First Records of Aspergillus Porphyreostipitatus and Aspergillus Carlsbadensis Since Their Original Descriptions

CZECH MYCOLOGY 70(1): 67–82, MAY 29, 2018 (ONLINE VERSION, ISSN 1805-1421)

First records of Aspergillus porphyreostipitatus and Aspergillus carlsbadensis since their original descriptions

1,2 1,2 2 ABDEL-AAL H. MOUBASHER *, MOHAMED A. ABDEL-SATER ,ZEINAB S.M. SOLIMAN

1 Department of Botany and Microbiology, Faculty of Science, Assiut University, P.O. Box 71526, Assiut, Egypt 2 Assiut University Mycological Centre, Assiut University, P.O.Box 71526, Assiut, Egypt *corresponding author: [email protected]

Moubasher A.H., Abdel-Sater M.A., Soliman Z.S.M. (2018): First records of Asper- gillus porphyreostipitatus and Aspergillus carlsbadensis since their original descriptions. – Czech Mycol. 70(1): 67–82.

During a survey of phyllosphere and non-rhizosphere soil fungi of orange plantations in the Assiut area, Egypt, several isolates of species of Aspergillus belonging to the section Usti were isolated at 25 °C. These were identified using phenotypic and genotypic characters as Aspergillus porphyreostipitatus and Aspergillus carlsbadensis. To the best of our knowledge, these are the first global records since their original descriptions and indicate their probable wide distribution. The strains of both species could grow at 37 °C (a character contrasting to that of the original description of A. carlsbadensis), but both were not able to grow on CYA at 5 °C or 45 °C or to produce acid on creatine. It is interesting to report that both strains produced the urease enzyme (however weakly in A. porphyreostipitatus) and failed to grow on G25N at 25 °C, characters not examined in the original descriptions.

Key words: Aspergillus, section Usti, orange plantations, Assiut, Egypt, phenotypic and genotypic characterisation.

Article history: submitted 23 November 2017, revised 25 February 2018, accepted 3 May 2018, pub- lished online 29 May 2018.

Moubasher A.H., Abdel-Sater M.A., Soliman Z.S.M. (2018): První nálezy Aspergillus porphyreostipitatus a Aspergillus carlsbadensis od jejich originálního popisu. – Czech Mycol. 70(1): 67–82.

Během výzkumu společenstev půdních hub mimo rhizosféru a fylosférních hub v pomerančo- vých plantážích v okolí Asijútu (Egypt) byly při 25 °C izolovány dva druhy rodu Aspergillus, patřící do sekce Usti. Na základě fenotypových a genotypových znaků byly určeny jako Aspergillus porphyreostipitatus a Aspergillus carlsbadensis, přičemž podle našich poznatků se jedná o první záznamy o výskytu těchto druhů od doby, kdy byly popsány; tyto záznamy svědčí o jejich širším rozšíření. U kmenů obou druhů byl zjistěn růst při 37 °C (oproti původnímu popisu A. carlsbadensis), ale ani jeden nebyl schopný růst na CYA při 5 °C a 45 °C ani nebyla zaznamenána tvorba kyseliny na kreatinovém agaru. Zajímavé je, že oba druhy produkují ureázu (i když A. porphyreostipitatus jen slabě) a nerostly na G25N při 25 °C, což jsou znaky, které nebyly zjištěny v rámci originálního popisu.

67 CZECH MYCOLOGY 70(1): 67–82, MAY 29, 2018 (ONLINE VERSION, ISSN 1805-1421)

INTRODUCTION

Raper & Fennell (1965) classified Aspergillus ustus (together with A. conjunctus, A. deflectus, A. panamensis and A. puniceus)totheAspergillus ustus species group (Aspergillus section Usti according to Gams et al. 1985). Later, Kozakiewicz (1989) revised the group, and included A. ustus, A. conjunctus, A. granulosus, A. panamensis, A. pseudodeflectus and A. puniceus in the A. ustus species group, and established the A. deflectus species group including A. deflectus, A. pulvinus and A. silvaticus, based on morphological studies. Klich (1993) treated A. granulosus as a member of section Versicolores, and found that A. pseudodeflectus is only weakly related to this section based on a morphological treatment of section Versicolores. Peterson (2000) transferred A. conjunctus, A. funiculosus, A. panamensis, A. silvaticus and A. anthodesmis to section Sparsi. More recently, Peterson (2008) examined the relationships of the Asper- gillus genus using a phylogenetic analysis of sequences of four loci, and assigned 15 species to this section. In 2011, Samson et al. described, based on a phylogenetic analysis of sequence data, five new species, proposed one new combination, and included 21 species in section Usti, at least two of which are able to repro- duce sexually: Aspergillus heterothallicus (º Emericella heterothallica)and Aspergillus monodii (º Fennellia monodii). On 2012, Nováková et al. described two more species, namely A. baeticus and A. thesauricus in section Usti,from Spanish caves. On 2014, Visagie et al. added another novel species, A. porphyreostipitatus and on 2016, Jurjević & Peterson described two new species A. asper and A. collinsii in the section. In 2016, Hubka et al. showed that sect. Usti is not monophyletic and designated four Usti members “incertae sedis”. As a conse- quence, Chen et al. (2016) introduced the new section Cavernicolus for the four members (A. cavernicola, A. egyptiacus, A. kassunensis, A. subsessilis)that were designated “incertae sedis” by Hubka et al. (2016) in addition to A. cali- fornicus and accepted 23 species in sect. Usti. Recently, a new member was described, A. contaminans, by Crous et al. (2017), therewith increasing the number of species in the section to 24. Species of Aspergillus section Usti are common in foods, stored maize, soil, dung and indoor air environments (Moubasher 1993, Samson et al. 2004, 2011). However, a species like A. calidoustus is considered a rare human pathogen which can cause invasive infection in immunocompromised hosts (Houbraken et al. 2007, Varga et al. 2008, Balajee et al. 2009, Peláez et al. 2013), A. granulosus has been demonstrated to cause disseminated infection in a cardiac transplant patient (Fakih et al. 1995), and A. deflectus can cause disseminated mycosis in dogs (Jang et al. 1986, Robinson et al. 2000, Schultz et al. 2008, Krockenberger et al. 2011).

