Proposal of Four New Aureobasidium Species for Exopolysaccharide Production

In this study, 99 strains of Aureobasidium species were isolated from various samples collected from different locations in China, among which 14 isolates showed different morphological characteristics to other strains identified as known Aureobasidium species. Based on morphological characteristics, those 14 strains were classified into four groups, represented by stains of KCL139, MDSC−10, XZY411−4, and MQL9−100, respectively. Molecular analysis of the internal transcriptional spacer (ITS) and part of the large ribosome subunit (D1/D2 domains) indicated that those four groups represent four new species in the Aureobasidium. Therefore, the names Aureobasidium insectorum sp. nov., A. planticola sp. nov., A. motuoense sp. nov., and A. intercalariosporum sp. nov. are proposed for KCL139, MDSC−10, XZY411−4, and MQL9−100, respectively. We also found that there were differences in the yield of exopolysaccharides (EPS) among and within species, indicating strain-related exopolysaccharide-producing diversity.

With the increasing number of Aureobasidium species, its functional activities have been explored. For example, Aureobasidium species are resistant to Botrytis cinerea and Rhizopus stolonifera as biological control agents, and they can also be used as sources of single-cell proteins [21,22]. Aureobasidium pullulans is often fermented to produce β-polymalic acid, laccase, liamocins, pullulan polysaccharides, and other commercial compounds [14,[23][24][25][26][27][28]. Liamocins display pharmacological activities including anti-Streptococcus and anticancer [29]. Pullulan polysaccharides are non-toxic, tasteless, harmless, degradable, water-soluble, stable, film-forming, and present other excellent properties. As raw materials for food and cosmetics, pullulan polysaccharides are used for immune regulation, anti-tumor and antimetastasis, relief of influenza and food allergy, and relief of stress [30].
Pullulan polysaccharides produced by A. pullulans have the properties of water retention, barrier formation, regeneration, whitening, hydrating, and repairing, and are added as an ingredient to cosmetic formulas. In order to screen an excellent A. pullulans to make a fermentation of excellent efficacy for cosmetics, we collected various samples from Tibet, Zhejiang, Yunnan, Shaanxi, Gansu, Hebei, and other areas in China for strain isolation. Ninety-nine strains belonging to Aureobasidium were obtained, among which 14 strains were identified as four new species, based on morphological characteristics and molecular analysis of the internal transcriptional spacer (ITS) and part of the large ribosome subunit (D1/D2 domains).

Sample Collection and Strain Isolation
Two hundred leaf samples of Sea buckthorn, willow, oak, crabapple, privet, camphor, and poplar collected from Motuo County in Tibet, and the Yunnan, Shaanxi, Gansu, and Zhoushan regions in Zhejiang Province, were cut into small pieces under sterile conditions and soaked in 0.1% Tween 80. The Tween solution from the leaves was diluted 10 times and directly spread on a potato dextrose agar medium (PDA; 200 g potatoes, 20 g glucose, 20 g agar per L) containing chloramphenicol (0.1 mg/mL) and cultured at 28 • C for 48 h.
Ten insect samples were collected from Xiaowutai Mountain, Zhangjiakou region, Hebei Province. Insect samples with 75% alcohol disinfection were removed from the head and ground with a grinder, then soaked in 0.1% Tween 80. The Tween solution from the inset was diluted 10 times and directly spread on PDA containing chloramphenicol (0.1 mg/mL) and cultured at 28 • C for 48 h.

Observation of Morphology
The isolates were cultured on PDA, oatmeal agar (OA; 30 g oatmeal, 20 g agar per L), and malt extract agar (MEA; 50 g malt extract, 20 g agar per L), and incubated in darkness at 25 • C for one week, in order to obtain morphological descriptions, including colony color and appearance. Fungal structures were transferred to microscope slides and mounted on 85% lactic acid drops. M40Y and M60Y media were prepared as described [15,19]. MEA media containing salt were prepared by adding analytical grade NaCl to the MEA prior to sterilization. Cultures were incubated at 25 • C unless otherwise noted.
The microscopes are equipped with LEICA DM2500 cameras (LECIA, Wetzlar, Germany) and use LASV4.13 software. At least 50 representative measurements were randomly selected and measured to calculate the average size.

