Three Novel Entomopathogenic Fungi From China and Thailand

Entomopathogenic fungi are ubiquitous in tropical rainforests and feature a high level of diversity. This group of fungi not only has important ecological value but also medicinal value. Nevertheless, they are often ignored, and many unknown species have yet to be discovered and described. The present study aims to contribute to the taxonomical and phylogenetic understanding of the genus Paraisaria by describing three new species collected from Guizhou and Yunnan Provinces in China and Krabi Province in Thailand. The three novel species named Paraisaria alba, P. arcta, and P. rosea share similar morphologies as those in the genus Paraisaria, containing solitary, simple, fleshy stroma, completely immersed perithecia and cylindrical asci with thickened caps and filiform ascospores that often disarticulate at maturity. Phylogenetic analyses of combined LSU, SSU, TEF1-α, RPB1, RPB2, and ITS sequence data confirm their placement in the genus Paraisaria. In this study, the three entomopathogenic taxa are comprehensively described with color photographs and phylogenetic analyses. A synopsis table and a key to all treated species of Paraisaria are also included.


INTRODUCTION
Entomopathogenic fungi are a group of unicellular or multicellular, heterotrophic, eukaryotic microorganisms that can enter into a parasitic relationship with parasitized insects, killing or otherwise disabling their hosts (Samson et al., 1988). They reproduce via sexual or asexual spores, or both (Mora et al., 2017). It is of global importance to survey and describe insect pathogens (Hyde et al., 2019). Entomopathogenic fungi can act as natural enemies of agricultural pests and play an important role in maintaining ecological balance (Fernández-Grandon et al., 2020;Sobczak et al., 2020). For example, fungal pathogens such as, Coelomomyces, Culicinomyces, and Lagenidium have the capacity to kill larva and adult mosquitoes, reducing their host population (Scholte et al., 2004). Some entomopathogenic fungi, e.g., Beauveria bassiana, Beauveria brongniartii, Metarhizium anisopliae, and Verticillium lecanii, have been developed as biocontrol agents usable against agricultural pests like aphids, locusts, grasshoppers and cockchafer in Africa and Europe (Roberts and Hajek, 1992;Shah and Pell, 2003). Beauveria bassiana and B. brongniartii were found to be especially safe bioinsecticides (Zimmermann, 2007). Additionally, some insect pathogens with pharmacological activities are frequently studied, such as Cordyceps militaris extract, which exhibits antitumor properties (Li et al., 2020). Cordyceps spp. have been utilized as therapeutic agents for metabolic-related disorders (Cao et al., 2020). Cordyceps cicadae has renoprotective effects on hypertensive renal injuries (Huang et al., 2020). Entomopathogenic fungi have important biotechnological applications (Hyde et al., 2019) and Paraisaria is no exception. Several studies have explored the importance of Paraisaria species, such as their antioxidative activity (Ma et al., 2012), nucleoside components (Suo et al., 2013), intracellular polysaccharide composition (Wang et al., 2019) and AGS gastric cancer cells anti-proliferation effects (Ye et al., 2015). Additionally, P. heteropoda reportedly produces anti-bacterial and anti-fungal compounds (Krasnoff et al., 2005). Experiments into optimal cultural conditions and nutritional sources were conducted by Sung et al. (2011). Applications of other species in this genus have been poorly studied.
This study is part of a larger survey of fungi in the Greater Mekong Subregion where we came across numerous new taxa . In this study, three specimens of entomopathogenic fungi were collected from disturbed forests in China and Thailand, and the typical macro-and micro-morphological characteristics indicate that they are of the Paraisaria species. The multigene phylogenetic analysis of LSU, SSU, TEF1-α, RPB1, RPB2, and ITS confirmed their placement within Paraisaria as three distinct new species.