68 MOUBASHER A.H., ABDEL-SATER M.A., SOLIMAN Z.S.M.: FIRST RECORDS OF ASPERGILLI

Various molecular methods have been used for genotypic studies of aspergilli (Rinyu et al. 2000, Varga et al. 2000). The internal transcribed spacer (ITS) region, located between the 18S and 28S rRNA genes, is an area of particular importance in discriminating between closely related species or at intraspecific level and has been used to identify Aspergillus species (Henry et al. 2000). However, many au- thors (Varga et al. 2011, Visagie et al. 2014, Hubka et al. 2014, 2016, Chen et al. 2016, 2017) have revealed that ITS has only limited discriminatory power in the genus Aspergillus in contrast to beta-tubulin, calmodulin and RPB2 loci. Several Aspergillus isolates were obtained from orange plantations in the Assiut area. Our research aimed at identifying some of these isolates to species level, using phenotypic and molecular methods and this work also provides interesting records of two rare species, contributing to the knowledge of their global distribution. These two species are described in detail and their features and various growth characteristics are compared with related species.

MATERIAL AND METHODS

S t r a i n s e x a m i n e d. During the course of a survey of mycobiota of Citrus sinensis (L.) Osbeck (orange) plantations in the town of Sahel-Saleem approxi- mately 25 km south-east of the city of Assiut, Egypt, several isolates of species of Aspergillus were isolated at 25 °C on plates with dichloran rose Bengal chloramphenicol agar, DRBC (King et al. 1979) and dichloran yeast extract malt extract agar, DYM (Wickerham 1951 and modified by Moubasher et al. 2016). The strains examined were isolated from the phyllosphere in October 2008 and non- rhizosphere soil of the orange plantation in August 2008. They were isolated by Zeinab Soliman in a laboratory of Assiut University Mycological Centre (AUMC), Assiut, Egypt. The macro- and micro-morphological characteristics of the isolates proved the species to be related to section Usti. Morphology.Formacromorphological observations, the strain was grown in the dark on the following standard media: Czapek yeast extract agar (CYA; Samson & Pitt 1985), Czapek’s agar (CZ; Raper & Thom 1949), Czapek’s agar with 20% sucrose (CZ20S; Raper & Fennell 1965), malt extract agar (MEA; Blakeslee 1915), malt yeast with 40% sucrose agar (M40Y; Raper & Fennell 1965), glycerol 25% nitrate agar (G25N; Pitt 1973), mannitol agar (MAN; Brayford & Bridge 1989), tannin sucrose agar (TAN; Thrane 1986), creatine sucrose agar (CREA; Frisvad 1985) and Christensen’s urea agar (UREA; Christensen 1946). Three replicate plates of 3-pointed inoculation of all media were incubated at 25 °C, but CYA plates were incubated at 5 °C, 25 °C, 37 °C and 45 °C for 7 days. Growth rates were recorded on CYA, CZ and MEA after 7 days of incubation. Assessment of

69 CZECH MYCOLOGY 70(1): 67–82, MAY 29, 2018 (ONLINE VERSION, ISSN 1805-1421) growth on media with reduced water activity (CZ20S, G25N and M40Y) was also carried out. The change of colour to pink on the UREA medium was assessed as urease positive. Results of MAN were assessed by growth and acid production, turning the phenol red pH indicator from red to yellow. Growth and base production on CREA were also recorded by visible colour change of the medium from purple to yellow. Colony colours were identified according to Kornerup & Wanscher (1978). A Sony Cybershot DSCW5 5.1MP Digital Camera with 3× Opti- cal Zoom was used for plate photography. For micromorphological observations, microscopic mounts were made in lactophenol cotton blue from CYA colonies after 7–10 days of cultivation. A Carl Zeiss, Axiostar Plus microscope (Microimaging GmbH, Göttingen, Germany), magnification up to 1000× connected with a Canon Powershot G6 7.1MP Digital Camera was used for examination and microscopic photography. Growth of the fungus and DNA extraction and sequencing. The fungus was grown on CYA plates and incubated at 25 °C for 7 days. A small amount of fungal biomass was scraped off and resuspended in 100 μl of distilled water and boiled at 100 °C for 15 minutes, then sent to SolGent Co., Ltd. (Daejeon, South Korea) for DNA extraction and sequencing. The DNA was ex- tracted using SolGent purification bead. Internal transcribed spacer (ITS) sequences of nuclear rDNA were amplified using universal primers ITS1 (5'- TCC GTA GGT GAA CCT GCG G -3') and ITS4 (5'- TCC TCC GCT TAT TGA TAT GC -3'). Then amplification was performed using the polymerase chain reaction (PCR) (GeneAmp® PCR System 9700 thermal cycler, Applied Biosystems, Foster City, California, USA). The PCR reaction mixtures were prepared using SolGent EF- Taq as follows: 10X EF-Taq buffer 2.5 μl, 10 mM dNTP (T) 0.5 μl, forward primer (10 pmol/μl) 1.0 μl, reverse primer (10 pmol/μl) 1.0 μl, EF-Taq (2.5 U) 0.25 μl, tem- plate 1.0 μl, distilled water up to 25 μl. Then the amplification was carried out using the following PCR reaction conditions: one round of amplification consisting of denaturation at 95 °C for 15 min. followed by 30 cycles of denaturation at 95 °C for20s,annealingat50°Cfor40sandextensionat72°Cfor1min.,withafinal extension step of 72 °C for 5 min. The PCR products were then purified with the SolGent PCR Purification Kit-Ultra prior to sequencing. After that, the purified PCR products were reconfirmed (using size markers) by electrophoreses of the PCR products on 1% agarose gel. These bands were then eluted and sequenced. Each sample was sequenced in sense and antisense direction. Contigs were cre- ated from the sequence data using the CLCBio Main Workbench program. The sequence obtained from each isolate was further analysed using BLAST from the National Center of Biotechnology Information (NCBI) website. Sequences obtained together with those retrieved from the GenBank database were subjected to the Clustal W analysis using MegAlign software version 5.05 (DNASTAR Inc.,

