Use of universal primers for the 18S ribosomal RNA gene and whole soil DNAs to reveal the taxonomic structures of soil nematodes by high-throughput amplicon sequencing.

Nematodes are abundant metazoans that play crucial roles in nutrient recycle in the pedosphere. Although high-throughput amplicon sequencing is a powerful tool for the taxonomic profiling of soil nematodes, polymerase chain reaction (PCR) primers for amplification of the 18S ribosomal RNA (SSU) gene and preparation of template DNAs have not been sufficiently evaluated. We investigated nematode community structure in copse soil using four nematode-specific (regions 1-4) and two universal (regions U1 and U2) primer sets for the SSU gene regions with two DNAs prepared from copse-derived mixed nematodes and whole soil. The major nematode-derived sequence variants (SVs) identified in each region was detected in both template DNAs. Order level taxonomy and feeding type of identified nematode-derived SVs were distantly related between the two DNA preparations, and the region U2 was closely related to region 4 in the non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarity. Thus, the universal primers for region U2 could be used to analyze soil nematode communities. We further applied this method to analyze the nematodes living in two sampling sites of a sweet potato-cultivated field, where the plants were differently growing. The structure of nematode-derived SVs from the two sites was distantly related in the principal coordinate analysis (PCoA) with weighted unifrac distances, suggesting their distinct soil environments. The resultant ecophysiological status of the nematode communities in the copse and field on the basis of feeding behavior and maturity indices was fairly consistent with those of the copse- and the cultivated house garden-derived nematodes in prior studies. These findings will be useful for the DNA metabarcoding of soil eukaryotes, including nematodes, using soil DNAs.

Characterization of Nucleotide Binding -Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses.

Sweetpotato, Ipomoea batatas (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes' expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that IbNBS75, IbNBS219, and IbNBS256 respond to stem nematode infection; Ib-NBS240, IbNBS90, and IbNBS80 respond to cold stress, while IbNBS208, IbNBS71, and IbNBS159 respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

Discovery of a major QTL for root-knot nematode (Meloidogyne incognita) resistance in cultivated sweetpotato (Ipomoea batatas).

KEY MESSAGE: Utilizing a high-density integrated genetic linkage map of hexaploid sweetpotato, we discovered a major dominant QTL for root-knot nematode (RKN) resistance and modeled its effects. This discovery is useful for development of a modern sweetpotato breeding program that utilizes marker-assisted selection and genomic selection approaches for faster genetic gain of RKN resistance. The root-knot nematode [Meloidogyne incognita (Kofoid & White) Chitwood] (RKN) causes significant storage root quality reduction and yields losses in cultivated sweetpotato [Ipomoea batatas (L.) Lam.]. In this study, resistance to RKN was examined in a mapping population consisting of 244 progenies derived from a cross (TB) between 'Tanzania,' a predominant African landrace cultivar with resistance to RKN, and 'Beauregard,' an RKN susceptible major cultivar in the USA. We performed quantitative trait loci (QTL) analysis using a random-effect QTL mapping model on the TB genetic map. An RKN bioassay incorporating potted cuttings of each genotype was conducted in the greenhouse and replicated five times over a period of 10 weeks. For each replication, each genotype was inoculated with ca. 20,000 RKN eggs, and root-knot galls were counted ~62 days after inoculation. Resistance to RKN in the progeny was highly skewed toward the resistant parent, exhibiting medium to high levels of resistance. We identified one major QTL on linkage group 7, dominant in nature, which explained 58.3% of the phenotypic variation in RKN counts. This work represents a significant step forward in our understanding of the genetic architecture of RKN resistance and sets the stage for future utilization of genomics-assisted breeding in sweetpotato breeding programs.

Identification of Sweet Potato Germplasm Resistant to Pathotypically Distinct Isolates of Meloidogyne enterolobii from the Carolinas.

