isolates and specimens were deposited in theculture collection of the Iranian Research Institute of PlantProtection (IRAN, Tehran, Iran) and Centraalbureau voorSchimmelcultures (CBS, Utrecht, the Netherlands). Isolatesused for morphological and phylogenetic analyses arepresented (TABLE I).

Morphological characterization.—To enhance sporulationisolates were cultured on 2% water agar (WA) withdouble-autoclaved pine needles on the agar surface. Theplates were incubated at 25 C under mixed near-UV andcool-white fluorescent light in a 12 h light-dark regime 2–6 wk. Conidiomata were dissected and mounted in 100%

lactic acid. Digital images were made with a Leica DFC 320camera on a Leica DMR HC microscope, and measurementswere made with the Leica IM500 measurement module. Themean, standard deviation and 95% confidence intervals werecalculated from measurements of 50 conidia. Dimensions aregiven as the range of measurements with extremes in bracketsfollowed by 95% confidence limits and mean 6 standarddeviation. Dimensions of other structures are given as therange of at least 20 measurements. Cultural characteristics,growth rates and cardinal temperatures for growth weredetermined on 2% malt extract agar (MEA, Difco Laborato-ries) 5–35 C at 5 C intervals in the dark. Colony colors wereassessed for isolates on 2% MEA at 25 C in the dark bycomparison with the color chart of Rayner (1970).

DNA isolation and purification.—Genomic DNA was ex-tracted from 4–7-d old cultures grown in 2% malt extractbroth (MEB) incubated at room temperature using themethod of Raeder and Broda (1985) with modifications asdescribed by Abdollahzadeh et al. (2009).

MSP-PCR.—MSP-PCR profiles were generated with theprimers (GTG)5 and the phage M13 core sequence.Amplification conditions were annealing 53 C (1 min) andextension 72 C (2 min). PCR reactions were performed in25 mL mixtures containing 20 pmol each primer and 50–100 ng template DNA. PCR products were separated byelectrophoresis in 2% agarose gels at 80 V for 4 h in tris-borate-EDTA buffer 0.53. A GeneRulerTM DNA Ladder Mix(100–10 000 bp) was run at both sides of each gel. The gelswere stained with GelRedTM Nucleic Acid Gel Stain (BiotiumInc., USA) and visualized on a UV transilluminator to assessPCR amplification. Agarose gels were scanned and the DNAprofiles were analyzed with GelCompar II 4.1 (Applied Math,Kortrijk, Belgium). Similarities between the profiles wereestimated with Pearson’s correlation coefficient. Clusteranalysis of similarity matrices was performed in UPGMA.

Phylogenetic characterization.—Representative isolates fromall separated clusters in the MSP-PCR profiles were selected

for sequencing and phylogenetic analyses (TABLE I). PCRreaction mixtures were prepared according to Alves et al.(2004), with the addition of 5% DMSO to improve theamplification of some difficult DNA templates. The ITS1-5.8S-ITS2 plus D1/D2 domain of the 28S rDNA gene andthe translation elongation factor1-alpha (tef1-a) wereamplified with the primer pairs ITS1 (White et al. 1990)/NL4 (O’Donnell 1993) and EF1-688F/EF1-1251R (Alves etal. 2008) respectively. PCR conditions, purification andsequencing were as described by Abdollahzadeh et al.(2009).

Nucleotide sequences were read and edited with BioEditSequence Alignment Editor 7.0.9.0 (Hall 2006). The ITSand tef1-a sequences were assembled and added to out-groups (Diplodia mutila CBS 112553 and D. seriata CBS11255) and sequences of additional isolates retrieved fromGenBank (TABLE I). Sequences were aligned with Clustal X1.83 (Thompson et al. 1997), using these parameters:pairwise alignment (gap opening 5 10, gap extension 5

0.1) and multiple alignment (gap opening 5 10, gapextension 5 0.2, transition weight 5 0.5, delay divergentsequences 5 25%). Alignments were checked and manualadjustments were made where necessary. Phylogeneticinformation contained in indels (gaps) was incorporatedinto the phylogenetic analyses with simple indel coding asimplemented by GapCoder (Young and Healy 2003). Apartition homogeneity test determined the possibility ofcombining the ITS and tef1-a datasets (Farris et al. 1995;Huelsenbeck et al. 1996).

