Bhushaniella gen. nov. (Cordycipitaceae) on spider eggs sac: a new genus from Thailand and its bioactive secondary metabolites

Fungal specimens parasitic on spider egg sacs (Araneidae sensu lato) were collected, isolated, and identified based on molecular phylogenetic analyses of five nuclear loci (ITS, LSU, TEF1, RPB1 and RPB2) combined with morphological data. In this study, one novel monotypic genus is described, Bhushaniella rubra for Thailand. Bhushaniella rubra is characterized by producing superficial perithecia. Its anamorph has a unique character by producing verticillate phialides with a slightly curved neck. A concurrent evaluation of the secondary metabolites of the mycelial extracts of the new fungus revealed the presence of picoline alkaloids of the penicolinate type, for which we propose the trivial names penicolinates F and G. Their chemical structures were elucidated by two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and high resolution mass spectrometry (HR-MS). They only showed weak to no antibiotic activity and were devoid of significant cytotoxic effects.

Previous studies employing molecular phylogenetics revealed that members of Cordycipitaceae occur on a wide range of hosts and substrates. They are well-known attacking multiple orders of insects from larvae to adult states and spiders. Some species in Beauveria Vuill. and Cordyceps Fr. could be isolated from soil or be established as endophytes in plant seedlings (Zimmermann 2008;Alali et al. 2019;Ramakuwela et al. 2020). Furthermore, there is evidence that some species in this family like Simplicillium lanosoniveum (J.F.H. Beyma) Zare & W. Gams and Niveomyces coronatus J.P.M. Araújo & de Bekker are mycoparasites of Ophiocordyceps species pathogenic on ants. (Shrestha et al. 2016(Shrestha et al. , 2019Wei et al 2019;Araújo et al. 2022). Many studies have shown that some species in Cordycipitaceae have important economic value being used as biocontrol agents for agricultural insect pests or are sources of bioactive compounds. For instance, Beauveria bassiana (Bals.-Criv.) Vuill. can be used as a biocontrol agent to reduce the population of Odoiporus longicollis Olivier, which has a severe impact on banana production (Alagesan et al. 2019). Strains of Cordyceps fumosorosea (Wize) Kepler, B. Shrestha & Spatafora (= Isaria fumosorosea Wize) are frequently used for whitefly control (Avery et al. 2004;2008). In addition, several species in Cordycipitaceae produce secondary metabolites with bioactivities that have the potential for medicines or nutriment e.g. C. militaris (L.) Fr., C. cicadae (Miq.) Massee, and C. tenuipes (Peck) Kepler, B. Shrestha & Spatafora (Zhang et al. 2018;Jędrejko et al. 2021). Two bioactive compounds, gibellamines A (1) and B (2) were recently isolated from Gibellula gamsii Kuephadungphan, Tasan. & Luangsa-ard showing anti-biofilm activity against Staphylococcus aureus . The exploration of bioactive compounds from species of invertebrate-pathogenic fungi has recently received increasing interest (Helaly et al. 2019;Zhang et al. 2020;Mongkolsamrit et al. 2021).
In surveys of arthropod pathogenic fungi in Thailand's national parks, collections of pathogens on spider egg sacs were found on the underside of leaves of forest plants. Based on the macroscopic features of the teleomorph, specimens possess superficial perithecia on the spider eggs in a sac, which is similar to the teleomorph in Gibellula. Nonetheless, asexually reproductive species produce cylindrical synnema with verticillate phialides along the synnema and on hosts. These studies aim to elucidate the phylogenetic and taxonomic placement of these collections of parasitic fungi on spider eggs in a sac through multilocus molecular phylogenetic analyses to known members of Cordycipitaceae and an investigation of the bioactivity of secondary metabolites produced by these fungi is presented.

