Development and validation of common data model-based fracture prediction model using machine learning algorithm.

The need for an accurate country-specific real-world-based fracture prediction model is increasing. Thus, we developed scoring systems for osteoporotic fractures from hospital-based cohorts and validated them in an independent cohort in Korea. The model includes history of fracture, age, lumbar spine and total hip T-score, and cardiovascular disease. PURPOSE: Osteoporotic fractures are substantial health and economic burden. Therefore, the need for an accurate real-world-based fracture prediction model is increasing. We aimed to develop and validate an accurate and user-friendly model to predict major osteoporotic and hip fractures using a common data model database. METHODS: The study included 20,107 and 13,353 participants aged ≥ 50 years with data on bone mineral density using dual-energy X-ray absorptiometry from the CDM database between 2008 and 2011 from the discovery and validation cohort, respectively. The main outcomes were major osteoporotic and hip fracture events. DeepHit and Cox proportional hazard models were used to identify predictors of fractures and to build scoring systems, respectively. RESULTS: The mean age was 64.5 years, and 84.3% were women. During a mean of 7.6 years of follow-up, 1990 major osteoporotic and 309 hip fracture events were observed. In the final scoring model, history of fracture, age, lumbar spine T-score, total hip T-score, and cardiovascular disease were selected as predictors for major osteoporotic fractures. For hip fractures, history of fracture, age, total hip T-score, cerebrovascular disease, and diabetes mellitus were selected. Harrell's C-index for osteoporotic and hip fractures were 0.789 and 0.860 in the discovery cohort and 0.762 and 0.773 in the validation cohort, respectively. The estimated 10-year risks of major osteoporotic and hip fractures were 2.0%, 0.2% at score 0 and 68.8%, 18.8% at their maximum scores, respectively. CONCLUSION: We developed scoring systems for osteoporotic fractures from hospital-based cohorts and validated them in an independent cohort. These simple scoring models may help predict fracture risks in real-world practice.

Application of metagenomics for diagnosis of broilers displaying neurological symptoms.

BACKGROUND: Thirty-two-day-old broiler chickens at a farm located in northwestern South Korea displayed adverse neurological symptoms including limping, lying down, and head shaking. Approximately 2.1% of chickens died or were culled due to severe symptoms. Five carcasses were submitted to the Avian Disease Division of the Animal and Plant Quarantine Agency (APQA) for disease diagnosis. RESULTS: Broilers displayed severe pericarditis and perihepatitis associated with gross lesions. Broilers also displayed microscopic lesions in the cerebrum and in the granular layer of the cerebellum, which were associated with multifocal perivascular cuffing and purulent necrosis in the cerebrum, and severe meningitis with heterophil and lymphocyte infiltration. Staphylococcus spp. were identified in the liver and heart using bacteriological culture. PCR/RT-PCR assays revealed that broilers were negative for avian Clostridium botulinum, Newcastle disease virus, and avian encephalomyelitis virus. Bacterial and viral metagenomic analysis of brain sample further revealed the presence of Pseudomonas spp. and Marek's disease virus, which are known etiological agents of chicken meningoencephalitis. CONCLUSIONS: This study reports a diagnostic analysis of gross and histopathological lesions from 32-day-old broilers displaying unique neurological symptoms that revealed the presence of the several neurological diseases including meningoencephalitis. The causative agents associated with meningoencephalitis of broilers that had not been identified by routine diagnostic methods could be diagnosed by metagenomics, which proves the usefulness of metagenomics as a diagnostic tool for unknown neurological diseases in broilers.

Identification of Campylobacter jejuni and Chlamydia psittaci from cockatiel (Nymphicus hollandicus) using metagenomics.

