Abstract: During routine wheat disease surveys in. Hungary in 2007 Alternaria was isolated from leaf samples collected in Debrecen. Macro- and micro-.
Mycologia, 103(1), 2011, pp. 94–100. DOI: 10.3852/09-196 # 2011 by The Mycological Society of America, Lawrence, KS 66044-8897
Alternaria hungarica sp. nov., a minor foliar pathogen of wheat in Hungary economic importance (Simmons 2007). Only A. triticina Prasada & Prabhu has been reported to cause significant yield losses in wheat in parts of the world including India and Argentina (Prasada and Prabhu 1962, Perello´ and Sisterna 2006). We examined Alternaria isolated from wheat leaf lesions collected 2005–2007 in Hungary. Morphological examinations and sequence analysis of the ITS region (internal transcribed spacer regions 1 and 2 with the 5.8 S rRNA gene) of the isolates revealed that some Alternaria isolates did not fit any known species of this genus. In this paper we propose a new species, Alternaria hungarica To´th, Varga, Cso ˝sz, Simmons & Samson, to accommodate these isolates. Pathogenicity tests indicated that this species is a minor pathogen of wheat in Hungary.
Bea´ta To´th Ma´ria Cso ˝sz ´ gnes Szabo´-Heve´r A Cereal Research Non-profit Ltd. Company, P.O. Box 391, H-6701 Szeged, Hungary
Emory G. Simmons 717 Thornwood Road, Crawfordsville, Indiana 47933
Robert A. Samson CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands
Ja´nos Varga1 Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Ko¨ze´p fasor 52, H-6726 Szeged, Hungary
Abstract: During routine wheat disease surveys in Hungary in 2007 Alternaria was isolated from leaf samples collected in Debrecen. Macro- and micromorphological examinations and ITS sequence analyses indicated that the isolates represented a new Alternaria species, which we described as A. hungarica. The usually solitary conidia of A. hungarica resemble those of A. mouchaccae and A. molesta. However growth and sporulation pattern are more like those of A. geniostomatis and A. soliaridae. Phylogenetic analysis of ITS sequences indicated that this new species can be distinguished from all other examined Alternaria and Embellisia species. Pathogenicity tests indicated that A. hungarica can be considered a minor pathogen of wheat. Key words: Embellisia, ITS, phylogeny
MATERIALS AND METHODS
Morphological examinations.—Diseased leaf samples were collected from wheat cultivars grown in different parts of Hungary. Single conidia were isolated from necrotic tissue fragments under a stereomicroscope and transferred onto potato dextrose agar (PDA). Four isolates collected in 2007 in Debrecen were studied further. The isolates were cultivated as three-point inoculations on oatmeal (OA), PDA, unfiltered potato carrot agar (PCA) and 3% malt extract agar (MEA, Oxoid, Cambridge, UK) media at 25 C 5–7 d under alternating light and dark (Simmons 2007). For micromorphological examinations light microscopy Zeiss Axioskop 2 Plus (Carl Zeiss B.V., Sliedrecht, the Netherlands) was employed. Pathogenicity tests.—To determine the pathogenicity of the Alternaria isolates to wheat tests were conducted in the greenhouse by spraying wheat leaves with conidial suspensions of 3 3 103 conidia mL21 of the isolates at two-leaf stage (Zadoks stage 12, Zadoks et al. 1974). The pathogenicity of the isolates was tested against 12 wheat cultivars and lines. Disease symptoms were recorded after 12 d.
