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Department of Plant Pathology, Iowa State University, Ames, Iowa 50011
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Sooty blotch and flyspeck (SBFS) is a late-season disease of apple and pear fruit that cosmetically damages the cuticle, resulting in produce that is unacceptable to consumers. Previous studies reported that four species of fungi comprise the SBFS complex. We examined fungal morphology and the internal transcriber spacer (ITS) and large subunit (LSU) regions of rDNA of 422 fungal isolates within the SBFS complex from nine orchards in four Midwestern states (USA) and compared them to previously identified species. We used LSU sequences to phylogenetically place the isolates at the order or genus level and then used ITS sequences to identify lineages that could be species. We used mycelial and conidial morphology on apple and in culture to delimit putative species. Thirty putative species found among the Midwest samples were shown to cause SBFS lesions on apple fruit in inoculation field trials. Among them Peltaster fructicola and Zygophiala jamaicensis have been associated previously with SBFS in North Carolina. The LSU analyses inferred that all 30 SBFS fungi from Midwestern orchards were Dothideomycetes; one putative species was within the Pleosporales, 27 were within Dothideales, and two putative species could not be placed at the ordinal level. The LSU sequences of 17 Dothideales species clustered with LSU sequences of known species of Mycosphaerella.
Key words: Colletogloeum, Dissoconium, Dothideomycetes, Gloeodes pomigena, Passalora, plant pathology, Pseudocercospora, Pseudocercosporella, Ramularia, SBFS, Xenostigmina
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Batzer et al 2002b). Apple growers control SBFS by applying fungicides every 10–14 d, from soon after bloom until shortly before harvest (Hartman 1995). Without frequent protectant fungicide sprays most of the apples grown in the eastern and midwestern U.S. would be affected. Even with an intensive fungicide spray program, sporadic control failures occur (Williamson and Sutton 2000).
Sooty blotch and flyspeck initially were described as having a single causal agent, Dothidea pomigena Schwein. (Schweintz 1832). Colby (1920) determined that sooty blotch and flyspeck were caused respectively by Gloeodes pomigena (Schwein.) Colby and Schizothyrium pomi (Mont. & Fr.) Arx and also described the appearance of three mycelial types of sooty blotch on apple fruit. Groves (1933) described distinct mycelial types (signs) of sooty blotch as ramose, punctate, fuliginous and rimate. Subsequent researchers (Hickey 1960, Sutton and Sutton 1994) observed a wide range of variation of sooty blotch signs on apple fruit. Johnson and Sutton (1994) and Johnson et al (1997) recognized three sooty blotch species from ramose (Geastrumia polystigmatis Batista & M.L. Farr), punctate (Peltaster fructicola E.M. Johnson et al) and fuliginous (Leptodontium elatius [G. Mangenot] de Hoog) mycelial types. Considerable morphological variation also has been observed for flyspeck (Schizothyrium pomi, anamorph Zygophiala jamaicensis E. Mason) on apple fruit and in culture (Baker 1977, Nasu et al 1987, Lerner 1999).
Progress in understanding the taxonomy and ecology of the SBFS complex has been slowed by difficulty in isolating, maintaining and identifying these fungi in culture, as well as scarcity of fruiting structures on apple peels (Hickey 1960). Molecular identification has been used to elucidate the etiology of plant pathogenic fungi that otherwise were difficult to identify. For example, colony morphology and ITS sequences were used to characterize pathogenic and nonpathogenic fungi isolated from the vascular tissues of soybeans in the north central United States (Harrington et al 2000). Unknown basidiomycetes associated with bark beetles also were identified to putative species using the mitochondrial small subunit and the spacer regions of the rDNA (Hsiau and Harrington 2003). Using a similar strategy we examined the morphology and partial rDNA sequences of fungi within the SBFS complex from nine orchards in the Midwest to test the hypothesis that many species cause the same signs on apple. We used the LSU of nuclear rDNA to phylogenetically place the SBFS isolates at the order and genus levels, used ITS sequences to identify lineages that could be species, and phenotypically compared isolates from these lineages to delineate putative species (Harrington and Rizzo 1999).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
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Polymerase chain reaction and sequencing.— – The ITS region (ITS1, 5.8 S rDNA gene, ITS2) from one to three isolates of each distinct colony morphology type on PDA from each sampled orchard was sequenced for a total of 228 isolates. Isolates with identical ITS sequences were grouped; a portion of the LSU 28 S gene then was sequenced for one to three representative isolates within each group, totaling 117 LSU sequences.
