Four Unrecorded Aspergillus Species from the Rhizosphere Soil in South Korea

Abstract

The genus Aspergillus is commonly isolated from various marine and terrestrial environments; however, only a few species have been studied in rhizosphere soil. As part of the Korean indigenous fungal excavation project, we investigated fungal diversity from rhizosphere soil, focusing on Aspergillus species. A total of 13 strains were isolated from the rhizosphere soil of three different plants. Based on phylogenetic analysis of β-tubulin and calmodulin and morphological characteristics, we identified five Aspergillus species. A. calidoustus and A. pseudodeflectus were commonly isolated from the rhizosphere soil. Four species were confirmed as unrecorded species in Korea: A. calidoustus, A. dimorphicus, A. germanicus, and A. pseudodeflecuts. The detailed morphological descriptions of these unrecorded species are provided.

1. Introduction

Aspergillus is one of the most common fungi in various environments worldwide. Aspergillus is known as plant and human pathogen, mycotoxin producer, and food spoiler. However, it plays important roles in the ecological and industrial systems by producing antibiotics and organic acids, and degrading starches, celluloses, and other polysaccharides [1–5]. Morphological characters such as growth rate, color of the colony, thermotolerance, and size of conidial heads and conidia are known to be important features for initial identification of Aspergillus [6]. However, morphological feature is not enough to recognize species because their morphological characteristics vary by their ecological habitats [6,7]. For accurate identification of Aspergillus, standardized methods including morphology, molecular analysis, and extrolite profiling have been proposed. DNA markers such as the internal transcribed spacer region, calmodulin (CaM), β–tubulin (BenA), and the RNA polymerase II second largest subunit (RPB2) have been used in Aspergillus identification and phylogeny [8]. According to the current research, the genus consists of six subgenera, 27 sections, and 446 species worldwide [9]. In Korea, 69 Aspergillus species have been reported [10–14]. Although some species have been reported from soil in terrestrial, marine, and clinical environments [15–18], many Aspergillus were isolated from food fermentation such as meju and nuruk [12,19–21]. Nonetheless, study on Aspergillus from rhizosphere soil in Korea is limited [22].

Fungi in rhizosphere environments play an important role in plant growth and adaptation [23,24]. Aspergillus is one of the common fungi in rhizosphere soil [25–31]. Some Aspergillus are known to produce plant promoting chemicals such as gibberellic acid and indole acetic acid [27,28]. Many Aspergillus strains were only identified at the genus level, as previous studies mainly focused on bioactive compounds [27–30]. Therefore, the diversity of Aspergillus in in rhizosphere soil is unclear.

This project is organized by the National Institute of Biological Resources to excavate Korean indigenous fungi from the rhizosphere soil. We explored fungal diversity from rhizosphere soil of various plants; Aspergillus, Penicillium, Trichoderma, and Fusarium were common genera. Recently, we reported on diversity of Penicillium, revealing eight unrecorded species in Korea [32]. The main purpose of this study was to focus on Aspergillus in the rhizosphere of various plants and to identify them based on BenA and CaM loci. We discovered four unrecorded species: A. calidoustus, A. dimorphicus, A. germanicus, and A. pseudodeflectus.

2. Materials and methods

2.1. Sample collections and isolation

Rhizosphere soil of three plants (Calystegia soldanella, Orobanche coerulescens, and Sorbus commixta) were collected from five sites in Korea in 2019 (Table 1). Five grams of soil for each sample was diluted tenfold in sterile water. A 100 µL of each dilute was plated on dichloran rose bengal chloramphenicol agar (DRBC; Difco, Becton Dickinson). All plates were incubated at 25 °C for 7 days. Based on morphology, Aspergillus-like strains were transferred to potato dextrose agar (PDA; Difco, Becton Dickinson) plate. Strains were stored in 20% glycerol at −80 °C at the Seoul National University Fungus Collection (SFC).

Table 1.

The information of Aspergillus strains isolated from rhizosphere soil.

