Volatile Organic Compounds Mediate Host Selection of Wheat Midge, Sitodiplosis Mosellana (Géhin) (Diptera: Cecidomyiidae) between Preanthesis and Postanthesis Stages of Wheat.

The orange wheat blossom midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), is a significant wheat pest in the Prairie Provinces of Canada and northern regions of the USA. Wheat phenology plays a critical role in wheat midge oviposition. We hypothesized that S. mosellana oviposition behaviour is influenced by volatile organic compounds (VOCs) emitted by wheat at two adjacent wheat growth stages: preanthesis and postanthesis. A higher number of S. mosellana eggs laid on preanthesis than postanthesis spikes in an oviposition choice experiment using the susceptible spring wheat cultivar 'Roblin'. In preanthesis, wheat emitted higher amounts of Z-3-hexenyl acetate (Z3-06:OAc) than at the postanthesis stage. Higher amounts of methyl ketones such as 2-tridecanone, 2-pentadecanone, and 2-undecanone were emitted by wheat in the postanthesis stage and these VOCs were sensitive to S. mosellana antennae used in the Gas Chromatography-Electroantennographic Detection. Females were attracted to synthetic Z3-06:OAc but were deterred by 2-tridecanone relative to the solvent control in the vertical Y-tube olfactometer. 2-Undecanone and 2-pentadecanone did not show any attractiveness or deterrence. In a no-choice oviposition experiment, fewer eggs were laid in preanthesis wheat exposed to a synthetic VOC blend of Z3-06:OAc, 2-undecanone, 2-tridecanone, and 2-pentadecanone at the concentrations released by postanthesis spikes. This study shows that the reduction of Z3-06:OAc, in the VOC mix, and possibly the increase in 2-tridecanone, are likely responsible for the reduction in oviposition on postanthesis wheat. These results elucidate for the first time the role of specific VOCs mediating S. mosellana oviposition in preanthesis and postanthesis wheat.

The Role of Plant Defense Signaling Pathways in Phytoplasma-Infected and Uninfected Aster Leafhoppers' Oviposition, Development, and Settling Behavior.

In plant-microbe-insect systems, plant-mediated responses involve the regulation and interactions of plant defense signaling pathways of phytohormones jasmonic acid (JA), ethylene (ET), and salicylic acid (SA). Phytoplasma subgroup 16SrI is the causal agent of Aster Yellows (AY) disease and is primarily transmitted by populations of aster leafhoppers (Macrosteles quadrilineatus Forbes). Aster Yellows infection in plants is associated with the downregulation of the JA pathway and increased leafhopper oviposition. The extent to which the presence of intact phytohormone-mediated defensive pathways regulates aster leafhopper behavioral responses, such as oviposition or settling preferences, remains unknown. We conducted no-choice and two-choice bioassays using a selection of Arabidopsis thaliana lines that vary in their defense pathways and repeated the experiments using AY-infected aster leafhoppers to evaluate possible differences associated with phytoplasma infection. While nymphal development was similar among the different lines and groups of AY-uninfected and AY-infected insects, the number of offspring and individual female egg load of AY-uninfected and AY-infected insects differed in lines with mutated components of the JA and SA signaling pathways. In most cases, AY-uninfected insects preferred to settle on wild-type (WT) plants over mutant lines; no clear pattern was observed in the settling preference of AY-infected insects. These findings support previous observations in other plant pathosystems and suggest that plant signaling pathways and infection with a plant pathogen can affect insect behavioral responses in more than one manner. Potential differences with previous work on AY could be related to the specific subgroup of phytoplasma involved in each case.

Characterization and correlation of the probing behaviors of Macrosteles quadrilineatus (Hemiptera: Cicadellidae) with electropenetrography (EPG) waveforms.

