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

Phylogeny and of Botryosphaeria and Neofusicoccum species in , with description of Botryosphaeria scharifii sp. nov.

Jafar Abdollahzadeh1 are well known fungi associated with these symptoms Department of Plant Protection, Faculty of Agriculture, worldwide. University of Kurdistan, PO Box 416, Sanandaj, Iran Based on extensive phylogenetic studies 18 Rasoul Zare different genera including Botryosphaeria (anam. Department of Botany, Iranian Research Institute of Fusicoccum)andNeofusicoccum have been described Plant Protection, PO Box 1454, Tehran 19395, Iran in this family (Phillips et al. 2008; Rojas et al. 2008; Phillips and Alves 2009). The genus Botryosphaeria Alan J.L. Phillips was described 150 y ago (Cesati and de Notaris Centro de Recursos Microbiolo´gicos, Departamento de 1863). Phylogenetic analysis based on LSU sequence Cieˆncias da Vida, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, data together with morphological characteristics by Portugal Crous et al. (2006) divided Botryosphaeria into 10 lineages representing 10 distinct genera including Neofusicoccum and Pseudofusicoccum.Mostofthe Abstract: Species of are impor- species were transferred to Neofusicoccum. Fusicoc- tant pathogens and endophytes associated with woody cum stromaticum was transferred to Pseudofusicoc- plants. Botryosphaeria and Neofusicoccum are two well cum,andonlyBotryosphaeria dothidea, B. mamane known genera of the family. In this study 125 isolates and B. corticis were retained in Botryosphaeria.More morphologically resembling members of this family recently F. ramosum (Pavlic et al. 2008), F. atrovirens were collected from about 20 different fruit and forest (Mehl et al. 2011) and F. fabicercianum (Chen et al. trees in Iran. Based on morphology, MSP-PCR profile 2011) were described in the Botryosphaeria s.s. and DNA sequence data (ITS and tef1-a), four species clade. were identified. Of these, Botryosphaeria dothidea, The genus Neofusicoccum was established by Crous Neofusicoccum mediterraneum and N. parvum are et al. (2006) to accommodate Botryosphaeria-like known while Botryosphaeria scharifii is described here species with Fusicoccum-like anamorphs and Dicho- as new. N. mediterraneum is a new record for Iran and mera-like synanamorphs. Botryosphaeria and Neofusi- is reported here for the first time on mango trees. coccum are similar morphologically, but they differ in High diversity within Iranian population of N. parvum producing a Dichomera synanamorph that occurs only suggests the need to revise and reassess the morpho- in some Neofusicoccum species. However, a Dichomera logical species description of N. parvum and closely anamorph is not produced by all Neofusicoccum related species. species or by all isolates of a species, and furthermore Key words: Botryosphaeria, Botryosphaeriaceae, some B. dothidea isolates occasionally may form a Fusicoccum, ITS, Neofusicoccum, Phylogeny, tef1-a Dichomera-like state (Phillips et al. 2005). Neverthe- less, the two genera are phylogenetically distinct (Crous et al. 2006; Phillips et al. 2008). INTRODUCTION Despite intensive mycological studies in the past In Iran forest and horticultural crops cover an area two decades, little is known about the ascomycetous estimated respectively at 14.2 and 2.6 million ha. The fungi, including Botryosphaeriaceae, associated with most common forest trees are oak, beech, spruce, cankers, dieback, gummosis and fruit rots on trees in elm, alder, maple, juniper, wild pistachio and cypress. Iran. Before 2009, studies on this family were limited Among the horticultural crops pistachio, grape, date, to Abdollahzadeh et al. (2007a, b) and listed reports apple, walnut, orange and almond are the main in Ershad (2009). Phylogenetic and morphological woody plants (http://www.maj.ir). All of these trees studies on the family Botryosphaeriaceae in Iran have great value in forest and horticultural industries of have resulted in the description of the country and are affected by a variety of biotic and iraniana and Phaeobotryon cupressi (Abdollahzadeh abiotic stresses. Dieback, canker and fruit rot are et al. 2009) and four Lasiodiplodia species (Abdol- frequently observed. Members of the Botryosphaeriaceae lahzadeh et al. 2010). In this paper new findings from morphological and molecular studies on Submitted 2 Apr 2012; accepted for publication 20 Jun 2012. Botryosphaeria and Neofusicoccum species are present- 1 Corresponding author. E-mail: [email protected] ed and discussed.

