Genome-Wide Association Studies on Resistance to Pea Weevil: Identification of Novel Sources of Resistance and Associated Markers.

Little resistance to the pea weevil insect pest (Bruchus pisorum) is available in pea (Pisum sativum) cultivars, highlighting the need to search for sources of resistance in Pisum germplasm and to decipher the genetic basis of resistance. To address this need, we screened the response to pea weevil in a Pisum germplasm collection (324 accession, previously genotyped) under field conditions over four environments. Significant variation for weevil seed infestation (SI) was identified, with resistance being frequent in P. fulvum, followed by P. sativum ssp. elatius, P. abyssinicum, and P. sativum ssp. humile. SI tended to be higher in accessions with lighter seed color. SI was also affected by environmental factors, being favored by high humidity during flowering and hampered by warm winter temperatures and high evapotranspiration during and after flowering. Merging the phenotypic and genotypic data allowed genome-wide association studies (GWAS) yielding 73 markers significantly associated with SI. Through the GWAS models, 23 candidate genes were found associated with weevil resistance, highlighting the interest of five genes located on chromosome 6. These included gene 127136761 encoding squalene epoxidase; gene 127091639 encoding a transcription factor MYB SRM1; gene 127097033 encoding a 60S ribosomal protein L14; gene 127092211, encoding a BolA-like family protein, which, interestingly, was located within QTL BpLD.I, earlier described as conferring resistance to weevil in pea; and gene 127096593 encoding a methyltransferase. These associated genes offer valuable potential for developing pea varieties resistant to Bruchus spp. and efficient utilization of genomic resources through marker-assisted selection (MAS).

Genomic insights into Spiroplasma endosymbionts that induce male-killing and protective phenotypes in the pea aphid.

The endosymbiotic bacteria Spiroplasma (Mollicutes) infect diverse plants and arthropods, and some of which induce male killing, where male hosts are killed during development. Male-killing Spiroplasma strains belong to either the phylogenetically distant Citri-Poulsonii or Ixodetis groups. In Drosophila flies, Spiroplasma poulsonii induces male killing via the Spaid toxin. While Spiroplasma ixodetis infects a wide range of insects and arachnids, little is known about the genetic basis of S. ixodetis-induced male killing. Here, we analyzed the genome of S. ixodetis strains in the pea aphid Acyrthosiphon pisum (Aphididae, Hemiptera). Genome sequencing constructed a complete genome of a male-killing strain, sAp269, consisting of a 1.5 Mb circular chromosome and an 80 Kb plasmid. sAp269 encoded putative virulence factors containing either ankyrin repeat, ovarian tumor-like deubiquitinase, or ribosome inactivating protein domains, but lacked the Spaid toxin. Further comparative genomics of Spiroplasma strains in A. pisum biotypes adapted to different host plants revealed their phylogenetic associations and the diversity of putative virulence factors. Although the mechanisms of S. ixodetis-induced male killing in pea aphids remain elusive, this study underlines the dynamic genome evolution of S. ixodetis and proposes independent acquisition events of male-killing mechanisms in insects.

Identification and Behavioral Assays of Alarm Pheromone in the Vetch Aphid Megoura viciae.

Aphids are destructive pests, and alarm pheromones play a key role in their chemical ecology. Here, we conducted a detailed analysis of terpenoids in the vetch aphid, Megoura viciae, and its host plant Pisum sativum using gas chromatography/mass spectrometry. Four major components, (-)-ß-pinene (49.74%), (E)-ß-farnesene (32.64%), (-)-α-pinene (9.42%) and ( +)-limonene (5.24%), along with trace amounts of ( +)-sabinene, camphene and α-terpineol) (3.14%) were found in the aphid. In contrast, few terpenoids were found in the host plant, consisting mainly of squalene (66.13%) and its analog 2,3-epoxysqualene (31.59%). Quantitative analysis of the four major terpenes in different developmental stages of the aphid showed that amounts of the monoterpenes increased with increasing stage, while the sesquiterpene amount peaked in the 3rd instar. (-)-ß-Pinene was the most abundant terpene at all developmental stages. Behavioral assays using a three-compartment olfactometer revealed that the repellency of single compounds varied in a concentration-dependent manner, but two mixtures [(-)-α-pinene: (-)-ß-pinene: (E)-ß-farnesene: ( +)-limonene = 1:44.4:6.5:2.2 or 1:18.4:1.3:0.8], were repellent at all concentrations tested. Our results suggest that (-)-α-pinene and (-)-ß-pinene are the major active components of the alarm pheromone of M. viciae, but that mixtures play a key role in the alarm response. Our study contributes to the understanding of the chemical ecology of aphids and may help design new control strategies against this aphid pest.

