http://informahealthcare.com/mcb ISSN: 1040-841X (print), 1549-7828 (electronic) Crit Rev Microbiol, Early Online: 1–14 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/1040841X.2012.755948 REVIEW ARTICLE Phoma-like fungi on soybeans György János Kövics1, Erzsébet Sándor2, Mahendra K. Rai3, and László Irinyi1 Plant Protection Institute, Debrecen University, Debrecen, Hungary, 2Institute of Food Science, Quality Assurance and Microbiology, Debrecen University, Debrecen, Hungary, and 3Department of Biotechnology, SGB Amravati University, Amravati, India Abstract Keywords Numerous coelomycetous fungi classified in Ascochyta, Phoma and Phyllosticta, and lately established and/or re-classified genera and species, namely Boeremia and Peyronellaea have been recorded from spots on leaves and pods of soybeans. These rarely observed pathogens are cosmopolitan, ubiquitous species on diseased and dead plant materials, and define frequently as weak or opportunistic parasites. Based on the Genealogical Concordance Phylogenetic Species Recognition, the authors summarize the re-evaluation of the taxonomic status of Phoma sojicola (syn. Ascochyta sojicola) and Phyllosticta sojicola. Inspite of the former delimitation of Ph. sojicola based on small differences in morphological features, it has proved to be identical to Peyronellaea pinodella (syn. Phoma pinodella). Similarly, it was also confirmed that Ph. sojicola was identical to Boeremia exigua var. exigua (syn. Phoma exigua var. exigua). The authors and co-workers contributed to the identification of Phoma-like fungi by combined conventional and molecular methods. Protein-encoding genes (TEF1 and b-tubulin) were successfully applied within the Phoma genus to infer phylogenetic relationships. b-tubulin, Boeremia exigua var. exigua, Peyronellaea pinodella, Phoma sojicola, TEF1 sequences Introduction Different species of hyaline-spore pycnidial fungi have been associated with leaf and pod spot diseases of soybean (Glycine max/L./Merr.) from different parts of the world. These diseases might periodically occur, causing yield losses under favorable environmental conditions. Coelomycetous fungi (Grove, 1935), including Phoma-like species, are geographically widespread and are found in numerous ecological niches. This anamorphic, soil, group inhabiting organic debris and water, as well as species that parasitize other fungi, lichens, insects and vertebrates. Specimens have been isolated mainly from soil and from a wide range of plant hosts where they reside as primary pathogens, opportunists, saprobes or endophytes (Aveskamp et al., 2008). Pathogenicity Comparison of herbarium materials and pathogenicity tests made with various isolates have demonstrated that Phoma sojicola was identified as one of the specific pathogens of soybean (Kövics et al., 1999). This fungus, which had been described earlier as Ascochyta sojicola (as ‘‘sojaecola’’) by Abramov (1931) resembles some plurivorous Phoma species which may also be pathogenic on soybean, namely Phoma exigua Desm. var. exigua (listed as ‘‘A. phaseolorum’’, Wallace & Wallace, 1947, 1949 / ¼ Boeremia exigua var. Address for correspondence: György János Kövics, PhD, Plant Protection Institute, Debrecen University, Böszörményi út 138, Debrecen H-4032, Hungary. E-mail: kovics@agr.unideb.hu History Received 23 July 2012 Revised 30 November 2012 Accepted 3 December 2012 Published online 31 January 2013 exigua/) and Phoma pinodella (L.K. Jones) Morgan-Jones and Burch (as ‘‘A. pinodella’’, Noll, 1939 / ¼ Peyronellaea pinodella/). Finally, Phyllosticta sojicola Massalongo (1900) has been reported causing similar disease symptoms (Böning, 1938; Walters & Martin, 1981) on soybean leaves and pods. Further complicating the situation, other species of Ascochyta and Phoma have also been found on soybean which occurrences were registered only sporadically and mainly as opportunistic parasites (Kövics et al., 1999). As reported in previous studies (Faris-Mokaiesh et al., 1996; Fatehi et al., 2003; Onfroy et al., 1999; Peever et al., 2007), Ph. pinodella appears to be very closely related to Didymella pinodes (anam. Ascochyta pinodes) and because these species share the same host range, they are often confused. However, both species can easily be differentiated in terms of the amount of septate conidia formed in vitro, abundantly in A. pinodes, and in very small numbers in Ph. pinodella (¼Pey. pinodella). Noll (1939) isolated three strains of Ph. pinodella (¼Pey. pinodella) as Ascochyta pinodella from peas, broad beans and soybeans, and proved all of them to be pathogenic on soybean. Darpoux (1945) reported a hyaline-spore soybean pathogen pycnidial fungus as Ascochyta pisi, however its morphological and cultural characteristics closely resembled to Ph. pinodella (¼Pey. pinodella). On the basis of detailed cultural morphological and pathological tests made by Bondartzeva-Monterverde & Vassiljevskiy (1940), it could not be proven that A. pisi is pathogenic to soybean, in contrast with A. pinodella (¼Pey. pinodella). The pathogenicity of Ph. pinodella (¼Pey. pinodella), A. sojicola (suggested synonym of Ph. pinodella and ¼ Pey. pinodella now) and 20 13 Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. 1 2 G. J. Kövics et al. Ph. exigua var. exigua (¼B. exigua var. exigua) isolates was also confirmed by a wide range pathogenicity tests on soybean (Kövics et al., 1999). These findings also strengthen the likelihood of Darpoux’s (1945) misidentification. Bondartzeva-Monterverde & Vassiljevskiy (1940) sharply differentiated Ascochyta phaseolorum (¼B. exigua var. exigua) and A. sojicola (¼Pey. pinodella) as distinct species as the former one possesses higher pathogenicity on soybean. Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. Taxonomy The original genus concept of Saccardo (1880) was emended by Boerema & Bollen (1975) and after more than 40 years of taxonomical research, the admitted Phoma species were arranged in nine sections (cit. Boerema et al., 2004) which are mainly based on a single or just a few morphological characters and have not been confirmed as biologically realistic by molecular biological studies (Aveskamp et al., 2010). There is a convention of using a single generic name, based on priority but regardless of whether the genus is ‘‘anamorphic’’ or ‘‘teleomorphic’’. This classification is used for all unambiguous monophyletic phylogenetic lineages (Aveskamp et al., 2010; Crous et al., 2006, 2009a,b). These Phoma sections have teleomorph relations described in the genera Didymella, Mycosphaerella, Leptosphaeria and Pleospora (Boerema, 1997) indicating that Phoma anamorphs represent a polyphyletic group. For a long time, the genera Phoma and Ascochyta, both classified in the Pleosporales, have already been considered as closely related (de Gruyter et al., 2009). Based on the molecular phylogeny of the type species of the Phoma sections Phoma, Phyllostictoides, Sclerophomella, Macrospora and Peyronellaea a new teleomorph family, Didymellaceae has been introduced, besides sect. Paraphoma and sect. Heterospora assigned to the Phaeosphaeriaceae and Leptosphaeriaceae, respectively (de Gruyter et al., 2009). In terms of sequence data, it is estimated that approximately 70% of the species recognized by Boerema et al. (2004) can be associated with the Didymellaceae (Aveskamp et al., 2010). Currently, Ascochyta has teleomorphs described in both Mycosphaerella and Didymella genera (Corlett, 1981; Peever et al., 2007). However, the type species of the latest teleomorph genus, Didymella exigua is not associated with a Phoma anamorph state. The allied anamorph genera Ascochyta, Coniothyrium and Pyrenochaeta proved to be polyphyletic in the Pleosporales (de Gruyter et al., 2009). Lately, as a taxonomic novelty a new genus: Boeremia Aveskamp, Gruyter & Verkley has been established, and a lot of new species and new combinations have been introduced (Aveskamp et al., 2010). Morphological delimitation of species For identification and delimitation of Phoma-like species, it is suitable to study them in pure culture and using an integrated approach based on cultural, morphological, physiological, determination of ontogeny, biochemical evaluation or in ambiguous cases molecular approaches should be applied (Irinyi et al., 2009a,b; Monte et al., 1991). Crit Rev Microbiol, Early Online: 1–14 Morphological characteristics must be studied in vitro in order to establish a more realistic and practical classification of Phoma. in pure culture, a large number of fungi previously assigned to several species actually represent a restricted number of morphological types (Irinyi et al., 2009a). Phoma sensu lato can be easily differentiated from its allied genus Phyllosticta by the formation of relatively smaller pycnospores. In addition, the latter produces a hyaline sheath around pycnospores. A special appendage is also produced by the conidia, which can be visible in aquaceous condition; however, this feature may disappear on dry herbarium materials. The fungus is characterized by the formation of single-celled, hyaline pycnospores borne inside a fruiting body referred to as pycnidia, which vary from globose, sub-globose to coalescence forms (Irinyi et al., 2009a). As these fungi can only be differentiated with certainty in vitro (van der Aa et al., 1990), the early literature pertaining to them is very confusing. Boerema et al. (2004) published a monograph dealing with 223 specific and infraspecific taxa of Phoma Sacc. emend. Boerema and G.J. Bollen, with 1146 synonyms in various Coelomycetes genera, mainly on morphological basis. Additional biochemical characteristics Little work has been done on biochemical aspects of taxonomic criteria for species differentiation in Phoma. The NaOH spot test was introduced in Phoma taxonomy as a probe for checking E-metabolite (after Ph. exigua) production near the growing margins of cultures with a drop of NaOH. The metabolite E-producing culture results, within about 10 min, in a greenish spot or ring (pigment a), which changes to red (pigment b) after 1 h (Boerema & Höweler, 1967) and is reffered to as spot test. Pigments are sometimes restricted to the cytoplasm or guttules in the hyphae, and usually diffusing into the agar media is involved. These pigments are composed of anthraquinone components (pachybasin, chrysophanol, emodin, phomarin; Bick & Rhee, 1966). Some species can produce very characteristic dendritic crystals in the agar media which may be brefeldin A, pinodellalide A and B and radicin (Noordeloos et al., 1993). Species pathogenic to soybean To delimitate the closely related Phoma-like fungi, the review includes descriptions in vitro, disease symptoms in vivo, pathogenicity tests and differentiating characters of the species. Nowadays these data of species are complemented, confirmed and/or modified by molecular tools and re-classified in many cases. Boeremia exigua var. exigua (Desm.) Aveskamp, Gruyter & Verkley Boeremia exigua var. exigua (Desm.) Aveskamp, Gruyter & Verkley in Stud. Mycol. 65(1) 1–60. 2010. Basionym: Phoma exigua Desm. in Annals Sci.Nat. (Bot.) III, 11: 282. 1849, var. exigua (as ‘‘Var. a’’) Synonyms: This fungus has numerous, about 150 synonyms, invalid later homonyms, and nomina nuda (for details see in Boerema et al., 2004). Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. DOI: 10.3109/1040841X.2012.755948 Phyllosticta sojicola (as ‘‘Ph. sojaecola’’) Massal. in Atti del Reale Istituto Veneto di Scienze, Lettere ed Arti 59: 688. 1900. Phyllosticta glycineum (as ‘‘glycinea’’) Tehon & E.Y. Daniels in Mycologia 19: 117–118. 1927. This fungus is a cosmopolitan, ubiquitous species on diseased and dead plant materials (Morgan-Jones & Burch, 1988). The recently established genus (Aveskamp et al., 2010) was named after Gerhard H. Boerema, who made great contributions to our understanding of the taxonomy of phomoid fungi. Within the Ph. exigua (¼B. exigua) species, at least nine varieties of subtaxa (varietas, var.) were admitted (Boerema et al., 2004) and confirmed by molecular methods (Abeln et al., 2002). In addition to type species, B. exigua, recently Aveskamp et al. (2010) described two novel varieties, namely B. exigua var. gilvescens and B. exigua var. pseudolilacis. Some species of Phoma, including Ph. exigua var. exigua (¼B. exigua var. exigua), secrete phytotoxins as secondary metabolites, which have great potential for the biological control of weeds, and can be exploited for the production of mycopesticides (Rai et al., 2009). Rothweiler & Tomm (1966, 1970) purified and characterized the phomin and dehydrophomin from the culture filtrate of Ph. exigua Desm. var. exigua (¼B. exigua var. exigua) and phytotoxicity of phomin has been demonstrated. Other phytotoxic cytochalasins