Introduction

Fusarium is a large genus of filamentous fungi that is widely distributed in soil, plants and animals. This fungal genus is one of the most important plant pathogens and can cause a variety of plant diseases (Bai et al. 2002; Dean et al. 2012; Coleman 2016), thereby affecting the growth of crops and other plants and reducing their economic value. In addition, it also harbors mycotoxin producers that can produce several extremely important mycotoxins, such as trichothecenes and fumonisins (Ilgen et al. 2008; Woloshuk and Shim 2013), and opportunistic human pathogens (Desjardins 2006; Tupaki-Sreepurna et al. 2018). Although Fusarium species are of great economic importance because of their beneficial and harmful effects, they are difficult to identify.

In 1809, the genus Fusarium, with Fusarium roseum as the model species, was first described by Link, Heinrich Friedrich. In 1935, the first complete taxonomic system of Fusarium was proposed, and it divided Fusarium into 16 groups and 65 species and became the basis for the taxonomic study of Fusarium (Wollenweber and Reinking 1935). From 1945 to 1983, scholars have proposed 10 different classification systems (Snyder and Hansen 1940; Booth 1971; Gerlach and Nirenberg 1982; Nelson et al. 1983), and the most influential system was proposed by Nelson et al. (1983). To date, more than 300 different Fusarium species have been discovered, and nearly half have not been formally described (O’Donnell et al. 2018; Summerell 2019). Nevertheless, some Fusarium species have been isolated, identified and characterized, such as F. oxysporum, which could cause a variety of root-rot diseases (Pérez-Hernández et al. 2014; Liu et al. 2016); and F. graminearum, which could lead to head blight of wheat (Duan et al. 2019; Rojas et al. 2020). Most plant species have one or more fusarium diseases that affect their production. Fusarium spp. have both the asexual and sexual states in their life cycles. In morphological taxonomy, some morphological characteristics could provide a useful reference for the identification of Fusarium spp. such as the presence of the typical banana-shaped macroconidia, chlamydospores formed from hyphae, as well as microconidia and sexual reproductive structures. However, because the morphology of Fusarium spp. is complex and susceptible to environmental impacts, results based on morphological characteristics are not very accurate. Currently, molecular classification techniques, such as DNA markers to distinguish species and molecular phylogenetic analyses, have been successfully applied to the identification of the species of genus Fusarium.

In this study, soil samples were collected from a poplar plantation (32°52’28.45’’N, 120°49’47.63’’E) located in Jiangsu Province, Eastern China. The poplar plantation is close to the Yellow Sea State Forest Park and belongs to the transition region between the subtropics and warm-temperate zones. It has obvious transitional, oceanic and monsoon climates. The soil is classified as a Fluvisol according to the World Reference Base (WRB), and the soil pH is alkaline. From these collected soil samples, some interesting filamentous fungi were isolated. According to the morphological characteristics and molecular identification, a new species of the genus Fusarium is identified and described in this paper.

Materials and Methods

Isolation and culture condition

Soil samples were collected from the Dongtai Poplar Plantation (32°52’28.45’’N, 120°49’47.63’’E) of Jiangsu Province in eastern China. The soil had a sandy texture and was alkaline, and no fertilization or other treatments were conducted. Martin's plate (10 g of dextrose, 5 g of peptone, 0.5 g of MgSO₄·7H₂O, 1 g of KH₂PO₄, 3.3 mL of 0.1% Bengal Red Solution, 20 g of agar, 3.3 mL of 1000 UmL^-1 streptomycin solution, 20 mL of 2% sodium deoxycholate solution and 1 L of water) was used as the fungal isolation medium. Subsequently, 0.5 g soil samples were diluted along a gradient (10^-1, 10^-2, 10^-3, 10^-4, 10^-5) with sterile distilled water and then coated on the fungi isolation medium. After 3–10 days of incubation at 30ºC, single colonies were selected for further isolation and purification.

Morphological, physiological, and biochemical characterization

The isolated strain was cultivated on synthetic low nutrient agar (SNA; Elite-Media, China), Potato dextrose agar (PDA; BD Difco, Sparks, M.D., U.S.A.) and Czapek yeast agar (CYA; Kalang, Shanghai, China) and incubated at 30ºC for 7 days to obtain colony growth. The morphology of the strain was observed during this period. The strain was cultured on a VBC plate (0.5 g of dextrose, 1 g of KH₂PO₄, 1 g of NaNO₃, vitamin B, vitamin C, 20 g of agar, and 1 L of water) (Wang and Chen 1994) and incubated in a constant temperature incubator at 30ºC for 7 days. Spore production was observed during incubation. An inverted microscope (IX73, Olympus, Tokyo, Japan) was used for microscopic observation, and an advanced scientific camera control (Digital Optics, Ltd., Auckland, New Zealand) equipped with OCULAR software (Digital Optics, Ltd.) was used for image analysis.

