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Cercosporella acroptili and Cercosporella centaureicola sp. nov.—potential biological control agents of Russian knapweed and yellow starthistle, respectively

     USDA, ARS, Foreign Disease-Weed Science Research Unit, 1301 Ditto Avenue, Fort Detrick, Maryland 21702

U. Braun

     Martin-Luther-Universität, Institut für Geobotanik und Botanischer Garten, Herbarium, Neuwerk 21 D-06099 Halle, Germany

M.B. McMahon
D.G. Luster

     USDA, ARS, Foreign Disease-Weed Science Research Unit, 1301 Ditto Avenue, Fort Detrick, Maryland 21702

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 

Russian knapweed (Acroptilon repens [L.] DC.) and yellow starthistle (Centaurea solstitialis L.) are invasive weeds in the western United States, and both weeds are targeted for biological control. Cercosporella acroptili (Bremer) U. Braun was identified as a possible biological control agent for A. repens, and a morphologically similar Cercosporella sp. recently was found damaging to C. solstitialis in the field. Because both fungi are potentially important for biological control of the respective weeds, studies were undertaken to ascertain whether the isolates were identical based on morphology, pathogenicity, growth and spore production, and genetics (molecular characterization of the internal transcribed spacer regions of the ribosomal RNA genes). Differences in these variables between the two isolates were sufficient to indicate that the isolate from C. solstitialis was distinct and justified a new description at the species level: Cercosporella centaureicola sp. nov.

Key words: Acroptilon repens, Centaurea soltitialis, Ramularia acroptili

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 
Acroptilon repens (L.) DC. (Russian knapweed, ACREP, = Centaurea repens L.), and Centaurea solstitialis L. (yellow starthistle, YST), family Asteraceae, are invasive weeds in the western United States. Both weeds are targeted for biological control. In Sep 1997 R. Sobhian found epidemic disease on ACREP near Asparta, Turkey. The causal agent was identified tentatively as Ramularia acroptili Bremer, based on comparisons with specimens of R. acroptili from the USDA-ARS Mycology Laboratory in Beltsville, Maryland. After studying our specimens, U. Braun (Martin-Luther-University, Halle/Saale, Germany) and N. Ale-Agha, and G. B. Feige (University Duisburg-Essen, Germany) indicated that the fungus was Cercosporella acroptili (Bremer) U. Braun (Braun 1993). C. acroptili is known on Acroptilon repens from Kazakhstan, Kyrgystan, Turkey (Braun 1995a) and Germany (Jage and Braun 2004). In Jun 2001 D. Berner returned to this site (37°52'26''N, 30°41'41''E, 853 m elevation) and found a similar epidemic in a ca 0.5 ha field of an almost solid stand of ACREP plants. All lower leaves on all plants were diseased and covered in leaf spots bearing obvious fructifications of a fungus. The causal organism was isolated, identified as C. acroptili, and Koch’s postulates fulfilled at the quarantine facility of the Foreign Disease-Weed Science Research Unit (FDWSRU), USDA, ARS, Fort Detrick, Maryland. A voucher specimen has been deposited with the U.S. National Fungus Collections (BPI 745883). Live cultures are being maintained at FDWSRU and the Plant Protection Central Research Institute, Ankara, Turkey.

In spring 2004 an epidemic of dying YST plants was found near Kozani, Greece (40°22'07''N, 21°52'35''E, 634 m elevation). Rosettes of YST had small, brown leaf spots on most of the lower leaves. These spots frequently coalesced and resulted in necrosis of many of the leaves and death of the rosette. Along the roadside where the disease was found >100 of the YST plants showed disease symptoms. The causal organism was isolated and Koch’s postulates fulfilled at the quarantine facility of FDWSRU (Eskandari et al 2004). The organism initially was identified as a Cercosporella sp., based on fungal morphology, and later as Cercosporella sp. cf. acroptili by U. Braun. A voucher specimen was deposited in the U.S. National Fungus Collections (BPI 844247). Live cultures are maintained at FDWSRU and the European Biological Control Laboratory (EBCL), Greece.

Because both isolates of this fungus are potentially important for biological control of the respective weeds, studies were undertaken at FDWSRU and Martin Luther University, Institute of Geobotany and Botanical Garden, Herbarium, Halle, Germany, to ascertain whether the isolates were identical based on morphology, pathogenicity, growth and spore production, and sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA genes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 
Isolation and maintenance.— – Symptomatic leaves of ACREP, with and without surface disinfestation, were placed in moist chambers and incubated 2 d at 21 C. Growth of white conidiophores and conidia was observed, especially on the lower surface of the leaves in the necrotic areas. After treatment in the moist chambers, single spore cultures of this isolate (98-001) were started on water agar. Germinated spores were transferred to potato-dextrose agar (PDA), potato-carrot agar (PCA) and V8 juice agar after 48 h at 21 C.

