Ophiocordyceps flavida sp. nov. (Ophiocordycipitaceae), a new species from Thailand associated with Pseudogibellula formicarum (Cordycipitaceae), and their bioactive secondary metabolites

During a diversity study of entomopathogenic fungi in an agricultural ecosystem, two fungi were collected, isolated, and identified based on molecular phylogenetic analyses of three nuclear loci (LSU, TEF1, and RPB1) combined with morphological data. In this study, one novel species is described, Ophiocordyceps flavida, and a new record of Pseudogibellula formicarum for Thailand. Ophiocordyceps flavida morphologically resembles the Hirsutella anamorph of Ophiocordyceps pruinosa by having a mononematous character of the conidiophores and the same insect host (Hemiptera: Cicadellidae). Pseudogibellula formicarum is found to occur simultaneously with O. flavida, producing white conidiophores on the host. Additionally, secondary metabolites of both fungi were investigated and the major compound in O. flavida was identified as 2-[2-(4-chlorophenyl)ethyl]-2-(1,1-dimethylethyl)-oxirane. Pseudogibellula formicarum from Ghana and Thailand produces 6-methoxy-1H-indole-3-carbonitrile as a main component. These compounds are known from chemical synthesis or as products of biotransformation, respectively. However, they were obtained in our study as genuine fungal metabolites for the first time and may even constitute chemotaxonomic markers for the respective species.


Introduction
The genus Ophiocordyceps was erected by Petch (1931) with O. blattae as type. Species in Ophiocordyceps have diverse morphologies, including fibrous, hard, pliant to wiry, and dark to light colored stromata with perithecia that are superficial or immersed with an ordinal or oblique arrangement (Kobayasi 1941;Mains 1958;Sung et al. 2007). The hosts of Ophiocordyceps are found in various stages of the insect's life cycle, occurring on orders Blattodea, Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, Megaloptera, and Odonata (Kobayasi 1941;Mains 1958;Sung et al. 2007;Sanjuan et al. 2015;Shrestha et al. 2016;Khonsanit et al. 2018;Luangsa-ard et al. 2018;Tasanathai et al. 2019Tasanathai et al. , 2020Thanakitpipattana et al. 2020). The dominant anamorph associated with Ophiocordyceps is Hirsutella Patouillard followed by Hymenostilbe Petch and Syngliocladium Petch. Paraisaria Samson & B.L. Brady was previously described as an anamorph of Ophiocordyceps, but this genus is now resurrected and segregated from Ophiocordyceps based on distinct morphological characters and molecular phylogeny .
Leafhoppers and planthoppers are plant feeders belonging to the order Hemiptera, which are considered insect pests in agriculture and forestry. Hirsutella was erected by Patouillard (1892), based on the type species H. entomophila Pat. Previous studies showed that Hirsutella species are commonly found associated with these hoppers. Most species are Section Editor: Gerhard Rambold * Janet Jennifer Luangsa-ard jajen@biotec.or.th mononematous (e.g., O. pruinosa D. Johnson, G.H. Sung, Hywel-Jones & Spatafora), and a few species occasionally produce synnemata (e.g., Hirsutella citriformis Speare). The conidiogenous cells of Hirsutella spp. are monophialidic or polyphialidic producing conidia on phialides. Teleomorph and anamorph stages occurring on hoppers have been reported co-existing on the same or on different insect hosts. For example, Hywel-Jones observed both teleomorph and anamorph (Hirsutella versicolor Petch) stages in O. pruinosa on the same insect host, and a single collection in which only the anamorph was present (Hywel-Jones 1997). Pseudogibellula was established by Samson and Evans (1973) for Gibellula formicarum Mains as Pseudogibellula formicarum. In the original description, the insect host of Gibellula formicarum was an ant collected from Liberia (Mains 1949). Pseudogibellula is morphologically similar to Gibellula in producing synnemata scattered on the insect cadaver, giving rise to white or brown conidiophores arising from mycelium covering the host or from synnemata. However, Pseudogibellula differs from Gibellula in the way its conidia are produced. The conidia in Pseudogibellula are produced singly from sympodial and polyblastic conidiogenous cells whereas Gibellula produces conidia in chains from phialides (Samson et al. 1988). Additionally, Pseudogibellula occurs on a wide range of insect hosts whereas Gibellula is found exclusively on spiders. Samson et al. (1989) described Torrubiella pseudogibellulae infecting Palthyreus tarsatus from Ghana as the teleomorph associated with P. formicarum. The hosts of P. formicarum from Ghana were reported as Hymenoptera and Hemiptera (Samson and Evans 1973). In a taxonomic paper on Ophiocordyceps (Ophiocordycipitaceae, Ascomycota), Spatafora et al. (2015) proposed Ophiocordyceps pseudogibellulae (Samson, Reenen & H.C. Evans) B. Shrestha, G.H. Sung & Spatafora as a new combination for Torrubiella pseudogibellulae S a m s o n , R e e n e n & H . C . E v a n s i n w h i c h Pseudogibellula formicarum was included as its synonym without including this species in the phylogenetic analyses of Ophiocordycipitaceae. However, there was no formal transfer of the genus from Cordycipitaceae to Ophiocordycipitaceae.
During a diversity study on entomopathogenic fungi in an organic orchard ecosystem, we discovered a mononematous Hirsutella sp. attacking an unidentified leafhopper on the underside of pomelo leaves (Citrus maxima). The macromorphologies of the natural samples of Hirsutella sp. resemble H. versicolor, the anamorph of O. pruinosa. Additionally, we also found another fungus developing white conidiophores together with the Hirsutella sp. on the host. We attempted to identify these two fungi by investigating their molecular phylogeny and morphological characters. Hirsutella sp. did not match Hirsutella versicolor or any Hirsutella species. The second fungus was identified as Pseudogibellula formicarum. The aims of this study are to describe the fungus producing Hirsutella anamorph as a new species, O. flavida and to establish a new record of P. formicarum from Thailand. We confirm the position of P. formicarum in the Cordycipitaceae. Finally, an investigation of the secondary metabolites produced by both fungi is presented.