210 ABDOLLAHZADEH ET AL.: BOTRYOSPHAERIACEAE IN IRAN 211

MATERIALS AND METHODS for sequencing and phylogenetic analyses (TABLE I). PCR reaction mixtures were prepared according to Alves et al. Fungal isolation.—Isolates were derived from conidia (2004), with the addition of 5% DMSO to improve the produced in pycnidia on rotting fruits or branches with amplification of some difficult DNA templates. The ITS1- canker or dieback symptoms. Some isolates were obtained 5.8S-ITS2 plus D1/D2 domain of the 28S rDNA gene and by transferring pieces of disinfested symptomatic tissues to the translation elongation factor1-alpha (tef1-a)were half-strength potato dextrose agar (1/2 PDA, Difco Labo- amplified with the primer pairs ITS1 (White et al. 1990)/ ratories) supplemented with chloramphenicol (100 mg/L). NL4 (O’Donnell 1993) and EF1-688F/EF1-1251R (Alves et Single conidium cultures were prepared for all isolates. al. 2008) respectively. PCR conditions, purification and Representative isolates and specimens were deposited in the sequencing were as described by Abdollahzadeh et al. culture collection of the Iranian Research Institute of Plant (2009). Protection (IRAN, Tehran, Iran) and Centraalbureau voor Nucleotide sequences were read and edited with BioEdit Schimmelcultures (CBS, Utrecht, the Netherlands). Isolates Sequence Alignment Editor 7.0.9.0 (Hall 2006). The ITS used for morphological and phylogenetic analyses are and tef1-a sequences were assembled and added to out- presented (TABLE I). groups (Diplodia mutila CBS 112553 and D. seriata CBS Morphological characterization.—To enhance sporulation 11255) and sequences of additional isolates retrieved from isolates were cultured on 2% water agar (WA) with GenBank (TABLE I). Sequences were aligned with Clustal X double-autoclaved pine needles on the agar surface. The 1.83 (Thompson et al. 1997), using these parameters: plates were incubated at 25 C under mixed near-UV and pairwise alignment (gap opening 5 10, gap extension 5 cool-white fluorescent light in a 12 h light-dark regime 2– 0.1) and multiple alignment (gap opening 5 10, gap 6 wk. Conidiomata were dissected and mounted in 100% extension 5 0.2, transition weight 5 0.5, delay divergent lactic acid. Digital images were made with a Leica DFC 320 sequences 5 25%). Alignments were checked and manual camera on a Leica DMR HC microscope, and measurements adjustments were made where necessary. Phylogenetic were made with the Leica IM500 measurement module. The information contained in indels (gaps) was incorporated mean, standard deviation and 95% confidence intervals were into the phylogenetic analyses with simple indel coding as calculated from measurements of 50 conidia. Dimensions are implemented by GapCoder (Young and Healy 2003). A given as the range of measurements with extremes in brackets partition homogeneity test determined the possibility of followed by 95% confidence limits and mean 6 standard combining the ITS and tef1-a datasets (Farris et al. 1995; deviation. Dimensions of other structures are given as the Huelsenbeck et al. 1996). range of at least 20 measurements. Cultural characteristics, Phylogenetic analyses were performed with PAUP 4.0b10 growth rates and cardinal temperatures for growth were (Swofford 2003) for maximum parsimony (MP) analysis and determined on 2% malt extract agar (MEA, Difco Laborato- MrBayes 3.0b4 (Ronquist and Huelsenbeck 2003) for the ries) 5–35 C at 5 C intervals in the dark. Colony colors were Bayesian analysis. Maximum parsimony analysis was per- assessed for isolates on 2% MEA at 25 C in the dark by formed with the heuristic search option with 1000 random comparison with the color chart of Rayner (1970). taxon additions and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All characters were DNA isolation and purification.—Genomic DNA was ex- unordered and of equal weight and gaps were treated as tracted from 4–7-d old cultures grown in 2% malt extract missing data. Branches of zero length were collapsed and all broth (MEB) incubated at room temperature using the multiple, equally parsimonious trees were saved. The method of Raeder and Broda (1985) with modifications as robustness of the most parsimonious trees was evaluated described by Abdollahzadeh et al. (2009). by 1000 bootstrap replications (Hillis and Bull 1993). Other measures were consistency index (CI), retention index (RI) MSP-PCR.—MSP-PCR profiles were generated with the and homoplasy index (HI). primers (GTG) and the phage M13 core sequence. 5 Bayesian analyses employing a Markov chain Monte Carlo Amplification conditions were annealing 53 C (1 min) and (MCMC) method were performed. The general time extension 72 C (2 min). PCR reactions were performed in reversible model of evolution (Rodriguez et al. 1990), 25 mL mixtures containing 20 pmol each primer and 50– including estimation of invariable sites and assuming a 100 ng template DNA. PCR products were separated by discrete gamma distribution with six rate categories (GTR + electrophoresis in 2% agarose gels at 80 V for 4 h in tris- I + C), was used. Four MCMC chains were run simulta- borate-EDTA buffer 0.53. A GeneRulerTM DNA Ladder Mix neously, starting from random trees, for 106 generations. (100–10 000 bp) was run at both sides of each gel. The gels 4 TM Trees were sampled every 100th generation for a total of 10 were stained with GelRed Nucleic Acid Gel Stain (Biotium 3 Inc., USA) and visualized on a UV transilluminator to assess trees. The first 10 trees were discarded as the burn-in phase PCR amplification. Agarose gels were scanned and the DNA of each analysis. Posterior probabilities (Rannala and Yang profiles were analyzed with GelCompar II 4.1 (Applied Math, 1996) were determined from a majority rule consensus tree Kortrijk, Belgium). Similarities between the profiles were generated from the remaining 9000 trees. The analysis was estimated with Pearson’s correlation coefficient. Cluster repeated three times starting from different random trees analysis of similarity matrices was performed in UPGMA. to ensure trees from the same tree space were being sampled during each analysis. New sequences were depos- Phylogenetic characterization.—Representative isolates from ited in GenBank (TABLE I) and the alignment in TreeBASE all separated clusters in the MSP-PCR profiles were selected (S12532). 212 MYCOLOGIA