70 MOUBASHER A.H., ABDEL-SATER M.A., SOLIMAN Z.S.M.: FIRST RECORDS OF ASPERGILLI

Fig. 1. Phylogenetic tree of Aspergillus porphyreostipitatus AUMC 6930 and A. carlsbadensis AUMC 6717 together with closely related species.

Madison, Wisconsin, USA) for phylogenetic analysis (Thompson et al. 1994). The phylogenetic tree was constructed based on the neighbour-joining method within the DNASTAR software package. The bar below the tree indicates the number of substitutions per site. The sequences of other Aspergillus species used for com- parison were retrieved from the GenBank database (http://www.ncbi.nlm.nih.gov).

RESULTS AND DISCUSSION

Aspergillus porphyreostipitatus Visagie, Hirooka & Samson 2014 This species was isolated infrequently from a phyllosphere sample of orange plantations on DRBC at 25 °C. The strain was first identified as Aspergillus ustus (Soliman 2012). The strain was deposited at the culture collection of Assiut Uni- versity Mycological Centre (AUMC) and assigned to AUMC 6930. The ITS gene sequence of the strain is registered under GenBank accession number JQ425378 (Tab.1,Fig.1). G r o w t h c h a r a c t e r i s t i c s. Colony diameters (range and mean ± SD) after 7 days at 25 °C on CYA, CZ, MEA, CREA, UREA, MAN, TAN and low water activity media CZ20S, M40Y and G25N, and at 5 °C, 37 °C and 45 °C on CYA are shown in Tab. 2. No growth was detected on CYA at 5 °C or at 45 °C nor on G25N at 25 °C, and no acid was produced on CREA or MAN agar at 25 °C, but urease was produced

71 CZECH MYCOLOGY 70(1): 67–82, MAY 29, 2018 (ONLINE VERSION, ISSN 1805-1421) Visagie et al. 2014 Varga et al. 2008 Nováková et al. 2012 Rakeman et al. 2005 Peterson 2008 Peterson 2008 Samson et al. 2011 Jurjević & Peterson 2016 Samson et al. 2011 Nováková et al. 2012 Nováková et al. 2012 Peterson 2008 Peterson 2008 Peterson 2008 Peterson 2008 Rakeman et al. 2005 Nováková et al. 2012 Visagie et al. 2014 Houbraken et al. 2007 Varga et al. 2008 members based on BLAST similarity Usti Species References A. porphyreostipitatus A. ustus A. baeticus A. puniceus A. heterothallicus A. granulosus A. carlsbadensis A. asper A. germanicus A. calidoustus A. thesauricus A. pseudodeflectus A. insuetus A. granulosus A. heterothallicus A. puniceus A. baeticus A. porphyreostipitatus A. keveii A. ustus section Aspergillus Sequencing similarity (%) 526/527 (99.81) 509/514 (99.20) 550/556 (98.92) 559/577 (96.88) 547/567 (96.47) 543/562 (96.61) 464/464 (100) 518/526 (98.47) 454/464 (97.84) 454/464 (97.84) 541/554 (97.65) 545/559 (97.49) 544/559 (97.31) 540/562 (96.81) 542/567 (95.59) 541/568 (95.25) 535/561 (95.36) 511/531 (96.23) 495/510 (97.05) 498/518 (96.13) T T T T T T T T T T T T T T T T T T T T CBS 138203 Culture collection code IBT 14493 CBS 261.67 CCF 4226 CBS 495.65 NRRL 5096 NRRL 1932 NRRL 35910 CBS 123887 CBS 121601 CCF 4166 NRRL 6135 NRRL 279 NRRL 1932 NRRL 5096 CBS 495.65 CCF 4226 CBS 138203 CBS 209.92 CBS 261.67 NR_131284 NR_135431 NR_103579 NR_135368 NR_135348 GenBank match#ITS KT698840 NR_137507 NR_135435 NR_135432 NR_135372 NR_131292 NR_135348 NR_135368 NR_103579 NR_135431 NR_135461 NR_137492 NR_131284 isolates and the ex-type isolates of Length (bp) Closest accession number Genetic similarities between examined 6930 JQ425378 573 NR_135461 6717 JQ425406 816 NR_137522 AUMC number GenBank Tab. 1. searches.

