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Re-examination of Peltaster fructicola, a member of the apple sooty blotch complex

     Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695-7616

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 

Peltaster fructicola is one of several fungi that causes sooty blotch on apple. Johnson et al (1996, 1997) correctly described P. fructicola but illustrated two different fungi. One is P. fructicola and the other is an unidentified ascomycete. In this paper, P. fructicola is more completely described and accurately illustrated.

Key words: fruit diseases, plant pathology

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Sooty blotch is one of the most common diseases of apple (Malus x domestica Borkh.) in moist, temperate regions of the world (Hickey 1960, Sutton 1990). In the United States, sooty blotch is caused by several fungi including Leptodontium elatius (Mangenot) de Hoog, Geastrumia polystigmatis Batista & M.L. Farr and Peltaster fructicola Johnson et al (Johnson and Sutton 1994, Johnson et al 1996, 1997). Additional fungi recently have been associated with the disease in Iowa in the United States (Batzer et al 2002a, b) and in Germany and Poland (Simone Kern and Beata Wrona, pers comm).

Colby (1920) and Groves (1933) described colony types of what was considered the sole causal agent of sooty blotch on apple, Gloeodes pomigena (Schwein.) Colby. Groves (1933) categorized colonies into four groups: punctate, ramose, fuliginous and rimate, based on mycelial patterns and characteristics of fruiting bodies. Johnson et al (1996, 1997) found that these groups represent different fungal species rather than variation within G. pomigena. For example, Johnson et al (1996, 1997) associated L. elatius with fuliginous colonies and G. polystigmatis and P. fructicola with ramose colonies.

Studies in our laboratory indicate that Johnson et al (1996, 1997) correctly described P. fructicola, but the published pictures of P. fructicola on apple fruit included P. fructicola and an unidentified ascomycete. In addition, the authors incorrectly described colonies of P. fructicola as a ramose colony type instead of the punctate colony type. The purpose of this paper is to describe more completely P. fructicola and correctly illustrate P. fructicola on apple fruit.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Terminology. – We use the terms ramose and punctate to describe the two fungal colonies as Johnson et al (1996, 1997) did to relate this study to theirs. Herbarium abbreviations are taken from Holmgren et al (1990). Specimens and isolates of P. fructicola and the unidentified ascomycete collected during this study are maintained in the collection of Dr. Turner Sutton, North Carolina State University (NCSU).

Freshly picked apples. – Golden Delicious apples were obtained from North Carolina, Alabama, Michigan, New York, Ohio, Pennsylvania, Virginia and Wisconsin from Aug to Nov 1996. Fruit were examined immediately or stored at 4 C until observation. Punctate and ramose colonies similar to those shown in FIG. 1 (FIG. 1 from Johnson et al 1996) and in Johnson et al (1997) were examined. Dissecting and compound microscopes were used to aid in examination of colonies. A sterile dissecting needle was used to transfer mycelium and fruiting bodies from punctate and ramose colonies to acid water agar (AWA, 2% agar). Cultures were grown in the dark at 24 C in environmental chambers (Percival, Boone, Iowa). After about 1 wk, isolates were transferred to AWA, incubated 1–2 wk, then transferred to potato-dextrose agar (PDA, Difco Laboratories, Detroit, Michigan) and incubated about 2 wk.

View larger version (136K): [in this window] [in a new window]   FIG. 1. Ramose colony in the center with punctate colony upper right from Johnson et al (1996). Bar = 2 mm.

Overwintered apples. – Golden Delicious apples with punctate and ramose colonies were collected from North Carolina during late summer and fall 1996 and stored at 4 C before being placed in Saran cloth cages in the shade in Raleigh on 7 and 8 Jan 1997. Colonies were examined in April and May for the presence of spores. Fruit were taken to the laboratory on 3 Jun 1997, and punctate and ramose colonies were excised, pressed and dried between paper towels. Single-spore isolations from punctate colonies were made using the method previously described. Single-spore isolations were not made from ramose colonies because of the scarcity of spores.

Morphological characterization in vivo. – Squash mounts were made by placing fruiting bodies from punctate and ramose colonies into a drop of cotton blue in lactophenol. Whole mounts were prepared by excising the section of apple peel with the colony and mounting it in lactophenol. For histological examination, sections of fruit or peel containing the colony were fixed in formalin-2-propanol-pro-pionic acid (FPP), dehydrated in an isopropyl alcohol series and embedded in Paraplast-Plus (Monoject Scientific, St. Louis, Missouri) (Daykin and Hussey 1985). Sections were cut on a rotary microtome at 12 µm, mounted on microscope slides with Haupt’s adhesive and cleared with xylene. Permount was used to seal the slides.