Collection and isolation
Two species of fungi occurring simultaneously on leafhoppers (Hemiptera) were collected on the underside of pomelo leaves (Citrus maxima) in an organic orchard ecosystem in Samut Songkhram Province, Thailand. The specimens were collected in two different seasons, the first collection was during the rainy season in June 2016, and the second was during the dry period in January 2017. They were collected carefully so as not to lose the host, and were put in small plastic boxes and transported to the laboratory for isolation. The materials were examined under a stereo microscope (Olympus SZ61). For the isolation from anamorphs, a flamesterilized inoculation needle was used to remove conidia from sporulating structures to potato dextrose agar plates (PDA; freshly diced potato 200 g, dextrose 20 g, agar 15 g, in 1 L distilled water). Pure cultures were deposited at the BIOTEC Culture Collection (BCC). The leafhopper hosts were identified by morphology. The fungal specimens were dried in an electric food dryer (50-55°C) overnight and stored in plastic boxes before storage at the BIOTEC Bangkok Herbarium (BBH), National Biobank of Thailand.

Morphological observation
Fungal structures, such as phialides and conidia, were mounted in lactophenol cotton blue solution and measured using a light microscope (Olympus CX31). They were photographed using an Olympus DP70 Digital Camera mounted on an Olympus BX51 (Olympus) compound microscope and SZX12 (Olympus) stereo microscope as well as a Hitachi scanning electron microscope (Model SU8020). The cultures were grown on PDA for study of important morphological characters such as conidia, phialides, and colony coloration. The color of fresh specimens and cultures incubated on PDA for 21 days at 25°C was described and codified following the Sixth Royal Horticultural Society (R.H.S.) Colour Chart.