Host and symbiont genetic determinants of nutritional phenotype in a natural population of the pea aphid.

A defining feature of the nutritional ecology of plant sap-feeding insects is that the dietary deficit of essential amino acids (EAAs) in plant sap is supplemented by EAA-provisioning microbial symbionts in the insect. Here, we demonstrated substantial variation in the nutritional phenotype of 208 genotypes of the pea aphid Acyrthosiphon pisum collected from a natural population. Specifically, the genotypes varied in performance (larval growth rates) on four test diets lacking the EAAs arginine, histidine and methionine or aromatic EAAs (phenylalanine and tryptophan), relative to the diet containing all EAAs. These data indicate that EAA supply from the symbiotic bacteria Buchnera can meet total aphid nutritional demand for only a subset of the EAA/aphid genotype combinations. We then correlated single nucleotide polymorphisms (SNPs) identified in the aphid and Buchnera genomes by reduced genome sequencing against aphid performance for each EAA deletion diet. This yielded significant associations between performance on the histidine-free diet and Buchnera SNPs, including metabolism genes predicted to influence histidine biosynthesis. Aphid genetic correlates of performance were obtained for all four deletion diets, with associations on the arginine-free diet and aromatic-free diets dominated by genes functioning in the regulation of metabolic and cellular processes. The specific aphid genes associated with performance on different EAA deletion diets are largely nonoverlapping, indicating some independence in the regulatory circuits determining aphid phenotype for the different EAAs. This study demonstrates how variation in the phenotype of associations collected from natural populations can be applied to elucidate the genetic basis of ecologically important traits in systems intractable to traditional forward/reverse genetic techniques.

Identification of quantitative trait loci (QTL) controlling resistance to pea weevil (Bruchus pisorum) in a high-density integrated DArTseq SNP-based genetic map of pea.

Pea weevil (Bruchus pisorum) is a damaging insect pest affecting pea (Pisum sativum) production worldwide. No resistant cultivars are available, although some levels of incomplete resistance have been identified in Pisum germplasm. To decipher the genetic control underlying the resistance previously identify in P. sativum ssp. syriacum, a recombinant inbred line (RIL F8:9) population was developed. The RIL was genotyped through Diversity Arrays Technology PL's DArTseq platform and screened under field conditions for weevil seed infestation and larval development along 5 environments. A newly integrated genetic linkage map was generated with a subset of 6,540 markers, assembled into seven linkage groups, equivalent to the number of haploid pea chromosomes. An accumulated distance of 2,503 cM was covered with an average density of 2.61 markers cM-1. The linkage map allowed the identification of three QTLs associated to reduced seed infestation along LGs I, II and IV. In addition, a QTL for reduced larval development was also identified in LGIV. Expression of these QTLs varied with the environment, being particularly interesting QTL BpSI.III that was detected in most of the environments studied. This high-saturated pea genetic map has also allowed the identification of seven potential candidate genes co-located with QTLs for marker-assisted selection, providing an opportunity for breeders to generate effective and sustainable strategies for weevil control.

A Symbiotic Virus Facilitates Aphid Adaptation to Host Plants by Suppressing Jasmonic Acid Responses.