have also been extracted from this pathogen and Cimmino et al. (2008) found these to be effective herbicide agents which serve as potential biocontrol agents. Deoxaphomin demonstrated the highest level of toxicity on leaves of Sonchus arvensis. Cultural characteristics Colonies are gray to black with irregularly scalloped margins, attaining a diameter of 6–7 cm on oat-meal agar (OA) in 7 d; pycnidia black, abundant, sub-globose to globose, some coalesce to form irregular fructifications, ostiolate, parenchymatous, 62–290 mm; conidia hyaline, one to twocelled, ovoid on malt agar (MA) and rice-meal agar, ellipsoid on OA, 3.7–11.1  2–3.7 mm (Irinyi et al., 2009b). In spot tests, the greenish-blue discoloration of the agar medium on application of a drop of NaOH takes place, which is an important diagnostic characteristic. This greenish color gradually becomes brown-reddish. This feature was wrongly described as a misprint in Rai (1998). Phoma sojicola (Abramov) Kövics, Gruyter & Aa Phoma sojicola (Abramov) Kövics, Gruyter & Aa in Mycol. Res. 103: 1066. 1999. Basionym: Ascochyta sojicola Abramov (as ‘‘sojaecola’’) in Bolezni i vrediteli sojevich bobov na Dal’nem Vostoke: 68–70. 1931. Superfluous name: Ascochyta sojicola Nelen (as ‘‘sojaecola’’), Nov. Sist. niz. Rast. 14: 104 (1977) ¼ Ascochyta sojicola Abramov ex Nelen (as ‘‘sojaecola’’) in Naumova & Uspenskaya: Nomenclature vozbuditelja askochytoza soii. Mikol. Fitopatol. 22: 105–111. 1988. This name is not in use anymore, now it should be considered synonymous with Peyronellaea pinodella. Phoma-like fungi on soybeans 3 Ascochyta sojicola was described and validly published by Abramov (1931) (also often transliterated as Abramoff or Abramow) from soybean, causing necrotic spots on leaves and pods. Nelen (1977), following an earlier publication (Abramov, 1938), again described the fungus A. sojicola Nelen (as ‘‘sojaecola’’) giving a Latin diagnosis. This superfluous nomenclature was taken over by Naumova & Uspenskaya (1988) as ‘‘A. sojaecola Abramov ex Nelen’’. At the same time, when Abramov was studying this pathogen, Loukyanovits et al. (1931) reported a soybean Ascochyta species from West-Siberia which also might have been A. sojicola. The name A. sojicola (as ‘‘sojaecola’’) was often used for pycnidial fungi on soybean, especially in the East-European countries, namely former Czechoslovakia (Nováková-Pfeiferová, 1958, 1959; Ondrej, 1968), Yugoslavia (Numic, 1962), the former German Democratic Republic (Klinkowski et al., 1966), Hungary (Tóth & Kövics, 1978; Tóthné-Zahorecz, 1970), Romania (Rádulescu & Paulian, 1973), Poland (Marcinkowska, 1984, 1985; Marcinkowska et al., 1982; Opiola, 1981; Pietkiewicz, 1959) and the Soviet Union (Naumova, 1988). Ascochyta sojicola has also been recorded from Japan (Endo, 1963; Ishiyama, 1936; Kurata, 1960) and the former Belgian Congo (Hendricks, 1939). Hunt (1946) listed A. sojicola in his ‘‘Destructive plant diseases not yet established in North America’’. Frandsen (1953) described A. sojicola (as ‘‘sojaecola’’) for the first time in Western Europe from Germany, and deposited an isolate in the CBS culture collection (CBS 113.53). Enken (1959) considered the Ascochyta disease as being wide-spread in the Far-East Region of Russia, Japan, North-East China, and also occurring in the Ukraine, the North-Caucasus, Georgian, Voronezh and Moscow Regions. Kungurtseva (2008) compiled distribution countries as ‘‘number of countries’’ of Western Europe, Japan, China, Georgia, Ukraine and Moldova. In Russia, it meets in the Far East, Central European part, Northern Caucasus, Krasnodar Territory for Ph. sojicola (¼Pey. pinodella) as ‘‘A. sojaecola Abramov’’. Naumova (1988) listed A. sojicola (as ‘‘sojaecola’’) with Septoria glycines Hemmi, Fusarium spp., Peronospora manshurica (Naumov) Syd., Ph. sojicola (as ‘‘sojaecola’’) [?teleomorph: Pleosphaerulina sojicola Miura], Sclerotinia sclerotiorum (Lib.) de Bary, and Botrytis cinerea Pers.:Fr. as the most harmful and wide-spread soybean pathogens in the former Soviet Union. According to Naumova & Uspenskaya (1989), A. sojicola has been considered as a distinct species by Abramov (1931), Bondartzeva-Monteverde & Vassiljevskiy (1940), Frandsen (1953), Nováková-Pfeiferová (1959), Kurata (1960), Naumova (1988), Naumova & Uspenskaya (1988, 1989), while other authors considered the fungus synonymous with A. phaseolorum Sacc. (Kungurtseva, 2008; Marcinkowska, 1984; Melnik, 1977; Ondrej, 1976; Zukovskaya, 1979), which is a synonym of Ph. exigua var. exigua (¼B. exigua var. exigua) (Boerema, 1972). Naumova & Uspenskaya (1989) referred to intraspecific diversity of fungus. The leaf and pod spot of soybeans can affect cultivated and wild soybeans (Glycine hispida, Glycine ussuriensis /¼G. 4 G. J. Kövics et al. max/), Pisum arvense, Pisum sativum and Vicia faba. It can cause reduced seed germination by 25–40%, losses of shoots and adult plants, reduction of assimilating leaf surface, and decrease of grain yield and deterioration of its quality. The yield losses can sometimes reach 15–20% and more (Kungurtseva, 2008). Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. Cultural characteristics Pycnidia scarce on OA, 86–345 mm in diameter, (sub-) globose or irregular, solitary or confluent, glabrous, with one or more ostioles, olivaceous to olivaceous black, walls made up of 2–5 layers of cells, outer layer(s) pigmented, on the surface and in the agar at the margin of the colony, sometimes aggregated in distinct sectors; conidial exudate rosy buff to vinaceous buff. Conidiogenous cells globose to doliiform, ampulliform or lageniform, 3–7  3–7 mm. Conidia mainly aseptate, 4.8–8.1  1.9–3.6 mm, av. 6.3– 2.9 mm, oblong to ellipsoidal, Q ¼ 1.6–3.0, av. Q ¼ 2.3, with small guttules, occasionally one-septate, 8.0– 12.4  3.4–4.8 mm. Chlamydospores 8–16  7–8 mm, (sub-) globose to cylindrical, single or in chains, sometimes aggregated, intercalary or terminal, gray olivaceous to olivaceous with greenish guttules. Crystals are usually absent, only once dendritic crystals were produced in a colony sector of CBS 567.97 (D/056); after storage at 6  C for 3 months, dendritic crystals were also formed in most of the fresh isolates. NaOH spot test: negative on MA, weakly brownish red, not specific E-metabolite (Kövics et al., 1999). Symptoms in vivo Seedlings developing from infected seeds showed sharply delimited necrotic