72 MOUBASHER A.H., ABDEL-SATER M.A., SOLIMAN Z.S.M.: FIRST RECORDS OF ASPERGILLI Urease creatine Aspergillus porphyreostipitatus AUMC 6717. The values represent colony diame- 14–22 8–28 19–26 18–22 0 20–28 15–25 25–32 18–24 0 3.5 20–33 × 16–27 No acid + A. carlsbadensis .53 18.06 ± 2.07 17.67 ± 7.52 22.61 ± 2.28 20.22 ± 1.31 .72 23.39 ± 2.25 20.39 ± 2.62 28.78 ± 2.05 19.56 ± 1.76 am. Conidia size Hülle cell size Acid from AUMC 6930 and ings, in μm) and physiological characteristics of 26.72 ± 11.34 he lowest and infrequent values. 22.00 ± 7.87 AUMC 6717. Aspergillus porphyreostipitatus 008–340 16–45 0 105–245 5–6 (6)10–15 3.5–4 × 3– 26–38 12–22 14–27 20–28 25–32 34–42 24–35 25–40 25–32 34–43 (20)50–125 (3)3.5–5.5 (5)7–15 3–4 × 3–4 26–92 × 10–13 No acid w 31.11 ± 3.31 17.94 ± 2.99 21.33 ± 4.07 24.28 ± 2.69 29.06 ± 2 38.50 ± 2.01 27.39 ± 3.38 31.39 ± 3.76 26.67 ± 2.72 37.67 ± 2 A. carlsbadensis Microscopic measurements (ranges of at least 10 read Growth measurements of Growth temperatureCultivation medium 5A. °C porphyreostipitatus CYA 37 °C CYA 45 °C CYA A. carlsbadensis A. carlsbadensis Growth temperatureCultivation mediumA. porphyreostipitatus CYA CZ MEA CREA UREA MAN TANSpecies 25 °C CZ20S M40Y Stipe length G25N Stipe width Vesicle di A. porphyreostipitatus A. carlsbadensis ter in mm (range and mean ± SD of 9 readings). AUMC 6930 and Tab. 2. Tab. 3. Numbers in parentheses represent t Symbols: w = weak, + = positive.

73 CZECH MYCOLOGY 70(1): 67–82, MAY 29, 2018 (ONLINE VERSION, ISSN 1805-1421)

74 MOUBASHER A.H., ABDEL-SATER M.A., SOLIMAN Z.S.M.: FIRST RECORDS OF ASPERGILLI

Fig. 3. Microscopic structures of Aspergillus porphyreostipitatus AUMC 6930. A–C – conidial heads; D–G – hülle cells. Bars = 10 μm. Photos by A.M. Moharram. £ Fig. 2. Aspergillus porphyreostipitatus AUMC 6930 grown at 25 °C on CYA, CZ, MEA, TAN, CREA, MAN, UREA, CZ20S, M40Y and G25N, and at 5 °C, 37 °C and 45 °C on CYA. Photos by M.A. Ismail. weakly just beneath the colonies (Fig. 2). The current strain is able to grow at 37 °C just as those in the original description (Visagie et al. 2014). C o l o n y f e a t u r e s . C o l o u r: on CYA yellowish grey to brownish grey in the centre (4B–E2), mixed with orange grey to greyish orange (5B2–3), on CZ orange grey to greyish orange (6B2–3) with edge white, on MEA grey to brownish grey (3–4C–E1–2, with some sectors formed), on CZ20S grey to brownish grey in the centre (5C–D1–2) to brownish grey to light brown in the medium (6C–D2–4, with edge white), on MAN grey to dull green (28D–E1–3). Reverse:whiteattheedgeandorangeyellowtoolivebrowninthecentre (4–5B–D7–8) on CYA, white at the edge and pale to dull yellow in the centre (3A–B3–4) on CZ, white at the edge and brownish orange (6–7C4–6) to dark brown (7F4–6) in the centre on CZ20S, orange yellow to light brown (4–5B–D7–8) on MEA. No reverse colouration on MAN agar (Fig. 2).

