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Article

Four New Species of Aspergillus Subgenus Nidulantes from China

by
Bingda Sun
1,
Chunling Luo
2,
Gerald F. Bills
3,
Jibing Li
2,
Panpan Huang
4,
Lin Wang
4,
Xianzhi Jiang
4,* and
Amanda Juan Chen
4,*
1
China General Microbiological Culture Collection Centre, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
2
State Key Laboratory of Organic Geochemistry and Guangdong-Hong Kong-Macao Joint Laboratory for Environmental Pollution and Control, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China
3
Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, TX 77054, USA
4
Microbiome Research Center, Moon (Guangzhou) Biotech Ltd., Guangzhou 510535, China
*
Authors to whom correspondence should be addressed.
J. Fungi 2022, 8(11), 1205; https://doi.org/10.3390/jof8111205
Submission received: 30 July 2022 / Revised: 10 November 2022 / Accepted: 11 November 2022 / Published: 15 November 2022
(This article belongs to the Special Issue Diversity, Classification and Taxonomy of Aspergillus, Penicillium, Talaromyces and related genera (Eurotiales))
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Abstract

:
Aspergillus subgenus Nidulantes includes species with emericella-like ascomata and asexual species. Subgenus Nidulantes is the second largest subgenus of Aspergillus and consists of nine sections. In this study, agricultural soils were sampled from 12 provinces and autonomous regions in China. Based on primary BLAST analyses, seven of 445 Aspergillus isolates showed low similarity with existing species. A polyphasic investigation, including phylogenetic analysis of partial ITS, β-tubulin, calmodulin, and RNA polymerase II second largest subunit genes, provided evidence that these isolates were distributed among four new species (Aspergillus guangdongensis, A. guangxiensis, A. sichuanensis and A. tibetensis) in sections Aenei, Ochraceorosei, and Sparsi of subgenus Nidulantes. Illustrated morphological descriptions are provided for each new taxon.

1. Introduction

Aspergillus is a monophyletic genus comprising six subgenera: subgenus Aspergillus, Cremei, Circumdati, Fumigati, Nidulantes, Polypaecilum [1,2,3]. Subgenus Nidulantes (type species: Aspergillus nidulans) is the second largest subgenus after Circumdati and includes over 100 species [4]. However, only a few species have been reported from China. Most of species in this subgenus exhibit biseriate conidial heads, brown-pigmented conidiophores, globose and echinulate conidia. For some species (mostly in sections Aenei, Nidulantes and Usti), sexual states with reddish brown, violet or dark brown ascomata embedded in masses of Hülle cells are produced. Sexual species previously were named Emericella under the dual nomenclature system [5]. Ascospores produced by species in subgenus Nidulantes often bear equatorial ridges, reticulations and appendages that are morphologically distinct from the sexual states of other subgenera in Aspergillus [4,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24].
Initially, five sections were established in subgenus Nidulantes based on morphological features: Nidulantes, Versicolores, Usti, Terrei, and Flavipedes [25]. Subsequently, DNA sequences of the β-tubulin (BenA), calmodulin (CaM), RNA polymerase II genes (RPB2), and the ITS, large subunit rDNA sequences (LSU) were analyzed in combination to determine infrageneric relationships in Aspergillus. Sections Nidulantes, Sparsi, Ochraceorosei, and Usti formed monophyletic groups along with other outlying species, e.g., A. raperi, A. ivoriensis, A. silvaticus, and A. bisporus [26,27]. Section Aenei was later introduced based on phylogenetic analyses and included additional species [28]. Most recently, application of a comprehensive polyphasic approach defined nine sections in the subgenus, leading to the addition of section Cavernicolarum (as ‘Cavernicola’) [4]. Later, A. pepii was described in section Versicolores [29]; A. amethystinus, A. askiburgiensis, A. coloradensis, A. croceus, A. croceiaffinis, A. dipodomyus, A. longistipitatus, A. stelliformis, and A. tumidus were described in section Nidulantes [24,30,31]; A. contaminans and A. sigarelli were described in section Usti [32,33]. Houbraken et al. gave an overview of families and genera of the Eurotiales and introduced an updated subgeneric, sectional and series classification [34]. Recently, a broad species concept with only four species, namely A. versicolor, A. creber, A. sydowii, and A. subversicolor, was introduced in Aspergillus series Versicolores [35].
Temperature growth profiles are useful characters for distinguishing species in subgenus Nidulantes [4]. In section Nidulantes, most of the sexual species with globose ascospores grow optimally at 37 °C, while most of asexual and sexual species with stellate ascospores grow optimally at 27 to 37 °C [4]. In section Aenei, all species fail to grow at or above 40 °C [28]. Ascomata and ascospores are generally produced by members in sections Nidulantes and Aenei. Hülle cells that surround ascomata are specialized thick-walled cells associated with subgenus Nidulantes. In some species, emericella-like ascomata may be absent, yet the mycelium may give rise to masses or embedded Hülle cells, e.g., species in sections Aenei, Usti, Sparsi, and occasionally in others [4,17,28,36,37,38].
Species of subgenus Nidulantes are widespread, although they tend to be more common in warm, arid, and saline environments [15,17,19,21,39,40]. A few species in subgenus Nidulantes have been reported to cause human infections, including A. nidulans, A. quadrilineatus, A. sublatus, and A. delacroxii (=A. spinulosporus), all of which grow well at 37 °C and have been reported as etiologic agents of aspergillosis [41,42]. Other species, such as A. protuberus, A. unguis, A. sydowii, and A. hongkongensis, which grow poorly or do not grow at 37 °C were mostly responsible for superficial infections, such as onychomycosis, keratitis, and scalp mycosis [42,43,44,45,46,47,48,49].
Aspergillus nidulans is the model fungus for understanding eukaryotic cell biology and molecular processes [50]. Its whole genome was sequenced in 2005 [51] to study a wide range of cellular attributes, including recombination, genome editing, DNA repair, mutation, cell cycle control, nucleokinesis, pathogenesis, secondary metabolism, and experimental evolution [52,53,54,55,56,57,58]. In addition, species of subgenus Nidulantes are well known for their complex secondary metabolism. Aspergillus nidulans FGSC A4 is arguably the most intensively studied fungal strain with regard to its secondary metabolome [59]. Aspergillus pachycristatus is the commercial source of echinocandin B, the natural product precursor for the antifungal drug anidulafungin [60] and the clinical candidate rezafungin [61].
In this study, soils were sampled from 11 provinces (Anhui, Guangdong, Guangxi, Guizhou, Hainan, Henan, Jiangsu, Shandong, Shanxi, Sichuan, Yunnan) and the Tibet autonomous region in China during 2017. After selecting strains from soil isolation plates, all isolates were sequenced, and sequences were interrogated by public database searches. Based on sequence analysis, seven isolates (CGMCC 3.19704, 3.19705, 3.19706, 3.19707, 3.19708, 3.19709, 3.19710) showed low similarity to previously sequenced species, and further phylogenetic analysis placed these strains in subgenus Nidulantes. A multi-gene phylogeny was constructed from the ITS, BenA, CaM, and RPB2 gene sequences. Morphological features have been used to corroborate their phylogenetic distinctions and to define and describe four new species.