Meloidogyne enterolobii (syn. mayaguensis) is an emergent species of root-knot nematode that has become a serious threat to sweet potato (Ipomoea batatas) production in the southeastern United States. The most popular sweet potato cultivars grown in this region are highly susceptible to M. enterolobii. As a result, this pest has spread across most of the sweet potato growing counties in the Carolinas, threatening the industry as well as other crops in the region. The development and release of new sweet potato cultivars with resistance to M. enterolobii would help to manage and slow the spread of this pest. To support sweet potato resistance breeding efforts, 93 accessions selected from the U.S. Department of Agriculture germplasm collection and breeding programs in the United States were screened to identify 19 lines with strong resistance to M. enterolobii. The resistance in these accessions was tested against two M. enterolobii isolates that were collected from sweet potato production fields in the Carolinas. These isolates were found to have distinct pathotypes, with galling and nematode reproduction differences observed on cotton as well as sweet potato. This study is the first report of intraspecific pathotypic variation in M. enterolobii, and it identifies sweet potato germplasm with resistance against both pathogenic variants of this nematode.

Root-knot nematode genetic diversity associated with host compatibility to sweetpotato cultivars.

Plant parasitic root-knot nematodes (RKN) such as Meloidogyne incognita cause significant crop losses worldwide. Although RKN are polyphagous, with wide host ranges, races with differing host compatibilities have evolved. Associations between genotype and infection phenotype in M. incognita have not yet been discovered. In this study, 48 M. incognita isolates were collected from geographically diverse fields in Japan and their genomes sequenced. The isolates exhibited various infection compatibilities to five sweetpotato (SP) cultivars and were assigned to SP races. Genome-wide association analysis identified 743 SNPs affecting gene coding sequences, a large number of which (575) were located on a single 1 Mb region. To examine how this polymorphic region evolved, nucleotide diversity (Pi) was scanned at the whole genome scale. The SNP-rich 1 Mb region exhibited high Pi values and was clearly associated with the SP races. SP1 and 2 races showed high Pi values in this region whereas the Pi values of SP3, 4, and 6 were low. Principal component analysis of isolates from this study and globally collected isolates showed selective divergence in this 1 Mb region. Our results suggest for the first time that the host could be a key determining factor stimulating the genomic divergence of M. incognita.

Organic mulches reduce crop attack by sweetpotato weevil (Cylas formicarius).

Mulching with organic materials is a management practice with long history for weed suppression, soil water conservation and erosion control. Its potential impact on crop pests is less well explored. Here we report its utility for reducing crop damage by the serious pest, sweetpotato weevil (Cylas formicarius). Laboratory bioassays measured the response of adult female weevils to sweetpotato storage roots beneath mulches of fresh or dried plant materials. Weevils were significant repelled by fresh basil, catnip, basil lime and dry eucalyptus, cypress, lucerne and sugarcane. A subsequent field study found that mulches of dry cypress, eucalyptus and lucerne reduced movement of weevils from a release point to reach sweetpotato plants and lowered level of damage to storage roots. Results demonstrate that mulching with organic materials merits further testing as part of the integrated management of sweetpotato weevil, particularly to protect developing storage roots during dry periods when soil cracking can facilitate access by pests.

Development of molecular markers associated with resistance to Meloidogyne incognita by performing quantitative trait locus analysis and genome-wide association study in sweetpotato.

The southern root-knot nematode, Meloidogyne incognita, is a pest that decreases yield and the quality of sweetpotato [Ipomoea batatas (L.) Lam.]. There is a demand to produce resistant cultivars and develop DNA markers to select this trait. However, sweetpotato is hexaploid, highly heterozygous, and has an enormous genome (∼3 Gb), which makes genetic linkage analysis difficult. In this study, a high-density linkage map was constructed based on retrotransposon insertion polymorphism, simple sequence repeat, and single nucleotide polymorphism markers. The markers were developed using F1 progeny between J-Red, which exhibits resistance to multiple races of M. incognita, and Choshu, which is susceptible to multiple races of such pest. Quantitative trait locus (QTL) analysis and a genome-wide association study detected highly effective QTLs for resistance against three races, namely, SP1, SP4, and SP6-1, in the Ib01-6 J-Red linkage group. A polymerase chain reaction marker that can identify genotypes based on single nucleotide polymorphisms located in this QTL region can discriminate resistance from susceptibility in the F1 progeny at a rate of 70%. Thus, this marker could be helpful in selecting sweetpotato cultivars that are resistant to multiple races of M. incognita.

Population genetic structure of Bemisia tabaci MED (Hemiptera: Aleyrodidae) in Korea.