75 CZECH MYCOLOGY 70(1): 67–82, MAY 29, 2018 (ONLINE VERSION, ISSN 1805-1421)

Te x t u r e: floccose on CYA, CZ, CZ20S, CREA, MAN and UREA at 25 °C, slightly floccose on MEA at 25 °C and on CYA at 37 °C, slightly floccose with whitish powdery patches on TAN. M i c r o s c o p i c f e a t u r e s (Tab. 3, Fig. 3). Conidial heads radiate to short columnar; conidiophores biseriate; stipes sinuate, hyaline to brownish, mostly smooth, but some areas show roughness, (20)50–125 μm long, (3)3.5–5.5 μm wide; vesicles globose, sometimes slightly elongated, (5)7–15 μm; metulae 7 × 2.5 μm, covering 75% of head; phialides ampulliform, 6–10 × 2.5–3.0 μm; conidia globose to subglobose, 3–4 × 3–4 μm, spiny to tuberculate. Hülle cells on CYA, thick-walled, predominantly elongate, twisted, 26–92 μm long × 10–13 μm wide. Sclerotia absent, sexual stage not observed on any growth media incubated for up to 2 months. M o l e c u l a r i d e n t i f i c a t i o n. Phylogenetically our current strain AUMC 6930 matched with the type strains of A. porphyreostipitatus (99.81% sequence similarity), A. ustus (99.20%), A. baeticus (98.92%), A. puniceus (96.88%), A. granulosus (96.61%) and A. heterothallicus (96.47%) (Tab. 1, Fig. 1). N o t e s. This species was first isolated in 2010 from dust from a church, Sayulita, Mexico (type CBS 138203T = DTO 266D9), and from house dust, Song- khla, Thailand (CBS 138202 = DTO 132D1) by Ed Whitfield & Kalima Mwange, and described as a new species (A. porphyreostipitatus) by Visagie et al. (2014). The micro-morphological features of A. porphyreostipitatus are similar to those of A. baeticus, A. ustus, A. puniceus,andA. pseudoustus. These species are similar in producing brownish colours in colonies (Visagie et al. 2014). Vesicle shapes and diameter both show significant differences between A. porphyreostipitatus and A. ustus (hemispherical to subglobose, 7–15 μm in diameter) on the one hand and A. baeticus (elliptical to elongate, 9.8–15.4 × 16.8–19.6 μm in diameter), A. pseudoustus (globose, 10–14 μm) and A. puniceus (subglobose, 8–16 μm in diam. to elliptical, 15–18 × 13–15 μm) on the other. Additionally, the width of the stipes is smaller in A. porphyreostipitatus (3.5–6.5 μm), A. ustus (3–6 μm) and A. pseudoustus (3.5–5 μm) than in A. baeticus (5–8 μm) and A. puniceus (5.5–8 μm). The ability of A. porphyreostipitatus to grow on CYA at 37 °C easily distinguishes it from its morphologically similar relatives (A. baeticus, A. ustus, A. puniceus,andA. pseudoustus).

Aspergillus carlsbadensis Frisvad, Varga & Samson 2011 In 2008–2009 and during a survey of filamentous fungi of orange and grapevine plantations in the Assiut area, several isolates of Aspergillus related to section Usti were recovered from non-rhizosphere soil, in high frequency from orange and in moderate and low frequency from grapevine plantations on DRBC (King et al. 1979) and DYM (Moubasher et al. 2016). These isolates were initially

76 MOUBASHER A.H., ABDEL-SATER M.A., SOLIMAN Z.S.M.: FIRST RECORDS OF ASPERGILLI identified as Aspergillus calidoustus (Soliman 2012, Abdel-Sater et al. 2016). A representative isolate was deposited at the culture collection of Assiut Univer- sity Mycological Centre and assigned code AUMC 6717. The ITS gene sequence of the strain is registered under GenBank accession number JQ425406 (Tab. 1, Fig. 1). The strain was also deposited in the Culture Collection of Fungi at the Depart- ment of Botany, Prague under no. CCF 5180. G r o w t h c h a r a c t e r i s t i c s. Colony diameters (range and mean ± SD) after 7 days at 25 °C on CYA, CZ, MEA, CREA, UREA, MAN, TAN and the low water activity media CZ20S, M40Y and G25N, and at 5 °C, 37 °C and 45 °C on CYA are shown in Tab. 2. No growth was detected on CYA at 5 °C or at 45 °C nor on G25N at 25 °C, and no acid was produced on CREA or MAN agar at 25 °C, but the urease enzyme was produced (Fig. 4). Growth of the current strain at 37 °C is in contrast to that of the original description (Samson et al. 2011). C o l o n y f e a t u r e s . C o l o u r: whitish on CYA, CZ, CREA (3A1–2), pale yellow (3A1–3) to yellowish grey in the centre (2B2) on CZ20S, white to pale yellow (3–4A1–3) on UREA, dull green to greyish green (27E4–5) on M40Y, grey to greenish grey (26B–C1–2) on MEA and greenish grey to dull green (27C–E2–3) on MAN. Reverse:edgeandcentrepaleyellow(3A4–5) with a brown ring near edge (6F4–6) on CYA, pale yellow edge (3A3–4), yellow brown centre (5F5–8), with a yellowish brown ring in between (5F5–8) on CZ, brownish orange to golden brown (5B–D5–7) on MEA, pale yellow edge (3A2–3) to yellowish brown centre (5D–F5–8) on CZ20S at 25 °C, and pale to light yellow edge (3A3–5) to greyish yellow to amber yellow in the centre (4B6–7) on CYA at 37 °C. No reverse colouration on MY40S, CREA, MAN and TAN. Te x t u r e: velutinous on CYA, MEA, M40Y, TAN, CREA, MAN, UREA at 25 °C and on CYA at 37 °C, slightly floccose on CZ and CZ20S at 25 °C. M i c r o s c o p i c f e a t u r e s (Tab. 3, Fig. 5). Conidiophores biseriate with typ- ical smooth-walled, brown stipes 105–245 μm long, 5–6 μm wide, sometimes con- stricted below the vesicle; vesicles globose (6)10–15 μm in diam.; conidia dis- tinctly ornamented with spines or echinulations, ellipsoidal 3.5–4.0 × 3.0–3.5 μm. Hülle cells hyaline, thick-walled, globose to broadly ellipsoidal, 20–33 μm long to 16–27 μm wide. Sclerotia not observed after 7 days on any growth media incubated at different temperatures; sexual stage not observed on any growth media incubated for up to 2 months. M o l e c u l a r i d e n t i f i c a t i o n. The ribosomal DNA sequence of our strain matched with the type strains of A. carlsbadensis (100% sequence similarity), A. asper (98.47%), A. germanicus (97.84%), A. calidoustus (97.84%), A. thesauricus (97.65%), A. pseudodeflectus (97.49%), A. insuetus (97.31%) and A. keveii (97.05%) (Tab.1,Fig.1).