2. Materials and Methods

2.1. Soil Collection

Soils were sampledfrom farmlands and forests from 11 provinces in China, including Anhui, Guangdong, Guangxi, Guizhou, Hainan, Henan, Jiangsu, Shandong, Shanxi, Sichuan, Yunnan, and the Tibet autonomous region during 2017. At each sampling site, the superficial soil layer (approximately top 5 cm) was removed, and deeper soil was carefully excavated and sealed in a sterile plastic bag. Longitude and latitude of sampling locations and vegetation information were recorded. Soil samples were transported to the lab within three days.

2.2. Fungal Isolation and Preliminary Identification

From each soil collection, approximately 10 g were sieved to remove large particles, rocks, and debris, and then suspended in 90 mL sterile water. Aliquots of 10−3 dilutions were spread onto potato dextrose agar (PDA, Guangdong Huankai Microbiological Technology Co., Ltd. (Guangzhou, China)) and rose bengal medium (RBM, Beijing Luqiao Technology Co., Ltd. (Beijing, China)) with tetracycline hydrochloride and chloramphenicol at 100 μg/mL added after autoclaving. After three to five days, emerging colonies were removed and transferred to new PDA plates. After 1 to 2 weeks of growth, strains were sorted into approximate morphological types and identified further by microscopy. Strains belonging to the genus Aspergillus were submitted for DNA extraction using the UltracleanTM Microbial DNA isolation Kit (MoBio Laboratories Inc., Solana Beach, CA, USA) and CaM gene sequencing for accurate identification. The living ex-type strains of described new species were deposited in China General Microbiological Culture Collection Centre, Institute of Microbiology, Chinese Academy of Sciences (CGMCC, https://cgmcc.net/english/ accessed on 1 July 2022) and dried cultures were deposited in herbarium (HMAS) located in same institute (accessed on 1st July 2022).

2.3. Phylogenetic Analysis

For strains (Table 1) unassignable to a known species, BenA, CaM, and RPB2 gene fragments were amplified using previously described primers and programs [62] and sequenced via ABI 3730XL DNA sequencer (Applied Biosystems). Reference sequences from strains of subgenus Nidulantes were used for phylogenetic reconstruction and placement of unknown isolates [4,24,29,31,32,33]. Aligned sequences were analyzed with FindModel (http://hiv.lanl.gov/content/sequence/findmodel/findmodel.html, accessed on 1 January 2022) to select the most appropriate model of nucleotide substitution [63]. Maximum likelihood analyses with 1000 bootstrap replicates were run using RAxML [64]. Bayesian analyses were run with MrBayes v. 3.2 [65]. The sample frequency was set to 100, and the first 25% of trees were removed as a burn-in. BenA, CaM and RPB2 sequences from A. flavipes NRRL 302 were used as outgroup. The resulting trees were analyzed with FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 1 January 2022) and visualized using Adobe Illustrator CS5. BI posterior probabilities (pp) values and bootstrap (bs) percentages of analysis were labeled at the branch nodes. Values less than 0.95 pp and less than 70% bs were not shown. Branches with values more than 1.00 pp and 95% bs are thickened.

