Cercosporoid hyphomycetes associated with Tibouchina herbacea (Melastomataceae) in Brazil

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1 Mycol Progress (2014) 13: DOI /s ORIGINAL ARTICLE Cercosporoid hyphomycetes associated with Tibouchina herbacea (Melastomataceae) in Brazil Douglas Ferreira Parreira & Meiriele da Silva & Olinto Liparini Pereira & Dartanha José Soares & Robert Weingart Barreto Received: 19 June 2010 /Revised: 5 December 2013 /Accepted: 9 December 2013 /Published online: 16 January 2014 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2014 Abstract A survey of fungal pathogens associated with cane tibouchina, Tibouchina herbacea (Melastomataceae), was conducted in the neotropics, concentrated in south and southeastern Brazil and aimed at finding potential biological control agents for this herbaceous invasive weed for introduction into Hawaii. Numerous fungal species were found, including five cercosporoid fungi. Four new taxa are described and illustrated here: Passalora tibouchinae, Pseudocercospora subsynnematosa, Pseudocercospora tibouchina-herbaceae and Pseudocercospora tibouchinicola. A phylogenetic analysis of the ITS and TEF-1α regions of the three new species of Pseudocercospora was conducted and confirmed their separation as distinct taxa as indicated by morphological and cultural features. Additionally, Cercospora apii is reported for the first time on T. herbacea. Among the species of fungi described in this study, two appear particularly promising for use in a biological control program because they were found Taxonomical novelty: Passalora tibouchinae D.F. Parreira, O.L. Pereira & R. W. Barreto Pseudocercospora subsynnematosa D.F. Parreira, D.J. Soares & R. W. Barreto, Pseudocercospora tibouchina-herbaceae D.F. Parreira & R.W. Barreto, Pseudocercospora tibouchinicola D.F. Parreira, D.J. Soares & R. W. Barreto. D. F. Parreira: M. da Silva : O. L. Pereira : D. J. Soares : R. W. Barreto (*) Departamento de Fitopatologia, Universidade Federal de Viçosa (UFV), Viçosa , MG, Brazil rbarreto@ufv.br D. F. Parreira douglas.parreira@ufv.br M. da Silva meiriele.silva@ufv.br O. L. Pereira oliparini@ufv.br D. J. Soares dartanha.soares@embrapa.br to cause severe disease on T. herbacea: Passalora tibouchinae and Pseudocercospora subsynnematosa. Additional tests, including host-specificity screening, will be required in order to confirm this potential. Keywords Classical biological control. Cercospora. Fungal survey. New taxa. Passalora. Pseudocercospora Introduction Tibouchina herbacea (DC.) Cogn. (Melastomataceae), known as cane tibouchina (local names in Brazil: quaresmeirinha, quaresmeira do brejo), is a herbaceous plant of humid habitats that is native to South America, namely Argentina, Brazil, Paraguay and Uruguay (Wagner et al. 1999). It was introduced into the Hawaiian Islands as an ornamental for its showy violet flowers and has since become a noxious invasive weed. It is presently found in the islands of Maui, Hawaii, Molokai, Oahu and Lanai ( tibouchina_herbacea.htm). There are no native species of Melastomataceae in Hawaii and all species in this family that have been introduced are noxious weeds, including Clidemia hirta (L.) D. Don and Miconia calvescens DC., which are ranked amongst the worst weeds in that archipelago (DeWalt et al. 2003). In its native habitat, T. herbacea reaches a maximumheightof1.5m,whereasinhawaiitcanreachupto4m, forming dense stands in pastures and disturbed areas (Almasi 2000; Motooka et al. 2003). It is included in the State of Hawaii noxious weed list ( herbacea.htm) and its management has been made mainly through herbicide applications. However, classical biological control has been recognized as the sole sustainable method of control for exotic weeds invading natural ecosystems, as is the case of the weedy melastomes in the Pacific (Wikler and Souza 2005).

