European Journal of Plant Pathology

, Volume 135, Issue 4, pp 805–815

Diversity of sooty blotch and flyspeck fungi from apples in northeastern Turkey

Authors

  • Derrick A. Mayfield
    • Department of Plant Pathology and MicrobiologyIowa State University
  • Aziz Karakaya
    • Department of Plant Protection, Faculty of AgricultureUniversity of Ankara
  • Jean C. Batzer
    • Department of Plant Pathology and MicrobiologyIowa State University
  • Jennifer M. Blaser
    • Department of Plant Pathology and MicrobiologyIowa State University
    • Department of Plant Pathology and MicrobiologyIowa State University
Article

DOI: 10.1007/s10658-012-0123-1

Cite this article as:
Mayfield, D.A., Karakaya, A., Batzer, J.C. et al. Eur J Plant Pathol (2013) 135: 805. doi:10.1007/s10658-012-0123-1

Abstract

Sooty blotch and flyspeck (SBFS) is a mid- to late-season fungal disease complex that blemishes apples and other tree fruit crops in humid regions worldwide. SBFS is a concern for apple growers in northeastern Turkey, but the composition of the SBFS species assemblage in Turkey is unknown. In this study, SBFS fungi were isolated from apples collected in 2008 in the Rize Province of northeastern Turkey. Pressed SBFS colonies with subtending apple cuticle were shipped to Iowa State University for isolation. Of 633 primary isolates from 148 apple peels, 87 cultures were purified, 67 isolates were genetically characterized and 33 isolates examined for morphology. The internal transcriber spacer (ITS) ribosomal DNA and a portion of the 28S large subunit region (LSU) were compared to previously identified fungi using parsimony analysis. Putative species were delineated from ITS sequences as well as morphology on apple and in culture. Twelve species were delineated based on parsimony analysis of ITS sequences and morphology. A newly discovered and described species from the survey was Scleroramularia abundans; newly discovered putative species included Zygophiala sp. FS3.3, Stomiopeltis spp. RS7.1 and RS7.2, and Chaetothyriales sp. F1; previously recovered putative species included Zygophiala sp. FS6 and Stomiopeltis sp. RS4.1; and previously discovered and named SBFS species included Schizothyrium pomi, Zygophiala wisconsinensis, Microcyclosporella mali, Microcyclospora tardicrescens, and Peltaster fructicola. Based on parsimony analysis of the LSU, one species was placed in the subclass Chaetothyriomycetidae and 11 species were placed in subclass Dothideomycetidae. Ten species were placed in the order Capnodiales. These findings expand the documented extent of genetic diversity within the worldwide SBFS complex and are the first published description of the composition of the SBFS complex from Turkey.

Keywords

Black Sea RegionEpiphytic fungiMalusTaxonomySBFS

Introduction

Sooty blotch and flyspeck (SBFS) is a fungal disease complex that impacts apple (Malus x domestica Borkh.) production in humid regions worldwide (Williamson and Sutton 2000; Gleason et al. 2011). These fungi colonize the epicuticular wax layer of apple fruit and usually become visible during the mid- to late growing season (Gleason et al. 2011). Fungal thalli appear as a continuum of colony morphologies from dark irregular blotches of mycelia to clusters of sclerotium-like bodies (Gleason et al. 2011). Although SBFS damage is superficial, colonies on the fruit prevent fresh market sales, resulting in a reduction of crop value that can exceed 90 % in North America (Williamson and Sutton 2000).