Phylogenetic analyses were performed with PAUP 4.0b10(Swofford 2003) for maximum parsimony (MP) analysis andMrBayes 3.0b4 (Ronquist and Huelsenbeck 2003) for theBayesian analysis. Maximum parsimony analysis was per-formed with the heuristic search option with 1000 randomtaxon additions and tree bisection and reconnection (TBR)as the branch-swapping algorithm. All characters wereunordered and of equal weight and gaps were treated asmissing data. Branches of zero length were collapsed and allmultiple, equally parsimonious trees were saved. Therobustness of the most parsimonious trees was evaluatedby 1000 bootstrap replications (Hillis and Bull 1993). Othermeasures were consistency index (CI), retention index (RI)and homoplasy index (HI).

Bayesian analyses employing a Markov chain Monte Carlo(MCMC) method were performed. The general timereversible model of evolution (Rodriguez et al. 1990),including estimation of invariable sites and assuming adiscrete gamma distribution with six rate categories (GTR +I + C), was used. Four MCMC chains were run simulta-neously, starting from random trees, for 106 generations.Trees were sampled every 100th generation for a total of 104

trees. The first 103 trees were discarded as the burn-in phaseof each analysis. Posterior probabilities (Rannala and Yang1996) were determined from a majority rule consensus treegenerated from the remaining 9000 trees. The analysis wasrepeated three times starting from different random treesto ensure trees from the same tree space were beingsampled during each analysis. New sequences were depos-ited in GenBank (TABLE I) and the alignment in TreeBASE(S12532).

ABDOLLAHZADEH ET AL.: BOTRYOSPHAERIACEAE IN IRAN 211

RESULTS

Morphology.—The 125 isolates resembling members ofthe Botryosphaeriaceae were obtained from differentwoody hosts showing various disease symptoms includ-ing dieback, canker and fruit rots. All isolates producedpycnidia on pine needles on 2% WA within 3–4 wk. Noascomata were observed in culture. Based on culturaland conidial characteristics isolates were considered tobelong to Botryosphaeria or Neofusicoccum (TABLE II).

MSP-PCR.—Reproducibility for both primers wasestimated as 80–85% by comparing the DNA profilesresulting from independent amplifications for 10%

of the isolates. Four major clusters were identifiedwith each primer, and, based on the host, localityand some morphological features, representativeisolates were selected for phylogenetic analysis.MSP-PCR fingerprinting technique discriminatedall the species recognized in this study, and clusteranalysis of DNA profiles corresponded and

TABLE I. Isolates sequenced in this study

Isolate no. Identity Host Location Collector

GenBank accession no.

ITS EF1-a

IRAN1527C Botryosphaeriadothidea

Acer sp. Iran, Golestan R. Zare JQ772025 JQ772062

IRAN1531C B. dothidea Juglans regia Iran, Golestan A. Javadi JQ772022 JQ772059IRAN1561C B. dothidea J. regia Iran, Zanjan A. Javadi JQ772024 JQ772061IRAN1563C B. dothidea Fagus sp. Iran, Guilan A. Javadi JQ772027 JQ772064IRAN1566C B. dothidea J. regia Iran, Golestan A. Javadi JQ772021 JQ772058CJA175 B. dothidea Cupressus

sempervirensIran, Golestan M.A. Aghajani JQ772026 JQ772063

CJA283 B. dothidea V. vinifera Iran, Kurdistan J. Abdollahzadeh JQ772023 JQ772060IRAN1529C B. scharifii Mangifera indica Iran, Tehran J. Abdollahzadeh JQ772020 JQ772057IRAN1543C B. scharifii M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772019 JQ772056CJA290 B. scharifii M. indica Iran, Kurdistan J. Abdollahzadeh n.s.a n.s.IRAN1526C Neofusicoccum

mediterraneumUnknown Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772052 JQ772089