Symbiotic viruses exist in many insects; however, their functions in host insects are not well understood. In this study, we explored the role of acyrthosiphon pisum virus (APV) in the interaction of its host aphid Acyrthosiphon pisum with plants. APV is primarily located in aphid salivary glands and gut and propagated in the insect. APV is horizontally transmitted to host plants during aphid feeding, but the virus does not replicate in the host plant. When the pea host race of aphids colonized two low-fitness plants, Medicago truncatula and Vicia villosa, the virus titers in both the aphids and plants significantly increased. Furthermore, APV infection strongly promoted the survival rate of the pea host race on V. villosa. Transcriptomic analysis showed that only 0.85% of aphid genes responded to APV infection when aphids fed on V. villosa, with a fold change in transcript levels of no more than fourfold. The improved survival due to APV infection was apparently related to the inhibitory effect of the virus on levels of phytohormone jasmonic acid (JA) and JA-isoleucine. Our data suggest a benefit of the symbiotic virus to its aphid host and demonstrate a novel case of symbiotic virus-mediated three-species interaction.

Reproduction Ability and Growth Effect of Pin Nematode, Paratylenchus nanus, With Selected Field Pea Cultivars.

Greenhouse experiments were conducted to determine the reproductive ability and effect of the pin nematode Paratylenchus nanus from North Dakota on field pea cultivars. Reproduction of P. nanus was determined on seven field pea cultivars using naturally infested field soils at low (1,500/kg of soil) and high (4,500/kg soil) initial pin nematode densities. Nematode effect on plant growth and seed yield was evaluated at 4,500 P. nanus per 1 kg of soil by artificially inoculating P. nanus on six field pea cultivars. Reproductive factor (RF) of P. nanus was observed to be greater at the low density than the high density of the nematode. At the low population density, RF values ranged from 1.10 to 11.20, whereas at the high density, RF ranged from 1.20 to 2.50. In experiments evaluating P. nanus effects on cultivar growth, the nematode (4,500 P. nanus per 1 kg soil) caused reduction (P < 0.05) of plant height in most cultivars tested, and it also significantly impacted dry shoot weight and dry seed weight in some experiments. Plant height and shoot weight reductions were the highest in the cultivar Arcadia (up to 37 and 53%, respectively), with a dry seed weight reduction up to 32%. This research demonstrated for the first time the negative impact of P. nanus on field peas in controlled greenhouse conditions, which is an important step toward developing effective management strategies to improve the productivity of this leguminous crop.

Bioactivity-guided isolation of rosmarinic acid as the principle bioactive compound from the butanol extract of Isodon rugosus against the pea aphid, Acyrthosiphon pisum.

Aphids are agricultural pest insects that transmit viruses and cause feeding damage on a global scale. Current pest control practices involving the excessive use of synthetic insecticides over many years have resulted in aphid resistance to a number of pesticides. In nature, plants produce secondary metabolites during their interaction with insects and these metabolites can act as toxicants, antifeedants, anti-oviposition agents and deterrents towards the insects. In a previous study, we demonstrated that the butanol fraction from a crude methanolic extract of an important plant species, Isodon rugosus showed strong insecticidal activity against the pea aphid, Acyrthosiphon pisum. To further explore this finding, the current study aimed to exploit a bioactivity-guided strategy to isolate and identify the active compound in the butanol fraction of I. rugosus. As such, reversed-phase flash chromatography, acidic extraction and different spectroscopic techniques were used to isolate and identify the new compound, rosmarinic acid, as the bioactive compound in I. rugosus. Insecticidal potential of rosmarinic acid against A. pisum was evaluated using standard protocols and the data obtained was analyzed using qualitative and quantitative statistical approaches. Considering that a very low concentration of this compound (LC90 = 5.4 ppm) causes significant mortality in A. pisum within 24 h, rosmarinic acid could be exploited as a potent insecticide against this important pest insect. Furthermore, I. rugosus is already used for medicinal purposes and rosmarinic acid is known to reduce genotoxic effects induced by chemicals, hence it is expected to be safer compared to the current conventional pesticides. While this study highlights the potential of I. rugosus as a possible biopesticide source against A. pisum, it also provides the basis for further exploration and development of formulations for effective field application.

Limited effects of the maternal rearing environment on the behaviour and fitness of an insect herbivore and its natural enemy.

