Diversity of sooty blotch and flyspeck fungi from apples in northeastern Turkey
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- Mayfield, D.A., Karakaya, A., Batzer, J.C. et al. Eur J Plant Pathol (2013) 135: 805. doi:10.1007/s10658-012-0123-1
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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.
KeywordsBlack Sea RegionEpiphytic fungiMalusTaxonomySBFS
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
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.
Species, number of isolates, and distinguishing morphological characters of sooty blotch and flyspeck fungi isolated from apples from Rize Province in northeastern Turkey
GenBank accession No. b
Diameter growth (mm)d
Chaetothyriales sp. F1a
20.5–28.5 × 2.5–3
dark brownish/sooty black, loosely convoluted, black margins
24.5–33 × 1.5–2
white to pale grey, surface cracks showing black interior, pale white-pink feathered margins, sclerotia produced
6–8.5 × 2–3
dark olive, mounded, brownish to cream yeast-like undergrowth, pale yellow pigment produced
16–20 × 1.5–2.0
iron grey, bumpy, ridged, irregular margin
Stomiopeltis-like sp. RS4.1a
dark olive grey, mounded, hard, irregular margins, yellow pigment produced
Stomiopeltis-like sp. RS7.1a
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
grayish olive, velvety, flat, light olive gray margin, clay colored pigment produced
19–22.5 × 5–5.5
dark olive gray, irregular cream margin
Zygophiala sp. FS6 a
16.5–20 × 4.5–5.5
olive gray, deep cream margins
15–18 × 6–7
tea green, thin
Zygophiala sp. FS3.3 a
16–18 × 6.5–7.5
dark, thin, spreading with white aerial mycelium, pale yellow pigment produced
22–31 × 2–3
light grayish olive aerial mycelia, gelatinous, zonate, convoluted, margin is olive gray, sucked up media from plate
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
Class Dothideomycetes; Genus: Scleroramularia (anamorph)
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
Order Capnodiales; Family Schizothyriaceae; Genus: Schizothyrium (anamorph Zygophiala)
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.
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.