Screening of Phosphate-Solubilizing Fungi From Air and Soil in Yunnan, China: Four Novel Species in Aspergillus, Gongronella, Penicillium, and Talaromyces

Phosphate-solubilizing fungi (PSF) play an important role in increasing the bioavailability of phosphorus in soils for plants. Thirteen fungal strains, one collected from air and 12 from soil, were screened and described here in detail. These fungal strains were tested for their ability to solubilize tricalcium phosphate (TCP) on both solid and liquid Pikovskaya (PVK) media in vitro. The airborne fungal strain KUMCC 18-0196 (Aspergillus hydei sp. nov.) showed the most significant phosphate solubilizing activity on a solid PVK medium with the solubilization index (SI) (2.58 ± 0.04 cm) and the highest solubilized phosphates (1523.33 ± 47.87 μg/mL) on a liquid PVK medium. To the best of our knowledge, A. hydei sp. nov. is the first phosphate-solubilizing fungus reported from air. We also provide the identification especially for Aspergillus, Penicillium and Talaromyces, generally reported as PSF. It is important to not only screen for PSF but also identify species properly so that researchers have a clearer taxonomic picture for identifying potential taxa for future plant growth-promoting applications. Herein, A. hydei (section Nigri), Gongronella hydei, Penicillium soli (section Lanata-Divaricata) and Talaromyces yunnanensis (section Talaromyces) are fully described and introduced as new to science. These four new species are identified based on both morphological characteristics and multigene phylogenetic analyses, including the genealogical concordance phylogenetic species recognition method where necessary. Penicillium austrosinense is considered to be a synonym of P. guaibinense.


INTRODUCTION
Fungi are widely distributed across terrestrial, marine, and freshwater environments Phookamsak et al., 2019;Dayarathne et al., 2020;Dong et al., 2020). Fungi play important roles in both economics and ecology as sources of food and medicine that also provide decomposition services (De Silva et al., 2012a,b;Lange, 2014;Hyde et al., 2019b). Moreover, fungal research can lead to breakthroughs in microbial biotechnology and other industries Solomon et al., 2019). However, fungi can also cause diseases in humans, animals, and plants, posing numerous health challenges in need of addressing (Giacomazzi et al., 2016;Hyde et al., 2018). Given the overlooked position of studies on phosphate-solubilizing fungi (PSF), there are likely many novel fungal species still awaiting discovery.
Phosphate-solubilizing fungi (PSF) are able to enhance the solubilization of insoluble phosphate (P) compounds. They also have the capacity to mobilize and increase nutrient uptake, produce organic acids and increase the efficiency of phosphate fertilizers, such as superphosphate and rock phosphate (Jyoti et al., 2013;Li et al., 2015). Some PSF promote plant growth by secreting indole-3-acetic acid (IAA) and siderophore (Rahi et al., 2009;Zhang et al., 2018). They have enormous potential in enhancing the release of phosphorous from fertilizer (Zhang et al., 2018). PSF in soils, particularly filamentous fungi such as Aspergillus and Penicillium, have been widely investigated (Elias et al., 2016;Li Z. et al., 2016), but airborne fungi remain to be explored. In addition, previous identifications of PSF (e.g., Aspergillus, Penicillium, and Talaromyces) have been based on morphology, nucleotide similarities in MegaBLAST searches of NCBI's GenBank nucleotide database and/or phylogenetic analysis of ITS sequence data (e.g., Vyas et al., 2007;Nelofer et al., 2015;Qiao et al., 2019).
In this paper, 13 fungal strains from air and soil samples were screened for their ability to solubilize phosphate. Known species are identified on the basis of phylogenetic analyses. New species are introduced based on morphological characteristics and phylogenetic analyses. This study provides new scientific insights into novel PSF isolated from the air. In addition, new fungal taxa isolated from the soil of cultivated oak seedlings (Quercus rubra) in Honghe County, Yunnan Province, China are introduced.