The maternal rearing environment can affect offspring fitness or phenotype indirectly via 'maternal effects' and can also influence a mother's behaviour and fecundity directly. However, it remains uncertain how the effects of the maternal rearing environment cascade through multiple trophic levels, such as in plant-insect herbivore-natural enemy interactions. Pea aphids (Acyrthosiphon pisum) show differential fitness on host legume species, while generalist aphid parasitoids can show variable fitness on different host aphid species, suggesting that maternal effects could operate in a plant-aphid-parasitoid system. We tested whether the maternal rearing environment affected the behaviour and fitness of aphids by rearing aphids on two plant hosts that were either the same as or different from those experienced by the mothers. A similar approach was used to test the behaviour and fitness of parasitoid wasps in response to maternal rearing environment. Here, the host environment was manipulated at the plant or plant and aphid trophic levels for parasitoid wasps. We also quantified the quality of host plants for aphids and host aphids for parasitoid wasps. In choice tests, aphids and parasitoid wasps had no preference for the plant nor plant and aphid host environment on which they were reared. Aphid offspring experienced 50.8% higher intrinsic rates of population growth, 43.4% heavier offspring and lived 14.9% longer when feeding on bean plants compared to aphids feeding on pea plants, with little effect of the maternal rearing environment. Plant tissue nitrogen concentration varied by 21.3% in response to aphid mothers' rearing environment, and these differences correlated with offspring fitness. Maternal effects in parasitoid wasps were only observed when both the plant and aphid host environment was changed: wasp offspring were heaviest by 10.9-73.5% when both they and their mothers developed in bean-reared pea aphids. Also, parasitoid wasp fecundity was highest by 38.4% when offspring were oviposited in the maternal rearing environment. These findings indicate that maternal effects have a relatively small contribution towards the outcome of plant-aphid-parasitoid interactions.

Molecular Characterization and Identification of Stubby Root Nematode Species From Multiple States in the United States.

Stubby root nematodes (SRN) are important plant parasites infecting many crops and widely distributed in many regions of the United States. SRN transmit Tobacco rattle virus, which causes potato corky ringspot disease, thereby having a significant economic impact on the potato industry. In 2015 to 2017, 184 soil samples and 16 nematode suspensions from North Dakota, Minnesota, Idaho, Oregon, Washington, South Carolina, North Carolina, and Florida were assayed for the presence of SRN. SRN were found in 106 soil samples with population densities of 10 to 320 SRN per 200 g of soil and in eight of the nematode suspensions. Sequencing of ribosomal DNA (rDNA) or species-specific polymerase chain reaction assays revealed the presence of four SRN species, including Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus. Accordingly, their rDNA sequences were characterized by analyzing D2-D3 of 28S rDNA, 18S rDNA, and internal transcribed spacer (ITS) rDNA obtained in this study and retrieved from GenBank. Both intra- and interspecies variations were higher in ITS rDNA than 18S rDNA and D2-D3 of 28S rDNA. Based on phylogenetic analysis, the four SRN species formed a monophyletic group, with P. allius more closely related to P. porosus than P. minor and T. obtusus. Indel variation of ITS2 rDNA was present in P. allius populations from the same geographic regions. This study documented the occurrence of SRN species across multiple states. The intra- and interspecies genetic diversity of rDNA in this study will provide more information for understanding the evolutionary relationships of SRN and will be valuable for future studies of SRN species identification and management.

Context-dependent vertical transmission shapes strong endosymbiont community structure in the pea aphid, Acyrthosiphon pisum.

