Genomic analyses of Purpureocillium lilacinum reveal unique DNA regions for developing isolate-specific molecular markers.

Background: The Purpureocillium lilacinum NCHU-NPUST-175 (Pl-NCHU-NPUST-175) strain has been previously reported as an entomopathogenic fungus (EPF) with potential for controlling Forcipomyia taiwana (little black mosquito). In this study, genome sequencing of Pl-NCHU-NPUST-175 was performed via long-read sequencing (Oxford Nanopore Technologies, ONT). The comparative genomic analysis was conducted to identify isolate- and species-specific DNA regions for developing markers for rapid molecular identification, in order to detect the fungus in an environment and to confirm species identity before practical field applications.

Results: The genome assembly of Pl-NCHU-NPUST-175 is 36.55 Mb, consisting of 13 contigs with a GC content of 58.64%. The largest contig of the Pl-NCHU-NPUST-175 genome was 6.79 Mb, and the N50 value was 4.08 Mb. The genome contains 14,069 putative protein-coding genes, with a gene density of 384.90 genes per Mb and a median number of three exons per gene. Additionally, one mitogenome, Pl-NCHU-NPUST-175, was assembled with a size of 0.23 Mb and contained 16 protein-coding genes. In the Pl-NCHU-NPUST-175 genome, repeat regions accounted for 3.70% of the genome. Most repetitive sequences exhibit a nucleotide sequence divergence of less than 30%. Phylogenetic analysis revealed that Pl-NCHU-NPUST-175 is most closely related to other P. lilacinum strains. To identify distinctive genomic regions (DGRs), a comparative genomics analysis was conducted between Pl-NCHU-NPUST-175, P. takamizusanense and P. lilacinum. A total of 1,154 unaligned fragments were identified by genome-wide alignment, ten DGRs were randomly selected for specific primer design, and the specificity of the primer sets was tested. The results showed that all primer sets could serve as specific molecular markers for Pl-NCHU-NPUST-175. Among the ten primer sets, three primer sets were selected for the soil-sample detection test. The qualitative PCR results indicate that all three isolate-specific primer sets (pl_1, pl_8, and pl_10) detected 10⁵ conidia g^-1 soil in the natural soil samples for up to 14 days. However, qPCR improve the accuracy and sensitivity of detecting Low fungal concentrations 10³ conidia g^-1 soil in complex environmental matrices like soil. These findings suggest that these primers set have potential for future applications in monitoring specific fungal isolates in the field over period of time.

Conclusions: The genome sequence of Pl-NCHU-NPUST-175 was decoded and compared with those of closely related isolates and species. Based on a developed comparative genomics approach, we highlight the feasibility of developing specific molecular markers of EPF, and this method could be further applied to commercialize EPF products to address sustainability in the natural environment.