Collection and Isolation of Fungi From Air Using the Settle Plate Method
Five Petri plates (9 cm diam.) with opened lids containing PVK agar with 5% tricalcium phosphate [TCP, Ca 3 (PO 4 ) 2 ] were placed under a Quercus variabilis tree in Kunming City, Yunnan Province, China and left for 20 min. Quercus variabilis is a selected host in this study as it is a native oak in China. Lids were then closed over the plates and carefully transferred to the laboratory where they were incubated for 2 days at room temperature. The fungal colony with the widest clear zone around the colony on PVK agar was selected and transferred to fresh potato dextrose agar (PDA) to obtain a phosphatesolubilizing fungus.

Collection and Isolation of Fungi From Soil Using Soil Dilutions
Twenty grams of rhizospheric soil samples (10 cm depth) were collected from Quercus rubra in Honghe County, Yunnan Province, China. Quercus rubra is also a selected host herein as it is the most valuable timber species in red oak group. Soil samples were separated from roots, stored in ziplock bags and carefully transferred to the laboratory. One gram of each soil sample was dissolved into 9 mL of sterilized distilled water in 15 mL test tubes and mixed well. The serial soil dilutions were made for 10 −4 , 10 −5 , 10 −6 . Soil dilutions were used to isolate fungi from soil samples. Half a milliliter (0.5 mL) of each dilution was drawn using a micropipette and placed onto a PDA plate supplemented with 100 mg/mL streptomycin and spread using a sterile L-shaped spreader. The Petri plates were then inoculated for 2-5 days at room temperature. Fungal colonies were purified by transferring a single hyphal tip to PDA. Pure cultures of each fungal isolate were preserved on PDA agar slants in 2 mL vial tubes at 4 • C for further screening.

Determination of Solubilization Index on PVK Agar
Thirteen fungal isolates obtained from pure cultures were preliminarily screened for their potential to solubilize TCP as insoluble inorganic phosphate sources on PVK agar. One liter (1 L) of PVK agar contained the following (g/L): 0.5 g (NH 4 ) 2 SO 4 , 0.1 g MgSO 4 ·7H 2 O, 0.02 g NaCl, 0.02 g KCl, 0.003 g FeSO 4 ·7H 2 O, 0.003 g MnSO 4 ·H 2 O, 5 g Ca 3 (PO 4 ) 2 , 10 g glucose, 0.5 g yeast extract, 15 g agar, and 1000 mL distilled water (Pikovskaya, 1948). The medium was autoclaved at 121 • C for 15 min. Sterilized PVK agar was poured into sterilized Petri plates. Fungal mycelium plugs of each isolate grown on PDA at 28 • C for 7 days were cut from the edges of each actively growing colony using a sterile cork borer (5 mm 3 ). Fungal mycelium plugs were then placed on Petri plates containing PVK agar supplemented with 0.5% TCP for 7 days at 28 • C. Plugs of sterile PDA were used as controls. Three replicates were tested for each fungal isolate. The diameter of clear zones around the colony of each isolate was measured after the 1 st , 3 rd , 5 th , and 7 th day of incubation. The phosphate solubilization index was calculated according to the formula below (Premono et al., 1996). The phosphate solubilization activity test was carried out in a 150 mL conical flask with 100 mL PVK broth supplemented with 0.5% TCP. The initial pH of the medium was adjusted to 7.0 before sterilization. The preparation of spore suspensions from each fungal isolate was done according to the method described in Elias et al. (2016). Ten milliliter (10 mL) spore suspensions of each fungal culture (10 7 spores/mL) were inoculated in the sterilized PVK broth of each conical flask. Ten milliliters (10 mL) of sterile distilled water in the sterilized PVK broth was treated as the control. Three replicates were maintained for each fungal test. The cultures were incubated on a rotary shaker (Sanco, India) at 28 • C at 130 rpm for 7 days. Aliquots of 1.5 mL culture supernatant were aseptically collected from each conical flask on the 1 st , 3 rd , 5 th , and 7 th day. The samples were centrifuged (Sigma, Germany) at 12,000 rpm for 2 min to discard any suspended solids and mycelial fragments. Supernatants (0.1 mL) of each culture were then taken out to estimate the amount of phosphorus released from TCP of fungal strains. The available soluble phosphate in culture supernatants was estimated using the molybdenum blue method under 700 nm (Ryan et al., 2001). The pH of the culture supernatant in PVK broth was also measured with a digital pH meter FE 28 equipped with a glass electrode (Shanghai, Co., Ltd.).

