A role of mulberry leaves in improving resistance to virus‐mediated disease in Allomyrina dichotoma

The purpose of this study was to develop a safe and effective method for preventing Allomyrina dichotoma nudivirus (AdNV) infection in the Korean horned beetle, Allomyrina dichotoma, found on the farms in the Republic of Korea. Mulberry leaf powder was added to fermented oak sawdust to minimize mortality in AdNV‐infected A. dichotoma. Mulberry leaves were found to contain 1‐deoxynojirimycin, which has anti‐inflammatory, antiviral, and anti‐tumor effects. Based on the proposed antiviral effects of mulberry leaves, a feed of fermented sawdust combined with 1% or 5% mulberry leaf powder was fed to AdNV‐infected second or third stage A. dichotoma larvae. The larval mortality rate was recorded over 10 weeks. The second and third instar larvae that were fed with the sawdust mixture with 5% mulberry leaf powder had mortality rates of 60% and 30%, respectively. In contrast, the control group that was fed with the sawdust without mulberry leaf powder had a mortality rate of 100%. Also, we confirmed that AdNV was not detected in the experimental group that was subjected to an outdoor application test for 8 months with mulberry leaf powder treatment. A reduced mortality rate after treatment with 1% mulberry leaf powder was observed in the field application. In addition, a comparison of the control colony and mulberry leaf treated group showed a statistical difference in growth of larvae at various states, and demonstrated the efficiency of mulberry leaf powder combined with fermented sawdust for treatment of AdNV‐ infected A. dichotoma.     

The first report on the characterization of a virus isolated from Allomyrina dichotoma in the Republic of Korea

This report reveals the structure of a virus extracted from the Korean horn beetle Allomyrina dichotoma. The purified virus particle was 100% identical to Allomyrina virus lef‐8 sequence registered as KM_233709.1. The structure of this virus was confirmed to be closely related to that of the Nudiviridae family, and it was rod shaped and enveloped, and observed to be of approximately the mean length of a single viral nucleocapsid of 200–210 nm and mean diameter of 100–110 nm. These results provide an insight into the structural characteristics of the Nudiviridae family that can be used for nudiviral identification.     

Effects of solvent fractions of Allomyrina dichotoma larvae through the inhibition of in vitro BACE1 and β‐amyloid(25–35)‐induced toxicity in rat pheochromocytoma PC12 cells

Amyloid‐β peptide (Aβ) generation initiated by β‐site amyloid precursor protein cleaving enzyme 1 BACE1 is a critical cause of Alzheimer's disease. In the course of our ongoing investigation of natural anti‐dementia resources, the ethyl acetate (EtOAc) fraction exerted strong BACE1‐specific inhibition with the half maximal inhibitory concentration (IC₅₀) value of 9.2 × 10^?5 μg/mL. Furthermore, Aβ(25–35)‐induced cell death was predominantly prevented by the EtOAc fraction of Allomyrina dichotoma larvae through diminishing of cellular oxidative stress and attenuating apoptosis by inhibiting caspase‐3 activity. Taken together, the present study demonstrated that A. dichotoma larvae possess novel neuroprotective properties not only via the selective and specific inhibition of BACE1 activity but also through the alleviation of Aβ(25–35)‐induced toxicity, which may raise the possibility of therapeutic application of A. dichotoma larvae for preventing and/or treating dementia.     

Development of a loop‐mediated isothermal amplification assay for rapid and sensitive detection of Sporisorium scitamineum in sugarcane

Smut disease caused by Sporisorium scitamineum is one of the most destructive sugarcane diseases worldwide. The pathogen spreads primarily through infected sugarcane setts, and hence, the use of disease‐free planting materials is essential for preventing disease development in the field. In this study, a species‐specific loop‐mediated isothermal amplification (LAMP) assay was developed for rapid and accurate detection of S. scitamineum. Based on the differences in internal transcribed spacer (ITS) sequences of S. scitamineum, a set of four species‐specific primers, F3, B3, FIP and BIP, were designed by using a panel of fungal and bacterial species as controls. After optimization of the reaction conditions, the detection limit of LAMP assay was about 2 fg of the S. scitamineum genomic DNA in 25 µL reaction solution, 100‐fold lower than that of conventional polymerase chain reaction. The assay showed high specificity to discriminate all S. scitamineum isolates from nine other fungal and bacterial pathogens. The LAMP assay also detected smut infection from young sugarcane leaves with no visible smut‐disease symptoms. The findings from this study provide a simple, highly sensitive, rapid and reliable technique for early detection of S. scitamineum, which may be useful for sugarcane quarantine and production of smut‐free seedcanes. This is the first report of LAMP‐based assay for the detection of S. scitamineum in sugarcane.     

