The PCR-amplified sequences (BenA, CaM, and RPB2) of the isolates are compared and analyzed with other species available on the NCBI website using the Blast-n program (Altschul et al. 1990). The results showed that the strains were most similar to the Penicillium genus. Sequences were downloaded from the database of NCBI website according to the alignment results and used to construct phylogenetic trees based on single gene (CaM, RPB2) and combined gene (BenA-CaM-RPB2) analyses. Strains and their sequences accession numbers used in this study are listed in Table 1.
Phylogenetic position of two strains and their phylogenetic relationship with other strains were determined by analysis using neighbour-joining method. According to the analysis, P. dongtaiense sp. novT (AF2021001) was clustered into section Sclerotiora of genus Penicillium. In the phylogenetic tree based on RPB2 gene sequences, P. dongtaiense sp. novT forms a clade with Penicillium exsudans (HMAS248735) and Penicillium acidum (EML-DLW4-1). In addition, Penicillium mallochii (DAOM239917) appeared as sister species to this clade with a high bootstrap value of 99 (Fig. 1A). In the CaM NJ tree, these four strains were also clustered into a clade with a high bootstrap of 84 (Fig. 1B). The combined analysis of multiple gene sequence (CaM+RPB2+BenA) further indicated that the strain P. dongtaiense sp. novT formed a monophyletic clade that also contains P. mallochii, P. exsudans and P. acidum (Fig. 1C). The branches of this clade are supported by bootstraps with value of 100, indicating that this phylogenetic tree is strongly resolved.
Three strains AF2021051, AF2021080 and AF2021081 were clustered into a clade without distinguish in three NJ trees. They were most closely related to the two Penicillium species, P. oxalicum (CBS219.30) and P. asturianum (CBS173.81) according to three phylogenetic trees. In the phylogenetic trees based on RPB2 or CaM genes, the three isolates were grouped with other species belonged to section Lanata-Divaricata (Visagie et al. 2015a), including P. oxalicum (CBS219.30), P. asturianum (CBS173.81), P. ludwigii (CBS 417.68), P. cremeogriseum (CBS 223.66), P. glaucoroseum (NRRL 908), P. janthinellum (CBS 340.48), P. curticaule (CV2842), P. limosum (CBS 339.97), P. brefiedianum (NRRL710) and P. coeruleum (CBS 141.45). The result indicated that the isolates should be grouped into the section Lanata-Divaricata with certainty. When the three gene (RPB2, BenA and CaM) regions were combined to construct a phylogenetic tree, the isolates are clearly distinct from P. oxalicum and P. asturianum, indicating that these strains are a new species belonged to genus Penicillium based on DNA analysis.
Overall, these molecular phylogenetic analyses indicated that the isolate AF2021001 may be a new species belonged to section section Sclerotiora and described here as P. dongtaiense sp. novT. Other three strains AF2021051, AF2021080 and AF2021081 are belonged to section Lanata-Divaricata and described as P. yanchengense sp. novT. The novelty of new species was further supported by morphological observations.
After incubation at 26 ℃ (± 0.5 ℃) for 2 days, color changes of each well of FF MicroPlate™ were recorded and listed in Table 2. Available carbon source to P. dongtaiense sp. novT. isolate as follows: N-Acetyl-D-glucosamine, D-Arabinose, L-Arabinose, Arbutin, D-Gluconic Acid, Glucose-1-phosphate, D-Glucuronic Acid, Glycogen, 2-Keto-D-gluconic Acid, D-Mannitol, D-Ribose, Salicin,D-Xylose, γ-Hydroxy-butyric Acid,α-Keto-glutaric Acid, D-Malic Acid, L-Malic Acid, Quinic Acid, D-Saccharic Acid, Succinic Acid, L-Alanine, L-Alanyl-glycine, L-Asparagine, L-Aspartic Acid, L-Glutamic Acid, Glycyl-L-glutamic Acid, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, Adenosine (Table 2.A). For the isolate P. yanchengsnse sp. novT, the carbon sources available are γ-Hydroxy-butyric Acid, Quinic Acid, Alaninamide, L-Alanine, L-Alanyl-glycine, L-Asparagine, L-Aspartic Acid, L-Glutamic Acid, L-Proline, L-Serine, L-Threonine (Table 2B).
Penicillium dongtaiense sp. novT
ITS barcode: MZ298467
Alternative markers: RPB2 = MZ327629; BenA = MZ313190; CaM =MZ476797
Etymology: dongtaiense, named after Dongtai, China where this strain was originally isolated.