2 692 Mycol Progress (2014) 13: Insect surveys in Brazil yielded a series of potential biocontrol agents to be deployed against T. herbacea, but Syphraea uberabensis (Bechhyné, 1956) (Coleoptera: Chrysomelidae) was demonstrated to have the greatest potential (Wikler and Souza 2005; Souder 2008). Studies aimed at introducing this particular insect are well advanced. Syphraea uberabensis is now under quarantine in Hawaii for its final evaluation (T. Johnson, personal communication). Parallel to this, systematic field surveys of the mycobiota on T. herbacea were also conducted. This publication represents the first study of the mycobiota of T. herbacea in Brazil and is the first account of fungi associated with this plant species worldwide. No records of fungi associated with T. herbacea were found in the literature and it was expected that a significant proportion of the fungi collected on this host may represent taxonomic novelties and that some could be potential biocontrol agents for use against this host. Sixteen fungal species have been identified, thus far. This paper concentrates on the descriptions and a discussion of five cercosporoid hyphomycetes. The remaining mycobiota will be covered in a separate publication. Tibouchina herbacea is regarded as a species complex that remains unresolved (R. Goldenberg, personal communication), and the records provided here include fungi collected on herbaceous forms of Tibouchina which may belong to more than one host species. More precise information on host identity will await further clarification of species delimitation in this complex. Material and methods Prior to the field survey, a list of collecting localities from herbarium records of T. herbacea was compiled from the following Brazilian herbaria: Herb. IAC, Herbário Fanerogâmico e Criptogâmico do Instituto Agronômico; Herb. SP, Herbário Maria Eneyda P. K. Fidalgo; Herb. RB, Instituto de Pesquisas Jardim Botânico do Rio de Janeiro; Herb. R, Herbário do Museu Nacional do Rio de Janeiro; Herb. VIC, Universidade Federal de Viçosa; Herb. MBM, Museu Botânico Municipal; Herb. OUPR, Herbário José Badini. A range of sites in southeastern and southern Brazil was explored based on the herbarium records. Details of the survey and the laboratory methodology can be found in Barreto and Evans (1994). Morphological studies Diseased T. herbacea plants found in the field were photographed (Fig. 1) and samples were collected and dried in a plant press and taken to the laboratory for further observation. Each sample was examined under a stereomicroscope (Olympus SZ X7). Sections containing the fungal structures and fungal structures scraped with a scalpel from the plant surfaces were mounted in lactophenol. Observations, measurements and line drawings were prepared using an Olympus BX 51 light microscope fitted with a drawing tube and an Olympus E330 camera. The collections examined were deposited in the herbarium of the Universidade Federal de Viçosa (VIC). Isolates Pure cultures were obtained by transfer of conidia from conidiophore fascicles on leaves onto potato dextrose agar (PDA) plates with a sterile fine pointed needle. The cultures obtained were deposited in Coleção de Culturas de Fungos Fitopatogênicos Prof. Maria Menezes (CMM) (Table 1)and in the culture collection of the Universidade Federal de Viçosa (COAD). Plates were seeded with a 7-mm culture disk obtained from the margin of actively growing cultures and placed in the center of each Petri plate. Colony characters and pigment production on PDA and V8 juice-agar (V8) were observed in colonies incubated at 25 C under two conditions: complete darkness and 12/12 h light/dark. Descriptions were prepared after 20 days of incubation. Colony colors were rated according to Rayner (1970). Phylogenetic analysis Mycelium was scraped from cultures grown on PDA plates, placed in a sterile 1.5-mL microcentrifuge tube and ground into fine powder using liquid nitrogen. Genomic DNA of the isolates was extracted using the Wizard Genomic DNA Purification kit (Promega) according to the manufacturer s instructions. Two nuclear gene regions were targeted for PCR amplification. The internal transcribed spacer region (ITS) and the partial gene regions of the translation elongation factor 1- alpha (TEF-1α). These genes were amplified and sequenced using the primer pair ITS-1 and ITS-4 (White et al. 1990)and EF1-728 F and EF1-986R (Carbone and Kohn 1999). PCR conditions for each 25 μl reaction were as follows: 2 μl of genomic DNA (25 ng/μl), 12.5 μlofdreamtaqtm PCR Master Mix 2X (MBI Fermentas), 1 μl ofeachprimer synthesized by Invitrogen, 1 μl of dimethyl sulfixide (DMSO, Sigma-Aldrich), 5 μl of 100 (10 mg/ml) bovine serum albumin (BSA; Sigma-Aldrich) and nuclease-free water to complete the volume. The amplifications were carried out starting with a BIO RAD C1000 (Thermal Cycler) with initial denaturation at 95 C for 5 min, followed by 35 cycles of 94 C for 60 s, annealing at 60 C for ITS and at 56 C for EF for 45 s, 72 C for 60 s and a final extension of 72 C at 7 min. Amplified products were visualized on 1 % agarose gel stained with GelRed and viewed under UV light to check for product size and purity. PCR products

3 Mycol Progress (2014) 13: Fig. 1 Selected leaf spot symptoms on Tibouchina herbaceaeto which cercosporoids collected in the survey were associated: 1 Passalora tibouchinae; 2 Pseudocercospora subsynnematosa; 3 Pseudocercospora tibouchinaherbaceae; 4 Pseudocercospora tibouchinicola were purified and sequenced by Macrogen, South Korea ( The DNA sequences obtained from forward and reverse primers were used to obtain consensus sequences using DNA Dragon software (Hepperle 2011). The sequences obtained in this study were deposited in GenBank and compared against other sequences of ITS and TEF1-1α in NCBI s GenBank nucleotide (nr) database using a megablast search program to identify their closest species and alignments were constructed based on these results (Table 1). Sequences for relevant taxa for this study such as Pseudocercospora dissotidis (Chupp & Doidge) Crous & U. Braun and Pseudocercospora tamoneae (Chupp) U. Braun & R.F. Castañeda have not been included because there are no sequences available for these in public databases. The hit sequences were then downloaded in FASTA format and aligned using multiple sequence alignment program MUSCLE (Edgar 2004). Alignments were manually adjusted when necessary in MEGA v.5 software (Tamura et al. 2011). Bayesian inference analyses (BI) were performed with single and concatenated gene regions (ITS and FE-1α). MrMODELTEST 2.3 (Posada and Buckley 2004) was used to select the models of nucleotide substitution for each gene region and included in the BI analysis. Once the likelihood scores were calculated, the models were selected according to the Akaike Information Criterion (AIC) applying a SYM+I+G for ITS and HKY+G for FE-1α. A phylogenenetic analysis of the concatenated aligment was performed on CIPRES webportal (Miller et al. 2010) using MrBayes v (Ronquist and Huelsenbeck 2003). Analyses of four Markov chain Monte Carlo (MCMC) were run from random trees for generations and sampled every 1,000 generations, resulting in 10,000 saved trees. The first 2,500 trees, which represented the burn-in phase of the analyses, were discarded and posterior probabilities (Rannala and Yang 1996)

4 694 Mycol Progress (2014) 13: Table 1 GenBank accession numbers of DNA sequences of isolates of Pseudocercospora and Pseudocercospora-like used in phylogenetic analyses The specimens obtained in this study are highlighted in bold CBS Centraalbureau voor Schimmelcultures, Utrecht, Netherlands; CPC culture collection of Dr. Pedro Crous, housed at CBS; MUCC Murdoch University culture collection, Australia; CMM Coleção de Culturas de Fungos Fitopatogênicos Prof. Maria Menezes, Brazil Spicies Isolate Genbank acession no. References ITS EF Mycosphaerella africana CMW3026 DQ DQ Hunter et al Mycosphaerella aurantia CBS AY DQ Crous et al Mycosphaerella ellipsoidea CBS AY JX Quaedvlieg et al Mycosphaerella laricina CBS GU