Growth temperature of the strain: The strain was grown at different temperatures (4, 15, 25, 30 and 35ºC), and PDA (BD Difco) was used to investigate the temperature range of strain growth. Growth was observed during culturing at different temperatures for 7 days.

The carbon source utilization of the strain was investigated by using the carbon source identification plate FF Micro Plate™ (Biolog Inc., Hayward, CA, USA).

Molecular characterization

The high-fidelity PCR enzyme KOD FX DNA Polymerase (TOYOBO, Osaka, Japan) was used to amplify the target genes from the fungal mycelia directly. The translation elongation factor 1-alpha gene (EF-1α), the largest subunit of the RNA polymerase gene (RPB1), the sec largest subunit of the RNA polymerase gene (RPB2) and 28 S large subunit (LSU) sequences were amplified in a Gene Amp 9700 system (Applied Biosystems, Foster City, CA, USA) with primer pairs EF1/EF2 (O’Donnell et al. 2008), Fa/G2R (O’Donnell et al. 2010), 5F2/7cR (O’Donnell et al. 2007), and NL1/NL4 (Kwiatkowski et al. 2012). The PCRs were performed as follows: initial denaturation at 94ºC for 4 min; followed by 40 cycles at 98ºC for 10 s, 50ºC for 30 s, and 68ºC for 30 s; and a final extension at 72ºC for 7 min. The PCR products were isolated using agarose gel electrophoresis and then purified using a TaKaRa MiniBEST DNA Fragment Purification Kit (TaKaRa, Otsu, Japan). The purified PCR products were further sequenced and analyzed. The DNA sequence data were deposited in GenBank under accession numbers MN848239, MN848237, MN848238 and MN809346. Previously published sequences included in this study are available from the GenBank database (Table 1).

DNA sequences were aligned with ClustalX (Thompson et al. 1997), and then edited and trimmed by using BioEdit software (Hall 1999). The EF-1α, RPB1, RPB2 and LSU sequences of the strain isolated in this study and similar model strains were used to construct the phylogenetic tree by using the Neighbor-joining method (Saitou and Nei 1987) in MEGA 5.0 software (Tamura et al. 2011). The evolutionary distances were computed using the Kimura 2-parameter method (Kimura 1980), and the unit is the number of base substitutions per site. Gaps and missing data were taken into consideration when > 95% unambiguity was encountered. One thousand bootstrap methods were used (Felsenstein 1985).

Results

Morphological, physiological and biochemical characteristics

The strain PD2^T isolated from a soil sample was spot-cultured on PDA, SNA, and CYA plates at 30℃ for 7 days to obtain colony growth and observe the morphology of the strain (Table 1). The diameter of a 7-day old single colony on PDA medium was 67-73 mm, on CYA medium was 85-86 mm, and on SNA medium was 48–53 mm. On PDA medium, the edge of the colony was light brown and irregular, the aerial hyphae were white, the spores were transparent to white, and the reverse colony was light orange. On CYA medium, the surface of the colony was wrinkled, the aerial hyphae were white, the spores were transparent to white, and the reverse colony was light yellow. On the SNA medium, the positive and negative sides of the colony were white and translucent, the aerial hyphae were white, and the spores were transparent to white (Fig. 1 a–c).