Animal-associated microbiomes are often comprised of structured, multispecies communities, with particular microbes showing trends of co-occurrence or exclusion. Such structure suggests variable community stability, or variable costs and benefits-possibilities with implications for symbiont-driven host adaptation. In this study, we performed systematic screening for maternally transmitted, facultative endosymbionts of the pea aphid, Acyrthosiphon pisum. Sampling across six locales, with up to 5 years of collection in each, netted significant and consistent trends of community structure. Co-infections between Serratia symbiotica and Rickettsiella viridis were more common than expected, while Rickettsia and X-type symbionts colonized aphids with Hamiltonella defensa more often than expected. Spiroplasma co-infected with other endosymbionts quite rarely, showing tendencies to colonize as a single species monoculture. Field estimates of maternal transmission rates help to explain our findings: while Serratia and Rickettsiella improved each other's transmission, Spiroplasma reduced transmission rates of co-infecting endosymbionts. In summary, our findings show that North American pea aphids harbour recurring combinations of facultative endosymbionts. Common symbiont partners play distinct roles in pea aphid biology, suggesting the creation of "generalist" aphids receiving symbiont-based defence against multiple ecological stressors. Multimodal selection, at the host level, may thus partially explain our results. But more conclusively, our findings show that within-host microbe interactions, and their resulting impacts on transmission rates, are an important determinant of community structure. Widespread distributions of heritable symbionts across plants and invertebrates hint at the far-reaching implications for these findings, and our work further shows the benefits of symbiosis research within a natural context.

Elevated CO2 increases R gene-dependent resistance of Medicago truncatula against the pea aphid by up-regulating a heat shock gene.

Resistance against pathogens and herbivorous insects in many plant results from the expression of resistance (R) genes. Few reports, however, have considered the effects of elevated CO2 on R gene-based resistance in plants. The current study determined the responses of two near isogenic Medicago truncatula genotypes (Jester has an R gene and A17 does not) to the pea aphid and elevated CO2 in open-top chambers in the field. Aphid abundance, mean relative growth rate and feeding efficiency were increased by elevated CO2 on A17 plants but were reduced on Jester plants. According to proteomic and gene expression data, elevated CO2 enhanced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) but decreased the effector-triggered immunity (ETI) in aphid-infested A17 plants. For aphid-infested Jester plants, by contrast, elevated CO2 enhanced the ETI-related heat shock protein (HSP) 90 and its co-chaperones, the jasmonic acid (JA) signaling pathway, and ubiquitin-mediated proteolysis. In a loss-of-function experiment, silencing of the HSP90 gene in Jester plants impaired the JA signaling pathway and ubiquitin-mediated proteolysis against the aphid under ambient CO2 , and negated the increased resistance against the aphid under elevated CO2 . Our results suggest that increases in expression of HSP90 are responsible for the enhanced resistance against the aphid under elevated CO2 .

2015 Outbreak of Cyclosporiasis Linked to the Consumption of Imported Sugar Snap Peas in Ontario, Canada.

An outbreak of cyclosporiasis in Ontario, Canada, was investigated in the fall of 2015. Thirty-five confirmed and 10 probable cases were linked to the investigation. Epidemiological and food safety evidence implicated fresh sugar snap peas imported from Guatemala as the source of the outbreak. We describe here the first documented cyclosporiasis outbreak in Canada involving the consumption of sugar snap peas.

The Influence of Lead on Generation of Signalling Molecules and Accumulation of Flavonoids in Pea Seedlings in Response to Pea Aphid Infestation.

The aim of this study was to investigate the effect of an abiotic factor, i.e., lead at various concentrations (low causing a hormesis effect and causing high toxicity effects), on the generation of signalling molecules in pea (Pisum sativum L. cv. Cysterski) seedlings and then during infestation by the pea aphid (Acyrthosiphon pisum Harris). The second objective was to verify whether the presence of lead in pea seedling organs and induction of signalling pathways dependent on the concentration of this metal trigger defense responses to A. pisum. Therefore, the profile of flavonoids and expression levels of genes encoding enzymes of the flavonoid biosynthesis pathway (phenylalanine ammonialyase and chalcone synthase) were determined. A significant accumulation of total salicylic acid (TSA) and abscisic acid (ABA) was recorded in the roots and leaves of pea seedlings growing on lead-supplemented medium and next during infestation by aphids. Increased generation of these phytohormones strongly enhanced the biosynthesis of flavonoids, including a phytoalexin, pisatin. This research provides insights into the cross-talk between the abiotic (lead) and biotic factor (aphid infestation) on the level of the generation of signalling molecules and their role in the induction of flavonoid biosynthesis.

