Chrystel Olivier

Detection of aster yellows phytoplasma DNA in seed and seedlings of canola (Brassica napus and B. rapa) and AY strain identification

Abstract A study was conducted to identify the strains of aster yellows (AY) disease present in crops of Brassica napus and B. rapa grown near Medstead, Saskatchewan. AY phytoplasma DNA was detected in midrib, stem and root tissues of several symptomless plants as well as plants exhibiting typical AY disease symptoms. Most symptomatic and symptomless, AY-infected plants produced normal-looking and misshapen seeds. However, for both Brassica species, symptomatic plants produced significantly more seeds containing phytoplasma DNA than symptomless, AY-infected plants. Also, significantly more misshapen seeds contained phytoplasma DNA than normal seeds. Phytoplasma DNA belonging to subgroups 16SrI-A and 16SrI-B was detected in symptomatic and symptomless, AY-infected plants and in seed of these plants. The new AY strain sequences were registered in Genbank. The study reports for the first time the detection of AY strains in seedling tissues of both Brassica species. The research also showed that spiral cleaning has the potential to remove seeds that contain phytoplasma DNA in B. napus.

Molecular Diagnostic Tools for Detection and Differentiation of Phytoplasmas Based on Chaperonin-60 Reveal Differences in Host Plant Infection Patterns

Phytoplasmas (‘Candidatus Phytoplasma’ spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S–23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60) gene sequence from a diverse array of ‘Ca.Phytoplasma’ spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from ‘Ca.Phytoplasma’ spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns.

Maize bushy stunt phytoplasma affects native corn at high elevations in Southeast Mexico

In the 2013–2014 growing season, field surveys were conducted in native corn fields located in high altitude agricultural communities in the ‘Sierra Norte de Puebla’ in Mexico. Symptoms typical of maize bushy stunt (MBS) disease were observed and DNA extracted from symptomatic native corn plants was used as template to confirm the presence of phytoplasmas. Amplification and sequencing of 16S rRNA-encoding sequences and chaperonin 60 universal target (cpn60 UT) sequences followed by in vitro restriction fragment length polymorphism and phylogenetic analyses revealed that the phytoplasma detected belongs to the subgroup 16SrI-B, ‘Candidatus Phytoplasma asteris’. Based on 16S rRNA-encoding gene sequence analysis and on a single nucleotide polymorphism within the cpn60 UT sequence, two MBS strains, MBS-Puebla and MBS-Veracruz, were identified. This is the first detection of MBS phytoplasma (MBSP) affecting native corn and the first molecular survey made in corn fields in Mexico to detect and characterize MBSP. We discuss these results in light of the potential evolutionary relationship between corn and MBSP.

Leafhoppers and Planthoppers: Their Bionomics, Pathogen Transmission and Management in Vineyards

Auchenorrhyncha is the hemipteran suborder that includes cicadas, leafhoppers, froghoppers or spittlebugs, planthoppers and treehoppers. Leafhoppers (Cicadellidae) are cosmopolitan and one of the largest insect families with approximately 22,000 described species (Forero 2008). Planthoppers (infra-order Fulgoromorpha) are mainly tropical with approximately 20 described families (Urban and Cryan 2007). Leafhoppers and planthoppers have piercing-sucking mouthparts that cause direct damage to plants by feeding in mesophyll cells or on xylem and/or phloem sap, and indirect damage by transmitting pathogens. Because pathogens are not easily managed in plants, the most common control methods rely on the use of insecticides to manage insect populations. This chapter will provide an overview of the life cycles, feeding behavior and vector abilities of leafhoppers and planthoppers causing damage in vineyards. Present and future management methods will be presented in a viticultural context.