Phylogenetic Analyses
For phylogenetic analyses, 28 new ITS and LSU sequences were obtained from the present study, and 56 reference sequences from GenBank (Table 1).
ITS and D1/D2 sequences were aligned with the Muscle program in MEGA7 [34], and minor gaps in all alignments were manually deleted. The most appropriate model of DNA substitution was searched with MEGA7 [35]. The model GTR + I + G was selected for Maximum likelihood (ML) and Bayesian inference (BI) analyses. ML analysis was carried out using MEGA7 [35] with 1000 bootstrap replicates. Bayesian inference (BI) analysis was conducted using MrBayes 3.1.2 [34] with 10,000,000 generations, and parameter settings were proposed by Wang et al. [36]. The phylogenetic tree and the alignments were deposited in TreeBASE (www.treebase.org, accessed on 21 December 2022, No. 30010).

Exopolysaccharides Production
The starter cultures of the 29 strains (Table 2) were prepared by cultivating the strains in 50 mL of inoculum medium containing 10 g yeast extract, 20 g peptone, and 20 g glucose per liter of distilled water at 25 • C for 3 days on a 150-rpm rotary shaker. Then, 5 mL of the starter culture was transferred to 100 mL synthetic medium containing 100 g sucrose, 1.7 g yeast extract, 5 g K 2 HPO 4 , 0.2 g MgSO 4 .7H 2 O, 0.6 g (NH 4 ) 2 SO 4 and 1.0 g NaCl per liter of deionized water at 28 • C for 6 days on a 150-rpm rotary shaker, the culture obtained was centrifuged at 7104 × g for 5 min, and the supernatant was collected. In order to precipitate exopolysaccharides, the cold ethanol was added to the obtained solution at a volumetric ratio of 2:1 v/v and the resulting mixture was kept in a refrigerator at 4 • C for 16 h [37]. The cold mixture was further centrifuged at 7104× g at 4 • C for 15 min and the pellet was dried overnight at 45 • C and then weighed to determine the yield of crude EPS production [38].

