Polyphasic Characterisation of Three New Phyllosticta Spp
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Persoonia 28, 2012: 76–84 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE http://dx.doi.org/10.3767/003158512X645334 Polyphasic characterisation of three new Phyllosticta spp. Y.Y. Su1, L. Cai1 Key words Abstract Three new species of Phyllosticta, P. hostae on Hosta plantaginea (China), P. schimae on Schima superba (China), and P. ilicisaquifolii on Ilex aquifolium (UK), are described and illustrated in this study. They are molecular compared with morphologically similar and phylogenetically closely related species. A polyphasic approach using morphology phylogeny, host association, disease symptoms, colony and morphological characteristics, is employed to justify phylogeny the introduction of the new taxa. Phylogenetic relationships of the new species with other Phyllosticta species are systematics revealed by DNA sequence analyses based on the nrDNA-internal transcribed spacer (ITS) regions and a combined taxonomy multilocus alignment of the ITS, partial translation elongation factor 1-alpha (TEF1), actin (ACT), and glyceraldehyde 3-phosphate dehydrogenase (GPDH) gene regions. Article info Received: 14 December 2011; Accepted: 2 April 2012; Published: 27 April 2012. INTRODUCTION unreliable use of host-association based nomenclature. Poly- phasic approaches combining morphological characters and Many Phyllosticta (teleomorph Guignardia) species cause plant phylogenetic relationships can resolve species relationships, diseases such as leaf spots, leaf blotch, as well as black spots based on which a natural classification could be established and lesions on fruits of various plants (van der Aa & Vanev (Wulandari et al. 2009, Glienke et al. 2011). Although the rDNA- 2002). These plant pathogenic fungi may cause serious dam- internal transcribed spacer (ITS) locus has some resolution at age to the host plant through reduced photosynthetic ability and species level, it is insufficient for separating cryptic species in premature leaf or fruit fall (Glienke-Blanco et al. 2002, Baldas- Phyllosticta (Wulandari et al. 2009, Glienke et al. 2011). There- sari et al. 2008). Phyllosticta species have also been recorded fore, multilocus phylogenetic analyses have been increasingly as endophytes and saprobes on a wide range of host plants used for species discrimination in this genus (Wulandari et al. (Baayen et al. 2002, van der Aa & Vanev 2002, Okane et al. 2009, Glienke et al. 2011, Wang et al. 2012). For example, it 2003, Wulandari et al. 2009, Glienke et al. 2011). was shown that G. mangiferae is a distinct taxon from P. capi The generic circumscription of Phyllosticta as defined by van der talensis, which is a species complex awaiting more detailed Aa (1973) has been widely accepted (Bissett 1979, 1986, Yip phylogenetic study (Glienke et al. 2011). 1989, Crous et al. 2006, Motohashi et al. 2008, 2009, Glienke et In the present study, three new species of Phyllosticta are de- al. 2011, Wikee et al. 2011). The main characters are: pycnidial scribed based on morphological characters and phylogenies conidiomata, holoblastic conidiogenous cells with percurrent derived from ITS and combined multilocus gene sequences. proliferation, conidia aseptate, surrounded by a mucilaginous sheath, and provided with an apical extracellular appendage, a Guignardia sexual state, and Leptodothiorella spermatial state MATERIALS AND METHODS (van der Aa 1973, van der Aa & Vanev 2002). According to these criteria, van der Aa & Vanev (2002) reconsidered 2 936 Isolates names in Phyllosticta, accepting 141 species based on original Phyllosticta species were isolated from diseased leaves of literature and a re-examination of herbarium specimens. About ornamental or forest plant species from China and the United 50 % of the species were reclassified in Phoma, 20 % in Astero Kingdom. Infected leaves were incubated in moist chambers mella, 5 % in Phomopsis and c. 18 % in other coelomycetous at room temperature to induce sporulation. Pure cultures were genera or other taxonomic groups. Some Phyllosticta species obtained by single spore isolation as described by Choi et al. have been linked to their teleomorph states, for example, (1999). Alternatively, 5 × 5 mm pieces of surface-sterilised P. ampelicida is the anamorph of G. bidwellii (van der Aa 1973), tissue were taken from the margin of