Table1: Strains used in the molecular phylogenetic analysis in this study

Species	Source	Substrate/Host	Origin	GenBank accession number					Reference
				EF-1α	RPB1	RPB2	LSU	ITS
PD2	—	Soil	China	MN848239	MN848237	MN848238	MN809346	MN559538	In this study
Fusarium convolutans	CBS 144207^T	Kyphocarpa angustifolia rhizosphere	South Africa	LT996094	LT996193	LT996141	MN749523	—	Sandoval-Denis et al. (2018)
F. sublunatum	CBS 189.34^T=BBA 62431^T	Soil of banana plantation	Costa Rica	—	JX171451	JX171565	KM231680	NR111606	Nelson et al. (1983); Gräfenhan et al. (2011); Lombard et al. (2015)
	NRRL 20897	Unknown	Unknown	KX302919	KX302927	KX302935	—	—	—
F. algeriense	NRRL 66647^T	Durum, wheat	Algeria	MF120510	MF120488	MF120499	—	NR158423	Laraba et al (2017)
F. concolor	NRRL 13994^T	Hordeum vulgare	Uruguay	MH742650	MH742492	MH742569	—	—	Jacobs-Venter et al. (2018)
F. beomiforme	NRRL 13606^T	Soil	Australia	MF120507	MF120485	MF120496	U34553	—	Laraba et al. (2017)
F. coffeatum	CBS 635.76^T	Cynodonlemfuensis	South Africa	MN120755	MN120717	MN120736	NG057718	—	Lombard et al. (2019)
F. napiforme	NRRL 13604^T	Pennisetumtyphoides	Namibia	AF160266	HM347136	EF470117	U34541	—	Nirenberg and O'Donnell (1998)
F. inflexum	NRRL 20433^T=CBS 716.74^T	Viciafaba vascular bundle, wilting plant	Germany	AF008479	JX171469	JX171583	U34548	—	O’Donnell et al. (1998)
F. globosum	NRRL 26131^T	Zea mays	South Africa	KF466417	KF466396	KF466406	AY249384	—	Proctor et al. (2013)
F. petersiae	CBS 143231^T	Garden soil	Netherlands	MG386159	MG386138	MG386149	NG058528	NR156397	Crous et al. (2017)
F. ananatum	CBS 118516^T	Ananas comosus fruit	South Africa	LT996091	LT996188	LT996137	KU604065	—	Sandoval-Denis et al. (2018)
F. transvaalense	CBS 144211^T	Sidacordifolia rhizosphere	South Africa	LT996099	LT996210	LT996157	—	—	Sandoval-Denis et al. (2018)
F. concentricum	CBS 450.97^T	Musa sapientum fruit	Costa Rica	AF160282	LT996192	LT575063	U61652	—	Nirenberg and O'Donnell (1998)
F. bulbicola	CBS 220.76^T = NRRL 13618^T	Nerine bowdenii	Germany	KF466415	KF466394	KF466404	U61650	—	Proctor et al. (2013)
F. babinda	CBS 397.96^T	Soil in Nothofagus forest	Victoria	—	—	—	MH874204	NR159861	O’Donnell et al. (2013, 2015)
F. domesticum	CBS 434.34^T	Cheese	Belgium	—	—	—	NG057952	NR145050	Bachmann et al .(2005); Ropars et al. (2012)
F. penzigii	CBS 317.34^T	Fagus sylvatica decayed wood	England	EU926324	KM232211	KM232362	KM231661	NR137707	Schroers et al. (2009)
F. nurragi	CBS 393.96^T	Soil in heath land	Victoria	—	—	—	—	NR159860	O’Donnell et al. (2013)
F. biseptatum	CBS 110311^T	Ex soil	South Africa,Transkei	—	—	—	—	NR137706	Schroers et al. (2009)
Fusicolla acetilerea	IMI 181488^T	Polluted soil	Japan	—	—	—	—	NR111603	Tubaki et al. (1976)
F. violacea	NRRL 20896^T	Quadraspidiotusperniciosus on dying twig of Prunus domestica	Iran	—	—	—	—	NR137617	Gräfenhan et al. (2011)
Neonectria lugdunensis	CBS 250.58^T	Ilex aquifolium submerged decaying leaf	U.K.	—	—	—	—	NR155466	Chaverri et al. (2011)
N. major	CBS 240.29^T	Canker on Alnusincana	Norway	—	—	—	—	NR121496	Chaverri et al. (2011)
Paracremonium inflatum	CBS 485.77^T	Man	India	—	—	—	—	NR154312	Lombard et al. (2015)
P. binnewijzendii	CBS 143277^T	Soil	Netherlands	—	—	—	—	NR157491	Crous et al. (2017)
Pseudocosmos poraeutypellae	CBS 133966^T	Eutypella sp.	USA,Maryland, Beltsville	—	—	—	—	NR158888	Herrera et al. (2013)
P. porametajoca	BPI 879088^T	Eutypa sp.	New Zealand	—	—	—	—	NR155633	Herrera et al. (2013)
P. porarogersonii	BPI 1107121^T	Eutypella sp.	U.S.A.	—	—	—	—	NR154295	Herrera et al. (2013)
Rectifusarium robinianum	NRRL 25729^T	Robiniapseudoacacia twig	Germany	—	—	—	—	NR154410	Lombard et al. (2015)

The morphological structure of the strain was further observed by microscopy (Fig. 1 d–m). Microconidia were columnar; macroconidia were moderate in number, sickle-shaped, and segregated and most had 3–6 septate; the sporogenesis cell type was single-bottle-stalk sporogenesis; the conidia stalk was lateral; the chlamydospore was spherical, single or catenulate and showed intercalary and clustered growth and a high quantity. Sclerotial bodies were not observed. These morphological characteristics, especially the typical sickle-shaped and multicellular macroconidia as well as the spherical intercalary chlamydospores are very similar to those of Fusarium species, suggesting that this new isolate PD2^T may be a member of the genus Fusarium. The growth temperature of the strain PD2^T was investigated by cultivation on PDA plates at different temperatures (4, 15, 25, 30 and 35ºC) for 7 days. The results indicated that the temperature range of the strain growth on PDA medium is 15–30ºC.

Furthermore, the carbon source utilization of the strain was examined using the Biolog FF Micro Plate. After 48 h of incubation on the carbon source identification plate, the available carbon sources of the isolate were as follows: i-erythritol, glucose-1-phosphate, glycerol, γ-hydroxy-butyric Acid, p-hydroxyphenyl acetic acid, α-keto-glutaric acid, D-saccharicacid, L-alanine, L-alanyl-glycine, L-asparagine, L-aspartic acid, L-glutamic acid, L-ornithine, L-phenylalanine, L-serine, L-threonine, and adenosine-5'-monophosphate (Table 2).