The chemical signatures underlying host plant discrimination by aphids.

The diversity of phytophagous insects is largely attributable to speciation involving shifts between host plants. These shifts are mediated by the close interaction between insects and plant metabolites. However, there has been limited progress in understanding the chemical signatures that underlie host preferences. We use the pea aphid (Acyrthosiphon pisum) to address this problem. Host-associated races of pea aphid discriminate between plant species in race-specific ways. We combined metabolomic profiling of multiple plant species with behavioural tests on two A. pisum races, to identify metabolites that explain variation in either acceptance or discrimination. Candidate compounds were identified using tandem mass spectrometry. Our results reveal a small number of compounds that explain a large proportion of variation in the differential acceptability of plants to A. pisum races. Two of these were identified as L-phenylalanine and L-tyrosine but it may be that metabolically-related compounds directly influence insect behaviour. The compounds implicated in differential acceptability were not related to the set correlated with general acceptability of plants to aphids, regardless of host race. Small changes in response to common metabolites may underlie host shifts. This study opens new opportunities for understanding the mechanistic basis of host discrimination and host shifts in insects.

The interaction of the bioinsecticide PA1b (Pea Albumin 1 subunit b) with the insect V-ATPase triggers apoptosis.

PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin, extracted from Legume seeds, with a lethal activity towards several insect pests, such as mosquitoes, some aphids and cereal weevils. This toxin acts by binding to the subunits c and e of the plasma membrane H+-ATPase (V-ATPase) in the insect midgut. In this study, two cereal weevils, the sensitive Sitophilus oryzae strain WAA42, the resistance Sitophilus oryzae strain ISOR3 and the insensitive red flour beetle Tribolium castaneum, were used in biochemical and histological experiments to demonstrate that a PA1b/V-ATPase interaction triggers the apoptosis mechanism, resulting in insect death. Upon intoxication with PA1b, apoptotic bodies are formed in the cells of the insect midgut. In addition, caspase-3 enzyme activity occurs in the midgut of sensitive weevils after intoxication with active PA1b, but not in the midgut of resistant weevils. These biochemical data were confirmed by immuno-histochemical detection of the caspase-3 active form in the midgut of sensitive weevils. Immuno-labelling experiments also revealed that the caspase-3 active form and V-ATPase are close-localized in the insect midgut. The results concerning this unique peptidic V-ATPase inhibitor pave the way for the utilization of PA1b as a promising, more selective and eco-friendly insecticide.

Degenerate ITS7 primer enhances oomycete community coverage and PCR sensitivity to Aphanomyces species, economically important plant pathogens.

Metagenomic analysis of oomycetes through deep amplicon sequencing has been conducted primarily using the ITS6-ITS7 primer set that targets the ITS1 region. While this primer set shows a perfect match to most oomycete taxa, ITS7 contains 3 mismatches to the corresponding binding site of plant pathogens within the genus Aphanomyces. Polymerase chain reaction (PCR) efficiency differs for taxa with uneven primer matching characteristics, which may explain why previous studies have detected this genus at low abundance. To overcome the impact of these mismatches on PCR sensitivity, the mismatched nucleotides were replaced with degenerate nucleotides. Oomycete communities from 35 soil samples collected from asymptomatic and root rot diseased sites in pea fields across Alberta were analyzed simultaneously using ITS6-ITS7 and ITS6-ITS7-a.e. (modified version of ITS7) primer sets on 1 Illumina MiSeq run. The number of high-quality reads obtained by ITS6-ITS7-a.e. was more than twice that of ITS6-ITS7. The relative abundance of Pythium spp. was reduced and Aphanomyces spp. increased. Aphanomyces cf. cladogamus and Aphanomyces euteiches were the second and third most abundant species, respectively, in the pea rhizosphere using the ITS7-a.e. primer, but were rare using the ITS7 primer. These results indicate that use of ITS7-a.e. provides a more accurate picture of oomycete communities than ITS7 by enhancing PCR sensitivity to Aphanomyces.