Phylogeny
The phylogenetic tree, based on a combined dataset of the ITS region and D1/D2 domain of the LSU sequences, was used to resolve the taxonomic position of the newly collected strains within Aureobasidium.
Fourteen newly isolated strains in this study were formed into four separate groups (  Table 3. A. insectorum can grow at 4-30 °C, and the optimum growth temperature is 28 °C. On MEA supplemented with 15% (w/v) NaCl, the diameters of A. insectorum attained 8-9 mm, while A. mangrovei and A. pullulans were 5 mm and 7 mm, respectively [10,19]. Thus, A. insectorum grew stronger on concentrations of 15% NaCl than its closely related species. A. insectorum grew stronger on moderate-sugar-level media (PDA) and M40Y, and weaker on M60Y. This species is suitable to grow on M40Y and PDA.
Notes-This species is phylogenetically related to A. thailandense and A. castaneae. These three species can be distinguished by the hyphae color (hyaline or brown in A. castaneae, vs. hyaline in A. thailandense, vs. dark black in A. planticola) [12,19].  Table 3. A. motuoense can grow at 17-37 °C, and the optimum growth temperature is 30 °C. At 37 °C, the diameters of A. motuoense were 7-8 mm, while its relative A. mangrovei was 5 mm [15]. A. motuoense can tolerate concentrations of up to 15% NaCl. A. motuoense grew stronger on moderatesugar-level media (PDA), and on both M40Y and M60Y. This species is suitable to grow on M40Y.
Mycelium were hyaline to dark brown, smooth-walled, branched, and 1.3-12.7 µm (av. =8.3 µm) wide. Conidiogenous cells were 6.4-12.2 × 3.6-4.5 µm (av. = 8.7× 3.9 µm). Conidia were hyaline, smooth-walled, terminal, and mono-or bipolar budding. Chlamydospores were 12. Notes-This species is phylogenetically related to A. melanogenum and A. acericola. The colonies of A. melanogenum on MEA/PDA at 25 °C attained 25 mm diameters after 7 d, appearing smooth and slimy due to abundant sporulation and EPS formation, olivebrown to black in the centre, mustard yellow towards the margin, and at the margin were yellowish white. The colony morphology of A. acericola is fast-growing, attaining  Table 3. A. motuoense can grow at 17-37 • C, and the optimum growth temperature is 30 • C. At 37 • C, the diameters of A. motuoense were 7-8 mm, while its relative A. mangrovei was 5 mm [15]. A. motuoense can tolerate concentrations of up to 15% NaCl. A. motuoense grew stronger on moderatesugar-level media (PDA), and on both M40Y and M60Y. This species is suitable to grow on M40Y.
Mycelium were hyaline to dark brown, smooth-walled, branched, and 1.3-12.7 µm (av. =8.3 µm) wide. Conidiogenous cells were 6.4-12.2 × 3.6-4.5 µm (av. = 8.7× 3.9 µm). Conidia were hyaline, smooth-walled, terminal, and mono-or bipolar budding. Chlamydospores were 12. Notes-This species is phylogenetically related to A. melanogenum and A. acericola. The colonies of A. melanogenum on MEA/PDA at 25 • C attained 25 mm diameters after 7 d, appearing smooth and slimy due to abundant sporulation and EPS formation, olive-brown to black in the centre, mustard yellow towards the margin, and at the margin were yellowish white. The colony morphology of A. acericola is fast-growing, attaining diameters of 65 mm in 14 days, rapidly turning to olivaceous black, with dark green, irregular margins, covered with slimy masses of conidia, and mycelium immersed or no aerial mycelium [8,10] diameters of 65 mm in 14 days, rapidly turning to olivaceous black, with dark green, irregular margins, covered with slimy masses of conidia, and mycelium immersed or no aerial mycelium [8,10].  (Figure 2), attaining 34 mm, 27 mm, and 28 mm diameters after 7 days of incubation at 25 °C, respectively. Colonies on PDA had an entire margin, and were floccose, greenish black, and white at the edge. Colonies on MEA had an undulate margin, and were flat, brownish olivaceous, grey at the centre, and white near the edge, with sparse aerial mycelium. Colonies on OA were flat, pale, and olivaceous brown to white from the middle to the edge.
The growth diameters, sugar and salt tolerance, and different cardinal growth temperature of A. intercalariosporum are shown in Table 3. A. intercalariosporum can grow at 17-30 °C, and the optimum growth temperature is 28 °C. On MEA supplemented with 15% (w/v) NaCl, the diameters of A. intercalariosporum attained 9-13 mm, while A. mangrovei and A. pullulans were 5 mm and 7 mm, respectively [8,16]. Therefore A. intercalariosporum grew stronger on concentrations of 15% NaCl than its closely related species. A. planticola grew on moderate-sugar-level media (PDA), and on both M40Y and M60Y. This species is suitable to grow on M40Y.
Notes-This species is phylogenetically related to A. pini, A. planticol, A. khasianum, A. leucospermi, A. namibiae, A. pullulans, and A. proteae. Colonies grew moderately on PDA, MEA, and OA (Figure 2), attaining 34 mm, 27 mm, and 28 mm diameters after 7 days of incubation at 25 • C, respectively. Colonies on PDA had an entire margin, and were floccose, greenish black, and white at the edge. Colonies on MEA had an undulate margin, and were flat, brownish olivaceous, grey at the centre, and white near the edge, with sparse aerial mycelium. Colonies on OA were flat, pale, and olivaceous brown to white from the middle to the edge.
The growth diameters, sugar and salt tolerance, and different cardinal growth temperature of A. intercalariosporum are shown in Table 3. A. intercalariosporum can grow at 17-30 • C, and the optimum growth temperature is 28 • C. On MEA supplemented with 15% (w/v) NaCl, the diameters of A. intercalariosporum attained 9-13 mm, while A. mangrovei and A. pullulans were 5 mm and 7 mm, respectively [8,16]. Therefore A. intercalariosporum grew stronger on concentrations of 15% NaCl than its closely related species. A. planticola grew on moderate-sugar-level media (PDA), and on both M40Y and M60Y. This species is suitable to grow on M40Y.