TABLE I. Isolates sequenced in this study

GenBank accession no. Isolate no. Identity Host Location Collector ITS EF1-a IRAN1527C Botryosphaeria Acer sp. Iran, Golestan R. Zare JQ772025 JQ772062 dothidea IRAN1531C B. dothidea Juglans regia Iran, Golestan A. Javadi JQ772022 JQ772059 IRAN1561C B. dothidea J. regia Iran, Zanjan A. Javadi JQ772024 JQ772061 IRAN1563C B. dothidea Fagus sp. Iran, Guilan A. Javadi JQ772027 JQ772064 IRAN1566C B. dothidea J. regia Iran, Golestan A. Javadi JQ772021 JQ772058 CJA175 B. dothidea Cupressus Iran, Golestan M.A. Aghajani JQ772026 JQ772063 sempervirens CJA283 B. dothidea V. vinifera Iran, Kurdistan J. Abdollahzadeh JQ772023 JQ772060 IRAN1529C B. scharifii Mangifera Iran, Tehran J. Abdollahzadeh JQ772020 JQ772057 IRAN1543C B. scharifii M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772019 JQ772056 CJA290 B. scharifii M. indica Iran, Kurdistan J. Abdollahzadeh n.s.a n.s. IRAN1526C Neofusicoccum Unknown Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772052 JQ772089 mediterraneum IRAN1549C N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772053 JQ772090 IRAN1550C N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772047 JQ772084 IRAN1554C N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772048 JQ772085 IRAN1555C N. mediterraneum sp. Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772055 JQ772092 CJA13 N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772049 JQ772086 CJA144 N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772051 JQ772088 CJA154 N. mediterraneum Unknown Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772050 JQ772087 CJA266 N. mediterraneum M. indica Iran, Hormozgan J. Abdollahzadeh/A. Javadi JQ772054 JQ772091 IRAN1524C N. parvum J. regia Iran, Mazandaran A. Javadi JQ772033 JQ772070 IRAN1528C N. parvum J. regia Iran, Golestan A. Javadi JQ772031 JQ772068 IRAN1533C N. parvum Unknown Iran, Mazandaran J. Abdollahzadeh JQ772034 JQ772071 IRAN1535C N. parvum Salix sp. Iran, Mazandaran J. Abdollahzadeh JQ772045 JQ772082 IRAN1536C N. parvum J. regia Iran, Golestan A. Javadi JQ772032 JQ772069 CJA8 N. parvum J. regia Iran, Golestan A. Javadi JQ772040 JQ772077 CJA21 N. parvum Unknown Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772041 JQ772078 CJA30 N. parvum J. regia Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772030 JQ772067 CJA55 N. parvum Rubus fruticosus Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772037 JQ772074 CJA56 N. parvum Actinidia Iran, Mazandaran J. Abdollahzadeh JQ772036 JQ772073 deliciosa CJA63 N. parvum Unknown Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772028 JQ772065 CJA73 N. parvum J. regia Iran, Guilan J. Abdollahzadeh/A. Javadi JQ772029 JQ772066 CJA108 N. parvum J. regia Iran, Mazandaran A. Javadi JQ772042 JQ772079 CJA114 N. parvum Salix sp. Iran, Mazandaran A. Javadi JQ772038 JQ772075 CJA133 N. parvum A. deliciosa Iran, Mazandaran J. Abdollahzadeh JQ772039 JQ772076 CJA136 N. parvum Salix sp. Iran, Mazandaran J. Abdollahzadeh JQ772044 JQ772081 CJA203 N. parvum J. regia Iran, Mazandaran A. Javadi JQ772035 JQ772072 CJA204 N. parvum A. deliciosa Iran, Mazandaran F. Beigi JQ772043 JQ772080

a n.s. 5 not sequenced.

RESULTS MSP-PCR.—Reproducibility for both primers was estimated as 80–85% by comparing the DNA profiles Morphology.—The 125 isolates resembling members of resulting from independent amplifications for 10% theBotryosphaeriaceaewereobtainedfromdifferent of the isolates. Four major clusters were identified woody hosts showing various disease symptoms includ- with each primer, and, based on the host, locality ing dieback, canker and fruit rots. All isolates produced and some morphological features, representative pycnidia on pine needles on 2% WA within 3–4 wk. No isolates were selected for phylogenetic analysis. ascomata were observed in culture. Based on cultural MSP-PCR fingerprinting technique discriminated and conidial characteristics isolates were considered to all the species recognized in this study, and cluster belong to Botryosphaeria or Neofusicoccum (TABLE II). analysis of DNA profiles corresponded and ABDOLLAHZADEH ET AL.: BOTRYOSPHAERIACEAE IN IRAN 213