Statistical Analysis
Data from different treatments were calculated and statistically analyzed with a one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) for Windows version 22 (SPSS, Inc., Chicago, IL, United States). Duncan's multiple range tests were used to determine significant differences at p < 0.05 between the mean values.

Morphological Studies
The fungi were identified using morphological characteristics and phylogenetic analyses.

DNA Extraction, PCR Amplification, and Sequencing
The DNA was extracted from the mycelium grown on PDA at 25 • C for 7 days using a Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux R , China) following the manufacturer's protocol. The 5.8S nrRNA gene with the two flanking internal transcribed spacers (ITS) regions were first amplified and sequenced using the primers ITS1/ITS4 (White et al., 1990) for all fungal strains. They were preliminarily identified to genera level as Aspergillus, Penicillium, Talaromyces, Gongronella, and Fusarium based on the BLAST searches of ITS in the nucleotide database of GenBank 1 . Partial calmodulin gene (CaM) using primers CMD5/CMD6 (Hong et al., 2005), partial beta-tubulin gene (BenA) using primers Bt2a/Bt2b (Glass and Donaldson, 1995) and partial RNA polymerase II second largest subunit (RPB2) using primers 5F/7CR (Liu et al., 1999) were then amplified and sequenced for strains of Aspergillus, Penicillium, and Talaromyces. The LSU gene region was amplified and sequenced using the primers LR0R and LR5 (Vilgalys and Hester, 1990;Rehner and Samuels, 1994) for the strains of Gongronella. Translation elongation factor 1-alpha (TEF1-α) was amplified and sequenced using primers TEF1/TEF2 (O'Donnell et al., 1998) for the strain of Fusarium but was unsuccessful even after several efforts.
The PCR amplifications were conducted in 25 µL final volumes which consisted of 12.5 µL of 2 × Power Taq PCR MasterMix (a premix and ready to use solution, including 0.1 Units/µL Taq DNA Polymerase, 500 µM dNTP Mixture each (dATP, dCTP, dGTP, dTTP), 20 mM Tris-HCl pH 8.3, 100 mM KCl, 3 mM MgCl 2 , stabilizer and enhancer), 1 µL of each primer (10 µM), 1 µL genomic DNA extract and 9.5 µL distilled-deionized water. Thermocycling was completed on a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, United States) under the following conditions: initial denaturation of 5 min at 95 • C, followed by 35 cycles of 1 min at 95 • C, 2 min at 52 • C, 90 s at 72 • C and a final extension period of 10 min at 72 • C for ITS and RPB2. The annealing temperature was changed to 55 • C for CaM, LSU, TEF, and BenA. The amplified PCR products were purified and sequenced by BGI Tech Solutions, Co. Ltd. (BGITech), China.

Phylogenetic Analyses
Consensus sequences were generated using Geneious R R7 (Biomatters, Ltd., New Zealand) and BioEdit (Hall, 1999). Sequences were BLAST searched in the nucleotide database of GenBank 1 to examine their closely related taxa. The reference sequences and outgroups were selected from recent relevant literature and GenBank (Supplementary Table S1). The datasets of genomic regions were aligned individually using the MAFFT online version 7.450 server 2 . Phylogenetic trees were inferred with maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI), according to the details described in Wanasinghe et al. (2018).

Genealogical Concordance Phylogenetic Species Recognition (GR)
The new Aspergillus taxon and closely related species were analyzed separately using the GCPSR model. The pairwise homoplasy index (PHI) was determined using SplitsTree v. 4.15.1 (Huson and Bryant, 2006 3 ) as described by Taylor et al. (2000) and Quaedvlieg et al. (2014) to determine the recombination level within phylogenetically closely related species. A PHI value below 0.05 ( w < 0.05) indicates the presence of significant recombination in the dataset. Split graphs were constructed to ease visualization of the relationship between closely related species. The relationships between this new taxon and closely related species were visualized by constructing splits graphs using both the LogDet transformation and splits decomposition options.