Conidia were measured from punctate colonies collected during the summer and fall, and from overwintered apples from North Carolina, and from punctate colonies collected from Alabama, Pennsylvania, Michigan, Virginia and Wisconsin during the fall. Diameters of mature pycnothyria were measured from whole mounts of punctate colonies collected from North Carolina during the summer, and from Pennsylvania and Virginia during the fall. Pycnothyria were determined to be mature if conidia were present, or if the pycnothyrium had dehisced revealing an empty fruiting body or conidia inside. Pycnothyria from punctate colonies obtained from overwintered apples from North Carolina were also measured. Pycnothyria on whole mounts of overwintered material were difficult to measure, so they were removed using a dissecting needle, mounted in lactophenol, measured, then crushed to confirm the presence of conidia. In the process of removing pycnothyria, especially in the case of larger pycnothyria, it was possible that 5–10 µm of the periphery of the structure was left behind and not included in the measurements. Diameters of pycnothyria were determined by averaging two measurements taken at 90° angles. Measurements of conidia and pycnothyria were subjected to an analysis of variance for a hierarchical sampling design, accounting for seasons, colonies within seasons, and individuals (pycnothyria or conidia) within colonies (SAS Institute, Cary, North Carolina). Measurements are presented as minimum, mean, standard deviation and maximum.

Morphological characterization in vitro. – Isolates of P. fructicola were grown on PDA (TABLE I). A plug of each isolate was transferred from the edge of actively growing cultures to the center of two dishes each of PDA, 2% WA poured in a thick layer, and 2% WA poured in a thin layer in the Petri dish, and incubated in the dark at 25 C. PDA and WA cultures were examined and the diameter of each colony was measured after 21 d. Diameters were determined by averaging two measurements taken at 90° angles. Isolates grown on thin WA were incubated 14 d. Sections of one colony per isolate were removed from 0.5 to 2 mm behind the margin of the colony and mounted in lactophenol. Conidia were measured at 1000x, using phase contrast microscopy. Measurements are presented as minimum, mean and maximum.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
As a result of this study, we have determined that two different fungi are present in FIG. 1, which is reproduced from Johnson et al (1996). The colony in the center of their figure, which they identified as P. fructicola and described as ramose, is an unidentified ascomycete observed on apple fruit in North Carolina from midsummer through fall. Colonies of this fungus were olivaceous to black and consisted of a network of hyphae and numerous thyriothecia. Ascal initials and immature asci were observed in late summer and fall. Immature and empty asci and a few ascospores were observed in overwintered colonies, however, in insufficient numbers to identify the fungus. Sixty-one mycelial isolates obtained from these colonies collected from freshly picked apples remained sterile. Isolates were not obtained from overwintered apples.

The second fungus pictured by Johnson et al (1996) is P. fructicola (FIG. 1, upper center-right). One hundred twenty-one mycelial isolates of P. fructicola were obtained from punctate colonies from freshly picked apples. Six isolates of P. fructicola (listed in TABLE I) were derived from single conidia from these punctate colonies. All isolates produced unicellular, hyaline, ellipsoidal to ovoidal conidia in clusters along the hyphae on WA typical of P. fructicola. Nothing grew from isolations made from overwintered punctate colonies although conidia were abundant. Following is a detailed description of P. fructicola.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Peltaster fructicola Johnson, Sutton et Hodges, Mycologia 88:120. 1996.

Colonies olivaceous to black, on surface of apple fruit, circular or irregular in shape (FIGS. 2–4). Individual colonies 1–6 mm diam, multiple colonies and secondary spread covering up to 40% of the fruit surface. Hyphae dark, superficial, abundant, tightly adherent to the apple cuticle, septate, nonhyphopodiate, radiating outward from the center of colony, profusely branched, and anastomosing giving a more or less reticulate appearance. Hyphae 1.3–4.5 µm wide, primary hyphae 2.1–4.5 µm wide. Sometimes two or more main branches of adjacent hyphae parallel to each other. Hyphal branching at the colony margin arborescent. Pycnothyria randomly scattered or arranged in concentric rings on the colony, superficial, single but sometimes fused, dimidiate, brown, and circular, (19–)104.5 ± 58(–282) µm diam (FIG. 5). Shield of the pycnothyrium composed of irregularly shaped, nonradiate, isodiametrical cells. Upper portion of the pycnothyrium composed of up to several layers of parenchyma, the outermost being the darkest. No hypostroma or basal membrane. No conidiophores. Sessile conidia borne on an inverted hymenium and released through cracks in the surface of the shield (FIGS. 6–8). Ontogeny of conidia could not be determined conclusively. Conidia unicellular, hyaline, and ellipsoidal to ovoidal, (2.9–)4.2 ± 0.7(–7.1) x (0.8–)1.4 ± 0.3(–2.4) µm (FIG. 8).