IRAN1549C N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772053 JQ772090IRAN1550C N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772047 JQ772084IRAN1554C N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772048 JQ772085IRAN1555C N. mediterraneum Citrus sp. Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772055 JQ772092CJA13 N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772049 JQ772086CJA144 N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772051 JQ772088CJA154 N. mediterraneum Unknown Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772050 JQ772087CJA266 N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772054 JQ772091IRAN1524C N. parvum J. regia Iran, Mazandaran A. Javadi JQ772033 JQ772070IRAN1528C N. parvum J. regia Iran, Golestan A. Javadi JQ772031 JQ772068IRAN1533C N. parvum Unknown Iran, Mazandaran J. Abdollahzadeh JQ772034 JQ772071IRAN1535C N. parvum Salix sp. Iran, Mazandaran J. Abdollahzadeh JQ772045 JQ772082IRAN1536C N. parvum J. regia Iran, Golestan A. Javadi JQ772032 JQ772069CJA8 N. parvum J. regia Iran, Golestan A. Javadi JQ772040 JQ772077CJA21 N. parvum Unknown Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772041 JQ772078CJA30 N. parvum J. regia Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772030 JQ772067CJA55 N. parvum Rubus fruticosus Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772037 JQ772074CJA56 N. parvum Actinidia

deliciosaIran, Mazandaran J. Abdollahzadeh JQ772036 JQ772073

CJA63 N. parvum Unknown Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772028 JQ772065CJA73 N. parvum J. regia Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772029 JQ772066CJA108 N. parvum J. regia Iran, Mazandaran A. Javadi JQ772042 JQ772079CJA114 N. parvum Salix sp. Iran, Mazandaran A. Javadi JQ772038 JQ772075CJA133 N. parvum A. deliciosa Iran, Mazandaran J. Abdollahzadeh JQ772039 JQ772076CJA136 N. parvum Salix sp. Iran, Mazandaran J. Abdollahzadeh JQ772044 JQ772081CJA203 N. parvum J. regia Iran, Mazandaran A. Javadi JQ772035 JQ772072CJA204 N. parvum A. deliciosa Iran, Mazandaran F. Beigi JQ772043 JQ772080

a n.s. 5 not sequenced.

212 MYCOLOGIA

confirmed well with species differentiation based onphylogenetic analysis (FIG. 1a, b).

Phylogenetic analysis.—The partition homogeneity testin PAUP was not significant (P 5 0.26), indicating thatthe ITS (655 characters) and tef1-a (415 characters)datasets were congruent and produced similar phylog-enies. Phylogenies resulting from the individual locialso were compared visually and no differences couldbe detected. Therefore the two datasets were com-bined in a single analysis. ITS and tef1-a sequences forthe 37 isolates were combined and aligned with 39sequences of 21 taxa retrieved from GenBank. Incom-plete portions at the ends of the sequences wereexcluded from analyses. Of the 1184 characters(including gaps) in the aligned dataset, 225 wereexcluded, 607 were constant and 68 were variable andparsimony uninformative. A heuristic search of theremaining 284 parsimony informative characters re-sulted in 1000 most parsimonious trees of 535 steps (CI5 0.78, HI 5 0.22, RI 5 0.96), each with the sameoverall topology varying only in the arrangement oftaxa within the terminal clades.

The Bayesian analysis resulted in a tree with thesame topology as the trees resulting from MP analysis.One of the MP trees (FIG. 2) had MP bootstrapsupport values above and posterior probabilities forBayesian analysis below the branches. In this tree 13Neofusicoccum and seven Botryosphaeria species couldbe distinguished. Four species could be distinguishedamong the isolates from Iran. Three of these areknown (B. dothidea, N. mediterraneum, N. parvum)and the fourth, being morphologically (TABLE III)and phylogenetically distinct from all other knownspecies, is described here as a new species.

TAXONOMY

Botryosphaeria scharifii Abdollahzadeh, Zare, A.J.L.Phillips, sp. nov. FIG. 3

MycoBank MB564800Conidiomata pycnidial, produced on pine needles

on WA within 2–4 wk, solitary or aggregated, darkbrown to black, globose, up to 760 mm diam,

superficial, mostly uniloculate, thick-walled, non-pa-pillate with a central ostiole. Conidiophores cylindri-cal, 7.5–33.4 3 1.9–4.3 mm, hyaline, smooth, thin-walled, septate, branched at apex, lining the entireinner surface of the conidiomata. Conidiogenous cellscylindrical to lageniform, 6.9–15 3 1.6–3.7 mm, hyaline,thin-walled, smooth, holoblastic, phialidic with peri-clinal thickening. Conidia fusiform, (11.7–)13–17(–18.9) 3 4–6.3 mm, 95% confidence limits 5 15.2–15.63 5.2–5.4 mm (av. 6 SD 5 15.4 6 1.4 3 5.2 6 0.5 mm, l/w ratio 5 2.7 6 0.4), unicellular, hyaline, thin-walled,smooth, apex obtuse, base subtruncate to bluntlyrounded.