BACKGROUND: In July 2015, the carcasses of 11 cockatiels were submitted for disease diagnosis to the Avian Disease Division of the Animal and Plant Quarantine Agency of Korea. The cockatiels, which appeared dehydrated and underweight, had exhibited severe diarrhea and 22 % mortality over 2 weeks. Traditional diagnosis did not reveal the causes of these symptoms. METHODS: We conducted metagenomics analysis on intestines and livers from the dead cockatiels using Illumina high-throughput sequencing. To obtain more accurate and longer contigs, which are required for further genetic characterization, we compared the results of three de novo assembly tools (metaSPAdes, MEGAHIT, and IDBA-UD). RESULTS: Sequence reads of Campylobacter jejuni (C. jejuni) and Chlamydia psittaci (C. psittaci) were present in most of the cockatiel samples. Either of these bacteria could cause the reported symptoms in psittaciformes. metaSPAdes (ver.3.14.1) identified the 1152 bp flaA gene of C. jejuni and the 1096 bp ompA gene of C. psittaci. Genetic analysis revealed that flaA of C. jejuni was recombinant between C. jejuni and Campylobacter coli, and that ompA of C. psittaci isolated from cockatiel was closely related to strains isolated from humans. CONCLUSIONS: C. jejuni and C. psittaci were detected in cockatiels in the Republic of Korea using metagenomic analysis. This approach is useful for understanding pathogens of pet birds. Three de novo assemblers were compared to obtain accurate contigs from large quantities of reads, and sequences of C. jejuni and C. psittaci generated by metaSPAdes were analyzed.

Gene-disruption of the entomopathogenic fungus Beauveria bassiana incubated with dsRNA.

The species of Beauveria bassiana is widely used for the management of agricultural insect pests. In this study, we integrated egfp-double-stranded RNA (dsRNA) to a previously generated egfp-expressing B. bassiana transformant (Bb-egfp#3) using a protoplast integration method. The Bb-egfp#3 protoplast was mixed with the dsRNA under PEG/CaCl2 conditions and liquid-cultured in Sabouraud dextrose broth for 5 days. A control culture followed the same procedure without dsRNA. Bb-egfp#3/egfp-dsRNA cultures showed very low fungal growth (OD630 = 0.2) compared to the control culture, Bb-egfp#3 only (OD630 = 1.1). Screening of possible transformants on Sabouraud dextrose agar revealed a transformant T3, without egfp signal. T3 was confirmed as B. bassiana through sequencing of conserved genes and insect bioassays. Interestingly, the genomic egfp fragment of T3 was disrupted, and the egfp signal was not detected over four subcultures, which was also confirmed by RNA-seq of Bb-egfp#3 and T3. This study provides an interesting observation that protoplast integration with dsRNA could possibly generate significantly reduced gene expression in B. bassiana and it is stable across several generations.

Beauveria bassiana ERL836 and JEF-007 with similar virulence show different gene expression when interacting with cuticles of western flower thrips, Frankniella occidentalis.

BACKGROUND: Insect-killing fungal species, Beauveria bassiana, is as an environment-friendly pest management tool, and many isolates are on the track of industrialization. However, some of B. bassiana isolates show similar morphology and virulence against insect pests, and so it is hard to differentiate them. Herein we used two patented isolates, ERL836 and JEF-007, and investigated their virulence against western flower thrips, Frankliniella occidentalis, and further analyzed genome structures and transcriptional responses when interacting with cuticles of thrips to see possible differences on the initial step of fungal infection. RESULTS: The two isolates showed no significant differences in fungal growth, conidial production, and virulence against thrips, and they were structurally similar in genome. But, in transcription level, ERL836 appeared to infect thrips easily, while JEF-007 appeared to have more difficulty. In the GO analysis of ERL836 DEGs (differentially expressed genes), the number of up-regulated genes was much larger than that of down-regulated genes, when compared to JEF-007 DEGs (more genes down-regulated). Interestingly, in the enrichment analysis using shared DEGs between two infecting isolates, plasma membrane-mediated transporter activity and fatty acid degradation pathway including cytochrome P450 were more active in infecting ERL836. CONCLUSION: The two B. bassiana isolates had similar morphology and virulence as well as genome structure, but in transcription level they differently interacted with the cuticle of western flower thrips. This comparative approach using shared DEG analysis could be easily applied to characterize the difference of the two B. bassiana isolates, JEF-007 and ERL836.

Use of Metarhizum aniopliae s.l. to control soil-dwelling longhorned tick, Haemaphysalis longicornis.