View larger version (57K): [in this window] [in a new window]   FIGS. 2–4. Variation in P. fructicola colonies on apple. 2. A colony of another fungus adjacent to P. fructicola (center) affects the shape of the colony. Bar = 1 mm. 3. Two or more punctate colonies coalescing. Bar = 1 mm. 4. Secondary spread of P. fructicola (arrow).

View larger version (136K): [in this window] [in a new window]   FIGS. 5–8. 5. Pycnothyria of P. fructicola on apple fruit. The smaller pycnothyria at the top of the picture are mature as indicated by the presence of conidia and cracks in the shield. Bar = 200 µm. 6. Section through a pycnothyrium showing conidia borne on an inverted hymenium which lines the inside of the top of the pycnothyrium. Bar = 15 µm. 7. Pycnothyrium that has dehisced exposing the conidia inside. Bar = 15 µm. 8. Conidia released from a crushed pycnothyrium from apple. Bar = 15 µm.

Colonies on WA (8–)14(–22) mm diam after 21 d, spreading with an indeterminate margin, white to pale brown. Conidia produced in basipetal order from a single fertile locus that developed as a short stub at the midpoint of intercalary cells of undifferentiated hyphae. Clusters of conidia observed at each locus. Conidia unicellular, hyaline, ellipsoidal to ovoidal, 3.2–7.1 x 1.1–2.4 µm.

Habitat and distribution. – On peel of apple from North Carolina, Alabama, Michigan, New York, Ohio, Pennsylvania, Virginia, Illinois, Wisconsin, Kentucky, Missouri, Massachusetts. Summer, fall.

Specimens examined. – Thirty-nine specimens were examined. The following are representative. UNITED STATES. NORTH CAROLINA: Johnston County, Clayton, whole mounts of peel of Malus x domestica, Jul 1993, E.M. Johnson (HOLOTY PE. IMI 358519); squash slides of pycnothyria from Malus x domestica, Jul 1993, E.M. Johnson (IMI 358520); on dried peels of Malus x domestica, 3 Aug 1995, T.B. Sutton (BPI 803019); on dried peels of Malus x domestica, 3 Aug 1995, T.B. Sutton (Mycological Herbarium, North Carolina State University, Raleigh, North Carolina); on peel of Malus x domestica, 13 Aug 1996, S.M. Williamson 1P (NCSU); S.M. Williamson 3P (NCSU); on peel of Malus x domestica, 28 Aug 1996, S.M. Williamson 3P (NCSU); on peel of overwintered Malus x domestica, 3 Jun 1997, S.M. Williamson 3 (NCSU); S.M. Williamson 5 (NCSU); S.M. Williamson 9 (NCSU); on peel of Malus x domestica, Fall 1997, S.M. Williamson 100 (NCSU); S.M. Williamson 112 (NCSU); S.M. Williamson 115 (NCSU); Windsor, on dried peels of Malus x domestica, 1 Aug 1995, T.B. Sutton (PARATY PE. BPI 803018); Mountain Horticultural Crops Research Station, Fletcher, Henderson County, on peel of Malus x domestica, 11 Sep 1996, S.M. Williamson 3P (NCSU); on peel of Malus x domestica, Fall 1997, S.M. Williamson 112 (NCSU); S.M. Williamson 113 (NCSU); Henderson County, on peel of Malus x domestica, 25 Sep 1996, S.M. Williamson 3P (NCSU); Bertie County, on peel of Malus x domestica, 28 Aug 1996, S.M. Williamson 6P (NCSU); ALABAMA: on peel of Malus x domestica, 25 Oct 1996, S.M. Williamson 9P (NCSU); PENNSYLVANIA: on peel of Malus x domestica, 4 Oct 1996, S.M. Williamson 1P (NCSU); S.M. Williamson 5P (NCSU); MICHIGAN: on peel of Malus x domestica, 25 Oct 1996, S.M. Williamson 1P (NCSU); VIRGINIA: on peel of Malus x domestica, 6 Nov 1996, S.M. Williamson 9P (NCSU); S.M. Williamson 10P (NCSU); WISCONSIN: on peel of Malus x domestica, 10 Oct 1996, S.M. Williamson 5P (NCSU); S.M. Williamson 6P (NCSU).