spots on the cotyledons, often with pycnidia in concentric rings, being 3–8 mm in diameter. Pycnidia were (86–) 125–140 (–260) mm diameter, producing both aseptate conidia, 6.4–10.4  2.8–4.8 mm, and as septate conidia 7.2–12.0  3.4–4.8 mm. Infected seedlings developed slowly and usually decayed. Depressed spots form on cotyledons, sometimes deep through reddish-brown ulcers form on cotyledons and caulicle. Young leaves became infected at the tips or margins, showing irregular or V-shaped brownish areas. Leaf spots on older leaves were grayish to light brown, surrounded by a narrow brownish area. Later these spots turned dull gray to tan, up to 10–15 mm diameter, finally necrotic, occasionally falling out. These spots were observed on the whole leaf surface, often covered with black pycnidia. Infected leaves fell off early. On stems light brown, elliptical necrotic lesions with a purplish to brownish margin, 3–4 mm wide and up to several centimeter long, were formed. On the youngest pods, the spots were scattered, brownish with a darker margin of 5–10 mm diameter. On the ripe pods, the lesions were light brown to gray with a reddish margin, circular, usually with numerous pycnidia. Seeds from infected pods were brownish, shrunken or remained undeveloped. The pathogen can overwinter in seeds just under the seed-coat and on infected plant debris (Kövics et al., 1999). Crit Rev Microbiol, Early Online: 1–14 Peyronellaea pinodella (L.K. Jones) Aveskamp, Gruyter & Verkley Peyronellaea pinodella (L.K. Jones) Aveskamp, Gruyter & Verkley in Stud. Mycol. 65(1): 1–60. 2010. Basionym: Ascochyta pinodella L.K. Jones in Bull. N.Y.St. Agric. Exp. Stn. 547: 10. 1927. Synonyms: Phoma medicaginis Malbr. & Roum. var. pinodella (L.K. Jones) Boerema apud Boerema, Dorenbosch & Leffring, in Neth. J. Pl. Path. 71: 88. 1965. Phoma trifolii E.M. Johnson & Valleau, in Bull. Ky Agric. Exp. Stn. 339: 73–74. 1933. Ascochyta sojicola (as ‘‘sojaecola’’) Abramov, Bolezni i Vrediteli Soievykh Bobov na Dal’nem Vostoke. Vladivostok: Dal’stazva. 62. 1931. (Homonym: A. sojicola Nelen /as ‘‘sojaecola’’/ in Nov. Sist. Niz. Rast. 14: 105. 1977. Erroneously also listed with the author citation ‘‘Abramov ex Nelen’’.) Phoma pinodella (L.K. Jones) Morgan-Jones & K.B. Burch in Mycotaxon 29: 485. 1987. Phoma sojicola (Abramov) Kövics, Gruyter & Aa in Mycol. Res. 103: 1066. 1999. Because Ph. pinodella is morphologically so similar to Phoma medicaginis, it was once regarded as a variety of this species by Boerema et al. (1965). The variety was elevated to species rank after careful observation (White & MorganJones, 1987), but the varietal name is, however, currently still in common use (e.g. Bowen et al., 1997; Fatehi et al., 2003; Onfroy et al., 1999). The results reviewed in this article however, illustrate a substantial phylogenetical distance to Ph. medicaginis, and warrant recognition at species level, in the re-instated genus Peyronellaea. Peyronellaea group comprises many of the chlamydospore forming anamorph species, including the majority of the species that were accommodated in Phoma section Peyronellaea (Boerema et al., 2004). Also Phoma glomerata, type species of this section is accommodated here (Boerema, 1997). However, as section, Peyronellaea has a polyphyletic nature (Aveskamp et al., 2009a). Because of this, the genus name has to be emended as Peyronellaea Goid. ex Togliani (in Ann. Sperim. Agrar. II 6:93. 1952) emend. Aveskamp, Gruyter & Verkley (in Stud. Mycol. 65/1/:31/1–60/2010). According to this re-establishment, unicellular chlamydospores often abundantly formed in and on the agar and in the aerial mycelium, globose, intercalary, brown or olivaceous pigmented, measuring 5–22 mm diameter. Multicellular chlamydospores mainly alternarioid, terminal or intercalary, often in chains, brown or olivaceous pigmented, 10–50  7–25 mm. The high posterior probability for this group justified the recognition of a separate genus in the Didymellaceae. Therefore, the genus name Peyronellaea Goid is reestablished, and the associated species are recombined into this genus (Aveskamp et al., 2010). The sexual state of Ph. pinodella (as Ph. medicaginis var. pinodella, ¼ Pey. pinodella) that is reported and described by Bowen et al. (1997), but that has not been named thus far. From a phylogenetic point of view, this record is very plausible as all species in the subclade in which Ph. pinodella is embedded, do form a Didymella-like teleomorph Phoma-like fungi on soybeans DOI: 10.3109/1040841X.2012.755948 (Aveskamp et al., 2010). However, the species is probably heterothallic (Bowen et al., 1997), and the formal name for the teleomorph of Pey. pinodella is still missing (a Didymella sp., Pleosporales, Ascomycota). Recently, Woudenberg et al. (2012) analyzed the mating-type genes to distinguish the homothallic Pey. pinodes and the heterothallic Pey. pinodella both responsible for ‘‘Ascochyta’’ blight on pea and indicated the close phylogenetic relationship between these two species. Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. Cultural characteristics Colonies are greenish or yellowish olivaceous, margin pale, flat, radially filamentous, attaining a diameter of 5–6.2 cm on OA in 7 d, reverse on MA shows conspicuous white, dendritic crystals; aerial mycelium practically absent; pycnidia gray to black, scattered, some in radial rows, globose to irregular, parenchymatous, (80–) 96–175 (320) mm, sometimes micropycnidia occur near the apex of a pycnidia, 30–50 mm; conidia hyaline, one-celled, sometimes 1-septate, mostly ellipsoid, few ovoid, variable in size, 4–8.7  2.5–3.5 mm; chlamydospores solitary or in chains, confluent, gray to black globose to subcylindrical, highly guttulated, 8–20 mm diameter. Formation of crystals on MA is one of the most important characteristic features of this fungus (pinodellalide A and B, Noordeloos et al., 1993), however, it sometimes might be missing. Phoma pinodella is a plurivorous species and isolated from wide range of plants, especially on Leguminosae (Irinyi et al., 2009a). The main properties (colony morphology, pycnidia, conidia and chlamydospores) overlap among isolates of the two closely related species since these characteristics are rather variable. Kövics et al. (1999) separated Ph. pinodella and Ph. sojicola partly by the production of dendritic crystals on MA which characterize Ph. pinodella (Noordeloos et al., 1993). Earlier both were members of Phoma section Phyllostictoides Žerbele ex Boerema (1997). However, in later studies, production of dendritic crystals (pinodellalide A and B) was not observed among the examined isolates of Ph. pinodella in contrast with previous observations at the same isolates (Kövics et al., 1999). Data of comparative morphological studies in vitro show that Ph. sojicola cannot be definitely differentiated from Ph. pinodella either on the basis of morphological characters or molecular data, therefore it was proposed to synonymize Ph. sojicola with Ph. pinodella ( ¼ Pey. pinodella) (Irinyi et al., 2009b). Recently, on the base of molecular data of two Hungarian soybean isolates (CBS 567.97 and another one ‘‘CBS 100580’’/?