Rapid analysis of insecticidal metabolites from the entomopathogenic fungus Beauveria bassiana 331R using UPLC-Q-Orbitrap MS.

Beauveria bassiana, a representative entomopathogenic fungus, is increasingly being utilized as an eco-friendly pest management alternative to chemical insecticides. This fungus produces a range of insecticidal secondary metabolites that act as antimicrobial and immunosuppressive agents. However, detailed qualitative and quantitative analysis related to these compounds remains scarce, we developed a method for the rapid analysis of these metabolites. Eight secondary metabolites (bassianin, bassianolide, beauvericin, beauveriolide I, enniatin A, A1, and B, and tenellin) were efficiently extracted when B. bassiana-infected Tenebrio molitor larvae were ground in 70% EtOH extraction solvent and subsequently subjected to ultrasonic treatment for 30 min. The eight metabolites were rapidly and simultaneously analyzed using ultra-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap MS). Bassianolide (20.6-51.1 µg/g) and beauvericin (63.6-109.8 µg/g) were identified as the main metabolites in B. basssiana-infected larvae, indicating that they are likely major toxins of B. bassiana. Validation of the method exhibited recovery rates in the range of 80-115% and precision in the range of 0.1-8.0%, indicating no significant interference from compounds in the matrix. We developed a method to rapidly analyze eight insecticidal metabolites using UPLC-Q-Orbitrap MS. This can be extensively utilized for detecting and producing insecticidal fungal secondary metabolites.

Screening of Entomopathogenic Fungal Culture Extracts with Honeybee Nosemosis Inhibitory Activity.

This study aimed to select the most effective culture extracts for controlling honeybee nosemosis using 342 entomopathogenic fungi of 24 species from 18 genera. The germination inhibitory activity of the fungal culture extract on Nosema ceranae spores was evaluated using an in vitro germination assay method. Among 89 fungal culture extracts showing germination inhibitory activity of approximately 80% or more, 44 fungal culture extracts that maintained their inhibitory activity even at a concentration of 1% were selected. Finally, the honeybee nosemosis inhibitory activity was evaluated using the cultured extracts of five fungal isolates having a Nosema inhibitory activity of approximately 60% or more, even when the extract was removed after treatment. As a result, the proliferation of Nosema spores was reduced by all fungal culture extract treatments. However, only the treatment of the culture extracts from Paecilomyces marquandii 364 and Pochonia bulbillosa 60 showed a reduction in honeybee mortality due to nosemosis. In particular, the extracts of these two fungal isolates also increased the survival of honeybees.

Incidence of putative RNA mycoviruses in entomopathogenic fungi in Korea.

Entomopathogenic fungi have potential as biocontrol agents against insect pests, and mycovirus-mediated hypervirulence may enhance their efficacy. Before initiating research on hypervirulence, the presence or absence of double-stranded (ds) RNA elements was determined in 94 Korean entomopathogenic fungi. dsRNA elements varying in size from ca. 0.8 to 7 kbp were found in 14.9% (14/94) of the strains examined, including Beauveria bassiana, Metarhizium pemphigi, M. pinghaense, M. rileyi, and Cordyceps fumosorosea. This study provides information on the incidence and electrophoretic banding patterns of dsRNA elements and is the first report of mycoviruses entomopathogenic fungi in Korea.

The complete mitogenome of the entomopathogenic fungus Metarhizium pinghaense 15R.

In this study, the complete mitogenome of the entomopathogenic fungus Metarhizium pinghaense 15 R, which is highly virulent to aphids and was isolated from Korean soil, was assembled and annotated for three ATP synthase subunits (atp6, atp8, and atp9), three cytochrome oxidase subunits (cox1, cox2, and cox3), apocytochrome b (cob), seven subunits of NADH dehydrogenase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), two ribosomal RNAs (rnl and rns), and 19 tRNA genes. Five genes were carrying a total of eight introns, and they may encode ribosomal protein S3, LAGLIDADG and GIY-YIG endonucleases. Phylogenetic analysis based on the mitochondrial nucleotide sequence confirmed that the M. pinghaense 15 R is a member of the Clavicipitaceae, and is closely related to the species M. anisopliae, M. robertsii, and M. brunneum. The mtDNA base sequence of the M. pinghaense 15 R strain reported in this study is thought to be useful for biological resource genetic data.