2.4. Morphology

Macroscopic and microscopic characters were observed and measured as previously described. The isolates were inoculated and incubated for 7 d on the agar media Czapek yeast autolysate agar (CYA), yeast extract sucrose agar (YES), creatine sucrose agar (CREA), dichloran 18% glycerol agar (DG18), oatmeal agar (OA), and malt extract agar (MEA; Oxoid CM0059) [4]. Colony colors were referenced to numbered color codes in parentheses [66]. Light microscope preparations were made from one wk old colonies grown on MEA. To induce ascomata formation, colonies were incubated for more than two weeks. A Zeiss Stereo Discovery V20 dissecting microscope and a Zeiss AX10 Imager A2 light microscope, both equipped with an Axiocam 506 color camera and ZEN v.2.0 software (made in Germany), were used to capture digital images.

3. Results

3.1. Phylogeny

We used concatenated sequence data combining the ITS, BenA, CaM and RPB2 regions for defining relationships within subgenus Nidulantes. The total length of the aligned data set was 2772 characters, containing 626, 576, 651, and 919 bp for ITS, BenA, CaM, and RPB2 respectively. K2P + G model was used for ITS, GTR+G model was used for BenA, CaM and RPB2. The alignment sequences and the tree file were deposited in TreeBASE (S29781, https://www.treebase.org accessed on 14 October 2022).
Based on multi-gene phylogeny, subgenus Nidulantes was classified into nine sections (Aenei, Bispori, Cavernicolarum, Nidulantes, Ochraceorosei, Raperorum, Silvatici, Sparsi and Usti). The four new species were resolved into sections Aenei, Ochraceorosei, and Sparsi, respectively (Figure 1).
Section Aenei (1.00 pp, 100 bs) includes A. aeneus, A. bicolor, A. crustosus, A. discophorus, A. eburneocremeus, A. foeniculicola, A. heyangensis, A. karnatakaensis, A. spectabilis, and two new species, A. sichuanensis and A. tibetensis. Aspergillus sichuanensis is represented by three strains isolated from soils under pea cultivation, Sichuan province, China. They formed a strongly supported clade closely related with A. heyangensis and A. crustosus in the multi-gene phylogeny and the BenA, CaM and RPB2 single-gene trees (Figure 1 and Figures S2–S4). In the ITS phylogeny, A. sichuanensis clusters outside the clade containing A. crustosus, A. heyangensis and A. tibetensis (Figure S1). Aspergillus tibetensis clustered outside the groups with A. crustotus, A. heyanensis and A. sichuanensis in the multi-gene phylogeny and in the BenA, CaM and RPB2 single-gene trees (Figure 1 and Figures S2–S4). In the ITS phylogeny, A. sichuanensis formed a statistically unsupported clade with A. crustosus and A. heyanensis (Figure S1).
Section Sparsi (1 pp, 100 bs) includes A. amazonicus, A. anthodesmis, A. biplanus, A. conjunctus, A. diversus, A. haitiensis, A. implicatus, A. panamensis, A. sparsus, and the new species A. guangxiensis. This species formed a highly supported clade with A. amazonicus, A. anthodesmis, A. conjunctus, and A. panamensis in the multi-gene phylogeny and the CaM and RPB2 single-gene trees (Figures S3 and S4). In the ITS and BenA phylogenies, A. biplanus and A. diversus were also included in this clade, but the statistical support for this clade was low. The last new species, A. guangdongensis, fell in section Ochraceorosei and is closely related to A. funiculosus in the multi-gene phylogeny and all single-gene trees (Figure 1 and Figures S1–S4).