/) the identity of Ph. pinodella (¼Pey. pinodella) has been confirmed by Aveskamp et al. (2010) who also agreed that Ph. sojicola and Ph. pinodella had been synonymized on the base of morphological observations and sequence data of internal transcribed spacer (ITS), b-tubulin and translation elongation factor (TEF) 1-a (Irinyi et al., 2009b). The isolate D/063 (¼Ph 58; MYA-408) is actually a Ph. pinodella (¼Pey. pinodella) and has been misidentified as ‘‘Ph. exigua var. exigua’’ from Petroselinum crispum, listed among the available hosts of Ph. pinodella (¼Pey. pinodella) (Kinsey, 2002) and confirmed by Kövics et al. (1999) by morphological, physiological and molecular data. 5 Phyllosticta sojicola Massalongo Phyllosticta sojicola Massalongo (as ‘‘sojaecola’’), Atti Ist. Veneto Sci. 59: 688. 1900. Synonym: Phyllosticta glycinea (as ‘‘glycineum’’), Tehon & E.Y. Daniels, Mycologia 19: 110–129. 1927. [Not Phyllosticta glycines Thümen, Revista Scientifica e litteraria Ciombra 28: 504. 1880  1881. fide Saccardo, 1884 on leaves of Glycine violacea (¼ Hardenbergia violacea /Schneev./ Stearn.)] ? Phyllosticta sojicola (Massal.) Miura (as cited e.g. by Kurata, 1960). This name is not in use anymore, now it should be considered synonymous with Boeremia exigua var. exigua. Böning (1938) gave a description of the fungus in vitro, and deposited a culture in the CBS collection (CBS 301.39), which resembles Phoma subglomerata (Corda) Wollenw. & Hochapf., producing sparse aerial mycelium, but abundant pycnidia. The typical multicellular chlamydospores were not formed, but in his description Böning (l.c.) mentioned the occurrence of ‘‘zahlreiche Mikrosklerotien mit rundlichen Hyphenzellen’’ in the aerial mycelium and multicellular chlamydospores. Walters & Martin (1981) described this fungus in the USA under this name, as the cause of severe symptoms on pods. The isolate deposited by the authors, ATCC 44494, produces multicellular chlamydospores. In vitro characteristics correspond with those of Phoma pomorum var. calorpreferens Boerema et al. (Kövics et al., 1999). On the basis of study of the type material of Phyllosticta sojicola on leaves of G. max (Massalongo, 1900), Kövics et al. (1999) assumed to represent the plurivorous Phoma exigua var. exigua (¼B. exigua var. exigua). Van der Aa & Vanev (2002) considered the taxonomic status of P. sojicola uncertain, but accepted that Phyllosticta glycineum (as ‘‘glycinea’’) Tehon & E.Y. Daniels as an additional synonym. In the literature, the teleomorph is described as Pleosphaerulina sojicola Miura (1921) (as ‘‘sojaecola’’); however, no ascomata were present on the type material of P. sojicola (Kövics et al., 1999). Von Arx & Müller (1975) listed the genus name Pleosphaerulina among the synonyms of Pringsheimia Schulzer. Barr (1972) and Hawksworth et al. (1995) considered it as a synonym of Saccothecium Fr. These are dothidiaceous genera which do not have connections with the anamorph genus Phoma. Ascochyta sojae Miura Ascochyta sojae Miura, Flora of Manchuria and East Mongolia, III Cryptogams, Fungi. Industr. Contr. S. Manch. Rly 27: 443–444. 1928 Synonym: A. glycines Miura, in Arata Ideta, Suppl. Handb. Pl. Dis. Japan, Vol.II: 682 (1926) nomen nudum. The original description corresponds to an Ascochyta species, with mainly septate, rather large conidia, 12–18  4–4.5 mm in size. Ideta (1926) referred to an Ascochyta species (as ‘‘A. glycines Miura’’) recorded by Miura (1921) on soybean. Later, Miura (1928) formally described this species as Ascochyta sojae. In the literature, this fungus has been considered as a separate species (e.g. Bondartzeva-Monteverde & Vassiljevskiy, 1940, or as a synonym of A. sojaecola 6 G. J. Kövics et al. Crit Rev Microbiol, Early Online: 1–14 Table 1. Pathogenicity of Ph. sojicola and other Phoma-like species on soybean in artificial inoculation trials (Kövics et al., 1999). Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. Average number of spots per trifoliate leaf Phoma sojicola Phoma sojicola Phoma pinodella Phoma pinodella Phoma exigua var. exigua Phoma exigua var. exigua Phoma exigua var. exigua Phoma exigua var. exigua Phoma pomorum var. calorpreferens (as Phyllosticta sojicola) Phoma subglomerata (as Phyllosticta sojicola) Control Isolate Origin Year of isolation Intact leaf* Injured leafy D/054 CBS 113.53 D/082 PD 77/165 D/059 D/060 Ph 58 Ph 75 ATCC 44494 Glycine max, Hungary Glycine max, Germany Pisum sativum, Hungary Pisum sativum, the Netherlands Glycine max, Hungary Glycine max, Hungary Petroselinum crispum, Poland Rubus sp., Poland Glycine max, USA 1993 1953 1993 1977 1993 1993 1990 1993 1979 4.5a 5.1ab 5.2ab 5.0ab 5.9b 5.6ab 5.6ab 5.9b 5.0ab 4.9 5.0 5.0 4.8 6.0 5.5 5.7 6.0 4.9 CBS 301.39 Glycine max, Germany 1939 5.0ab 0 5.0 0 *Figures followed by the same letter(s) do not differ significantly, according to Student’s t-test (p ¼ 0.01). yNo significant differences. (¼Pey. pinodella), e.g. Kurata, 1960; Nelen, 1977). Its occurrence has been reported sporadically from Japan (Iwadare et al., 1943), China (Ling, 1948), Taiwan (Sawada, 1959), Amursk and Khabarovsk Territories of the former Soviet Union (Melnik, 1977) and Costa Rica (Gamboa, 1989). Herbarium material was not available for study (Kövics et al., 1999). Coniothyrium glycines (R.B. Stewart) Verkley & Gruyter Basionym: Pyrenochaeta glycines R.B. Stewart, Mycologia 49: 115. 1957. Synonyms: Dactuliochaeta glycines (R.B. Stewart) G.L. Hartm. & J.B. Sinclair, Mycologia 80 (5): 699. 1988. Dactuliophora glycines C.L. Leakey, Trans. Br. mycol. Soc. 47 (3): 346. 1964. Phoma glycinicola Gruyter & Boerema, Persoonia 17 (4): 554. 