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a major agricultural pest that causes economic damages worldwide. In particular, B. tabaci MED (Mediterranean) has resulted in serious economic losses in tomato production of Korea. In this study, 1,145 B. tabaci MED females from 35 tomato greenhouses in different geographic regions were collected from 2016 to 2018 (17 populations in 2016, 13 in 2017, and five in 2018) and analyzed to investigate their population genetic structures using eight microsatellite markers. The average number of alleles per population (NA) ranged from 2.000 to 5.875, the expected heterozygosity (HE) ranged from 0.218 to 0.600, the observed heterozygosity (HO) ranged from 0.061 to 0.580, and the fixation index inbreeding coefficient (FIS) ranged from -0.391 to 0.872 over the three years of the study. Some significant correlation (p < 0.05) was present between genetic differentiations (FST) and geographical distance, and a comparatively high proportion of variation was found among the B. tabaci MED populations. The B. tabaci MED populations were divided into two well-differentiated genetic clusters within different geographic regions. Interestingly, its genetic structures converged into one genetic cluster during just one year. The reasons for this genetic change were speculated to arise from different fitness, insecticide resistance, and insect movement by human activities.

Preference of red mite Tetranychus ludeni Zacher (Acari: Tetranychidae) to sweet potato genotypes / Preferência do ácaro vermelho Tetranychus ludeni Zacher (Acari: Tetranychidae) por genótipos de batata-doce

Abstract Tetranychus ludeni damages the sweet potato. Pest development can vary between plant genotypes. The objective was to identify the preference of Tetranychus ludeni for Ipomoea batatas genotypes, from the germplasm bank at the Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM). Natural infestations of this mite were observed on 54 sweet potato genotypes in potted, in a greenhouse. Three mite-infested leafs of each genotype were collected and analyzed. The red mite showed different population density rate in genotypes. The BD 29 genotype was found to be highly susceptible, the BD 08, BD 57, BD 17 and Espanhola genotypes were moderately susceptible, and the others forty-nine genotypes showed low susceptibility to the mite.

Resumo Tetranychus ludeni danifica plantas de batata-doce. O desenvolvimento de pragas pode variar entre genótipos de plantas. O objetivo foi identificar a preferência de T. ludeni para genótipos de Ipomoea batatas do banco de germoplasma da Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM). Infestações naturais deste ácaro foram observadas em 54 genótipos de batata doce plantados em vasos e mantidos em estufa. Três folhas infestadas por ácaros, de cada genótipo, foram coletadas e analisadas. Tetranychus ludeni mostrou diferentes taxas de crescimento populacional entres os genótipos. O genótipo BD 29 foi altamente suscetível, os BD 08, BD 57, BD 17 e Espanhola foram moderadamente suscetíveis e os outros 49 genótipos mostraram baixa suscetibilidade ao ácaro.

Preference of red mite Tetranychus ludeni Zacher (Acari: Tetranychidae) to sweet potato genotypes.

Tetranychus ludeni damages the sweet potato. Pest development can vary between plant genotypes. The objective was to identify the preference of Tetranychus ludeni for Ipomoea batatas genotypes, from the germplasm bank at the Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM). Natural infestations of this mite were observed on 54 sweet potato genotypes in potted, in a greenhouse. Three mite-infested leafs of each genotype were collected and analyzed. The red mite showed different population density rate in genotypes. The BD 29 genotype was found to be highly susceptible, the BD 08, BD 57, BD 17 and Espanhola genotypes were moderately susceptible, and the others forty-nine genotypes showed low susceptibility to the mite.

Transcriptome analysis of root-knot nematode (Meloidogyne incognita)-resistant and susceptible sweetpotato cultivars.

MAIN CONCLUSION: Transcriptome analysis was performed on the roots of susceptible and resistant sweetpotato cultivars infected with the major root-knot nematode species Meloidogyne incognita. In addition, we identified a transcription factor-mediated defense signaling pathway that might function in sweetpotato-nematode interactions. Root-knot nematodes (RKNs, Meloidogyne spp.) are important sedentary endoparasites of many agricultural crop plants that significantly reduce production in field-grown sweetpotato. To date, no studies involving gene expression profiling in sweetpotato during RKN infection have been reported. Therefore, in the present study, transcriptome analysis was performed on the roots of susceptible (cv. Yulmi) and resistant (cv. Juhwangmi) sweetpotato cultivars infected with the widespread, major RKN species Meloidogyne incognita. Using the Illumina HiSeq 2000 platform, we generated 455,295,628 pair-end reads from the fibrous roots of both cultivars, which were assembled into 74,733 transcripts. A number of common and unique genes were differentially expressed in susceptible vs. resistant cultivars as a result of RKN infection. We assigned the differentially expressed genes into gene ontology categories and used MapMan annotation to predict their functional roles and associated biological processes. The candidate genes including hormonal signaling-related transcription factors and pathogenesis-related genes that could contribute to protection against RKN infection in sweetpotato roots were identified and sweetpotato-nematode interactions involved in resistance are discussed.