Rapid Detection of Banana Streak Virus by Loop‐mediated Isothermal Amplification Assay in South China

Banana streak virus (BSV) is a significant constraint to banana production and genetic improvement. It is necessary to develop and use BSV detection strategies that are both reliable and sensitive for the management of the virus. A loop‐mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of BSV. Four primers matching a total of six sequences of the conserved ORF III polyprotein genes were synthesized for developing a specific and sensitive LAMP for DNA extracts from field‐infected banana plants. LAMP assay could detect as low as 1 pg/μl template DNA. Test results of all field samples collected from different regions of South China showed that LAMP is more sensitive than PCR. This relatively simple and sensitive technique showed excellent potential with field‐collected samples and for routine screening of tissue culture materials in South China.     

Larval nutritional environment determines adult size in Japanese horned beetles <Emphasis Type="Italic">Allomyrina dichotoma</Emphasis>

The effects of larval nutrition and parental size on offspring horn (male) and body size (male and female) were examined in the Japanese horned beetle Allomyrina dichotoma L. (Coleoptera: Scarabaeidae). Offspring-parent regressions for both horn size and body size of males show no heritable effect, and the magnitudes of these traits were primarily determined by the larval nutritional condition. Male Allomyrina dichotoma also displayed dimorphic horn size-body size allometry, that is, larger males had longer horns relative to their body size and vice versa. Because it has been suggested that males of different body sizes adopt different reproductive tactics, the dimorphic horn size–body size allometry and male reproductive tactics are also a result of the larval environment. Similarly, female body size was determined by larval nutrition, and, thus, larval condition might influence future female fecundity. Females under low nutrition treatment spent longer duration of the third larval instar than females under high nutrition. Females under poor nutrition treatment probably attempted to be as large as possible by the extent of larval duration. Since horn and/or body sizes of males and females affect their fitness, this suggests the evolution of female choice for better oviposition site.     

Effects of single and mixed infections with wild type and genetically modified Helicoverpa armigera nucleopolyhedrovirus on movement behaviour of cotton bollworm larvae

Naturally occurring insect viruses can modify the behaviour of infected insects and thereby modulate virus transmission. Modifications of the virus genome could alter these behavioural effects. We studied the distance moved and the position of virus‐killed cadavers of fourth instars of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) infected with a wild‐type genotype of H. armigera nucleopolyhedrovirus (HaSNPV) or with one of two recombinant genotypes of this virus on cotton plants. The behavioural effects of virus infection were examined both in larvae infected with a single virus genotype, and in larvae challenged with mixtures of the wild‐type and one of the recombinant viruses. An egt‐negative virus variant caused more rapid death and lower virus yield in fourth instars, but egt‐deletion did not produce consistent behavioural effects over three experiments, two under controlled glasshouse conditions and one in field cages. A recombinant virus containing the AaIT‐(Androctonus australis Hector) insect‐selective toxin gene, which expresses a neurotoxin derived from a scorpion, caused faster death and cadavers were found lower down the plant than insects infected with unmodified virus. Larvae that died from mixed infections of the AaIT‐expressing recombinant and the wild‐type virus died at positions significantly lower, compared to infection with the pure wild‐type viral strain. The results indicate that transmission of egt‐negative variants of HaSNPV are likely to be affected by lower virus yield, but not by behavioural effects of egt gene deletion. By contrast, the AaIT recombinant will produce lower virus yields as well as modified behaviour, which together can contribute to reduced virus transmission under field conditions. In addition, larvae infected with both the wild‐type virus and the toxin recombinant behaved as larvae infected with the toxin recombinant only, which might be a positive factor for the risk assessment of such toxin recombinants in the environment.     

Exosome isolation from hemolymph of Korean rhinoceros beetle,Allomyrina dichotoma (Coleoptera: Scarabaeidae)

Exosomes are 30–150 nm vesicles that are secreted from a range of cells. Recently, exosomes have been the subject of considerable research because there is mounting awareness of their diverse functions, including a role in cell–cell communication and presenting pathogens for immune responses. Exosomes contain diverse nucleic acid and protein cargos, derived not only from the organism but also from pathogens, making them suitable for use in disease diagnosis. The Korean rhinoceros beetle, Allomyrina dichotoma (Coleoptera: Scarabaeidae), is commercially reared in Korea for the pet trade and is used in traditional medicine for liver‐related diseases. However, several insect diseases caused by bacteria, fungi and viruses have been reported in A. dichotoma mass‐rearing facilities. Identifying these diseases with accuracy and in a timely manner is of paramount importance. Such diagnosis can be accomplished by identifying the nucleic acid or amino acid fragments from these disease‐causing pathogens in the exosome of A. dichotoma. We isolated exosomes from the hemolymph of A. dichotoma and used them to analyze exosome RNA and proteins. We confirmed the isolation of exosomes through RNA profiling, protein analysis and Western blotting. Our research established a solid foundation for using insect exosome protein and RNA analyses for the accurate diagnosis of insect diseases. To our knowledge, this is the first report of exosome isolation from insect hemolymph.     

