Detection of Fusarium oxysporum f. sp. fragariae by Using Loop-Mediated Isothermal Amplification.

We developed a loop-mediated isothermal amplification (LAMP) assay for detecting Fusarium oxysporum f. sp. fragariae, the causal agent of wilt in strawberry plants. This assay was based on genomic regions between the portions of transposable elements Han and Skippy of the fungus. The LAMP assay allowed the efficient detection of F. oxysporum f. sp. fragariae DNA by visual inspection, without requiring gel electrophoresis. The detection limit was 100 pg of genomic DNA, which is comparable to that of PCR. The LAMP primers successfully discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and other fungi. The LAMP assay at 63°C, which was found to be the optimal treatment temperature, for 1.5 h successfully detected F. oxysporum f. sp. fragariae California strains GL1270 and GL1385. When the assay was performed using a Genelyzer FIII portable fluorometer, these California strains were successfully detected in 1 h. The assay facilitated the detection of conidia in soil samples after they were precultured on a selective medium for F. oxysporum (FoG2) as well as latent infection in strawberry plants after preculturing. The LAMP assay for visual inspection of DNA required only a heating block and an incubator, reducing the cost of this assay. Thus, it could be suitable for the detection of F. oxysporum f. sp. fragariae strains in centers that store prefoundation and foundation stocks of strawberry, including plant nurseries.

Host plant and humidity effects on phytoseiid mite, Gynaeseius liturivorus (Acari: Phytoseiidae) egg hatchability.

Gynaeseius liturivorus (Ehara) (Acari: Phytoseiidae) is a promising biological control agent for tiny arthropod pests, including Thrips tabaci Lindeman (Thysanoptera: Thripidae) known as a major pest of Welsh onion. In fields during summer, G. liturivorus is observed on soybean, but not on Welsh onion, even when numerous T. tabaci are present. To elucidate G. liturivorus's occurrence on soybean and Welsh onion in relation to relative humidity (RH), we examined its egg hatching on their seedlings under low RH conditions. Then we estimated the moisture transpiration from both plants. Egg hatching occurred only on soybean plants exhibiting greater moisture transpiration. Aiming at utilizing G. liturivorus as a biological control agent for Welsh onion production in summer, evaluation of its relative tolerance and compensation potential for drought injury is necessary. Therefore, we used five phytoseiid species including G. liturivorus and Neoseiulus californicus to estimate the RH and vapor pressure deficit (VPD) at which 50% of eggs hatch (RH50 and VPD50). Furthermore, we examined G. liturivorus and N. californicus egg hatching under different RH oscillation conditions. Results show G. liturivorus as the most drought-sensitive among the five species tested, but G. liturivorus is able to compensate for lethal low-RH effects on egg hatching in part by periodic exposure to high RH conditions, as observed for N. californicus.

Molecular monitoring of Neoseiulus californicus released from sheltered slow-release sachets for spider mite control in a Japanese pear greenhouse.

A novel system for spider mite control was developed with a slow-release sachet containing Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) protected by a waterproof shelter. Monitoring the efficacy of the predator release system for spider mite control at a Japanese pear greenhouse requires discrimination of N. californicus from other indigenous phytoseiid mite species inhabiting the study site and subsequent identification of the released N. californicus. The report of our earlier study described a PCR-based method for discrimination of N. californicus species. For the present study, we first examined phytoseiid mite species composition in the greenhouse. Subsequently, we developed microsatellite markers to identify the released N. californicus. Finally, we installed the predator release system in the greenhouse and conducted a population survey of phytoseiid and spider mites. Results demonstrated that approximately 1 month is necessary for distribution of the released N. californicus on the leaves.

Management of apple orchards to conserve generalist phytoseiid mites suppresses two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae).