3.2. Taxonomy

Aspergillus guangdongensis B.D. Sun, X.Z. Jiang & A.J. Chen, sp. nov. Figure 2.
MycoBank: MB837898.
Diagnosis: Aspergillus guangdongensis produces uniseriate sterigmata, globose vesicles measuring 25–35 μm, ellipsoidal, smooth conidia, orange in mass.
Typification: CHINA, Guangdong, isolated from farmland soil under bean cultivation, 2017, P.P. Huang (holotype HMAS 248373). Ex-type culture CGMCC3.19704 = MN014767. GenBank: ITS = MN640760; BenA = MN635246; CaM = MN635257; RPB2 = MN635269.
Etymology: Latin, name refers to its origin, isolated from Guangdong province, China.
Description: Colony diam—7 d (mm): CYA 30–33; CYA 37 °C Microcolonies 3–4; MEA 43–44; OA 43–44; YES 51–54; CREA 23–24; DG18 28–29. Colony characters—CYA 25 °C, 7 d: Colonies deep, plane; margins entire; mycelium sulphur yellow (15); texture floccose; sporulation sparse; conidia en masse greyish yellow-green (68); soluble pigments absent; exudates absent; reverse rust (39) and orange (7). MEA 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium greyish yellow-green (68); texture floccose; sporulation dense; conidia en masse primrose (66); soluble pigments absent; exudates absent; reverse primrose (66). YES 25 °C, 7 d: Colonies deep, sulcate; margins entire; mycelium sulphur yellow (15); texture floccose; sporulation sparse; conidia en masse greyish yellow-green (68); soluble pigments absent; exudates absent; reverse rust (39) and orange (7). DG18 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium sulphur yellow (15); texture floccose; sporulation sparse; conidia en masse greyish yellow-green (68); soluble pigments fawn (87); exudates absent; reverse orange (7) at middle, pale luteous (11) at edge. OA 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium sulphur yellow (15); texture floccose, crust-like in middle; sporulation sparse; conidia en masse greyish yellow-green (68); soluble pigments fawn (87); exudates clear droplets; reverse saffron (10). CREA 25 °C, 7 d: Moderate growth, acid production absent.
Ascomata and Hülle cells not observed. Conidiophores with smooth stipes, light brown, 230–450 × 9–11.5 μm; vesicles light brown, globose, 25–35 μm wide, fertile over the two thirds, uniseriate, phialides hyaline, flask-shaped, 7.5–8 × 2.5–3 μm. Conidia smooth, orange in mass, subglobose to ellipsoidal, 3–4 × 2–3 μm.
Notes: Morphologically and phylogenetically, A. guangdongensis is closely related to A. funiculosus (Table 2). Both species produce yellow colonies, uniseriate and globose vesicles, however A. funiculosus produces two kinds of conidia: smooth, elliptical, faintly colored conidia and coarsely rugulose, globose, yellow-brown conidia [17]. According to BLAST analyses, A. guangdongensis is differ from A. funiculosus in ITS (96% similarity), BenA (93% similarity), CaM (91% similarity) and RPB2 (95% similarity) sequences.
Aspergillus guangxiensis B.D. Sun, X.Z. Jiang & A.J. Chen, sp. nov. Figure 3.
MycoBank: MB837899.
Diagnosis: Aspergillus guangxiensis grows restrictedly, produces globose vesicles measuring 17–28 μm, produces subglobose to globose, finely roughened conidia measuring 3–4 μm.
Typification: CHINA, Guangxi, isolated from farmland soil under maize cultivation, 2017, P.P. Huang (holotype HMAS 248372). Ex-type culture CGMCC3.19709 = MN115635. GenBank: ITS = MN640765; BenA = MN635251; CaM = MN635262; RPB2 = MN635274.
Etymology: Latin, name refers to its origin, isolated from Guangxi province, China.
Description: Colony diam—7 d (mm): CYA 11–13; CYA 37 °C No growth; MEA 9–11; OA 13–14; YES 12–14; CREA 6–8; DG18 4–5. Colony characters—CYA 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium buff (45); texture floccose; sporulation dense; conidia en masse greyish yellow-green (68); soluble pigments absent; exudates absent; reverse buff (45). MEA 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium white; texture floccose; sporulation dense; conidia en masse greyish yellow-green (68); soluble pigments absent; exudates absent; reverse buff (45). YES 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium white; texture floccose; sporulation dense; conidia en masse greyish glaucous (91); soluble pigments absent; exudates absent; reverse pale luteous (11). DG18 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium white; texture floccose; sporulation absent; soluble pigments absent; exudates absent; reverse orange (7). OA 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium white and straw (46); texture floccose; sporulation moderately dense; conidia en masse dark green (21); soluble pigments absent; exudates clear droplets; reverse pure yellow (14) CREA 25 °C, 7 d: Acid production absent.
Ascomata and Hülle cells not observed. Conidiophores with smooth stipes, light brown, 350–600 × 5.5–10 μm; vesicles hyaline, globose, 17–28 μm wide, fertile over the entire surface, metulae hyaline, 6.5–7.5 × 4–6 μm; phialides hyaline, flask-shaped, 7–9 × 2.5–3.5 μm. Conidia finely roughened, pale green in mass, subglobose to globose, 3–4 μm.
Notes: In the phylogenetic analyses, the two A. guangxiensis strains were positioned at a unique branch basal to branches with A. conjunctus, A. panamensis and A. anthodesmis (Figure 1).
Morphologically, A. guangxiensis resembles A. conjunctus and A. haitiensis because all three species produce globose vesicles measuring around 15 to 30 μm, however, A. conjunctus grows faster, reaching 30 mm in 10 days on CA [17], while A. haitiensis produces larger globose to ellipsoidal smooth conidia measuring 4–5.6 × 5–6 μm [38]. According to BLAST analyses, ITS of A. guangxiensis is close to A. haitiensis and A. sparsus (92% similarity), BenA sequence is close to A. amazonicus and A. conjunctus (88% similarity), CaM sequence is close to A. anthodesmis (87% similarity), RPB2 sequence is close to A. heyangensis (94% similarity).