Commentary. – Colonies of P. fructicola on apple fruit were circular or irregular in shape due to an inability to grow on russetted areas, limitations by neighboring fungi, colonies coalescing, or secondary spread by conidia (FIGS. 2–4). Colonies were often observed "dripping" down the side of apple fruit as a result of secondary spread by conidia (FIG. 4).

Mature pycnothyria and conidia of P. fructicola were observed throughout the growing season; however, all pycnothyria did not mature by the end of the growing season. An individual colony had none to many mature pycnothyria at a given time. The size of pycnothyria ranged widely with small mature pycnothyria often interspersed among larger immature ones on colonies in the summer and fall (FIG. 5). There were more mature pycnothyria and conidia on overwintered colonies than on colonies collected in the summer. Mature pycnothyria from overwintered fruit were significantly larger, (33–)132.4 ± 53.4 (–282) µm, compared to those on colonies collected in the summer, (19–)58 ± 27.4(–124) µm (P = 0.0001). Smaller pycnothyria appeared to mature in the fall and the larger pycnothyria matured after overwintering.

Johnson et al (1996, 1997) described pycnothyria from colonies of P. fructicola on apples from North Carolina and other states and on brambles (Rubus sp.) from North Carolina collected throughout the year and on overwintered colonies. Those authors measured mature pycnothyria that appear to have included pycnothyria with conidia or cracks in the pycnothyrium, however this was not defined clearly. In this study, we defined mature pycnothyria as those in which conidia were present or the pycnothyrium had dehisced, revealing an empty fruiting body or conidia inside. Our measurements of mature pycnothyria from colonies of P. fructicola on apples collected in the fall were similar to those reported by Johnson et al (1996, 1997) [(81–)97.3 ± 11.3(–113) µm]. The larger size of mature pycnothyria from overwintered colonies on apples from North Carolina, however, increases the size range of pycnothyria of P. fructicola.

Conidia from colonies collected in North Carolina on apple fruit during the summer, fall, and after overwintering, and from other states, were similar in size. These conidia also were similar to the size and shape reported by Johnson et al (1996, 1997) [(3.6–)6.4 ± 2.6(–7.2) x (0.7–)0.8 ± 0.1(–1.1) µm].

Growth of P. fructicola on WA and PDA, and conidial production on WA was similar to that described by Johnson et al (1996) with a few exceptions. Those authors found that colonies on WA and PDA reached 15 and 10 mm diam after 21 d, respectively, but in our study some colonies reached 22 and 23 mm diam on WA and PDA, respectively, after 21 d. However, the average for all isolates was 14 mm diam on both media. Johnson et al (1996) observed that colonies of P. fructicola were dark brown to black on WA and that some isolates produced an extracellular pigment visible in the agar; however, we found that growth on WA was white to pale brown and that the pigment was visible only on PDA. In our studies, conidia on WA were similar in size and shape to conidia from fruit and also similar to the size and shape Johnson et al (1996) reported for conidia on WA.

IMI 358519 and 358520, labeled as Gloeodes sp., represent P. fructicola. BPI 803018, BPI 803019, and specimens from the North Carolina State University Mycological Herbarium, labeled as P. fructicola, were the as yet unidentified ascomycete discovered in this study. New specimens of P. fructicola from apple from North Carolina, including apple peels, whole mounts, and squash slides, were deposited as IMI 385257, IMI 385258, IMI 385362, BPI 747569, BPI 747568 and BPI 747832 and at the North Carolina State University Mycological Herbarium.

ATCC 76560 was labeled incorrectly as P. fructicola and has been de-accessioned. ATCC 96996 was confirmed as P. fructicola based on growth and conidial production on PDA and WA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
In 1920, Colby described Gloeodes pomigena, which he associated with the apple sooty blotch disease. Colby (1920) and Groves (1933) noted considerable variation in the morphology of colonies of G. pomigena on apple fruit. Groves (1933) established four groups: punctate, ramose, fuliginous and rimate, as an aid to differentiation. Johnson et al (1997) did not find G. pomigena in any of their sooty blotch collections but did identify several fungi associated with sooty blotch symptoms. Johnson et al (1996, 1997) associated L. elatius with the fuliginous colony type, and G. polystigmatis and P. fructicola with the ramose colony type. All apple sooty blotch fungi have not been identified, so G. pomigena cannot be excluded as a possible member of the sooty blotch complex.