In 1832, Schweinitz described SBFS as being caused by a single species, Dothidea pomigena. Colby (1920) identified Gloeodes pomigena as the causal agent of sooty blotch and Schizothyrium pomi as the cause of flyspeck. Sooty blotch was subsequently divided into distinct mycelial types by Colby (1920) and further described by Groves (1933) as variants of Gloeodes pomigena (Hickey 1960, Sutton and Sutton 1994). More recently, it was shown that several different SBFS mycelial types on apple fruit were caused by different species; for example, Peltaster fructicola exhibited a punctate mycelial type, Leptodontidium elatius exhibited a fuliginous mycelial type, and Geastrumia polystigmatis caused a ramose mycelial type on apples. Subsequent studies of SBFS biodiversity around the globe have led to the discovery of many additional species. Using traditional morphology coupled with rDNA analysis, studies in Serbia (Ivanović et al. 2010), Poland (Wrona and Grabowski 2004), China (Li et al. 2010; Li et al. 2011; Yang et al. 2010), Germany, Slovenia (Frank et al. 2010), and the United States (Batzer et al. 2005; Díaz Arias et al. 2010) found that the SBFS complex was comprised of at least 80 putative and named species. On apple fruit, each SBFS species exhibits only a single mycelial type, but multiple species produce similar phenotypes on apple (Batzer et al. 2005; Gleason et al. 2011). Although individual SBFS species from Asia, Europe, and North America have been described, comprehensive regional biodiversity surveys have been limited to the United States and Serbia (Batzer et al. 2005; Díaz Arias et al. 2010; Ivanović et al. 2010). These multi-orchard surveys have shown that some SBFS species are cosmopolitan in distribution, whereas others are limited to a single geographic region.

Turkey is the third largest apple producer in the world after China and the United States, producing 2.6 million tons of fruit in 2010 (FAO 2012). Turkey is located relatively near the centre of apple domestication in central Asia and is home to the wild Malus species M. orientalis, a close genetic relative of domesticated apple cultivars (Gharghani et al. 2009). Therefore, characterizing SBFS diversity in this region is an important component to understanding worldwide biodiversity patterns in the SBFS complex. Rize Province, located in the Black Sea Region of northeast Turkey, experiences higher rainfall than other apple-producing regions in the country, and therefore is at greatest risk of SBFS outbreaks. The objective of this study was to characterize diversity in the SBFS complex from the Black Sea Region of Turkey and describe the taxonomic relationship of SBFS fungi of Turkey to previously identified species worldwide. A preliminary report has been published (Blaser et al. 2010).

Materials and methods

Isolates

Harvested apples showing SBFS signs were collected in September 2008 from markets, orchards, and warehouses from Rize Province (41°01′N 40°31′E), in the Black Sea Region of Turkey, in order to obtain as wide a range of regional diversity as possible. Apple peels exhibiting multiple colonies of SBFS were excised, pressed, and photographed at University of Ankara (Ankara, Turkey), then mailed in November 2008 to Iowa State University (Ames, Iowa, USA) for isolation. From 148 peels, 633 primary isolations were made from non-overlapping SBFS colonies (Batzer et al. 2005). Under a dissecting microscope, each SBFS colony was subdivided into four quadrants and a subsample of mycelium was transferred to water agar amended with 1.25 ml/l of 50 % lactic acid (AWA) (Becton Dickinson, Sparks, Maryland, USA) and incubated for 3 to 6 weeks at 21 to 24 °C under ambient light. After quadrants from each colony on AWA were inspected for uniformity, 238 isolates were transferred to potato dextrose agar (Difco Laboratories Inc., Detroit, Michigan, USA) acidified with 40 drops of 50 % lactic acid per liter (PDAL). A total of 87 purified isolates were stored in 15 % glycerol at −80 °C.

Polymerase chain reaction and sequencing

Template DNA for PCR was extracted from 4- to 6-week-old cultures grown on PDA by scraping mycelia with a sterile pipette tip and transferring the mycelia into 50 ul of PrepMan Ultra Sample Reagent (Life Technologies, Carlsbad, California). The internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1, 5.8S rDNA gene, ITS2) from one to three isolates of each mycelial type on PDA was amplified by PCR (Batzer et al. 2005) and sequenced using primer pair ITS-1 F/ITS4 (White et al. 1990); a total of 67 sequences were obtained. After isolates with identical ITS sequences were grouped, one or two representative isolates from each clade were selected for further examination of conidial characters and colony morphology. In addition, for each clade identified by parsimony analysis of the ITS region, a portion of the 28S (large subunit, LSU) rDNA gene was amplified and sequenced using primer pair LROR/LR5 (Vilgalys and Hester 1990). PCR reaction conditions, purification, and sequencing of PCR products were as described in Batzer et al. (2005).