Phylogenetic analyses

The sequences of EF-1α, RPB1, RPB2 and LSU genes of the isolate PD2^T were subjected to a BLAST sequence alignment on the NCBI website, and the results showed that the genus Fusarium had the highest similarity with this strain. Some sequences from the alignment results were selected to construct phylogenetic trees. The strains used in the molecular phylogenetic study are listed in Table 1. To analyze the phylogenetic relationship, we selected the following strains and used them for phylogenetic tree construction: the new isolate and 14 Fusarium species belonging to the F. buharicum (FBSC), F. fujikuroi (FFSC), F. tricinctum (FTSC), F. sambucinum (FSAMSC), F. incarnatum-equiseti (FIESC), F. oxysporum (FOSC), F. burgessii and F. concolor species complexes (Fig. 2). According to the phylogenetic tree, the new isolate can be clustered within the FBSC clade (Vu et al. 2018) based on the combined sequences of partial EF-1α, RPB1 and RPB2 genes (Fig. 2). In addition to strain PD2^T, F. convolutans (CBS 144207) and F. sublunatum (CBS 189.34) are also clustered in the FBSC clade. The similarity analysis of the combined sequences (partial sequences of EF-1α, RPB1 and RPB2 genes) showed that the PD2^Tstrain had the highest sequence similarity of 98.89% with F. convolutans, followed by F. sublunatum (94.18%). Despite the high sequence similarity (99.28%, 97.89% and 99.43% identical to F. convolutans for EF-1α, RPB1, and RPB2), a further BLAST analysis indicated that the LSU sequence of the PD2^T strain is 81.17% identical to that of F. convolutans, implying that the PD2^T strain may not belong to F. convolutans.

Fig. 1: Fusarium soli (PD2^T). a-c. Colony morphology grown on PDA, CYA, and SNA after 7 days at 30 ºC in the dark. d-e. Phialides and microconidia on PDA. f. Phialides and microconidia on CD. g. Macroconidia on SNA. h-i. Macroconidia on VBC. j-k. Chlamydospores on CYA. l-m. Chlamydospores on PDA

Fig. 2: Phylogenetic tree based on the sequences combined with the EF-1α, RPB1 and RPB2 genes of 15 strains. The evolutionary history was inferred using the neighbour-joining method in MEGA software version 5. Bars, 0.02 expected nucleotide substitutions per site. Only bootstrap values above 50 % are shown (1000 replicates) at branching points. The strains used here belong to F. buharicum (FBSC), F. fujikuroi (FFSC), F. tricinctum (FTSC), F. sambucinum (FSAMSC), F. incarnatum-equiseti (FIESC), F. oxysporum (FOSC), F. burgessii and F. concolor species complexes

To further study the phylogenetic relationship of the isolate and other Fusarium species, the phylogenetic tree was generated based on a combination of partial sequences, including EF-1α and LSU (Fig. 3). Twelve strains were selected for further construction and analysis of a phylogenetic tree, and the results showed that the new isolate PD2^T is distributed in the FBSC clade while the other 11 species belong to the FBSC, FFSC, FTSC, FIESC, FOSC, F. dimerum (FDSC) and F. burgessii species complexes. The EF-1α plus LSU sequence similarity of the new isolate PD2^T is 96.36% identical to that of F. convolutans (Fig. 3).

Another phylogenetic tree constructed using the combined partial sequences of RPB1, RPB2 and LSU genes (Fig. 4) also showed that the strain PD2^T is still clustered in the FBSC clade. The combined sequence of RPB1,