Developmental and reproductive response of Brachmia macroscopa (Lepidoptera: Gelechiidae) to three host plants.

The sweet potato leaf folder, Brachmia macroscopa Meyrick (Lepidoptera: Gelechiidae), which is a significant pest of plants in the family Convolvulaceae, is rapidly expanding its range in South China and other subtropical regions. Studies were designed to examine the effects of three different host plants (sweet potato, Ipomoea batatas (L.) Lam.; water spinach, I. aquatica Forsskål; and morning glory, Pharbitis purpurea (L.)) on the development and life table parameters of B. macroscopa under laboratory conditions. We found that the intrinsic rates of increase of B. macroscopa were 0.17 ± 0.004, 0.21 ± 0.005 and 0.16 ± 0.004 on I. batatas, I. aquatica and P. purpurea, respectively. The highest net reproduction rate was 158.06 ± 18.22 per female reared on I. aquatica. The larvae had five instars when reared on I. batatas and I. aquatica, but required six instars on P. purpurea. The mean generation lengths of B. macroscopa ranged from 24.32 ± 0.18 days when reared on I. aquatica to 29.40 ± 0.24 days on P. purpurea. The survival of all stage and fecundity curves was intuitively manipulated using the age-stage-structured and two-sex population life table method, to enable comprehensive descriptions of the stage and population trends of B. macroscopa on the three Convolvulaceae plants. Our results indicated that I. batatas and I. aquatica were more suitable host plants than P. purpurea.

Antennal transcriptome and expression analyses of olfactory genes in the sweetpotato weevil Cylas formicarius.

The sweetpotato weevil, Cylas formicarius (Fabricius), is a serious pest of sweetpotato. Olfaction-based approaches, such as use of synthetic sex pheromones to monitor populations and the bait-and-kill method to eliminate males, have been applied successfully for population management of C. formicarius. However, the molecular basis of olfaction in C. formicarius remains unknown. In this study, we produced antennal transcriptomes from males and females of C. formicarius using high-throughput sequencing to identify gene families associated with odorant detection. A total of 54 odorant receptors (ORs), 11 gustatory receptors (GRs), 15 ionotropic receptors (IRs), 3 sensory neuron membrane proteins (SNMPs), 33 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSPs) were identified. Tissue-specific expression patterns revealed that all 54 ORs and 11 antennal IRs, one SNMP, and three OBPs were primarily expressed in antennae, suggesting their putative roles in olfaction. Sex-specific expression patterns of these antenna-predominant genes suggest that they have potential functions in sexual behaviors. This study provides a framework for understanding olfaction in coleopterans as well as future strategies for controlling the sweetpotato weevil pest.

Limited mobility of target pests crucially lowers controllability when sterile insect releases are spatiotemporally biased.

The sterile insect technique (SIT) is a genetic pest control method wherein mass-reared sterile insects are periodically released into the wild, thereby impeding the successful reproduction of fertile pests. In Okinawa Prefecture, Japan, the SIT has been implemented to eradicate the West Indian sweet potato weevil Euscepes postfasciatus (Fairmaire), which is a flightless agricultural pest of sweet potatoes. It is known that E. postfasciatus is much less mobile than other insects to which the SIT has been applied. However, previous theoretical studies have rarely examined effects of low mobility of target pests and variation in the spatiotemporal evenness of sterile insect releases. To theoretically examine the effects of spatiotemporal evenness on the regional eradication of less mobile pests, we constructed a simple two-patch population model comprised of a pest and sterile insect moving between two habitats, and numerically simulated different release strategies (varying the number of released sterile insects and release intervals). We found that spatially biased releases allowed the pest to spatially escape from the sterile insect, and thus intensively lowered its controllability. However, we showed that the temporally counterbalancing spatially biased releases by swapping the number of released insects in the two habitats at every release (called temporal balancing) could greatly mitigate this negative effect and promote the controllability. We also showed that the negative effect of spatiotemporally biased releases was a result of the limited mobility of the target insect. Although directed dispersal of the insects in response to habitats of differing quality could lower the controllability in the more productive habitat, the temporal balancing could promote and eventually maximize the controllability as released insects increased.