GU Crous et al Paracercospora egenula CPC GU GU Crous et al Paracercospora egenula MUCC 883 GU GU Crous et al Pseudocercospora cercidis-chinensis CBS14481 GU GU Crous et al Pseudocercospora cordiana CBS GU GU Crous et al Pseudocercospora exosporioides MUCC 893 GU GU Crous et al Pseudocercospora fukuokaensis CPC GU GU Crous et al Pseudocercospora fukuokaensis MUCC 887 GU GU Crous et al Pseudocercospora glauca CPC GU GU Crous et al Pseudocercospora kaki MUCC 900 GU GU Crous et al Pseudocercospora lythracearum CPC GU GU Crous et al Pseudocercospora lythracearum MUCC 890 GU GU Crous et al Pseudocercospora mali MUCC 886 GU GU Crous et al Pseudocercospora pancratii CBS GU GU Crous et al Pseudocercospora paraguayensis CBS JQ GU Crous et al Pseudocercospora paraguayensis CBS DQ JX Hunter et al Pseudocercospora pini-densiflorae MUCC 534 GU GU Crous et al Pseudocercospora pyracanthigena CPC GU JX Quaedvlieg et al Pseudocercospora schizolobii CBS KF KF Verkley et al Pseudocercospora sp. CPC JQ JQ Crous et al Pseudocercospora sp. CBS GU GU Crous et al Pseudocercospora subsynnematosa CMM 2892 KF KF Present work Pseudocercospora tibouchina-herbaceae CMM 2894 KF Present work Pseudocercospora tibouchinicola CMM 2813 KF Present work Pseudocercospora tibouchinigena CBS GU GU Crous et al Pseudocercospora viticicola MUCC 777 GU GU Crous et al Septoria rubi CPC GU GU Crous et al determined from the remaining trees. New sequences generated in this study were deposited in NCBI s GenBank nucleotide database ( Table 1). Phylogenetic trees were visualized in FigTree (Rambaut 2009) and resulting alignment was deposited into TreeBASE (TreeBASE study S14808). ( treebase/index.html). Results Phylogeny For the combined analysis, DNA sequence data from the ITS and TEF1-α gene regions were utilized. The final alignment contained 29 ingroup taxa with a total of 812 characters and Septoria rubi (CPC 12331; GenBank Acession: GU269865, GU483576) served as outgroup taxon. From the combined alignement of 812 characters, 177 were parsimony informative, 256 were variable, and 527 were conserved. The molecular analysis (Fig. 2) confirmed that the three Pseudocercospora species found on Tibouchina herbaceae,as indicated by the morphology data, are distinct and warrant recognition as separate and novel species. Taxonomy Cercospora apii Fresen. sensu lato, emend. Crous and Braun, Mycosphaerella and its anamorphs: 1. Names published in Cercospora and Passalora. CBS Biodiversity Ser. 1: (2003). (Fig. 3)

5 Mycol Progress (2014) 13: Fig. 2 Multilocus phylogenetic tree inferred from Bayesian analysis based on the combined sequences of the ITS and TEF-1α. Bayesian posterior probabilities are indicated above the nodes. Septoria ribi represents the outgroup taxon. The species obtained in this study are in bold Lesions on living leaves, necrotic, initially circular to ellipsoid, later coalescing to form large spots, mm, with indistinct margins, brown. Internal hyphae μm diam, branched, septate, brown. External hyphae not observed. Stromata reduced to few cells in the substomatal cavities. Conidiophores hypophyllous, arising through stomata, fasciculate, erect, subcylindrical, μm, 2 5- septate, unbranched, brown, paler at apex, thin-walled, smooth. Conidiogenous cells terminal and intercalary, integrated, subcylindrical, proliferating sympodially, μm, light brown. Conidiogenous loci conspicuous, 1 3 per cell, μm diam., thickened, darkened. Conidia solitary, ranging from obclavate-cylindrical to (mostly) acicular-filiform, straight to curved, μm, apex acute, base truncate, 2 7-septate, guttulate, hyaline, thinwalled, smooth, hila thickened and darkened. KnowndistributiononlivingleavesofTibouchina herbacea in Brazil: Espírito Santo, Minas Gerais, Paraná and Rio Grande do Sul. Material examined: Minas Gerais Viçosa, 02 Feb. 2001, R.W. Barreto (VIC 30569); Paraná, Curitiba, 30 Mar 1998, R.W. Barreto (VIC 30620); Rio Grande do Sul, Nova Petrópolis, 18 Jan 2001, R.W. Barreto (VIC 30695); Rio Grande do Sul, Nova Petrópolis, 19 Jan 2001, R.W. Barreto (VIC 30696); Minas Gerais, Viçosa, road to Cajuri, 16 Mar 1996, R.W. Barreto (VIC 30660); Minas Gerais, Jequeri, 21 Apr 1996, R.W. Barreto (VIC 30668); Minas Gerais, Juiz de Fora, 06 Nov 1998, R.W. Barreto (VIC 30673); Espírito

