Morphological And Molecular Phylogenetic Studies Of Ulocladium, Stemphylium And Allied Genera

Species of Ulocladium, Stemphylium and similar genera are among the most commonly isolated fungi worldwide. Some species in this fungal group occur in soil as saprophytes, playing an important ecological role in the decomposition and recycling of materials in natural ecosystems. They are common contaminants in buildings following water damage. As cellulolytic fungi, they often grow on damp wood and paper. However some species are plant pathogens causing a range of diseases on important agricultural crops and fruit trees. As competitive saprophytic colonizers, they have been reported to suppress sporulation of some pathogens of different plants. Based on clinical symptoms, some species also cause some human diseases. For a long time, species classification in these five fungal genera is primarily based on morphological characters of conidia and conidiophores. Numerous discrepencies hinder the development of a phenotype-based taxonomy largely because some of the described morphological characterstics are unstable and overlapped, which are easily affected by different substrates and at different temperatures. Some species in this genus occasionally manifest atypical, aberrant conidiophore formation. Additionally, anamorphic developmental patterns have not been effective for determining the evolutionary relationships among Ulocladium and four similar genera.Based on my previous research, I further collected more than 600 diseased leaf samples from Urumchi, Aksu, Korla, Kashi, Yili in Sinkiang provinces during 2007 to 2009. From these samples we gained 42 fungal isolates of Ulocladium and Stemphylium, which enriched Chinese fungal resource. The taxonomy of the genus Ulocladium was mainly based on morphology, including conidial shape, size range, septation, ornamentation, and presence or absence of catenulation by means of apical secondary conidiophores, which were still indispensable in identifying a new taxon. The assignment to species within Stemphylium was primarily based on the morphological characteristics of conidia and conidiophores, such as variation in conidial shape, size range, length/width, color, septation, and ornamentation, as well as the length of conidiophore and diameter of terminal swollen apical cell of conidiophore as well as upon the development time of the corresponding telemorphs. In this study, all isolates were grown under standardized conditions, and our taxonomic analyses combined morphological comparisons and molecular phylogeny. On the basis of the careful identifications, 1 new genus, 11 new species, 1 new combinations and 1 Chinese new record were described and illustrated, respectively. Among them, 1 new genus was Sinomyces Yong Wang bis & X.G. Zhang, 11 new species were Ulocladium solani Yong Wang bis & X.G. Zhang,Ulocladium cantlous Yong Wang bis & X.G. Zhang,Ulocladium lycopersici Yong Wang bis & X.G. Zhang,Ulocladium porri Yong Wang bis & X.G. Zhang,Ulocladium pseudoatrum Yong Wang bis & X.G. Zhang; Stemphylium mali Yong Wang bis & X.G. Zhang,Stemphylium pyrinum Yong Wang bis & X.G. Zhang, Stemphylium phaseolina Yong Wang bis & X.G. Zhang,Stemphylium variabilis Yong Wang bis & X.G. Zhang; Sinomyces fusiodeus Yong Wang bis & X.G. Zhang, Sinomyces obovoideus Yong Wang bis & X.G. Zhang, one new combination was Sinomyces alternariae (E.G. Simmons) Yong Wang bis & X.G. Zhang and 1 Chinese new record was Ulocladium microsporum Moub. & Abdel-Hafez. In order to better resolve the taxonomic questions of Ulocladium, morphological studies and phylogenetic analyses of DNA sequences from Alt a 1,β-tubulin, EF-1α, gpd and RPB 2 were conducted. By doing so, we hoped 1) to re-evaluate the taxonomic criteria and frame of Ulocladium, 2) to illuminate the association between Ulocladium species delimitation and different cultures, 3) to accurately identify some ambiguous or problematic taxa, and 4) to rebuild the key of Ulocladium. The multi-gene analyses indicated the Ulocladium species were monophyletic except E. indefessa. This monophyletic group was composed of 26 species, which was resolved into two independent clades (Clade 1 and Clade 2). Clade 1, included the greatest number of Ulocladium spp., which possessed the typical morphological characters of Ulocladium. This clade accommodated four species-groups (atrum species-group, botrytis species-group, solani species-group and consortiale species-group). Clade 2, including U. chartarum, U. capsicuma, U. oudemansii, U. arborescens and U. septosporum, had conidiophores with less or not obvious geniculations. The taxonomic positions of three new species were also determined based on morphological comparison and molecular phylogeny. We summarized 6 points of Ulocladium as follows after finishing the analyses of results in earnest: 1) For investigating the taxonomic problem of Ulocladium, it was very essential to describe and illustrate all Ulocladium species under standardized condition; 2) We supported Simmons'opinion that the standardized conditions was on potato-carrot agar (PCA) in plastic petri dishes, under ambient temperature of 20–23°C and under cool-white fluorescent light 35–40 cm above the culture surface, with an 8 h on and 16 h off light cycle; 3) About multiplex conidium morphology, Simmons believed it was a valuable approach for identifying Ulocladium spp., but the phylogenetic analyses did not support the this idea because four species of U. atrum group were separated into three species-groups. Thus, multiplex conidial morphology might not be a reasonable criterion for identification of Ulocladium species; 4) The degree of geniculation in conidiophores was essential for delimition on the level of genus and species; 5) The conidial size range and the number of transverse or longitudinal septa were very important for species delimitation in Ulocladium, but the ornamentation of conidial wall and dimension of conidiophores were not very useful in identifying different Ulocladium species; 6) Multi-gene analyses could generally reflect the morphological characters, and it was very reasonable and necessary to combine the morphological comparison and molecular phylogeny together in investigating the taxonomy of Hyphomycetes.We also researched the phylogenetic relationship of Ulocladium and four similar genera (Alternaria, Embellisia, Nimbya and Stemphylium) by multi-gene analyses. The results indicated that among these five genera, Alternaria, Embellisia, Nimbya and Ulocladium cluster together as a large monophyletic clade, which displayed that these four genera possessed a close relationship. However, Stemphylium was as the sister taxon of this large monophyletic clade. The phylogenetic analyses supported the morphological characters. The degree of geniculations of conidiophores was an important criterion in delimiting different genera in this fungal group. The phylogenetic results possessed some differences to morphological comparisons. We proposed that the traditional Alternaria genus should be divided into two independent genera, and some Alternaria species should be accommodated to Embellisia and Nimbya. The morphological criteria among Alternaria, Embellisia and Nimbya should be clarified and re-evaluated.