Template DNA for polymerase chain reaction (PCR) was obtained directly by scraping mycelia with a pipette tip from 4–6 wk old cultures grown on PDA (Harrington and Wingfield 1995). Primer pairs used for amplification and sequencing of the ITS region were ITS-1F/ITS4 (White et al 1990), and primer pairs used for amplification and sequencing of LSU were LROR/LR5 and LROR/LR3, respectively (Vilgalys and Hester 1990). Amplification reactions consisted of 4 mM MgCl2, 5% DMSO, 1x Sigma buffer, 200 µM dNTPSs, 0.5 µM of the forward and reverse primers, and 3 units of Taq polymerase (Sigma Chemical Co., St Louis, Missouri). Cycling conditions (MJ Research Inc. thermocycler, PTC-100 Waltham, Massachusetts) for amplifications were an initial denaturation at 94 C for 95 s, followed by 35 cycles of denaturation at 94 C for 35 s, annealing at 49 C for LSU and at 52 C for ITS for 60 s and extension at 72 C for 2 min. The PCR product was purified with a QIAquick DNA Purification Kit (QIAgen, Valencia, California) and quantified on a Hoefer DyNA Quant 200 Fluorometer (Amersham Pharmacia Biotech, San Francisco, California). Automated sequencing was performed at the Iowa State University DNA Sequencing and Synthesis Facility.
The ITS and LSU database.— –
To identify possible relatives of the isolated SBFS fungi, BLAST searches (version 2.2.6, National Center for Biotechnical Information, Bethesda, Maryland) were conducted with representative ITS and LSU sequences. Partial sequences of taxa with high homology to SBFS fungal sequences were downloaded for phylogenetic comparisons. Additional ITS and LSU sequences of representative ascomycete taxa also were downloaded and used in analyses (TABLE II).
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), and the 5'- and the 3'- ends were trimmed to aid alignment. Lengths of the ITS sequences analyzed ranged from 458 to 534 base pairs, and those of the partial LSU sequences ranged from 582 to 609 base pairs. Preliminary alignments of the ITS and LSU matrices were generated using Clustal X (Thompson et al 1997) with gap opening and gap extension parameters of 50 : 5; these alignments were optimized manually. For LSU analysis ambiguously aligned regions associated with six indels were eliminated, resulting in a matrix of 435 characters, including gaps. Taxa with redundant LSU sequences were eliminated from the data block, reducing the number of taxa in the analysis from 117 to 77. Peziza ampelina was used as outgroup in the LSU phylogenetic analysis. The numerous, large insertion/deletions in the ITS dataset prevented unambiguous alignment of all sequences. Therefore ITS sequences of subsets of taxa with similar sequences were analyzed separately to delineate species.
Maximum parsimony analysis was performed with PAUP* version 4.0b10 for 32-bit Microsoft Windows (Swofford 2002). Heuristic searches 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 a "fifth base." All characters were given equal weight. To assess the robustness of clades and internal branches, a strict consensus of the most parsimonious trees was generated and a bootstrap analysis of 1000 replications was performed for ITS datasets and 200 replications for the LSU dataset. Alignments and strict consensus trees were deposited in TreeBASE. To test alternative taxa clustering relationships based on the minimum evolution principle (Nei and Kumar 2000), a nearest neighbor joining (NJ) tree also was generated from the LSU dataset with PAUP (Swofford 2002), and a bootstrap analysis of 1000 replicates was performed. Classification of taxa was based on the work of Barr (1987).
Putative species designation.— –
Isolates were grouped into putative species based on ITS parsimony analysis, conidial characters and colony morphology on apples and artificial media. Genus designations were determined by anamorph morphology. Putative species within each genus were assigned letter designations based on their appearance on apple, including flyspeck (FS), compact speck (CS), discrete speck (DS), ramose (RS), punctate (P), ridged honeycomb (RH) and fuliginous (FG) (FIGS. 1–9) (Colby 1920, Groves 1933, Batzer et al 2002b). Letter designations of putative species were followed by numbers based on unique ITS sequences and phenotypic differences in culture.