6 696 Mycol Progress (2014) 13: Fig. 3 Cercospora apii(vic 30569) 5Acicular conidia. 6 Conidiophores arising through stomata either, solitary or fasciculate. Bar 10 μm Fig. 4 Passalora tibouchinae (VIC 30568). 7 Conidia. 8 Conidiophore fascicles arising through stomata. Bars 10 μm Santo, Venda Nova do Imigrante, entrance of Parque Estadual Pedra Azul, 09 Dec 2004, O.L. Pereira (VIC 30682). Passalora tibouchinae D.F. Parreira & O.L. Pereira sp. nov. (Fig. 4) MycoBank: Etymology: named in reference to the host genus. Stromata μm. Conidiophores μm, 0 6-septate. Conidia solitary, acicular to obclavate, μm, 2 7-septate. Leaf spots initially small becoming circular, brown with an outer reddish margin and light brown center, mm diam. Internal hyphae μm diam., branched, septate, light brown. External hyphae absent. Stromata variable in size and shape, immersed either reduced to few cells filling the substomatal cavity or well developed, μm, composed of dark brown textura angularis. Conidiophores amphigenous, arising through stomata, fasciculate, erect, straight to slightly curved or sinuose, subcylindrical, μm, 0 6-septate, unbranched, brown, becoming paler at apices, thin-walled, smooth. Conidiogenous cells terminal, integrated, cylindrical, conspicuously geniculate, proliferating sympodially, μm, light brown. Conidiogenous loci conspicuous, 1 5 per cell, 1 2 μm diam, thickened and darkened. Conidia solitary, cylindrical to filiform, straight to curved, μm, apex obtuse or subacute, base subtruncate, 2 7-septate, guttulate, light brown, thin-walled, smooth, hila thickened and darkened. In culture (CMM 2816): Colonies slow-growing (10 18 mm diam after 20 days), flat, somewhat lobate, sometimes appressed, aerial mycelium velvety, iron gray to olivaceousblack sometimes greenish black in the center followed by an intermediate iron gray to olivaceous-gray area and greenish black margin, reverse olivaceous-gray; diurnal zonation observed under light/darkness alternation regime; no sporulation. KnowndistributiononlivingleavesofTibouchina herbacea in Brazil: Espirito Santo, Minas Gerais, Paraná, Rio de Janeiro, Rio Grande do Sul and São Paulo. Material examined: Espirito Santo, Ibatiba, 9 Dec 2004, O.L. Pereira (VIC 30568, holotype; CMM 2816, ex type culture); Espírito Santo, Venda Nova do Imigrante, entrance of Parque Estadual da Pedra Azul, 9 Dec 2004, O.L. Pereira (VIC 30680); Espírito Santo, Venda Nova do Imigrante, entrance of Parque Estadual Pedra Azul, 9 Dec 2004, O.L. Pereira (VIC 30681); Minas Gerais, Lagoa da Prata, 5 Feb 2005, O.L. Pereira (VIC 30683); Paraná, Curitiba, Road

7 Mycol Progress (2014) 13: Curitiba-Paranaguá Km 51, 31 Mar 1998, R.W. Barreto (VIC 30619); Rio de Janeiro, Nova Friburgo, 24 Feb 1998, R.W. Barreto (VIC 30621); Rio de Janeiro, Vila do Grama, 24 Feb 1998, R.W. Barreto (VIC 30625); Minas Gerais, Rio Pomba, 19 Oct 1998, R.W. Barreto (VIC 30628); Minas Gerais, Santa Barbara do Tugúrio, 21 Nov 1998, R.W. Barreto (VIC 30629); Rio Grande do Sul, Nova Petrópolis, 19 Jan 2001, R.W. Barreto (VIC 30696); Minas Gerais, Coronel Pacheco, 13 Mar 2002, R.W. Barreto (VIC 30641); Rio de Janeiro, Nova Friburgo, 6 Apr 2008, R.W. Barreto (VIC 30643); São Paulo, Bananal, Serra da Bocaina, 28 Dec 1995, R.W. Barreto (VIC 30656); Minas Gerais, Araponga, Cachoeira do Estouro, 9 Mar 1996, R.W. Barreto (VIC 30659); Minas Gerais, Alto Caparaó, near Hotel Caparaó, river margin, 31 Mar 1996, R.W. Barreto (VIC 30665); Rio de Janeiro, Nova Friburgo, 19 Oct 1996, R.W. Barreto (VIC 30671). Pseudocercospora subsynnematosa D.F. Parreira & D.J. Soares sp. nov. (Fig. 5) MycoBank: Differing from all species of Pseudocercospora described on members of Melastomataceae by having conidiophores aggregated in short compact synnemata. Leaf spots initially small and discrete brown surrounded by