Putative species designation

Sequences were manually aligned in BioEdit (Hall 1999) using all ITS sequences obtained from this survey as well as previously identified ascomycete taxa, including SBFS fungi identified during past surveys (Batzer et al. 2005; Díaz Arias et al. 2010; Frank et al. 2010; Ivanović et al. 2010; Li et al. 2010; Li et al. 2011; Wrona and Grabowski 2004; Yang et al. 2010) were downloaded from NCBI GenBank sequence database using a BLASTn search. Previously isolated taxa from Mark Gleason’s personal collection (GPC) at Iowa State University were also included. In order to obtain unambiguous alignments, sequences were placed into one of six groups, each of which aligned sequences of species within a taxonomic family. Maximum parsimony analysis (PAUP version 4.0b10; Swofford 2002) trees were used to cluster isolates into clades. Alignable gaps were treated as a “fifth base.” Clades were designated as putative species based on strict consensus and bootstrap analysis of 1,000 replications.

Putative species delineated from ITS parsimony analysis were corroborated using phenotypic characters. Morphology of one or two representatives from each putative species was examined, for a total of 33 isolates. Mean diameter growth of colonies was determined on 1.5 % malt extract agar (MEA) after 4 weeks at 24 C in darkness (Batzer et al. 2005). Colony color and appearance were described after 4 weeks of growth in ambient light on PDA at ambient room temperature. Colony colour was described using the colour plates of Ridgway (1912). Surface texture, margins, colony shape, presence of aerial mycelium, and presence of soluble pigment was also noted. Conidia were obtained from 5- to 10-day-old growth on carnation leaf agar (CLA) under fluorescent light at ambient room temperature (Fisher et al. 1982). Samples were mounted in lactic acid and 95th percentiles were recorded of 20 measurements (wherever possible at ×1000 magnification) of conidia length and width, with range of measurements given in brackets.

Putative species were placed in subclass, order and sometimes family based on analysis of the 28S large subunit (LSU) region of rDNA. Partial sequences (844 bp) of the LSU from selected isolates of each putative species were aligned with previously identified SBFS species and other fungal species obtained from GenBank. The tree was rooted to Peziza vesiculosa Bull. Taxa with identical LSU sequences were eliminated from the data block, reducing the number of distinct taxa to 12. Heuristic searches in PAUP were conducted with random sequence addition and tree bisection-reconnection (TBR) branch swapping algorithms, collapsing zero-length branches, and saving all minimal length trees. Maxtrees was set at 10,000. Alignable gaps were treated as missing data. Unalignable regions totalling 42 bp were eliminated from the data set. All characters were given equal weight. To assess the robustness of clades and internal branches for data sets, a strict consensus of the most parsimonious trees was generated and a bootstrap analysis of 1,000 replications was performed.

Results

Twelve SBFS species were delineated from this survey; five were newly discovered, whereas the remaining seven had been isolated previously from SBFS infected apples from other geographic regions (Table 1). Parsimony analysis of the LSU region of the rDNA revealed that 11 species were in the Dothideomycetes and one species resided in the Eurotiomycetes. The LSU data set contained 76 taxa, including outgroup Peziza vesiculosa (NG027570), and 844 characters of which 264 were parsimoniously informative, 59 were variable and uninformative, and 480 were constant. Parsimony analysis of the LSU resulted in 1,064 equally informative trees, one of which is shown in Fig. 1a and b. Chaetothyriales sp. F1 grouped with an undescribed Chaetothyriales species with 100 % bootstrap support. Two other previously identified species of SBFS, Phialophora sessilus, and Leptodontidium elatius, were also placed in the order Chaetothyriales with 100 % bootstrap support (Fig. 1a).
Table 1

