International Journal of Systematic and Evolutionary Microbiology (2013), 63, 790–795 DOI 10.1099/ijs.0.047613-0
Aureobasidium thailandense sp. nov. isolated from leaves and wooden surfaces
Stephen W. Peterson,1 Pennapa Manitchotpisit2 and Timothy D. Leathers3
Correspondence 1Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Stephen W. Peterson Utilization Research, US Department of Agriculture, 1815 North University Street, Peoria, [email protected] IL 61604, USA 2Biochemistry Unit, Department of Medical Sciences, Faculty of Science, Rangsit University, Muangake, Lakhok, Patumthani 12000, Thailand 3Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, US Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA
Aureobasidium thailandense sp. nov. is described from cultures of material collected on leaves and wooden surfaces in Thailand and the type isolate is NRRL 58539T. Phylogenetically it is distinct from other species of the genus Aureobasidium. Phenotypically it is distinguished by its cardinal growth temperatures, salt tolerance and production of reddish brown hyphal pigmentation in PDA cultures, but micro-morphologically it is not clearly distinguishable from Aureobasidium pullulans. Unlike A. pullulans, A. thailandense sp. nov. produces a non-pullulan extracellular polysaccharide whose characteristics are unknown. The two known isolates of A. thailandense sp. nov. possess an approx. 500 bp type I intron in the 18S rRNA gene that is present in ITS amplifications using primers ITS4 and ITS5. A. pullulans isolates uniformly lack this intron.
Aureobasidium pullulans is a yeast-like fungus in the The taxonomy of the genus Aureobasidium as viewed Ascomycota class Dothideomycetes (Schoch et al., 2006) by different authors has consisted of its division into that is related to leaf-spot fungi from grasses, shrubs and phenotypic varieties of A. pullulans (Hermanides-Nijhof, trees (Hermanides-Nijhof, 1977; de Hoog & Yurlova, 1994; 1977; Zalar et al., 2008), descriptions of monophyletic Nichols et al., 2010). It has been isolated from the clades in A. pullulans without specific Linnaean ranking phylloplanes of diverse plants, maize silks, brackish water, (Manitchotpisit et al., 2009) or descriptions of distinct mangrove swamps, glacial ice in Norway and wooden or species (Crous et al., 2011). The broad clade containing the painted structures (Arfi et al., 2012; Manitchotpisit et al., genus Aureobasidium (Schoch et al., 2006; Bills et al., 2012) 2009; Santo et al., 2012; Zalar et al., 2008). Aureobasidium includes other genera, such as Kabatiella and Selenophoma proteae was reported to cause chronic fungal meningitis that express Aureobasidium and other anamorphic states (Kutlesˇa et al., 2012) as well as a leaf spot disease of under different growth conditions (synanamorphy). Proteaceae (Crous et al., 2011). Some isolates of A. pullulans Manitchotpisit et al. (2009) isolated two putative Aureo- produce semiochemicals (Davis et al., 2012) that influence basidium pullulans strains whose phylogenetic position was wasp behaviour in nature and that may be involved in not fully resolved. These two isolates contain an 18S rRNA dispersal of members of the genus Aureobasidium, while intron not found in other A. pullulans isolates and produce other isolates produce large amounts of the extracellular an uncharacterized EPS. We conducted phylogenetic polysaccharide (EPS) pullulan (Manitchotpisit et al., 2009), analysis of these isolates to resolve their relationship to a polysaccharide with many valuable industrial and medical A. pullulans and, as they form a monophyletic clade uses (Leathers, 2002; Cheng et al., 2011). Still other isolates distinct from other species, we provide a description and biosynthesize products that have commercial value such as name for these two isolates. laccase (Rich et al., 2011), xylanase (Manitchotpisit et al., 2009) or poly b-L-malic acid (Manitchotpisit et al., 2012). Culture growth and examination Abbreviation: EPS, exopolysaccharide. Cultures used in this study and their provenance are The GenBank/EMBL/DDBJ accession numbers for the nucleotide listed in Table 1 and are available from the Agricul- sequences reported in this paper are JX462671–JX462675. tural Research Service (NRRL, Peoria, IL, USA; http:// The Mycobank number for Aureobasidium thailandense sp. nov. is nrrl.ncaur.usda.gov/). Numerous DNA sequences were MB801148. obtained from GenBank and their GenBank and culture