Formation of Clear Zones and Phosphate Solubilization Index (SI) on PVK Agar
The clear zone became visible in the isolates KUMCC 18-0196 and KUMCC 18-0206 on the 1 st day. The clear zone of the other fungal isolates became visible on the 3 rd day and did not show the clear zone around colonies after the 7 th day but solubilized TCP by removing white spots on PVK agar (Figure 1). The isolate KUMCC 18-0196 showed the widest clear zone around the fungal colony (Figure 1).
The phosphate solubilization index began increasing in isolates KUMCC 18-0196 and KUMCC 18-0206 on the 1 st day, while in other fungal isolates it began increasing on the 3 rd day (Figure 2). The highest phosphate solubilization index was seen in KUMCC 18-0196 (SI = 2.58 ± 0.04 cm) compared with other fungal strains and the control on the 7 th day, followed by KUMCC 18-0198 with SI = 1.97 ± 0.15 cm and KUMCC 18-0203 with SI = 1.90 ± 0.01 cm (Supplementary Table S2). It was observed that isolate KUMCC 18-0197 had the smallest SI = 1.10 ± 0.17 cm. The phosphate solubilization of isolate KUMCC 18-0196 was significantly different (p < 0.05) from other fungal strains (Supplementary Table S2).
The pH values of all 13 fungal isolates decreased from 7.01 after the 1 st day of incubation. The pH values continued to decrease after the 3 rd day and increased or decreased to variable levels in the PVK broth containing TCP during the 5 th and 7 th days of incubation, depending on the fungal isolates (Figure 4). The isolate KUMCC 18-0196 showed the highest decrease in pH from an initial pH of 7.01 to 3.33 ± 0.01 on the 7 th day of incubation, which was significantly different compared to the control and other fungal strains (Supplementary Table S2).

