Integrated analysis of the lncRNA/circRNA-miRNA-mRNA expression profiles reveals novel insights into potential mechanisms in response to root-knot nematodes in peanut.

BACKGROUND: Peanut is the most essential oil and food crop globally due to its high oil and protein content. Root-knot nematode infects peanut roots, causing poor development and severely limiting peanut yields worldwide. The discovery of peanut genome identified a considerable number of genetic loci controlling the peanut root-knot nematode; however, the molecular mechanism of root-knot nematode remains unknown. RESULTS: The heterogeneous response to root-knot nematode stress in peanut roots was identified using whole-transcriptome RNA-seq. A total of 430 mRNAs, 111 miRNAs, 4453 lncRNAs, and 123 circRNAs were found to have differential expression between infected and non-infected peanuts. The expression profiles of the lncRNA/circRNA-miRNA-mRNA network were developed to understand the potential pathways that lead to root-knot nematodes in peanut roots. During root-knot nematodes stress, a total of 10 lncRNAs, 4 circRNAs, 5 miRNAs, and 13 mRNAs can create competing endogenous RNA and participate in the oxidation-reduction process as well as other biological metabolism processes in peanuts. The findings will highlight the role of peanut ceRNAs in response to root-knot nematodes. CONCLUSION: The GO classification and KEGG pathway enrichment study of core regulatory networks revealed that ceRNAs are involved in oxidation-reduction, peroxidase activity, lignin synthesis in the xylem, and flavonoid synthesis. Overall, these findings may help researchers better understand the role of non-coding RNAs in response to root-knot nematodes.

Defining the combined stress response in wild Arachis.

Nematodes and drought are major constraints in tropical agriculture and often occur simultaneously. Plant responses to these stresses are complex and require crosstalk between biotic and abiotic signaling pathways. In this study, we explored the transcriptome data of wild Arachis species subjected to drought (A-metaDEG) and the root-knot nematode Meloidogyne arenaria (B-metaDEG) via meta-analysis, to identify core-stress responsive genes to each individual and concurrent stresses in these species. Transcriptome analysis of a nematode/drought bioassay (cross-stress) showed that the set of stress responsive DEGs to concurrent stress is distinct from those resulting from overlapping A- and B-metaDEGs, indicating a specialized and unique response to combined stresses in wild Arachis. Whilst individual biotic and abiotic stresses elicit hormone-responsive genes, most notably in the jasmonic and abscisic acid pathways, combined stresses seem to trigger mainly the ethylene hormone pathway. The overexpression of a cross-stress tolerance candidate gene identified here, an endochitinase-encoding gene (AsECHI) from Arachis stenosperma, reduced up to 30% of M. incognita infection and increased post-drought recovery in Arabidopsis plants submitted to both stresses. The elucidation of the network of cross-stress responsive genes in Arachis contributes to better understanding the complex regulation of biotic and abiotic responses in plants facilitating more adequate crop breeding for combined stress tolerance.

Effective and Sustained Control of Soil-Borne Plant Diseases by Biodegradable Polyhydroxybutyrate Mulch Films Embedded with Fungicide of Prothioconazole.

Soil-borne diseases and plant rhizosphere nematode have caused many crop yield losses. Increased environmental awareness is leading to more restrictions on the use of certain fumigants and root irrigation methods due to their impact on human health and soil system. Therefore, it is necessary to find alternative treatments to maintain crop economic yields and environmental sustainability. In the present work, biodegradable antifungal mulches were prepared by blending poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (PHB) with fungicide of prothioconazole (PRO), which were used for effective and sustained control of soil-borne plant diseases. To reveal the application prospect of the PHB/PRO composite films in the management of soilborne plant diseases, some physical and biological properties were evaluated. The proper mulch film of PHB/PRO was assessed based on its mechanical and optical properties, while water solubility and the film micromorphology was further characterized. The release patterns of composite films under different pH levels were investigated. Moreover, the in vitro antifungal bioassay and pot experiment showed satisfactory bioactivity of the PHB/PRO films against Sclerotium rolfsii Sacc., a soil-borne disease in peanut fields. This study demonstrated that the biodegradable mulch films containing PRO fungicide are capable of inhibiting soil-borne plant pathogenic fungi effectively, and this facile but powerful strategy may find wide applicability in sustainable plant and horticulture protection.