The longhorned tick (bush tick),Haemaphysalis longicornis (Ixodida: Ixodidae), is a serious pest; it transmits the severe fever with thrombocytopenia syndrome (SFTS) virus to humans and has a wide distribution. The use of chemical control is not favored for environmental and health reasons, so more environmentally sound management methods need to be developed. Herein, we describe the use of an entomopathogenic fungal library to develop a fungus-mediated tick management system. Field-collected nymphs were assayed for their susceptibility to entomopathogenic fungi belonging to genera Beauveria, Metarhizium, Cordyceps, and Akanthomyces. Three M. anisopliae s.l. isolates, JEF-214, -279, and -290 showed high virulence in a dose-dependent manner. One Cordyceps isolate was pathogenic but virulence was much lower than the M. anisopliae isolates. Beauveria isolates were not pathogenic to the tick. Because the longhorned tick dwells on the soil surface except for blood-feeding periods, the soil surface was sprayed with conidial suspensions of the isolates after the release of longhorned ticks. The treatments resulted in 60-90% mortality after 30 days. M. anisopliae s.l. isolates were highly virulent against longhorned tick, and the application of fungus-based biopesticides on the soil surface could be an effective control strategy to reduce the tick population for long-term tick management.

Conidiogenesis-related DNA photolyase gene in Beauveria bassiana.

Beauveria bassiana is an entomopathogenic fungi used in environmentally mindful pest management. Its main active ingredient, conidia, is commercially available as a fungal biopesticide. Many studies of conidia production have focused on how to optimize culture conditions for maximum productivity and stability against unfavorable abiotic factors. However, understanding of how conidiogenesis-related genes provide improved conidial production remains unclear. In this study, we focus on identifying conidiogenesis-related genes in B. bassiana ERL1170 using a random mutagenesis technique. Transformation of ERL1170 using restriction enzyme-mediated integration generated one morphologically different transformant, ERL1170-pABeG #163. The transformant was confirmed to represent B. bassiana, and the binary vector was successfully integrated into the genome of ERL1170. Compared to the wild type, transformant #163 showed very slow hyphal growth and within 6 days only produced <1 × 106 conidia/0.28 cm2 agar block (wild type: 6.2 × 107 conidia/agar block). Transformant #163 also exhibited different morphology than the wild type, including thicker hyphae with some club-shaped parts. In contrast, the typical morphology of wild type B. bassiana exhibits thread-like hyphae and conidiophore structures and circular conidia. To determine the location of the randomly inserted DNA, we conducted thermal asymmetric interlaced (TAIL) PCR and Escherichia coli cloning to clearly sequence the disrupted region. We identified one colony (colony No. 7) with an insertion site identified as DNA photolyase. This was confirmed through a gene knock-out study. It is possible the gene that encodes for DNA photolyase was disrupted during the insertion process and might be involved in fungal conidiogenesis. This work serves as a platform for exploring the function of a variety of B. bassiana genes involved in pest management and their downstream processing.

Characterization of T-DNA insertion mutants with decreased virulence in the entomopathogenic fungus Beauveria bassiana JEF-007.

The bean bug, Riptortus pedestris, is a major agricultural pest that reduces crop quality and value. Chemical pesticides have contributed to pest management, but resistance to these chemicals has significantly limited their use. Alternative strategies with different modes of action, such as entomopathogenic fungi, are therefore of great interest. Herein, we explored how entomopathogenic fungi can potentially be used to control the bean bug and focused on identifying virulence-related genes. Beauveria bassiana (JEF isolates) were assayed against bean bugs under laboratory conditions. One isolate, JEF-007, showed >80 % virulence by both spray and contact exposure methods. Agrobacterium tumefaciens-mediated transformation (AtMT) of JEF-007 generated 249 random transformants, two of which (B1-06 and C1-49) showed significantly reduced virulence against Tenebrio molitor and R. pedestris immatures. Both species were used for rapid screening of virulence-reduced mutants. The two transformants had different morphologies, conidial production, and thermotolerance than the wild type. To determine the localization of the randomly inserted T-DNA, thermal asymmetric interlaced (TAIL) PCR was conducted and analysis of the two clones found multiple T-DNA insertions (two in B1-06 and three in C1-49). Genes encoding complex I intermediate-associated protein 30 (CIA30) and the autophagy protein (Atg22) were possibly disrupted by the T-DNA insertion and might be involved in the virulence. This work provides a strong platform for future functional genetic studies of bean bug-pathogenic B. bassiana. The genes putatively involved in fungal virulence should be experimentally validated by knockdown in future studies.

A novel picorna-like virus, Riptortus pedestris virus-1 (RiPV-1), found in the bean bug, R. pedestris, after fungal infection.