Taxonomic and Phylogenic Studies
In total, 13 fungal strains screened for TCP solubilization on both solid and liquid PVK media are listed in Supplementary Table  S3. Five strains are introduced as new species viz. Aspergillus hydei, Gongronella hydei, Penicillium soli and Talaromyces yunnanensis, based on morphological characteristics and phylogenetic analyses. Penicillium guaibinense collected from soil in China is also illustrated and described. Their morphological characteristics and phylogenetic analyses are provided below.
Notes: Aspergillus section Nigri is the black aspergilli. This section includes species with smooth conidiophores and hyaline or pigmented below the vesicle, globose, subglobose to pyriform vesicles, typically radiate conidial heads, or divergent columns in some species, and black conidia (Gams et al., 1986). They have been isolated from various habitats, such as plants, contaminated materials, indoor air environments and soil samples (Sørensen et al., 2011;Visagie et al., 2014a;Fungaro et al., 2017;Serra et al., 2017;this study). Many strains of Aspergillus niger are used in biotechnology for the production of various metabolites, such as in antibiotics, organic acids, bioenergy or as microbial fermentation in food (Hasegawa et al., 2007;Show et al., 2015;Chuppa-Tostain et al., 2018). They have also been reported as the pathogen responsible for invasive pulmonary aspergillosis (Walsh et al., 2007) and other plant pathogens (Gautam et al., 2011;Sharma, 2012;Guo et al., 2017). Black aspergilli are hard to differentiate solely by morphological characteristics due to high species variation. We introduce the new species A. hydei (KUMCC 18-0196) from air based on morphological characteristics and multi-loci phylogenetic analyses, including the GCPSR method.
Holotype: MFLU 20-0430 Macromorphology: Colonies on MEA reaching 50-55 mm diam. after 7 days in the dark at 25 • C, circular to filamentous, undulate to filamentous, flat or effuse to raise, dense, compact white basal felt covered by a dense layer of dark brown to black conidial heads in the center, white at the edge from above; yellow or creamy, wrinkled and cracked from below. Colonies on Czapeck Yeast Agar (CYA) reaching 75-80 mm diam. after 7 days in the dark at 25 • C, irregular in shape, curled to undulate, flat or effuse, compact white basal felt covered by a dense layer of black conidial heads, with concentric rings from above; white from below. Colonies on PDA reaching 35-40 mm diam. after 7 days in the dark at 25 • C, filamentous shape, undulate to filamentous, flat or effuse to raise, dense, compact white basal felt covered by a dense layer of dark brown to black conidial heads, white at the edge from above; white or creamy from below. Colonies on WA reaching 50-55 mm diam. after 7 days FIGURE 1 | Colony features and clear zones formation for TCP solubilization on PVK agar after 7 days Except isolate KUMCC 18-0196 after 5 days (left = above view, right = below).
Frontiers in Microbiology | www.frontiersin.org FIGURE 2 | Solubilization index (SI) of 13 fungal strains and control on PVK agar containing TCP after the 1 st , 3 rd , 5 th , and 7 th day of incubation (each value represents the mean of the three replicates).
FIGURE 3 | Solubilized P concentrations of 13 fungal strains and control in PVK broth containing TCP after the 1 st , 3 rd , 5 th , and 7 th day of incubation (each value represents the mean of the S3). pH values of PVK broth containing TCP inoculated with each 13 fungal isolate and control after the 1 st , 3 rd , 5 th , and 7 th day of incubation (each value represents the mean of the three replicates).
in the dark at 25 • C, slightly circular, edge entire to undulate, flat, loose, white with black conidial heads at the center (mycelium plug) from above; white from below. Colonies on Rosebengal reaching 60-65 mm diam. after 7 days in the dark at 25 • C, slightly circular, edge entire to undulate, flat, loose, compact white basal felt covered by a lose layer of cream conidial heads with gray conidial heads at the center (mycelium plug), with wrinkled from the center to the edge, white at the edge from above; white to cream from below.
Notes: Colony diameter after 7 days of incubation at 25 • C on CYA of A. hydei is obviously smaller than A. brunneoviolaceus (75-80 vs. > 85 mm) (Jurjević et al., 2012). A. hydei (MFLUCC 18-0196) had a close phylogenetic affinity with A. brunneoviolaceus based on individual ITS and CaM sequence data (Supplementary Figures S1, S2). However, A. hydei formed an independent lineage to the ex-type (NRRL 4912) and other strains of A. brunneoviolaceus based on combined CaM, BenA, ITS and RPB2 sequence data with significant support (78% MP, 82% ML and 1.00 PP) (Figure 6). A. hydei also had close phylogenetic affinity with A. aculeatinus, A. aculeatus, A. floridensis, and A. trinidadensis (Supplementary Figures  S3, S4) but formed a distinct lineage from these four species (Figure 6). The GCPSR test was applied to a combined dataset including A. hydei and its closely related A. brunneoviolaceus, containing eight isolates. The result revealed that there was no significant recombination between A. brunneoviolaceus and A. hydei ( w = 1.00) (Figure 7). Thus, A. hydei can be recognized as a new species.