Aster leafhopper (Hemiptera: Cicadellidae: Macrosteles quadrilineatus Forbes) is a polyphagous insect species that migrates into the upper Midwest of the United States and the Western Canadian Prairies. Populations of this insect are associated with the transmission of a plant pathogen (Candidatus Phytoplasma asteris, 16SrI) to several annual crops and perennial plant species. Previous studies suggest that aster leafhoppers can sometimes prefer less suitable hosts for their development and survival, yet it is unclear if this lower performance on certain plant species is associated with reduced or impaired probing behaviors due to characteristics of the plants. To characterize the probing behaviors of aster leafhoppers, direct current electropenetrography recordings of male and female adults on barley (Polaes: Poaceae: Hordeum vulgare L.) were combined with plant histology, allowing the identification of nine waveforms and their proposed biological meanings. For each waveform, the number of waveform events per insect (NWEI), the waveform duration per insect (WDI), the waveform duration per event per insect (WDEI), and the percentage of recording time were calculated and statistically compared between sexes. Male and female aster leafhoppers exhibited similar behavioral responses for most of these variables, except for the NWEI for waveforms associated with nonprobing activities and the pathway phase. In these cases, male aster leafhoppers exhibited a higher number of events than females. Comparison of the proposed waveforms in this study with previous work on other hemipteran species provided additional support to the interpretation of the biological activities associated with each waveform.

Evolutionary genomics of three agricultural pest moths reveals rapid evolution of host adaptation and immune-related genes.

BACKGROUND: Understanding the genotype of pest species provides an important baseline for designing integrated pest management (IPM) strategies. Recently developed long-read sequence technologies make it possible to compare genomic features of nonmodel pest species to disclose the evolutionary path underlying the pest species profiles. Here we sequenced and assembled genomes for 3 agricultural pest gelechiid moths: Phthorimaea absoluta (tomato leafminer), Keiferia lycopersicella (tomato pinworm), and Scrobipalpa atriplicella (goosefoot groundling moth). We also compared genomes of tomato leafminer and tomato pinworm with published genomes of Phthorimaea operculella and Pectinophora gossypiella to investigate the gene family evolution related to the pest species profiles. RESULTS: We found that the 3 solanaceous feeding species, P. absoluta, K. lycopersicella, and P. operculella, are clustered together. Gene family evolution analyses with the 4 species show clear gene family expansions on host plant-associated genes for the 3 solanaceous feeding species. These genes are involved in host compound sensing (e.g., gustatory receptors), detoxification (e.g., ABC transporter C family, cytochrome P450, glucose-methanol-choline oxidoreductase, insect cuticle proteins, and UDP-glucuronosyl), and digestion (e.g., serine proteases and peptidase family S1). A gene ontology enrichment analysis of rapid evolving genes also suggests enriched functions in host sensing and immunity. CONCLUSIONS: Our results of family evolution analyses indicate that host plant adaptation and pathogen defense could be important drivers in species diversification among gelechiid moths.

Development of economic thresholds for pea aphid (Hemiptera: Aphididae) management in lentil (Fabaceae) based on in-field insecticide efficacy trials.

Pea aphid (Acyrthosiphom pisum Harris, Hemiptera: Aphididae) presents a significant economic challenge to lentil (Lens culinaris Medik.) production in the major growing region of Saskatchewan, Canada. During 2019-2020, field experiments were conducted to optimize the management tools for pea aphid control on lentils. A randomized split-plot design was used with main plots consisting of different pea aphid pressures and subplots consisting of different insecticide treatments. The main plot design was aimed to assess the impact of A. pisum feeding on lentil yields during the late vegetative to early reproductive stages. Subplots of the study evaluated the efficacy of 3 insecticides in suppressing pea aphid populations on lentils. Lentil is susceptible to A. pisum feeding and requires management at low pest densities. The economic threshold for pea aphids on lentil crops varied depending on environmental conditions, ranging from 20 to 66 aphids per sweep, calculated using a discrete daily growth rate of 1.116. The estimated economic thresholds provided a 7-day lead time before aphid populations achieved the economic injury level (EIL). The EIL was defined as 78 ± 14 aphids per sweep net sample or 743 ± 137 cumulative aphid days from the first aphid present in the field. In addition, the results of the study found that, on average, foliar applications of insecticides containing the pyrethroid active ingredient lambda-cyhalothrin (IRAC group: 3A) reduced pea aphid populations by 83% compared with untreated control.