Exopolysaccharides Production
Ninety-nine Aureobasidium strains were isolated from different leaf and insect samples. Twenty-nine strains among them were selected to evaluate the exopolysaccharides production capacity (Table 3). PTSL19-104 and PTSL20-104 had the highest exopolysaccharide yield, which was 54.8 g/L and 52.33 g/L, respectively. The exopolysaccharides production capacity of Aureobasidium strains was different between species. A. melanogenum had a strong exopolysaccharide production capacity, with an average exopolysaccharide production of 39.06 g/L. A. thailandense was the weakest, with an average exopolysaccharide production of 6.17 g/L. As the interspecific hetero, the intraspecific differences also existed in the production capacity of exopolysaccharides. For example, the lowest sugar production of A. melanogenum strain was 1.53 g/L, and the highest sugar production was 54.58 g/L. The strains in the same branch of the phylogenetic tree have the same apparent and microscopic morphology, but the exopolysaccharides yields produced by fermentation are different. For example, the exopolysaccharides yields of four strains among A. motuoense are 15.74 g/L, 21.39 g/L, 31.72 g/L, and 37.43 g/L, respectively. In summary, there were differences in exopolysaccharides production capacity among species. Although strain morphology and molecular sequence were consistent within species, there were differences in physiological functions such as exopolysaccharides production capacity.

Discussion
The species of Aureobasidium are widely distributed globally in various habitats, such as house dust, air, tree surfaces (such as needles of Pinus tabuliformis, Acer pseudosieboldianum, Bintaro plants, Castanea henryi, and Castanea mollissima), plant interiors, seawater, sea ice and glacial meltwaters, water and sediment samples, soil, and subcutaneous phaeohyphomycosis from the US, Canada, Korea, Indonesia, China, the Arctic coast, and Brittany (France) [1,8,10,11,18,21,23]. In this study, many strains were isolated from soil and plant leaves, but strain KCL139 was isolated from the surface of a spittle insect. Yeasts occur commonly on insect species, and the number of insect-related yeasts had increased in the

Exopolysaccharides Production
Ninety-nine Aureobasidium strains were isolated from different leaf and insect samples. Twenty-nine strains among them were selected to evaluate the exopolysaccharides production capacity (Table 3). PTSL19-104 and PTSL20-104 had the highest exopolysaccharide yield, which was 54.8 g/L and 52.33 g/L, respectively. The exopolysaccharides production capacity of Aureobasidium strains was different between species. A. melanogenum had a strong exopolysaccharide production capacity, with an average exopolysaccharide production of 39.06 g/L. A. thailandense was the weakest, with an average exopolysaccharide production of 6.17 g/L. As the interspecific hetero, the intraspecific differences also existed in the production capacity of exopolysaccharides. For example, the lowest sugar production of A. melanogenum strain was 1.53 g/L, and the highest sugar production was 54.58 g/L. The strains in the same branch of the phylogenetic tree have the same apparent and microscopic morphology, but the exopolysaccharides yields produced by fermentation are different. For example, the exopolysaccharides yields of four strains among A. motuoense are 15.74 g/L, 21.39 g/L, 31.72 g/L, and 37.43 g/L, respectively. In summary, there were differences in exopolysaccharides production capacity among species. Although strain morphology and molecular sequence were consistent within species, there were differences in physiological functions such as exopolysaccharides production capacity.