Species, number of isolates, and distinguishing morphological characters of sooty blotch and flyspeck fungi isolated from apples from Rize Province in northeastern Turkey

Species

GenBank accession No. b

Morphology

Conidia (μm)c

Diameter growth (mm)d

Colony

Chaetothyriales sp. F1a

JX014309 JX042486

20.5–28.5 × 2.5–3

9.0–22.0

dark brownish/sooty black, loosely convoluted, black margins

Scleroramularia abundans

FR716676 FR716667

24.5–33 × 1.5–2

19.0–21.5

white to pale grey, surface cracks showing black interior, pale white-pink feathered margins, sclerotia produced

Peltaster fructicola

JQ358792 JX042485

6–8.5 × 2–3

19.0–23.0

dark olive, mounded, brownish to cream yeast-like undergrowth, pale yellow pigment produced

Microcyclospora tardicrescens

JQ358790 JX042484

16–20 × 1.5–2.0

14.0–17.0

iron grey, bumpy, ridged, irregular margin

Stomiopeltis-like sp. RS4.1a

JQ358787 JX042482

Sterile

12.0–16.5

dark olive grey, mounded, hard, irregular margins, yellow pigment produced

Stomiopeltis-like sp. RS7.1a

JQ358788 JX042481

Sterile

20.0–22.0

citrine drab color, velvety, flat, light olive gray margin with thin outer brownish olive ring, clay colored pigment produced

Stomiopeltis-like sp. RS7.2 a

JQ358789 JX042483

Sterile

25.0–29.0

grayish olive, velvety, flat, light olive gray margin, clay colored pigment produced

Schizothyrium pomi

JQ358786 JX042476

19–22.5 × 5–5.5

19.0–25.0

dark olive gray, irregular cream margin

Zygophiala sp. FS6 a

JQ358783 JX042479

16.5–20 × 4.5–5.5

14.0–17.0

olive gray, deep cream margins

Zygophiala wisconsinensis

JQ358785 JX042478

15–18 × 6–7

35.0–50.0

tea green, thin

Zygophiala sp. FS3.3 a

JQ358784 JX042477

16–18 × 6.5–7.5

22.0–28.0

dark, thin, spreading with white aerial mycelium, pale yellow pigment produced

Microcyclosporella mali

JQ358791 JX042480

22–31 × 2–3

6.0–21.0

light grayish olive aerial mycelia, gelatinous, zonate, convoluted, margin is olive gray, sucked up media from plate

aPutative species

bGenBank accession numbers for the rDNA ITS and LSU regions of representative isolates

cConidia were obtained from 5-to10-day-old growth under fluorescent light on CLA at 24 °C

dDiameter of colonies measured after 4 weeks in darkness on MEA at 24 °C

eColony appearance and colour described after 4 weeks of growth in ambient light on PDA at 24 °C

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Fig. 1

One of 1,064 most parsimonious trees using partial LSU sequences (844 bp) from SBFS species and other ascomycetes obtained from GenBank. Taxa in bold were obtained from infected apples from the Rize region of NE Turkey. Taxa designated by * were identified previously as members of the SBFS complex. Tree is rooted to Peziza vesiculosa (NG027570). Bootstrap values >70 % derived from the 1,000 replications are indicated above branches. Strict consensus branches are thickened. Gaps were treated as missing data. Parsimony informative characters = 264; Tree length (TL) = 1290; Consistency index (CI) = 0.4116; Homoplasy index (HI) = 0.5884; Retention index (RI) = 0.7549. Peziza vesiculosa (NG027570) was used as the outgroup taxon. The tree was divided into two parts, 1a and 1b, to permit sufficient resolution for reading taxon names