Increased survival of the honey bee Apis mellifera infected with the microsporidian Nosema ceranae by effective gene silencing.

This study examined the control of nosemosis caused by Nosema ceranae, one of the hard-to-control diseases of honey bees, using RNA interference (RNAi) technology. Double-stranded RNA (dsRNA) for RNAi application targeted the mitosome-related genes of N. ceranae. Among the various mitosome-related genes, NCER_100882, NCER_101456, NCER_100157, and NCER_100686 exhibited relatively low homologies with the orthologs of Apis mellifera. Four gene-specific dsRNAs were prepared against the target genes and applied to the infected A. mellifera to analyze Nosema proliferation and honey bee survival. Two dsRNAs specifics to NCER_101456 and NCER_100157 showed high inhibitory effects on spore production by exhibiting only 62% and 67%, respectively, compared with the control. In addition, these dsRNA treatments significantly rescued the honey bees from the fatal nosemosis. It was confirmed that the inhibition of Nosema spore proliferation and the increase in the survival rate of honey bees were resulted from a decrease in the expression level of each target gene by dsRNA treatment. However, dsRNA mixture treatment was no more effective than single treatments in the rescue from the nosemosis. It is expected that the four newly identified mitosome-related target genes in this study can be effectively used for nosemosis control using RNAi technology.

Beauveria bassiana for the simultaneous control of Aedes albopictus and Culex pipiens mosquito adults shows high conidia persistence and productivity.

This study was conducted to determine the optimal entomopathogenic fungus for the simultaneous control of the adults of two mosquito species, Aedes albopictus and Culex pipiens. The pathogenicity and virulence against the two species of mosquitoes were evaluated by using 30 isolates of Beauveria bassiana, an entomopathogenic fungus isolated from Korea that has high thermotolerance and UV-B tolerance. Regarding pathogenicity, 23 isolates were pathogenic to Ae. albopictus and 12 isolates were pathogenic to Cx. pipiens; Ae. albopictus adults were more susceptible to B. bassiana than Cx. pipiens adults. Among the isolates, 6 isolates that were simultaneously pathogenic to the two species of mosquitoes were used to evaluate virulence and conidia productivity. B. bassiana CN6T1W2 and JN5R1W1 had higher virulence than the other isolates, and they were more virulent in Ae. albopictus than inCx. pipiens. The conidia productivity of B. bassiana JN5R1W1 on millet grain medium was higher than that of B. bassiana CN6T1W2. Based on these results, B. bassiana JN5R1W1 was selected as the most efficient isolate for the simultaneous control of the two mosquito species. B. bassiana JN5R1W1 can be used effectively in the development of fungal insecticides to simultaneously control Ae. albopictus and Cx. pipiens adults with similar distribution areas.

Baculovirus entire ORF1629 is not essential for viral replication.

It is generally accepted that ORF1629 is essential for baculovirus replication, which has enabled isolation of recombinant viruses in a baculovirus expression system using linearized viral DNA. ORF1629-defective viruses cannot replicate in insect cells; only recombinant virus with complete ORF1629 restoration by recombination can propagate, allowing for pure isolation and the development of bacmids for easy selection of recombinant viruses. We inadvertently found proliferation in insect cells of a bacmid lacking a complete ORF1629. PCR indicated no other viruses but a lack of complete ORF1629 in the proliferated bacmid, suggesting that the baculovirus propagated without a complete ORF1629. Lack of ORF1629 decreased the virus growth rate and yield; it also increased the occlusion body (OB) size but decreased its yield. These results were confirmed for Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV). Thus, entire ORF1629 is not essential for viral replication, though it does affect the virus growth rate, yield, and size and OB production.