Table 2: Carbon source utilization of the species

Carbon source	Reaction	Carbon source	Reaction	Carbon source	Reaction
Water	—	Lactulose	—	γ-Hydroxy-butyric Acid	+++
Tween 80	+	Maltitol	—	p-Hydroxyphenylacetic Acid	++
N-Acetyl-D-galactosamine	—	Maltose	—	α-Keto-glutaric Acid	++
N-Acetyl-D-glucosamine	—	Maltotriose	—	D-Lactic Acid Methyl Ester	—
N-Acetyl-D-mannosamine	—	D-Mannitol	+	L-Lactic Acid	—
Adonitol	—	D-Mannose	—	D-Malic Acid	+
Amygdalin	—	D-Melezitose	—	L-Malic Acid	+
D-Arabinose	—	D-Melibiose	—	Quinic Acid	+
L-Arabinose	—	α-Methyl-D-galactoside	—	D-Saccharic Acid	+++
D-Arabitol	+	β-Methyl-D-galactoside	+	Sebacic Acid	—
Arbutin	+	α-Methyl-D-glucoside	—	Succinamic Acid	—
D-Cellobiose	—	β-Methyl-D-glucoside	—	Succinic Acid	++
α-Cyclodextrin	—	Palatinose	—	Succinic Acid Mono-mMethyl Ester	—
β-Cyclodextrin	—	D-Psicose	—	N-Acetyl-L-glutamic Acid	—
Dextrin	+	D-Raffinose	—	Alaninamide	+
i-Erythritol	++	L-Rhamnose	—	L-Alanine	+++
D-Fructose	—	D-Ribose	—	L-Alanyl-glycine	+++
L-Fucose	—	Salicin	+	L-Asparagine	+++
D-Galactose	—	Sedoheptulosan	—	L-Aspartic Acid	++
D-Galacturonic Acid	—	D-Sorbitol	+	L-Glutamic Acid	++
Gentiobiose	—	L-Sorbose	+	Glycyl-L-glutamic Acid	+
D-Gluconic Acid	—	Stachyose	+	L-Ornithine	+++
D-Glucosamine	—	Sucrose	—	L-Phenylalanine	+++
α-D-Glucose	—	D-Tagatose	—	L-Proline	—
Glucose-1-phosphate	+++	D-Trehalose	—	L-Pyroglutamic Acid	—
Glucuronamide	—	Turanose	—	L-Serine	+++
D-Glucuronic Acid	+	Xylitol	—	L-Threonine	+++
Glycerol	+++	D-Xylose	—	2-Amino Ethanol	—
Glycogen	+	γ-Amino-butyric Acid	—	Putrescine	—
m-Inositol	—	Bromosuccinic Acid	—	Adenosine	—
2-Keto-D-gluconic Acid	—	Fumaric Acid	—	Uridine	—
α-D-Lactose	—	β-Hydroxy-butyric Acid	—	Adenosine-5'-Monophosphate	+++

Growth reactions: —, no growth; +, weak growth; ++, moderate growth; +++, strong growth

RPB2 and LSU genes shared 97.54% and 94.22% similarity with those of F. convolutans and F. sublunatum, respectively. In addition to these two strains, the DNA sequences of other Fusarium species showed less similarity with this strain. Overall, these molecular phylogenetic analyses of the above mentioned genes demonstrated that this new discovered strain PD2^T is a new species distributed in the FBSC, and it is named Fusarium soli spp. nov.

Description of F. soli spp. nov

F. soli spp. nov: The temperature range for strain growth on PDA medium is 15–30°C. On PDA, the diameter of a single colony was 67–73 mm after 7 days at 30°C, the edge of the colony was light brown, the aerial hyphae were white, the spores were transparent to white, and the reverse colony was light orange. On CYA, the diameter of a single colony was 85–86 mm after 7 days at 30°C, the surface of the colony was wrinkled, the aerial hyphae were white, the spores were transparent to white, and the reverse colony was light yellow. On SNA, the diameter of a single colony was 48–53 mm after 7 days at 30°C, the positive and negative sides of the colony were white and translucent, the aerial hyphae were white, and the spores were transparent to white. Microconidia were columnar; macroconidia were moderate in number, sickle-shaped, and segregated and most had 3–6 septate; the sporogenesis cell type was single-bottle-stalk; the conidia stalk was lateral; the chlamydospore was spherical, single or catenulate and showed intercalary and clustered growth and a high quantity.

The type strain PD2^T was isolated from the upper layer of soil in a poplar plantation (32°52’28.45’’N, 120°49’47.63’’E) of Jiangsu Province in eastern China.

Discussion

The new species described in this paper was identified to belong to the genus Fusarium based on the presence of typical morphological features, such assickle-shaped macroconidia and intercalary chlamydospores. In the study by O’Donnell (2015), EF-1α, RPB1 and RPB2 genes could be used for the accurate identification of the genus Fusarium. According to the phylogenetic trees (Fig. 2–4), the strain PD2^T has the closest relationship with F. convolutans (Sandoval-Denis et al. 2018). The minimum and maximum temperatures for growth of this strain on PDA are 12°C and 36°C, respectively. The surface of the colony is white to cream colored, with short aerial mycelium; and the margin of colony is irregular to rhizoid, with abundant white to gray submerged mycelium. The reverse side is white with straw to yellow diffusible pigment. Sporulation is scant from conidiophores formed on aerial mycelia, and sporodochia are not observed. Conidiophores on the aerial mycelia are straight or curved, smooth and thin-walled, and simple, and most of them degenerate into conidia cells; phialides are subulate to subcylindrical, and smooth- and thin-walled; and the conidia are lunate to falcate shaped and curved or somewhat straight, with (1–2–)3-septa. Chlamydospores are abundant, globose to sub globose, terminal or intercalary in the hyphae or conidia, and they are often borne laterally at the tip of elongated, cylindrical, stalk-like projections and found alone or in small clusters.