Pest scenario of Spodoptera litura (Fab.) on groundnut under representative concentration pathways (RCPs) based climate change scenarios.

Multi-model ensemble of Maximum (Tmax) and Minimum (Tmin) temperature data of four Representative Concentration Pathways viz., RCP 2.6, RCP 4.5, RCP 6.0 and RCP 8.5 of Coupled Model Intercomparison Project 5 (CMIP5) models were generated for ten major groundnut growing locations of the India to predict the number of generations of Spodoptera litura (Fab.) using Growing Degree Days approach during three future climate viz., Near (NF), Distant (DF) and Very Distant (VDF) periods and were compared over 1976-2005 baseline period (BL). Projections indicate significant increase in Tmax (0.7-4.7 °C) and Tmin (0.7-5.1 °C) in NF, DF and VDF periods under the four RCP scenarios at the ten groundnut growing locations. Higher percent increase of the number of generations of S. litura was predicted to occur in VDF (6-38%) over baseline, followed by DF (5-22%) and NF (4-9%) periods with reduction of generation time (5-26%) across the four RCP scenarios. Reduction of crop duration was higher (12-22 days) in long duration groundnut than in medium and short duration groundnut. Decrease in crop duration was higher in VDF (12.1-20.8 days) than DF (8.26-13.15 days) and NF (4.46-6.15 days) climate change periods under RCP 8.5 scenario. Increase in number of generations of S. litura was predicted even with altered crop duration of groundnut. Among locations, more number of generations of S. litura with reduced generation time are likely at Vridhachalam and Tirupathi locations. Geographical location (74-77%) and climate period (15-19%), together explained over 90 percent of the total variation in the number of generations and generation time of S. litura. These findings suggest that the incidence of S. litura on groundnut could be higher in future.

Evolutionarily conserved plant genes responsive to root-knot nematodes identified by comparative genomics.

Root-knot nematodes (RKNs, genus Meloidogyne) affect a large number of crops causing severe yield losses worldwide, more specifically in tropical and sub-tropical regions. Several plant species display high resistance levels to Meloidogyne, but a general view of the plant immune molecular responses underlying resistance to RKNs is still lacking. Combining comparative genomics with differential gene expression analysis may allow the identification of widely conserved plant genes involved in RKN resistance. To identify genes that are evolutionary conserved across plant species, we used OrthoFinder to compared the predicted proteome of 22 plant species, including important crops, spanning 214 Myr of plant evolution. Overall, we identified 35,238 protein orthogroups, of which 6,132 were evolutionarily conserved and universal to all the 22 plant species (PLAnts Common Orthogroups-PLACO). To identify host genes responsive to RKN infection, we analyzed the RNA-seq transcriptome data from RKN-resistant genotypes of a peanut wild relative (Arachis stenosperma), coffee (Coffea arabica L.), soybean (Glycine max L.), and African rice (Oryza glaberrima Steud.) challenged by Meloidogyne spp. using EdgeR and DESeq tools, and we found 2,597 (O. glaberrima), 743 (C. arabica), 665 (A. stenosperma), and 653 (G. max) differentially expressed genes (DEGs) during the resistance response to the nematode. DEGs' classification into the previously characterized 35,238 protein orthogroups allowed identifying 17 orthogroups containing at least one DEG of each resistant Arachis, coffee, soybean, and rice genotype analyzed. Orthogroups contain 364 DEGs related to signaling, secondary metabolite production, cell wall-related functions, peptide transport, transcription regulation, and plant defense, thus revealing evolutionarily conserved RKN-responsive genes. Interestingly, the 17 DEGs-containing orthogroups (belonging to the PLACO) were also universal to the 22 plant species studied, suggesting that these core genes may be involved in ancestrally conserved immune responses triggered by RKN infection. The comparative genomic approach that we used here represents a promising predictive tool for the identification of other core plant defense-related genes of broad interest that are involved in different plant-pathogen interactions.