Toxicological and biochemical basis of synergism between the entomopathogenic fungus Lecanicillium muscarium and the insecticide matrine against Bemisia tabaci (Gennadius).

The sweetpotato whitefly Bemisia tabaci (Gennadius) was challenged with different combinations of matrine (insecticide) and Lecanicillium muscarium (entomopathogenic fungus). Our results revealed a synergistic relationship between matrine and L. muscarium on mortality and enzyme activities of B. tabaci. To illustrate the biochemical mechanisms involved in detoxification and immune responses of B. tabaci against both control agents, activities of different detoxifying and antioxidant enzymes were quantified. After combined application of matrine and L. muscarium, activities of carboxylestrease (CarE), glutathione-s-transferase (GSTs) and chitinase (CHI) decreased during the initial infection period. Acetylcholinestrase (AChE) activities increased during the entire experimental period, whereas those of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) decreased during the later infection period. The increased mortality and suppression of enzymatic response of B. tabaci following matrine and L. muscarium application suggests a strong synergistic effect between both agents. The strong synergistic effect is possibly related to the disturbance of acetylcholine balance and changes in AchE activities of the whitefly as both matrine and L. muscarium target insect acetylcholine (Ach) receptors which in turn effects AchE production. Therefore, our results have revealed the complex biochemical processes involved in the synergistic action of matrine and L. muscarium against B. tabaci.

Mitochondrial genome of the sweet potato hornworm, Agrius convolvuli (Lepidoptera: Sphingidae), and comparison with other Lepidoptera species.

In the present study, we sequenced the complete mitochondrial genome (mitogenome) of Agrius convolvuli (Lepidoptera: Sphingidae) and compared it with previously sequenced mitogenomes of lepidopteran species. The mitogenome was a circular molecule, 15 349 base pairs (bp) long, containing 37 genes. The order and orientation of genes in the A. convolvuli mitogenome were similar to those in sequenced mitogenomes of other lepidopterans. All 13 protein-coding genes (PCGs) were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which seemed to be initiated by the codon CGA, as observed in other lepidopterans. Three of the 13 PCGs had the incomplete termination codon T, while the remainder terminated with TAA. Additionally, the codon distributions of the 13 PCGs revealed that Asn, Ile, Leu2, Lys, Phe, and Tyr were the most frequently used codon families. All transfer RNAs were folded into the expected cloverleaf structure except for tRNASer(AGN), which lacked a stable dihydrouridine arm. The length of the adenine (A) + thymine (T)-rich region was 331 bp. This region included the motif ATAGA followed by a 19-bp poly-T stretch and a microsatellite-like (TA)8 element next to the motif ATTTA. Phylogenetic analyses (maximum likelihood and Bayesian methods) showed that A. convolvuli belongs to the family Sphingidae.

A myo-inositol-1-phosphate synthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematode resistance in transgenic sweet potato.

Myo-inositol-1-phosphate synthase (MIPS) is a key rate limiting enzyme in myo-inositol biosynthesis. The MIPS gene has been shown to improve tolerance to abiotic stresses in several plant species. However, its role in resistance to biotic stresses has not been reported. In this study, we found that expression of the sweet potato IbMIPS1 gene was induced by NaCl, polyethylene glycol (PEG), abscisic acid (ABA) and stem nematodes. Its overexpression significantly enhanced stem nematode resistance as well as salt and drought tolerance in transgenic sweet potato under field conditions. Transcriptome and real-time quantitative PCR analyses showed that overexpression of IbMIPS1 up-regulated the genes involved in inositol biosynthesis, phosphatidylinositol (PI) and ABA signalling pathways, stress responses, photosynthesis and ROS-scavenging system under salt, drought and stem nematode stresses. Inositol, inositol-1,4,5-trisphosphate (IP3 ), phosphatidic acid (PA), Ca(2+) , ABA, K(+) , proline and trehalose content was significantly increased, whereas malonaldehyde (MDA), Na(+) and H2 O2 content was significantly decreased in the transgenic plants under salt and drought stresses. After stem nematode infection, the significant increase of inositol, IP3 , PA, Ca(2+) , ABA, callose and lignin content and significant reduction of MDA content were found, and a rapid increase of H2 O2 levels was observed, peaked at 1 to 2 days and thereafter declined in the transgenic plants. This study indicates that the IbMIPS1 gene has the potential to be used to improve the resistance to biotic and abiotic stresses in plants.