Sample Collection, Isolation, and Morphological Studies
In this study, a total of four fungal specimens were collected. One specimen (HKAS 102484) was collected from Krabi Province in Thailand on an adult cricket. Two specimens (HKAS 102553 and HKAS 102552) on dead larvae of Lepidoptera sp. were collected from Guizhou Province of China. One specimen (HKAS 102546) was collected from Yunnan Province in China on Coleoptera sp. larva. Among them, the hosts of specimens HKAS 102484, HKAS 102553 and HKAS 102552 were found completely immersed into soil with the stroma protruding from the ground in a forest. Specimen HKAS 102546 was found in a similar condition, but differed in that it was found under a karst stone formation. Macro-morphological characteristics of fresh collections were recorded with a camera (iPhone XS Max) in the field and then the specimens were transported to the laboratory in plastic boxes for subsequent studies. The culture of the specimen HKAS 102546 was created by transferring a small mass of mycelium inside the body of the host into potato dextrose agar (PDA, 1% w/v peptone) using a burned needle and incubated at room temperature (25 • C). The pure culture was stored in twice-sterilized water, a 15% glycerinum solution and PDA medium, and deposited in the KUMCC culture collection of the Kunming Institute of Botany (KIB), Chinese Academy of Sciences (CAS). The fruiting bodies were dried with allochroic silica gel and deposited in KUN herbarium of KIB. Facesoffungi numbers were registered as outlined in Jayasiri et al. (2015).
The fresh fruiting bodies were examined and hand-sectioned under an Optec SZ660 stereo dissecting microscope. The key fungal structures viz. ascomata, perithecia, peridium, asci and ascospores were mounted in sterilized water or cotton blue solution slides and observed and photographed using a compound microscope (Nikon ECLIPSE Ni) with a digital camera (Canon EOS 600D) fitted on to the top of the microscope. These important fungal structures were measured with the Tarosoft (R) Image Frame Work program and the images used were processed with Adobe Photoshop CS3 Extended v. 10.0 (Adobe R , San Jose, CA, United States).

DNA Extraction, PCR Amplification, and Sequencing
The total DNA was extracted from stromal tissue of specimens HKAS 102552, HKAS 102553, HKAS 102484 and from fresh mycelium of KUMCC 20-0001 (ex-type culture of isolate HKAS 102546) using DNA extraction kit (Omega Fungus Genomic DNA Extraction Kit, China), following the protocol of the manufacturer. The obtained DNA was stored at −20 • C in a refrigerator. The PCR amplification was performed in 25 µL volumes consisting 12.5 µL PCR mixture (2 × Taq PCR Master Mix, red dye) which contains Taq DNA polymerase, dNTPs, MgCl 2 , a reaction buffer, a PCR reaction enhancer, an optimizer and stabilizer, 8.5 µL of twice-sterilized water, 1 µL of each primer and 2 µL of 30 µg/µl DNA template. The internal transcribed spacer (ITS1-5.8S-ITS2, ITS), large subunit ribosomal RNA (LSU rRNA), small subunit ribosomal RNA (SSU rRNA), translation elongation factor 1-alpha gene (TEF1-α) and RNA polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) were amplified with the primers and procedures mentioned in Table 1. The PCR products were sent to Tsingke company, Yunnan Province, China, for sequencing the above genes. The generated sequences were submitted to GenBank, and the accession numbers have been shown in Table 2.  (Liu et al., 1999;Sung et al., 2007b) RPB2-5F RPB2-7cR GAYGAYMGWGATCAYTTYGG CCCATRGCTTGTYYRCCCAT (1) Initialization at 95 • C for 3 min.
(2) 40 cycles of denaturation at 95 • C for 1 min, annealing at 52 • C for 2 min, and extension at 72 • C for 90 s.
(3) final elongation at 72 • C for 10 min and (4) storage at 4 • C.  The new species generated in this study are in black bold.

Sequence Alignment and Phylogenetic Analyses
The generated sequences were assembled with Sequencing Project Management (SeqMan) (Clewley, 1995). The sequences for the combined alignment were selected based on the blast results of LSU, SSU, ITS, TEF, RPB1, and RPB2 as well as the recent references listed in Table 2. The individual gene alignment was aligned in MAFFT v. 7 web server 1 (Kuraku et al., 2013;Katoh et al., 2019). The alignments of each locus were improved by manually removing uninformative gaps and ambiguous regions using BioEdit v. 7.0.9.1 (Hall, 1999) and were concatenated in Sequence Matrix v. 1.7.8 (Vaidya et al., 2011). The final combined alignment was converted to a NEXUS file (.nex) with ClustalX2 v. 1.83 (Thompson et al., 1997) (Miller et al., 2010), with default algorithm and bootstrap iterations were set to 1,000 and substitution model was set to GTRGAMMA + I. Maximum parsimony analysis was implemented in PAUP v. 4.0b10 (Swofford, 2002) through heuristic search with 1,000 random replicates of stepwise addition and tree-bisection-reconnection (TBR) of branchswapping algorithm. Gaps were treated as missing data and max trees was set to 1,000. Branches collapsed when minimum branch length was zero. The consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for the maximum parsimony tree. For the delimitation of new species based on nucleotide comparison, we follow the suggestion of Jeewon and Hyde (2016). The tree topologies were visualized in FigTree v1.4.0 (Rambaut, 2006)
In the phylogenetic analyses (Figure 1), eight genera are included in Ophiocordycipitaceae labeled on the tree. With the exception of Ophiocordyceps, the other remaining genera are monophyletic and individually they received strong statistical support. The three novel entomopathogenic fungi grouped with the taxa in Paraisaria with significant statistical support (1.00 PP/100% ML/98% MP). Paraisaria alba (HKAS 102484) constitutes a sister phylogenetic affiliation to P. yodhathaii with 0.96 PP/98% MP statistical support. Paraisaria rosea (HKAS 102546) is closely related to P. amazonica and P. blattarioides, but this is statistically not supported in all three formats. Two strains of P. arcta grouped as an intermediate clade with close phylogenetic connection to P. coenomyiae, P. gracilioides, and P. heteropoda.