Enhanced Production of Recombinant Protein by Fusion Expression with Ssp DnaB Mini-Intein in the Baculovirus Expression System.

The baculovirus expression system (BES) is considered to be a very powerful tool for the expression of numerous difficult to express vertebrate proteins. Ssp DnaB mini-intein is a useful fusion partner for the production of recombinant proteins because it can be self-cleaved by controlling the pH and temperature, without additional treatment. To evaluate the utility of Ssp DnaB mini-intein in the BES, recombinant viruses were generated to express the enhanced green fluorescent protein, the VP2 protein of porcine parvovirus, and the E2 protein of classical swine fever virus fused to a mini-intein. As expected, intracellular self-cleavage of the mini-intein occurred during virus infection, but the cleavage initiation time varied depending on the target protein. Significantly enhanced protein production was observed for all of the target proteins that were fused to the mini-intein. This increase was enough to overcome the decrease in the fusion protein due to intracellular self-cleavage. The mini-intein in all of the recombinant fusion proteins was successfully cleaved by controlling the pH and temperature. These results suggest that the Ssp DnaB mini-intein is a useful fusion partner in the BES for easy purification and enhanced production of target proteins.

Entomopathogenic Fungi as Dual Control Agents against Both the Pest Myzus persicae and Phytopathogen Botrytis cinerea.

The green peach aphid (Myzus persicae), a plant pest, and gray mold disease, caused by Botrytis cinerea, affect vegetables and fruit crops all over the world. To control this aphid and mold, farmers typically rely on the use of chemical insecticides or fungicides. However, intensive use of these chemicals over many years has led to the development of resistance. To overcome this problem, there is a need to develop alternative control methods to suppress populations of this plant pest and pathogen. Recently, potential roles have been demonstrated for entomopathogenic fungi in endophytism, phytopathogen antagonism, plant growth promotion, and rhizosphere colonization. Here, the antifungal activities of selected fungi with high virulence against green peach aphids were tested to explore their potential for the dual control of B. cinerea and M. persicae. Antifungal activities against B. cinerea were evaluated by dual culture assays using both aerial conidia and cultural filtrates of entomopathogenic fungi. Two fungal isolates, Beauveria bassiana SD15 and Metarhizium anisopliae SD3, were identified as having both virulence against aphids and antifungal activity. The virulence of these isolates against aphids was further tested using cultural filtrates, blastospores, and aerial conidia. The most virulence was observed in the simultaneous treatment with blastospores and cultural filtrate. These results suggest that the two fungal isolates selected in this study could be used effectively for the dual control of green peach aphids and gray mold for crop protection.

Efficient Method for the Rapid Purification of Nosema ceranae Spores.

Nosema ceranae is an obligate intracellular fungal parasite that causes mortality in honey bees and enhances the susceptibility of honey bees to other pathogens. Efficient purification of Nosema spores from the midgut of infected honey bees is very important because Nosema is non-culturable and only seasonably available. To achieve a higher yield of spores from honey bees, in this study, we considered that the initial release of spores from the midgut tissues was the most critical step. The use of 2 mm beads along with enzymatic treatment with collagenase and trypsin enhanced the homogenization of tissues and the yield of released spores by approximately 2.95 times compared with the use of common 3 mm beads alone. The optimal time for the enzyme treatment was determined to be 1 hr as measured by the yield and viability of the spores. A one-step filtration using a filter paper with an 8-11 µm pore size was sufficient for removing cell debris. This method may be useful to purify not only N. ceranae spores but also other Nosema spp. spores.

Autographa californica multiple nucleopolyhedrovirus ORF11 is essential for budded-virus production and occlusion-derived-virus envelopment.