Diseased YST leaves collected in Greece were surface disinfested and placed on moist filter paper in Petri dishes. After 48 h conidiophores and conidia, from which isolate 04-011 was derived, were observed. The fungus then was grown on modified potato-carrot agar (MPCA): 140 g cut up carrot (not peeled), 140 g cut up potato (unpeeled), 1 L water. Carrot and potato pieces were boiled 10 min and filtered. Water was adjusted to 1 L, 20 g agar were added and the medium was autoclaved. Conidia for plant inoculations were harvested from 2 wk old cultures.

Morphological examinations.— – Morphological examinations in vivo and in vitro were carried out with standard light microcopy (Olympus BX50, Hamburg, Germany), based on preparations stained with cotton blue. Phase contrast was used to examine the structure of the conidiogenous loci in detail. These collections have been examined: Cercosporella acroptili, on Acroptilon repens, Turkey, Ankara, 14 Jul 1947, Bremer, Reliquiae Petrakianae 363 (B, GZU, W 11177), type material of C. acroptili; Turkey, 20 km east of Asparta, 1 Sep 1997, R. Sobhian (BPI 745883); Germany, Sachsen-Anhalt, Eisleben, SO Rollsdorf, S Kerner Lake, 21 Sep 2001, H. Jage (HAL 1840). Cercosporella sp., on Centaurea solstitialis, Greece, Macedonia region, Kozani prefecture, Kozani, 28 Apr 2004, D. Berner (BPI 844247, HAL 1841).

Pathogenicity tests.— – Pathogenicity tests were performed in a quarantine greenhouse at FDWSRU by spray-inoculating the foliage of 4 wk old YST and ACREP rosettes with aqueous 1 x 10⁶/mL conidial suspensions from isolates 98-001 or 04-011. Twenty-five plants each of YST and ACREP were inoculated with isolate 04-011 and seven plants each were inoculated with isolate 98-001. Inoculated plants were placed in an environmental chamber at 23 C with 8 h of daily light and continuous dew for 48 h. Inoculated and control plants were moved to a greenhouse bench at 20–25 C with 12 h light daily and watered twice daily. Because air exchange in the quarantine greenhouse was regulated by constantly filtered air conditioning, the relative humidity in the greenhouse was always low and averaged 30–50%. After 12 d leaf spots were observed first on lower leaves. The number of leaves, leaves with spots and spots per leaf were recorded for each plant. These data were recorded again after 16 d for plants inoculated with isolate 04-011. Data on these variables were analyzed by SAS (Statistical Analysis System Software, Cary, North Carolina) as a completely randomized design with isolates as the independent variables. Least squares means and mean comparisons were generated from the SAS analysis of variance.

Fungal growth studies.— – A drop of conidial suspension (10⁶ conidia/mL) from each isolate was placed on the middle of a sterile Millipore^® membrane filter (cellulose nitrate with a pore size of 0.2 µm and 25 mm diam) placed at the center of a 60 x 15 mm plastic Petri dish containing MPCA. Four Petri dishes of each isolate were placed on individual metallic strips set at different temperatures on a temperature gradient. Thirteen temperature strips were used, and the temperatures of the culture media on the strips were: 7, 10, 13, 15, 17, 20, 22, 24, 26, 27, 28, 29, 30 C. Two Petri dishes of each isolate at each temperature were covered with aluminum foil so that they remained in the dark, and the other two Petri dishes of each isolate at each temperature received 24 h of light (two black lights, General Electric-BLB fluorescent near UV, 40 W; 1220 mm long). The dishes were incubated at each temperature and lighting regime for 10 d. After removal from the temperature gradient, inoculum sites (fungal growth) were weighed fresh, dried on a laboratory bench for 48 h at ambient temperature (ca 20 C) and reweighed. The dried fungal growth then was homogenized and suspended in 5 mL sterile distilled water. Spore concentrations in the suspensions were determined with a hemocytometer. These data were analyzed by SAS as an analysis of covariance with isolates as independent variables and temperature within isolates fitted as linear and quadratic covariates. Least squares means and mean comparisons were generated from the SAS analysis of covariance. Overall fit of the temperature response of each isolate to a polynomial (quadratic) regression was obtained by polynomial regression analyses in SAS.