A viral genome was assembled de novo from next-generation sequencing (NGS) data from bean bugs, Riptortus pedestris, infected with an entomopathogenic fungus, Beauveria bassiana (Bb), and was further confirmed via the RACE method. This is a novel insect positive-sense single-stranded RNA virus, which we named Riptortus pedestris virus-1 (RiPV-1) (GenBank accession no. KU958718). The genome of RiPV-1 consists of 10,554 nucleotides (nt), excluding the poly(A) tail, which contains a single large open reading frame (ORF) of 10,371 nt encoding a polyprotein (3456 aa) and flanked by 71 and 112 nt at the 5' and 3' untranslated regions (UTR), respectively. RiPV-1 genome organization from the 5' end contains a consensus organization of picorna-like RNA virus helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp), in addition to two putative structural proteins located at the 3' region and a poly(A) tail at the 3' end. The viral particles were approximately 30nm in diameter with some dispersal distinctive surface projections. Based on the phylogenetic analysis of the RdRp sequences, RiPV-1 was clustered in the unassigned insect RNA viruses with two other viruses, APV and KFV. These three viruses were suggested to constitute a new group of insect RNA viruses. RiPV-1 could be found in all stages of lab-reared bean bugs and was detected abundantly in the thorax, abdomen, midgut and fat body, but not in the reproductive organs and muscle. Interestingly, RiPV-1 replication was increased dramatically in bean bugs 2-6days after fungal infection. In conclusion, a novel insect RNA virus was found by NGS data assembly. This virus can provide further insight into the interaction between virus, fungus and the host.

Histopathological Findings and Metagenomic Analysis of Esophageal Papillary Proliferation Identified in Laying Broiler Breeders.

White or pale-yellow nodules 2-7 mm in length were observed in the esophageal lumen in a number of laying broiler breeders with reduced laying rates. Metaplasia of the mucosal epithelial layer and mucous gland, as well as lymphocyte infiltration under the esophageal mucous gland and epithelial cell layer, were observed, which we found were caused by vitamin A deficiency. In one chicken, however, the stratified squamous epithelial cells of the esophagus were completely replaced by increased numbers of ducts/ductules, lymphocytes, and connective tissue, resulting in a papillary morphology. The ducts were surrounded by a fibrous stroma. Multiple hyperplasia of the esophageal gland was also observed and the esophageal glands were lined by a single layer of columnar cells, and a large number of lymphocytes were infiltrated into the submucosal layer. Based on the gross findings, this papillary proliferation was considered papilloma, but histopathologically, a mass composed of squamous epithelium was not observed. Therefore, the papillary lesion appeared as adenoma with squamous metaplasia of the esophageal gland and ectasia, or mucosal epithelial papillary hyperplasia, associated with chronic esophagitis. A metagenomic analysis of the esophagus samples from this chicken was performed to determine the infectious etiology. No viral cause was identified; however, a contributing role of Bradyrhizobium sp. could not be excluded. In this study, we report the first histopathological examination of a rare case of esophageal papillary proliferation in a chicken and highlight the importance of histopathological results for a definitive diagnosis of such cases.

Genetic Diversity of Fowlpox Virus and Putative Genes Involved in Its Pathogenicity.

To determine the genomic variations of fowlpox virus (FPV)-the largest, very ancient, and still harmful avian virus-the complete genomes of 21 FPVs were analyzed. The genomes showed low genetic diversity relative to their overall size. Our studies revealed that FPVs could phylogenetically be divided into two clades, based on their regional distribution, and comparative analysis showed that 40 putative proteins of FPV were associated with geographic differences in viruses, viral pathogenicity, or the onset of diphtheritic lesions. The strain, classified into a subgroup different from others in the genomic analysis, showed relatively low pathogenicity in chickens, and the onset of diphtheritic lesions was observed to be caused only by the specific strain. Despite genetic differences, some commercial vaccines are protective against virulent strains, and intact reticuloendotheliosis virus inserted into field FPV strains was activated but there was no enhancement of the pathogenicity of FPV. These findings will expand our knowledge of the viral proteome and help us understand the pathogenicity of FPV. IMPORTANCE This study aims at determining molecular candidates using comparative genomics to differentiate between the diphtheritic and cutaneous forms of FPV infection, in addition to their association with the pathogenicity of the virus. Full-genomic analyses of multiple fowlpox strains, including field viruses, isolated between 1960s and 2019, and vaccine strains showed the genetic diversity due to regional differences. Comparative genomic analysis offered the clues related to viral virulence. We believe that our study makes a significant contribution to the literature because we are the first to perform such an elaborate study that compares 21 FPVs to study and highlight their diversity, despite the high level of homology between them. Our results shall help provide insights for tackling FPV that has been taking a toll on the poultry for years now.