Eleven species were placed in the class Dothideomycetes. Scleroramularia abundans could not be placed at the subclass level (Fig. 1a). The remaining 10 species resided in subclass Dothideomycetidae and clustered with the order Capnodiales with bootstrap support of 91 %. Within Capnodiales, Peltaster spp. formed a strongly supported clade (99 % bootstrap support) (Fig. 1a). The remaining Capnodiales species were placed in four families (94 % bootstrap support) (Fig. 1b). Microcyclospora spp. formed a distinct clade (100 % bootstrap support) with the family Teratosphaeriaceae (94 % bootstrap support). Schizothyrium pomi and four Zygophiala anamorphs formed a strong clade (100 % bootstrap support) in the family Schizothyriaceae. Three Stomiopeltis-like spp. grouped within the family Micropeltidaceae (91 % bootstrap support). Microcyclosporella spp. grouped with 84 % bootstrap support within the family Mycosphaerellaceae. Based on the LSU analysis, the 67 isolates in this study were placed into one of six clades and ITS sequences were used to delineate putative species.

Subclass Chaetothyriomycetidae; Order Chaetothyriales

Since the ITS sequence of isolate T65D1b could not be unambiguously aligned with sequences of known fungal species and previously identified SBFS isolates, a parsimony tree was not generated. A BLASTn search revealed the closest identities to T65D1b were Chaetothyriales sp. NY6956a (HM992800) (588/604 bp (97 %)) and Chaetothyriales sp. CN-Cre-Bol-4 (HQ634614) (636/707 bp (90 %)). These fungi were previously described as an endophyte from rain forest plants and an ant-associated black yeast from Cameroon, respectively (Voglmayr et al. 2011). Colonies on PDA were dark brownish/sooty black, loosely convoluted with black margins with edges that depressed the media 1–2 mm (Fig. 2). Colony diameter on MEA ranged between 9.0 and 22.0 mm after 4 weeks (Table 1). Conidia of isolate T65D1b, designated as Chaetothyriales sp. F1, were septate scolecospores with a blunt apex, narrow base, subhyaline, and thick walled (13) 20.5–28.5 (45) × (2) 2.5–3 (3.5) μm (Fig. 2).
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Fig. 2

Chaetothyriales sp. F1. a Colony on PDA. b Budding conidia. ce Scolecospores on CLA. Scale bars: bd =5 μm, e =10 μm

Class Dothideomycetes; Genus: Scleroramularia (anamorph)

Isolates obtained in the present study were recently described as a new species (Li et al. 2011). Isolates from this anamorph genus have been recovered from the fruit surface of many crops, including winter squash, pawpaw and apple, in China and the U.S. Six most parsimonious trees determined from ITS sequences grouped the two isolates of Scleroramularia abundans with 99 % bootstrap support (Fig. 3). On apple, S. abundans formed a compact speck mycelial type comprised of round to irregular, flattened, black sclerotium-like bodies. Cultures on PDA were white to pale pink, surface cracks showed black interior, pale white-pink feathered margins and produced black sclerotia on the underside of the colony against the bottom of the plate (Fig. 4). Diameter on MEA reached 19.0–21.5 mm after 4 weeks on MEA (Table 1). The conidia of S. abundans were hyaline, straight to slightly curved, formed in chains, 0–3 septate, (16) 24.5–33 (50) × 1.5–2 (3) μm; reduced conidiophores developed from erect, solitary, subcylindrical conidiogenous cells and could be distinguished by intercalary and terminal conidia (Fig. 4).
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Fig. 3

One of five most parsimonious trees determined from ITS sequences (555 bp) from Scleroramularia anamorph isolates obtained from SBFS signs on fruit. Tree is rooted to Guignardia mangiferae (AB454360). Gaps were treated as a fifth base. Parsimony informative characters = 30; TL = 231; CI = 0.9524; HI = 0.0476; RI = 0.8830. Strict consensus branches are thickened. Bootstrap values >60 % from 1000 replicates are indicated above branches. Taxa in bold were recovered from apples infected with SBFS in Turkey. Other isolates were recovered from SBFS colonies from the U.S. and China