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

Species No. of isolates Frequency (%) Distribution Hosts

B. dothidea 74 60 north, south olive, oak, mango, beech, raspberry, northwest, west walnut, poplar, peach, spruce, lemon, maple, kiwi, willow, grapevine B. scharifii 3 2 south mango N. mediterraneum 14 11 south mango, citrus N. parvum 34 27 north raspberry, pine, willow, kiwi, pear, cherry, walnut confirmed well with species differentiation based on superficial, mostly uniloculate, thick-walled, non-pa- phylogenetic analysis (FIG. 1a, b). pillate with a central ostiole. Conidiophores cylindri- cal, 7.5–33.4 3 1.9–4.3 mm, hyaline, smooth, thin- Phylogenetic analysis.—The partition homogeneity test walled, septate, branched at apex, lining the entire in PAUP was not significant (P 5 0.26), indicating that the ITS (655 characters) and tef1-a (415 characters) inner surface of the conidiomata. Conidiogenous cells datasets were congruent and produced similar phylog- cylindrical to lageniform, 6.9–15 3 1.6–3.7 mm, hyaline, enies. Phylogenies resulting from the individual loci thin-walled, smooth, holoblastic, phialidic with peri- also were compared visually and no differences could clinal thickening. Conidia fusiform, (11.7–)13–17(– be detected. Therefore the two datasets were com- 18.9) 3 4–6.3 mm, 95% confidence limits 5 15.2–15.6 bined in a single analysis. ITS and tef1-a sequences for 3 5.2–5.4 mm(av.6 SD 5 15.4 6 1.4 3 5.2 6 0.5 mm, l/ the 37 isolates were combined and aligned with 39 w ratio 5 2.7 6 0.4), unicellular, hyaline, thin-walled, sequences of 21 taxa retrieved from GenBank. Incom- smooth, apex obtuse, base subtruncate to bluntly plete portions at the ends of the sequences were rounded. excluded from analyses. Of the 1184 characters Culture characteristics: Colonies with abundant (including gaps) in the aligned dataset, 225 were aerial mycelium reaching the lid of Petri plate, aerial excluded, 607 were constant and 68 were variable and mycelium becoming smoke gray (21’’’’f) to olivaceous parsimony uninformative. A heuristic search of the gray (21’’’’i) at the surface and greenish olivaceous remaining 284 parsimony informative characters re- (23’’’i) to dull green (27’’m) at the reverse after 2 wk sulted in 1000 most parsimonious trees of 535 steps (CI in the dark at 25 C. Colonies reaching 84 mm on MEA 5 0.78, HI 5 0.22, RI 5 0.96), each with the same after 3 d in the dark at 25 C. Cardinal temperatures overall topology varying only in the arrangement of for growth; min # 5 C, max $ 35 C, opt 25 C. taxa within the terminal clades. Etymology: Named to honor the late Dr Ghavame- The Bayesian analysis resulted in a tree with the din Scharif, who significantly contributed to the same topology as the trees resulting from MP analysis. knowledge of mycology and plant pathology in Iran. One of the MP trees (FIG. 2) had MP bootstrap Teleomorph: Not observed. support values above and posterior probabilities for Habitat: Twigs and fruits of Mangifera indica. Bayesian analysis below the branches. In this tree 13 Known distribution: southern Iran and Pakistan. Neofusicoccum and seven Botryosphaeria species could HOLOTYPE. IRAN. Tehran, on fruits of Mangifera be distinguished. Four species could be distinguished indica imported from Pakistan, Aug 2006, J. Abdol- among the isolates from Iran. Three of these are lahzadeh (a dry culture ex IRAN 14275F on pine known (B. dothidea, N. mediterraneum, N. parvum) needles; ex-type culture IRAN 1529C 5 CBS 124703). and the fourth, being morphologically (TABLE III) Additional specimens examined: IRAN. HORMOZGAN and phylogenetically distinct from all other known PROVINCE, Minab, on twigs of Mangifera indica, 27 Feb species, is described here as a new species. 2007, J. Abdollahzadeh & A. Javadi (IRAN 1543C 5 CBS 124702); KURDISTAN PROVINCE, Sanandaj, on fruits of Mangifera indica imported from Pakistan, 16 Oct 2011, J. TAXONOMY Abdollahzadeh (CJA290). Botryosphaeria scharifii Abdollahzadeh, Zare, A.J.L. Notes: Botryosphaeria scharifii is phylogenetically Phillips, sp. nov. FIG.3 most closely related to Fusicoccum ramosum. Conidia MycoBank MB564800 of B. scharifii and F. ramosum are considerably shorter Conidiomata pycnidial, produced on pine needles than all other species in the Botryosphaeria clade. on WA within 2–4 wk, solitary or aggregated, dark However, the slightly longer conidia of B. scharifii brown to black, globose, up to 760 mmdiam, distinguishes it from F. ramosum (TABLE III). 214 MYCOLOGIA

FIG. 1. Dendrograms resulting from MSP-PCR profiles of (a) M13 and (b) (GTG)5 analyzed with GelCompar II. Similarity between the profiles were estimated with Pearson’s correlation coefficient. Cluster analysis of similarity matrices was done with UPGMA. ABDOLLAHZADEH ET AL.: BOTRYOSPHAERIACEAE IN IRAN 215