Molecular phylogenetic analyses
Genomic DNA was harvested from mycelial mass on PDA using a modified cetyltrimethyl-ammonium bromide ( C T A B ) m e t h o d a s d e s c r i b e d p r e v i o u s l y i n Mongkolsamrit et al. (2009). The partial gene regions of three nuclear loci, including nuc 28S rDNA (large subunit ribosomal DNA: LSU), translation elongation factor-1α gene (TEF1), and the largest subunit of RNA polymerase II (RPB1), were sequenced. The primer pairs and thermocycler conditions for PCR amplifications used in this study followed Mongkolsamrit et al. (2018). 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 2004) and then submitted to GenBank. Table 1 shows the list of LSU, TEF1, and RPB1 sequences generated in this study as well as those of other taxa from previous studies. Phylogenetic analyses were performed, including maximum parsimony (MP), Bayesian inference (BI), and maximum likelihood (ML). The MP analysis was conducted on the combined dataset using PAUP 4.0a168 (Swofford 2003) (http://paup.phylosolutions.com/) adopting random addition sequences (10 replications) with gaps being treated as missing data. The nodes in the best MP topologies were evaluated via 1000 bootstrap replicates. The Bayesian inference was performed using MrBayes v. 3.2.7a (Ronquist et al. 2012) with the GTR model. Four Markov chains were run from random starting trees for five million generations using a sampled frequency of 100 generations and a burn-in of 25% from the total run. The maximum likelihood (ML) analysis was performed with RAxML-v 8.2.12 (Stamatakis 2014) using the GTR-GAMMA model of evolution with 1000 bootstrap replicates. RAxML and BI were run on XSEDE in CIPRES portal (www.phylo.org). Phylogenetic trees were visualized in TreeView v.1.6.6 (Page 1996). The sequence alignment for the dataset used in this study is provided in Supplementary Information.
The broth and fungal mycelia obtained from culture were extracted together with ethyl acetate (100 mL) to yield a crude analyte. The fungal extracts from O. flavida and P. formicarum were chemically analyzed on a Dionex Ultimate 3000 HPLC system with diode array detector using a reverse phase column with gradient condition (Puropher® C18, 2 × 55 mm, 3 μm; 0-100% MeCN/H 2 O over 10 min, 100% MeCN over 2 min, then reversed back to 100% H 2 O in 1 min and equilibrated at 100% H 2 O over 2 min). For the mobile phase, 0.05% formic acid was added to both acetonitrile and deionized water.

Isolation of secondary metabolites
Pseudogibellula formicarum BCC 81493 was grown on YMG medium (200 mL) for 8 days under static condition. The culture broth was obtained after the separation from the fungal mycelia and extracted three times with ethyl acetate (3 × 300 mL) to give a crude material (3.8 mg). Fungal mycelia were extracted with methanol and sonicated for 1 hour to obtain a crude substance (6 mg). Both fractions were analyzed on an Agilent 1260 UHPLC Infinity Systems using a previously described gradient condition method (Noumeur et al. 2017), and were discovered to possess very similar HPLC chromatograms. Isolation of pure compounds was performed on a Waters HPLC system with the mobile phase composed of acid-free deionized water and acid-free acetonitrile. The combined broth and mycelial extracts were purified by semipreparative HPLC using a reversed-phase column (Phenomenex Luna C18, 21.2 × 250 mm, 10 μm; a flow rate  as follows. Culture broth and mycelia were combined and then extracted three times with ethyl acetate (80 mL) to yield a crude extract. Crude extracts from three fungal strains were analyzed on a Dionex Ultimate 3000 HPLC system with a previously mentioned analytical gradient condition method. HPLC profiles from these three strains were shown to be similar with one major and a few minor peaks. The isolation of the major component was performed on a Waters HPLC system and the mobile phase was composed of acid-free solvents. The combined broth and mycelial extracts (22.9 mg) were purified by semipreparative HPLC using a reversed-phase column (VDSpher PUR 100 C18-E, 20 × 250 mm, 10 μm; a flow rate 10 mL/min, 30% MeCN/H 2 O for 7 min, 30-80%

Biological characterization
The compound 6-methoxy-1H-indole-3-carbonitrile was tested for antimicrobial affects against several bacteria and fungi and against mammalian cells as reported previously, using the same protocols (Cheng et al. 2019;Rupcic et al. 2018;Sandargo et al. 2020). The other compound 2-[2-(4-chlorophenyl)ethyl]-2-(1,1-dimethylethyl)-oxirane (2.4 mg) unfortunately decayed before the bioassays could be carried out. Hence, no antimicrobial and cytotoxic activities are reported. List of test microorganisms used are in Supplementary Information. The accession numbers marked in bold font refer to sequences new in this study or have been generated by our group in Thailand 1T