To determine whether the isolates were vegetatively compatible, an MPCA agar plate was divided in two sections by drawing a line on the outside of a plate from top to bottom through the center. Three drops of 04-011 spore suspension were placed, one after another, on half of the plate along the drawn line and three drops of 98-011 were placed on the other half. Growth of the isolates was monitored weekly for 6 wk to determine whether the developing colonies grew together.

DNA sequence analyses.— – Genomic DNA was extracted from 7 d old mycelial cultures of isolates 98-001 and 04-011 with the DNEasy Plant Mini kit (QIAGEN, Valencia, California). Approximately 100 mg of mycelium was ground in liquid nitrogen by mortar and pestle for the extraction. The genomic DNA was quantified on a fluorometer with Pico Green as a fluorescent dye (Molecular Probes, Eugene, Oregon). DNA, at a concentration of 10 ng/100 uL was used to amplify the internal transcribed spacer (ITS) 1, the 5.8 ribosomal RNA gene and the ITS spacer 2. Polymerase chain reaction was carried out on the GeneAmp 9700 (Applied Biosystems, Foster City, California) at these parameters: denaturing at 94 C for 2 min; followed by 94 C for 30 s, annealing at 58 C for 30 s, extension at 72 C for 1 min for a total of 30 cycles; then a final extension of 72 C for 10 min. The primers used in the amplification were ITS5 and ITS4 (White et al 1990) at a final concentration of 1 µM. PCR products were sequenced directly with Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI 310 Genetic Analyzer (Applied Biosystems) according to manufacturers’ guidelines in 20 µL reactions containing 100 ng of PCR template. The ITS5 and ITS4 primers were used in the sequencing reaction at a concentration of 3.2 pM. The sequences generated from reactions with the ITS5/ITS4 primer set were aligned with the BLAST algorithm of the National Center for Biotechnology Information.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 
Morphological comparisons.— – Based on type material of Cercosporella acroptili, a recent collection of this species from Germany (Jage and Braun 2004) and isolate 98-001 on inoculated ACREP, in vivo this fungus can be described as: disease symptoms amphigenous, leaf spots circular to angular-irregular, occasionally oblong, 1–20 mm wide, margin indefinite or spots surrounded by a narrow, purple to rose-colored border. Caespituli amphigenous, punctiform to subeffuse, fine, grayish white. Mycelium internal. Stromata lacking or substomatal, 10–30(–40) µm diam, colorless, later at most pale yellowish. Conidiophores in small to moderately large fascicles, loosely to densely associated, arising from internal hyphae or stromata, emerging through stomata, erect, straight, subcylindrical to somewhat geniculate-sinuous, unbranched, 10–100 x (2–)2.5–5(–6) µm, 0–2-septate, hyaline, thin-walled, smooth; conidiogenous cells integrated, terminal, 10–40 µm long; conidiogenous loci cercosporella-like (umbonate, thickened, but not darkened, at most somewhat refractive, 1.5–2 µm diam, occasionally with a minute frill and central papilla). Conidia solitary or in secondary short chains, with shed conidia forming secondary conidia, ellipsoid-ovoid, obovoid, subcylindrical, fusoid, short subclavate, (10–)15–40(–50) x (4–) 5–8(–10) µm, 0–3(–5)-septate, hyaline, thin-walled, smooth or almost so to verruculose, apex obtuse, broadly rounded in solitary conidia or attenuated and truncate in catenate conidia, base obconically truncate, 1.5–2 µm diam, hila umbonate, somewhat thickened and refractive, but not darkened. Microcyclic conidiogenesis occasionally occurring with shed conidia often germinating, forming terminal, subterminal or subbasal germ tubes.

Infections of the isolate 04-011 on YST were phenotypically similar to those on ACREP, although some minor differences could be observed: leaf spots amphigenous, subcircular to somewhat irregular, 1–5 mm wide, brownish, with distinct dark green margins. Caespituli amphigenous, punctiform, grayish white. Mycelium internal. Stromata substomatal, 15–75 µm diam, at first colorless, but yellowish-ochraceous to brownish with age. Conidiophores 10–95 x 2.5–4 µm. Conidia solitary or in short secondary chains, small, aseptate conidia obovoid, septate conidia cylindrical, short clavate, occasionally subcylindrical with somewhat swollen apical cell, (10–)15–50 x 3–10 µm, 0–5-septate, hyaline, thin-walled, smooth to faintly rough walled (on MPCB 11.2–39.2 x 4.2–7 µm).