77 CZECH MYCOLOGY 70(1): 67–82, MAY 29, 2018 (ONLINE VERSION, ISSN 1805-1421)

78 MOUBASHER A.H., ABDEL-SATER M.A., SOLIMAN Z.S.M.: FIRST RECORDS OF ASPERGILLI

Fig. 5. Microscopic structures of Aspergillus carlsbadensis AUMC 6717. A–C – conidial heads; D–G – hülle cells. Bars = 10 μm (A–C), 20 μm (D–G). Photos by A.M. Moharram. £ Fig. 4. Aspergillus carlsbadensis AUMC 6717 grown at 25 °C on CYA, CZ, MEA, TAN, CREA, MAN, UREA, CZ20S, M40Y and G25N, and at 5 °C, 37 °C and 45 °C on CYA. Photos by M.A. Ismail.

N o t e s. This species was first isolated in 1992 from soil, Lechuguilla Cave, Carlsbad Caverns National Park, New Mexico, USA, by D.E. Northup, soil from Galapagos Islands, Ecuador, and soil from Carthage, Tunesia (Samson et al. 2011). Samson et al. (2011) revealed that A. carlsbadensis is related to but clearly distinct from a clade including A. calidoustus, A. pseudodeflectus, A. insuetus and A. keveii in all three phylogenetic trees presented, and it is unable to grow at 37 °C, while acid production was not observed on CREA. However, our strain could grow at 37 °C, but does not produce organic acid on CREA. The micro-morphological features of A. carlsbadensis are similar to those of A. contaminans, A. calidoustus, A. keveii, A. pseudodeflectus, A. insuetus and A. granulosus. The vesicle shapes and diameter of these species (A. carlsbadensis globose, 10–14 μm, A. ustus hemispherical to subglobose 7–15 μm diam., A. insuetus hemispherical to subglobose, 11–16 μm diam., A. keveii pyriform, 9–13 μm diam., A. pseudodeflectus globose to elevate, 4–12 μm and A. calidoustus

79 CZECH MYCOLOGY 70(1): 67–82, MAY 29, 2018 (ONLINE VERSION, ISSN 1805-1421) pyriform to broadly spathulate, 7–20 μm) are significantly different from those of A. contaminans (globose or pyriform, 12–28 μm diam.) and A. granulosus (ovoid to elliptical, 15–25 × 12–18 μm). Additionally, the width of the stipes is smaller in A. carlsbadensis (4–5 μm), A. pseudodeflectus (2.5–3.5 μm), A. ustus (3–6 μm), and A. keveii (4–6 μm) than in A. contaminans, A. insuetus (4–8 μm), A. granulosus (5.5–8 μm) and A. calidoustus (4–7 μm). The conidia sizes can be used as a reliable feature for distinguishing three groups of species – conidia less 3 μm: A keveii; conidia exceeding 3 μm but less than 5 μm: A. carlsbadensis, A. calidoustus, A. contaminans, A. insuetus, A. ustus; up to 5–5.5 μm: A. pseudodeflectus and A. granulosus. Aspergillus carlsbadensis strain AUMC 6717 (contrasting to the originally described strains) shares with A. calidoustus, A. peseudodeflecus and A. granulosus the ability to grow on CYA at 37 °C, which easily distinguishes these species from morphologically similar relatives (A. contaminans, A. keveii, A. insuetus and A. ustus) (Houbraken et al. 2007, Samson et al. 2011, Crous et al. 2017). An added value of the current study was the use of media such as UREA, G25N, TAN and MAN, which enabled us to find more cultural or physiological characters that might be beneficial for differentiating these or other related species (ability of urease production, growing on G25N and TAN, growing and acid production on mannitol).

REFERENCES

ABDEL-SATER M.A., MOUBASHER A.H., SOLIMAN Z.S.M. (2016): Biodiversity of filamentous and yeast fungi in soil of citrus and grapevine plantations in Assiut area, Egypt. – Czech Mycology 68(2): 183–214. BALAJEE S.A., KANO R., BADDLEY J.W., MOSER S.A., MARR K.A., ALEXANDER B.D., ANDES D., KONTO- YIANNIS D.P., PERRONE G., PETERSON S., BRANDT M.E., PAPPAS P.G ., C HILLER T. (2009): Molecular identification of Aspergillus species collected for the transplant-associated infection surveil- lance network. – Journal of Clinical Microbiology 47: 3138–3141. DOI: 10.1128/JCM.01070-09. BLAKESLEE A. (1915): Lindner’s roll tube method of separation cultures. – Phytopathology 5: 68–69. BRAYFORD D., BRIDGE P.D. (1989): Differentiation of Fusarium oxysporum from Fusarium solani by growth and pigmentation on media containing sugar alcohols. – Letters in Applied Microbiol- ogy 9(1): 9–12. CHEN A.J., FRISVAD J.C., SUN B.D., VARGA J., KOCSUBE S., DIJKSTERHUIS J., KIM D.H., HONG S.-B., HOUBRAKEN J., SAMSON R.A. (2016): Aspergillus section Nidulantes (formerly Emericella): polyphasic taxonomy, chemistry and biology. – Studies in Mycology 84: 1–118. DOI: 10.1016/j.simyco.2016.10.001. CHEN A.J., HUBKA V., FRISVAD J.C., VISAGIE C.M., HOUBRAKEN J., MEIJER M., VARGA J., DEMIRE R., JURJEVIĆ Ž., KUBÁTOVÁ A., SKLENÁŘ F., Z HOU Y.G., SAMSON R.A. (2017): Polyphasic taxonomy of Aspergillus section Aspergillus (formerly Eurotium), and its occurrence in indoor environments and food. – Studies in Mycology 88: 37–135. DOI: 10.1016/j.simyco.2017.07.001.