Molecular phylogeny
We generated 6 LSU, 8 TEF1, and 8 RPB1 sequences from three strains of O. flavida, two strains of O. blattae, two strains of P. formicarum from Thailand, and two strains of P. formicarum from Ghana (CBS 433.73 and CBS 871.72) ( Table 1). Purpureomyces khaoyaiensis (Hywel-Jones) Luangsa-ard, Samson & Thanakitpipattana (BCC 1375 and BCC 14290) in the Clavicipitaceae was used as the outgroup. Phylogenetic analyses (Fig. 1) strongly support P. formicarum from Thailand (BCC 81493, BCC 84257) and from Ghana (CBS 433.73, CBS 871.72) as a monophyletic clade and as members of Cordycipitaceae with strong support (RAxML 100%, MP 100%, BPP 100%). O. flavida is nested in Ophiocordyceps with strong support (RAxML 100%, MP 100%, BPP 100%). Additionally, analyses of O. flavida with related species in Ophiocordyceps strongly support (RAxML100%, MP 100%, BPP 100%) O. flavida as a distinct clade that formed independently and did not group with any known species occurring on leafhoppers or planthoppers (Fig. 2). The species descriptions based on morphological characters of O. flavida as a novel species and P. formicarum from Thailand are given below. Comparison of LSU sequence of the only available sequence data for Hirsutella homalodiscae nom. prov., which was reported to cause outbreaks on the same cicadellid hosts in the USA (Boucias et al. 2006), confirms that O. flavida is distinct from the latter species (Supplementary Information).
Etymology: Describes the pale yellow color of the fresh specimens.
Anamorph: Mycelium covering loosely the leafhopper with compact pale yellow to orange (22A) hyphae and surrounded by a radiating mat over the leaf. Conidiogenous structures phialidic, borne directly and singly on hyphae.
Distribution: Found in western Thailand. Additional specimens examined: Thailand, Samut Songkhram Province, Boonmee Orchard, on leafhopper (Hemiptera: Cicadellidae: Tartessus  in having yellow to brown mycelium and fusiform to globose conidia occurring on leafhoppers (Hemiptera) that can also be found on the underside of leaves. However, our phylogenetic analyses (Fig. 2) clearly show that O. flavida formed its own and separate clade from Hirsutella versicolor ARSEF 1037. Another species reported on cicadellid hosts is Hirsutella homalodiscae nom. prov. occurring on Homalodisca coagulata (glassy-winged sharpshooter) causing epizootics in southeastern USA. This species differs from O. flavida in having longer phialides (30.9 ± 4.5 μm) and in the citriform to amygdaliform shape of its conidia (Boucias et al. 2006).
Pseudogibellula Samson & H.C. Evans, Acta Bot. Neerl. 22: 524 (1973) Circumscription: The genus is emended here consisting of one species in the node defined in Fig. 1 as the reference phylogeny for the Pseudogibellula clade in Cordycipitaceae, which contains P. formicarum. The descriptions from the teleomorph are taken from Samson et al. (1989). The morphological character associated with the teleomorph includes cream to orange mycelium. Synnemata arising from the mycelium, bearing one or more perithecia terminally or subterminally. Perithecia surrounded with mycelium at the base, ovoid to conoid or flasked-shaped, smooth, brownish. Asci cylindrical with hyaline cap. Ascospores filiform, not breaking into part-spores. The morphologies associated with the anamorph reproductive stage include the presence of distinct white powdery conidia from scattered conidiophores arising from mycelium covering the host. Conidiogenous cells  (2015).
The description and illustrations provided herein are based on Pseudogibellula formicarum specimens collected from Thailand (Fig. 4).
Distribution: Found in western Thailand. Notes: Based on the macromorphologies of the natural specimens, Pseudogibellula formicarum collected from Thailand closely resembles Beauveria by producing powdery white conidia covering its host. In Thailand, P. formicarum grows with O. flavida, possibly as a mycoparasite. Samson and Evans (1973), who reported P. formicarum found in Ghana as a parasitic fungus on insect hosts (Hemiptera: Cercopidae, Ricaniidae and Hymenoptera: Ponerinae, Myrmicinae), also recognized it as a strongly competitive fungus on insect substrates that frequently exploits ant cadavers killed by other fungal pathogens. It was found to be especially