a reddish brown margin, circular to elliptic, 2 7 mm diam, later developing into a larger spots, light brown in the center, with a reddish brown margin, vein-delimited. Internal hyphae thin-walled, μm diam, branched, septate, light brown. External hyphae absent. Stromata epiphyllous, welldeveloped, immersed to erumpent, subsphaerical, μm, composed of dark brown textura angularis. Conidiophores epigenous, aggregated in dense short synnemata, erect, straight to slightly sinuose, subcylindrical, μm, 1 5-septate, unbranched, brown, becoming paler at the apex, thin-walled, smooth. Conidiogenous cells terminal, integrated, cylindrical, μm, light brown, smooth. Conidiogenous loci often protruding as a short stalk, 1 2 per cell, μm diam, unthickened, not darkened. Conidia solitary, obclavate-cylindrical to subcylindrical, attenuating gradually towards the apex, straight to slightly curved, μm, apex subacute to occasionally obtuse, base either subtruncate or sometimes protruding as a short stalk, 4 15-septate, guttulate, pale olivaceous to pale brown, smooth, thin-walled, hila unthickened, not darkened. In culture (CMM-2892): Colonies slowgrowing (9 13 mm diam. after 20 days), appressed, flat, with lobate margins, felty, olivaceous-gray to iron gray in the center becoming olivaceous-gray to pale olivaceous-gray towards the margins, reverse iron gray to greenish black, with diurnal zonation under light/dark alternation regime; sporulation abundant on V8 and scarce to absent on PDA. KnowndistributiononlivingleavesofTibouchina herbacea in Brazil: Minas Gerais. Material examined: Minas Gerais, Tabuleiro, 15 Oct 2007, D.F. Parreira and D.J. Soares (VIC 30717). Minas Gerais, Tabuleiro, 26 Sep 2009, D.F. Parreira and R.W. Barreto (VIC 30717, holotype; CMM-2892, COAD-276, ex type culture). Pseudocercospora tibouchinicola D.F. Parreira & D.J. Soares sp. nov. (Fig. 6) MycoBank: Similar to Pseudocercospora dissotidis but having smaller conidiophores ( μm) and longer and narrower conidia ( μm). Colonies on living leaves, not forming conspicuous leaf spots, growing mainly abaxially, forming a dense olivaceous mat composed of conidia and conidiophores, colonies somewhat vein-delimited abaxially; adaxially colonies sparse and less commonly formed; on senescing leaves with green islands forming around colonized areas, whereas the rest of the lamina becomes necrotic and red or brown colored. Internal hyphae μm diam, branched, septate, hyaline, thinwalled. External hyphae absent. Stromata absent. Conidiophores arising through stomata, sparsely fasciculate, erect, straight, subcylindrical, μm, 1 4-septate, mostly unbranched, light brown, thin-walled, smooth. Conidiogenous cells terminal, integrated, μm, pale olivaceous, smooth. Conidiogenous loci protruding, 1 2 per cell, 1 2 μm diam, unthickened, not darkened. Conidia solitary, obclavate-cylindrical to subcylindrical, straight to curved, occasionally somewhat distorted by one infra-median heeled cell, μm, apex subacute, occasionally obtuse, with a strongly protruding hilum at the base, 1 16-septate, guttulate, pale olivaceous, thin-walled, smooth, hila unthickened, not darkened. In culture (CMM 2813): Colonies slowgrowing (10 13 mm diam. after 20 days), flat, aerial mycelium velvety gray-olivaceous, olivaceous-gray, smoke gray to iron gray centrally, pale olivaceous to olivaceous-gray or greenish black at periphery, reverse olivaceous-gray to iron gray, in the middle iron gray at the periphery; no sporulation. KnowndistributiononlivingleavesofTibouchina herbacea in Brazil: Minas Gerais. Material examined: Minas Gerais, Tabuleiro, 15 Oct. 2007, D.F. Parreira and D.J. Soares (VIC 30564, holotype; CMM 2813, COAD-127 ex type culture). Pseudocercospora tibouchina-herbaceae D.F. Parreira & R.W. Barreto sp. nov. (Fig. 7) MycoBank: Similar to Pseudocercospora tamoneae but having narrower conidiophores aggregated in sporodochia, longer and narrower conidia which are 3-11 septate and also differing from P. subsynnematosa by the absence of synnemata and a truncate conidial base.

