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.

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.