80 MOUBASHER A.H., ABDEL-SATER M.A., SOLIMAN Z.S.M.: FIRST RECORDS OF ASPERGILLI

CHRISTENSEN W.B. (1946): Urea decomposition as a means of differentiating Proteus and paracolon cultures from each other and from Salmonella and Shigella types. – Journal of Bacteriology 52(4): 461–466. CROUS P. W. , W INGFIELD M.J., BURGESS T.I., CARNEGIE A.J., HARDY G.E.St.J., SMITH D., SUMMERELL B.A., CANO-LIRA J.F., GUARRO J., HOUBRAKEN J., LOMBARD L., MARTÍN M.P., SANDOVAL-DENIS M. et al. (2017): Fungal Planet description sheets: 625–715. – Persoonia 39: 270–467. DOI: 10.3767/persoonia.2017.39.11. FAKIH M.G., BARDEN G.E., OAKES C.A., BERENSON C.S. (1995): First reported case of Aspergillus granulosus infection in a cardiac transplant patient. – Journal of Clinical Microbiology 33: 471–473. FRISVAD J.F. (1985): Creatine sucrose agar, a differential medium for mycotoxin producing terverti- cillate Penicillium species. – Letters in Applied Microbiology 1: 109-113. GAMS W., CHRISTENSEN M., ONIONS A.H., PITT J.I., SAMSON R.A. (1985): Infrageneric taxa of Asper- gillus. – In: Samson R.A., Pitt J.I., eds., Advances in Penicillium and Aspergillus Systematics, pp. 55–62. Plenum Press, New York. HENRY T., IWEN P.C ., H INRICHS S.H. (2000): Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. – Journal of Clinical Microbiology 38: 1510–1515. HOUBRAKEN J., DUE M., VARGA J., MEIJER M., FRISVAD J.C., SAMSON R.A. (2007): Polyphasic taxonomy of Aspergillus section Usti. – Studies in Mycology 59: 107–128. DOI: 10.3114/sim.2007.59.12. HUBKA V., LYSKOVÁ P., F RISVAD J.C., PETERSON S.W., SKOŘEPOVÁ M., KOLAŘÍK M. (2014): Aspergillus pragensis sp. nov. discovered during molecular re-identification of clinical isolates belonging to Aspergillus section Candidi. – Medical Mycology 52: 565–576. DOI: 10.1093/mmy/myu022. HUBKA V., NOVÁKOVÁ A., PETERSON S.W., FRISVAD J.C., SKLENÁŘ F., M ATSUZAWA T., KUBÁTOVÁ A., KOLAŘÍK M. (2016): A reappraisal of Aspergillus section Nidulantes with descriptions of two new sterigmatocystin-producing species. – Plant Systematics and Evolution 302: 1267–1299. DOI: 10.1007/s00606-016-1331-5. JANG S.S., DORR T.E., BIBERSTEIN E.L., WONG A. (1986): Aspergillus deflectus infection in four dogs. – Journal of Medical and Veterinary Mycology 24: 95–104. JURJEVIĆ Ž., PETERSON S.W. (2016): Aspergillus asper sp. nov. and Asperillus collinsii sp. nov., from Aspergillus secion Usti. – International Journal of Systematic and Evolutionary Microbiology 66: 2566–2572. DOI: 10.1099/ijsem.0.001094. KING D.A., HOCKING A.D., PITT J.I. (1979): Dichloran rose Bengal medium for enumeration and isola- tion of molds from foods. – Applied and Environmental Microbiology 37: 959–964. KLICH M.A. (1993): Morphological studies of Aspergillus section Versicolores and related species. – Mycologia 85: 100–107. KORNERUP A., WANSCHER J.H. (1978): Methuen Handbook of Colour, 3rd ed. – 252 pp., London. KOZAKIEWICZ Z. (1989): Aspergillus species in stored products. – Mycological Papers 161: 1–188. KROCKENBERGER M.B., SWINNEY G., MARTIN P., R OTHWELL T.R., MALIK R. (2011): Sequential opportu- nistic infections in two German Shepherd dogs. – Australian Veterinary Journal 89: 9–14. DOI: 10.1111/j.1751-0813.2010.00666.x. MOUBASHER A.H. (1993): Soil fungi in Qatar and other Arab countries. – 566 pp., Scientific and Ap- plied Research Center, Qatar University, Doha. MOUBASHER A.H., ABDEL-SATER M.A., SOLIMAN Z.S.M. (2016): Biodiversity and molecular character- ization of yeast and filamentous fungi in the air of citrus and grapevine plantations in Assiut area, Egypt. – Mycosphere 7(3): 236–261. DOI: 10.5943/mycosphere/7/3/1. NOVÁKOVÁ A., HUBKA V., SAIZ-JIMENEZ C., KOLAŘÍK M. (2012): Aspergillus baeticus sp. nov. and Asper- gillus thesauricus sp. nov., two species in section Usti from Spanish caves. – International Jour- nal of Systematic and Evolutionary Microbiology 62: 2778–2785. DOI: 10.1099/ijs.0.041004-0. PELÁEZ T., ÁLVAREZ-PÉREZ S., MELLADO E., SERRANO D., VALERIO M., BLANCO J.L., GARCIA M.E., MUŃOZ P., C UENCA-ESTRELLA M., BOUZA E. (2013): Invasive aspergillosis caused by cryptic

