In our previous publication, we have tentatively concluded that our fungal specimen DL0038A and DL0038B are Cordyceps takaomontana. In order to further support the identification, we continued to analyse the nrSSU (nuclear ribosomal small subunit) and rpb1 (largest subunit of RNA polymerase II) genes, as well as combined analysis the nrLSU (largest subunit of RNA polymerase II) together with nrSSU and rpb2 genes on these specimens in order to support the morphological identification of those fungi. The results show that we successfully amplified all genes. Sequencing method was then adopted and proofread before molecular phylogenetic analysis was applied with reference sequences obtained from the publication of Sung et al. (2007). Once again, this analysis strongly supports the DL0038A and DL0038B specimen as Cordyceps takaomontana.
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.
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