Observations were recorded from colonies grown at 25 ℃ for 7 days unless stated otherwise.
Colony diameter, 7d, in mm—CYA 25 ℃, 35–38; CYA 5 ℃, 7–10; CYA 15 ℃, 24–26; CYA 30 ℃, 39–40; CYA 37 ℃, no growth; YES 25 ℃, 34–37; CREA 25 ℃, 6–10; MEA 25 ℃, 28–32.
Colony descriptions: On CYA 25 ℃, 7 d: colony margin on the surface but sunken at center, radially and concentrically sulcate, texture floccose, blue green at center fading to grey green until white at margin, moderate sporulation, plentiful exudate centrally present as small droplets from yellow to orange, no soluble pigments, reverse yellowish cream.
On YES 25 ℃, 7 d: colony in velvety texture, cerebriform grooves, colony margin on the surface, white, narrow, sporulation moderate, conidia dark green, centrally slight fasciculate hypha growth, no exudate, soluble pigments absent, reverse orange centrally extending to yellow.
On MEA 25 ℃, 7 d: colonies radially sulcate, plane, lightly sunk centrally, texture velutinous, floccose at margin, colored dark green to white marginal ring, moderate sporulation, prolific exudate presents as clear droplets, soluble pigments absent, reverse cream.
On CREA 25 ℃, 7 d: colonies are plane and thin, velutinous, poor acid production, reverse yellow.
Conidiophores strictly monoverticillate similar to most species in section Sclerotiora, stipes slightly rough walled, 200–237.5 µm×4.17–6.25 µm, ramus/branch appressed, Phialides ampulliform, 6.67–9.3 µm×28–32 µm. Conidia broadly subglobose to ellipsoidal, 10.77–11.54×12.31-16.92 µm, conidia finely smooth (Fig. 2).
Enrich test: No reaction.
Penicillium yanchengense sp. novT.
ITS Barcode: MW487229
Alternative markers: BenA = MZ327630, RPB2 = MZ327631, CaM = MZ416920
Etymology: yanchengense, named after a city of Yancheng, China where the fungus was collected originally.
Diagnosis: A faster growth rate on CYA at 37 ℃ and on CREA at 25 ℃, relative strong production of acid on CREA. The shape of conidia is almost circular compared with other relatively close species.
Colony diameter: 7 days, in mm—(descriptions based on the growth on medium at 25 ℃ unless otherwise stated) CYA 45–52; CYA 5 ℃, 5–6; CYA 15 ℃, 26–27; CYA 30 ℃, 64–65; CYA 37 ℃, 68–74; CYA 42 ℃, 7–13; MEA 36–38; YES 41–45; CREA 18–21.
On CYA at 25 ℃, 7 d: colonies nearly circular, plain, gently radially sulcate in the center, floccose in texture, colored dark green with a peripheral white band of hyphae; margin moderately wide, entire; moderate sporulation; plentiful exudates present as small clear droplets, soluble pigment absent, reverse pale yellow.
On MEA at 25 ℃, 7 d: colonies highly and centrally sulcate, relatively plane, texture floccose, mycelia dark green but white at the margin; margin moderately wide, entire; moderate sporulation, exudates droplets clear and prolific; soluble pigment absent; reverse cream.
On YES at 25 ℃, 7 d: colonies moderately deep, highly and randomly sulcate, slightly sunken at the center; colony dark green fading to blue green, margin white, narrow and entire; texture velvety, sporulation dense; no exudate, soluble pigment absent, reverse yellow.
On CREA at 25 ℃, 7 d: Moderate growth, colony plane, strong acid produced. Reverse luminous yellow.
Conidiophores mostly monoverticillate, sometimes divaricate or biverticillate, stipes slightly rough walled, 146–397×4.8–10 µm, ramus/branch appressed, 0.49–0.85 µm×2.93×3.17 1–2 branch/ramus per stipe. Phialides ampulliform, 3–6 per branch/metula, 17-24×5-7 µm (19.5±2.5×6.1±1.3 µm). Conidia broadly near-spherical and some ellipsoidal, 7.5–9×7–9.5 µm smooth walled (Fig. 3).
The reaction is strong.
Additional strains examined: AF2021080 isolated from Yancheng, JiangSu Province. ITS barcode: OK090909 (alternative markers: CaM = OK127813; BenA = OK127816; RPB2 = OK513275); AF2021081 isolated from Yancheng, JiangSu Province. ITS barcode: OK090910 (alternative markers: CaM = OK127817; BenA = OK127815; RPB2 = OK127814)