2002. [‘2001’] [Not Phoma glycines Sawada in Spec.Publ.Coll.Agric., Nat.Taiwan Univ. 8: 129. 1959. nom. inval. (¼ Phoma glycines Sawada ex J.K. Bai & G.Z. Lu, Fl. Fungorum Sin. 15: 33. 2003.)] This is considered as a serious pathogen recorded on Glycine spp. causing leaf spot in Africa (Ethiopia, Zambia and Zimbabwe) but reliable isolates were not available to examine. The primary host is probably Glycine javanica, but G. max appears to be very susceptible. The leaf spots are reddish-brown, later become necrotic and fall out, giving a ragged appearance (Boerema et al., 2004). Pathogenicity tests The pathogenicity of two isolates of Ph. sojicola was compared with two isolates of Ph. pinodella, four of Ph. exigua var. exigua and two of P. sojicola on 3-week-old plants of G. max cv. McCall (Table 1). The experiment was performed on plants kept intact as well as on plants gently wounded by rubbing with cotton and abrasive (SiC – 400 mesh) before inoculation. The plants, which had five to six trifoliate leaves, were inoculated with a conidial suspension in distilled water. The inoculum concentration was 5  106 conidia mL1. Five plants were grown per 25 cm pot, with five replicates (pots) per treatment (Kövics et al., 1999). Pathogenicity tests have shown that Ph. sojicola causes rather similar disease symptoms to those of other Phoma species occurring on soybean (Table 1). Pycnidia and conidia are also rather similar. Ph. sojicola forms in vitro glabrous, thin-walled pycnidia, with mainly aseptate, but some septate, conidia. Chlamydospores are unicellular, single or in chains, sometimes clustered. It closely resembles Ph. pinodella (¼Pey. pinodella) which, however, produces abundant pycnidia and readily forms dendritic crystals on MA after 1 week (Noordeloos et al., 1993). However, reports of Ph. pinodella from soybean are rare. There were no significant differences in pathogenicity among the Ph. sojicola, Ph. pinodella (¼Pey. pinodella), Ph. exigua var. exigua (¼B. exigua var. exigua) and P. sojicola (¼B. exigua var. exigua) isolates. All isolates produced similar disease symptoms on intact as well as on wounded leaves. More taxa (including A. sojae and Ph. glycinicola) to involve were not available because shortage of living isolates. In Table 2, three other Phoma species are included, which also are reported from soybean, but do not cause characteristic leaf spotting but are well known as soilinhabitants (non-pathogenic ones). The isolate CBS 310.39, deposited and described on soybean by Böning (1938) as P. sojicola, resembles Ph. subglomerata Boerema, de Gruyter & Noordeloos (Boerema, 1993), producing sparse aerial mycelium with abundant pycnidia. Walters & Martin (1981) described P. sojicola in the USA as the cause of severe symptoms on pods. The isolate ATCC 44494 (CBS 568.97) deposited by the authors, producing multicellular chlamydospores, corresponds with Ph. pomorum var. calorpreferens Boerema et al. (Kövics et al., 1999). Phoma multirostrata (Mathur, S.K. Menon & Thirum.) Dorenb. & Boerema has been obtained from necrotic lesions on leaves and stems of many herbaceous plants including soybean. In vitro, the fungus can be differentiated from Negative Positive (Eþ) Negative Negative Negative Sometimes positive (Eþ) Negative Negative Absent Absent Absent Absent Vinaceous Reddish Absent Absent Unicellular Absent Unicellular Unicellular Unicellular Dictyochlamydospores Dictyochlamydospores Dictyochlamydospores Phoma sojicola Phoma exigua var. exigua Phoma pinodella Phoma multirostrata Phoma terrestris Phoma sorghina Phoma subglomerata Phoma pomorum var. calorpreferens Glabrous Glabrous Glabrous Glabrous Setose Glabrous Glabrous Glabrous Aseptate, some septate, 6  5  12  2  5  5 mm Aseptate, some septate, 3  10  1  5  2  5 mm Aseptate, some septate, 4  8  2  3  5 mm Only aseptate, 2  5  7  5  1  5  3 mm Only aseptate, 3  5  7  5  1  5  2  5 mm Only aseptate, 4  8  5  1  5  3  5 mm Aseptate, some septate, 5  12(17)  2  4  5 mm Aseptate, 4  12  2  3  5 mm Occasional, dendritic Absent Present, dendritic Absent Absent Occasional, needle-like Absent Absent Diffusable pigment on OA Crystals Chlamydospores Conidia Pycnidia Table 2. Differential in vitro characters of Phoma-like species recorded from Glycine max (Kövics et al., 1999). Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. NaOH spot test DOI: 10.3109/1040841X.2012.755948 Phoma-like fungi on soybeans 7 Ph. sojicola by the absence of septate conidia and the relatively fast growth of the colonies (Boerema, 1986; Dorenbosch & Boerema, 1973). Phoma terrestris H.N. Hansen (¼Setophoma terrestris /H.N. Hansen/Gruyter, Aveskamp & Verkley, 2010), causing pink rot disease of onions in the warmer regions of the world, can also infect other hosts as shown by inoculation tests of seedlings (Kreutzer, 1941). On soybean, no disease symptoms were observed, but isolations from symptomless root pieces yielded cultures of Ph. terrestris (¼S. terrestris). Thornberry & Anderson (1940) cited unpublished data on the host range of Ph. terrestris supplied by Dr. E.I. Melhus based on root infection inoculation experiments, in which soybean is included. The fungus can be differentiated by its setose pycnidia, characteristic of Phoma sect. Paraphoma (MorganJones & J.F. White) Boerema (Boerema, 1997). Colonies on oatmeal agar are distinctly vinaceous, and the conidia are always aseptate, up to 7  2.5 mm, with two or more distinct guttules. An isolate of Phoma sorghina (Sacc.) Boerema, Dorenb. & Kesteren (¼Leptosphaeria sacchari Breda de Haan, 1892), obtained from seeds of soybean in Nepal, has been deposited in the IMI culture collection (IMI 333584). This fungus is a common soil inhabitant in the tropics and subtropics and may occur as an opportunistic parasite, mainly on Poaceae as Oryza sativa, Sorghum vulgare, Saccharum officinarum and Triticum aestivum, causing spots on leaves and glumes, root rot and dying off. The fungus can also be found on seeds. The best differentiating characteristic is the production of dictyochlamydospores (Boerema et al., 1973, 1977). Ascochyta pisi Lib., also producing larger, mainly septate conidia, has been recorded on soybean from Zimbabwe (Hopkins, 1939, 1950; Whiteside, 1960) and Serbia (Numic, 1962). Inoculation experiments by Bondartzeva-Monteverde & Vassiljevskiy (1940) showed, however, that A. pisi is not pathogenic on soybean. Evolutionary comparisons