Pathogenicity tests.— – Disease reactions of the two isolates on YST and ACREP are presented (TABLE I). The isolates produced disease only on the plants from which they were isolated. In terms of leaf spots per plant and proportion of leaves with spots, the isolate 98-001 from ACREP was more aggressive on ACREP than was isolate 04-011 on YST (P = 0.07).

View larger version (112K): [in this window] [in a new window]   FIG. 1. Culture appearances of Cercosporella isolates 98-001, left (from Acroptilon repens), and 04-011, right (from Centaurea solstitialis), grown on modified potato-carrot agar.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 
Morphological comparisons between isolates of Cercosporella sp. on YST and C. acroptili on ACREP, including type material of the latter species, showed them both to be phenotypically closely allied taxa. Lesions, caepituli, mycelium, conidiophores and conidia are barely distinct. However the stromata on ACREP are lacking or small, 10–40 µm diam, and colorless, at most somewhat yellowish with age, whereas the stromata on YST are larger, up to 80 µm diam and yellowish to brownish with age. Septate conidia of C. acroptili are ellipsoid-ovoid, subcylindrical to short subclavate (vs. cylindrical to short clavate, occasionally with swollen tip in Cercosporella sp. on YST). Furthermore there were obvious differences in the appearance of the ACREP and YST colonies and significant differences in culture dry weight and spore production. The isolates were also not vegetatively compatible. Pathogenicity tests revealed that the two taxa from ACREP and YST are pathologically clearly different. In any case this distinct host preference would justify delineation of the two pathogens as separate formae speciales.

However the question arose concerning whether the biological specialization combined with the minor morphological differences in vivo and in vitro are sufficient to consider the two isolates members of different species. The difference in host specificity between the two isolates is a character that separates them. Acroptilon and Centaurea are allied genera belonging to the Asteraceae (Cardueae, Centaureinae). Comprehensive systematic work has shown that Acroptilon is a distinct genus not belonging to the closest relatives of Centaurea. Acroptilon is a member of the Rhaponticum group that constitutes a basal assemblage within the Centaureinae (Garcia-Jacas et al 2000, Greuter 2003, Hellwig 2004). Centaurea solstitialis usually is placed in a separate section (viz. sect. Solstitialis (Wagenitz 1975)).

Sequence analyses of the ITS region are useful taxonomic tools. Goodwin et al (2001) published a key paper in this respect for cercosporoid Mycosphaerella anamorphs, indicating that taxa differing by two or more nucleotides may be distinct species. Sequences of the ITS region of Cercosporella acroptili and Cercosporella sp. on Centaurea solstitialis have a similarity of 99% (three base pair difference). Multilocus approaches are usually necessary for genetic differentiation of species, but in combination with the biological, morphological and cultural differences discussed above the ITS sequences provide additional circumstantial evidence that the two taxa are allied closely, but nevertheless are two distinct species.

Genetically clearly distinguished but morphologically indistinguishable or little differentiated taxa (cryptic species) are not uncommon in anamorphic fungi (Braun et al 2001), but constitute a new challenge in the taxonomy of hyphomycetes, above all with regard to species concepts. It has to be taken into consideration that cercosporoid anamorphs may be anaholomorphs having permanently lost the ability to form teleomorphs, or they represent only one stage within the life cycle of Mycosphaerella species, in which morphological differences could only be manifested in the teleomorph (i.e. morphologically indistinguishable anamorphs can be different species belonging to morphologically differentiated holomorphs). If the ability to form teleomorphs has been lost permanently (anaholomorphs), morphologically indistinguishable but genetically differentiated anamorphs may represent distinct species. Anamorphs of powdery mildew fungi are obvious examples. Within Podosphaera sect. Sphaerotheca subsect. Magnicellulatae (U. Braun) U. Braun & N. Shishkoff (Podosphaera fuliginea (Schltdl.: Fr.) U. Braun & S. Takam. Sphaerotheca fuliginea (Schltdl.: Fr.) Pollacci complex), the anamorphs are morphologically uniform and little diagnostic for the differentiation of species. The discrimination of species is based mainly on teleomorphic features (Braun 1987, 1995b, Takamatsu et al 2000, Braun et al 2001). On the other hand, in the genus Leveillula Arnaud, teleomorphs are uniform and little diagnostic, but the anamorphs, above all the characteristics of the conidia, represent a useful basis for taxonomic diagnosis (Braun 1987, 1995b; Khodaparast et al 2001). There is also a strong tendency in various other groups of plant pathogenic fungi to return to a narrower species concept, discriminating closely allied, morphologically little differentiated species, based on biological specializations and molecular data (e.g. within the downy mildews [Göker et al 2004]). This tendency has been discussed for fungi in general by Hawksworth (2004).