Species	Section	Strain	Location	Substrate
A. calidoustusa	Usti	SFC20191113-NB113	Heunghae-eup, Buk-gu, Pohang-si, Gyeongsangbuk-do	Calystegia soldanella
		SFC20191113-NB116	Heunghae-eup, Buk-gu, Pohang-si, Gyeongsangbuk-do	Calystegia soldanella
		SFC20191113-NB135	Heunghae-eup, Buk-gu, Pohang-si, Gyeongsangbuk-do	Calystegia soldanella
		SFC20191113-NB146, NIBRFG0000509071	Heunghae-eup, Buk-gu, Pohang-si, Gyeongsangbuk-do	Calystegia soldanella
		SFC20191113-NB197, NIBRFG0000509286	Sannae-myeon, Miryang-si, Gyeongsangnam-do	Sorbus commixta
A. dimorphicus	Cremei	SFC20191113-NB100, NIBRFG0000509072	Guryongpo-eup, Nam-gu, Pohang-si, Gyeongsangbuk-do	Orobanche coerulescens
A. germanicus	Usti	SFC20191113-NB098, NIBRFG0000509073	Guryongpo-eup, Nam-gu, Pohang-si, Gyeongsangbuk-do	Orobanche coerulescens
A. insuetus	Usti	SFC20191113-NB013	Guryongpo-eup, Nam-gu, Pohang-si, Gyeongsangbuk-do	Calystegia soldanella
A. pseudodeflectus	Usti	SFC20191113-NB114, NIBRFG0000509074	Heunghae-eup, Buk-gu, Pohang-si, Gyeongsangbuk-do	Calystegia soldanella
		SFC20191113-NB115	Heunghae-eup, Buk-gu, Pohang-si, Gyeongsangbuk-do	Calystegia soldanella
		SFC20191113-NB136	Heunghae-eup, Buk-gu, Pohang-si, Gyeongsangbuk-do	Calystegia soldanella
		SFC20191113-NB156	Heunghae-eup, Buk-gu, Pohang-si, Gyeongsangbuk-do	Calystegia soldanella
		SFC20191113-NB199, NIBRFG0000509287	Sannae-myeon, Miryang-si, Gyeongsangnam-do	Sorbus commixta

^aThe unrecorded Aspergillus species in Korea are represented in bold.

2.2. DNA extraction, amplification, and sequencing

Genomic DNA was extracted from isolated Aspergillus using a modified cetyltrimethylammonium bromide extraction protocol [33]. For the primer sets, Bt2a/Bt2b for BenA and CF1/CF4 or cmd5/cmd6 for CaM, were used [34–36]. PCR was performed in a C1000 thermal cycler (Bio-Rad, Richmond, CA, USA) with previously described methods [37]. The PCR products were purified using the ExpinTM PCR Purification Kit (GeneAll Biotechnology, Seoul, Korea), according to the guideline. DNA sequencing was performed using the PCR primers at Macrogen (Seoul, Korea), using an ABI Prism 3730 genetic analyzer (Life Technologies, Gaithersburg, MD, USA).

2.3. Phylogenetic analysis

The sequences were assembled, proofread, and aligned using MEGA7 [38] and were deposited in GenBank (accession numbers in Table 2). Hamigera avellanea CBS 295.48 was used as the outgroup [39]. Multiple alignments were performed using the default settings of the Multiple Alignment Fast Fourier Transform (MAFFT ver. 7) [40]. Then, each sequence was manually checked and adjusted. Maximum likelihood (ML) phylogenetic tree was performed with RAxML [41] implemented on CIPRES web portal [42], using the GTR + GAMMA model of evolution with 1000 bootstrap replicates.

Table 2.

Strains used for phylogenetic analyses in this study.