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Morphology of SBFS isolates on apple and in vitro.— – Signs of SBFS preserved on apple peels were described, including mycelial growth patterns and fruiting body size and density. One to five representative isolates from each putative species were grown on 1.5% malt-extract agar (MEA), PDA and carnation leaf agar (CLA) (Fischer et al 1982). Colony descriptions were made after 1 mo of growth on PDA and MEA at 21–24 C under intermittent ambient light. Washed and autoclaved pieces of cellulose membrane (Flexel Inc., Covington, Indiana) were placed on CLA plates, mycelial plugs were transferred to the edge of the cellulose pieces and hyphae were allowed to grow over the cellulose. When fungal structures were evident, the cellulose pieces were transferred from the agar, mounted on glass slides, and examined at 400x and 1000x magnification. Twenty measurements of each type of structure were taken if sufficient material was available.
Diameter growth of colonies was determined on MEA. Nine mycelial plugs (6 mm diam) from 3 wk old colonies on MEA were placed upside down onto three plates (three plugs per plate). After plates had been incubated 4 wk at 25 C in the dark two perpendicular measurements of colony diameters were made and the diameter of the plug was subtracted to determine the extent of diameter growth. Average diameters and ranges for each isolate were recorded.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mycelial types on apple fruit.— –
Not all putative species could be distinguished based on mycelial type on apple fruit, but each putative species was associated with a single mycelial type. Mycelial types described by Colby (1920) and Groves (1932) were employed when appropriate, but additional mycelial types were encountered (FIGS. 1–9
). In some species conidiomata or spermogonia were observed to produce conidia or spermatia, respectively, on inoculated apple fruit. The term sclerotium-like bodies was employed to indicate dark hyphal structures that may have been sterile conidiomata, spermogonia, or pseudothecia-like structures. Ascospores were never observed in pseudothecia-like structures on apples, either in fall or spring after incubation in wire cages on the ground during winter, although ascospores of Schizothyrium pomi and Stomiopeltis versicolor have been reported to occur on apple fruit (Baker et al 1977, Turner B. Sutton pers comm). Mycelial types were classified as blotch and speck based respectively on the presence or absence of dark mycelial mats.
Three speck types were noted on apple fruit. Flyspeck (FS) had dark, shiny, round to oval, flattened sclerotium-like bodies ranging from 109–600 µm diam at a density of 0.5–2 structures/mm2 (FIG. 1). Compact speck (CS) also had shiny, black, flattened sclerotium-like bodies, but these were round to irregular (35–418 µm diam) and densely arranged (5–22/mm2) (FIG. 2). In contrast, discrete speck (DS) appeared as groupings (5–20 sclerotium-like bodies/mm2) of tiny spheres (35–170 µm diam), with numerous setae that were visible only with magnification (FIG. 3).
Sooty blotch mycelial mats with sclerotium-like bodies varied in color, density and margin type. Ramose (RS) colonies had sclerotium-like bodies (45–278 µm diam) that appeared larger, shinier and more convex at the center of the colony than at the margin (FIGS. 4, 5) and were arranged at a density of 3–10/mm2. Sclerotium-like bodies of ramose mycelial types were composed of interlocking cells. In contrast sclerotium-like bodies of punctate (P) mycelial types (FIGS. 6, 7) were dull brown, comprising several overlapping hyphal strands and appeared similar throughout the mycelial mat. Sclerotium-like bodies of punctate types ranged from 18 to 216 µm diam and were arranged at a density of 12–33/mm2.
Color of sooty blotch mycelial mats with no sclerotium-like bodies ranged from pale olive to black. Ridged-honeycomb (RH) mycelial types were characterized by clumps and ridges of mycelia (FIG. 8). These mycelial clumps occasionally resembled sclerotium-like bodies, but were not well organized when observed through a compound microscope. All fuliginous mycelial types exhibited uniform mats of mycelia, but edges of the colonies varied from abrupt to feathered (FIG. 9).