790 047613 Printed in Great Britain Aureobasidium thailandense sp. nov. collection accession numbers are listed in the phylogenetic trimmed to equal length. Phylograms and bootstrap analysis tree. were generated using PAUP* (Swofford, 2003) version 4.0 b10 with parsimony criterion. Maximum-parsimony analysis was Cultures were initiated from lyophilized preparations and conducted with the dataset initially subjected to a heuristic maintained on potato dextrose agar (PDA; Difco) during the course of this study. Malt extract agar (MEA), Hay search with random sequence addition (500 repetitions), infusion agar, Harrold’s M40Y and M60Y media were NNI (nearest neighbour interchange) branch swapping, and prepared as described (Raper & Fennell, 1965). MEA media maximum trees set to 5000. Subsequently, the random containing salt were prepared by adding analytical grade addition trees were used as the starting point for a heuristic NaCl to the MEA prior to sterilization. Cultures were search using ‘as-is’ sequence addition, TBR (tree bisection- incubated at 25 uC unless otherwise noted. The mature reconnection) branch swapping, and maximum trees set at culture appearance was recorded and culture plates were 5000. Bootstrap analysis was performed in PAUP* using the photographed with a Kodak 14n digital camera. parsimony criterion, TBR and 1000 repetitions with maximum trees set to 100. Tree diagrams were viewed using Microscopy was conducted on cellular material mounted TreeView (Page, 1996) and redrawn using CorelDraw X3. in 0.1 % Triton X-100 on a microscope slide. The Zeiss axioscope was fitted with a Kodak 14n camera and had phase-contrast, DIF contrast and plain light illumination. Results Photographs of culture plates and micromorphology were Growth characteristics of A. pullulans and A. thailandense sp. sized and fitted into composite illustrations using nov. isolates on media with varying sugar or salt concentra- Photoshop Elements 10 (Adobe). tions and at different temperatures are shown in Table 2. Amplification and sequencing of the ITS region (primers DNA sequencing ITS5, ITS4) of A. thailandense sp. nov. demonstrated an Each isolate was grown in 2 ml YM broth (Difco) approx. 500 bp insert in the 39 end of the 18S rRNA gene T overnight, and cells were collected by centrifugation and from isolates NRRL 58539 and NRRL 58543, but resuspended in 1 ml TE buffer, pH 8.0. A 0.5 ml volume of sequencing of numerous A. pullulans isolates revealed no glass beads (0.5 mm Zirconia/Silica beads, BioSpec introns (Manitchotpisit et al., 2009). In a BLAST search of Products, Inc.) was added and cells were then disrupted the GenBank database, this rRNA insert segment demon- using a FastPrep instrument (Qbiogene), set at number 4, strated 100 % similarity to the comparable region from for 30 s. Total DNA was isolated and purified using the Neoscytalidium dimidiatum over 68 % of the segment QIAquick PCR purification kit (Qiagen). length and probably represents a group I intron in the nuclear SSU-rRNA gene (Bhattacharya et al., 2005). The ITS and LSU-rRNA gene were amplified as previously described (Zalar et al., 2008; Manitchotpisit et al., 2009). The nuclear LSU-rRNA gene alignment included 529 nt The amplified fragments were sequenced using the positions. Four base positions were indels and these amplification primers and BigDye v3 Terminator Cycle positions were eliminated from further analysis. Out of Sequencing kit (Applied Biosystems) and an ABI 3730 the 525 remaining positions analysed, 467 were constant (capillary) DNA Analyzer (Applied Biosystems). and 61 were variable with 41 of those positions being phylogenetically informative. Parsimony analysis produced four equally most parsimonious trees of 77 steps. The Phylogenetic analyses consistency index was 0.8701 and the rescaled consistency All sequences were determined from bidirectional sequen- index was 0.7951. One of those trees is shown in Fig. 1. cing, and errors were viewed and corrected using Sequencher Bootstrap values above 70 % are listed on the tree diagram. (http://www.genecodes.com). Corrected sequences were The tree diagram is composed of four clades. Two isolates T aligned with CLUSTAL W (Thompson et al., 1994) and of A. thailandense sp. nov. (NRRL 58539 and NRRL 58543)
Table 1. Provenance of Aureobasidium thailandense sp. nov. and A. pullulans isolates
NRRL accession number Provenance
Aureobasidium pullulans MB 508998 58012T France, Beaujolais, isol. ex Vitis vinifera, 1974, E. J. Hermanides-Nijhof, ex neotype Y-7703 USA, isol. ex corn silks, 1974, R. Bothast Y-12974 Bahama, isol. ex seaweed, 1985, T.D. Leathers Aureobasidium thailandense MB 801148 58539T5CBS 133856T Thailand, Nakhonratchasima, isol. ex leaf of Cerbera odollum, 2006, P. Manitchotpisit, type 585435CBS 133857 Thailand, Prachuapkhirikhan, isol. ex wood surface, 2006, P. Manitchotpisit
http://ijs.sgmjournals.org 791 S. W. Peterson, P. Manitchotpisit and T. D. Leathers
Table 2. Diameter (mm) of A. pullulans and A. thailandense sp. nov. colonies under different conditions and on different media
Growth temperature was 25 uC unless noted otherwise and incubation was for 7 d.