Specimen collection and isolation
The fungal specimens were collected from different forests in Thailand, located in Chumphon Province. They were searched on the underside of leaves and placed in plastic boxes. The protocol for isolating ascospores and conidia followed a previous study (Mongkolsamrit et al. 2018) using potato dextrose agar (PDA) plates (PDA: freshly diced potato 200 g/L, dextrose 20 g/L, agar 15 g/L). After the inoculated medium was incubated overnight at room temperature, it was examined with a stereomicroscope to locate germinated ascospores and conidia. The germinated ascospores and conidia were transferred to fresh PDA plated and then incubated for 14 days at 25 °C under light/dark conditions (L:D 14:10). The cultures were deposited at the BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology, Thailand. All fungal specimens were dried in an electric food dryer (50-55 °C) overnight and accessioned in the BIOTEC Bangkok Herbarium (BBH), National Biobank of Thailand.

Morphological observation
Macroscopic characters were observed based on natural specimens and pure cultures. Microscopic characters of perithecia, asci, ascospores, phialides and conidia were mounted on a microscope slide containing a drop of lactophenol cotton blue solution. The shapes, sizes, and colours of individual characters were determined and measured according to Mongkolsamrit et al. (2020). Fungal strains were grown on oatmeal agar (OA, Difco: oatmeal 60 g/L, agar 12.5 g/L) and PDA agar plates at 25 °C under light/dark conditions (L:D 14:10) for 14 days. The cultures were observed to compare morphological characters including conidia, phialides, and colony pigmentation. The colours of fresh specimens and cultures incubated on OA and PDA were described and codified following the Royal Horticultural Society colour chart (RHS Colour Chart 2015).

Molecular phylogenetic analyses
Genomic DNA was harvested from mycelial mass on PDA using a modified cetyltrimethyl-ammonium bromide (CTAB) (Doyle and Doyle 1987) as previously described by Mongkolsamrit et al. (2020). Nuclear loci, including the nuc rDNA region encompassing the internal transcribed spacer (ITS) regions ITS1 and ITS2, nc LSU rDNA (large subunit of the ribosomal DNA), the translation elongation factor-1α (TEF1), and the partial gene regions of the largest and second-largest subunits of the RNA polymerase II (RPB1 and RPB2), were amplified and sequenced. The primer pairs and thermocycler conditions for PCR amplifications used in this study followed Mongkolsamrit et al. (2023) and Thanakitpipattana et al. (2022). The purified PCR products were sequenced with the same PCR amplification primers for Sanger dideoxy sequencing. The DNA sequences generated in this study were checked for ambiguous bases using BioEdit v. 7.2.5 (Hall 1999) and then submitted to GenBank. Table 1 shows the list of ITS, LSU, TEF1, RPB1 and RPB2 sequences generated in this study as well as those of other taxa from previous studies. Phylogenetic analyses were performed using RAxMLHPC2 on XSEDE v 8.2.12 (Stamatakis 2014) in the CIPRES Science Gateway portal, using the GTRGAMMA + I model with 1000 bootstrap iterations. Bayesian inference (BI) of phylogenetic relationships was performed in MrBayes v. 3.2.7a (Ronquist et al. 2012), with best-fit models selected using MrModeltest v. 2.2 (Nylander 2004). The best model was GTR + G + I. Markov chain Monte Carlo (MCMC) simulations were run for 5,000,000 The accession numbers marked in bold font refer to sequences new in this study or have been generated by our group in Thailand. T = type specimens generations, sampling every 1000 and discarding the first 10% as burn-in. RAxML and BI output were imported into TreeView version 1.6.6 to visualize the phylogenetic trees (Page 1996).

Fermentation and Extraction
For 4 L fermentation, twenty 500 mL Erlenmeyer flasks, each containing 200 mL YMG media (4 g yeast extract; 10 g malt extract; 4 g D-glucose; ad 1000 ml distilled water) were inoculated with 7 mycelial plugs (approx. 1 cm × 1 cm) from an actively growing colony of strain BCC 47541. After incubation at 23 °C on a rotary shaker (140 rpm) for 7 to 8 days, until free glucose was depleted in every flask, the culture broth was separated from the mycelia by vacuum filtration and the mycelia subsequently extracted with acetone, followed by EtOAc to afford roughly 300 mg of mycelial extract as brown gum.