Phylogenetic placement based on LSU sequences.— –
Maximum parsimony (MP) analysis of the LSU sequences resulted in 766 equally most parsimonious trees. A MP tree (FIG. 10) with a topology similar to that of the neighbor joining (NJ) tree (FIG. 11) was selected to determine phylogenetic placement. Results of parsimony and distance analyses indicated that all Midwest species were Dothideomycetes sensu Barr (1987). Both MP and NJ trees placed sterile mycelium sp. RS3 within the Pleosporales (FIGS. 10, 11). Peltaster sp. CS1 and Ramularia sp. CS2 did not clearly fall within the Pleosporales, Dothideales or Chaetothyriales. The NJ tree grouped Peltaster sp. CS1 and Ramularia sp. CS2 together, but the MP tree did not group these fungi with bootstrap support (FIGS. 10, 11). This grouping might have been based on long-branch attraction (Nei and Kumar 2000). The remainder of the Midwestern putative species, as well as the four species from North Carolina (Schizothyrium pomi, Peltaster fructicola and two species of Stomiopeltis) were grouped with the Dothideales, which had bootstrap support of 81%. The LSU analyses clustered most of the SBFS anamorph genera (Peltaster, Colletogloeum, Pseudocercosporella, Dissoconium, Passalora and Xenostigmina) with 10 Mycosphaerella species, and this branch had a bootstrap support of 96% (FIGS. 10, 11). The teleomorph Schizothyrium pomi and the three putative species of the anamorph genus Zygophiala, the teleomorph Piedraia hortae, and the three putative species of the anamorph genus Pseudocercospora also grouped with these Mycosphaerella species. Within the Dothideales Peltaster fructicola and Peltaster spp. P2.1 and P2.2 formed a strongly supported clade, with bootstrap support of 100% (FIGS. 10, 11).
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) and morphology (TABLE III). Zygophiala spp. were isolated from each of the nine orchards surveyed. The 23 Midwestern isolates of Zygophiala sp. FS1 had LSU and rDNA sequences similar to those of Z. jamaicensis (teleomorph Schizothyrium pomi) (FIGS. 10–12) from North Carolina.
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), but colony morphology was not associated with the minor differences in ITS sequences (FIG. 12). Colonies of all seven isolates of Zygophiala sp. FS2 isolates were green and white and produced a deep red soluble pigment on PDA (TABLE III). Cultures of Zygophiala spp. FS3.1 and FS3.2 were flat and zonate, whereas those of Zygophiala spp. FS1 and FS2 were thick, lumpy and flocculate (TABLE III). Conidia of Zygophiala spp. FS3.1 and FS3.2 were smaller than those of Zygophiala spp. FS1 and FS2 (TABLE III). Growth rate of Zygophiala sp. FS3.1 was faster than Zygophiala sp. FS3.2 (TABLE III).
On apple sclerotium-like bodies of Zygophiala jamaciensis (sp. FS1) were round, 290(155–409) µm diam at a density of 1.5/mm2 (FIG. 1). Sclerotium-like bodies of Zygophiala sp. FS2 also were round, but smaller (mean diam 195[109–364] µm) and at a density of 2.0/mm2. Zygophiala spp. FS3.1 and FS3.2 sclerotium-like bodies were ovoid, larger (380[300–450] µm x 500[425–600] µm) and were more sparsely arranged at a density of 0.5/mm2.
Pseudocercospora..
Three SBFS species were identified as belonging to Pseudocercospora on the basis of their morphology, but ITS and LSU sequences differed from those deposited for any Pseudocercospora species. Analyses of the LSU clustered Pseudocercospora sp. FG1.1, FG1.2 and FS4, and this clade was near Piedraia hortae (FIGS 10, 11). An anamorph of Piedraia hortae has not been reported. The ITS sequences of SBFS species FG1.1 and FS4 were similar (98% base-pair homology, 465/475 identity), and their closest match in a BLAST search was with the sequence from Trimmatostroma abietis (219 of 231 bases for Pseudocercospora sp. FS4 and 218 of 231 bases for Pseudocercospora sp. FG1.1). The ITS region of the single available isolate of Pseudocercospora sp. FG1.2 did not amplify after two attempts.