Condition A. pullulans A. thailandense sp. nov.
Y-12974 NRRL 58012T NRRL 58039T NRRL 58043
PDA 30 30 25 24 Hay infusion 17 19 15 M40Y 30 28 26 M60Y 26 23 24 MEA+5 % NaCl 16 15 13 MEA+10 % NaCl 9 8 9 MEA+15 % NaCl 7 0 0 MEA+20 % NaCl 0 0 0 MEA at 4 uC8 00 MEA at 15 uC231714 MEA at 25 uC382727 MEA at 28 uC363027 MEA at 30 uC9 99 MEA at 37 uC0 00 formed a distinct clade with a 92 % bootstrap value. Phenotypic recognition Aureobasidium thailandense isolates formed a sister clade to A. thailandense sp. nov. resembles A. pullulans in the trichotomy of Selenophoma mahoniae; A. leucospermi producing initially pale yeast-like colonies with glistening and A. pullulans varieties; and A. pullulans, A. proteae, moist surfaces where spores and EPS are produced. After Discosphaeria fagi and Kabatiella microsticta. Sydowia poly- 7 d growth, most of the A. thailandense sp. nov. colony is spora was chosen as the outgroup species based on prior reddish brown coloured (Fig. 2a). A. pullulans colonies on studies (Schoch et al., 2006; Crous et al., 2011; Bills et al., PDA remain white to pink coloured for at least a week, 2012) showing that the genus Sydowia is representative of a strongly supported clade sister to the Aureobasidium clade. usually 2–3 weeks. A. pullulans var. melanogenum isolates become melanized early in colonial growth appearing greenish black. A. pullulans var. subglaciale isolates remain Description of Aureobasidium thailandense pinkish at 7 d but are melanized by 14 d. A. pullulans var. sp. nov. S. W. Peterson, Manitchotpisit & Leathers namibiae isolates become melanized in the first week Figs 2, 3 producing an olive–brown colour. Mycobank number: MB 801148. A. thailandense sp. nov. has different cardinal growth Holotype: NRRL 58539T was isolated from leaves of Cerbera temperatures from A. pullulans (Table 2), failing to grow at odollum Gaertn. 2006, Nakhonratchasima, Thailand by 4 uC while some growth is seen in A. pullulans at that Pennapa Manitchotpisit, and is kept metabolically inactive temperature. A. pullulans grows on MEA supplemented as a lyophilized culture (article 8.4) in the Agricultural with 15 % (w/v) NaCl (Table 2, Zalar et al., 2008), but A. Research Service Culture Collection, Peoria, IL. thailandense sp. nov. can only tolerate concentrations of up to 10 % NaCl. The optimum growth temperature for both Diagnosis: Aureobasidium thailandense sp. nov. differs species is in the 25–28 uC range. There was little difference from A. pullulans by displaying reddish brown melanized in growth of the two species on very weak media (hay hyphae after 4–5 days growth on PDA medium, while A. infusion agar), on moderate sugar level media (PDA) or pullulans remains white to pinkish for 2–3 weeks and its media with high sugar levels (M40Y, M60Y, Table 2). varieties darken in different colours. Micromorphologically A. thailandense sp. nov. is difficult to Description: Aureobasidium thailandense sp. nov. colonies distinguish from A. pullulans because of overlapping spore on PDA (Fig. 2a, b) attain 24–25 mm diam after 7 d sizes and shapes, and similar types of conidium production. growth at 25 uC, are reddish brown centrally, appearing slimy from spore and EPS production, margins irregular, reverse white peripherally to pale brown centrally, Genotypic recognition produces non-pullulan EPS. Microscopically (Fig. 3a, b, c, d) composed of short broad hyphae, 7–10620–40 mm The A. thailandense sp. nov. DNA sequences from b- producing blastoconidia terminally or laterally, conidia tubulin, calmodulin, ITS, LSU-rRNA and RPB2 are distinct subglobose, ovoid, or pyriform 3–1065–12 mm, smooth from those of A. pullulans and all other sequences available walled, individual cells proliferating by polar budding. either in our laboratory or in the GenBank databases.