Specific and Sensitive Detection of Phytophthora nicotianae by Nested PCR and Loop‐mediated Isothermal Amplification Assays

Phytophthora nicotianae is an important soilborne plant pathogen. It causes black shank in tobacco and other commercially important crop diseases. Early and accurate detection of P. nicotianae is essential for controlling these diseases. In this study, primers based on the Ras‐related protein gene (Ypt1) of P. nicotianae were tested for their specific detection of the pathogen using nested PCR and LAMP assays. For specificity testing, DNA extracts from 47 P. nicotianae isolates, 45 isolates of 16 different oomycetes and 25 isolates of other fungal species were used; no cross‐reaction with other pathogens was observed. The sensitivity assay showed that the nested PCR and LAMP assays had detection limits of 100 fg and 10 fg genomic DNA per 25‐μl reaction, respectively. Furthermore, the nested PCR and LAMP assays were used for the detection of DNA from naturally P. nicotianae‐infected tobacco tissues and soil. Our results suggest that the LAMP assay has the greatest potential for the specific detection of P. nicotianae in regions that are at risk of contracting tobacco black shank disease and that the Ypt1 gene is a novel and effective target of P. nicotianae LAMP visual detection.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Detection of Botrytis cinerea by loop-mediated isothermal amplification

Aims: To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop‐mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. Methods and Results: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross‐reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real‐time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. Conclusions: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. Significance and Impact of the Study: The LAMP method combines the sensitivity and specificity of nucleic acid‐based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on‐site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.     

Nucleopolyhedrovirus infection and/or parasitism by Microplitis pallidipes affected haemolymph titre of 20‐hydroxyecdysone in Spodoptera exigua larvae

This study examined the physiological effects of joint and separate parasitism and infection by the endoparasitoid Microplitis pallidipes Szépligeti and the nucleopolyhedrovirus (NPV), respectively, on haemolymph 20‐hydroxyecdysone (20‐E) titre in Spodoptera exigua (Hübner) larvae. The results indicated that in parasitized larvae, virus‐infected larvae (5.7 × 10³ and 5.7 × 10⁵ OB/ml) and parasitized larvae infected with virus at 5.7 × 10⁵ OB/ml, compared to healthy larvae, the 20‐E all declined during the first 3 days but began to increase from day 4 after treatment, while in jointly parasitized and infected larvae (5.7 × 10³ OB/ml), the 20‐E declined during the first 4 days but began to increase on day 5 after treatment. Meanwhile, compared to parasitized larvae, the 20‐E declined during the first 4 days but significantly increased on day 5 in jointly parasitized and infected larvae (5.7 × 10³ OB/ml), while significantly increased during the first 2 days but began to decrease from day 3 after treatment in jointly parasitized and infected larvae (5.7 × 10⁵ OB/ml). Finally, in larvae that were both parasitized and virus infected (5.7 × 10³ OB/ml), compared to just virus‐infected larvae (5.7 × 10³ OB/ml), the 20‐E was lower on days 3 and 4 but higher on other days after treatment; in larvae that were both parasitized and virus infected (5.7 × 10⁵ OB/ml), compared to just virus‐infected larvae (5.7 × 10⁵ OB/ml), the 20‐E was significantly higher at the first 2 days but lower from day 3 after treatment. Our results revealed that 2nd instar larval M. pallidipes in host bodies may release 20‐E into the haemolymph of S. exigua larvae and that NPV infection may stimulate S. exigua to release more 20‐E during its third to fourth instar larval moulting. We found that this stimulatory effect was greater with higher virus concentrations.     

Rapid and sensitive detection of Acidovorax citrulli in cucurbit seeds by visual loop‐mediated isothermal amplification assay

A rapid, sensitive and visual loop‐mediated isothermal amplification (LAMP) method for detecting Acidovorax citrulli in cucurbit seed was developed in this study. The LAMP primers were designed to recognize the non‐ribosomal peptide synthetase (NRPS) gene (locus tag: Aave_4658) from A. citrulli. The LAMP assay was conducted at 64°C in 1 hr with calcein as an indicator. The sensitivity and specificity of the LAMP assay were further compared with those of a conventional polymerase chain reaction (PCR). The LAMP assay is highly specific to A. citrulli, and no cross‐reaction was observed with other bacterial pathogen. The sensitivity of the LAMP assay was 100‐fold higher than that of conventional PCR with a detection limit of 1 pg of genomic DNA. Using the LAMP assay, 7 of 12 cantaloupe seedlots collected from Xinjiang province were determined to be positive for A. citrulli. In contrast, only 2 of 12 seedlots showed positive for the pathogen with conventional PCR. Moreover, A. citrulli was detected in 100% of artificially infested seedlots with 0.01% infestation or greater. Our results demonstrated that the LAMP assay was simple, visual and sensitive for detecting A. citrulli, especially in seed health testing. Hence, this method has great potential application in routine detecting seed‐borne pathogens and reducing the risk of epidemics.