by phylogenetic markers Up to now, molecular-based phylogenetic analyses within Phoma genus have only been used for defining phylogenetic relationships among isolates within one or closely related species (Balmas et al., 2005; Fatehi et al., 2003; MendesPereira et al., 2003; Voigt et al., 2005). It seems reasonable to combine tools for identification and delimitation of Phoma-like fungi of soybean which could be applicable in Phoma taxonomy. In addition to conventional morphological criteria, there are some other supplementary methods which can contribute to comparison and proper delimitation of Phoma-like fungi. Recently, serial publications came out with reclassification consequences of Phoma-like fungi on the basis of molecular data (Aveskamp et al., 2009a,b, 2010; de Gruyter et al., 2009, 2010; Woudenberg et al., 2009). Furthermore, molecular markers were applied on phylogenetic-based delimitation of the specific Phoma-like species associated with soybeans (Irinyi et al., 2009a,b). The complete sequence of the ITS region, fragments of the TEF1 and b-tubulin genes were obtained from representative isolates of Ph. sojicola (CBS 567.97) and P. sojicola (CBS Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. 8 G. J. Kövics et al. 301.39). Sequences from Ph. pinodella (¼Pey. pinodella), Ph. exigua var. exigua (¼B. exigua var. exigua) and Ph. glomerata isolated from soybean and other hosts; moreover, non-soybean pathogenic Phoma and Ascochyta species were also used in the comparative analyses. Sequences of ITS containing part of the regions 1, 2 and the 5.8S rDNA, the large intron of the TEF1 gene, and a part of b-tubulin gene (tub1) (Irinyi et al., 2009b) were analyzed as phylogenetic markers. The phylogenetic analysis of multiple protein-encoding genes with the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) is proposed as a more robust method of determining and recognizing species than analysis based on morphology or mating behavior (Taylor et al., 2000). Phylogenetic analysis of the two protein-encoding genes, TEF1 and b-tubulin together with the complete ITS sequences yielded consensus results. Ph. sojicola isolates always formed one clade with all the examined Ph. pinodella isolates, while representative isolate of P. sojicola grouped together with Ph. exigua var. exigua (Irinyi et al., 2009b). The highest resolution could be revealed with the analysis of the TEF1 gene, while sequences of b-tubulin showed the lowest polymorphism among the examined Phoma species. Data analysis Primers used to amplify approximately 520 bp of the ITS region containing the ITS regions 1 and 2 and the 5.8S rDNA are based on published composite sequences, SR6R and LR1 (White et al., 1990). The large intron (approximately 300 bp) of the TEF1 gene was amplified by the EF1-728F and EF1-986R primer pair (Druzhinina & Kubicek, 2005). Primers Bt2a and Bt2b (Glass & Donaldson, 1995) were used to amplify a 300 bp fragment of b-tubulin gene. The obtained DNA sequences were aligned first with ClustalX (Thompson et al., 1997) and manually checked for ambiguities and adjusted when necessary using Genedoc (Nicholas et al., 1997). Single gaps were treated either as missing data or as the fifth base and multistate characters were treated as uncertain. For the Bayesian analysis, models of sequence evolution were evaluated for each dataset and model parameter estimates obtained with Modeltest v.3.7 (Posada & Grandall, 1998) using Bayesian Information Criterion. Markov chain Monte Carlo (Larget & Simon, 1999; Mau et al., 1999) was performed with the computer program MRBAYES 3.1.1 (Huelsenbeck & Ronquist, 2001). Markov chains were run 1 M generations from which every 100th tree was sampled. Phylogenetic analyses were performed in PAUP*4.0b (Swofford, 2002). Parsimony analysis (Farris, 1970; Fitch, 1971; Kluge & Farris, 1969) consisted of heuristic searches with 1000 random addition sequences and tree bisectionreconnection branch swapping. Crit Rev Microbiol, Early Online: 1–14 similarity of Phoma TEF1 sequences with the closely related Ascochyta species. Some representative sequences of the different Ascochyta species were chosen for the phylogenetic analysis. A 50% majority-rule consensus tree from a Bayesian analysis of nucleotides is shown (Figure 1). Ascochyta species are grouped together into a strongly supported group (100% posterior probabilities, PP). The two soybean specific Phoma-like species, Ph. sojicola and P. sojicola are grouped together with different plurivorous Phoma species. Phoma sojicola isolates are grouped together with Ph. pinodella (¼Pey. pinodella) isolates from different hosts into a distinct clade (100% PP). The two Ph. exigua var. exigua isolated from soybean and the representative isolate of P. sojicola formed a separate clade within the Ph. exigua (¼B. exigua) vars. (varietas) group (100% PP; Figure 1). Phoma species represented by more than one isolates constitute clades which are well separated from each other like Ph. pinodella and Ph. exigua var. exigua group, which prove that the TEF1 sequences are well suited for delineating phylogenetic relationships within the Phoma genus. The Phoma species are well separated from their closely related Ascochyta taxa. Most of Phoma species (Ph. pinodella, Ph. exigua, Ph. glomerata and Ph. plurivora) are well separated from the other tested Phoma species. Some Phoma species constitute clades but there are some species, which cannot be distinguished on the basis of TEF1 sequences (Ph. pinodella and Ph. sojicola, P. sojicola and Ph. exigua as well as Ph. foveata and Ph. multirostrata). The topology of the most parsimonious tree was similar to the Bayesian consensus tree. Ascochyta isolates formed a separate clade with 100% bootstrap values from the examined Phoma species. The support of both the Ph. pinodella (¼Pey. pinodella) group containing the Ph. sojicola isolated, and the Ph. exigua (¼B. exigua) vars. group, containing the P. sojicola isolate was 100%. b-Tubulin sequences b-tubulin sequences were edited to 298 bp to aid alignment with sequences downloaded from GenBank. Parsimony analysis of b-tubulin (298 bp) revealed 49 parsimony informative sites, 20 polymorphic sites and 229 sites were constant among all isolates. The phylogenetic tree obtained by the analysis of b-tubulin sequences (Figure 2) is really similar to that of the TEF1 and ITS (Figures 1 and 