FIG. 2. One of the most parsimonious trees obtained from combined ITS and tef-1a sequence data. MP bootstrap values based on 1000 pseudoreplicates and posterior probabilities from Bayesian analysis are above and below the branches respectively. The species characterized in this study are in boldface. Bar 5 10 changes. Diplodia mutila (CBS112553) and Diplodia seriata (CBS112555) were included as outgroups. Phylogeny deposited in TreeBASE (S12532). 216 MYCOLOGIA

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

DISCUSSION various disease symptoms on a broad range of woody hosts including canker, dieback, trunk diseases and In this survey a large collection of Botryosphaeria and fruit rots (Ma et al. 2001). However, before the study Neofusicoccum isolates from woody hosts in Iran were of Slippers et al. (2004) B. dothidea was considered to studied. The isolates initially were grouped according be a complex of species including N. ribis and N. to their MSP-PCR profiles, and representative isolates parvum and therefore earlier reports could be of each group were selected for sequencing of the ITS unreliable. Our findings showed that of the four and tef1-a loci. As a result four species (B. dothidea, B. species isolated B. dothidea has the broadest distribu- scharifii, N. mediterraneum, N. parvum) were identi- tion in Iran and was found in a variety of climates fied. ranging from temperate and humid in the north near The most frequently encountered species was B. the Caspian Sea to the semi-arid regions in the west dothidea, which represented 60% of the isolates and along the Zagros Mountains and the humid tropical was found on 14 different forest and fruit trees from regions of the south near the . It is different regions of Iran (TABLE II). This species is a important to note that most of the isolates (92%) well known fungal pathogen from temperate regions were found in the north while only 8% were from the worldwide and has been reported in association with south and west. This species has been reported on