UNLABELLED: ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene with unknown function. To determine the role of ac11 in the baculovirus life cycle, an ac11 knockout mutant of AcMNPV, Ac11KO, was constructed. Northern blot and 5' rapid amplification of cDNA ends (RACE) analyses revealed that ac11 is an early gene in the life cycle. Microscopy, titration assays, and Western blot analysis revealed that budded viruses (BVs) were not produced in Ac11KO-transfected Sf9 cells. However, quantitative PCR (qPCR) analysis demonstrated that the deletion of ac11 did not affect viral DNA replication. Furthermore, electron microscopy revealed that there was no nucleocapsid in the cytoplasm or plasma membrane of Ac11KO-transfected cells, which demonstrates that the defect in BV production in Ac11KO-transfected cells is due to the inefficient egress of nucleocapsids from the nucleus to the cytoplasm. In addition, electron microscopy observations showed that the nucleocapsids in the nucleus were not enveloped to form occlusion-derived viruses (ODVs) and that their subsequent embedding into occlusion bodies (OBs) was also blocked in Ac11KO-transfected cells, demonstrating that ac11 is required for ODV envelopment. These results therefore demonstrate that ac11 is an early gene that is essential for BV production and ODV envelopment. IMPORTANCE: Baculoviruses have been extensively used not only as specific, environmentally benign insecticides but also as helper-independent protein expression vectors. Although the function of baculovirus genes in viral replication has been studied by using gene knockout technology, the functions of more than one-third of viral genes, which include some highly conserved genes, are still unknown. In this study, ac11 was proven to play a crucial role in BV production and ODV envelopment. These results will lead to a better understanding of baculovirus infection cycles.

The Autographa californica multiple nucleopolyhedrovirus ORF78 is essential for budded virus production and general occlusion body formation.

ORF78 (ac78) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in the baculovirus life cycle, an AcMNPV mutant with ac78 deleted, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype, indicating that no infectious budded viruses (BVs) were produced. The defect in BV production was also confirmed by both viral titration and Western blotting. However, viral DNA replication was unaffected, and occlusion bodies were formed. An analysis of BVs and occlusion-derived viruses (ODVs) revealed that AC78 is associated with both forms of the virions and is an envelope structural protein. Electron microscopy revealed that AC78 also plays an important role in the embedding of ODV into the occlusion body. The results of this study demonstrate that AC78 is a late virion-associated protein and is essential for the viral life cycle.

Complete genomic sequences and comparative analysis of Mamestra brassicae nucleopolyhedrovirus isolated in Korea.

Mamestra brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) was isolated from naturally infected M. brassicae (Lepidoptera: Noctuidae) larvae in Korea. The full genome sequences of MabrNPV-K1 were determined, analysed and compared to those of other baculoviruses. The MabrNPV-K1 genome consisted of 152,710 bp and had an overall G + C content of 39.9%. Computer-assisted analysis predicted 158 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. Two inhibitor of apoptosis (iap) and six baculovirus repeated ORFs were interspersed in the MabrNPV-K1 genome. The unique MabrNPV-K1 ORF133 was identified in the MabrNPV-K1 genome that was not previously reported in baculoviruses. The gene content and arrangement in MabrNPV-K1 had the highest similarity with those of Helicoverpa armigera MNPV (HearMNPV) and Mamestra configurata NPV-B (MacoNPV-B), and their shared homologous genes were 99% collinear. The MabrNPV-K1 genome contained four homologous repeat regions (hr1, hr2, hr3 and hr4) that accounted for 3.3% of the genome. The genomic positions of the four MabrNPV-K1 hr regions were conserved among those of HearMNPV and MacoNPV-B. The gene parity plot, percent identity of the gene homologues and a phylogenetic analysis suggested that these three viruses are closely related not only to each other but also to the same virus strains rather than different virus species.

Hyper-enhanced production of foreign recombinant protein by fusion with the partial polyhedrin of nucleopolyhedrovirus.

To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused polyhedrin fragment. In addition, when the polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreign recombinant protein by using the partial polyhedrin in baculovirus.

NeuroBactrus, a novel, highly effective, and environmentally friendly recombinant baculovirus insecticide.