Section of Aspergillus	Species	Strain	GenBank accession no.
			BenA	CaM
Cremei	A. arxii	CBS 525.83T	MN969365	MN969223
	A. brunneouniseriatus	NRRL 4273T	EF652123	EF652138
	A. chaetosartoryae	NRRL 5501T	EF652117	EF652129
	A. chrysellus	NRRL 5084T	EF652109	EF652136
	A. citocrescens	CBS 140566T	FR775317	LN878969
	A. cremeus	NRRL 5081T	EF652120	EF652125
	A. dimorphicus	NRRL 3650T	EF652111	EF652135
		SFC20191113-NB100	MW711172	MW711185
		CMV012C9	MK451246	MK451357
		NRRL 35052	EU021672	EU021685
	A. europaeus	CBS 134393T	LN909006	LN909007
	A. flaschentraegeri	NRRL 5042T	EF652113	EF652130
	A. gorakhpurensis	NRRL 3649T	EF652114	EF652126
	A. inflatus	CBS 682.70T	FJ531008	FJ531090
	A. itaconicus	NRRL 161T	EF652118	EF652140
	A. koreanus	EML-GSNP1-1T	KX216530	KX216528
	A. pulvinus	NRRL 5078T	EF652121	EF652139
	A. stromatoides	CBS 500.65T	FJ531038	EF652127
	A. tardus	CBS 433.93T	FJ531001	FJ531084
	A. wentii	NRRL 375T	EF652106	EF652131
Usti	A. asper	CBS 140842T	KT698838	KT698839
	A. baeticus	NRRL 62501T	HE615092	HE615117
	A. calidoustus	CBS 121601T	FJ624456	HE616559
		SFC20191113-NB113	MW711167	MW711176
		SFC20191113-NB116	MW711161	MW711173
		SFC20191113-NB135	MW711160	MW711175
		SFC20191113-NB146	MW711162	MW711174
		SFC20191113-NB197	MW711163	MW711177
		E449/MI09	HG931688	HG931695
		E460	HG964949	HG964950
	A. carlsbadensis	IBT 14493T	FJ531179	FJ531126
	A. collinsii	CBS 140843T	KT698843	KT698844
	A. contaminans	CBS 142451T	LT594443	LT594425
	A. deflectus	NRRL 2206T	EF652261	EF652349
	A. elongatus	NRRL 5176T	EF652326	EF652414
	A. germanicus	DTO 27-D9T	FJ531172	FJ531141
		SFC20191113-NB098	MW711171	MW711184
	A. granulosus	NRRL 1932T	EF652254	EF652342
	A. heterothallicus	NRRL 5096T	EF652323	EF652411
	A. insuetus	NRRL 279T	EF652281	EF652369
	A. insuetus	SFC20191113-NB013	MW711170	MW711183
	A. keveii	CBS 209.92T	EU076376	EU076365
	A. keveioides	CBS 132737T	JN982694	JN982684
	A. lucknowensis	NRRL 3491T	EF652283	EF652371
	A. monodii	CBS 435.93T	FJ531171	FJ531142
	A. porphyreostipitatus	DTO 266-D9T	KJ775080	KJ775338
	A. pseudodeflectus	ET1611	KY853416	KY853415
		NRRL 6135T	EF652331	EF652419
		SFC20191113-NB114	MW711169	MW711178
		SFC20191113-NB115	MW711166	MW711179
		SFC20191113-NB136	MW711168	MW711182
		SFC20191113-NB156	MW711164	MW711181
		SFC20191113-NB199	MW711165	MW711180
		AS3 15308	JN982689	JN982679
		NRRL 278	EF652280	EF652368
	A. pseudoustus	IBT 28161T	FJ531168	FJ531129
	A. puniceus	NRRL 5077T	EF652322	EF652410
	A. sigurros	CMV005I4T	MK451066	MK451512
	A. thesauricus	NRRL 62487T	HE615095	HE615120
	A. turkensis	CBS 504.65T	FJ531191	FJ531145
	A. ustus	NRRL 275T	EF652279	EF652367

^“T” indicates the ex-type strains.

Sequences produced in this study are presented in bold letters.

2.4. Morphological analysis

Morphological analysis of the four unrecorded species was performed on three different culture media using previously described methods: Czapek yeast autolysate agar (CYA; yeast extract, Difco), malt extract agar (MEA; Oxoid), and yeast extract sucrose agar (YES; yeast extract, Difco). The Methuen Handbook of Color was used for the color names and alphanumeric codes for macromorphological characteristics [43]. The microscopic observation was processed under a light microscope (Eclipse 80i, Nikon, Tokyo, Japan) using the samples grown on MEA and CYA.