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. ABDOLLAHZADEH ET AL.: BOTRYOSPHAERIACEAE IN IRAN 217 walnut, rose and almond in Iran (Ershad 2009). Apart Lasiodiplodia separated L. parva and L. pseudotheo- from walnut, this is the first report of B. dothidea on bromae from L. theobromae based on the ability of the the other hosts (TABLE II) in Iran and to our first two species to produce a pink pigment in PDA at knowledge this is the first time it has been reported 35 C while Abdollahzadeh et al. (2010) did not on spruce (Picea sp.). identify any differences between these species and The second most common species, N. parvum, questioned the taxonomic significance of this char- comprising 27% of the isolates on seven plant species acter. Furthermore, Pennycook and Samuels (1985) was restricted to the temperate northern part of the distinguished N. luteum from N. parvum and N. ribis country. Neofusicoccum parvum has been reported based on the ability of N. luteum to produce a yellow commonly as an important pathogen of many woody pigment in PDA at 25 C. However, in the present plants in temperate regions around the world study some of our isolates of N. parvum produced a (Phillips 2002; van Niekerk et al. 2004; Inderbitzin yellow pigment in PDA at 25 C. et al. 2010; Chen et al. 2011). It has been recorded on High variation in cultural characteristics, morphol- 71 host species across six continents and 21 countries ogy and MSP-PCR fingerprinting among our isolates (Sakalidis 2011). N. parvum has been reported on of N. parvum led us to sequence 18 isolates. Vitis vinifera in Iran (Mohammadi et al. 2008) and Phylogenetic analysis showed that they all belong to this is the first report of this species on all hosts listed the same species and these variations reflect high in this study in Iran. genetic diversity among populations and indicate the We should point out that some ambiguities remain need to revise and reassess the morphological species about these earlier reports because some species such description. as N. ribis (5 B. ribis) have been regarded as All isolates of Neofusicoccum mediterraneum were synonyms of B. dothidea (von Arx and Mu¨ller 1954; found here on citrus, mango and on unknown trees Pennycook and Samuels 1985; Smith et al. 1994) and from southern Iran. N. mediterraneum first was N. parvum (5 B. parva) often has not been described from Eucalyptus in (Crous et al. differentiated from N. ribis (Smith and Stanosz 2007). Subsequently it was found on olive drupes in 2001; Zhou and Stanosz 2001). Furthermore, five Italy (Lazzizera et al. 2008), grapevine in Spain cryptic species are recognized within the N. parvum/ (Martin et al. 2011) and various woody plants N. ribis complex (Pavlic et al. 2009; Bogoude et al. including most notably Persian walnut, pistachio, 2010; Sakalidis et al. 2011). Despite these ambiguities, citrus, grapevine and almond in the United States to our knowledge this is the first report of N. parvum (Trouillas et al. 2010; Inderbitzin et al. 2010; U´ rbez- on willow, cherry and raspberry. Torres et al. 2010). In a BLAST query of our Because Slippers et al. (2004) differentiated B. sequences we found many N. mediterraneum isolates dothidea, N. ribis and N. parvum with morphological with notable differences in ITS and tef1-a sequences. and combined sequence datasets of ITS, tef1-a and b- Thus, it is possible that N. mediterraneum is a complex tubulin, these species are now clearly defined. of species. This species is a new record for Iran, and to Patterns of