Multilocus sequence typing of diverse phytoplasmas using hybridization probe-based sequence capture provides high resolution strain differentiation.

Phytoplasmas are insect-vectored, difficult-to-culture bacterial pathogens that infect a wide variety of crop and non-crop plants, and are associated with diseases that can lead to significant yield losses in agricultural production worldwide. Phytoplasmas are currently grouped in the provisional genus 'Candidatus Phytoplasma', which includes 49 'Candidatus' species. Further differentiation of phytoplasmas into ribosomal groups is based on the restriction fragment length polymorphism (RFLP) pattern of the 16S rRNA-encoding operon, with more than 36 ribosomal groups (16Sr) and over 100 subgroups reported. Since disease symptoms on plants are not associated with phytoplasma identity, accurate diagnostics is of critical importance to manage disease associated with these microorganisms. Phytoplasmas are typically detected from plant and insect tissue using PCR-based methods targeting universal taxonomic markers. Although these methods are relatively sensitive, specific and are widely used, they have limitations, since they provide limited resolution of phytoplasma strains, thus necessitating further assessment of biological properties and delaying implementation of mitigation measures. Moreover, the design of PCR primers that can target multiple loci from phytoplasmas that differ at the sequence level can be a significant challenge. To overcome these limitations, a PCR-independent, multilocus sequence typing (MLST) assay to characterize an array of phytoplasmas was developed. Hybridization probe s targeting cpn60, tuf, secA, secY, and nusA genes, as well as 16S and rp operons, were designed and used to enrich DNA extracts from phytoplasma-infected samples for DNA fragments corresponding to these markers prior to Illumina sequencing. This method was tested using different phytoplasmas including 'Ca. P. asteris' (16SrI-B), 'Ca. P. pruni' (16SrIII-A),'Ca. P. prunorum' (16SrX-B), 'Ca. P. pyri' (16SrX-C), 'Ca. P. mali' (16SrX-A), and 'Ca. P. solani' (16SrXII-A). Thousands of reads were obtained for each gene with multiple overlapping fragments, which were assembled to generate full-length (typically >2 kb), high-quality sequences. Phytoplasma groups and subgroups were accurately determined based on 16S ribosomal RNA and cpn60 gene sequences. Hybridization-based MLST facilitates the enrichment of target genes of phytoplasmas and allows the simultaneous determination of sequences corresponding to seven different markers. In this proof-of-concept study, hybridization-based MLST was demonstrated to be an efficient way to generate data regarding 'Ca. Phytoplasma' species/strain differentiation.

Rapid Molecular Diagnostics in the Field and Laboratory to Detect Plant Pathogen DNA in Potential Insect Vectors.

A variety of sensitive and specific molecular diagnostic assays has been described for detecting nucleic acids in biological samples that may harbor pathogens of interest. These methods include very rapid, isothermal nucleic acid amplification methods that can be deployed outside of the laboratory environment, such as loop-mediated isothermal DNA amplification (LAMP) and recombinase-polymerase amplification (RPA). However, all molecular diagnostic assays must be preceded by nucleic acid extraction from the biological samples of interest, which provides suitable template molecules for the assays. To exploit the features of the amplification assays and be utilized outside of the lab, these methods must be rapid and avoid the need for typical laboratory chemicals and equipment. We describe a protocol for the extraction of DNA from field-collected insects that can be implemented at the point of collection and used to detect the presence of DNA sequences from potential plant pathogens that may be vectored by the insects. This protocol provides template DNA that is suitable for PCR, LAMP, and RPA. The FTA PlantSaver card-based DNA extraction product was also confirmed to amplify the mitochondrial cytochrome oxidase 1 (CO1) universal barcode that could later be sequenced to identify any insect. Lastly, we provide an example using field-collected insects, Neokolla (Graphocephala) heiroglyphica, and demonstrate the detection of the plant pathogen Xylella fastidiosa in carrier insects using PCR, RPA, and LAMP.