3. Results

3.1. Species identification

A total of 13 Aspergillus strains were isolated from rhizosphere of three plants. Based on the combined dataset of BenA and CaM sequences, they were identified as five species in two sections with four unrecorded species in Korea (Table 1 and Figures 1 and 2). Twelve strains were included in section Usti and were identified as four species: A. calidoustus (5 strains), A. germanicus (1), A. insuetus (1), and A. pseudodeflectus (5). A. calidoustus, A. germanicus, and A. pseudodeflectus were unrecorded species in Korea. For section Cremei, one strain was discovered and identified as A. dimorphicus, which was unrecorded species in Korea.

Figure 1.

Maximum likelihood (ML) phylogenetic tree of Aspergillus sect. Cremei based on the combined data set of BenA and CaM sequences. Bootstrap values >70 are presented at the nodes. The scale bar represents the number of nucleotide substitutions per site. “T” indicates the ex-type strains. Aspergillus reported in this study are represented in bold. The unrecorded Aspergillus species are accented in color box.

Figure 2.

Maximum likelihood (ML) phylogenetic tree of Aspergillus sect. Usti based on the combined data set of BenA and CaM sequences. Bootstrap values >70 are presented at the nodes. The scale bar represents the number of nucleotide substitutions per site. “T” indicates the ex-type strains. Aspergillus reported in this study are represented in bold. The unrecorded Aspergillus species are accented in color box.

A. calidoustus and A. pseudodeflectus were commonly isolated from the rhizosphere soil (Table 1). Aspergillus diversity was higher in rhizosphere soil of Calytegia soldanella compared to others. Although A. pseudodeflectus was commonly isolated from C. soldanella and Sorbus commixta, generally, the Aspergillus diversity was found unique for each plant.

3.2. Taxonomy

Aspergillus calidoustus Varga, Houbraken, & Samson (2008)

Description: Colony diameter, at 25 °C for 7 days, in mm: CYA 50–51; CYA 15 °C 12–13; CYA 30 °C 60–66; CYA 37 °C 8–12; MEA 51–54; YES 50–51 (Figure 3).

Figure 3.

The unrecorded Aspergillus species in Korea: A. calidoustus (SFC20191113-NB146), A. dimorphicus (SFC20191113-NB100), A. germanicus (SFC20191113-NB098) and A. pseudodeflectus (SFC20191113-NB114). (a–c) Colonies grown on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), and yeast extract sucrose agar (YES) from left to right (top = obverse, bottom = reverse). (d–f): Conidiophores; (g) Conidia (scale bar = 10 μm).

Colonies on CYA, lightly sulcate, moderate to good sporulation, floccose, greenish gray (27D2) elsewhere with 1 mm white margin, exudate brownish orange (6D8) to dark brown (6F8) droplets, soluble pigment absent, reverse color olive brown (4D3) at center and light yellow (1A4) elsewhere. Colonies on MEA, lightly sulcate, moderate sporulation, velvety with floccose at center, central part gray (27B1) at center and greenish gray (27E2) elsewhere with 1 mm white margin, no exudates, soluble pigment absent, reverse color olive brown (4E4) and orange yellow (4B8) elsewhere. Colonies on YES, lightly sulcate, moderated sporulation, central floccose, gray (3C1) to yellowish gray (3C2) elsewhere with 1 mm white margin, no exudates, soluble pigment light yellow (3A4), reverse color olive (3D3) to yellow (3A7).

Conidiophores biseriate with thick, smooth-walled, brown, (2.4–) 3.3 (–4.9) µm wide; vesicles pyriform to broadly spathulate, (8.6–) 9.3 × 9.8 (–10.5) µm; conidial heads loosely columnar; metulae covering the upper half to three-fourths of upper surface, (3.1–) 4.3 × 5.6 (–7.1) µm; phialides (2.8–) 3.2 × 5.4 (–6.7) µm; conidia rough walls, inner and outer wall visible, globose, (3.2–) 3.6 to 3.7 (–4.1) µm.

