Chemical pesticides have been used for pest control for many decades, but they cause serious problems, including insecticide resistance, secondary pest resurgence, and negative environmental impacts. Therefore, sustainable alternatives to chemical pesticides are necessary for pest control. Entomopathogenic fungi (EPF) are natural epizootic pathogens of insects, and some of them have been used as microbial biocontrol agents. Herein, we attempted to construct an entomopathogenic fungal library (EFLib) via the “Tenebrio molitor pathogenicity-based fungal collection method (TmPC)” to select EPF for control of the serious agricultural pest Spodoptera litura. A total of 172 soil samples were collected in northern and central Taiwan for the EFLib construction. The isolation efficiency of TmPC was 64.02%. The EFLib consisted of 101 isolates and was designated as the National Chung Hsing University (NCHU) EFLib. Among these isolates, 26 showed high virulence (mortality = 100%) to T. molitor larvae. Based on the results of molecular identification, the highly virulent isolates belonged to seven genera, including Beauveria, Clonostachys, Fusarium, Cordyceps, Penicillium, Purpureocillium, and Metarhizium. To evaluate the potential of these isolates for Spodoptera litura control, 12 isolates were selected for pathogenicity screening against S. litura larvae. A total of six EPF isolates belonging to the genera Beauveria and Metarhizium showed rapid eradication of the S. litura larvae. To rank the potential of these fungal strains for pest management, the six isolates were subjected to thermotolerance and conidial production assays, and a novel effective conidia number (ECN) formula was applied. The results indicated that the ECN index of Beauveria australis (NCHU-113) was much higher than that of Metarhizium isolates. However, among the Metarhizium isolates, NCHU-95 showed the highest ECN index. Altogether, NCHU-69 and NCHU-113 should be further tested in field trials. To our knowledge, this is the first attempt to integrate pathogenicity or virulence and ECN data into EPF screening and ranking, providing a baseline for mass selection of potential EPF strains for further applications.
Since 2016, Apis cerana sacbrood virus (AcSBV) has been recorded in Taiwan. It is epizootic in Apis cerana (Hymenoptera: Apidae) and causing serious loss of A. cerana. Herein, we performed a long-term survey of AcSBV prevalence in the populations of A. cerana in Northern Taiwan from January 2017 to July 2018. The surveillance of AcSBV prevalence in A. mellifera (Hymenoptera: Apidae) populations was starting and further confirmed by sequencing since April 2017; thus, these data were also included in this survey. In our survey, the average prevalence rates of AcSBV were 72 and 53% in A. cerana and A. mellifera, respectively, in 2017, which decreased to 45 and 27% in 2018. For the spatial analysis of AcSBV in two honey bee populations, Hsinchu showed the highest prevalence, followed by New Taipei, Yilan, Taipei, and Keelung, suggesting that AcSBV might have come from the southern part of Taiwan. Interestingly, the AcSBV prevalence rates from A. cerana and A. mellifera cocultured apiaries gradually synchronized. The result of phylogenetic analysis and comparison of the annual AcSBV prevalence in A. cerana-only, A. mellifera-only, and A. cerana/A. mellifera cocultured sample sites indicate cross-infection between A. cerana and A. mellifera; however, AcSBV may lose the advantage of virulence in A. mellifera. The evidence suggested that the transmission of AcSBV might occur among these two honey bee species in the field. Therefore, A. mellifera may serve as a guard species to monitor AcSBV in A. cerana, but the cross-infection still needs to be surveyed.
Sacbrood virus (SBV) was the first identified bee virus and shown to cause serious epizootic infections in the population of Apis cerana in Taiwan in 2015. Herein, the whole genome sequences of SBVs in A. cerana and A. mellifera were decoded and designated AcSBV-TW and AmSBV-TW, respectively. The whole genomes of AcSBV-TW and AmSBV-TW were 8776 and 8885 bp, respectively, and shared 90% identity. Each viral genome encoded a polyprotein, which consisted of 2841 aa in AcSBV-TW and 2859 aa in AmSBV-TW, and these sequences shared 95% identity. Compared to 54 other SBVs, the structural protein and protease regions showed high variation, while the helicase was the most highly conserved region among SBVs. Moreover, a 17-amino-acid deletion was found in viral protein 1 (VP1) region of AcSBV-TW compared to AmSBV-TW. The phylogenetic analysis based on the polyprotein sequences and partial VP1 region indicated that AcSBV-TW was grouped into the SBV clade with the AC-genotype (17-aa deletion) and was closely related to AmSBV-SDLY and CSBV-FZ, while AmSBV-TW was grouped into the AM-genotype clade but branched independently from other AmSBVs, indicating that the divergent genomic characteristics of AmSBV-TW might be a consequence of geographic distance driving evolution, and AcSBV-TW was closely related to CSBV-FZ, which originated from China. This 17-amino-acid deletion could be found in either AcSBV or AmSBV in Taiwan, indicating cross-infection between the two viruses. Our data revealed geographic and host specificities between SBVs. The amino acid difference in the VP1 region might serve as a molecular marker for describing SBV cross-infection.
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