Aspergillus sichuanensis B.D. Sun, X.Z. Jiang & A.J. Chen, sp. nov. Figure 4.
MycoBank: MB837900.
Diagnosis: Aspergillus sichuanensis produces Hülle cells, long conidiophores measuring 200–500 μm and echinulate green conidia measuring 3–4 μm.
Typification: CHINA, Sichuan, isolated from farmland soil under pea cultivation, 2017, P.P. Huang (holotype HMAS 248374). Ex-type culture CGMCC3.19706 = MN18437. GenBank: ITS = MN640762; BenA = MN635248; CaM = MN635259; RPB2 = MN635271.
Etymology: Latin, name refers to its origin, isolated from Sichuan province, China.
Description: Colony diam—7 d (mm): CYA 14–23; CYA 37 °C 5–6; MEA 13–16; OA 14–20; YES 18–28; CREA 9–20; DG18 13–18. Colony characters—CYA 25 °C, 7 d: Colonies moderately deep, sulcate; margins slightly irregular; mycelium white; texture floccose; sporulation sparse; conidia en masse dark green (21); soluble pigments absent; exudates absent; reverse primrose (66). MEA 25 °C, 7 d: Colonies moderately deep, plane; margins slightly irregular; mycelium white; texture floccose; sporulation moderately dense; conidia en masse dark green (21); soluble pigments absent; exudates absent; reverse buff (45). YES 25 °C, 7 d: Colonies deep, sulcate; margins slightly irregular; mycelium white; texture floccose; sporulation sparse; soluble pigments absent; exudates absent; reverse primrose (66). DG18 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium white; texture floccose; sporulation sparse; soluble pigments absent; exudates absent; reverse buff (45). OA 25 °C, 7 d: Colonies low, plane; margins entire; mycelium white; texture floccose; sporulation moderately dense, dark green (21); soluble pigments absent; exudates absent; reverse white. CREA 25 °C, 7 d: Acid production absent.
Ascomata not observed. Hülle cells hyaline, globose to ovoid, 11–18 μm wide. Conidiophores with smooth stipes, hyaline to light brown, 200–500 × 4–5.5 μm; vesicles hyaline, subclavate, 10–17 μm wide, fertile over the upper two thirds, metulae hyaline, 7–8.5 × 3.5–4.5 μm; phialides hyaline, flask-shaped, 6–8.5 × 2.5–3.5 μm. Conidia echinulate, green in mass, globose, 3–4 μm.
Notes: Aspergillus sichuanensis is phylogenetically close to A. heyangensis, a species discovered and named after HeYang county, ShanXi province, China. Aspergillus sichuanensis can be differentiated from A. heyangensis by ITS (96% similarity, 421/435 bp), BenA (91% similarity, 378/414 bp), CaM (89% similarity, 409/458 bp) and RPB2 (94% similarity, 713/757 bp) sequences. The distinguishing morphological features of A. heyangensis are its brown conidia in mass and lack of Hülle cells [67]. The echinulate, green conidia resemble those of A. crustosus, but in A. crustosus the vesicles are narrower, (5.5)–7–10–(12) μm, and the conidiophores are shorter (150–250 μm) [17] (Table 2).
Aspergillus tibetensis B.D. Sun, X.Z. Jiang & A.J. Chen, sp. nov. Figure 5.
MycoBank: MB837901.
Diagnosis: Aspergillus tibetensis grows restrictedly on all media and produces orange pigmented mycelia after one week of incubation.
Typification: CHINA, Tibet, Nyingchi, isolated from rhizosphere soil of willow, 2017, P.P. Huang (holotype HMAS 248371). Ex-type culture CGMCC3.19707 = MN110445. GenBank: ITS = MN640763; BenA = MN635249; CaM = MN635260; RPB2 = MN635272.
Etymology: Latin, name refers to its origin, isolated from Tibet, China.
Description: Colony diam—7 d (mm): CYA 13–17; CYA 37 °C No growth; MEA 20–22; OA 19–21; YES 28–31; CREA 14–17; DG18 7–9. Colony characters—CYA 25 °C, 7 d: Colonies low, plane; margins entire; mycelium white; texture floccose; sporulation sparse; conidia en masse pale green (19); soluble pigments absent; exudates absent; reverse buff (45). MEA 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium white; texture floccose; sporulation sparse; conidia en masse pale green (19); soluble pigments absent; exudates absent; reverse buff (45). YES 25 °C, 7 d: Colonies deep, plane; margins entire; mycelium white; texture floccose; sporulation sparse; soluble pigments absent; exudates absent; reverse honey (11) in the middle, buff (45) at edge. DG18 25 °C, 7 d: Colonies moderately deep, plane; margins entire; mycelium white; texture floccose; sporulation absent; soluble pigments absent; exudates absent; reverse buff (45). OA 25 °C, 7 d: Colonies low, plane; margins entire; mycelium white; texture floccose; sporulation absent; soluble pigments absent; exudates absent; reverse pale luteous in the middle, buff (45) at edge. CREA 25 °C, 7 d: Acid production absent.
Ascomata not observed. Hülle cells hyaline, globose to ovoid, 20–26 μm wide. Hyphae hyaline when young, orange pigmented after one wk. Conidiophores with smooth stipes, hyaline, 125–300 × 3.5–4.5 μm; vesicles hyaline, subclavate, 7–12 μm wide, fertile over the upper half to two thirds, metulae hyaline, 2.5–3.5 × 1.5–2.5 μm; phialides hyaline, flask-shaped, 3.5–4.5 × 1.5–2 μm. Conidia smooth, pale green in mass, globose, 2.5–3 μm.
Notes: Aspergillus tibetensis grows restrictedly, and its hyphae develop orange pigment after one week, features that differentiate it from other species of section Aenei. According to BLAST analyses, ITS of A. tibetensis is close to A. crustosus (97% similarity), BenA, CaM and RPB2 sequences of A. tibetensis are close to A. spectabilis (94%, 86%, 97% respectively).