A new source of root-knot nematode resistance from Arachis stenosperma incorporated into allotetraploid peanut (Arachis hypogaea).

Root-knot nematode is a very destructive pathogen, to which most peanut cultivars are highly susceptible. Strong resistance is present in the wild diploid peanut relatives. Previously, QTLs controlling nematode resistance were identified on chromosomes A02, A04 and A09 of Arachis stenosperma. Here, to study the inheritance of these resistance alleles within the genetic background of tetraploid peanut, an F2 population was developed from a cross between peanut and an induced allotetraploid that incorporated A. stenosperma, [Arachis batizocoi x A. stenosperma]4×. This population was genotyped using a SNP array and phenotyped for nematode resistance. QTL analysis allowed us to verify the major-effect QTL on chromosome A02 and a secondary QTL on A09, each contributing to a percentage reduction in nematode multiplication up to 98.2%. These were validated in selected F2:3 lines. The genome location of the large-effect QTL on A02 is rich in genes encoding TIR-NBS-LRR protein domains that are involved in plant defenses. We conclude that the strong resistance to RKN, derived from the diploid A. stenosperma, is transferrable and expressed in tetraploid peanut. Currently it is being used in breeding programs for introgressing a new source of nematode resistance and to widen the genetic basis of agronomically adapted peanut lines.

The characteristic of Arachis duranensis-specific genes and their potential function.

Arachis species produce flowers aerially, and then grow into the ground, where they develop into fruits; a feature that is unique to Arachis species. We hypothesized that Arachis species evolved genes specifically involved in the control of aerial flowers and the formation of underground fruits. Arachis duranensis is more resistant to biotic and abiotic stressors. Here, we compared different legume species and identified Arachis duranensis-specific genes. We analyzed gene expression patterns, base substitution patterns and sequence features between genes that are conserved across legume plants and A. duranensis-specific genes. Furthermore, we tested the role of A. duranensis-specific genes during seed development, response to nematode Meloidogyne arenaria infection and drought stress. We found that A. duranensis-specific genes had characteristics of young genes. The gene expression level and breadth were lower in the A. duranensis-specific genes compared to conserved genes. The A. duranensis-specific genes had higher codon usage bias than conserved genes, and the polypeptide length and GC content at the three codon sites were lower compared to conserved genes. Of the A. duranensis-specific genes, single-copy and duplicated genes had different features. The RNA-seq result showed A. duranensis-specific genes were involved in seed development, as well as response to nematode infection and drought stress. In addition, we detected asymmetric functions in A. duranensis-specific duplicated genes in response to nematode infection and drought stress.

Bioturbation of peanut worms Sipunculus nudus on the composition of prokaryotic communities in a tidal flat as revealed by 16S rRNA gene sequences.

To understand the impacts of peanut worms Sipunculus nudus on the prokaryotic community composition in a tidal flat, an onsite investigation was conducted in Suixi in the Beibu Gulf (109.82E, 21.35N) in the burrow sediments, non-burrow sediments and the sediments without peanut worm disturbance (control). The16S rRNA gene Illumina MiSeq sequencing was used to investigate the microbial communities and their response to bioturbation by S. nudus in a sandy tidal flat. A total of 18 bacteria phyla were detected, and Proteobacteria and Cyanobacteria constituted the majority of the prokaryotic community in the samples. The distribution of the relative abundances of genera showed that approximately 6.99%-17% of the reads in the samples were classified into 25 known genera. Sulfate-reducing bacteria (Desulfococcus and Desulfosarcina) were the most abundant taxa, followed by Thermodesulfovibrionaceae LCP-6, indicating that sulfate reduction is the main process in the sandy tidal flat. The abundances of Desulfococcus, LCP-6 and Cyanobacterium in the non-burrow sediment were greater than in the burrow sediment, suggesting that the anoxic condition is more suitable for Desulfococcus and LCP-6 when the activity of S. nudus is absent. The biomass of Cyanobacterium was decreased by the feeding bioturbation of S. nudus. Meanwhile, the relative abundance of the Bacteroidetes Luteimonas in the burrow sediments was significantly greater than in the non-burrow sediment, and there was a strong relationship between S. nudus bioturbation and increased in oxygen contents and oxidation-reduction potentials in the burrow sediment. The abundances of Desulfococcus and LCP-6 were greater in the middle layer (20-30 cm) than in the top layer in the non-burrow sediment. However, the middle and bottom layers (20-30, 30-40 cm) had higher abundances of these genera than did the upper layers (0-10, 10-20 cm) in the burrow sediments. The abundances of the Fusobacteria Propionigenium and the Spirochaetes Spirochaeta were greater in the middle and bottom layers (20-30 cm, 30-40 cm) than in the top layers (0-10, 10-20 cm) in the burrow sediment, but this phenomenon was not found in the non-burrow sediment. This study demonstrates that bioturbation by S. nudus plays an important role in reshaping the bacterial community composition in intertidal regions.