Viral Metagenomic Analysis of Japanese Quail (Coturnix japonica) with Enteritis in the Republic of Korea.

We performed viral metagenomics analysis of Japanese quail affected with enteritis to elucidate the viral etiology. Metagenomics generated 21,066,442 sequence reads via high-throughput sequencing, with a mean length of 136 nt. Enrichment in viral sequences suggested that at least three viruses were present in quail samples. Coronavirus and picornavirus were identified and are known as pathogens causing quail enteritis that match the observed morphology. Abundant reads of coronavirus from quail samples yielded four fragment sequences exhibiting six genomes of avian coronavirus. Sequence analysis showed that this quail coronavirus was related to turkey coronavirus and chicken infectious bronchitis virus. Quail picornavirus 8177 bp in size was identified and was similar to the QPV1/HUN/01 virus detected in quails without clinical symptoms in Hungary with 84.6% nucleotide and 94.6% amino acid identity. Our results are useful for understanding the genetic diversity of quail viruses. Further studies must be performed to determine whether quail coronavirus and quail picornavirus are pathogens of the digestive tract of quails.

Artículo regular­Análisis metagenómico viral de la codorniz japonesa (Coturnix japonica) con enteritis en la República de Corea. Se realizó un análisis de metagenómica viral de codornices japonesas afectadas con enteritis para dilucidar la etiología viral. La metagenómica generó 21,066,442 lecturas de secuencia mediante secuenciación de alto rendimiento, con una longitud media de 136 nucleótidos. El enriquecimiento en secuencias virales sugirió que al menos tres virus estaban presentes en las muestras de codorniz. Se identificaron coronavirus y picornavirus que son conocidos como patógenos que causan enteritis de codornices que coinciden con la morfología observada. Las lecturas abundantes de coronavirus de muestras de codorniz produjeron cuatro secuencias de fragmentos que exhibían seis genomas de coronavirus aviar. El análisis de secuencia mostró que este coronavirus de codorniz estaba relacionado con el coronavirus del pavo y con el virus de la bronquitis infecciosa del pollo. Se identificó un picornavirus de codorniz de 8177 pares de bases de tamaño y fue similar al virus QPV1/HUN/01 detectado en codornices sin signos clínicos en Hungría con 84.6% de nucleótidos y 94.6% de identidad de aminoácidos. Estos resultados son útiles para comprender la diversidad genética de los virus de la codorniz. Se deben realizar más estudios para determinar si el coronavirus y el picornavirus de las codornices son patógenos del tracto digestivo de las codornices.

Research Note: Simultaneous detection of infectious laryngotracheitis virus, fowlpox virus, and reticuloendotheliosis virus in chicken specimens.

Infectious laryngotracheitis (ILT), fowlpox (FP), and reticuloendotheliosis are important poultry diseases caused by gallid herpesvirus 1 (ILTV), fowlpox virus (FWPV), and reticuloendotheliosis virus (REV), respectively. Coinfections with ILTV and FWPV occur naturally in chickens, and FP in its more virulent wet form is characterized by diphtheritic lesions and easily confused with ILT. Moreover, the insertion of only partial REV-LTR or a nearly full-length REV into the FWPV genome, located between the ORF 201 and ORF 203, has increased recently in wild-type field FWPV isolates. Therefore, it is critical to detect ILTV, FWPV, REV-integrated FWPV, and REV early and accurately. In this study, we successfully developed a multiplex PCR assay for the simultaneous detection of ILTV, FWPV, REV-integrated FWPV, and REV, and the detection limits was 1 × 54 copies/tube. When used to test clinical samples, the results of the multiplex PCR were in 100% agreement with singleplex PCRs and sequencing. This new multiplex PCR is a simple, rapid, sensitive, specific, and cost-effective method for detection of 4 viruses in clinical specimens.

Transcriptome Analysis of the Japanese Pine Sawyer Beetle, Monochamus alternatus, Infected with the Entomopathogenic Fungus Metarhizium anisopliae JEF-197.