81 CZECH MYCOLOGY 70(1): 67–82, MAY 29, 2018 (ONLINE VERSION, ISSN 1805-1421)

Aspergillus species: a report of two consecutive episodes in a patient with leukaemia. – Journal of Medical Microbiology 62: 474–478. DOI: 10.1099/jmm.0.044867-0. PETERSON S.W. (2000): Phylogenetic relationships in Aspergillus based on rDNA sequence analysis. – In: Samson R.A., Pitt J.I., eds., Integration of modern taxonomic methods for Penicillium and Aspergillus classification, pp. 323–355. Harwood Academic Publishers, Amsterdam. PETERSON S.W. (2008): Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. – Mycologia 100: 205–226. PITT J.I. (1973): An appraisal of identification methods for Penicillium species: novel taxonomic cri- teria based on temperature and water relations. – Mycologia 65: 1135–1157. DOI: 10.1002/jobm.19810210822. RAKEMAN J.L., BUI U., LAFE K., CHEN Y.-C., HONEYCUTT R.J., COOKSON B.T. (2005): Multilocus DNA sequence comparisons rapidly identify pathogenic molds. – Journal of Clinical Microbiology 43(7): 3324–3333. DOI: 10.1128/JCM.43.7.3324-3333.2005. RAPER K.B., FENNELL D.I. (1965): The genus Aspergillus. – 686 pp., Williams & Wilkins, Baltimore. RAPER K.B., THOM C. (1949): A manual of the Penicillia. – 878 pp., Williams & Wilkins, Baltimore. RINYU E., VARGA J., FERENCZY L., KOZAKIEWICZ Z. (2000): Phenotypic and genotypic variability within Aspergillus section Fumigati. – In: Samson R.A., Pitt J.I., eds., Integration of modern taxonomic methods for Penicillium and Aspergillus classification, pp. 483–490. Harwood Academic Pub- lishers, Amsterdam. ROBINSON W.F., CONNOLE M.D., KING T.J., PITT J.I., MOSS S.M. (2000): Systemic mycosis due to Aspergillus deflectus in a dog. – Australian Veterinary Journal 78: 600–602. SAMSON R.A., PITT J.I. (1985): General recommendations. – In: Samson R.A., Pitt J.I., eds., Advances in Penicillium and Aspergillus systematics, pp. 455–460. Plenum Press, New York. SAMSON R.A., HOEKSTRA E.S., FRISVAD J.C., eds. (2004): Introduction to food and airborne fungi. 7th ed. – Centraal Bureau voor Schimmelcultures, Utrecht. SAMSON R.A., VARGA J., MEIJER M., FRISVAD J.C. (2011): New taxa in Aspergillus section Usti. – Stud- ies in Mycology 69: 81–97. DOI: 10.3114/sim.2011.69.06. SCHULTZ R.M., JOHNSON E.G., WISNER E.R., BROWN N.A., BYRNE B.A., SYKES J.E. (2008): Clinico- pathologic and diagnostic imaging characteristics of systemic aspergillosis in 30 dogs. – Journal of Veterinary Internal Medicine 22: 851–859. SOLIMAN Z.S.M. (2012): Studies on yeast and filamentous fungi in citrus and grapevine plantations. – M.Sc. Thesis [depon. in Faculty of Science, Assiut University, Assiut, Egypt]. THOMPSON J.D., HIGGINS D.G., GIBSON T.J. (1994): CLUSTAL W: improving the sensitivity of progres- sive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. – Nucleic Acids Research 22: 4673–4680. DOI: 10.1093/nar/22.22.4673. THRANE U. (1986): The ability of common Fusarium species to grow on tannin-sucrose agar. – Let- ters in Applied Microbiology 2: 33–36. VARGA J., HOUBRAKEN J., VAN DER LEE H.A., VERWEIJ P.E., SAMSON R.A. (2008): Aspergillus calidoustus sp. nov., causative agent of human infections previously assigned to Aspergillus ustus. – Eukaryotic Cell 7: 630–638. VARGA J., TÓTH B., RIGÓ K., TÉREN J., HOEKSTRA R.F., KOZAKIEWICZ Z. (2000): Phylogenetic analysis of Aspergillus section Circumdati based on sequences of the internal transcribed spacer regions of the 5.8 S rRNA gene. – Fungal Genetics and Biology 30: 71–80. DOI: 10.1006/fgbi.2000.1204. VARGA J., FRISVAD J.C., SAMSON R.A. (2011): Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. – Studies in Mycology 69: 57–80. DOI: 10.3114/sim.2011.69.05. VISAGIE C.M., HIROOKA Y., TANNEY J.B., WHITFIELD E., MWANGE K., MEIJER M., AMEND A.S., SEIFERT K.A., SAMSON R.A. (2014): Aspergillus, Penicillium and Talaromyces isolated from house dust sam- ples collected around the world. – Studies in Mycology 78: 63–139. WICKERHAM L.J. (1951): Taxonomy of yeasts. – Technical Bulletin No. 1029, U.S. Department of Agri- culture, Washington D.C.