Culture characteristics: Colonies with abundantaerial mycelium reaching the lid of Petri plate, aerialmycelium becoming smoke gray (21’’’’f) to olivaceousgray (21’’’’i) at the surface and greenish olivaceous(23’’’i) to dull green (27’’m) at the reverse after 2 wkin the dark at 25 C. Colonies reaching 84 mm on MEAafter 3 d in the dark at 25 C. Cardinal temperaturesfor growth; min # 5 C, max $ 35 C, opt 25 C.

Etymology: Named to honor the late Dr Ghavame-din Scharif, who significantly contributed to theknowledge of mycology and plant pathology in Iran.

Teleomorph: Not observed.Habitat: Twigs and fruits of Mangifera indica.Known distribution: southern Iran and Pakistan.HOLOTYPE. IRAN. Tehran, on fruits of Mangifera

indica imported from Pakistan, Aug 2006, J. Abdol-lahzadeh (a dry culture ex IRAN 14275F on pineneedles; ex-type culture IRAN 1529C 5 CBS 124703).

Additional specimens examined: IRAN. HORMOZGANPROVINCE, Minab, on twigs of Mangifera indica, 27 Feb2007, J. Abdollahzadeh & A. Javadi (IRAN 1543C 5 CBS124702); KURDISTAN PROVINCE, Sanandaj, on fruits ofMangifera indica imported from Pakistan, 16 Oct 2011, J.Abdollahzadeh (CJA290).

Notes: Botryosphaeria scharifii is phylogeneticallymost closely related to Fusicoccum ramosum. Conidiaof B. scharifii and F. ramosum are considerably shorterthan all other species in the Botryosphaeria clade.However, the slightly longer conidia of B. scharifiidistinguishes it from F. ramosum (TABLE III).

TABLE II. Frequency, distribution and hosts of characterized species from Iran

Species No. of isolates Frequency (%) Distribution Hosts

B. dothidea 74 60 north, southnorthwest, west

olive, oak, mango, beech, raspberry,walnut, poplar, peach, spruce, lemon,maple, kiwi, willow, grapevine

B. scharifii 3 2 south mangoN. mediterraneum 14 11 south mango, citrusN. parvum 34 27 north raspberry, pine, willow, kiwi, pear,

cherry, walnut

ABDOLLAHZADEH ET AL.: BOTRYOSPHAERIACEAE IN IRAN 213

FIG. 1. Dendrograms resulting from MSP-PCR profiles of (a) M13 and (b) (GTG)5 analyzed with GelCompar II. Similaritybetween the profiles were estimated with Pearson’s correlation coefficient. Cluster analysis of similarity matrices was donewith UPGMA.

214 MYCOLOGIA

FIG. 2. One of the most parsimonious trees obtained from combined ITS and tef-1a sequence data. MP bootstrap valuesbased on 1000 pseudoreplicates and posterior probabilities from Bayesian analysis are above and below the branchesrespectively. The species characterized in this study are in boldface. Bar 5 10 changes. Diplodia mutila (CBS112553) andDiplodia seriata (CBS112555) were included as outgroups. Phylogeny deposited in TreeBASE (S12532).

ABDOLLAHZADEH ET AL.: BOTRYOSPHAERIACEAE IN IRAN 215

DISCUSSION

In this survey a large collection of Botryosphaeria andNeofusicoccum isolates from woody hosts in Iran werestudied. The isolates initially were grouped accordingto their MSP-PCR profiles, and representative isolatesof each group were selected for sequencing of the ITSand tef1-a loci. As a result four species (B. dothidea, B.scharifii, N. mediterraneum, N. parvum) were identi-fied.

The most frequently encountered species was B.dothidea, which represented 60% of the isolates andwas found on 14 different forest and fruit trees fromdifferent regions of Iran (TABLE II). This species is awell known fungal pathogen from temperate regionsworldwide and has been reported in association with

various disease symptoms on a broad range of woodyhosts including canker, dieback, trunk diseases andfruit rots (Ma et al. 2001). However, before the studyof Slippers et al. (2004) B. dothidea was considered tobe a complex of species including N. ribis and N.parvum and therefore earlier reports could beunreliable. Our findings showed that of the fourspecies isolated B. dothidea has the broadest distribu-tion in Iran and was found in a variety of climatesranging from temperate and humid in the north nearthe Caspian Sea to the semi-arid regions in the westalong the Zagros Mountains and the humid tropicalregions of the south near the Persian Gulf. It isimportant to note that most of the isolates (92%)were found in the north while only 8% were from thesouth and west. This species has been reported on