3) trees. Species represented by more than one isolates are also clustered in the same clades and they are well separated from each other. Ascochyta isolates constitute well-separated clades revealing that b-tubulin is also appropriate for inferring phylogenetic relationships in the genus. Translation elongation factor ITS sequences The amplified TEF1 gene fragments had similar size for all isolates. Alignment of TEF1 (310 bp) revealed 173 parsimony informative sites, 16 polymorphic sites and 121 sites are constant among all isolates. Blast analysis showed high The polymerase chain reaction amplification resulted in single fragments approximately 520 bp of the total ITS region. There was no size variation observed among amplified rDNA fragments. Similarly to the TEF1 and b-tubulin trees, the Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. DOI: 10.3109/1040841X.2012.755948 Phoma-like fungi on soybeans 9 Figure 1. Phylogenetic relationships of Phoma strains inferred by the Bayesian analysis of TEF1 sequences. Numbers above lines indicate Bayesian posterior probability values. Numbers given within parenthesis represent the bootstrap values from 1000 bootstrap samples in Parsimony analysis. The columns on the right side represent the Phoma section based on morphological characterization. *Ph. pinodella (D/063) misidentified as ‘‘Ph. exigua var. exigua’’ (Irinyi et al., 2009b). rDNA tree (Figure 3) is well resolved and groups are highly supported. Parsimony analysis of ITS revealed 32 parsimony informative sites, 5 polymorphic sites and 417 sites are constant among all isolates. Species represented by more than one isolates constitute clades which are well separated from each other. Phoma species are also well separated from their closely related Ascochyta taxa. Summary Peyronellaea pinodella (syn. Phoma pinodella) is a wellknown pathogen especially on leguminous plants like Pisum sativum and Trifolium pratense, causing foot rot and leaf spot of pea and black stem disease of red clover (Dorenbosch, 1970; White & Morgan-Jones, 1987). Reports of Ph. pinodella (¼Pey. pinodella) from soybean are rare, and similarity to Ph. sojicola was evident (Kövics et al., 1999). The morphological differences were small, and delimitation was made basically on absent crystal production on MA, in contrast to Ph. pinodella (¼Pey. pinodella). However, this feature seems not to be a stable character. All phylogenetic relationship analyses classified Ph. sojicola in the same clades of Ph. pinodella (¼Pey. pinodella). On the basis of presented GCPSR and morphological results, re-classification of Ph. sojicola was suggested as a synonymous of Ph. pinodella (¼Pey. pinodella) (Irinyi et al., 2009b), this opinion was confirmed by Aveskamp et al. (2010). Within B. exigua (syn. Phoma exigua) species at least nine variety subtaxa (vars.) were admitted (Boerema et al., 2004), Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. 10 G. J. Kövics et al. Crit Rev Microbiol, Early Online: 1–14 Figure 2. Phylogenetic relationships of Phoma strains inferred by the Bayesian analysis of b-tubulin sequences. Numbers above lines indicate Bayesian posterior probability values. Numbers given within parenthesis represent the bootstrap values from 1000 bootstrap samples in Parsimony analysis. The columns on the right side represent the Phoma section based on morphological characterization. *Ph. pinodella (D/063) misidentified as ‘‘Ph. exigua var. exigua’’ (Irinyi et al., 2009b). and confirmed supported by molecular methods (Abeln et al., 2002). Moreover, two new varieties (B. exigua var. gilvescens, B. exigua var. pseudolilacis) were established by Aveskamp et al. (2010). However, the phylogenetic analysis revealed that the current Boeremaean subdivision is incorrect from an evolutionary point of view, revealing the genus to be highly polyphyletic (Aveskamp et al., 2010). Boeremia exigua var. exigua (mentioned as ‘‘Ph. exigua’’), is mainly a weak parasite species (Sutton & Waterston, 1966), and can be pathogenic on soybeans. The E-metabolite production of B. exigua var. exigua is a frequent character but sometimes shows weak discoloration only (Boerema et al., 2004) or might be completely missing at some strains (van der Aa et al., 2000). Formerly, these strains were classified as Ph. exigua var. inoxidabilis (¼B. exigua var. heteromorpha). The incidence of Phyllosticta sojicola on soybeans occurs only sporadically, and examining living cultures is difficult. Morphological characters of the only authentic isolate (CBS 301.39, deposited by Böning, 1939) were available, and complemented by molecular features, and it proved that P. sojicola was synonymous with Ph. exigua var. exigua (¼B. exigua var. exigua) (Irinyi et al., 2009b). This also supports the former hypothesis based on examined type material of P. sojicola on leaves of G. max (Massalongo, 1900), represented the plurivorous Ph. exigua var. exigua (¼B. exigua var. exigua) (Kövics et al., 1999). Acknowledgements We thank Dr. Ildikó Tar, University of Debrecen (Hungary) and Michael McGuire, PhD student at University of Critical Reviews in Microbiology Downloaded from informahealthcare.com by UNIVERSITY OF DEBRECHEN on 02/04/13 For personal use only. DOI: 10.3109/1040841X.2012.755948 Phoma-like fungi on soybeans 11 Figure 3. Phylogenetic relationships of Phoma strains inferred by the Bayesian analysis of ITS sequences. Numbers above lines indicate Bayesian posterior probability values. Numbers given within parenthesis represent the bootstrap values from 1000 bootstrap samples in Parsimony analysis. The columns on the right side represent the Phoma sections based on morphological characterization. *Ph. pinodella (D/063) misidentified as ‘‘Ph. exigua var. exigua’’ (Irinyi et al., 2009b). Debrecen and Indiana University Bloomington (USA) for translation and native English control. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. References Aa HA van der, Boerema GH, Gruyter J de. (2000). Contributions towards a monograph of Phoma (Coelomycetes) VI-1. Section Phyllostictoides: characteristics and nomenclature of its type species Phoma exigua. Persoonia 17:435–56 (Errata in Persoonia 2002;18:53). Aa HA van der, Noordeloos ME, Gruyter J de. (1990). Species concepts in some larger genera of the Coelomycetes. Stud Mycol 32:3–19. Aa HA van der, Vanev S. (2002). 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