INTRODUCTION
Genus Alternaria Nees originally was described in 1816 with A. tenuis Nees as the type and only member of the genus (Nees von Esenbeck 1816–1817). Since then more than 1100 names have been published in Alternaria and Simmons (2007) accepted nearly 300 taxa. Species of Alternaria are widely distributed and infect a wide variety of economically important crops worldwide. Some species (e.g. A. solani Soraurer and A. brassicae [Berk.] Sacc.) are well known as destructive pathogens (Kucharek 1994, Simmons 2007), but the great majority are either saprophytic or have been described as occurring on hosts of little
Extraction and analysis of nucleic acids.—Cultures used for the molecular studies were grown on malt peptone (MP) broth with 10% (v/v) malt extract (Oxoid, Basingstoke, UK) and 0.1% (w/v) bacto peptone (Becton and Dickinson, LePont-de-Claix, France), 2 mL medium in 15 mL tubes. The cultures were incubated at 25 C 7 d. DNA was extracted with the MasterpureTM yeast DNA purification kit (Epicentre Biotechnol., Madison, Wisconsin) according to the instructions of the manufacturer. Fragments containing the ITS region were amplified with primers ITS1 and ITS4 (White et al. 1990). Sequence analysis was performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit for both strands, and sequences were aligned with the MT Navigator software (Applied Biosystems, Foster City, California). All
Submitted 29 Jul 2009; accepted for publication 7 Jun 2010. 1 Corresponding author. E-mail: jvarga@bio.u-szeged.hu
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TO´TH ET AL.: ALTERNARIA HUNGARICA TABLE I.
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Isolates examined in this study Species
Strain number
Alternaria mouchaccae
CBS 453.74
Alternaria mouchaccaeT Alternaria mouchaccae Alternaria molestaT Alternaria geniostomatisT Alternaria soliaridaeT Alternaria alternataT Alternaria limaciformisT Alternaria limaciformis Embellisia tumidaT Alternaria hungarica Alternaria hungaricaT Alternaria hungarica Alternaria hungarica
CBS CBS CBS CBS CBS CBS CBS CBS CBS CBS CBS CBS CBS
119671 208.74 548.81 118701 118387 916.96 481.81 121337 539.83 123924 123925 123926 123927
5 5 5 5
DKM DKM DKM DKM
GenBank accession number
Source
28.1 28.2 28.3 28.5
Carduus pycnocephalis, causing seedling death, Australia desert soil, Egypt salt-marsh soil, Kuwait skin lesion in Phocaena phocaena, Denmark Geniostoma sp., leafspot, New Zealand soil, sagebrush/grassland, Wyoming, USA Arachis hypogaea, India Soil, England Triglochin maritima, UK Triticum aestivum, rhizosphere, Australia Triticum aestivum, leaf lesions, Debrecen, Hungary Triticum aestivum, leaf lesions, Debrecen, Hungary Triticum aestivum, leaf lesions, Debrecen, Hungary Triticum aestivum, leaf lesions, Debrecen, Hungary
sequencing reactions were purified by gel filtration through Sephadex G-50 (Amersham Pharmacia Biotech, Piscataway, New Jersey) equilibrated in double-distilled water and analyzed on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, California). Sequences were deposited in GenBank (TABLE I). Data analysis.—Sequence data was optimized with the software package Seqman (DNAStar Inc., Madison, Wisconsin). Sequence alignments were performed by MEGA 4.0 (Tamura et al. 2007) and improved manually. PAUP* 4.0 software was used for parsimony analysis (Swofford 2000). Alignment gaps were treated as a fifth character state, and all characters were unordered and of equal weight. Maximum parsimony analysis was performed for all datasets with the heuristic search option with 100 random taxa additions and tree bisection and reconstruction (TBR) as the branchswapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees was evaluated with 1000 bootstrap replications (Hillis and Bull 1993). Bayesian p(MC)3 analyses were run in MrBayes 3.1 (Ronquist and Huelsenbeck 2003). Metropolis coupled Markov Chains with Monte Carlo simulations were run until likelihoods reached stationarity and the two independent runs converged. Accordingly the chains were run 4 000 000 generations with a sampling frequency of every 100th generation. Clades that received posterior probabilities . 0.95 were considered strongly supported. After plotting the likelihood values against generations an interval of 2 000 000 generations was established as burn-in. The remaining 40 000 trees were summarized in a 50% majority rule phylogram in MrBayes. ITS sequences of Alternaria and Embellisia species examined by Pryor and Bigelow (2003) and others available from GenBank were used for phylogenetic analysis. ITS region of several other Embellisia and Alternaria species that might have been related to these isolates based on morphological features also were sequenced and included in the analysis (TABLE I).