Strains examined: SFC20191113-NB113, SFC20191113-NB116, SFC20191113-NB135, SFC20191113-NB146, and SFC20191113-NB197

Remarks:A. calidoustus is morphologically similar to A. pseudodeflectus and A. ustus. A. calidoustus was able to grow at 37 °C, but A. ustus was not [44]. A. calidoustus can be distinguished from A. pseudodeflectus by narrow margin and moderate or good sporulation in CYA.

Aspergillus dimorphicus B.S. Mehrotra & R. Prasad (1969)

Description: Colony diameter, at 25 °C for 7 days, in mm: CYA 40–43; CYA 15 °C 10–12; CYA 30 °C 37–39; No growth at CYA 37 °C; MEA 30–31; YES 74–75 (Figure 3).

Colonies on CYA, moderately sulcate, moderate sporulation, floccose, yellowish gray (3C2) at center, pastel yellow (3A5) elsewhere with 1 mm white margin exudates yellowish white (3A2), soluble pigment absent, reverse color yellowish white (2A2). Colonies on MEA, lightly sulcate, moderate sporulation, floccose, pastel yellow (3A4) elsewhere with 1 mm white margin, exudates yellowish white (3A2), soluble pigment absent, reverse color light orange (5A5). Colonies on YES, lightly sulcate, good sporulation, floccose, olive yellow (3D6) elsewhere with 1 mm white margin, no exudates, soluble pigment absent, reverse color pastel yellow (3A4).

Conidiophores biseriate with smooth-walled, sinuous, light yellow, (3.7–) 4.4 (–6.1) µm wide; vesicles mostly globose to subglobose, (8.0–) 9.9 × 10.3 (–13.4) µm; conidial heads globose to loosely radiate, phialides (3.0–) 3.6 × 9.6 (–11.5) µm; conidia subglobose to globose with rough wall, 3.5 to 4.7 µm.

Strain examined: SFC20191113-NB100

Remarks:A. dimorphicus is morphologically similar to A. wentii, it can be distinguished from A. wentii by branched conidiophore with two vesicles [45].

Aspergillus germanicus Varga, Frisvad & Samson (2011)

Description: Colony diameter, at 25 °C for 7 days, in mm: CYA 42–43; CYA 15 °C 13–14; CYA 30 °C 47–48; CYA 37 °C 8–9; MEA 40–42; YES 45–48 (Figure 3).

Colonies on CYA, poor sporulation, floccose, orange gray (6B2) to white (26C1) at center, no exudates, soluble pigment yellowish gray (3B2), reverse color brownish gray (4E4) to pale yellow at margin (3A3). Colonies on MEA, poor sporulation, velvety, white, no exudates, soluble pigment absent, reverse color orange (5A7). Colonies on YES, poor sporulation, floccose to velvety, central part color white to grayish yellow (4B4) to white at center, no exudates, soluble pigment pale yellow (3A3), reverse color grayish orange (5B4) at center and grayish yellow (3C4) and light yellow (3A5).

Conidiophores biseriate with thick, smooth-walled, brown, (3.7–) 4.7 (–5.4) µm wide; vesicles septulate, (8.5–) 10.1 × 10.3 (–12.4) µm; conidial heads loosely columnar; metulae covering the upper half to three-fourths of upper surface, (2.3–) 3.2 × 5.2 (–5.8) µm; phialides (2.3–) 2.7 × 5.0 (–6.1) µm; conidia globose with brown smooth wall, (2.8–) 3.1 to 3.4 (–3.9) µm.

Strain examined: SFC20191113-NB098

Remarks:A. germanicus is morphologically similar to A. thesauricus, it can be distinguished from A. thesauricus by growth at 37 °C, thicker conidiophore, and smaller vesicle diameter [46].

Aspergillus pseudodeflectus Samson & Mouchacca (1975)

Description: Colony diameter, at 25 °C for 7 days, in mm: CYA 49–51; CYA 15 °C 10–13; CYA 30 °C 57–59; CYA 37 °C 14–18; MEA 47–48; YES 52–54 (Figure 3).