Fig. 3: Neighbour-joining phylogenetic tree based on the sequences combined with the EF-1α and LSU genes of 12 strains. Bars, 0.02 expected nucleotide substitutions per site. Only bootstrap values above 50% are shown (1000 replicates) at branching points. The strains used here belong to F. buharicum (FBSC), F. fujikuroi (FFSC), F. tricinctum (FTSC), F. incarnatum-equiseti (FIESC), F. oxysporum (FOSC), F. dimerum (FDSC) and F. burgessii species complexes.

Fig. 4: Neighbour-joining phylogenetic tree based on the sequences combined with the RPB1, RPB2 and LSU genes of 13 strains. Bars, 0.05 expected nucleotide substitutions per site. Only bootstrap values above 50% are shown (1000 replicates) at branching points. The strains used here belong to F. buharicum (FBSC), F. fujikuroi (FFSC), F. tricinctum (FTSC), F. incarnatum-equiseti (FIESC), F. oxysporum (FOSC), F. dimerum (FDSC) and F. burgessii species complexes

Although the morphological characteristics of the strain F. convolutans are partially similar to those of the new isolate F. soli, some distinct characteristics can be used to distinguish them from each other. For example, compared with F. convolutans, the new isolate F. soli has more sparse aerial hyphae on SNA, up to 6-septate macroconidia, catenulate chlamydospores and no curved sterile hypha. In addition, under the same culture conditions, the strain F. soli has a significantly faster growth rate than the strain F. convolutans. These different morphological characteristics combined with the molecular phylogenetic analysis results suggest that this isolate was a completely different species from F. convolutans.

The phylogenetic trees showed that in addition to the new isolate and F. convolutans, the strain F. sublunatum also belongs to the same FBSC clan. The aerial mycelia of F. sublunatum are sparse and white; the sporodochia are orange; the sclerotia are dark blue to blue-green; microconidia are rare; the macroconidia are sickle-shaped with a distinctly foot-shaped basal cell; and the chlamydospores are abundant (Nelson et al. 1983; Gräfenhan et al. 2011; Lombard et al. 2015). F. sublunatum was also isolated from the soil and the perfect state is still unknown. However, according to the phylogenetic trees and BLAST analysis, F. sublunatum has only a relatively low sequence similarity with the isolate F. soli, and the new isolate can be distinguished from F. sublunatum by transparent to white sporodochia and the lack of sclerotia.

F. petersiae (CBS 143231) (Crous et al. 2017) is another strain of the genus Fusarium with relative lower sequence similarity to PD2^T based on the phylogenetic tree analysis. The morphological characteristics of F. petersiae on SNA included hyphae that were hyaline and smooth and absent chlamydospores; sporulation was abundant only from sporodochia; no conidiophores were observed on the aerial mycelia; sporodochia were abundant only on the surface of carnation leaves; macroconidia were falcate and curved and showed a papillate and curved apical cell that tapered towards a foot-like basal cell. F. petersiae is a new member of the F. tricinctum species complex (FTSC) that is closely related to F. flocciferum (Booth 1971) and F. torulosum. There are obvious differences in the morphological characteristics between F. petersiae and the new isolate PD2^T.

Combining the results of the molecular phylogenetic analyses and morphological characteristics indicates that the strain F. soli isolated in this study is a new species clustered in the FBSC of the genus Fusarium.

Conclusion

The new isolate showed the closest relationship with F. convolutans which was clustered in the FBSC clade. However, sequence similarity analyses combined with different morphological characteristics, such as macroconidia with more septa, catenulate chlamydospores and no curved sterile hypha, demonstrated that the isolate is a new species of the genus Fusarium. The carbon source utilization of the new isolate F. soli was further examined in this study.

Acknowledgements

This study was supported by the Natural Science Foundation of China (31570107), the Six Talent Peaks Program of Jiangsu Province of China (TD-XYDXX-006) and Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Author Contributions

The main author of this work, Y.-J.Z.; design of experiments, L.J. and F.-J.J.; original draft preparation and references investigation, Y.-J.Z., X.-Y.Y. and B.-T.W.; review and editing, L.J. and F.-J.J. All authors have read and agreed to the published version of the manuscript.

References

Bachmann HP, C Bobst, U Bütikofer, MG Casey, MD Torre, MT Fröhlich-Wyder, M Fürst (2005). Occurrence and significance of Fusarium domesticum alias Anticollanti on smear-ripened cheeses. Food Sci Technol 38:399–407

Bai GH, AE Desjardins, RD Plattner (2002). Deoxynivalenol-nonproducing Fusarium graminearum causes initial infection, but does not cause disease spread in wheat spikes. Mycopathologia 153:91–98

Booth C (1971). The genus Fusarium, pp:1–237. Commonwealth Mycological Institute, Kew, UK

Chaverri P, C Salgado, Y Hirooka, AY Rossman, GJ Samuels (2011). Delimitation of Neonectria and Cylindrocarpon (Nectriaceae, Hypocreales, Ascomycota) and related genera with Cylindrocarpon-like anamorphs. Stud Mycol 68:57–78