Risk assessment of the occurrence of aflatoxin and fungi in peanuts and cashew nuts

In the present study, the occurrence of fungi and aflatoxins (AFs) in peanut and cashew nut samples was investigated. Mycological analysis revealed the presence of fungi in 58.8% of samples, and assessment of AFs by chromatographic methods revealed that 52.9% were contaminated by AFs. AFB1 was the principal component in all AF-contaminated samples, with a mean level of 14.0, and 1.08 µg/kg in peanut and cashew nut, respectively. Eleven samples (32.4%) exceeded the total AF maximum level (4 µg/kg) and 8 samples (23.5%) exceeded the AFB1 (2 µg/kg) established by the European Commission. Our findings suggest that the incidence of AFs emphasizes the need for regular monitoring and a more stringent food safety system to control AFs at the lowest possible levels in peanuts and cashew nuts. The hypothetical dietary exposure suggests that the food products evaluated may significantly contribute to the overall human exposure

Genome-wide identification of LRR-containing sequences and the response of these sequences to nematode infection in Arachis duranensis.

BACKGROUND: Leucine-rich repeat (LRR)-containing genes are involved in responses to various diseases. Recently, RNA-seq data from A. duranensis after nematode (Meloidogyne arenaria) infection were released. However, the number of LRR-containing genes present in A. duranensis and the response of LRR-containing genes to nematode infection are poorly understood. RESULTS: In this study, we found 509 amino acid sequences containing nine types of LRR domains in A. duranensis. The inferred phylogenetic relationships revealed that the nine types of LRR domains had two originations. The inferred selective pressure was mainly consistent with LRR domains undergoing purifying selection. Twenty-one LRR-containing genes were associated with possible resistance to nematode infection after 3, 6, and 9 days. Among them, Aradu.T5WNW, Aradu.JM17V, and Aradu.MKP1A were up-regulate at these three time points, while Aradu.QD5DS and Aradu.M0ENQ were up-regulated 6 and 9 days after nematode infection. The expression of the above mentioned five genes was significantly and negatively correlated with the number of LRR8 domain, indicating that fewer LRR8 domains are associated with the promotion of LRR-containing genes that resist nematode infection. Patterns of co-expression and cis-acting elements indicated that WRKY possibly regulate the responses of LRR-containing genes to nematode infection and that expansin genes may work together with LRR-containing genes in response to nematode infection. CONCLUSIONS: We identified the number and type of LRR-containing genes in A. duranensis. The LRR-containing genes that were found appear to be involved in responses to nematode infection. The number of LRR8 domains was negatively correlated with expression after nematode infection. The WRKY transcription factor may regulate resistance to nematode infection based on LRR-containing genes. Our results could improve the understanding of resistance to nematodes and molecular breeding in peanuts.

Comparative root transcriptome of wild Arachis reveals NBS-LRR genes related to nematode resistance.