Metagenomic analysis of oomycete communities from the rhizosphere of field pea on the Canadian prairies.

Oomycetes are a diverse group of microorganisms; however, little is known about their composition and biodiversity in agroecosystems. Illumina MiSeq was used to determine the type and abundance of oomycetes associated with pea root rot in the Canadian prairies. Additional objectives of the study were to identify differences in oomycete communities associated with pea root health and compare oomycete communities among the 3 prairie provinces, where field peas are commonly cultivated. Samples of soil from the rhizosphere of field pea (Pisum sativum L.) were collected from patches of asymptomatic or diseased plants from 26 commercial fields in 2013 and 2014. Oomycete communities were characterized using metagenomic analysis of the ITS1 region on Illumina MiSeq. From 105 identified operational taxonomic units (OTUs), 45 and 16 oomycete OTUs were identified at species and genus levels, respectively. Pythium was the most prevalent genus and Pythium heterothallicum the most prevalent species in all 3 provinces in both 2013 and 2014. Aphanomyces euteiches, a very important pea root rot pathogen in regions of the prairies, was detected in 57% of sites but at very low abundance (<0.2%). Multivariate analysis revealed differences in the relative abundance of species in oomycete communities between asymptomatic and diseased sites, and among years and provinces. This study demonstrated that deep amplicon sequencing can provide information on the composition and diversity of oomycete communities in agricultural soils.

The Dynamics of the Defense Strategy of Pea Induced by Exogenous Nitric Oxide in Response to Aphid Infestation.

The aim of this study was to investigate the effect of exogenous nitric oxide (NO), i.e., S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP), on the metabolic status of Pisum sativum L. cv. Cysterski leaves infested by Acyrthosiphon pisum Harris, population demographic parameters and A. pisum feeding activity. A reduction in the level of semiquinone radicals in pea seedling leaves pretreated with exogenous NO occurred 24 h after A. pisum infestation, which was earlier than in non-pretreated leaves. A decrease in the level of O2•- was observed in leaves pretreated with GSNO and infested by aphids at 48 and 72 h post-infestation (hpi). Directly after the pretreatment with GSNO, an increase in the level of metal ions was recorded. NO considerably induced the relative mRNA levels for phenylalanine ammonia-lyase in 24-h leaves pretreated with NO donors, both non-infested and infested. NO stimulated the accumulation of pisatin in leaves until 24 h. The Electrical Penetration Graph revealed a reduction in the feeding activity of the pea aphid on leaves pretreated with NO. The present study showed that foliar application of NO donors induced sequentially defense reactions of pea against A. pisum and had a deterrent effect on aphid feeding and limited the population growth rate.

Ecdysone signaling underlies the pea aphid transgenerational wing polyphenism.

The wing polyphenism of pea aphids is a compelling laboratory model with which to study the molecular mechanisms underlying phenotypic plasticity. In this polyphenism, environmental stressors such as high aphid density cause asexual, viviparous adult female aphids to alter the developmental fate of their embryos from wingless to winged morphs. This polyphenism is transgenerational, in that the pea aphid mother experiences the environmental signals, but it is her offspring that are affected. Previous research suggested that the steroid hormone ecdysone may play a role in this polyphenism. Here, we analyzed ecdysone-related gene expression patterns and found that they were consistent with a down-regulation of the ecdysone pathway being involved in the production of winged offspring. We therefore predicted that reduced ecdysone signaling would result in more winged offspring. Experimental injections of ecdysone or its analog resulted in a decreased production of winged offspring. Conversely, interfering with ecdysone signaling using an ecdysone receptor antagonist or knocking down the ecdysone receptor gene with RNAi resulted in an increased production of winged offspring. Our results are therefore consistent with the idea that ecdysone plays a causative role in the regulation of the proportion of winged offspring produced in response to crowding in this polyphenism. Our results also show that an environmentally regulated maternal hormone can mediate phenotype production in the next generation, as well as provide significant insight into the molecular mechanisms underlying the functioning of transgenerational phenotypic plasticity.