Isolation of Metabolites 1 and 2
The extract was separated in 3 runs through a Kromasil RP C18 column (250 mm × 20 mm, 7 µm, MZ-Analysentechnik, Mainz, Germany) using deionized water (Milli-Q Millipore) and acetonitrile (HPLC grade) as the mobile phase on an Agilent 1100 series HPLC system (Agilent Technologies, Wilmington, DE, USA). The separation was carried out according to the following gradient: from 20% -75% acetonitrile in 50 min, then rising to 100% ACN in 10 min, and maintaining 100% for 5 min. UV detection was performed at 220, 280, and 325 nm. Fractions were collected and pooled according to the observed peaks. The separation yielded two fractions including compound 1 (1.2 mg) and 2 (1.1 mg) at retention times t R 15-16 and 28-29 min, respectively.

Molecular phylogeny
We generated 14 new sequences (3 ITS, 3 LSU, 3 TEF1, 3 RPB1 and 2 RPB2) from living cultures ( Description: Spider eggs in a sac are covered with pale yellowish white to moderate yellow mycelium. Teleomorph: Perithecia produced on the mycelial mat covering the body of the hosts, superficial with mycelia covering the bottom half of the perithecium, ovoid narrowing towards the ostiole. Asci cylindrical with thickened caps, 8-spored. Ascospores filiform, hyaline, whole. Anamorph: Synnema produced from the mycelial mat-covering hosts, erect, unbranched, solitary, and cylindrical. Conidiophores erect arising along with the synnema, occasionally found on the mycelium covering the hosts, verticillate with phialides in whorls of two to five. Phialides comprising a cylindrical basal portion, tapering into a thin slightly curved neck. Conidia fusiform, slightly curved, aggregated at the apex of the phialides. Notes: Bhushaniella contains one species, Bh. rubra. The teleomorph state is morphologically similar to Gibellula spp. by producing superficial perithecia on the mycelial mat covering the body of the hosts. However, it differs from Gibellula spp. in producing whole ascospores. The ascospores of Gibellula spp. are multiseptate and disarticulate. Additionally, the anamorph in Bhushaniella produces verticillate phialides with a slightly curved neck. Mongkolsamrit, Noisripoom & Luangsa-ard, sp. nov. (Fig. 2) Etymology: The name refers to the pale red pigment produced by the fungus when growing on PDA and OA.