To improve the success of integrated pest management (IPM) in apple orchards, we investigated whether generalist phytoseiid mites have suppressed the occurrence of Tetranychus urticae. In Akita Prefecture, northern Japan, in 2012 and 2013, two types of experimental plot were compared. Conservation plots had been managed for the conservation of generalist phytoseiid mites by selective chemical spraying without mowing since 2009. Conventional plots were managed by non-selective chemical spraying with regular mowing. The conservation plots had significantly fewer T. urticae adult females per tree in both years. Two species of generalist phytoseiid mites-Typhlodromus vulgaris and Amblyseius tsugawai-were continuously present in the conservation plots, with only a few T. urticae. The conservation plots had significantly more A. tsugawai adult females in the undergrowth in both years, and significantly more T. vulgaris adult females on apple leaves in 2012. Typhlodromus vulgaris was continuously present in the conservation plots but was scarce from late May to early August in the conventional plots. In the presence of T. vulgaris, low numbers of T. urticae did not increase on apple leaves. These results indicate that the generalist phytoseiid mites serve as important biological control agents in IPM in apple orchards.

Estimation of soil-to-plant transfer factors of radiocesium in 99 wild plant species grown in arable lands 1 year after the Fukushima 1 Nuclear Power Plant accident.

One year after the deposition of radionuclides from the Fukushima 1 Nuclear Power Plant (A formal name is Fukushima Daiichi Nuclear Power Station) in March 2011, radiocesium (¹³4Cs, ¹³7Cs) concentrations ([Cs]) were comprehensively investigated in the wild plants of 99 species most of which were annual or summer green perennial herbs and started to grow from April 2012 at the heavily contaminated fields of paddy (three study sites) and upland (one study site) in Fukushima Prefecture. The survey was conducted three times (April, July and October) in the year. In each site, soils (soil cores of 5-cm depth) and plants (aerial shoots) were collected for determination of [Cs] on a dry weight basis, and then the transfer factor (TF) of radiocesium from soil to plant ([Cs]plant/[Cs]soil) was estimated in each species. The [Cs] values of both soils and plants largely varied. However, some species exhibited relatively high TF values (more than 0.4) (e.g., Athyrium yokoscense, Dryopteris tokyoensis, and Cyperus brevifolius), while others exhibited almost negligible values (less than 0.01) (e.g., Salix miyabeana, Humulus scandens, and Elymus tsukushiensis). In addition, judging from the 11 species grown in both paddy and upland fields, TF values were generally higher in the paddy fields. The estimation of phytoextraction efficiency of soil radiocesium by weed communities in the paddy fields suggests that the weed community is not a practical candidate for phytoremediation technique.

Genomic analyses of sodium channel α-subunit genes from strains of melon thrips, Thrips palmi, with different sensitivities to cypermethrin.

We examined the genomic organization of the sodium channel α-subunit gene in two strains of melon thrips, Thrips palmi, having differing sensitivity to cypermethrin. The nucleotide sequences of the strains included 18 or 16 putative exons which covered the entire coding region of the gene producing 2039 amino acid residues. Deduced amino acid sequences of both strains showed 80% homology with those of Periplaneta americana and Cimex lectularius. Comparison of deduced amino acid sequences of both strains showed no consistent amino acid difference. In addition to the previously reported resistant amino acid (Ile) at the T929I site, both strains encoded another resistant amino acids at two positions which are involved in pyrethroid resistance in other arthropods. These amino acids might also involve in the basal levels of resistance to pyrethroids of both strains.

Spinosad resistance of melon thrips, Thrips palmi, is conferred by G275E mutation in α6 subunit of nicotinic acetylcholine receptor and cytochrome P450 detoxification.