Taxonomy
Paraisaria alba D. P. Wei and K. D. Hyde, sp. nov. Figure 2 Etymology: alba refers to the white fertile head.

Notes
The multigene phylogenetic analysis showed that P. alba groups with P. yodhathaii with fairly good statistical support (0.96 PP/98% MP, Figure 1). This relationship is, however, not supported by the ML analysis. Paraisaria alba differs from P. yodhathaii in having solitary stroma, a white fertile head, and smaller perithecia, asci and secondary ascospores, whereas P. yodhathaii has paired stromata, grayish yellow fertile head, larger perithecia and larger asci and secondary ascospores ( Table 3). The comparison of the nucleotide sequences between P. alba and P. yodhathaii show 10 (including 6 gaps) out of 410 bp (2.4%), 6 out of 746 bp (0.8%), 5 out of 881 bp (0.56%) and 8 out of 534 bp differences (1.5%) in ITS, LSU, TEF1-α The new species generated in this study are in bold. a Ban et al., 2009;b Ban et al., 2015;c Hennings, 1904;d Kobayasi, 1941;e Mains, 1940;f Mongkolsamrit et al., 2019;g Samson and Brady, 1983;h Sanjuan et al., 2015;i Wen et al., 2016. Frontiers in Microbiology | www.frontiersin.org and RPB1 sequences, respectively. SSU and RPB2 sequences data of P. yodhathaii are not available in GenBank. Henceforth, we describe our collection as a new species in Paraisaria according to the guidelines of Jeewon and Hyde (2016).

Notes
Paraisaria arcta resembles P. alba found in Krabi Province, Thailand and P. tettigonia discovered in Guizhou Province, China in having white fertile heads but differs from P. alba in its associated host and number of stromata are distinct from P. tettigonia (Wen et al., 2016). Paraisaria arcta can also be distinguished from the other species in Paraisaria by the color and shape of its fertile head. A conspicuous ravine throughout the center of the fertile head is present in P. arcta, which is lacking in the other species in this genus. The detailed comparisons are shown in Table 3. Multigene phylogenetic analysis showed P. arcta constitutes a distant clade from other species in Paraisaria, with strong statistical support (100% ML, 100% MP, 1.00 PP, Figure 1). Herein, we introduce this collection as a new species of Paraisaria.

Culture characteristics
Culture was made from mycelium inside body of the host larva, slowly growing on PDA, reaching 1.3 cm in diam after incubated at room temperature (25 • C) for 50 days, convex, dense, with undulate edges, smooth surface become filamentous after forming aerial synnemata. The shooting conidia land on the surrounding culture and develop new colonies.

Notes
Paraisaria rosea is closely related to P. amazonica and P. blattarioides, without any statistical support (Figure 1). However, P. rosea can be distinguished from these related species based on the number of stromata, the color of the fertile head and the size of asci and secondary ascospores ( Table 3). The ITS sequence of P. amazonica and P. blattarioides are not available in GenBank database; the nucleotide differences in the TEF1α, RPB1 and RPB2 region between P. rosea and the two above species are greater than 1.5% (Table 4). Thereby, we introduced P. rosea as a new species in this genus based on the distinctive morphology and molecular support.

DISCUSSION
The sexual morph of Paraisaria species phenotypically share an erect or slightly flexuous, cylindrical, colorless, fleshy stipe that terminates in a subglobose to globose fertile head and completely immersed perithecia. Asci are cylindrical with a thickened apical cap. Ascospores are hyaline, multi-septate and usually break into numerous cylindrical, truncated fragments at maturity. However, they can be distinguished according to their associated host, the number of stroma and the color of the fertile head. Species in this genus usually infect several stages of insects, such as larvae of Coleoptera, Diptera, and Lepidoptera; nymphs of Hemiptera and Orthoptera; or adults of Dictyoptera, Hymenoptera (ant) and Orthoptera (Evans et al., 2010;Sanjuan et al., 2015;Mongkolsamrit et al., 2019). According to the number of stromata, species of Paraisaria can be divided into three groups: solitary stroma, paired stromata and multiple stromata (see the key below). The shape of their fertile head features little variation, though differing in color, ranging from white, pale pink, pale rufous, red ochreous to pale orange, chestnut, cinnamon buff, grayish, reddish brown to dark brown (see Table 3).