TABLE III. Conidial dimension of Botryosphaeria species investigated in this and previous studies

Species Conidial dimensions (mm) Average (mm) L/W ratio Reference

F. atrovirens 30.9–36 3 7.1–9.9 33.5 3 8.5 — Mehl et al. 2011B. corticis 23.5–32.5 3 5.5–7 28.9 3 6.4 4.5 Phillips et al. 2006B. dothidea 18–20 3 4–5 19.6 3 4.8 4.1 Slippers et al. 2004F. fabicercianum 19.6–24.4 3 5.2–6.4 22 3 5.8 3.8 Chen et al. 2011B. mamane 28–43 3 5–7 35.5 3 6.1 5.8 Mohali et al. 2007F. ramosum 12–15 3 5–6 13.4 3 5.7 2.3 Pavlic et al. 2008B. scharifii 13–17 3 4–6.3 15.4 3 5.2 2.7 This study

FIG. 3. Botryosphaeria scharifii. a. Conidiomata on pine needles in culture. b. Conidia developing on conidiogenous cells. c.Conidiogenous cells with periclinal thickening. d–e. Conidiogenous cells and conidiophores. f. Conidia. Bars: a 5 1000 mm, b–e 5 5 mm, f 5 10 mm.

216 MYCOLOGIA

walnut, rose and almond in Iran (Ershad 2009). Apartfrom walnut, this is the first report of B. dothidea onthe other hosts (TABLE II) in Iran and to ourknowledge this is the first time it has been reportedon spruce (Picea sp.).

The second most common species, N. parvum,comprising 27% of the isolates on seven plant specieswas restricted to the temperate northern part of thecountry. Neofusicoccum parvum has been reportedcommonly as an important pathogen of many woodyplants in temperate regions around the world(Phillips 2002; van Niekerk et al. 2004; Inderbitzinet al. 2010; Chen et al. 2011). It has been recorded on71 host species across six continents and 21 countries(Sakalidis 2011). N. parvum has been reported onVitis vinifera in Iran (Mohammadi et al. 2008) andthis is the first report of this species on all hosts listedin this study in Iran.

We should point out that some ambiguities remainabout these earlier reports because some species suchas N. ribis (5 B. ribis) have been regarded assynonyms of B. dothidea (von Arx and Muller 1954;Pennycook and Samuels 1985; Smith et al. 1994) andN. parvum (5 B. parva) often has not beendifferentiated from N. ribis (Smith and Stanosz2001; Zhou and Stanosz 2001). Furthermore, fivecryptic species are recognized within the N. parvum/N. ribis complex (Pavlic et al. 2009; Bogoude et al.2010; Sakalidis et al. 2011). Despite these ambiguities,to our knowledge this is the first report of N. parvumon willow, cherry and raspberry.

Because Slippers et al. (2004) differentiated B.dothidea, N. ribis and N. parvum with morphologicaland combined sequence datasets of ITS, tef1-a and b-tubulin, these species are now clearly defined.Patterns of septation and discoloration in olderconidia discharged from pycnidia was regarded as auseful morphological feature to distinguish N. ribisfrom N. parvum, and this is supported by moleculardata. Aging conidia of N. parvum become light brownwith 1–2 septa, frequently with a darker brown middlecell, while conidia of N. ribis rarely become lightbrown with 1–2 septa, according to Slippers et al.(2004). Careful observation is crucial because someconidia remain hyaline and aseptate even afterdischarge in 2 mo old cultures. After carefulobservation of our isolates of N. parvum we did notsee aging conidia turning light brown and all agedconidia remained hyaline with two septa even in 10 wkold cultures. The concept of aging has not beenclearly defined and thus changes in conidial charac-ters are difficult to apply in practice.

Cultural characteristics have been used to differen-tiate the species of the family Botryosphaeriaceae. Forinstance, Alves et al. (2008) in a phylogenetic study of

Lasiodiplodia separated L. parva and L. pseudotheo-bromae from L. theobromae based on the ability of thefirst two species to produce a pink pigment in PDA at35 C while Abdollahzadeh et al. (2010) did notidentify any differences between these species andquestioned the taxonomic significance of this char-acter. Furthermore, Pennycook and Samuels (1985)distinguished N. luteum from N. parvum and N. ribisbased on the ability of N. luteum to produce a yellowpigment in PDA at 25 C. However, in the presentstudy some of our isolates of N. parvum produced ayellow pigment in PDA at 25 C.