Both of our isolates of Cercosporella caused significant damage to their respective hosts, A. repens and C. solstitialis, in the field, and each has potential as a biological control agent for their respective hosts. Realization of this potential will depend on further evaluations of efficacy and safety (host range). Because these pathogens are exotic to the USA, these evaluations can be done only in quarantine at FDWSRU or in their countries of origin. If the pathogens are found to be effective in damaging the target weeds and have a sufficiently narrow host range to be deemed safe to native and agriculturally important plants in the USA, then petitions to release the pathogens for classical biological control will be sought.

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION TAXONOMY LITERATURE CITED

 
Cercosporella centaureicola D. Berner, U. Braun & F.

Eskandari, sp. nov FIG. 2

View larger version (36K): [in this window] [in a new window]   FIG. 2. Cercosporella centaureicola sp. nov. A. conidiophore fascicle. B. conidiophores. C. conidia. D. conidia with germ tubes. Bar = 10 µm. U. Braun del.

English description see Results.

HOLOTYPE. GREECE, MACEDONIA REGION: Kozani prefecture, Kozani, 40°22'07''N, 21°52'35''E, 634 m elevation, isolated from leaves of Centaurea solstitialis (Asteraceae), 28 Apr 2004, D. Berner 04-011 (U.S. National Fungus Collections, BPI 844247). ISOTYPE. HAL 1841.

	FOOTNOTES

 
Accepted for publication July 27, 2005.

¹ Corresponding author. E-mail: Dana.Berner{at}ars.usda.gov

	LITERATURE CITED

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———. 1993. Studies on Ramularia and allied genera VI. Nova Hedwigia 56:423–454.

———. 1995a. A Monograph of Cercosporella, Ramularia and Allied Genera (Phytopathogenic Hyphomycetes). Vol. 1. IHW-Verlag.

———. 1995b. The powdery mildews (Erysiphales) of Europe. G. Fischer-Verlag Jena.

———, Shishkoff N, Takamatsu S. 2001. Phylogeny of Podosphaera sect. Sphaerotheca subsect. Magnicellulatae (Sphaerotheca fuliginea auct. s. lat.) inferred from rDNA ITS sequences—a taxonomic interpretation. Schlechtendalia 7:45–52.

Eskandari FM, Berner DK, Kashefi J, Strieth L. 2004. First report of leaf spot caused by Cercosporella sp. on Centaurea solstitialis in Greece. Plant Dis 88:1382.

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Goodwin SB, Dunkle LD, Zismann VL. 2001. Phylogenetic analysis of Cercospora and Mycospharella based on the internal transcribed spacer region of ribosomal DNA. Phytopathology 91:648–658.[CrossRef]

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Hawksworth DL. 2004. Fungal diversity and its implications for genetic resource collections. Stud Mycol 50:9–18.

Hellwig FH. 2004. Centaureinae (Asteraceae) in the Mediterranean—history of ecogeographical radiation. Plant Sys Evol 246:137–162.

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Khodaparast SA, Takamatsu S, Hedjaroude G-A. 2001. Phylogenetic structure of the genus Leveillula (Erysiphales: Erysiphaceae) inferred from the nuclear sequences of the rDNA ITS region with special reference to the L. taurica species complex. Mycol Res 105:909–918.[CrossRef]

Takamatsu S, Hirata T, Sato Y. 2000. A parasitic transition from trees to herbs occurred at least two times in tribus Cystotheceae (Erysiphaceae): evidence from nuclear ribosomal DNA. Mycol Res 104:1304–1311.[CrossRef]

Wagenitz G. 1975. Centaurea. In: Davis PH, ed. Flora Turkey 5:465–485.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand JJ, Sninski JJ, White TJ, eds. PCR Protocols. San Diego: Academic Press. p 315–322.

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 Google Scholar Google Scholar Articles by Berner, D.K. Articles by Luster, D.G. Search for Related Content PubMed Articles by Berner, D.K. Articles by Luster, D.G. Agricola Articles by Berner, D.K. Articles by Luster, D.G.