Colonies on CYA, lightly sulcate, poor to moderate sporulation, floccose, grayish orange (5B3) at center and white elsewhere, exudates dark brown (8F4), soluble pigment pale yellow (3A3), reverse color olive (3D3) at center and light yellow (2A5) elsewhere. Colonies on MEA, moderately sulcate, poor to moderate sporulation, floccose, brownish gray (5C2) and white at margin, no exudates, soluble pigment absent, reverse color brown (6E5) and golden yellow at margin (5B7). Colonies on YES, radially sulcate and wrinkled at center, poor to moderate sporulation, floccose, yellowish gray (4B2) and white at margin, no exudates, soluble pigment pale yellow (3A3), reverse color deep yellow (4A8).

Conidiophores biseriate with short, curved, rough-walled, brown, (3.4–) 3.9 (–4.2) µm wide; vesicles globose to clavate, (6.7–) 7.9 × 9.5 (–10.5) µm; conidial heads brown, radiate; metulae more or less cylindrical, (2.4–) 3.6 × 4.6 (–5.4) µm; phialides (2.8–) 3.4 × 6.4 (–8.1) µm; conidia globose to ellipsoidal with thick-walled, brown, rough wall, (3.3–) 3.7 to 4.5 (–5.3) µm.

Strains examined: SFC20191113-NB114, SFC20191113-NB115, SFC20191113-NB136, SFC20191113-NB156, and SFC20191113-NB199

Remarks:A. pseudodeflectus is morphologically similar to A. calidoustus. A. pseudodeflectus can be distinguished from A. calidoustus by wide margin and poor sporulation in CYA.

4. Discussion

The rhizosphere soil is a complex and dynamic environment that provides a close relationship between plants and microbes. A total of 13 Aspergillus strains were isolated from rhizosphere soil of three plants and were identified as five species in two sections including four unrecorded species based on BenA and CaM sequences. Although 12 species in Aspergillus section Fumigati have been reported from arable soil [17], many previous studies only focused on limited environments, such as meju and nuruk [12,19–21]. Only Aspergillus terreus has previously been reported from rhizosphere soil of paprika plants in Korea [47]. Five additional species (A. calidoustus, A. dimorphicus, A. germaincus, A. insuetus, and A. pseudodeflectus) are reported for the first time in this study, from the rhizosphere soil in Korea.

Four species were unrecorded in Korea: A. calidoustus, A. dimorphicus, A. germanicus, and A. pseudodeflectus. A. calidoustus is commonly found in clinical environments, indoor air, and forest soil [44,48,49]. It has been isolated from Acanthospermum austral and is known for its antifungal and cytotoxic activity [50]. In this study, A. calidoustus was isolated from the rhizosphere soil of Calystegia soldanella and Sorbus commixta. A. pseudodeflectus was previously isolated from desert soil and seaweed [51,52]. It produced pseudodeflectusin, which exhibited cytotoxic activity [51]. In this study, A. pseudodeflectus strains were isolated from rhizosphere soil of Calystegia soldanella and Sorbus commixta. The A. pseudodeflectus strains isolated from Korea exhibited faster growth on YES compared to the other reported strains [53,54]. Some fungi isolated from different environments exhibit different metabolism and growth rates due to environment adaptation [55,56]. A. germanicus was first isolated from indoor air, but there are few reports of the species so far [57]. In this study, A. germanicus was isolated from the rhizosphere soil of Orobanche coerulescens. Our study is the first report of the species from rhizosphere soil. A. dimorphicus was isolated from garden soil, loess soil, and deep-sea sediment [58–61]. A. dimorphicus showed antitumor activities [62] and proteolytic activities [63]. A. dimorphicus strain was isolated from the rhizosphere soil of Orobanche coerulescens in this study.

Aspergillus species are well known for their potential for usage in industrial and medical compounds, but many strains remain at the genus level. Therefore, we believe that our study will provide the basis for the discovery of new compound based on accurate identification of Aspergillus. Although many Aspergillus species have been found in rhizosphere soils using the NGS method [64–66], the role of Aspergillus in rhizosphere soil is unclear. To understand the interaction between Aspergillus and plants, further studies are needed to investigate the function of Aspergillus in rhizosphere soil.

Funding Statement

This research was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea [grant number NIBR202002104 and NIBR202102107].

Disclosure statement

No potential conflict of interest was reported by the author(s).