The Japanese pine sawyer (JPS) beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae), damages pine trees and transmits the pine wilt nematode, Bursaphelenchus xylophilus Nickle. Chemical agents have been used to control JPS beetle, but due to various issues, efforts are being made to replace these chemical agents with entomopathogenic fungi. We investigated the expression of immune-related genes in JPS beetle in response to infection with JEF-197, a Metarhizium anisopliae isolate, using RNA-seq. RNA samples were obtained from JEF-197, JPS adults treated with JEF-197, and non-treated JPS adults on the 8th day after fungal treatment, and RNA-seq was performed using Illumina sequencing. JPS beetle transcriptome was assembled de novo and differentially expressed gene (DEG) analysis was performed. There were 719 and 1953 up- and downregulated unigenes upon JEF-197 infection, respectively. Upregulated contigs included genes involved in RNA transport, ribosome biogenesis in eukaryotes, spliceosome-related genes, and genes involved in immune-related signaling pathways such as the Toll and Imd pathways. Forty-two fungal DEGs related to energy and protein metabolism were upregulated, and genes involved in the stress response were also upregulated in the infected JPS beetles. Together, our results indicate that infection of JPS beetles by JEF-197 induces the expression of immune-related genes.

Gene diversity explains variation in biological features of insect killing fungus, Beauveria bassiana.

Beauveria bassiana is a species complex whose isolates show considerable natural genetic variability. However, little is known about how this genetic diversity affects the fungus performance. Herein, we characterized the diversity of genes involved in various mechanisms of the infective cycle of 42 isolates that have different growth rates, thermotolerance and virulence. The analysed genes showed general genetic diversity measured as non-synonymous changes (NSC) and copy number variation (CNV), with most of them being subjected to positive episodic diversifying selection. Correlation analyses between NSC or CNV and the isolate virulence, thermotolerance and growth rate revealed that various genes shaped the biological features of the fungus. Lectin-like, mucin signalling, Biotrophy associated and chitinase genes NSCs correlated with the three biological features of B. bassiana. In addition, other genes (i.e. DNA photolyase and cyclophilin B) that had relatively conserved sequences, had variable CNs across the isolates which were correlated with the variability of either virulence or thermotolerance of B. bassiana isolates. The data obtained is important for a better understanding of population structure, ecological and potential impact when isolates are used as mycoinsecticides and can justify industrialization of new isolates.

Soil Application of Metarhizium anisopliae JEF-314 Granules to Control, Flower Chafer Beetle, Protaetia brevitarsis seulensis.

Root-feeding Scarabaeidae, particularly white grubs are considered among the most harmful coleopteran insect pests in turfgrass. In this work, sixteen entomopathogenic fungal species were assayed against flower chafer beetle, Protaetia brevitarsis (Coleoptera: Scarabaeidae) and Metarhizium anisopliae JEF-314 showed high virulence. The control ability of the isolate JEF-314 has been in detail tested for a model insect flower chafer beetle. Further analyses showed insect stage-dependent virulence where the fungal virulence was the highest against smaller instar larvae. Additionally, we confirmed that millet-based solid cultured granule was effective against the soil-dwelling larval stage. The isolate also showed a similar ability for a representative pest (Popillia spp.) in laboratory conditions. Our results clearly suggest a high potential of M. anisopliae JEF-314 to control the flower chafer beetle, possibly resulting in controlling of root-feeding white grubs in turfgrass. Based on the insect life cycle and susceptibility to the fungus, late spring and summer time would be the optimum time to apply JEF-314 granules for an effective control. Further characterization of the efficacy of the fungus under field conditions against the Scarabaeidae beetles might provide an efficient tool to control this beetle in an environment-friendly way.

Colonization of Metarhizium anisopliae on the surface of pine tree logs: A promising biocontrol strategy for the Japanese pine sawyer, Monochamus alternatus.

We investigated the colonization potential of five Metarhizium anisopliae isolates on pine tree surfaces under laboratory conditions, determined the influence of the pine bark extract on fungal growth and evaluated the insecticidal activity following colonization on the Japanese pine sawyer. Finally, the effect of colonization on adults pine sawyer was evaluated using the top three performing isolates (JEF-197, JEF-271 and JEF-279) under laboratory and field conditions. As a result, isolate JEF-197 showed the highest conidial production on the pine surfaces, and five isolates, including JEF-197, showed higher hyphal growth on autoclaved pine bark extract agar, compared to a water agar. Pine bark treated with the isolates showed 40-70 % mortality of adults pine sawyer. Under mimicked overwintering conditions, in the JEF-197 treatment group, 40 % of the inserted larvae became adults and all were dead after 59 d. In a field test, colonized isolate JEF-197 also showed 37 % insecticidal activity against emerged adults from the pine logs as overwintering sites. This work suggests that M. anisopliae isolate JEF-197 possibly colonized the pine surface and application of a conidial suspension on the pine logs as overwintering sites could be an effective strategy to control the pine sawyer.