8 698 Mycol Progress (2014) 13: Fig. 5 Pseudocercospora subsynnematosa (VIC 30565). 9 Conidia (note hila varying from subtruncate to protuberant). 10 Conidiophores aggregated in dense, subsynnematous fascicle. 11 Close-up of conidiophores. Bars 10 μm Leaf spot similar to those caused by P. subsynnematosa,but larger, mm diam. Internal hyphae indistinct. External hyphae absent. Stromata subglobose to irregular, welldeveloped, sub-immersed to erumpent, μm, composed of dark brown textura angularis. Conidiophores aggregated in sporodochia, erect, straight to slightly curved or somewhat sinuose, subcylindrical, μm, 0 4-septate, unbranched, light brown, thinwalled, smooth. Conidiogenous cells terminal, integrated, subcylindrical, μm, light brown. Conidiogenous loci inconspicuous, 1 2 per cell, 1 3 μm diam, unthickened, not darkened. Conidia solitary, cylindrical to acicular, straight to slightly curved, sometimes distorted by one suprabasal to infra-median curved cells, μm, apex subacute, base truncate, 3 11-septate, guttulate, pale olivaceous, thinwalled, smooth, hila unthickened and not darkened. In culture (CMM-2894): Colonies slow-growing (13 18 mm diam. after 20 days), flat, sometimes ridged, aerial mycelium velvety, pale olivaceous gray, lavender gray, gray-olivaceous to olivaceousblack in the center with an olivaceous-gray, apricot or olivaceous-black intermediate area and iron gray margins, reverse olivaceous gray to pale olivaceous with iron gray margin; coloring the medium with a pink diffusate; no sporulation. KnowndistributiononlivingleavesofTibouchina herbacea in Brazil: Minas Gerais, Paraná, Rio de Janeiro, Rio Grande do Sul. Material examined: Rio de Janeiro, Itatiaia, Parque Nacional do Itatiaia, 27 Dec 1995, R.W. Barreto (VIC 30653, holotype; CMM-2894, COAD-48 ex type culture); Minas Gerais, Rio Pomba, 19 Oct 1998, R.W. Barreto (VIC 30628); Minas Gerais, Ouro Preto, Reserva Biológica do Tripuí, 1 Aug 1998, R.W. Barreto (VIC 30566); Minas Gerais, Ouro Preto, Rio Grande do Sul, Nova Petrópolis, 18 Jan 2001,

9 Mycol Progress (2014) 13: Fig. 6 Pseudocercospora tibouchinicola (VIC 30564). 12 Conidia (note geniculation on some conidia, wide range of sizes and shapes and protuberant hila). 13 Conidiophores arising from stomata. Bars 10 μm R.W. Barreto (VIC 30636); Rio de Janeiro, Minas Gerais, Manhuaçu, road Belo Horizonte-Vitória, 30 Mar 1996, R. W. Barreto (VIC 30661); Minas Gerais, Barão de Cocais, Santuário do Caraça, 3 May 1996, R.W. Barreto (VIC 30667). Discussion The cercosporoid fungi are among the most common of all plant pathogens (Crous and Braun 2003) and several papers have already been published dealing with fungi in this group from Brazil (Crous et al. 1997; Braun et al. 1999; Dornelo- Silva et al. 2007; Rocha et al. 2008). The Cercospora species collected on T. herbacea fits well within Cercospora apii sensu lato. Many species of Cercospora that were distinguished from C. apii based solely on host-association were reduced to synonymy with the latter species (Crous and Braun 2003). There are, nevertheless, still many unresolved issues regarding taxonomic delimitation at the species or infraspecific level within the C. apii complex, as revealed by later studies (Groenewald et al. 2005, 2006). This represents the first report of C. apii on a member of the Melastomataceae. Fig. 7 Pseudocercospora tibouchina-herbaceae (VIC 30653). 14 Conidia. 15 Conidiophores on a dense immersed stroma. Bars 10 μm There are no previous reports of a fungus in the genus Passalora on a member of the Melastomataceae, and it is clear that Passalora tibouchinae represents a new species for the genus. With the new additions included in this work, there are now 19 species of Pseudocercospora recorded on members of the Melastomataceae (Farr and Rossman 2013). Nevertheless, only two have been reported in association with Tibouchina spp: [Pseudocercospora tibouchinae (Viégas) Deighton and Pseudocercospora tibouchinigena Crous & U. Braun], and both differ from our species by having smaller conidiophores and conidia (Viégas 1945; Crous et al. 2013). Information on the morphology of these taxa is given in Table 2. The three new species of Pseudocercospora introduced here have clear morphological differences that allow their distinction as new and separate taxa. Pseudocercospora tibouchinicola is somewhat similar to Pseudocercopora dissotidis (Chupp & Doidge) Crous & U. Braun by having no stromata and forming indistinct leaf spots. All other Pseudocercospora species on the Melastomataceae are associated with leaf spots. Conidiophores of P. tibouchinicola are smaller and narrower and conidia are longer and narrower than those of P. dissotidis (Table 2). Pseudocercospora tibouchinicola grouped in a

