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 de- scription 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.

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