To examine the resistance mechanisms of Thrips palmi against spinosad, we cloned partial nucleotide sequences of the nicotinic acetylcholine receptor α6 subunit (TPα6) gene from susceptible (OK) and resistant (TS1 and TS5) strains and compared the deduced amino acid sequences among the three strains. The OK, TS1, and TS5 strains respectively showed LC50 values of 3.4mg/L, 2838.5mg/L, and 6655.5mg/L. The deduced amino acid sequence of TPα6 gene showed 96% identity with that of Frankliniella occidentalis. Comparison of the deduced amino acid sequences of TPα6 gene among the three strains showed that the TS1 and TS5 strains had a resistant amino acid, Glu, at amino acid position 275. On the other hand, a susceptible amino acid, Gly, was encoded at the corresponding amino acid position for the OK strain. The synergist, piperonyl butoxide, respectively caused 1.1-fold , 5.8-fold , and 9.0-fold decreases in the resistance ratios of the OK, TS1, and TS5 strains. These results suggest that spinosad resistance of T. palmi is conferred by reduced sensitivity of TPα6 and cytochrome P450-mediated detoxification.

Population survey of phytoseiid mites and spider mites on peach leaves and wild plants in Japanese peach orchard.

A population survey of phytoseiid mites and spider mites was conducted on peach leaves and wild plants in Japanese peach orchards having different pesticide practices. The phytoseiid mite species composition on peach leaves and wild plants, as estimated using quantitative sequencing, changed during the survey period. Moreover, it varied among study sites. The phytoseiid mite species compositions were similar between peach leaves and some wild plants, such as Veronica persica, Paederia foetida, Persicaria longiseta, and Oxalis corniculata with larger quantities of phytoseiid mites, especially after mid-summer. A PCR-based method to detect the ribosomal ITS sequences of Tetranychus kanzawai and Panonychus mori from phytoseiid mites was developed. Results showed that Euseius sojaensis (specialized pollen feeder/generalist predator) uses both spider mites as prey in the field.

Field survey of reproductive modes and sodium channel mutations associated with pyrethroid resistance in Thrips tabaci.

Using PCR-Restriction Fragment Length Polymorphism (RFLP) with mitochondrial cytochrome c oxidase subunit I sequences, we examined the reproductive modes of female adults of Thrips tabaci collected at 54 sites across Japan. Results showed the presence of heteroplasmic insects harboring mitochondria associated with arrhenotoky and thelytoky. Using the insects, we also applied PCR-RFLP to examine the genotypes for the amino acid mutation (T929I) site involved in pyrethroid resistance. Findings showed the presence of thelytokous heterozygotes under the circumstance that most arrhenotokous insects are resistant homozygotes, and many thelytokous insects are susceptible homozygotes. These results suggest that, in the field, genetic exchange occurs between insects through of both reproductive modes. A survey of the genotypes for the other amino acid mutations using nucleotide sequencing showed a decline of insects with an M918T and L1014F pair and an increase of insects with M918L. These results suggest the evolutional progression of amino acid mutations associated with pyrethroid resistance in T. tabaci.

Evolutionary origin and distribution of amino acid mutations associated with resistance to sodium channel modulators in onion thrips, Thrips tabaci.

In onion thrips Thrips tabaci, reduced sensitivity of the sodium channel caused by several sodium channel mutations have been correlated with pyrethroid resistance. For this study, using mitochondrial cytochrome c oxidase subunit I gene sequences, we examined the phylogenetic relation among a total of 52 thelytokous and arrhenotokous strains with different genotypes of the sodium channel mutations. Then, we used flow cytometry to estimate their ploidy. Results showed that the strains are divisible into three groups: diploid thelytoky, triploid thelytoky, and diploid arrhenotoky. Using 23 whole genome resequencing data obtained from 20 strains out of 52, we examined their genetic relation further using principal component analysis, admixture analysis, and a fixation index. Results showed that diploid and triploid thelytokous groups are further classifiable into two based on the sodium channel mutations harbored by the respective group members (strains). The greatest genetic divergence was observed between thelytokous and arrhenotokous groups with a pair of T929I and K1774N. Nevertheless, they shared a genomic region with virtually no polymorphism around the sodium channel gene loci, suggesting a hard selective sweep. Based on these findings, we discuss the evolutionary origin and distribution of the sodium channel mutations in T. tabaci.