Gongronella Ribaldi
Type: Gongronella urceolifera Ribaldi Notes: Species of Gongronella have mainly been isolated from soil (Akone et al., 2014;Adamčík et al., 2015;Ariyawansa et al., 2015). Morphological characteristics of Gongronella species are diverse in shape, making it difficult to clearly delineate the species belonging to this genus. In addition, Gongronella species have been described from different media Ellis, 1961, 1964;Adamčík et al., 2015;Ariyawansa et al., 2015;Zhang et al., 2019). However, Gongronella species can be separated by phylogenetic analysis based on ITS sequences as shown in Li G. J. et al. (2016) and Tibpromma et al. (2017) and this study. Multilocus gene regions would well-resolve to determine the species. The genus comprises nine epithets in Index Fungorum (2020). G. hydei is introduced as a new species herein.
Holotype: MFLU 20-0431 Macromorphology: Colonies on PDA reaching 60-65 mm diam. after 7 days in the dark at 25 • C, circular, edge entire, flat or effuse, to raise, dense, white from above and below. Colonies on CYA reaching 55-65 mm diam. after 7 days in the dark at 25 • C, irregular in shape, curled to undulate, flat or effuse, medium dense, white from above and below. Colonies on Rosebengal reaching 45-47 mm diam. after 7 days in the dark at 25 • C, slightly circular, edge entire to undulate, filamentous, flat, dense, cottony, concentric ring, white from above and below. Colonies on WA reaching 30-35 mm diam. after 7 days in the dark at 25 • C, irregular in shape, curled, flat, loose, with obviously white filamentous around the center (mycelium plug) from above; white from below.
Penicillium Section Lanata-Divaricata Raper and Thom ex Pitt Facesoffungi number: FoF 08839 Macromorphology: Colonies on MEA reaching 55-60 mm diam. after 7 days in the dark at 25 • C, circular to slightly undulate, with entire margin, flat or effuse, dense, with wrinkled from center to edge, white with pale yellow circle from above, pale yellow from below. Colonies on CYA reaching 30-35 mm diam. after 7 days in the dark at 25 • C, circular, filamentous, low convex in the center, flat at the edge, medium dense, cottony, white from above, orange-cream from below. Colonies on PDA reaching 25-32 mm diam. after 7 days in the dark at 25 • C, circular, entire, flat or effuse, dense, cottony, white from above, yellow to cream from below. Colonies on Rosebengal reaching 40-45 mm diam. after 7 days in the dark at 25 • C, circular, edge entire to filamentous, flat, dense, cottony, white from above and below.
Talaromyces Section Talaromyces C. R. Benj.  Notes: Talaromyces section Talaromyces was introduced by Stolk and Samson (1972) and characterized by yellow ascomata, as well as occasionally white, creamish, pinkish or reddish and yellow ascospores. Conidiophores are usually of the biverticillatesymmetrical type, and some species are reduced to conidiophores with solitary phialides. Phialides are usually acerose, with a minor proportion of species having wider bases (Stolk and Samson, 1972). Phylogenetic analyses of ITS, BenA, CaM, and RPB2 sequence data were used to determine species delimitation Guevara-Suarez et al., 2020). Section Talaromyces species are usually isolated from soil, indoor air environments and humans (Chen et al., 2016;Yilmaz et al., 2016;Lau et al., 2017;Pangging et al., 2019;Sun et al., 2020). We introduce the new species T. yunnanensis into the section.
Holotype: MFLU 20-0434 Macromorphology: Colonies on MEA reaching 50-53 mm diam. after 7 days in the dark at 25 • C, circular, edge entire, flat or effuse, dense, wrinkled from center to edge, green with orange mass (exudate) in the center, gradually yellow and white at the edge from above, yellow from below. Colonies on CYA reaching 35-37 mm diam. after 7 days in the dark at 25 • C, circular, edge entire to filamentous, flat, medium dense, yellow at the center and white at the edge from above, pale yellow at the center and white at the edge from below. Colonies on PDA reaching 30-40 mm diam. after 7 days in the dark at 25 • C, irregular in shape, curled, flat, dense, yellowish green in the center, yellow and white from above, yellowish cream from below. Colonies on Rosebengal reaching 35-38 mm diam. after 7 days in the dark at 25 • C, circular, edge entire, flat, dense, yellow in the center, white at the edge from above, white from below.