4. Discussion

In this study, soil samples were collected from 11 provinces and the Tibet autonomous region and ranged across temperate and tropical regions, high and low altitude areas of China. After sorting the isolates into morphological types, preliminary identification revealed 445 Aspergillus strains of 6840 fungal strains. These Aspergillus species belonged to six subgenera, and among them 93 isolates belonged to subgenus Nidulantes, and seven isolates were identified as four new specie belonging to sections Aenei, Ochraceorosei, and Sparsi of subgenus Nidulantes, respectively (Table S1).
Some of the species in subgenus Nidulantes produce a sexual state. These species with emericella-like ascomata mostly belong to section Nidulantes (A. stellatus, A. aurantiobrunneus, A. nidulans and A. multicolor clades) [4] and section Aenei (A. bicolor, A. discophorus, A. foeniculicola and A. spectabilis) [21,28,68]. Two new species (A. sichuanensis and A. tibetensis) assigned to section Aenei in this study only produced Hülle cells in culture, but not ascospores. In section Aenei, all species produce subglobose to subclavate, biseriate conidial heads, however, the length of conidiophores, size of vesicles and conidial ornamentation can be used to distinguish species in this section [17,28].
Species in sections Sparsi and Ochraceorosei generally originate from tropical or subtropical soils [17,36,37,38,69,70,71]. Two new species (A. guangdongensis and A. guangxiensis) reported in this study were isolated from adjacent subtropical provinces (Guangdong and Guangxi provinces). Section Sparsi species are characterized by thin, sparse and submerged mycelia [17], such as observed in A. guangxiensis. Other features common in this section include globose to subglobose, biseriate conidial heads that are fertile over the entire surface. Most species in section Sparsi produce smooth to finely roughened conidia. The only exception is A. biplanus, which produces conspicuously echinulate conidia [17].
Species assigned to section Ochraceorosei clustered in two main branches, A. guangdongensis clustered together with A. funiculosus, while the other two species, A. ochraceoroseus and A. rambellii, clustered together. The classification of A. funiculosus in a species group was originally doubtful, even though Raper and Fennell (1965) accepted A. funiculosus as the only uniseriate species in section Sparsi (A. sparsus group) [17]. Phylogenetic classifications showed that A. funiculosus belonged to section Ochraceorosei even though bootstrap and Bayesian support values were low [4,34]. Introducing a second uniseriate species, A. guangdongensis, from this study, has clarified and stabilized the phylogenetic position of A. funiculosus. The two species form a distinct pair of sister species both with uniseriate vesicles. Aspergillus guangdongensis produces microcolonies on CYA at 37 °C, however, the growth characteristics of A. funiculosus at 37 °C were not mentioned in its original description [72]. Shumi et al. (2004) reported that profuse aerial mycelium was present at 37 °C during an enzyme screening experiment [73]. Not all species in section Ochraceorosei species produce Hülle cells. Aspergillus ochraceoroseus and A. rambellii, which appeared as sister species on a separate branch (Figure 1), are biseriate and do not grow at 37 °C [36,37].
To date, thirty-six species of subgenus Nidulantes have been recorded in China. The China General Microbiological Culture Collection Centre (CGMCC) maintains 11 species [4,74,75] from 15 provinces, and most of them originated from soil, air, and moldy materials. The first extensive study of subgenus Nidulantes and related species in China [74] was based on morphological identifications and recorded 17 species in subgenus Nidulantes and eight species in Emericella (assigned in sections Aenei, Nidulantes, Covernicolarum according to current classification). Sun and Qi described Aspergillus heyangensis [67], Li et al. (1998) collected and examined 1402 soil samples from northeastern (Jiamusi and Harbin), northern (Beijing), and northwestern (Yunchuan and Xian) China, and recorded eight species and one variety: Emericella acristata, E. corrugate, E. foeniculicola, E. miyajii, E. nidulans var. lata, E. quadrilineata, E. rugulosa and E. undulata [76]. Later, Aspergillus keveioides and A. sigarelli in section Usti and Emericella miraensis in section Nidulantes were newly described [33,77,78]. With four new species described in this study, subgenus Nidulantes is now extended to 40 species in China and includes species in sections Sparsi and Ochraceorosei. The findings of this study will improve our understanding of the distribution of Aspergillus species in China and provide a basis for the further development and application of species in the subgenus Nidulantes.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/jof8111205/s1, Figure S1: Phylogenetic tree of subgenus Nidulantes inferred from ITS. Branches with values more than 1.00 pp and 95% bs are thickened. The phylogram is rooted with Aspergillus flavipes NRRL 302 T, Figure S2. Phylogenetic tree of subgenus Nidulantes inferred from BenA. Branches with values more than 1.00 pp and 95% bs are thickened. The phylogram is rooted with Aspergillus flavipes NRRL 302 T, Figure S3. Phylogenetic tree of subgenus Nidulantes inferred from CaM. Branches with values more than 1.00 pp and 95% bs are thickened. The phylogram is rooted with Aspergillus flavipes NRRL 302 T, Figure S4. Phylogenetic tree of subgenus Nidulantes inferred from RPB2. Branches with values more than 1.00 pp and 95% bs are thickened. The phylogram is rooted with Aspergillus flavipes NRRL 302 T. Table S1: Aspergillus species encountered in this study.