Do Options Matter? Settling Behavior, Stylet Sheath Counts, and Oviposition of Aster Leafhoppers (Hemiptera: Cicadellidae) in Two-Choice Bioassays.

Polyphagous insects are characterized by a broad diet comprising plant species from different taxonomic groups. Within these insects, migratory species are of particular interest, given that they encounter unpredictable environments, with abrupt spatial and temporal changes in plant availability and density. Aster leafhoppers (Hemiptera: Cicadellidae: Macrosteles quadrilineatus Forbes) arrive in the Canadian Prairies in spring and early summer and are the main vector of a prokaryotic plant pathogen known as Aster Yellows Phytoplasma (AYp) (Candidatus Phytoplasma asteris). Host choice selection behavior of Aster leafhoppers was evaluated through two-choice bioassays, using domesticated and wild plants species commonly found in the Canadian Prairies. Leaf tissues from these plants were collected and stained to quantify the number of stylet sheaths and eggs. To assess possible effects due to insect infection, two-choice bioassays were repeated using leafhoppers infected with AYp and a subset of plant species. When two domesticated or wild plant species were presented together, similar numbers of uninfected Aster leafhoppers were observed on both plant species in most combinations. In domesticated-wild plant bioassays, uninfected Aster leafhoppers preferred to settle on the domesticated species. There was little to no association between settling preferences and stylet sheath and egg counts. These findings provide a better understanding of AY epidemiology and suggest that after domesticated species germination, leafhoppers could move from nearby wild plants into the preferred cereals (Poales: Poaceae) to settle on them, influencing the risk of AYp infection in some of these species.

Detection and Identification of a 'Candidatus Liberibacter solanacearum' Species from Ash Tree Infesting Psyllids.

'Candidatus Liberibacter' species are associated with severe, economically important diseases. Nearly all known species are putatively insect transmitted, specifically by psyllids. Detection of 'Ca. Liberibacter' in plants is complicated by their uneven distribution in host plants and largely fastidius nature. The death of black (Fraxinus nigra) and mancana (Fraxinus mandshurica) ash trees in Saskatchewan, Canada has been associated with infestation by the cottony ash psyllid (Psyllopsis discrepans). A combination of conventional PCR amplification and Sanger sequencing of the 16S recombinant DNA was used to detect and identify 'Ca. Liberibacter' in psyllids collected from ash trees in Saskatchewan. BLAST analysis of two 16S sequences that were 1,058 and 1,085 bp long (NTHA 5, GenBank accession number MK942379 and NTHA 6, GenBank accession number MK937570, respectively) revealed they were 99 to 100% similar to a 'Ca. Liberibacter solanacearum' sequence (GenBank accession number KX197200) isolated from the Nearctic psyllid (Bactericera maculipennis) of U.S. provenance. Sequencing the psyllid genes CO1 and Cyt-b confirmed that the psyllids from which the bacterial DNA was isolated were P. discrepans, based on comparisons with sequences in GenBank and BOLD and a reference sample from the United Kingdom. These results provide the first evidence that 'Ca. Liberibacter solanacearum' species are associated with psyllids collected from ash trees and specifically P. discrepans. The recent episodes of dieback of ash in Saskatchewan associated with psyllid feeding are consistent with disease symptoms caused by 'Ca. Liberibacter' pathogens, and this possibility warrants further study.

Oviposition Behavior and Development of Aster Leafhoppers (Hemiptera: Cicadellidae) on Selected Host Plants From the Canadian Prairies.