BACKGROUND: The Root-Knot Nematode (RKN), Meloidogyne arenaria, significantly reduces peanut grain quality and yield worldwide. Whilst the cultivated species has low levels of resistance to RKN and other pests and diseases, peanut wild relatives (Arachis spp.) show rich genetic diversity and harbor high levels of resistance to many pathogens and environmental constraints. Comparative transcriptome analysis can be applied to identify candidate resistance genes. RESULTS: Transcriptome analysis during the early stages of RKN infection of two peanut wild relatives, the highly RKN resistant Arachis stenosperma and the moderately susceptible A. duranensis, revealed genes related to plant immunity with contrasting expression profiles. These included genes involved in hormone signaling and secondary metabolites production and also members of the NBS-LRR class of plant disease resistance (R) genes. From 345 NBS-LRRs identified in A.duranensis reference genome, 52 were differentially expressed between inoculated and control samples, with the majority occurring in physical clusters unevenly distributed on eight chromosomes with preferential tandem duplication. The majority of these NBS-LRR genes showed contrasting expression behaviour between A. duranensis and A. stenosperma, particularly at 6 days after nematode inoculation, coinciding with the onset of the Hypersensitive Response in the resistant species. The physical clustering of some of these NBS-LRR genes correlated with their expression patterns in the contrasting genotypes. Four NBS-LRR genes exclusively expressed in A. stenosperma are located within clusters on chromosome Aradu. A09, which harbors a QTL for RKN resistance, suggesting a functional role for their physical arrangement and their potential involvement in this defense response. CONCLUSION: The identification of functional novel R genes in wild Arachis species responsible for triggering effective defense cascades can contribute to the crop genetic improvement and enhance peanut resilience to RKN.

Subterranean infestation by Holotrichia parallela larvae is associated with changes in the peanut (Arachis hypogaea L.) rhizosphere microbiome.

Rhizosphere microorganisms contribute to the health and development of crops and these beneficial microbes are recruited to the root-zone when plants experience biotic/abiotic stress. The subterranean pests Holotrichia parallela cause severe crop loss in peanut (Arachis hypogaea L.) fields. Hypothesizing that infestation by H. parallela larva may influence the composition of rhizosphere microbial communities, deep sequencing of V3 and V4 hypervariable regions of 16S rRNA gene was used to characterize the rhizosphere bacteria of infested and uninfested peanuts. A total of 2,673,656 reads were generated and an average of 2558 OTUs were obtained for each sample. Comparisons of rhizosphere bacterial community structure of peanuts with those infested by H. parallela larva revealed that the relative abundance of Proteobacteria and Bacteroidetes increased, while that of Actinobacteria decreased in the rhizosphere with infestation. A significant shift in bacterial communities was observed within 24 h after infestation by principal coordinate analysis. For the 332 genera identified in 24 h treatment, infestation of white grubs led to the significant changes of abundance of 67 genera. An increase in the Pseudomonas genus of infested-samples for 24 h was verified by real-time qPCR. Our results indicate H. parallela larvae infestation can quickly leads to the change of peanut rhizosphere microbiome and enrichment of specific bacterial species. But the effects were not persistent. This study provides the insight into the function of rhizosphere microbiome in the interaction between subterranean pests and crops.

Gene expression profiling describes the genetic regulation of Meloidogyne arenaria resistance in Arachis hypogaea and reveals a candidate gene for resistance.

Resistance to root-knot nematode was introgressed into cultivated peanut Arachis hypogaea from a wild peanut relative, A. cardenasii and previously mapped to chromosome A09. The highly resistant recombinant inbred RIL 46 and moderately resistant RIL 48 were selected from a population with cv. Gregory (susceptible) and Tifguard (resistant) as female and male parents, respectively. RNA-seq analysis was performed on these four genotypes using root tissue harvested from root-knot nematode infected plants at 0, 3, 7 days after inoculation. Differential gene expression analysis provides evidence that root-knot nematodes modulate biological pathways involved in plant hormone, defense, cell signaling, cytoskeleton and cell wall metabolism in a susceptible reaction. Corresponding to resistance reaction, an effector-induced-immune response mediated by an R-gene was identified in Tifguard. Mapping of the introgressed region indicated that 92% of linkage group A09 was of A. cardenasii origin in Tifguard. RIL46 and RIL 48 possessed 3.6% and 83.5% of the introgression on A09, respectively. Within the small introgressed region carried by RIL 46, a constitutively expressed TIR-NBS-LRR gene was identified as the candidate for nematode resistance. Potential defense responsive pathways include effector endocytosis through clathrin-coated vesicle trafficking, defense signaling through membrane lipid metabolism and mucilage production.