Scolecospores differed among these three putative Pseudocercospora species (TABLE III), as did their appearance on apples and colony morphology on PDA and MEA. Pseudocercospora sp. FG1.1 and Pseudocercospora sp. FG1.2 had fuliginous mycelial types with no fruiting bodies on apple, whereas Pseudocercospora sp. FS4 had no mycelial mat and dark shields similar in size to flyspeck signs caused by Zygophiala spp. The soluble orange and brown pigments formed respectively on PDA by Pseudocercospora spp. FS4 and FG1.2 were not observed for Pseudocercospora sp. FG1.1 (TABLE III).
Pseudocercosporella..
Three species with ridged-honeycomb mycelial types (FIG. 8) on apple had Pseudocercosporella anamorphs. These species were found in eight of nine orchards surveyed. Isolates produced two-celled primary conidia with numerous secondary scolecospores in a slimy mass on PDA and MEA. Primary conidia varied in size and shape among the three putative species (TABLE III). They were formed on short conidiophores along rows of parallel hyphae; the primary conidia did not form secondary conidia until they separated from the conidiophore. The three species of Pseudocercosporella differed in colony morphology on MEA and PDA (TABLE III).
The 29 ITS sequences of Pseudocercosporella sp. RH1 grouped together with 87% bootstrap support (FIG. 13); sequence differences, of one to five base pairs, were not associated with the highly variable mycelial characters observed among the isolates. The four ITS sequences of Pseudocercosporella sp. RH2.1 grouped separately from other species of Pseudocercosporella with bootstrap support of 94% (FIG. 13). Although the ITS sequences placed in Pseudocercosporella sp. RH2.2 lacked support for a taxon, the RH2.2 isolates had similar conidial and colony morphology (TABLE III).
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). These three Colletogloeum spp. on apple fruit formed a dense, fuliginous mycelial mat with no sclerotium-like bodies (FG) (FIG. 9). All three putative species had thick-walled, ovoid to allantoid blastospores that were highly vacuolate, subhyaline, and truncate at the base. The NJ tree grouped the LSU sequences of the three Colletogloeum spp. with Mycosphaerella marksii with bootstrap support of 84% (FIG. 11), but the branch was not supported by parsimony analysis (FIG. 10). The MP tree of the ITS sequence analysis delimited Colletogloeum sp. FG2.3 with bootstrap support of 100% and separated Colletogloeum spp. FG2.1 and FG2.2 with only moderate bootstrap support of 61 % (FIG. 14). Mycelial color of Colletogloeum spp. FG2.1 and FG2.2 differed from that of Colletogloeum sp. FG2.3, and the growth rate of Colletogloeum sp. FG2.1 on MEA was less than that of Colletogloeum spp. FG2.2 and FG2.3 (TABLE III).
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Eight isolates of Ramularia sp. CS2 produced smooth pink colonies on PDA with black reverse. The compact speck on apple had sclerotium-like bodies 183(81–418) µm diam at a density of 6.8 per mm2 (FIG. 2) but had no visible mycelial mat. Conidiophores were distinctive; the tip of the condiophore had a dark ring near its apex and produced catenate conidia attached with a narrow, curved bridge (TABLE III).
Dissoconium..
Five putative species of SBFS were identified as members of the genus Dissoconium. LSU sequences of these species grouped with the teleomorph Mycosphaerella lateralis (anamorph Dissoconium aciculare) (FIGS. 10, 11). ITS sequences of Dissoconium spp. DS1.1 and DS1.2 grouped with D. aciculare with bootstrap support of 85% (FIG. 15). Although we were unable to amplify the ITS region for Dissoconium sp. DS2, the LSU sequences grouped Dissoconium sp. DS2 with Dissoconium spp. DS1.1, DS1.2 FG4, and FG5 with bootstrap support of 91 and 97% (FIGS. 10, 11). Thus the LSU analyses imply that these species are within the same genus, and the ITS sequences and morphology indicate that they are distinct species.