Identification of the major metabolite from Ophiocordyceps flavida
The HPLC profile of the crude extract of Ophiocordyceps flavida (BCC 84254,BCC 84255,and BCC 84256) showed one major metabolite peak (Rt = 8.7 min) with a few other minor metabolites evident in the chromatogram (Fig. 5a). Additional purification by semi-preparative HPLC led to the isolation of the major metabolite, which could be identified as 2-[2-(4-chlorophenyl)ethyl]-2-(1,1dimethylethyl)-oxirane (Fig. 6a). The structure of the isolated compound was elucidated based on the interpretation of 2D NMR spectroscopic data including 1 H-1 H correlation spectroscopy (COSY), distortionless enhancement by polarization transfer (DEPT), heteronuclear singlequantum correlation spectroscopy (HSQC), and heteronuclear multiple-bond correlation spectroscopy (HMBC). The core structure of the molecule could be established using proton-carbon correlations from these experiments. The molecular formula was shown to be C 14 H 19 ClO by HRESIMS and the pattern of mass spectrum peaks further confirmed the presence of a chloride atom in the molecule.

Identification of the major metabolite from
Pseudogibellula formicarum HPLC profiles of the broth and mycelial extract of Pseudogibellula formicarum (BCC 81493) showed one similar major metabolite peak (Rt = 6.4 min) with a UV absorption pattern characteristic for indoles chromophors (Fig. 7). Further purification by semi-preparative HPLC led to the isolation of this major metabolite as 6-methoxy-1H-indole-3carbonitrile (Fig. 6b). The structure of the isolated compound was elucidated from the interpretation of 2D NMR spectroscopic data including 1 H-1 H COSY, HSQC, and HMBC. Its molecular formula was confirmed as C 10 H 8 N 2 O by HRESIMS and the UV spectrum also matched that of the corresponding synthetic compound with characteristic absorption bands at 221, 271, and 290 nm. This substance has been reported to exhibit antifungal activity against Alternaria brassicicola (Pedras and Abdoli 2013).

New Ophiocordyceps on leafhoppers
Phylogenetic analyses based on multiple loci indicated that Ophiocordyceps flavida is a new species of parasitic fungus infecting leafhoppers belonging to Ophiocordyceps. Several species have been documented infecting leafhoppers and planthoppers (Hemiptera) in Ophiocordyceps (Johnson et al. 2009), including Hirsutella guyana Minter & B.L. Brady on immature stage nymphs of Saccharosydne saccharivora from Venezuela (Minter and Brady 1980) and Hirsutella strigosa Petch on an unknown species of leafhopper from Sri Lanka (Petch 1939). Ophiocordyceps pruinosa (Petch) D. Johnson, G.H. Sung, Hywel-Jones & Spatafora occurs on leafhoppers and its anamorph was identified as Hirsutella versicolor by Petch. This species was originally described from Sri Lanka (Petch 1932 From our phylogenetic analyses in Fig. 2, Ophiocordyceps flavida is closely related to Hirsutella versicolor and shows similar morphological characteristics. Both species occur on leafhoppers that are found on the underside of leaves. The phialides of both species are conoid, cylindrical to flaskshaped and their sizes are in the same range (7-20 × 2-4 μm vs. 8-20 × 2.5-3 μm). However, the conidia in O. flavida are fusiform to globose whereas the conidia of O. pruinosa (H. versicolor) are narrowly cymbiform or narrow-oval.