Recombinants from the crosses between amphidiploid and cultivated peanut (Arachis hypogaea) for pest-resistance breeding programs.

Peanut is a major oilseed crop worldwide. In the Brazilian peanut production, silvering thrips and red necked peanut worm are the most threatening pests. Resistant varieties are considered an alternative to pest control. Many wild diploid Arachis species have shown resistance to these pests, and these can be used in peanut breeding by obtaining hybrid of A and B genomes and subsequent polyploidization with colchicine, resulting in an AABB amphidiploid. This amphidiploid can be crossed with cultivated peanut (AABB) to provide genes of interest to the cultivar. In this study, the sterile diploid hybrids from A. magna V 13751 and A. kempff-mercadoi V 13250 were treated with colchicine for polyploidization, and the amphidiploids were crossed with A. hypogaea cv. IAC OL 4 to initiate the introgression of the wild genes into the cultivated peanut. The confirmation of the hybridity of the progenies was obtained by: (1) reproductive characterization through viability of pollen, (2) molecular characterization using microsatellite markers and (3) morphological characterization using 61 morphological traits with principal component analysis. The diploid hybrid individual was polyploidized, generating the amphidiploid An 13 (A. magna V 13751 x A. kempff-mercadoi V 13250)4x. Four F1 hybrid plants were obtained from IAC OL 4 × An 13, and 51 F2 seeds were obtained from these F1 plants. Using reproductive, molecular and morphological characterizations, it was possible to distinguish hybrid plants from selfed plants. In the cross between A. hypogaea and the amphidiploid, as the two parents are polyploid, the hybrid progeny and selves had the viability of the pollen grains as high as the parents. This fact turns the use of reproductive characteristics impossible for discriminating, in this case, the hybrid individuals from selfing. The hybrids between A. hypogaea and An 13 will be used in breeding programs seeking pest resistance, being subjected to successive backcrosses until recovering all traits of interest of A. hypogaea, keeping the pest resistance.

Comparative host selection responses of specialist (Helicoverpa assulta) and generalist (Helicoverpa armigera) moths in complex plant environments.

We tested the behavioral responses of ovipositing females and natal larvae of two sibling species, a generalist Helicoverpa armigera (Hübner) and a specialist Helicoverpa assulta (Guenée), to odor sources emitted from different combinations of six plant species (tobacco, Nicotiana tabacum; hot pepper, Capsicum annuum; tomato, Solanum esculentum; cotton, Gossypium hirsutum; peanut, Arachis hypogaea; maize, Zea mays). Under the conditions of plant materials versus corresponding controls, both stages of both species could find their corresponding host plants. However, H. assulta females and larvae exhibited a supersensitive and an insensitive response, respectively. Under the conditions of tobacco paired with each plant species, H. assulta females exhibited more specialized ovipositional response to tobacco than its sibling. When each plant species were combined with tobacco and tested against tobacco reference, peanut played an opposite role in the two species in their ovipositional responses to tobacco, and cotton can enhance the approaching response of H. armigera larvae when combined with tobacco. It seems that two attractive host plants also can act antagonistically with respect to host selection of the generalist via volatile exchange. Tomato should better be excluded from host list of H. assulta.

Genetic Mapping of Resistance to Meloidogyne arenaria in Arachis stenosperma: A New Source of Nematode Resistance for Peanut.

Root-knot nematodes (RKN; Meloidogyne sp.) are a major threat to crops in tropical and subtropical regions worldwide. The use of resistant crop varieties is the preferred method of control because nematicides are expensive, and hazardous to humans and the environment. Peanut (Arachis hypogaea) is infected by four species of RKN, the most damaging being M. arenaria, and commercial cultivars rely on a single source of resistance. In this study, we genetically characterize RKN resistance of the wild Arachis species A. stenosperma using a population of 93 recombinant inbred lines developed from a cross between A. duranensis and A. stenosperma. Four quantitative trait loci (QTL) located on linkage groups 02, 04, and 09 strongly influenced nematode root galling and egg production. Drought-related, domestication and agronomically relevant traits were also evaluated, revealing several QTL. Using the newly available Arachis genome sequence, easy-to-use KASP (kompetitive allele specific PCR) markers linked to the newly identified RKN resistance loci were developed and validated in a tetraploid context. Therefore, we consider that A. stenosperma has high potential as a new source of RKN resistance in peanut breeding programs.