Coleman JJ (2016). The Fusarium solani species complex: Ubiquitous pathogens of agricultural importance. Mol Plant Pathol 17:146–158

Crous PW, L Lombard, JZ Groenewaldet (2017). Fungal Planet description sheets. Persoonia 39:270–467

Dean R, JAV Kan, ZA Pretorius, KE Hammond-Kosack, AD Pietro, PD Spanu, JJ Rudd, M Dickman, R Kahmann, J Ellis, GD Foster (2012). The top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13:414–430

Desjardins AE (2006). Fusarium mycotoxins: Chemistry, Genetics and Biology. American Phytopathological Society, St. Paul, Minnesota, USA

Duan Y, X Tao, H Zhao, X Xiao, M Li, J Wang, M Zhou (2019). Activity of demethylation inhibitor fungicide metconazole on Chinese Fusarium graminearum species complex and its application in carbendazim-resistance management of Fusarium head blight in wheat. Plant Dis 103:929–937

Felsenstein J (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783–791

Gerlach W, HI Nirenberg (1982). The genus Fusarium - a pictorial atlas, pp:1–406. Berlin-Dahlem (Heft 209): Mitteilungenaus der Biologis-chenBundesanstaltfür Land-und Forstwirtschaft, Berlin, Germany

Gräfenhan T, HJ Schroers, HI Nirenberg, KA Seifert (2011). An overview of the taxonomy, phylogeny, and typification of nectriaceous fungi in Cosmospora, Acremonium, Fusarium, Stilbella and Volutella. Stud Mycol 68:79–113

Hall BG (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. In: Nucleic Acids Symposium Series, Vol. 41, pp:95–98. Information Retrieval Ltd., Oxford University, UK

Herrera CS, AY Rossman, GJ Samuels, P Chaverri (2013). Pseudocosmospora, a new genus to accommodate Cosmosporavilior and related species. Mycologia 105:1287–1305

Ilgen P, F Maier, W Sch¨afer (2008). Trichothecenes and lipases are host-induced and secreted virulence factors of Fusarium graminearum. Cereal Res Commun 36:421–428

Jacobs-Venter A, I Laraba, DM Geiser, M Busman, MM Vaughan, RH Proctor, SP McCormick, K O'Donnell (2018). Molecular systematics of two sister clades, the Fusarium concolor and F. babinda species complexes, and the discovery of a novel microcycle macroconidium–producing species from South Africa. Mycologia 110:1189–1204

Kimura M (1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120

Kwiatkowski NP, WM Babiker, WG Merz, KC Carroll, SX Zhang (2012). Evaluation of nucleic acid sequencing of the D1/D2 region of the large subunit of the 28S rDNA and the internal transcribed spacer region using SmartGene IDNS software for identification of filamentous fungi in a clinical laboratory. J Mol Diagn 14:393–401

Laraba I, A Keddad, H Boureghda, N Abdallah, MM Vaughan, RH Proctor, M Busman, K O'Donnell (2017). Fusarium algeriense, sp. nov., a novel toxigenic crown rot pathogen of durum wheat from Algeria is nested in the Fusarium burgessii species complex. Mycologia 109:935–950

Liu Y, M Wisniewski, JF Kennedy, Y Jiang, J Tang, J Liu (2016). Chitosan and oligochitosan enhance ginger (Zingiber officinale Roscoe) resistance to rhizome rot caused by Fusarium oxysporum in storage. Carbohydr Polym 151:474–479

Lombard L, RV Doorn, PW Crous (2019). Neotypification of Fusarium chlamydosporum-a reappraisal of a clinically important species complex. Fung Syst Evol 4:183–200

Lombard L, NAVD Merwe, JZ Groenewald, PW Crous (2015). Generic concepts in Nectriaceae. Stud Mycol 80:189–245

Nelson PE, TA Toussoun, WFO Marasas (1983). Fusarium species: An Illustrated Manual for Identification, pp:1–191. Pennsylvania State University Press, University Park, Philadelphia, USA

Nirenberg HI, K O’Donnell (1998). New Fusarium species and combinations within the Gibberella fujikuroi species complex. Mycologia 90:434–458

O'Donnell K, SP McCormick, M Busman, RH Proctor, TJ Ward, G Doehring, DM Geiser, JF Alberts, JP Rheeder (2018). Marasas et al. 1984 “Toxigenic Fusarium Species: Identity and Mycotoxicology” revisited. Mycologia 27:1058–1080

O’Donnell K, TJ Ward, VARG Robert, PW Crous, DM Geiser, S Kang (2015). DNA sequence-based identification of Fusarium: Current status and future directions. Phytoparasitica 43:583–595

O’Donnell K, AP Rooney, RH Proctor, DW Brown, SP McCormick, TJ Ward, RJN Frandsen, E Lysøe, SA Rehner, T Aoki, VARG Robert, PW Crous, JZ Groenewald, S Kang, DM Geiser (2013). Phylogenetic analyses of RPB1 and RPB2 support a middle Cretaceous origin for a clade comprising all agriculturally and medically important fusaria. Fung Gen Biol 52:20–31

