Sordariomycetes is the second largest class of Ascomycota  and includes 1,331 genera distributed across 105 families, 32 orders, and six subclasses . The Xylariaceae family includes 85 genera and more than 1,350 species, which render it one of the largest and most diverse families of Ascomycota [2,3]. Biscogniauxia is a genus of the Xylariaceae family that is characterized by stromatic ascomata, unitunicate asci with an amyloid apical apparatus, a brown germ slit of ascospores, and hyphomycetous asexual morphs with holoblastic conidiogenesis . The phylogenetic affinities between Biscogniauxia and Hypoxyloideae have been shown, as well as its clustering with Camillea on a sister clade to Annulohypoxylon, Daldinia, and Hypoxylon . Recently, a large-scale multilocus phylogenetic analysis of Xylariaceae revealed a well-supported Biscogniauxia clade within Hypoxyloideae, which includes Camillea and Obolarina . However, phylogenetic studies have placed it with the Graphostromataceae family, which is close to Xylariaceae, while genera such as Biscogniauxia and Camillea have been described to share similar morphological characters [6,7]. Therefore, the recognition of a separate family called Graphostromataceae was deemed doubtful based on the molecular data available at the time, and Graphostromataceae was synonymized with Xylariaceae . However, the family Graphostromataceae was resurrected and the Biscogniauxia, Camillea, and Obolarina genera were added to it based on additional molecular evidence .
During the screening of fungal species in Korea, the KNU-WDM2A2 strain was isolated from mosquitoes and identified as Biscogniauxia petrensis, which is a fungal species that had not been reported in Korea. In this study, molecular phylogenetic analyses were used to identify the undescribed fungal species, and its cultural and morphological characteristics were examined.
MATERIALS AND METHODS
Mosquito collection and fungal strain isolation
In 2019, mosquito samples were collected in Gimcheon (36°11'00.6"N, 128°07'05.8" E), Korea, using an insect net. The mosquito samples were placed in a 1.5 mL Eppendorf tube and transported to the laboratory for fungal strain isolation. The captured mosquitoes were identified as Ades albopictus based on morphological structures. Each mosquito sample was washed once with 70% ethanol, twice with sterile distilled water, and then grinded using a hand grinder. Subsequently, the samples were suspended in 1.0 mL of sterile distilled water, vortexed gently, and diluted serially, followed by the spreading of 100 μL of each sample onto PDA (Difco, Detroit, MI, USA) plates and incubation at 25℃ for 2-3 days. Single colonies were transferred to PDA plates and incubated at 25℃ for 5-7 days. The strain was selected for molecular analyses based on various morphological characteristics and the fungal strain was stored for further study in 20% glycerol at -80℃.
PDA culture medium and synthetic nutrient agar (SNA: agar 14.0 g/L of agar, 1.0 g/L of KH2PO4, 1.0 g/L of KNO3, 0.25 g/L of MgSO4.7H2O, 0.5 g/L of KCl, 0.2 g/L of glucose-, and 0.2 g/L of sucrose)  were used to study the cultural and morphological characteristics of the KNU-WDM2A2 strain after incubation at 25℃ for 14 days . Subsequently, the cultural characteristics, such as colony color, fungal growth, and texture, were recorded, and the morphology of the fungus was observed using a light microscope (BX-50; Olympus, Tokyo, Japan).
Genomic DNA extraction, PCR amplification, and sequencing
The genomic DNA was extracted from fungal mycelia grown on PDA for 5-7 days at 25℃. The HiGene Genomic DNA prep kit (BIOFACT, Daejeon, Korea) was used as per the manufacturer’s instructions. The PCR amplification process was carried out on the internal transcribed spacer (ITS) regions using the primer pairs ITS1F/ITS4 [11,12] and the 28S rDNA large subunit (LSU) with LROR/LR5 . Finally, the amplified PCR products were purified using EXOSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced by Solgent Co., Ltd. (Daejeon, Korea).
Molecular phylogenetic analysis
To perform the phylogenetic analysis, the consensus sequences were retrieved from the GenBank database of the National Center for Biotechnology Information (NCBI) (Table 1). Evolutionary distance matrices were generated based on neighbor-joining algorithm with the Kimura model . The phylogenetic analyses were performed using the MEGA 7.0 software with bootstrap analysis of 1,000 replications .