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.

Development of melamine-formaldehyde resin microcapsules with low formaldehyde emission suited for seed treatment.

To reduce the application frequency and improve the efficacy of insecticides, melamine-formaldehyde (MF) resin microcapsules suited for seed treatment containing a mixture of fipronil and chlorpyrifos were prepared by in situ polymerization. A formaldehyde/melamine molar ratio of 4:1 yielded microcapsules with the smallest size and the most narrow size distribution. The level of unreacted formaldehyde in the microcapsule suspension increased proportionally with the F/M molar ratio. When the MF resin microcapsule suspension was used as a seed treatment to coat peanut seeds, the unreacted formaldehyde did not significantly inhibit the seedling emergence, but the ongoing release of formaldehyde generated from the degradation of MF resins played an important role in inhibiting emergence. Melamine was shown to be an effective formaldehyde scavenger that mitigated this inhibition when it was incorporated within the microcapsule wall. Field experiments showed that MF-resin-encapsulated mixtures of fipronil and chlorpyrifos have much greater efficacies against white grubs than the conventional formulation.

Controlled release study on microencapsulated mixture of fipronil and chlorpyrifos for the management of white grubs (Holotrichia parallela) in peanuts (Arachis hypogaea L.).

This study was conducted to determine the release dynamics of a microencapsulated mixture of fipronil and chlorpyrifos in peanut fields and its efficacy against white grubs. The results indicated that microencapsulation significantly stabilized this mixture against degradation in the environment so that a single dose of this microencapsulated formulation applied through seed treatment effectively controlled white grubs throughout the entire growing season. During the experimental course, the concentration of chlorpyrifos in the soil with the microencapsulated formulation was 13.6 ± 9.9 (n = 6) times that of the conventional formulation, and the concentration of fipronil was at least 2.2 times that of the conventional formulation in the soil and peanut roots. However, the residue risks of chlorpyrifos and fipronil in the kernels were different. At harvest, there was a low risk that the residual chlorpyrifos in the kernels exceeded the MRLs (maximum residue limit). In contrast, the amount of residual fipronil in some kernel samples reached the statutory MRL set by the European Union, which suggested that a higher application rate or the repeated application of the microencapsulated fipronil formulation would not be acceptable.

Host plant resistance against tomato spotted wilt virus in peanut (Arachis hypogaea) and its impact on susceptibility to the virus, virus population genetics, and vector feeding behavior and survival.

Tomato spotted wilt virus (TSWV) severely affects peanut production in the southeastern United States. Breeding efforts over the last three decades resulted in the release of numerous peanut genotypes with field resistance to TSWV. The degree of field resistance in these genotypes has steadily increased over time, with recently released genotypes exhibiting a higher degree of field resistance than older genotypes. However, most new genotypes have never been evaluated in the greenhouse or laboratory against TSWV or thrips, and the mechanism of resistance is unknown. In this study, TSWV-resistant and -susceptible genotypes were subjected to TSWV mechanical inoculation. The incidence of TSWV infection was 71.7 to 87.2%. Estimation of TSWV nucleocapsid (N) gene copies did not reveal significant differences between resistant and susceptible genotypes. Parsimony and principal component analyses of N gene nucleotide sequences revealed inconsistent differences between virus isolates collected from resistant and susceptible genotypes and between old (collected in 1998) and new (2010) isolates. Amino acid sequence analyses indicated consistent differences between old and new isolates. In addition, we found evidence for overabundance of nonsynonymous substitutions. However, there was no evidence for positive selection. Purifying selection, population expansion, and differentiation seem to have influenced the TSWV populations temporally rather than positive selection induced by host resistance. Choice and no-choice tests indicated that resistant and susceptible genotypes differentially affected thrips feeding and survival. Thrips feeding and survival were suppressed on some resistant genotypes compared with susceptible genotypes. These findings reveal how TSWV resistance in peanut could influence evolution, epidemiology, and management of TSWV.