10 700 Mycol Progress (2014) 13: Table 2 Morphology of Pseudocercospora species recorded on the Melastomataceae Species Stromata Conidiophores Conidia References Size (μm) Size (μm) Septation Size (μm) P. aciotidis (Chupp) U. Braun & Crous Reduced to few cells Chupp 1954 P. curta (Syd.) U. Braun & Crous diam Absent Sydow 1939 P. dissotidis (Chupp & Doidge) Crous & U. Braun Absent Crous and Braun 1996 P. erythrogena (G. F. Atk.) U. Braun Absent or reduced to Braun 1999 few cells P. leandrae (Syd.) U. Braun diam Sydow 1939 P. melastomobia (W. Yamam.) Deighton diam Septate Hsieh andgoh 1990 P. miconiae (Gonz. Frag. & Cif.) U. Absent or reduced to 45 6 Septate Saccardo 1972 Braun & Crous few cells P. miconiicola (Chupp) U. Braun & Crous diam Chupp 1954 P. miconiigena U. Braun & R. Urtiaga Absent Septate Braun and Urtiaga 2008 P. mirandensis (Chupp) R.F. Castañeda &U.Braun diam Rarely Kirschner and Piepenbring 2006 P. monochaeticola (Chupp) U. Braun & Crous diam Rarely Chupp 1954 P. osbeckiae (Chona, Lall & Munjal) Kamal, or Septate Kamal and Khan 1990 M.K. Khan & R.K. Verma lacking P. oxysporae (A.K. Kar & M. Mandal) Deighton diam Deighton 1987 P. subsynnematosa D.F. Parreira & D.J. Soares present work P. tamoneae (Chupp) U. Braun & R.F. Castañeda Braun and Castañeda 1991 P. tibouchina-herbaceae D.F. Parreira & Present work R.W. Barreto P. tibouchinae (Viégas) Deighton Viégas, 1945 P. tibouchinicola D.F. Parreira & D.J. Soares Absent Present work P. tibouchinigena Crous & U. Braun Crous et al clade that accommodates species of Pseudocercospora s. l. and is phylogenetically distant from the other two new species, P. subsynnematosa and P. tibouchina-herbacea, both of which are leaf spot-causing pathogens (Fig. 5). Pseudocercospora subsynnematosa is easily distinguished from other Pseudocercospora on Melastomataceae, including the newly described species, by its conidiophore arrangement in short compact synnemata. Pseudocercospora tibouchinaherbaceae does not produce synnemata, as in the former species, and has longer and narrower conidia which have a truncate base, never ending in a strongly protruding hilum as commonly seen in P. subsynnematosa. Its morphology is somewhat similar to that of P. tibouchinicola but it has conidiophores arranged in sporodochia, whereas P. tibouchinicola does not produce sporodochia or stromata and does not cause leaf spots on T. herbaceae. Additionally, P. tibouchinaherbacea consistently produces a pink diffusate in the medium whereas P. tibouchinicola lacks pigments and grows more slowly. Finally, the two species occur on different forms of T. herbacea: P. tibouchina-herbaceae being found on the most common form of the weed, whereas P. tibouchinicola occurs only on the more shrubby rarer form of T. herbacea. Phylogenetically, P. subsynnematosa grouped in a clade that includes species of Pseudocercospora s.l. close to Pseudocercospora schizolobii (M.J. Wingf. & Crous) M.J. Wingf. & Crous (Fig. 5). Nevertheless, P. schizolobii is a pathogen of a distantly related host [Schizolobium parahybum (Vell. Conc.) S. F. Blake (Fabaceae)] and has a distinct morphology shorter and narrower conidia ((30) (80) (2.5) 3 (3.5) μm) and is clearly not conspecific with the fungus on Tibouchina. Pseudocercospora tibouchinaherbaceae is morphologically similar to P. tamoneae but differs from the latter by having narrower conidiophores and longer and narrower conidia. It grouped in a clade close to P. tibouchinigena which, according to Crous et al. (2013), is not well resolved because it does not have the typical features of Pseudocercospora s.str. It is thought that taxa in this subclade may eventually be recognized as a distinct genus, but until the clade has been more fully resolved, this species is retained in Pseudocercospora. Among the species of fungi described in this study, two appear to have potential for introduction as classical biological control agents against T. herbacea for being associated with severe disease symptoms: Passalora tibouchinae and Pseudocercospora subsynnematosa. Although the specificity of these fungi has not yet been tested, P. tibouchinae and

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