Molecular record for the first authentication of Isaria cicadae from Vietnam

Abstract The entomopathogenic fungus T011, parasitizing on nymph of Cicada, collected in the coffee garden in Dak Lak Province, Vietnam, was preliminarily morphologically identified as Isaria cicadae, belonged to order Hypocreales and family Clavicipitaceae. To ensure the authenticity of T011, phylogenetic analysis of the concatenated set of multiple genes including ITS, nrLSU, nrSSU, Rpb1, and Tef1 was applied to support the identification. Genomic DNA was isolated from dried sample T011. The PCR assay sequencing was applied to amplify ITS, nrLSU, nrSSU, Rpb1, and Tef1 gene. For phylogenetic analysis, the concatenated data of both target gens were constructed with MEGAX with a 1,000 replicate bootstrap based on the neighbor-joining, maximum likelihood, maximum parsimony method. As the result, the concatenated data containing 62 sequences belonged to order Hypocreales, families Clavicipitaceae, and 2 outgroup sequences belonged to order Hypocreales, genus Verticillium. The phylogenetic analysis results indicated that T011 was accepted at subclade Cordyceps and significantly formed the monophyletic group with referent Cordyceps cicadae (Telemorph of Isaria cicadae) with high bootstrap value. The phylogenetically analyzed result was strongly supported by our morphological analysis described as the Isaria cicadae. In summary, phylogenetic analyses based on the concatenated dataset were successfully applied to strengthen the identification of T011 as Isaria cicadae.

Due to their numerous bioactivities, I. cicadae, as well as C. cicadae, is considered the most valued traditional Chinese medicine. Its medicinal bioactive components, such as adenosine, cordycepin, ergosterol, etc., which have been used to relieve exhaustion remedy, treat numerous diseases, such as antitumor activities, and food source, have been recorded [3,[19][20][21][22]. To obtain precious valued herbal medicine, the exploration and collection of local I. cicadae (C. cicadae) play an important role to apply for further medicinal applications. During our expedition to validate the fungal diversity in Ea Knop Town -Ea Kar District (Latitude: 12°34′26″N-13°02′09″ N; Longitude: 108°22′08″E-108°43′2″E) located in Dak Lak Province, we collected the sample T011, parasitizing on the nymph of Cicada, which was classified and confirmed by the specialist on the entomologist, Faculty of Biotechnology, Ho Chi Minh City Open University, Ho Chi Minh City, Vietnam. In this paper, to ensure the origin and authenticity of T011 as I. cicadae, we conducted the morphology analysis and molecular phylogenetic analysis of the concatenated set genes including ITS, nrLSU, nrSSU, Rpb1, and Tef1.

Sample collection
The specimen T011, parasitizing on the nymph of Cicada, was collected in the coffee garden in Ea Knop Town -Ea Kar District, Dak Lak Province on the morning of June 24, 2018. In the laboratory, the specimen was conditioned to be dried at 60°C and stored for further analysis.

Morphology analysis
Macroscopic characteristics of the fresh body were carefully observed in the many macroscopic characteristics. For the microscopic analysis, a bunch of conidiogenous cells was cut into small species, then, soaked in the water for about 3 min. A sample of the synemata containing the conidiogenous cells was immersed in distilled water for 3 min. Asexual spores were removed using a clean brush. The fertile part was then analyzed under a microscope. Conidia size was recorded. According to the identification of conidia, phialides, and colony coloration, the isolate cultures were grown on YMG media, composed of 4 g/L Yeast extract, 10 g/L Malt extract, 4 g/L Glucose, incubated at 20°C within a period of 20 days.

DNA extraction, PCR amplification, target gene sequencing
Genomic DNA was extracted from dried material by using the phenol/chloroform method (pH = 8). The dried material was added to a lysis buffer (2.0% SDS, Tris-HCl pH 8.0, 150 mM NaCl, 10 mM EDTA, 0.1 mg/mL Proteinase K).
During the incubation at 65°C for overnight, it was mixed thoroughly by inverting the tube several times. Then, the supernatant was collected by centrifugation. About 700 μL of phenol/chloroform/isoamyl alcohol at a ratio of 25  Table 1). About 5 μL aliquots of amplification products were electrophoresed on a 2.0% agarose gel and visualized in a UV transilluminator. The amplified product was sequenced at Nam Khoa (Vietnam) company.