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). Macroconidia were hyaline, smooth, two-celled, fusiform, and with the septum at or slightly above the middle, whereas microconidia were single-celled and hyaline. Macro-conidial and microconidial size and shape differed among Dissoconium spp. DS1.1, DS1.2, DS2 and FG4 (TABLE III). In contrast Dissoconium sp. FG5 had a single conidial type. Conidia were one- or two-celled, ovoid, were produced singly in whorls along a needle-like, dark brown conidiophore and were not forcibly discharged.
On apple fruit Dissoconium spp. DS1.1, DS1.2 and DS2 appeared as the discrete speck mycelial type with spherical to globose sclerotium-like bodies (FIG. 3). On occasion, sclerotium-like bodies developed into spermogonia that discharged spermatia (5 x 3 µm) through an ostiole. In contrast Dissoconium spp. FG4 and FG5 appeared as the fuliginous mycelial type on apple (FIG. 9), having dense olivaceous mycelial mats with no sclerotium-like bodies.
Xenostigmina..
Two species of SBFS, Xenostigmina sp. P3 and P4, had a similar punctate appearance on apple, with dark olivaceous, sparse, thick, ropy mycelia with feathery margins and abundant sclerotium-like bodies (FIG. 6). Despite numerous attempts to isolate from this relatively common mycelial type, only two isolates of Xenostigmina sp. P3 and one isolate of Xenostigmina sp. P4 were recovered from two orchards in Illinois. These isolates rarely produced slow-growing, carbonaceous colonies with dark brown, smooth, thick-walled conidia that varied in size and shape (TABLE III). The ITS sequences of Xenostigmina spp. P3 and P4 were similar (98% base-pair homology, 474/481 identity). The closest matches from BLAST searches were to sequences of Mycosphaerella latebrosa (243/259 identity for Xenostigmina sp. P3 and 223/233 identity for Xenostigmina sp. P4). The NJ tree grouped Xenostigmina spp. P3 and P4 with two Stomiopeltis species from North Carolina, three species with sterile mycelium (RS1, RS2.1 and RS2.2), and Passalora sp. FG3, with high bootstrap support of 91% (FIG. 11). The MP analysis had the branch appearing in the strict consensus tree, but there was no bootstrap support.
Mycelia sterilia..
Fourteen isolates from three orchards produced sterile mycelia. LSU analysis (FIGS. 10, 11) grouped three isolates of Mycelia sterilia sp. RS3 with Shiraia bambusicola in the Pleosporales. LSU analyses grouped Mycelia sterilia spp. RS1 and RS2 with Stomiopeltis sp. obtained from North Carolina. ITS sequences separated Mycelia sterilia spp. RS1 and RS2 from 11 Stomiopeltis sp. Isolates obtained from North Carolina and 20 ITS sequences of Stomiopeltis spp. downloaded from GenBank (FIG. 16). Although species having sterile mycelium produced ramose mycelial types on fruit, with dark mycelial mats and shiny, black, convex sclerotium-like bodies (FIGS. 4, 5), isolates with similar ITS sequences had distinctive colony characters on PDA (TABLE III).
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). Passalora sp. FG3 produced a fuliginous mycelial type on apple fruit (FIG. 9). Both MP and NJ trees of LSU sequences grouped Passalora sp. FG3 with Mycelia sterilia spp. RS1 and RS2 and Stomiopeltis spp. with bootstrap support of 80 and 90%, respectively (FIGS. 10, 11). The ITS region of Passalora sp. FG3 was not amplified after two attempts.
Peltaster..
Anamorph morphology of four putative species was similar to Peltaster fructicola, having indistinct conidiophores, with single-celled ovoid conidia successively produced through a hyphal pore. Isolates with similar ITS sequences (FIG. 17) also were grouped according to colony morphology and growth rate (TABLE II). Isolates of P. fructicola (=Peltaster sp. P1) were among the most commonly found, although none were found in Iowa orchards. Colonies were velvety green on PDA. One isolate (MSTE4a) grew more slowly than other P. fructicola isolates (2.9 and 7.5 mm respectively). Peltaster spp. P2.1 and P2.2 formed zonate black and green colonies on PDA and grew more slowly than P. fructicola. The ITS sequences of Peltaster spp. P2.1 and P2.2 were 18 base pairs longer than P. fructicola and other sequences of SBFS isolates in the Dothideales.