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Fig. 4

Scleroramularia abundans. a Colony on PDA. bd Chains of conidia (note hyphal bridge in c on CLA). Scale bars = 5 μm

Order Capnodiales; Genus: Peltaster (anamorph)

The 12 most parsimonious trees grouped the six isolates with previously described SBFS species Peltaster fructicola with 98 % bootstrap support (data not shown). Isolates on PDA formed cream buff budding yeast with no visible mycelia during the first week of growth and became dark olive gray, wrinkly and velvety at 4 weeks. Colony diameter on MEA ranged from 19.0 to 23.0 mm after 4 weeks. Conidia were (3) 6–8.5 (11) × (1.5) 2–3 (4) μm (Table 1), hyaline, unicellular, elliptic, produced successively on indistinct conidiophores on short mycelial strands.

Order Capnodiales; Family Teratosphaeriaceae; Genus: Microcyclospora (anamorph)

The six isolates grouped with the previously identified Microcyclospora tardicrescens from Slovenia (GU570541) (data not shown). Colonies were iron grey in colour, bumpy or ridged with uneven margins and produced a pale pink soluble pigment on PDA. Isolate T78Dc1c reached 14.0–17.0 mm in diameter after 4 weeks on MEA at 24C (Table 1). Conidia were scolecosporous, cylindrical, truncated, smooth, transversely 1- to multiseptate, (12) 19.5–27.0 (37) × 1.5–2.0 (2.5) μm. Conidiophores were highly reduced to mono-polyblastic, light brown conidiogenous cells.

Order Capnodiales; Family Micropeltidaceae; Putative teleomorph genus: Stomiopeltis-like

The ITS sequences of these isolates grouped with previously identified species of uniloculate (AY160170) and multiloculate (AY160162, AY160165, AY160168 and AY160172) Stomiopeltis spp. (Fig. 5), and since the isolates were sterile in culture, a Stomiopeltis-like species designation was used. SBFS species in the Micropeltidaceae have all been associated with a ramose mycelial type on apple, and this was also observed for the present colonies on apple peels. The three most parsimonious trees clustered the 13 isolates into three well-supported putative species (Fig. 5). Seven isolates were grouped with a previously recovered putative species from Tennessee, Stomiopeltis-like sp. RS4.1, with bootstrap support of 75 % and clustered with a uniloculate Stomiopeltis species. The six remaining isolates grouped with 100 % bootstrap support, and within this cluster two isolates (T38A1c and T36A1b), designated as RS7.1, were sister to four isolates designated as RS7.2. Isolates of RS4.1 grew relatively slowly, reaching 12.0–16.5 mm in diameter on MEA after 4 weeks (Table 1). On PDA, cultures of Stomiopeltis-like sp. RS4.1 were smooth, tough, and deep olive gray and also produced a yellow pigment. In contrast, isolates of Stomiopeltis-like sp. RS7.1 and Stomiopeltis-like sp. RS7.2 produced reddish-clay coloured pigment on PDA (Fig. 6). Isolates of Stomiopeltis-like sp. RS7.1 were velvety, flat, citrine drab colour with light olive gray margins. The thin outer brownish olive ring that surrounded the margin of Stomiopeltis-like sp. RS7.1 was not present in Stomiopeltis-like sp. RS7.2, which was velvety, grayish olive with olive gray margins on PDA. These putative species were also distinguished by differences in colony diameter on MEA. Stomiopeltis-like sp. RS7.2 reached 25.0–29.0 mm in diameter on MEA after 4 weeks at 24 C, whereas Stomiopeltis-like sp. RS7.1 reached 20.0–22.0 mm (Table 1).
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Fig. 5

One of three most parsimonious trees determined from ITS sequences (527 bp) obtained from SBFS isolates with sterile mycelia. The tree is rooted to Mycosphaerella bixea. Gaps were treated as a fifth state. Characters 13 through 80 were eliminated due to insertions and deletions in the ITS1 region. Parsimony informative characters = 154. Tree length = 437; Consistency index (CI) = 0.6888; Homoplasy index (HI) = HI = 0.3112; Retention index (RI) = 0.9028. Strict consensus branches are shown in bold. Bootstrap values >50 % are indicated above branches. Taxa in bold were recovered from apples infected with SBFS in Turkey. Taxa were included from Mark Gleason’s personal collection (designated GPC) at Iowa State University. Other isolates were recovered from ramose SBFS mycelial type colonies on apples in the U.S.