DISCUSSION
We carried out phosphate solubilization of 13 fungal strains in both solid and liquid PVK media in vitro. This study indicates that the airborne isolate KUMCC 18-0196 (A. hydei sp. nov.) was a highly efficient strain in solubilizing phosphate. Isolate KUMCC 18-0196 also showed the greatest drop in pH in the PVK broth containing TCP, suggesting that it might produce organic acids. The A. hydei is the most efficient P-solubilizing than other strains tested in this study. Our results are similar to the study of Pradhan and Sukla (2006), which concluded that Aspergillus spp. show a much higher drop in pH in the PVK broth containing TCP and high P solubilization when compared to Penicillium spp. Many previous studies reported PSF mostly from soils, including rhizospheric and non-rhizospheric soil samples from cultivated plants and asymptomatic plant roots (Nelofer et al., 2015;Elias et al., 2016;Adhikari and Pandey, 2019;Qiao et al., 2019, Supplementary Table S5). To date, PSF from air have not yet been reported. Thus, A. hydei sp. nov. described herein is the first report of PSF from air. However, the determination of phosphate solubilization has been tested on solid and liquid media under laboratory conditions. The ability to solubilize phosphate under field conditions should be closely examined to confirm P dissolving activity. PSF did not lose P solubilizing ability under laboratory conditions, although sub-culturing was repeated (Kucey, 1983;Sharma et al., 2013).
Several phosphate-solubilizing microorganisms viz. actinomyces, bacteria, fungi, and yeasts have been screened for phosphate solubilization. They were shown to enhance the solubilization of insoluble P compounds (Gizaw et al., 2017;Nandimath et al., 2017;Mohamad et al., 2018;Wang et al., 2018;Chen and Liu, 2019;Liu et al., 2019). For fungi, Rhizopus stolonifer var. stolonifer, Aspergillus niger, and Alternaria alternata have been found in the literature to be the most effective strains with respect to the amount of TCP solubilization by Ceci et al. (2018). However, solubilization by PSF depends on the insoluble inorganic phosphate source, type of carbon, nitrogen and metal ions in soils and culture conditions (Richardson and Simpson, 2011;Jyoti et al., 2013). Vyas et al. (2007) tested the quantity of phosphate solubilized by Eupenicillium parvum (current name Penicillium parvum) from the different inorganic phosphates [TCP, aluminum phosphate (AP) and North Carolina phosphate (NCRP)]. Their results showed that the quantity of phosphate solubilized was by far the greatest for TCP among the inorganic phosphates, whereas the solubilization of iron phosphate was low. Phosphatesolubilizing microorganisms convert insoluble phosphates into a soluble form through the processes of acidification, chelation, exchange reaction, and production of organic acid (Behera et al., 2017).
Some studies of PSF identified fungal species names based on morphology, highest nucleotide similarity in MegaBLAST searches of NCBI's GenBank nucleotide database and/or phylogenetic analysis of ITS sequence data (Nelofer et al., 2015;Wang et al., 2018;Zhang et al., 2018, Supplementary  Table S5). However, these are insufficient to determine the species names, especially within species complexes (Samson et al., 2014;Doilom et al., 2017;Hyde et al., 2020a,b). A comparison of sequence data from fungal strains with the ex-type cultures of named species must be considered to confirm the species name (Dayarathne et al., 2016). Several taxonomic schemes and new molecular approaches have been proposed for fungal identification (Visagie et al., 2014b;Jeewon and Hyde, 2016;Zhao et al., 2018). We provide the fungal identification of PSF based on morphological characteristics and multi-loci phylogenetic analyses, including the GCPSR method where necessary. GenBank accession numbers of species in Aspergillus, Gongronella, Penicillium, and Talaromyces with our newly generated sequences are also provided in Supplementary Table  S1 to further facilitate species identification. Fungal taxonomy is discussed in each taxonomic note.
Applications of beneficial microbes, especially phosphatesolubilizing fungi, are useful toward plant growth-promoting strategies. The isolate KUMCC 18-0196 (A. hydei sp. nov.) was a highly efficient strain in solubilizing phosphate; accordingly, we are optimistic that it can play potential roles in supporting plant growth in practical applications. This research provides important information relevant to the application of beneficial fungi to promote plant growth. This will have critical implications for the development of agriculture, industry, restoration ecology and forestry going forward.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
MD, PM, J-CX, and J-WG designed the study. MD planned and conducted the experiments and wrote the manuscript. MD, KY, WD, C-FL, RP, KY, and DP conducted the experiments, analyzed the data, and revised the manuscript. PM, IP, NS, and SL contributed to research funds. All authors revised the manuscript.