Discussion
The species of Aureobasidium are widely distributed globally in various habitats, such as house dust, air, tree surfaces (such as needles of Pinus tabuliformis, Acer pseudosieboldianum, Bintaro plants, Castanea henryi, and Castanea mollissima), plant interiors, seawater, sea ice and glacial meltwaters, water and sediment samples, soil, and subcutaneous phaeohyphomycosis from the US, Canada, Korea, Indonesia, China, the Arctic coast, and Brittany (France) [1,8,10,11,18,21,23]. In this study, many strains were isolated from soil and plant leaves, but strain KCL139 was isolated from the surface of a spittle insect. Yeasts occur commonly on insect species, and the number of insect-related yeasts had increased in the last 10 years reaching a ratio of 7.25% [42][43][44], compared with the report of Boekhout [45]. However, according to our knowledge, Aureobasidium species isolated from insects are rare. Our finding not only expands the ecology niches of Aureobasidium, but also supports the 'dispersal-encounter' hypothesis proposed by Madden et al. [46]. The four new species described in this study have different growth temperatures from each other ( Table 2). They can grow at 17 • C, 28 • C and 30 • C; however, only A. motuoense can tolerate a high temperature of 37 • C, a feature associated with the climate characteristics of Motuo County where this new species was collected. Motuo County boasts a typical sub-tropical moist climate. The optimum growth temperature for A. planticola, A. intercalariosporum, and A. insectorum is 28 • C, whereas A. motuoens is 30 • C. A. planticola, A. intercalariosporum, and A. motuoense fail to grow at 4 • C, whereas A. insectorum grows at this temperature normally. Unfortunately, the psychrophilic character of A. insectorum cannot be explained based on the present data. A. planticola, A. motuoens, and A. insectorum can grow on MEA supplemented with 15% (w/v) NaCl, but A. planticola can only tolerate concentrations of up to 5% NaCl. All tested Aureobasidium species [10], can grow on 10% NaCl MEA, and most of them can tolerate concentrations of 10% NaCl. Only A. planticola is intolerant to more than 10% NaCl. A. planticola shows good growth on the M40Y medium, while the other strains grow well on the PDA medium.
The study of Zou et al. [47] showed that A. pullulans produced polymalic acid by fermentation, and strains ZD-3d, IP-1, Sp. P6, MCW, and ZX-10 produced 57.2 g/L, 57.4 g/L, 91.1 g/L, and 117.4 g/L, respectively, which indicated that different strains could produce different yields of polymalic acid at the same fermentation conditions. In this paper, we found that different strains of the same species, such as A. insectorum, A. leucospermi, A. motuoense, and A. melanogenum, produced pullulan ranging from 0.92 to 54.58 g/L (Table 3), which in agreement with the result from Haghighatpanah et al. [37], shows that there were differences in exopolysaccharides production within the same species. Our results also showed that different Aureobasidium species have great differences in their exopolysaccharides production capacity. Therefore, we should screen more diverse environments to isolate strains of certain species for comprehensive study in exploring Aureobasidium compounds for industrial application.

Conclusions
In this study, 14 strains of Aureobasidium isolated from Tibet, Hebei, and Zhejiang are proposed as four new species based on molecular analysis of their ITS and large ribosomal subunits of D1/D2 domains. The new species are named as Aureobasidium insectorum sp. nov., A. planticola sp. nov., A. motuoense sp. nov., and A. intercalariosporum sp. nov. We found differences in interspecific and intraspecific exopolysaccharides production between the species, indicating the diversity in strain-specific exopolysaccharides production. Aureobasidium has strong adaptability and wide distribution, and there may be many new Aureobasidium taxa in nature, which have the potential to produce new metabolites. Therefore, studies into the species diversity of this genus and the industrial application of its metabolites are of great significance.
Author Contributions: Q.W. conceived and designed the project. F.W. and Z.F. performed sampling and yeasts isolation. F.W., M.W., Z.F. and Q.W. performed phenotypic characterization and analyzed the molecular data. F.W., M.W. and Q.W. wrote the paper. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: All newly generated sequence data are available in NCBI GenBank. The phylogenetic tree and the alignments were deposited in TreeBASE.