Taxa and ITS, nrLSU, nrSSU, Rpb1, and
Tef1 sequences collection, DNA proofreading, and phylogeny analysis The data set of ITS, nrLSU, nrSSU, Rpb1, and Tef1 sequences were established by sequences downloaded from Genbank (NCBI) and based on the previous data published by Sung et al. (2007) [16]. The ITS, nrLSU, nrSSU, Rpb1, and Tef1 were noted with accession number, name of taxon, and locality. The multiple gene data used in the current study were established based on the combination of ITS, nrLSU, nrSSU, Rpb1, and Tef1 data. The amplified DNA sequences were proofread to remove ambiguous signals at both ends by different software, including Seaview 4.2.12 and Chromas Lite 2.1.1. The phylogenetic tree was constructed based on the neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) by using Molecular Evolutionary Genetics Analysis (MEGA) version X. Additionally, the best evolution model was predicted by using jModelTest.

Morphology analysis
The sample of T011 was collected in the soil of the coffee garden on the morning of June 24, 2018. The synnemata were emerging from the soil, while the host was in the soil. Host: unidentified cicada nymph. Synnemata: presence, branching, 15-60 mm in length × 1.0-2.5 mm in diameter. Synnemata originated from the head of cicada nymphs with the thick layer of mycelia (hiding under the ground). Color: white to cream. Form: simple, erect, and   smaller than those of from synnemata ( Figure 1).

3.3
The systematic concatenated ITS, nrLSU, nrSSU, Rpb1, and Tef1 dataset and phylogeny analysis Total of 62 sequences of ITS, nrLSU, nrSSU, Rpb1, and Tef1 belonged to order Hypocreales, families Clavicipitaceae (served as referent data), and 2 sequences belonged to order Hypocreales, genus Verticillium (served as outgroup) were collected from Genbank and listed in Table 3 and T011 sequence. According to 62 sequences, they were divided into three families (Cordycipitaceae, Clavicipitaceae, Ophiocordycipitaceae), and each family was also divided intro genus, including Cordycipitaceae .040. This model was used to construct phylogenetic trees using maximum likelihood from concatenated data set. Phylogenetic analysis was presented in Figure 2.
To confirm the authenticity of T011 as Isaria cicadae, the construction of ITS, nrLSU, nrSSU, Rpb1, and Tef1- based phylogeny was performed. According to Mitchell et al. (1995), they suggested that molecular phylogenetic approaches to fungal evolution have proved valuable information toward the goals of understanding the relationship among the specific fungal groups [8]. Additionally, the use of fungal molecular data, including ITS, nrLSU, nrSSU, Rpb1, and Tef1, for the identification of fungi ushered in a new era of molecular phylogenetic sequence identification in kingdom Fungi [1,12]. In this study, the combination of ITS, nrLSU, nrSSU, Rpb1, and Tef1 genes were applied to strongly strengthen the identification of T011, which was classified as I. cicadae. According to phylogenetic analysis, phylogenetic analysis of ITS, nrLSU, nrSSU, Rpb1, and Tef1 yielded consistent topology in different taxa of Clavicipitaceae. The phylogenetic position of T011 was obtained and accepted at subclade level: Cordyceps. Notably, within this clade, the highly supported monophyletic group with referent C. cicadae was obtained with high bootstrap value (Bootstrap >95: NJ: 100; MP: 100; ML: 100) and separated this group from other referent taxon in subclade Cordyceps, such as C. ninchukispora, C. pruinosa, and C. kyusyuensis. Additionally, T011 formed the group with referent C. cicadae, Cordyceps sp., and Isaria sp. Among them, Cordyceps sp. and Isaria sp. were proposed using the ancient Chinese name "chanhua" (Cordyceps chanhua) [25]. Therefore, based on the phylogenetic analysis, the T011 was identified as the Isaria cicadae (anamorph of C. cicadae), which was strongly similar to Cordyceps chanhua. Therefore, we have successfully applied the phylogenetic analyses based on the concatenated dataset to strengthen the identification of T011, collected in the local coffee garden in Ea Knop Town -Ea Kar District, as I. cicadae (anamorph of C. cicadae).

Conclusion
We have successfully applied the phylogenetic analysis of multiple genes of ITS, nrLSU, nrSSU, Rpb1, and Tef1 to demimit sample T011, which was collected in Ea Knop Town -Ea Kar District, Đak Lak Province, was strongly supported as Isaria cicadae (anamorph of C. cicadae), which was similar to our preliminary identification. This is the first molecular record of Isaria cicadae in Vietnam.