High variation in cultural characteristics, morphol-ogy and MSP-PCR fingerprinting among our isolatesof N. parvum led us to sequence 18 isolates.Phylogenetic analysis showed that they all belong tothe same species and these variations reflect highgenetic diversity among populations and indicate theneed to revise and reassess the morphological speciesdescription.

All isolates of Neofusicoccum mediterraneum werefound here on citrus, mango and on unknown treesfrom southern Iran. N. mediterraneum first wasdescribed from Eucalyptus in Greece (Crous et al.2007). Subsequently it was found on olive drupes inItaly (Lazzizera et al. 2008), grapevine in Spain(Martin et al. 2011) and various woody plantsincluding most notably Persian walnut, pistachio,citrus, grapevine and almond in the United States(Trouillas et al. 2010; Inderbitzin et al. 2010; Urbez-Torres et al. 2010). In a BLAST query of oursequences we found many N. mediterraneum isolateswith notable differences in ITS and tef1-a sequences.Thus, it is possible that N. mediterraneum is a complexof species. This species is a new record for Iran, and toour knowledge this is the first time it has beenreported on mango trees.

Species of Botryosphaeriaceae are common patho-gens associated with mangoes worldwide (Slipperset al. 2005). Recently studies on mango trees have ledto the characterization of eight species of Botryo-sphaeriaceae on mangoes in Australia and threespecies in Brazil (Sakalidis et al. 2011; Costa et al.2010). Despite extensive studies on Botryosphaeria-ceae associated with mango, in this study a newspecies named B. scharifii was characterized in Iran.In view of the recent changes to the ICBN ratified atthe Melbourne conference, priority of names de-pends on the publication date irrespective of the lifestage of the fungus. Thus, Fusicoccum (1829) haspriority over Botryosphaeria (1863). Although therehas been no official decision on which name (Botryo-sphaeria or Fusicoccum) will take priority, we recom-mend that the name Botryosphaeria should beconserved. Although the three species names

ABDOLLAHZADEH ET AL.: BOTRYOSPHAERIACEAE IN IRAN 217

currently in Fusicoccum will have to be transferred, wehave not done so but prefer to wait until a decisionhas been made. In our study B. scharifii was the leastabundant species with only three isolates, one frommango twigs from southern Iran with diebacksymptoms and the other two from mango fruits withend-rot symptoms imported from Pakistan. Moresampling and isolation is necessary to understandthe host range, distribution and variability of this newspecies.

Members of Botryosphaeriaceae are consideredendophytes and latent opportunistic pathogens thatpose a potentially significant threat to agricultural andforest trees, especially under stressed conditions(Slippers and Wingfield 2007). Thus, taking appropri-ate and efficient strategies to manage these pathogensdepends on a good knowledge of their taxonomy,diversity, ecology and pathogenicity. In this firstsystematic study of Botryosphaeria and Neofusicoccumin Iran, most isolates were obtained from trees withdieback symptoms, although pathogenicity studieswere not conducted. Host specificity of Botryosphaer-iaceae is debatable, but Slippers and Wingfield (2007)and our findings apparently suggest that host neutral-ism is common, especially in some well known speciesincluding B. dothidea and N. parvum. Thus, we thinkthat environmental factors are the most importantvariables affecting geographic distribution of thespecies characterized in this study. In general, inaddition to describing a new species, this study hasexpanded the known geographic distribution and hostrange of B dothidea, N. parvum and N. mediterraneum.

ACKNOWLEDGMENTS

This work was financed in part by the European RegionalDevelopment Fund and Fundacao para a Ciencia e aTecnologia (FCT) Portugal under project PPCDT/AGR/56140/2004. A.J.L. Phillips was supported by grantnumber SFRH/BCC/15810/2005 from FCT, and J. Abdol-lahzadeh received a grant from Studienstiftung Mykologie,Koln, Germany, and Kurdistan provincial office. We thankA. Javadi, F. Beigi and Dr M.A. Aghajani for sampling andthe two anonymous reviewers for their helpful commentsand suggestions for improving the manuscript.

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