Diversity and composition of flower-visiting insects and related factors in three fruit tree species.

Animal-mediated pollination is an essential ecosystem service for the production of many fruit trees. To reveal the community composition of flower-visiting wild insects which potentially contribute to fruit production and to examine the effects of geographic location, local meteorological conditions and locally introduced domesticated pollinators on them, we investigated the community composition of insects visiting the flowers (hereafter, "visitors") of apple, Japanese pear and Oriental persimmon for 1‒3 years at 20 sites around Japan. While most of the variation (82%) of the community composition was explained by tree species with a slight contribution by geographic distance (2%), maximum temperature and tree species contributed 62% and 41% of the variation in total abundance of the visitors, respectively. Though the dominant families of the visitors varied spatiotemporally, the community composition of the visitors of apple and Japanese pear clearly differed from that of Oriental persimmon. While Andrenidae and Syrphidae together accounted for 46%‒64% of the visitors of apple and Japanese pear, Apidae represented 57% of the visitors of Oriental persimmon. The taxonomic richness, diversity and evenness of the visitors were best predicted by locally introduced domesticated pollinators and local meteorological conditions of wind speed and maximum temperature. Amongst these selected factors, locally introduced domesticated pollinators could have the largest impact. It seemed to be strongly related to the reduction of taxonomic richness, diversity and evenness of the visitors, accounting for 41‒89% of the variation. Results suggested that the community composition and total abundance of potential pollinators were predominantly determined by tree species and temperature, but locally introduced domesticated pollinators could have a determinantal pressure on the taxonomic diversity of the community.

Phytoseiid mite species composition in Japanese peach orchards estimated using quantitative sequencing.

We attempted a population survey of spider mites and phytoseiid mites in Japanese peach orchards with different pesticide practices; however, we had difficulty discriminating phytoseiid mites. To estimate phytoseiid mite species composition, ribosomal gene fragments were amplified from genomic DNA of five phytoseiid mite species using PCR. Cloning and nucleotide sequencing of amplified fragments identified species-specific polymorphic sites. Newly amplified fragments from recombinant plasmids were mixed in various ratios to produce standard DNA template mixtures. After direct sequencing, the signal ratios between two nucleotides at each species-specific polymorphic site were calculated and shown against the corresponding expected ratios. Quadratic regression equations were used to estimate the phytoseiid mite species composition. Results showed that the phytoseiid mite species composition changed during the survey period and varied among study sites.

Estimating the proportion of resistance alleles from bulk Sanger sequencing, circumventing the variability of individual DNA.

Specimens should be examined as much as possible to obtain a precise estimate of the proportion of resistance alleles in agricultural fields. Monitoring traps that use semiochemicals on sticky sheets are helpful in this regard. However, insects captured by such traps are ordinarily left in the field until collection. Owing to DNA degradation, the amount of DNA greatly varies among insects, causing serious problems in obtaining maximum likelihood estimates and confidence intervals of the proportion of the resistance alleles. We propose a statistical procedure that can circumvent this degradation issue. R scripts for the calculation are provided for readers. We also propose the utilization of a Sanger sequencer. We demonstrate these procedures using field samples of diamide-resistant strains of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). The validity of the assumptions used in the statistical analysis is examined using the same data.

Sodium channel mutations (T929I and F1534S) found in pyrethroid-resistant strains of the cigarette beetle, Lasioderma serricorne (Coleoptera: Anobiidae).

RNA-seq data analysis of cigarette beetle (Lasioderma serricorne) strains having different sensitivities to pyrethroids identified sodium channel mutations in strains showing pyrethroid resistance: the T929I and F1534S mutations. These results suggest that reduced sensitivity of the sodium channel confers the pyrethroid resistance of L. serricorne. Results also showed that the F1534S mutation mostly occurred concurrently with the T929I mutation. The functional relation between both mutations for pyrethroid resistance is discussed.