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). On apples, the compact speck mycelial type of Peltaster sp. CS1 also differed from the punctate mycelial type of Peltaster anamorphs (FIGS. 2, 7). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Ocamb-Basu and Sutton 1998), were isolated from several Midwestern orchards, but the other 28 putative species were not recognized previously as being involved in the SBFS disease complex. Leptodontium elatius and Geastrumia polystigmina, which were commonly found in North Carolina (Williamson and Sutton 2000), were not recovered from Midwestern orchards. Although spores were not seen in Mycelia sterilia spp. RS1 and RS2, these two midwestern putative species might be related to Stomiopeltis species found in North Carolina.
All SBFS fungi were Dothideomycetes sensu Barr (1987) based on LSU analyses. However unambiguous placement of the SBFS fungi into the Dothideales could not be made because LSU sequences of Capnodium citri (Capnodiales) and Myriangium duriaei (Myrangiales) also clustered in the Dothideales. Extensive morphological homoplasy has led to confusion about the classification of the Dothideomycetes (Reynolds 1991), and orders within the Dothideomycetes have not been shown to be monophyletic in other studies (Erikkson 2003, Lumbsch and Lindemuth 2001). Eriksson and Winka (1998) performed phylogenetic analyses using 18 S rDNA sequences of Dothideomycetes and accepted the orders Dothideales, Patellariales, Pleosporales, Myriangiales and Capnodiales, and they elevated the Chaetothyriales to class status, but they did not classify 51 families, including Dothideaceae. Lumbsh and Lindemuth (2001) further investigated LSU and SSU sequences of isolates in the Pleosporales, Myriangiales, Melanommatales and Capnodiales, but they did not analyze the Dothideales.
The topology of the MP trees of partial sequences of the LSU was validated with the single NJ distance tree. However, when all 228 ITS sequences were aligned, parsimony analysis resulted in several thousands of trees with topologies conflicting with those of the LSU. Therefore subtrees of closely related species, based on LSU analyses, were constructed to delineate species. In turn the increased taxon sampling for the ITS subtrees let us discover evolutionary changes among these species that were not apparent when using a smaller sampling of taxa for the LSU trees. These newly discovered putative species were supported by unique culture characteristics and morphology. Although detailed descriptions were beyond the scope of this study, efforts are being made to name these putative species. As additional species are discovered (studies in China and Europe are under way), studies based on DNA and cultural characteristics and morphology will be required. Sequences for the elongation factor 1-
and the ß-tubulin gene regions, for example, have been used to compare a large number of Mycosphaerella species from Eucalyptus (Crous et al 2004) and might be a valuable part of future studies.
Peltaster fructicola P1, P2.1 and P2.2 had holoblastic conidia like those of Aureobasidium spp. (Dothideales, Dothioraceae) (de Hoog and McGinnis 1987). Other SBFS fungi appeared related to Mycosphaerella based on LSU and ITS sequences analysis (Stewart et al 1999, Crous et al 2001a). Crous et al (2001b) linked 23 anamorph genera to Mycosphaerella. We found seven anamorph genera in the SBFS complex that appeared to be related to Mycosphaerella (Braun 1998, Crous et al 2001a). Morphological features of anamorphs in vivo used by Stewart (1999) and Crous and Wingfield (1997) for the identification of leaf blotch complex of Eucalyptus spp. in Australia and South Africa were useful for identification of the many anamorphs of the SBFS complex. However colony diameter on MEA at 4 wk and color, texture and pigment production on PDA also were useful features for delimiting phenotypes of putative species.