septation and discoloration in older our knowledge this is the first time it has been conidia discharged from pycnidia was regarded as a reported on mango trees. useful morphological feature to distinguish N. ribis Species of Botryosphaeriaceae are common patho- from N. parvum, and this is supported by molecular gens associated with mangoes worldwide (Slippers data. Aging conidia of N. parvum become light brown et al. 2005). Recently studies on mango trees have led with 1–2 septa, frequently with a darker brown middle to the characterization of eight species of Botryo- cell, while conidia of N. ribis rarely become light sphaeriaceae on mangoes in Australia and three brown with 1–2 septa, according to Slippers et al. species in Brazil (Sakalidis et al. 2011; Costa et al. (2004). Careful observation is crucial because some 2010). Despite extensive studies on Botryosphaeria- conidia remain hyaline and aseptate even after ceae associated with mango, in this study a new discharge in 2 mo old cultures. After careful species named B. scharifii was characterized in Iran. observation of our isolates of N. parvum we did not In view of the recent changes to the ICBN ratified at see aging conidia turning light brown and all aged the Melbourne conference, priority of names de- conidia remained hyaline with two septa even in 10 wk pends on the publication date irrespective of the life old cultures. The concept of aging has not been stage of the . Thus, Fusicoccum (1829) has clearly defined and thus changes in conidial charac- priority over Botryosphaeria (1863). Although there ters are difficult to apply in practice. has been no official decision on which name (Botryo- Cultural characteristics have been used to differen- sphaeria or Fusicoccum) will take priority, we recom- tiate the species of the family Botryosphaeriaceae. For mend that the name Botryosphaeria should be instance, Alves et al. (2008) in a phylogenetic study of conserved. Although the three species names 218 MYCOLOGIA currently in Fusicoccum will have to be transferred, we Mycology Congress and International Marine and have not done so but prefer to wait until a decision Freshwater Mycology Symposium Malaysia, 136. has been made. In our study B. scharifii was the least ———, Mohammadi Goltapeh E, Javadi A, Shams-bakhsh abundant species with only three isolates, one from M, Zare R, Phillips AJL. 2009. Barriopsis iraniana and mango twigs from southern Iran with dieback Phaeobotryon cupressi: two new species of the Botryo- sphaeriaceae from trees in Iran. Persoonia 23:1–8, symptoms and the other two from mango fruits with doi:10.3767/003158509X467552 end-rot symptoms imported from Pakistan. More ———, ———, ———, Zare R, Phillips AJL. 2010. sampling and isolation is necessary to understand Phylogeny and morphology of four new species of the host range, distribution and variability of this new Lasiodiplodia from Iran. Persoonia 25:1–10, species. doi:10.3767/003158510X524150 Members of Botryosphaeriaceae are considered Alves A, Correia A, Luque J, Phillips AJL. 2004. Botryo- endophytes and latent opportunistic pathogens that sphaeria corticola sp. nov. on Quercus species, with notes pose a potentially significant threat to agricultural and and description of Botryosphaeria stevensii and its forest trees, especially under stressed conditions anamorph Diplodia mutila. Mycologia 96:598–613, (Slippers and Wingfield 2007). Thus, taking appropri- doi:10.2307/3762177 ———, Crous PW, Correia A, Phillips AJL. 2008. 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