Author Contributions

Conceptualization, X.J. and A.J.C.; methodology, B.S. and C.L.; validation, P.H., J.L. and L.W.; formal analysis, B.S. and C.L.; investigation, P.H.; resources, L.W.; data curation, J.L.; writing—original draft preparation, B.S.; writing—review and editing, A.J.C. and G.F.B.; supervision, X.J. and A.J.C.; funding acquisition, C.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Key-Area Research and Development Program of Guangdong Province 2022B0202110001 and 2020B1111530003.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The alignment sequences and the tree file were deposited in TreeBASE (S29781, https://www.treebase.org, accessed on 14 October 2022).

Conflicts of Interest

The authors declare no conflict of interest.

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Jof 08 01205 g001a 550Jof 08 01205 g001b 550
Figure 1. Phylogenetic tree of subgenus Nidulantes inferred from concatenated four loci: ITS, BenA, CaM and RPB2. Branches with values more than 1.00 pp and 95% bs are thickened. The phylogram is rooted with Aspergillus flavipes NRRL 302 T. T = ex-type.
Figure 1. Phylogenetic tree of subgenus Nidulantes inferred from concatenated four loci: ITS, BenA, CaM and RPB2. Branches with values more than 1.00 pp and 95% bs are thickened. The phylogram is rooted with Aspergillus flavipes NRRL 302 T. T = ex-type.
Jof 08 01205 g001aJof 08 01205 g001b
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Figure 2. Aspergillus guangdongensis CGMCC 3.19704 T. (a) Colonies: top row left to right, obverse CYA, obverse MEA, YES and OA; bottom row left to right, reverse CYA, reverse MEA, DG18 and CREA. (b–f) Conidiophores. (g) Conidia. Scale bars: (b) = 500 μm; (c–g) = 10 μm.
Figure 2. Aspergillus guangdongensis CGMCC 3.19704 T. (a) Colonies: top row left to right, obverse CYA, obverse MEA, YES and OA; bottom row left to right, reverse CYA, reverse MEA, DG18 and CREA. (b–f) Conidiophores. (g) Conidia. Scale bars: (b) = 500 μm; (c–g) = 10 μm.
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Figure 3. Aspergillus guangxiensis CGMCC 3.19709 T. (a) Colonies: top row left to right, obverse CYA, obverse MEA, YES and OA; bottom row left to right, reverse CYA, reverse MEA, DG18 and CREA. (b–f) Conidiophores. (g) Conidia. Scale bars: (b) = 20 μm; (c–g) = 10 μm.
Figure 3. Aspergillus guangxiensis CGMCC 3.19709 T. (a) Colonies: top row left to right, obverse CYA, obverse MEA, YES and OA; bottom row left to right, reverse CYA, reverse MEA, DG18 and CREA. (b–f) Conidiophores. (g) Conidia. Scale bars: (b) = 20 μm; (c–g) = 10 μm.
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Figure 4. Aspergillus sichuanensis CGMCC 3.19705 T. (a) Colonies: top row left to right, obverse CYA, obverse MEA, YES and OA; bottom row left to right, reverse CYA, reverse MEA, DG18 and CREA. (b,d–f) Conidiophores. (c) Hülle cells. (g) Conidia. Scale bars: (b–g) = 10 μm.
Figure 4. Aspergillus sichuanensis CGMCC 3.19705 T. (a) Colonies: top row left to right, obverse CYA, obverse MEA, YES and OA; bottom row left to right, reverse CYA, reverse MEA, DG18 and CREA. (b,d–f) Conidiophores. (c) Hülle cells. (g) Conidia. Scale bars: (b–g) = 10 μm.
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Figure 5. Aspergillus tibetensis CGMCC 3.19707 T. (a) Colonies: top row left to right, obverse CYA, obverse MEA, YES and OA; bottom row left to right, reverse CYA, reverse MEA, DG18 and CREA. (b,d–f) Conidiophores. (c) Hülle cells. (g) Conidia. Scale bars: (b–g) = 10 μm.