Johnson et al (1996, 1997) correctly identified and described P. fructicola. However, incorrect pictures of the fungus on apple fruit were published. Some of the figures identified as P. fructicola are an unidentified ascomycete and not P. fructicola. FIGURE 1 in Johnson et al (1996) shows the unidentified ascomycete in the center and P. fructicola in the upper right corner; it is difficult to know whether FIG. 2 depicts pycnothyria or thyriothecia. The other figures in the paper correctly depict characteristics of P. fructicola. FIGURES 1 and 5 in Johnson et al (1997) also illustrate the unidentified ascomycete. FIGURES 2A and 6 correctly depict a punctate colony, and pycnothyria and conidia of P. fructicola, respectively.

Johnson et al (1996, 1997) described P. fructicola colonies as ramose because they exhibited a strong radial growth pattern and arborescent branching at the colony margin, but they should have been described as punctate. Groves (1933) described punctate colonies as having large, conspicuous plectenchymal bodies. According to Groves, variations in the punctate group included: size and abundance of the fruiting bodies, density of the thallus and conspicuousness and abundance of the mycelium between fruiting bodies. Some colonies in this group exhibited a distinct ramose (much-branched) character. Colonies of the unidentified ascomycete also can be described as punctate. Because of the cultural differences of isolates of P. fructicola and the ascomycete, as well as colony differences on apples, it is unlikely that the ascomycete is the teleomorph of P. fructicola.

Johnson et al (1996, 1997) also observed colonies on apples that were similar to those caused by P. fructicola but had smaller pycnothyria and conidia. They speculated that these colonies might represent another new species of Peltaster. We found that the size of pycnothyria and conidia from these colonies were within the natural variation found in P. fructicola. Consequently there is no evidence currently that a second species of Peltaster is involved in the sooty blotch complex.

	ACKNOWLEDGMENTS

 
We thank D. Chapman, A. Jones, D. Rosenberger, M. Ellis, K. Hickey, K. Yoder, P. McManus, J. Hartman, W. Schaffer and D. Cooley for supplying disease specimens for our studies. We also thank J. Harrison for collection of specimens, C. Brownie for assistance with statistical analysis and L. Grand for review of the manuscript.

	FOOTNOTES

 
Accepted for publication March 1, 2004.

¹ Corresponding author. E-mail: turnerpsutton{at}ncsu.edu

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS TAXONOMY DISCUSSION LITERATURE CITED

 
Batzer JC, Gleason ML, Harrington T, Chen W. 2002a. Expansion of the sooty blotch and flyspeck complex on apple using ribosomal DNA. Phytopathology 92(Supplement):S6–7.

———, ———, Tiffany LH. 2002b. Discrete speck, a putative newly discovered fungus in the sooty blotch and flyspeck complex on apples. Phytopathology 92(Supplement):S6.

Colby AS. 1920. Sooty blotch of pomaceous fruits. Trans Ill Acad Sci 13:139–175.

Daykin ME, Hussey RS. 1985. Staining and histopathological techniques in nematology. In: Barker KR, Carter CC, Sasser JN, eds. An advanced treatise on Meloidogyne. Vol. 2. Methodology. Raleigh, North Carolina: North Carolina State University Graphics. p 39–48.

Groves AB. 1933. A study of the sooty blotch disease of apples and the causal fungus Gloeodes pomigena. Va Agric Exp Stn Bull 50:1–43.

Hickey KD. 1960. The sooty blotch and fly speck diseases of apple with emphasis on variation within Gloeodes pomigena (Schw.) Colby [Doctoral Dissertation]. University Park: Pennsylvania State University.

Holmgren PK, Holmgren NH, Barnett LC. 1990. Index herbariorum: Part 1: the herbaria of the world. Reg Veg 120:1–693.

Johnson EM, Sutton TB. 1994. First report of Geastrumia polystigmatis on apple and common blackberry in North America. Plant Dis 78:1219.

———, ———, Hodges CS. 1996. Peltaster fructicola: a new species in the complex of fungi causing apple sooty blotch disease. Mycologia 88:114–120.

———, ———, ———. 1997. Etiology of apple sooty blotch disease in North Carolina. Phytopathology 87:88–95.

Sutton TB. 1990. Sooty blotch and flyspeck. In: Jones AL, Aldwinkle HS, eds. Compendium of apple and pear diseases. St. Paul, Minnesota: American Phytopathological Society. p 20–22.

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This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Williamson, S.M. Articles by Sutton, T.B. Search for Related Content PubMed Articles by Williamson, S.M. Articles by Sutton, T.B. Agricola Articles by Williamson, S.M. Articles by Sutton, T.B.