Transcriptional response of bean bug (Riptortus pedestris) upon infection with entomopathogenic fungus, Beauveria bassiana JEF-007.

BACKGROUND: Entomopathogenic Beauveria bassiana has been used as a biocontrol agent for insect pests, but its effect at the molecular level on the hosts has not been studied in detail. Herein, we performed transcriptome analysis of bean bug, Riptortus pedestris (Hemiptera: Alydidae) in response to infection with a highly virulent strain of B. bassiana JEF-007 (Bb JEF-007). RESULTS: Based on RNA-seq data from R. pedestris infected with Bb JEF-007 compared with non-infected bean bugs, infection was assumed to strongly activate (i) the energy production pathway by expressing dehydrogenases, (ii) metabolic pathways by expressing secreted proteins, GTPase, MBF2 transcription factor family, pigment-dispersing factor, antioxidants, and cuticle proteins, and (iii) the immune response pathway by expressing serine-threonine kinase in Toll pathway of bean bug. CONCLUSION: We have established the platform for functional studies of the genes required for an immune response against entomopathogenic fungi like B. bassiana in the bean bug, R. pedestris. Moreover, this study also paves the way for genetic modification of B. bassiana to combat with the defense mechanism of R. pedestris. © 2018 Society of Chemical Industry.

Tenebrio molitor Gram-negative-binding protein 3 (TmGNBP3) is essential for inducing downstream antifungal Tenecin 1 gene expression against infection with Beauveria bassiana JEF-007.

The Toll signaling pathway is responsible for defense against both Gram-positive bacteria and fungi. Gram-negative binding protein 3 (GNBP3) has a strong affinity for the fungal cell wall component, ß-1,3-glucan, which can activate the prophenoloxidase (proPO) cascade and induce the Toll signaling pathway. Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in the Toll signaling pathway. In this study, we monitored the response of 5 key genes (TmGNBP3, TmMyD88, and Tenecin 1, 2, and 3) in the Toll pathway of the mealworm Tenebrio molitor immune system against the fungus Beauveria bassiana JEF-007 using RT-PCR. TmGNBP3, Tenecin 1, and Tenecin 2 were significantly upregulated after fungal infection. To better understand the roles of the Toll signaling pathway in the mealworm immune system, TmGNBP3 and TmMyD88 were knocked down by RNAi silencing. Target gene expression levels decreased at 2 d postknockdown and were dramatically reduced at 6 d post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 d post-dsRNA injection. Silencing of TmMyD88 and TmGNBP3 resulted in reduced resistance of the host to fungal infection. Particularly, reducing TmGNBP3 levels obviously downregulated Tenecin 1 and Tenecin 2 expression levels, whereas silencing TmMyD88 expression resulted in decreased Tenecin 2 expression. These results indicate that TmGNBP3 is essential to induce downstream antifungal peptide Tenecin 1 expression against B. bassiana JEF-007.

Genomic Analysis of the Insect-Killing Fungus Beauveria bassiana JEF-007 as a Biopesticide.

Insect-killing fungi have high potential in pest management. A deeper insight into the fungal genes at the whole genome level is necessary to understand the inter-species or intra-species genetic diversity of fungal genes, and to select excellent isolates. In this work, we conducted a whole genome sequencing of Beauveria bassiana (Bb) JEF-007 and characterized pathogenesis-related features and compared with other isolates including Bb ARSEF2860. A large number of Bb JEF-007 genes showed high identity with Bb ARSEF2860, but some genes showed moderate or low identity. The two Bb isolates showed a significant difference in vegetative growth, antibiotic-susceptibility, and virulence against Tenebrio molitor larvae. When highly identical genes between the two Bb isolates were subjected to real-time PCR, their transcription levels were different, particularly in heat shock protein 30 (hsp30) gene which is related to conidial thermotolerance. In several B. bassiana isolates, chitinases and trypsin-like protease genes involved in pathogenesis were highly conserved, but other genes showed noticeable sequence variation within the same species. Given the transcriptional and genetic diversity in B. bassiana, a selection of virulent isolates with industrial advantages is a pre-requisite, and this genetic approach could support the development of excellent biopesticides with intellectual property protection.