leaf lesions and were but most appear to be asexual. Recent changes to the rules consecutively immersed in 70 % ethanol solution for 1 min, that govern fungal nomenclature require that only one name for sodium hypochlorite solution with 3 % available chlorine for a single biological species should be used instead of different 2 min, rinsed in sterile distilled water, blotted dry in sterile pa- names for different morphs (Hawksworth et al. 2011, Wingfield per towels and incubated on 2 % potato-dextrose agar (PDA) et al. 2012). The earlier and well-known generic name Phyllo (Cai et al. 2009). sticta (Persoon 1818), thus has priority over Guignardia (Viala & Ravaz 1892), as followed by Glienke et al. (2011). Morphology The systematics of Phyllosticta species has long been problem- Cultures were grown on PDA for microscopic examination. atic because of the limited morphological characters and the Fungal structures were mounted on glass slides in clear lactic acid, and studied by means of a light microscope. Colony 1 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China; morphologies were assessed after 7 d growth on PDA, and corresponding author e-mail: [email protected]. colours rated according to the colour charts of Rayner (1970). © 2012 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. Y.Y. Su & L. Cai: Three new Phyllosticta spp. 77 DNA extraction, PCR amplification and sequencing Phylogenetic analyses were performed using PAUP v. 4.0b10 Mycelial discs were taken from actively sporulating areas near (Swofford 2003). Ambiguously aligned regions were excluded the growing edge of 10 d old cultures and transferred to PDA. from all analyses. An unweighted parsimony (UP) analysis was Genomic DNA was extracted with a Biospin Fungus Genomic performed. Trees were inferred using the heuristic search option DNA Extraction Kit (Bioer. Technology Co., Ltd., Hangzhou, with TBR branch swapping and 1 000 random sequence addi- P.R. China) according to the manufacturer’s protocol. Quality tions, branches of zero length were collapsed and all equally and quantity of DNA were estimated visually by staining with most parsimonious trees were saved. Descriptive tree statis- GelRed after 1 % agarose gel electrophoresis. The ITS1 and tics such as tree length [TL], consistency index [CI], retention ITS4 primer pair (White et al. 1990) was used to amplify the index [RI], rescaled consistency index [RC], and homoplasy ITS region following the procedure described by White et al. index [HI], were calculated for trees generated. Clade stability (1990). The primers EF1-728F and EF1-986R (Carbone & Kohn was assessed in a bootstrap analysis with 1 000 replicates, 1999) were used to amplify a partial fragment of the transla- each with 10 replicates of random stepwise addition of taxa. A tion elongation factor 1-α gene (TEF1); the primers ACT-512F Shimodaira-Hasegawa test (SH test) (Shimodaira & Hasegawa and ACT-783R (Carbone & Kohn 1999) were used to amplify 1999) was performed in order to determine whether trees were a partial fragment of the actin gene (ACT); the primers GDF1 significantly different. Trees were visualised in TreeView v. 1.6.6 (Guerber et al. 2003) and Gpd2-LM (Myllys et al. 2002) or (Page 1996). GDR1 (Guerber et al. 2003) were used to amplify a partial For the Bayesian analyses, the models of evolution were esti- fragment of the glyceraldehyde 3-phosphate dehydrogenase mated by using MrModeltest v. 2.3 (Nylander 2004). Posterior gene (GPDH). Amplification conditions followed Arzanlou et al. probabilities (PP) (Rannala & Yang 1996, Zhaxybayeva & (2008). DNA sequencing was performed at the SinoGenoMax Gogarten 2002) were determined by Markov Chain Monte Company Limited, Beijing. Carlo sampling (BMCMC) in MrBayes v. 3.0b4 (Huelsenbeck & Ronquist 2001), under the estimated model of evolution. Six Sequence alignment and phylogenetic analyses simultaneous Markov chains were run for 1 000 000 genera- Sequences from forward and reverse primers were aligned tions and trees were sampled every 100th generation (resulting to obtain a consensus sequence. Sequences of our isolates, in 10 000 total trees). The first 2 000 trees, representing the together with reference sequences obtained from GenBank burn-in phase of the analyses, were discarded and the remain- (Table 1), were aligned using Clustal X (Thompson et al. 1997). ing 8 000 trees were used for calculating posterior probabilities