Figure 5. Aspergillus tibetensis CGMCC 3.19707 T. (a) Colonies: top row left to right, obverse CYA, obverse MEA, YES and OA; bottom row left to right, reverse CYA, reverse MEA, DG18 and CREA. (b,d–f) Conidiophores. (c) Hülle cells. (g) Conidia. Scale bars: (b–g) = 10 μm.
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Table
Table 1. Strains used in this study.
Table 1. Strains used in this study.
SpeciesOriginStrain No.ITSBenACaMRPB2
Aspergillus guangdongensisFarmland soil, GuangdongCGMCC 3.19704 T MN640760MN635246MN635257MN635269
A. guangxiensisFarmland soil, GuangxiCGMCC 3.19709 T MN640765MN635251MN635262MN635274
A. guangxiensisFarmland soil, GuangxiCGMCC 3.19710MN640766MN635252MN635263MN635275
A. sichuanensisFarmland soil, SichuanCGMCC 3.19705 T MN640761MN635247MN635258MN635270
A. sichuanensisFarmland soil, SichuanCGMCC 3.19706 MN640762MN635248MN635259MN635271
A. sichuanensisFarmland soil, SichuanCGMCC 3.19708MN640764MN635250MN635261MN635273
A. tibetensisFarmland soil, TibetCGMCC 3.19707 TMN640763MN635249MN635260MN635272
Table
Table 2. Morphological comparisions of new species and their closely related species.
Table 2. Morphological comparisions of new species and their closely related species.
Macromorphology Colony Diam 25 °C, 7 d (mm)Micromorphology (μm)
SpeciesCYACYA 37 °CMEAConidial HeadVesicleStipe LengthConidia OrnamentationConidia Shape and SizeAscomataHülle CellsReferences
Aspergillus guangdongensis30–333–443–44Uniseriate25–35230–450SmoothSubglobose to ellipsoidal, 3–4 × 2–3NOBNOBThis study
A. funiculosus* 30–35 after 10–14 d on CA--Uniseriate8–35400–600Smooth to coarsely ruguloseElliptical to globose, 3–3.5 × 2–2.5NOBNOB[17]
A. guangxiensis11–13NG9–11Biseriate17–28350–600Finely roughenedSubglobose to globose, 3–4NOBNOBThis study
A. conjunctus* 30 after 10 d on CA -40 after 10 dBiseriate15–30500–700Smooth to finely roughenedSubglobose to globose, 2.5–4NOBelongate[17]
A. haitiensis30–35 after 14 d-50–60 after 14 dBiseriate10–25200–500SmoothGlobose to ellipsoidal, 4–5.6 × 5–6NOBNOB[38]
A. sichuanensis14–235–613–16Biseriate10–17200–500EchinulateGlobose, 3–4NOBglobose to ovoidThis study
A. heyangensis19–23NG-Biseriate8–1260–150–(200)RoughenedSubglobose to globose, 2.5–3.5NOBNOB[67]
A. crustosus* 20–25 after 14 d on CA --Biseriate(5.5)–7–10– (12)150–250EchinulateGranular to echinulate, 2.5–3NOBglobose to irregular[17]
A. tibetensis13–17NG20–22Biseriate20–26125–300SmoothGlobose, 2.5–3NOBglobose to ovoidThis study
* CA = Czapek’ solution agar; NG = No growth; NOB = not observed.
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MDPI and ACS Style

Sun, B.; Luo, C.; Bills, G.F.; Li, J.; Huang, P.; Wang, L.; Jiang, X.; Chen, A.J. Four New Species of Aspergillus Subgenus Nidulantes from China. J. Fungi 2022, 8, 1205. https://doi.org/10.3390/jof8111205

AMA Style

Sun B, Luo C, Bills GF, Li J, Huang P, Wang L, Jiang X, Chen AJ. Four New Species of Aspergillus Subgenus Nidulantes from China. Journal of Fungi. 2022; 8(11):1205. https://doi.org/10.3390/jof8111205

Chicago/Turabian Style

Sun, Bingda, Chunling Luo, Gerald F. Bills, Jibing Li, Panpan Huang, Lin Wang, Xianzhi Jiang, and Amanda Juan Chen. 2022. "Four New Species of Aspergillus Subgenus Nidulantes from China" Journal of Fungi 8, no. 11: 1205. https://doi.org/10.3390/jof8111205

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