Some of the newly identified putative species of SBFS fungi have characters similar to those ascribed by early researchers to Gloeodes pomigena. Descriptions and line drawings of G. pomigena (Colby 1920) show conidia on apple fruit similar to those of Colletogloeum spp. FG2.1, FG2.2 and FG2.3. Another description of G. pomigena made by Baines and Gardner (1932) depicted colonies grown on PDA as similar to those of Peltaster sp. CS1, which had distinctive pink, yeast-like production on PDA. In contrast Groves (1933) described G. pomigena as having abundant masses of constricted, septate, cigar-shaped, budding spores of variable size, similar to what we observed for Pseudocercosporella spp. RH1 and RH2.1. Groves also noted that G. pomigena was associated most frequently with the ridged-honeycomb mycelial type on apple, as we found for putative species of Pseudocercosporella. Hickey (1960) described a wide range of G. pomigena spore types and colony morphologies in culture and on fruit. Three members of the SBFS complex in North Carolina were delineated by Johnson and Sutton (1994) and Johnson et al (1996, 1997), but they did not report finding G. pomigena.
This study showed that several genetically distinct fungi in the SBFS complex may give rise to the same mycelial type on apple fruit. Therefore mycelial type alone is not a reliable character to identify a SBFS fungus to species. Mycelial type on apple was consistent within a putative species, however, and still should be considered a useful tool when observing SBFS in the field. Because results from our survey suggest that there is more than one type of speck (fruiting bodies with no mycelial mat), we described the additional mycelial types "compact speck" and "discrete speck" that differ from flyspeck. Although we occasionally recovered more than one putative species from a single colony on apple fruit, it subsequently was found that such colonies consisted of more than one mycelial type. This occurred four times in our study; in each case mixed colonies were observed when the preserved apple peel was reexamined. We frequently have observed several mycelial types growing on top of one another.
Although pseudothecium-like bodies were observed on many of the colonies, none of these structures were observed to produce asci, even when infected apples were allowed to overwinter outdoors. Mature ascospores have been observed in North Carolina for Stomiopeltis spp. and Schizothyrium pomi (T.B. Sutton pers comm). It is possible that the longer growing season in the southern U.S. is more suitable for ascocarp maturation on the fruit. Fungi in the SBFS complex are likely to have many suitable hosts among the wild plants surrounding orchards (Hickey 1960, Johnson and Sutton 1994, Williamson and Sutton 2000), and infestation of apple fruit might not be a primary means of survival for these fungi (Baines and Gardner 1932, Williamson and Sutton 2000).
It is reasonable to believe that there are undiscovered members of the SBFS complex in the Midwest because only nine orchards were sampled in a single year. Annual surveys of mycelial types from apples grown throughout the Midwest have indicated that prevalence and incidence of mycelial types in the SBFS complex vary according to location and year (Batzer unpubl). Because these fungi probably reside on plant species surrounding apple orchards (Groves 1933, Hickey 1960, Johnson et al 1997) the complex may vary from orchard to orchard depending on the local flora.
Environment might play a substantial role in the morphology of the fungi on apple fruit. In a preliminary experiment, for example, isolates from Dissoconium spp. DS1.1, DS1.2 and DS2, and Pseudo-cercosporella spp. RH1, RH2.1 and RH2.2 were inoculated on immature apple fruit and incubated in crispers in growth chambers at 25 C. After 4 wk, these isolates produced a light-colored, smooth-textured, mycelial mat covering the entire fruit surface, rather than the discrete speck or ridged honeycomb mycelial types found on orchard-grown apples. Because of this morphological variability on inoculated apple fruit, all Koch’s postulates evaluations were conducted on intact apple fruit grown in an orchard. These assays resulted in mycelial types similar to those observed on the original apple cuticle.
Strategies for management of SBFS might need to be reassessed according to the traits of the members of the SBFS complex predominating in particular orchards and geographic regions. Members of the SBFS complex may differ in ecology and thus require different management practices. Various SBFS mycelial types were removed from apples with different efficiency by postharvest dip treatments (Batzer et al 2002b). In a comparison of six SBFS putative species cultured on water agar amended with the fungicides thiophanate-methyl and ziram, fungicide sensitivity varied widely among putative species (Tarnowski et al 2003). Furthermore growth rates differed among putative species in response to nutrient composition of media (van deVoort et al 2003). Optimal temperatures for growth on water agar of six putative species of SBFS also varied significantly (Hernandez et al 2004). Understanding the ecology of local and regional SBFS complexes might lead to strategies that reduce the economic and environmental impact of SBFS management.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: mgleason{at}iastate.edu
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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