O'Donnell K, DA Sutton, MG Rinaldi, BA Sarver, SA Balajee, HJ Schroers, RC Summerbell, VA Robert, PW Crous, N Zhang, T Aoki, K Jung, J Park, YH Lee, S Kang, B Park, DM Geiser (2010). Internet-accessible DNA sequence database for identifying fusaria from human and animal infections. J Clin Microbiol 48:3708–3718

O'Donnell K, DA Sutton, A Fothergill, D McCarthy, MG Rinaldi, ME Brandt, N Zhang, DM Geiser (2008). Molecular phylogenetic diversity, multilocus haplotype nomenclature, and in vitro antifungal resistance within the Fusarium solani species complex. J Clin Microbiol 46:2477–2490

O'Donnell K, BA Sarver, M Brandt, DC Chang, J Noble-Wang, BJ Park, DA Sutton, L Benjamin, M Lindsley, A Padhye, DM Geiser, TJ Ward (2007). Phylogenetic diversity and microsphere array-based genotyping of human pathogenic fusaria, including isolates from the multistate contact lens-associated U.S. keratitis outbreaks of 2005 and 2006. J Clin Microbiol 45:2235–2248

O'Donnell K, HC Kistler, E Cigelnik, RC Ploetz (1998). Multiple evolutionary origins of the fungus causing Panama disease of banana: Concordant evidence from nuclear and mitochondrial gene genealogies. Proc Natl Acad Sci USA 95:2044–2049

Pérez-Hernández A, Y Serrano-Alonso, MI Aguilar-Pérez, R Gómez-Uroz, J Gómez-Vázquez (2014). Damping-off and root rot of pepper caused by Fusarium oxysporum in Almería province, Spain. Plant Dis 98:1159

Proctor RH, FV Hove, A Susca, G Stea, M Busman, TVD Lee, C Waalwijk, A Moretti, TJ Ward (2013). Birth, death and horizontal transfer of the fumonisin biosynthetic gene cluster during the evolutionary diversification of Fusarium. Mol Microbiol 90:290–306

Rojas EC, R Sapkota, B Jensen, HJL Jørgensen, T Henriksson, LN Jørgensen, M Nicolaisen, DB Collinge (2020). Fusarium head blight modifies fungal endophytic communities during infection of wheat spikes. Microb Ecol 79:397–408

Ropars J, C Cruaud, S Lacoste, J Dupont (2012). A taxonomic and ecologic overview of cheese fungi. Intl J Food Microbiol 155:199–210

Saitou N, M Nei (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

Sandoval-Denis M, WJ Swart, PW Crous (2018). New Fusarium species from the Kruger National Park, South Africa. MycoKeys 34:63–92

Schroers HJ, K O'Donnell, SC Lamprecht, PL Kammeyer, S Johnson, DA Sutton, MG Rinaldi, DM Geiser, RC Summerbell (2009). Taxonomy and phylogeny of the Fusarium dimerum species group. Mycologia 101:44–70

Snyder WC, HN Hansen (1940). The species concept in Fusarium. Amer J Bot 27:64–67

Summerell BA (2019). Resolving Fusarium: Current status of the genus. Annu Rev Phytopathol 57:323–339

Tamura K, D Peterson, N Peterson, G Stecher, M Nei, S Kumar (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

Thompson JD, TJ Gibson, F Plewniak, F Jeanmougin, DG Higgins (1997). The CLUSTAL X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools nucleic acids research. Nucl Acids Res 25:4876–4882

Tubaki K, C Booth, T Harada (1976). A new variety of Fusarium merismoides. Trans Braz Mycol Soc 66:355–366

Tupaki-Sreepurna A, V Thanneru, S Natarajan, S Sharma, A Gopi, M Sundaram, AJ Kindo (2018). Phylogenetic Diversity and in vitro susceptibility profiles of human pathogenic members of the Fusarium fujikuroi species complex isolated from South India. Mycopathologia 183:529–540

Vu D, M Groenewald, MD Vries, T Gehrmann, B Stielow, U Eberhardt, A Al-Hatmi, JZ Groenewald, G Cardinali, J Houbraken, T Boekhout, PW Crous, V Robert, GJM Verkley (2018). Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher tax on delimitation. Stud Mycol 92:135–154

Wang G, H Chen (1994). The medium for promoting sporulation of Fusarium. Acta Phytopathol Sin 25:165–166

Wollenweber HW, OA Reinking (1935). Die Fusarien, ihreBeschreibung, Schadwirkung und Bekämpfung [M]. Paul Parey, Berlin, Germany

Woloshuk CP, WB Shim (2013). Aflatoxins, fumonisins, and trichothecenes: A convergence of knowledge. FEMS Microbiol Rev 37:94–109