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Fig. 6

Stomiopeltis-like spp. aStomiopeltis-like sp. RS4.1 colony on PDA. bStomiopeltis-like sp. RS7.1 colony on PDA. cStomiopeltis-like sp. RS7.2 colony on PDA

Order Capnodiales; Family Schizothyriaceae; Genus: Schizothyrium (anamorph Zygophiala)

Based on the single most parsimonious tree obtained from ITS sequences, 13 isolates were delineated into four species (Fig. 7). All Zygophiala isolates produced conidia on conidiophores arising from a hyaline supporting cell that gave rise to a twisted, dark brown, smooth stipe, a subhyaline, finely verruculose apical cell, and two laterally divergent, finely verruculose, pale brown, ovate conidiogenous cells bearing prominent ellipsoidal scars. Conidia were solitary, smooth-walled, granular, aseptate to transversely 1-septate and produced in pairs (Fig. 8). A single isolate (T51A1a) grouped with a previously identified isolate of Schizothyrium pomi with strict consensus and a bootstrap support of 62 %. Morphology was consistent with that of the anamorph of S. pomi (Batzer et al. 2008); most notable was the long, tapered shape of the 2-celled conidia (Fig. 8b), in contrast to the cylindrical and spherical conidia for the other Zygophiala species found in this study. Six isolates of Zygophiala sp. FS6 grouped with an isolate obtained from Kentucky, U.S. with bootstrap support of 66 %, and clustered this putative species with Zygophiala cylindrica with 99 % bootstrap support. The growth of Zygophiala sp. FS6 isolates (14.0–17.0 mm in diameter) after 4 weeks on MEA differed from other closely related species (Table 1). Zygophiala sp. FS6 is morphologically distinct from Z. cylindrica by having smaller conidia (Fig. 8d) (12) 16.5–20 (25) × (3.5) 4.5–5.5 (7) μm (Table 1). Morphology on PDA was distinctive in having an olive gray centre with an abrupt, submerged, white/cream coloured margin and diffusible pale yellow-orange pigment (Fig. 8c). Three isolates of Zygophiala sp. FS3.3 differed from Z. wisconsinensis by a single base pair (Fig. 7); however, this species was readily distinguishable from Z. wisconsinensis by slower growth on MEA (22.0–28.0 mm) (Table 1). Growth on PDA was flat to mounded, spreading with white aerial mycelium with large, white, arborescent, submerged margins producing a diffusible pale yellow pigment; whereas growth of Z. wisconsinensis was flat with regular margins, and lacked aerial mycelium and diffusible pigment (Fig. 8).
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Fig. 7

One of four most parsimonious trees determined from ITS sequences (479 bp) obtained from Zygophiala anamorphs obtained from SBFS signs on fruit. The tree is rooted to Teratosphaeria nubilosa (AY534223). Gaps were treated as a fifth state. Parsimony informative characters = 36; TL = 154; CI = 0.9156; HI = 0.0844; RI = 0.9440. Strict consensus branches are thickened. Bootstrap values >60 % from 1000 replicates are indicated above branches. Taxa in bold were recovered from apples infected with SBFS in Turkey. Other isolates were recovered from SBFS colonies on apples in China and the U.S.