New host record for Pseudogibellula formicarum
This study is the first report of Pseudogibellula formicarum from Thailand. The results of our phylogenetic analyses (Fig.  1) indicated that Pseudogibellula is a strongly supported genus in the Cordycipitaceae. In a study by Samson and Evans (1973), P. formicarum in Ghana was documented as a generalist insect pathogen found on various insect hosts, such as Hymenoptera (Myrmicinae, Ponerinae) and Hemiptera (Ricaniidae, Cercopidae). Moreover, it is also recognized as a colonizer of insect debris in the soil. Kanga et al. (2004) collected glassy-wing sharpshooter (GWSS) Homalodisca coagulata (Cicadellidae) cadavers that had died on crape myrtle (Lagerstroemia indica L.) and holly (Ilex myrtifolia Walter) in Mississippi for identification of the primary pathogens of sharpshooters. They confirmed that P. formicarum was the cause of the epizootics in sharpshooters (Cicadellidae) in the field after conducting pathogenicity assay. Boucias et al. (2006) also studied outbreaks on the GWSS (H. coagulata) from north Florida and southern Georgia. They determined and identified that three fungi, including Hirsutella homalodiscae nom. prov., Pseudogibellula sp., and Sporothrix sp. are pathogens of the glassy-winged sharpshooter. However, there was no report of an association between Hirsutella homalodiscae and Pseudogibellula. Based on our study, we found the conidiophores of P. formicarum occurring simultaneously with O. flavida in the natural specimens on leafhoppers (Fig. 4a, 4b). More studies are needed to explain the association between O. flavida and P. formicarum and to clarify what fungal species caused the epizootics in the leafhopper.

Secondary metabolites from Ophiocordyceps flavida and Pseudogibellula formicarum
From the crude extract of Ophiocordyceps flavida (BCC 84254, BCC 84255, and BCC 84256), the major secondary metabolite could be isolated and identified as 2-[2-(4chlorophenyl)ethyl]-2-(1,1-dimethylethyl)-oxirane. The synthesized compound is commercially available, but no report regarding its original natural sources has been confirmed. Nevertheless, we report it here from a fungal source (and as a natural product) for the first time. There is a broad spectrum of usage for this metabolite. The compound is used as raw material in a variety of products and industries such as cosmetics and chemical manufacturing. In addition, agrochemical related products including fertilizer and pesticide also employ this substance as the raw material (Martins et al. 2013).
Pseudogibellula formicarum (BCC 81493) was discovered to produce a single metabolite as its major product. The structure elucidation process led to the identification of the isolated substance as the novel fungal metabolite, 6-methoxy-1H-indole-3-carbonitrile. This compound had previously only been reported as a derivative from the biotransformations of the phytoalexin camalexin with antifungal activity against Alternaria brassicicola (Pedras and Abdoli 2013). We confirmed its antifungal activity, although this is weaker than its activity against mammalian cells.
To further explore the metabolite production of P. formicarum BCC 81493, crude extracts from static cultures in YMG medium were also analyzed. The HPLC profiles were similar to previous results from cultures grown in shake flasks. The HPLC profiles from samples of five additional P. formicarum isolates (BCC 84247, BCC 84249, BCC 84251, BCC 84257, and BCC 84259) cultivated in PDB and Q6 media (Cheng et al. 2019) exhibited very similar chromatograms with 6-methoxy-1H-indole-3-carbonitrile as the dominant major metabolite. Moreover, the same major metabolite was identified from BCC 81493 cultivated under different conditions.
A great deal of work by natural product chemists has revealed a vast diversity of secondary metabolites from entomopathogenic fungi (Gibson et al. 2014;Helaly et al. 2019;Isaka et al. 2019;Kuephadungphan et al. 2019;Sonyot et al. 2020;Zhang et al. 2020). While the majority of these studies were aimed at the discovery of new biologically active compounds, some commonly found secondary metabolites have been reported to be suitable as chemotaxonomic markers. For instance, hopane triterpenes are specific for Hypocrella and Moelleriella species, and zeorin commonly occurs on Conoideocrella species on scale insects (Isaka et al. 2009;Isaka et al. 2011). Accordingly, all six strains of P. formicarum from Thailand which have been evaluated so far were found to produce 6-methoxy-1H-indole-3carbonitrile as their major component. In addition, commercially available P. formicarum CBS 871.72 and CBS 433.73 strains collected from Ghana in Western Africa were found to produce this metabolite. These data indicate that 6-methoxy-1H-indole-3-carbonitrile could be considered as a chemotaxonomic marker for P. formicarum, but additional strains including related species remain to be studied in order to assess whether it is species-specific.