Some plant pathogens are capable of manipulating their insect vectors and plant hosts in a way that disease transmission is enhanced. Aster leafhopper (Macrosteles quadrilineatus Forbes) (Hemiptera: Cicadellidae) is the main vector of Aster Yellows Phytoplasma (Candidatus Phytoplasma asteris) in the Canadian Prairies, which causes Aster Yellows (AY) disease in over 300 plant species including cereals and oilseeds. However, little is known about the host range of Aster leafhoppers or their host-choice selection behavior in this geographical region. Several crop and noncrop species commonly found in the Canadian Prairies were evaluated as food and reproductive hosts for Aster leafhoppers through no-choice bioassays. To study possible effects of pathogen infection, AY-uninfected and AY-infected insects were used. Cereals and some noncrops like fleabane were suitable reproductive hosts for Aster leafhoppers, with numbers of offspring observed in treatments using both AY-uninfected and AY-infected insects, suggesting an egg-laying preference on these plant species. Development was similar across the different plant species, except for canola and sowthistle, where growth indexes were lower. Sex-ratios of Aster leafhopper adults did not differ among the plant species or with respect to AY infection. Potential fecundity differed across plant species and was affected by the infection status of the insect. These findings have implications for AY epidemiology and suggest that while cereals can be suitable host plants for Aster leafhopper oviposition and development, some noncrop species could act as alternate hosts for leafhoppers that migrate into the Canadian Prairies before emergence of cereal and canola crops.

Genome Sequence of a Plant-Pathogenic Bacterium, "Candidatus Phytoplasma asteris" Strain TW1.

A draft genome sequence is presented for a strain of "Candidatus Phytoplasma asteris" affecting canola plants in Saskatoon, Canada. This phytopathogenic bacterium was determined to be a 16SrI strain and features 16S rRNA-encoding gene sequence heterogeneity.

State-Dependent Plasticity in Response to Host-Plant Volatiles in a Long-Lived Moth, Caloptilia fraxinella (Lepidoptera: Gracillariidae).

Volatile chemicals produced by plants mediate host location, mate-finding and oviposition behavior in insects. State-dependent response to plant cues allows for timing of foraging, mating and oviposition on ephemeral host plants or plant parts. Caloptilia fraxinella is a herbivorous specialist on the foliage of ash trees (Fraxinus). Adults are long-lived and undergo a nine-month reproductive diapause over the fall and winter. Mating and oviposition occur in the spring when volatile chemicals released by ash leaves mediate host location. This study tested the plasticity of olfactory response of C. fraxinella to host plant volatiles using both electroantennogram and behavioral bioassays. The effect of moth physiological state on olfactory response was tested on male and female moths in different nutritional, mating, and diapause states. Antennal responses to host plant volatiles were plastic and depended on moth physiological state, and were highest when moths were reproductively active and would be seeking oviposition hosts. Moth sex and nutritional status also impacted antennal response to host plant volatiles. Oriented flight of females to ash seedlings varied with physiological state and nutritional status, with fed, reproductively active females having the highest response. Physiological state impacted oriented flight of males to female-produced sex pheromone signals whether or not a host plant was present, and there was no increase in behavioral response to sex pheromone in the presence of an ash host.

Aster leafhopper survival and reproduction, and Aster yellows transmission under static and fluctuating temperatures, using ddPCR for phytoplasma quantification.

Aster yellows (AY) is an important disease of Brassica crops and is caused by Candidatus Phytoplasma asteris and transmitted by the insect vector, Aster leafhopper (Macrosteles quadrilineatus). Phytoplasma-infected Aster leafhoppers were incubated at various constant and fluctuating temperatures ranging from 0 to 35 °C with the reproductive host plant barley (Hordium vulgare). At 0 °C, leafhopper adults survived for 18 days, but failed to reproduce, whereas at 35 °C insects died within 18 days, but successfully reproduced before dying. Temperature fluctuation increased thermal tolerance in leafhoppers at 25 °C and increased fecundity of leafhoppers at 5 and 20 °C. Leafhopper adults successfully infected and produced AY-symptoms in canola plants after incubating for 18 days at 0-20 °C on barley, indicating that AY-phytoplasma maintains its virulence in this temperature range. The presence and number of AY-phytoplasma in insects and plants were confirmed by droplet digital PCR (ddPCR) quantification. The number of phytoplasma in leafhoppers increased over time, but did not differ among temperatures. The temperatures associated with a typical crop growing season on the Canadian Prairies will not limit the spread of AY disease by their predominant insect vector. Also, ddPCR quantification is a useful tool for early detection and accurate quantification of phytoplasma in plants and insects.