Applying insecticides through drip irrigation to reduce wireworm (Coleoptera: Elateridae) feeding damage in sweet potato.

BACKGROUND: A 2 year field study was conducted at multiple locations to determine whether insecticides or an entomopathogenic nematode, Steinernema carpocapsae Weiser, applied through drip irrigation in sweet potato reduced wireworm damage when compared with the non-treated check and/or insecticides applied conventionally. RESULTS: Wireworm damage was low in 2012, and there were no differences in the proportion of roots damaged or the severity of damage between treatments. In 2013, a preplant-incorporated (PPI) application of chlorpyrifos followed by either bifenthrin, imidacloprid, clothianidin, or oxamyl injected through drip irrigation significantly reduced the proportion of wireworm damage as well as the severity of wireworm damage when compared with the non-treated check. The incidence and severity of wireworm damage in these treatments did not differ significantly from those in the conventional management practice. The PPI application of chlorpyrifos followed by either cyantraniliprole or S. carpocapsae injected through drip irrigation was not significantly different from the non-treated check in the proportion of wireworm damage; however, both treatments reduced the severity of wireworm damage compared with the non-treated check. CONCLUSION: Applying insecticides through drip irrigation provides an alternative to conventionally applied insecticides. © 2015 Society of Chemical Industry.

Assembling of Holotrichia parallela (dark black chafer) midgut tissue transcriptome and identification of midgut proteins that bind to Cry8Ea toxin from Bacillus thuringiensis.

Holotrichia parallela is one of the most severe crop pests in China, affecting peanut, soybean, and sweet potato crops. Previous work showed that Cry8Ea toxin is highly effective against this insect. In order to identify Cry8Ea-binding proteins in the midgut cells of H. parallela larvae, we assembled a midgut tissue transcriptome by high-throughput sequencing and used this assembled transcriptome to identify Cry8Ea-binding proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). First, we obtained de novo sequences of cDNAs from midgut tissue of H. parallela larvae and used available cDNA data in the GenBank. In a parallel assay, we obtained 11 Cry8Ea-binding proteins by pull-down assays performed with midgut brush border membrane vesicles. Peptide sequences from these proteins were matched to the H. parallela newly assembled midgut transcriptome, and 10 proteins were identified. Some of the proteins were shown to be intracellular proteins forming part of the cell cytoskeleton and/or vesicle transport such as actin, myosin, clathrin, dynein, and tubulin among others. In addition, an apolipophorin, which is a protein involved in lipid metabolism, and a novel membrane-bound alanyl aminopeptidase were identified. Our results suggest that Cry8Ea-binding proteins could be different from those characterized for Cry1A toxins in lepidopteran insects.

Resistance to Ditylenchus destructor Infection in Sweet Potato by the Expression of Small Interfering RNAs Targeting unc-15, a Movement-Related Gene.

Stem nematode (Ditylenchus destructor) is one of most serious diseases that limit the productivity and quality of sweet potato (Ipomoea batatas), a root crop with worldwide importance for food security and nutrition improvement. Hence, there is a global demand for developing sweet potato varieties that are resistant to the disease. In this study, we have investigated the interference of stem nematode infectivity by the expression of small interfering RNAs (siRNAs) in transgenic sweet potato that are homologous to the unc-15 gene, which affects the muscle protein paramyosin of the pathogen. The production of double-stranded RNAs and siRNAs in transgenic lines with a single transgene integration event was verified by Northern blot analysis. The expression of unc-15 was reduced dramatically in stem nematodes collected from the inoculated storage roots of transgenic plants, and the infection areas of their storage roots were dramatically smaller than that of wild-type (WT). Compared with the WT, the transgenic plants showed increased yield in the stem nematode-infested field. Our results demonstrate that the expression of siRNAs targeting the unc-15 gene of D. destructor is an effective approach in improving stem nematode resistance in sweet potato, in adjunct with the global integrated pest management programs.