FJ196313 FJ196310 FJ196312 FJ196308 FJ196309 FJ196311 FJ196306 FJ196307 FJ196305 EU305602 EU305606 EU305603 EU305604 EU305605
RESULTS
Sequence analyses.—BLAST queries indicated weak relatedness to Alternaria thalictrigena Schub. & Crous (Schubert et al. 2007), Embellisia planifunda E.G. Simmons, E. conoidea E.G. Simmons, E. leptinellae E.G. Simmons & C.F. Hill and Lewia photistica E.G. Simmons (Simmons 1986). The aligned dataset included 481 nucleotides, 51 of which were parsimony informative. One of the 18 maximum parsimony trees based on ITS sequences is included (FIG. 1). Phylogenetic analysis of the dataset revealed that the Hungarian isolates are most closely related to A. thalictrigena, although this relationship was supported only by low bootstrap values (50%, FIG. 1). We propose the name Alternaria hungarica sp. nov. to accommodate these four isolates. Pathogenicity.—This species was able to cause weak nectoric lesions and/or chlorotic halo on wheat leaves of the examined cultivars (data not shown). TAXONOMY
Alternaria hungarica B. To´th, J. Varga, M. Cso ˝sz, E.G. Simmons & R.A. Samson, sp. nov. FIGS. 2, 3 MycoBank MB512907 Ex cultura in agaro PCA descripta. Sporulatio in conidiophoris 30–80 3 3–4 mm ex substrato et in aerio. Sporulatio aeria in ramis hypharum funicularium supra substratum radiantium et in aerie erigentium. Conidia solitaria vel in caespitibus parvulis. Conidia in maturitate ovoidea vel late ellipsoidea, 28–35 3 14–21 mm, 3–4(–6) transverse septata, 1–2(–3) longiseptata, laevia, obscure modice luteobrunnea, septis atrobrunneis.
Etymology. Name refers to the country where the fungus was found.
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FIG. 1. One of the MP trees based on ITS sequence data of the examined isolates. Numbers above branches are bootstrap values, while numbers below branches represent Bayesian posterior probabilities. Tree length: 179 steps, consistency index: 0.6480, retention index: 0.7623, rescaled consistency index: 0.4940.
TO´TH ET AL.: ALTERNARIA HUNGARICA
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FIG. 2. Alternaria hungarica sp. nov. A. Colony on potato carrot agar. B. Colonies on malt extract agar. C. Colonies on oatmeal agar. D–I. Conidiophores and conidia. Bar 5 10 mm.
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FIG. 3. Alternaria hungarica sp. nov. Conidia, conidiophores and sporulation pattern from a culture of ex-type isolate on PCA. Bar 5 50 mm.
TO´TH ET AL.: ALTERNARIA HUNGARICA Holotype: CBS H-20458 (dried culture preparation ex E.G.S. 52.199, ex isolate M. Cso ˝sz, from leaf of Triticum aestivum, Debrecen, 2007). Other material examined: CBS 123924 5 DKM 28.1, CBS 123925 5 DKM 28.2, CBS 123926 5 DKM 28.3, CBS 123927 5 DKM 28.5.