Evaluation of inflorescence visitors as pollinators of Echinacea angustifolia (Asteraceae): comparison of techniques.

Inflorescences (heads or capitula) of the putative self-incompatible species, purple coneflower (Echinacea angustifolia (DC) Cronq. (Asteraceae)), were visited by insects representing the Coleoptera, Diptera, Hymenoptera, and Lepidoptera, in accordance with a generalist pollination syndrome. Measurement of the effectiveness of insect species as pollinators was accomplished by permitting solitary visits to receptive, central disc florets of virgin (previously bagged) heads. Four parameters were quantified: total stigmatic pollen load and proportion of pollen grains germinated, numbers of pollen tubes at style bases, and percentages of total receptive florets that had retracted (shrivelled) styles. Quantifying total and germinated pollen grains proved ineffective, partly owing to the tendency of self-pollen to initiate pollen tubes. The most effective pollinators were Apidae, especially bumble bees (Bombus spp.) and the European honey bee (Apis mellifera L.) (mean: 39 - 61% of styles retracted). Other noteworthy pollinators were cloudless sulfur butterflies (Phoebis sennae L.--Pieridae; mean 47% of style bases with pollen tubes), golden blister beetles (Epicauta ferruginea Say--Meloidae; 44%), and grasshopper bee flies (Systoechus vulgaris Loew--Bombyliidae; 22%). Sunflower leafcutter bees (Megachile pugnata Say) were less effective (4% of styles retracted). Promisingly, analysis of the proportion of retracted styles provided similar results to the established technique of pollen-tube quantification, but had the significant advantages of being completed more rapidly, without a microscope, and in the field. The quantitative technique of retracted-style analysis appears well suited for prompt measurement of inflorescence-visiting insects as pollinators of many asteraceans.

Floral nectar production and nectary anatomy and ultrastructure of Echinacea purpurea (Asteraceae).

BACKGROUND AND AIMS: In spite of the impressive species diversity in the Asteraceae and their widespread appeal to many generalist pollinators, floral-nectary ultrastructure in the family has rarely been investigated. To redress this, a study using Echinacea purpurea, a plant of horticultural and nutraceutical value, was undertaken. Nectar secretion of disc florets was compared with floral nectary ultrastructure taking into account nectar's potential impact upon the reproductive success of this outcrossing species. METHODS: Micropipette collections of nectar in conjunction with refractometry were used to determine the volume and nectar-sugar quantities of disc florets throughout their phenology, from commencement of its production to cessation of secretion. Light, scanning-electron and transmission-electron microscopy were utilized to examine morphology, anatomy and ultrastructure of nectaries of the disc florets. KEY RESULTS: Florets were protandrous with nectar being secreted from anthesis until the third day of the pistillate phase. Nectar production per floret peaked on the first day of stigma receptivity, making the two innermost whorls of open florets most attractive to foraging visitors. Modified stomata were situated along the apical rim of the collar-like nectary, which surrounds the style base and sits on top of the inferior ovary. The floral nectary was supplied by phloem only, and both sieve elements and companion cells were found adjacent to the epidermis; the latter participated in the origin of some of the precursor cells that yielded these specialized cells of phloem. Companion cells possessed wall ingrowths (transfer cells). Lobed nuclei were a key feature of secretory parenchyma cells. CONCLUSIONS: The abundance of mitochondria suggests an eccrine mechanism of secretion, although dictyosomal vesicles may contribute to a granulocrine process. Phloem sap evidently is the main contributor of nectar carbohydrates. From the sieve elements and companion cells, an apoplastic route via intercellular spaces and cell walls, leading to the pores of modified stomata, is available. A symplastic pathway, via plasmodesmata connecting sieve elements to companion, parenchyma and epidermal cells, is also feasible. Uncollected nectar was reabsorbed, and the direct innervation of the nectary by sieve tubes potentially serves a second important route for nectar-sugar reclamation. Microchannels in the outer cuticle may facilitate both secretion and reabsorption.