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Fig. 8

Schizothyrium pomi and its Zygophiala anamorphs. aS. pomi colony on PDA. bS. pomi conidiophores and conidia on CLA. cZygophiala sp. FS6 colony on PDA dZygophiala sp. FS6 conidia on CLA. eZ. wisconsinensis colony on PDA. fZ. wisconsinensis conidia on CLA g. Zygophiala sp. FS3.3 colony on PDA. hZygophiala sp. FS3.3 conidiophores and conidia on CLA. Scale bars = 5 μm

Order Capnodiales; Family Mycosphaerellaceae; Genus: Microcyclosporella (anamorph)

The 18 isolates clustered into three moderately to strongly supported clades (bootstrap support from 69 to 92 %) and grouped with Microcyclosporella mali obtained from Slovenia, Serbia, Poland and the USA (data not shown). The highly variable phenotypic traits were not consistent with clades delineated by parsimony analysis of the ITS region. Most colonies on PDA were grayish olive, velvety, convoluted, mounded, pulling the media from the bottom of the plate, with olive grey to light yellow margins. Diameter growth ranged from 6.0 to 21.0 mm after 4 weeks on MEA (Table 1). Hyaline, intercalary conidiogenous cells formed from reduced conidiophores. Conidia were hyaline, smooth, tapered, 0–5 transversely septate, (15) 22–31 (48) × 2–3 (4) μm (Table 1). Scolecospores developed multiple side branches resulting in highly branched conidial masses on PDA.

Discussion

This study expands the documented range of genetic diversity within the SBFS complex and is the first information about the taxonomic classification of these fungi in the apple-producing regions of northeastern Turkey. This study also provides the first published evidence of five newly discovered SBFS putative species. Additionally, seven fungi from the SBFS complex found in northeastern Turkey have also been found in Central and Southeastern Europe, East Asia, or North America.

The most commonly isolated species in our survey was Microcyclosporella mali. This is among the most widely distributed species in northern and southeastern Europe as well as the eastern and central U.S. (Díaz Arias et al. 2010; Frank et al. 2010; Gleason et al. 2011; Ivanović et al. 2010). Peltaster fructicola is also widely distributed in these apple-growing regions. In contrast, Microcyclospora tardicresens has been reported only from Slovenia (Frank et al. 2010). Interestingly, four distinct species of the anamorph genus Zygophiala, the cause of the flyspeck mycelial type, were recovered from this survey. Previous surveys have suggested that Schizothyrium pomi is the predominant species in Europe and North America (Batzer et al. 2005; Díaz Arias et al. 2010; Frank et al. 2010; Gleason et al. 2011; Ivanović et al. 2010). A relatively large diversity of Schizothyrium spp. within a limited geographical region has not been observed previously. Besides Scleroramularia abundans reported here, Scleroramularia spp. had been documented previously only from China and North America (Li et al. 2011). Our study also reported three putative Stomiopeltis-like spp. which had been reported only in North America (Gleason et al. 2011). To confirm the pathogenicity of the putative species collected in the present study, we must complete a modified Koch’s postulates on apples as described in Batzer et al. (2005).

The present study thus adds to evidence that some SBFS taxa are restricted in geographic range whereas others are cosmopolitan. Batzer et al. (2012) recently reported that several SBFS species predominated for three consecutive years in apple orchards in Iowa in the central U.S., supporting the hypothesis that apple-producing regions can be characterized by distinct assemblages of major species. The discovery of five additional species in the present study highlights the importance of understanding a region’s species complex, since SBFS species can have unique environmental biology, including differences in sensitivity to fungicides (Batzer et al. 2010; Batzer et al. 2012; Tarnowski et al. 2003). Once the predominant SBFS species in a region are known and their environmental biology bas been characterized, SBFS management strategies for that geographic region may be modified to become more efficient and sustainable.

This survey provides a foundation for a more systematic, intensive survey across multiple orchards, years, and management regimes (Díaz Arias et al. 2010) which would clarify the biogeography and regional patterns of SBFS biodiversity in the Black Sea Region of Turkey. However, the present study revealed the prevalent as well as some of the less common species in the SBFS complex in northeastern Turkey.

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© KNPV 2012