RESULTS AND DISCUSSION
Morphology of the KNU-WDM2A2 strain
Colonies were whitish to light pink and cottony to wooly, with abundant production of aerial mycelia; moreover, they reached a fungal growth of 90 mm on PDA at 25℃ after 10 days of incubation. The colonies also produced red-droplet secretions after 14 days of incubation and their reverse side was yellowish to red-brown (Fig. 1A). In turn, the colonies were pink-white and cottony to wooly, with a fascicular shape on SNA media, and attained a size of 90 mm after 10 days of incubation at 25℃. The colonies exhibited a white to pink-white color on the reverse side (Fig. 1B). The strain also produced blackish conidiomata-like structures on the agar surface (Fig. 1C). Hyphae were hyaline to brown, branched, thin-walled, septate, and produced a great number of aerial mycelia. Conidiophores were hyaline to slightly yellowish and rough-walled with one or more branches, and produced conidiogenous cells terminally or laterally (Fig. 1D and E). Conidiogenous cells were hyaline to slightly yellowish, top swollen, thin, sometimes rough-walled, and cylindrical to oblong; they exhibited a size of 6.2-10.5×2.3-3.4 μm with an average diameter of 7.3-2.9 μm (Fig. 1F). Conidia were unicellular, hyaline to light brown, smooth, oval or ovoid to clavate, and measured 4.1-6.9×2.5-3.3 μm (mean, 5.3±0.6×2.9±0.2 μm, n=65) (Fig. 1G). The cultural and morphological characteristics were similar to those described previously for Biscogniauxia petrensis (Table 2). Therefore, the KNU-WDM2A2 fungal strain was closely related to B. petrensis.
Molecular phylogeny of the KNU-WDM2A2 strain
After the analysis of nucleotide sequences, the sequences of 686 and 846 bp were obtained from the ITS regions and 28S rDNA, respectively. The BLAST results of the ITS sequence exhibited a similarity of 100% with different strains of Biscogniauxia petrensis (MN844525, MN844517, MN844529, MN844513, and MN844542). The 28S rDNA of the large subunit displayed maximum similarity of 99.88% with the different strains of B. petrensis (KU746717, KU746716, KU746715, MK951680). A phylogenetic tree was constructed based on the combined sequences of ITS regions and partial sequences of the 28S rDNA using a neighbor-joining method. The generated phylogenetic tree revealed that the KNU-WDM2A2 strain was clustered with the previously identified B. petrensis (Fig. 2). Thus, the KNU-WDM2A2 fungal strain was identified as B. petrensis and deposited in the National Institute of Biological Resources (NIBRFG0000507057).
Some fungal species were reportedly associated with the larvae of several mosquitoes isolated from municipalities of the Brazilian Amazon: Acremonium kiliense, Penicillium oxalicum from Mansonia titillans; Gliocladium viride and Paecilomyces sp. from Anopheles darling . Also several species of Penicillium (such as P. decumbens, P. fellutanun, P. corylophilum, P. waksmanii, and P. janthinellum) were detected in some adults and larvae of mosquitoes (Aedes spp., Anopheles spp., Culex spp., and Mansonia spp.) collected in Minas Gerais, Rio de Janeiro, and Rondônia in Brazil . Though there are so many fungal species isolated from mosquitoes in different countries, but still now, there is no study in Korea. However, there are some fungal species namely P. brasilianum and Blakeslea trispora isolated from dead insect bodies and from gut of grasshopper in Korea, respectively [18,19]. Moreover, two Mucor species, i.e., Mucor irregularis and M. fragilis, were also isolated from the gut of soldier fly larvae inhabiting bulrush at a pond located in Gwangju, Korea . The member of Biscogniauxia called Biscogniauxia mediterranea transmitted through ambrosia beetle Platypus cylindrus and responsible for the charcoal canker disease of Quercus suber . Moreover, B. mangiferae was isolated from dead branch of Mangifera indica (Anacardiaceae) . B. rosacearum was isolated from rosaceous hosts in Italy. And Biscogniauxia spp. have been reported as endophytes or secondary invaders that attack only stressed plants . Even, B. nummularia, a xylariaceous fungus that causes strip canker and wood decay on European beech (Fagus sylvatica L.) . On the other hand, recently, B. petrensis strain was isolated from rocks in an unnamed karst cave located in Guizhou province, China . In case of B. petrensis was not reported from any other hosts as well as responsible for any diseases yet.
In this study, KNU-WDM2A2 strain was isolated from adult mosquitoes and identified as Biscogniauxia petrensis in Korea. Therefore, further research is required to provide in-depth knowledge about this species based on ecology and aspects of the agricultural field in Korea.