Secondary metabolites
Compound 1 was isolated as a colourless solid that revealed pseudomolecular ion peak at m/z 385.2 [M + H] + suggesting the molecular weight to be of 384 g/mol. Its molecular formula was established to be C 22 H 28 N 2 O 4 based on HRESIMS that exhibited a pseudomolecular ion peak at m/z 385.2120 [M + H] + (calculated for C 22 H 29 N 2 O 4 ; 385.2127) indicating ten degrees of unsaturation. The UV spectrum of 1 displayed three maximal absorption peaks (λmax) at 199, 226 and 273 nm. The 13 C NMR spectrum of 1 (Table 2, see supplementary materials Figure S7) showed the presence of ten carbon resonances suggesting that compound 1 is either a dimer of two identical monomers or a symmetric compound with each carbon peak represents two electromagnetically equivalent carbon atoms in 1. The ten carbon resonances can be recognized into one carbonyl carbon (δ c 166.1) and two quaternary carbons (δ C 146.0, 141.7), three olefinic carbons (δ C 149.4, 137.0, 124.4) along with five methylene carbons (δ C 32.0, 30.3, 28.9, 28.7, 28.5  available literature suggested that compound 1 is structurally related to picolinic acid fungal metabolites, penicolinates A-C (Intaraudom et al. 2013). By comparing the MS and NMR spectral data between metabolite 1 and penicolinate A (Intaraudom et al. 2013), it was obviously that 1 has less molecular weight by 28 amu compared to penicolinate A that has been reflected in 1 H NMR by the absence of methyl ester group at δ H 3.97 ppm and the appearance of a highly deshielded broad proton resonance at δ H 13.00 ppm assigned to the free carboxylic acid group. The aforementioned results suggested that compound 1 to be the free carboxylic acid derivative of penicolinate A featuring a symmetrical compound comprising two 2,5-disubstituted pyridine moieties bonded through a ten-methylene chain. Further confirmation of the ascribed chemical structure of 1 was provided by 2D NMR spectra including 1 H-1 H-COSY and HMBC (Table 2, Fig. 4, see supplementary materials Figure S5, S6). The 1 H-1 H-COSY (Table 2, Figs. 3 and 4) revealed two main spin systems, one between three olefinic protons at δ H 8.54, δ H 7.96 and δ H 7.80 assigned to H-6, H-3 and H-4, respectively, whereas the second spin system was among the five methylene protons at δ H 2.65 (t, J = 7.7 Hz), δ H 1.60 (quin, J = 7.7 Hz) and three methylenes at δ H 1.15-1.35 (m). In addition, a long-range COSY correlations (Table 2, Fig. 4) were also noticed between olefinic protons at δ H 8.54 and δ H 7.80 with the methylene protons at δ H 2.65 (t, J = 7.7 Hz) ascribed for H-4, H-6 and CH2-8, respectively. The HMBC spectrum (Table 2, Fig. 4) further confirmed the positions of the carboxylic acid moiety at C-2 via key correlations from H-3 (δ H 7.96, dd, J = 8.0 Hz) to the carboxylic acid carbon (δ C 166.1) along with the key correlations from H-4 and H-6 to the methylene carbon at (δ C 32.0) ascribed to C-8. In conclusion, compound 1 was unambiguously determined to be a new symmetric picolinic acid derivative that was given a trivial name, penicolinate F.
Based on the anamorph in the natural specimen, Bh. rubra has a solitary cylindrical synnema. The conidiophores in Bh. rubra are produced along the synnema and on the mycelium covering the hosts. Its phialides consist of a cylindrical basal portion, mostly verticillate and in whorls of two to five. Hence, its morphological characters clearly differ from those of several species in Hevansia, Jenniferia and Gibellula that produce multiple synnemata. Significantly, species in Gibellula produce the synnemata bearing predominantly aspergillus-like conidiophores or occasionally growing penicillate or granulomanus-like conidiophores. The anamorphs of Hevansia and Jenniferia are characterized by producing phialides with mono-or polyphialidic conidiogenous cells with cylindrical or swollen basal portions, and their conidia are not catenulate. Polystromomyces, the anamorph has not been seen in the field. (Kuephadungphan et al. 2020(Kuephadungphan et al. , 2022Mongkolsamrit et al. 2022).
Beauveria is one of the best-known entomopathogenic fungi with a global distribution . Samsoniella was established by Mongkolsamrit et al. (2018), there are currently 29 species Chen et al. 2021aChen et al. , b, 2022aWang et al. 2020Wang et al. , 2022) that have been recorded in the Index Fungorum (accessed in May 2023). Beauveria and Samsoniella have a wide host range, but were rarely reported from spiders. Currently, Beauveria araneola Wan H. Chen, Y.F. Han, Z.Q. Liang & D.C. Jin and S. farinosa Hong Yu bis, Yao Wang & Z.Q. Wang were found on spiders. Engyodontium rectidentatum (Matsush.) W. Gams, de Hoog, Samson & H.C. Evans is commonly isolated from soil, but can also be found on spiders (Meta menardi Latreille) in the Czech Republic (Gams et al. 1984;Kubátová 2017). Based on the morphological characters, the three aforementioned species produce white mycelium on spiders.
Bhushaniella rubra produces conidiophores bearing whorls of phialides, verticillate or solitary; conidia that are aggregated at the apex of the phialides, corresponding to what is described as the conidiogenesis in Lecanicillium  Currently, thirty-six species in Lecanicillium have been formally described and recorded in Index Fungorum (May 2023), and all these species are mostly associated with insects, spiders, plants, decayed wood, and soil. The phylogenetic studies confirmed that Lecanicillium is polyphyletic (Kepler et al. 2017;Zhou et al. 2022). Lecanicillium