Origin, selection, and spread of diamide insecticide resistance allele in field populations of diamondback moth in east and southeast Asia.

BACKGROUND: The investigation of molecular mechanisms and evolution of resistance to insecticides is an ongoing challenge, as researchers must provide guidance to manage the resistance to achieve sustainable production in agriculture. Predicting, monitoring, and managing insecticide resistance requires information on the origins, selection, and spread of resistance genes. The resistance of Plutella xylostella (L.) against diamide insecticides is becoming an increasingly severe problem in east and southeast Asia. In this study, the evolution of resistance was investigated using a resistance allele [ryanodine receptor (RyR); G4946E mutation] and its flanking regions, as well as mitochondrial cytochrome c oxidase subunit I (mtCOI). RESULTS: The sequences of the flanking region of the G4946E and mtCOI suggested that the G4946E mutation has a key role in resistance. Furthermore, the G4946E mutation has multiple origins, and congenic resistant mutations have spread across east and southeast Asia, despite substantial geographical barriers. In addition, the susceptibility of field populations partially recovered during winter, based on the observed decrease in the G4946E (resistant allele) frequency. Finally, the resistance level indexed by the frequency of the E4946 allele was significantly lower in non-overwintering regions than in overwintering regions. CONCLUSION: The information of the present study is useful to monitor resistance using molecular markers and to develop strategies to delay the evolution of diamide resistance.

Characterization of alternatively spliced transcripts encoding heat shock transcription factor in cultured cells of the cabbage armyworm, Mamestra brassicae.

A gene encoding heat shock transcription factor (HSF) was cloned and sequenced from cultured cells of the cabbage armyworm, Mamestra brassicae. The cDNA potentially encoded a 699-aa protein, with a calculated molecular weight of 77.8 kDa. Deduced amino acid identities to HSFs from Aedes aegypti and Drosophila melanogaster were 36 and 34%, respectively. Analysis of the genomic DNA revealed eight exons and three optional exons: a, b, and c. Exon a contained a premature in-frame stop codon that would generate a truncated protein. When the cells were exposed to high temperature or cadmium, no particular alternative transcripts showed significant up- or down-regulated expression relative to the total amount of the transcripts. These results suggest that alternative splicing may not be a principal mechanism for regulation of M. brassicae HSF gene expression in response to heat shock and cadmium.

Field survey of ryanodine receptor mutations (G4946E and I4790K) and their effects on biotic performance in the diamondback moth.

This study examined changes in the proportions of ryanodine receptor mutations (G4946E and I4790K) for Plutella xylostella populations in the field. Results show that the proportion of G4946E decreased during the survey years without diamide application and that insects with I4790K were observed less frequently during the survey period. This study also examined the biotic performances of diamide-resistant P. xylostella strains. The results show that the diamide-resistant strains exhibited hatchability, larval development, and fecundity equivalent to those of diamide-susceptible strains.

Ryanodine receptor mutations (G4946E and I4790K) differentially responsible for diamide insecticide resistance in diamondback moth, Plutella xylostella L.