The developing colony at 5d on PCA is ca. 4 cm diam and has 3–4 pairs of concentric rings of growth and sporulation. Although a delicate arachnoid layer of branching hyphae can be seen above the surface growth in young colonies (50 3 magnification), the growth is open and inconspicuous within the prolific population of short, simple and elongate-branched sporulating hyphae that arise within the rings of growth. The 7–10 d old colony that covers the substrate in a 60 mm diam plate is a dense, blackbrown layer of sporulation; it has no areas of nonsporulating growth. Conidium production is visible 2–3 d after growth begins. Primary conidiophores that sporulate near the agar surface are abundant throughout development of the colony. The enlarging colony concurrently produces a dense stand of long, procumbent, loosely intertwined aerial hyphae that radiate in successive rings and that branch and become conidiogenous near their aerial tips. These funiculose structures of 5–10 parallel hyphae become 4–5 mm long. Sporulation occurs on lateral conidiogenous branches that arise successively along the aerial elements as they lengthen. A fully extended funiculose element may have 10 or more lateral conidiophores. Both the primary conidiophores produced at the substrate surface and the aerial conidiogenous branches may lengthen by means of geniculate extensions; they commonly are variously curved and twisted, grow up to 30–80 3 3–4 mm, and produce small clusters of solitary conidia and short chains of conidia as they lengthen or branch. A high percentage of conidia in colonies of any age remain solitary. However as the colony ages short secondary conidiophores often are produced abruptly from the apical cell of primary conidia and frequently also laterally from basal or other body cells of the conidium. Solitary conidia or short chains of 2–4 secondary conidia are produced at each sporulation site. Juvenile conidia are ovoid before septation begins. They retain the shape or become broadly ellipsoid as they enlarge. Conidia have no definable beak structure; the apex of ovoid conidia is simply a conoid cell. Fully developed conidia are up to 28–35 3 14–21mm, although large numbers of conidia fall in a smaller range when they already have developed full pigmentation and are sufficiently mature to initiate chain development. Mature conidia have 3–4(–6)
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transepta and 1–2(–3) longitudinal septa in each of the transverse segments. The conoid apical cell of ovoid conidia commonly contains one long oblique septum and a shorter one at a right angle to the oblique wall. Conidium is a dull, medium yellowbrown; septa are dark brown in contrast. The conidium outer wall appears to be smooth at 780 3 magnification. (Photographs of colonies in FIG. 3 are taken from MEA, OA and PCA plates.) DISCUSSION
The simple, usually solitary conidia of A. hungarica resemble those of A. mouchaccae E.G. Simmons and A. molesta E.G. Simmons (Simmons 1981). However the growth and sporulation pattern, as well as many conidia, are more nearly like those of A. geniostomatis E.G. Simmons & C.F. Hill and A. soliaridae E.G. Simmons (Simmons 2007), which produce conspicuous layers of intertwined or funiculose aerial hyphae that sporulate abundantly on numerous lateral conidiophores. Neither the conidia nor the details of the sporulation apparatus of any of these four species are identifiable with those of A. hungarica, which is considered to represent a previously undescribed taxon. In addition this species is clearly distinguishable from any other Alternaria or Embellisia species with ITS sequence data (FIG. 1). Although A. thalictrigena was found to be the closest relative of A. hungarica based on ITS sequence data (FIG. 1), A. thalictrigena (Schubert et al. 2007) produces elongate conidia and is morphologically highly different from A. hungarica. Simmons (2007) did not consider A. thalictrigena to be an Alternaria species. Several other Alternaria species have been isolated from wheat and were considered as either saprophytes or as minor pathogens, including A. triticicola Rao (Rao 1964); A. californica E.G. Simmons & Koike, A. infectoria E.G. Simmons, A. metachromatica E.G. Simmons, A. oregonensis E.G. Simmons, A. triticimaculans E.G. Simmons & Perello´, all belonging to the A. infectoria species group (Simmons 2007); A. tenuissima (Kunze) Wiltshire (Gannibal et al. 2007, Simmons 2007); A. brevispora Zhang (Zhang 1999); and A. malorum (Ruehle) U. Braun, Crous & Dugan (Braun et al. 2003, Crous et al. 2006). The latter species was synonymized with Chalastospora gossypii (Jacz.) U. Braun & Crous (Crous et al. 2009). Among the Embellisia species E. chlamydospora (Hoes, G.W. Bruehl & C.G. Shaw) E.G. Simmons, E. tumida E.G. Simmons and E. planifunda were isolated from wheat root or seeds (Gonzales and Trevathan 1999, 2000; Taheri et al. 1994). Another species, E. allii (Campan.) E.G. Simmons, also has been suggested to be able to cause root and foot rot of wheat (Mohamma-
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dipour and Ilkhechi 2004). Alternaria hungarica produced only limited numbers of small lesions on wheat leaves, so this species can be considered only as a minor foliar pathogen of wheat with small economical importance. ACKNOWLEDGMENTS
This study was financially supported by grants NKTH GVOP3.1.1.-2004-05-0206/3.0 and DTR_2007. BT received a Bolyai fellowship grant. The technical assistance of Karolina Kis-Sebestye´n, Ka´lma´nne´ Gajda´cs and E´va Ko´tai is gratefully acknowledged. LITERATURE CITED
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