792 International Journal of Systematic and Evolutionary Microbiology 63 Aureobasidium thailandense sp. nov.
1 A. pullulans NRRL 58012T (JX462673)
100 A. proteae CPC 13701 (JN712490) Fig. 1. Parsimony analysis of A. thailandense A. proteae CPC 2824 (JN702491) sp. nov., A. pullulans and related taxa based on A. proteae CPC 2826 (JN712493) 98 partial LSU-rRNA sequences. The A. thailand- A. pullulans NRRL Y-7703 (JX462671) ense sp. nov. isolates form a distinct clade Discosphaerina fagi CBS 171.93 (AY016359) separate from A. pullulans. A. pullulans isolates occur on an unresolved trichotomy Kabatiella microsticta CBS 342.66 (FJ150945) that includes Aureobasidium species and 90 A. leucospermi CPC 15099 (JN712488) species of the genera Discosphaeria, Kabatiella 98 A. leucospermi CPC 15081 (JN712487) and Selenophoma. Sydowia polyspora is the A. pullulans v. namibiae CBS 147.97T (FJ150937) outgroup species (Schoch et al., 2006; Crous 92 et al., 2011). Bootstrap support .70 % is v. CBS 1233871T (FJ150913) A. pullulans subglaciale indicated on the branches. NRRL accession A. pullulans v. melanogenum CBS 105.22T (FJ150926) numbers refer to the Agricultural Research Selenophoma mahoniae CBS 388.92T (EU754213) Service Culture Collection; CBS refers to the Centraalbureau voor Schimmelcultures, Utrecht, 92 Aureobasidium thailandense NRRL 58543 (JX462675) The Netherlands; CPC refers to the culture Aureobasidium thailandense NRRL 58539T (JX462674) collection of Pedro Crous housed at the CBS. Sydowia polyspora CBS 750.71 (FJ150912) Bar, 1 substitution per nucleotide position.
Discussion learned that species from genera such as Kabatiella (Nichols et al., 2010) and Selenophoma (Ramaley, 1992) The genera Aureobasidium (de Bary) G. Arnaud (1891) and may display several anamorphic states, including Aureo- Kabatiella Buba´k (1907) were based on the different basidium states, under different growth conditions. anamorphic states seen in each. Hermanides-Nijhof Aureobasidium thailandense sp. nov. was isolated as part (1977) treated the different anamorphic states as repres- of a larger survey of pullulan production in A. pullulans entative of different form-genera, a situation no longer and initially was thought to be a ‘colour variant’ isolate of accepted under recent changes to the nomenclatural code A. pullulans (Manitchotpisit et al., 2009). The species was (Norvell, 2011; Hawksworth, 2012). We have subsequently never studied in situ so we do not know if synanamorphs in addition to the Aureobasidium state might be found in the leaf habitat, a common phenomenon (Nichols et al., 2010; Ramaley 1992; Crous et al., 2011). DNA studies (Manitchotpisit et al., 2009) demonstrated that A. thailandense sp. nov. could not be accommodated in a phylogenetically defined A. pullulans sensu Zalar et al. (2008) except by broadening the concept of an already variable species (Loncaric et al., 2009). Bills et al. (2012) showed that Aureobasidium thailandense sp. nov. (labelled Aureobasidium sp. in that study) occurs in the statistically supported Aureobasidium clade rather than the sister Sydowia clade (Schoch et al., 2006; Crous et al., 2011). Upon discovering the sibling relationship of K. proteae and A. pullulans, Crous et al. (2011) placed Kabatiella proteae in the genus Aureobasidium as A. proteae using the priority concept. Other genera and species in Kabatiella, Seleno- phoma or Discosphaeria also produce several anamorphic states and careful study may show that they should be transferred to the genus Aureobasidium. We found a type I intron in both isolates of A. thailandense sp. nov., while in A. pullulans type I introns were not found in any isolates examined. Coˆte´ et al. (2004) reported that in Monilinia Fig. 2. Obverse and reverse views of 7 d PDA cultures of: (a, b) fructicola isolates only some isolates of the species contained a Aureobasidium thailandense sp. nov. NRRL 58539T and (c, d) SSU-rRNA type I intron. For identification of A. thailandense Aureobasidium pullulans NRRL 58012T. sp. nov., we place more importance on the sequences from the http://ijs.sgmjournals.org 793 S. W. Peterson, P. Manitchotpisit and T. D. Leathers
Fig. 3. Aureobasidium thailandense sp. nov. NRRL 58539T. (a–d). Hyphal elements pro- ducing spores of various shape and size. Bar, 10 mm.
different loci than on the presence of the SSU-rRNA intron as Davis, T. S., Boundy-Mills, K. & Landolt, P. J. (2012). Volatile this may be a variable character. Bhattacharya et al. (2005) emissions from an epiphytic fungus are semiochemicals for eusocial reported that group I introns are absent from the family wasps. Microb Ecol 64, 1056–1063. Dothideomycetidae and this may represent a first report in this de Hoog, G. S. & Yurlova, N. A. (1994). Conidiogenesis, nutritional taxonomic group. physiology and taxonomy of Aureobasidium and Hormonema. Antonie van Leeuwenhoek 65, 41–54. The morphological diversity of synanamorphs found in the Hawksworth, D. L. (2012). Managing and coping with names of Aureobasidium clade species has been misleading about pleomorphic fungi in a period of transition. IMA Fungus 3, 15–24. phylogenetic relationships in the past. Current studies Hermanides-Nijhof, E. J. (1977). 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