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties, such as a higher insecticidal activity and improved recovery, compared to wild-type baculovirus. For the construction of NeuroBactrus, the Bacillus thuringiensis crystal protein gene (here termed cry1-5) was introduced into the Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of the polyhedrin-cry1-5-polyhedrin genes under the control of the polyhedrin promoter. In the opposite direction, an insect-specific neurotoxin gene, AaIT, from Androctonus australis was introduced under the control of an early promoter from Cotesia plutellae bracovirus by fusion of a partial fragment of orf603. The polyhedrin-Cry1-5-polyhedrin fusion protein expressed by the NeuroBactrus was not only occluded into the polyhedra, but it was also activated by treatment with trypsin, resulting in an ∼65-kDa active toxin. In addition, quantitative PCR revealed that the neurotoxin was expressed from the early phase of infection. NeuroBactrus showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the median lethal time against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Rerecombinant mutants derived from NeuroBactrus in which AaIT and/or cry1-5 were deleted were generated by serial passages in vitro. Expression of the foreign proteins (B. thuringiensis toxin and AaIT) was continuously reduced during the serial passage of the NeuroBactrus. Moreover, polyhedra collected from S. exigua larvae infected with the serially passaged NeuroBactrus showed insecticidal activity similar to that of wild-type AcMNPV. These results suggested that NeuroBactrus could be recovered to wild-type AcMNPV through serial passaging.

Fast and efficient generation of recombinant baculoviruses by in vitro transposition.

A novel recombinant bacmid, bEasyBac, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBac, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the non-recombinant background. The bEasyBac bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBac was transposed with pDualBac-EGFP, the resulting recombinant virus, AcEasy-EGFP, showed comparable levels of EGFP expression efficiency to the plaque-purified recombinant virus AcEGFP, which was constructed using the bAcGOZA system. In addition, no non-recombinant backgrounds were detected in unpurified AcEasy-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.

Production of classical swine fever virus envelope glycoprotein E2 as recombinant polyhedra in baculovirus-infected silkworm larvae.

Although, classical swine fever virus (CSFV) envelope glycoprotein E2 subunit vaccine has been developed using the baculovirus expression system, the expression of viral antigens in baculovirus-infected insect cells is often ineffective. Therefore, an alternative strategy to the traditional baculovirus expression system is needed that is more productive and effective. Here, we report a novel strategy for the large-scale production of a CSFV E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg/ml of hemolymph and 0.53 mg/larva at 6-days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedra elicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that this strategy can be used for the large-scale production of CSFV E2 antigen.

Characterization of Beauveria bassiana MsW1 isolated from pine sawyers, Monochamus saltuarius.

Monochamus saltuarius is a vector for pine wilt disease that causes enormous damage to native pine trees in Korea. To develop a biological control method for this pine wilt disease vector, an entomopathogenic fungus was isolated from the cadaver of an adult M. saltuarius supporting fungal conidiation. This fungus was named MsW1 and identified as Beauveria bassiana by microscopic examination, PCR amplification using B. bassiana -specific primers and genetic sequencing of the ITS and EF1-α regions. Virulence tests against M. saltuarius were conducted with conidial suspensions (1 × 10(8) conidia/ml) of B. bassiana MsW1 in laboratory conditions. The median lethal times (LT(50)) of adults and larvae were 7.2 and 7 days, and 100% mortality was observed at 11 and 13 days after inoculation, respectively. This is the first characterization of B. bassiana from M. saltuarius.

Simple purification of a foreign protein using polyhedrin fusion in a baculovirus expression system.

Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures, and that these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with the Polh gene at the N-terminus, including a linker and enterokinase (EK) site between Polh and EGFP, was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells in three steps: cell harvest, sonication, and EK digestion. Through final enterokinase digestion, EGFP presented mainly in the supernatant, and this supernatant fraction also showed a pure EGFP band. These results suggest that a combined procedure of Polh fusion expression and enterokinase digestion can be used for rapid and easy purification of other proteins.