This study examined diamondback moth (Plutella xylostella) strains showing high-level resistance to cyantraniliprole (KA17 strain) and to flubendiamide and chlorantraniliprole (KU13 strain). The LC50 value of the KA17 strain against cyantraniliprole was ca. 100-fold higher than that of the KU13 strain. The KA17 strain also exhibited high-level resistance to chlorantraniliprole and flubendiamide equivalent to those in the KU13 strain. The KU13 strain showed a higher LC50 value against cyantraniliprole than the susceptible strains. However, the LC50 value of the KU13 strain against cyantraniliprole was below the agriculturally recommended concentration. Subsequent QTL analysis using ddRAD-seq identified the resistance responsible regions of the KA17 and KU13 strains with different diamide resistance profiles. Ryanodine receptor (RyR) gene was included in the identified regions. Single nucleotide polymorphism calling in the RyR gene using RNA-seq found previously reported G4946E (amino acid mutation from glycine to glutamic acid at amino acid position 4946) and novel I4790K (amino acid mutation from isoleucine to lysine at amino acid position 4790) mutations, respectively, in the RyR of the KU13 and KA17 strains. Functional significance of I4790K in the resistance was confirmed in calcium imaging of the human embryonic kidney 293T cell line expressing Bombyx mori RyR with the mutation. This reporting is the first describing I4790K as a fundamental mechanism responsible for the resistance to the diamides including cyantraniliprole. From this study, we also report up-regulated expression of some degradation enzymes and that of the RyR gene in the KA17 and KU13 strains based on results of RNA-seq data analysis.

Long-term field insecticide susceptibility data and laboratory experiments reveal evidence for cross resistance to other neonicotinoids in the imidacloprid-resistant brown planthopper Nilaparvata lugens.

BACKGROUND: Long-term monitoring data is helpful to understand the fluctuation of susceptibility and pattern of cross resistance in insecticide resistance management. After the occurrence of imidacloprid resistance, the brown planthopper (BPH) has gradually developed resistance to thiamethoxam and clothianidin since 2010, but not to dinotefuran and nitenpyram. Here, we analyzed susceptibilities data of five neonicotinoids during 2005-2017 in East Asia and Vietnam to conduct cross-resistance patterns among neonicotinoids. To determine the factors of development of cross resistance in laboratory bioassays, we used the imidacloprid resistant and control strains that were selected from filed populations in the Philippines and Vietnam. RESULTS: The Linear Mixed Models (LMM) analyses of insecticide susceptibility data showed that the slope values of imidacloprid resistance effects were 0.68 and 1.09 for resistance to thiamethoxam and clothianidin, respectively. Laboratory bioassay results showed that the LD50 values for thiamethoxam and clothianidin in resistant strains (1.4-5.5 µg g-1 ) were 3.2-16.4 times higher than those in the control strains (0.28-1.5 µg g-1 ). However, the increase in the LD50 values for imidacloprid was not related to that for dinotefuran and nitenpyram based on the results of the LMM analysis and laboratory bioassay. CONCLUSION: Our results demonstrate that the development of imidacloprid resistance result in strong-cross resistance to some neonicotinoids, thiamethoxam and clothianidin, but not to others, dinotefuran and nitenpyram. We anticipate that our findings will be a starting point for understanding mechanism of the different trend of cross resistance by analyzing long-term susceptibility data and laboratory bioassays in insect pests. © 2019 Society of Chemical Industry.

Alternatively spliced sodium channel transcripts expressed in field strains of the diamondback moth.

The frequencies of the L1014F and T929I mutations, both of which are involved in nerve insensitive resistance to pyrethroids, were examined in field and laboratory strains of the diamondback moth, Plutella xylostella at DNA and RNA levels. Results showed that the resistance allele frequencies at the L1014F and T929I sites in the field strains were respectively, 82.8-100% and 72.9-94.4%. No posttranscriptional regulation of the L1014F mutation was observed. The examined insects were classifiable into four groups according to the expression patterns of mutually exclusive exons 18a and 18b. Most insects in the field strains expressed transcripts containing exon 18b more abundantly than those containing exon 18a, although both transcripts were expressed with similar proportions in all insects of the laboratory strains. Some other insects expressed a chimeric transcript comprising parts of exons 18a and 18b. Deduced amino acid sequences of the chimeric transcript encoded amino substitution from Met to Ile at the site corresponding to the super-kdr mutation (M918T) in Musca domestica. The frequencies of the M918I mutation in the field strains were 5.0-19.4%. Analyses of the genomic organization revealed that the chimeric sequences are encoded in the genome.