Diversity and Abundance of Leafhoppers in Canadian Vineyards

Abstract Leafhoppers (Hemiptera: Cicadellidae) are pests of many temperate crops, including grapevines ( Vitis species). Uncontrolled populations can induce direct and indirect damage to crops due to feeding that results in significant yield losses and increased mortality in infected vineyards due to virus, bacteria, or phytoplasmas vectored by leafhoppers. The main objective of this work was to determine the diversity of leafhoppers found in vineyards of the three main Canadian production provinces, i.e., in British Columbia, Ontario, and Quebec. Approximately 18,000 specimens were collected in 80 commercial vineyards from 2006 to 2008. We identified 54 genera and at least 110 different species associated with vineyards, among which 22 were predominant and represented more than 91% of all the leafhoppers. Species richness and diversity were estimated by both Shannon’s and Pielou’s indices. For each province, results indicated a temporal variation in species composition. Color photographs provide a tool to quickly identify 72 leafhoppers commonly associated with vineyards.

Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences.

Phytoplasmas are unculturable, phytopathogenic bacteria that cause economic losses worldwide. As unculturable micro-organisms, phytoplasma taxonomy has been based on the use of the 16S rRNA-encoding gene to establish 16Sr groups and subgroups based on the restriction fragment length polymorphism (RFLP) pattern resulting from the digestion of amplicon (in vitro) or sequence (in silico) with seventeen restriction enzymes. Problems such as heterogeneity of the ribosomal operon and the inability to differentiate closely related phytoplasma strains has motivated the search for additional markers capable of providing finer differentiation of phytoplasma strains. In this study we developed and validated a scheme to classify phytoplasmas based on the use of cpn60 universal target (cpn60 UT) sequences. Ninety-six cpn60 UT sequences from strains belonging to 19 16Sr subgroups were subjected to in silico RFLP using pDRAW32 software, resulting in 25 distinctive RFLP profiles. Based on these results we delineated cpn60 UT groups and subgroups, and established a threshold similarity coefficient for groups and subgroups classifying all the strains analysed in this study. The nucleotide identity among the reference strains, the correspondence between in vitro and in silico RFLP, and the phylogenetic relationships of phytoplasma strains based on cpn60 UT sequences are also discussed.

Characterization of the feeding behavior of three Erythroneura species on grapevine by histological and DC‐electrical penetration graph techniques

Feeding behavior of three leafhopper species – Erythroneura vitis (Harris), Erythroneura ziczac (Walsh), and Erythroneura elegantula (Say) (Hemiptera: Cicadellidae) – reared on grapevine, Vitis vinifera L. cv. ‘Seyval blanc’ (Vitaceae), was investigated using histological techniques and DC‐electrical penetration graphs (DC‐EPG). Histological studies revealed that the Erythroneura species induced white stipples on the leaves and that these leafhoppers produced thin salivary sheaths in grapevine leaf tissues. The DC‐EPG system allowed the characterization of five waveforms associated with stylet penetration and feeding in leaf tissues. These waveforms were characteristic of feeding phases corresponding to epidermis penetration pathway, salivation, and ingestion. We calculated 28 parameters (e.g., number of probes, duration of phases, and time spent in the various tissues) to describe and compare the feeding behavior of the Erythroneura species. We conclude that the three Erythroneura species are mainly mesophyll feeders but may probably also feed in other tissues such as xylem.

Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico

Geographically diverse samples from strawberry exhibiting symptoms of Strawberry Green Petal (SbGP), periwinkle plants with virescence, and blackberry, blueberry, and raspberry plants displaying yellowing and inedible fruits, were assayed for the presence of phytoplasma DNA. PCR targeting the 16S rRNA-encoding gene and chaperonin-60 (cpn60) showed that the plants were infected with phytoplasma subgroup16SrXIII-(A/I)I (SbGP/MPV). To examine the geographic distribution of this pathogen in Mexico, we designed an array of cpn60-targeted molecular diagnostic assays for SbGP/MPV phytoplasma. A fluorescent microsphere hybridization assay was designed that was capable of detecting SbGP/MPV phytoplasma in infected plant tissues, successfully differentiating it from other known phytoplasma cpn60 UT sequences, while identifying a double infection with SbGP/MPV and aster yellows (16SrI) phytoplasma. Two quantitative assays, quantitative real-time PCR (qRT-PCR) and droplet digital PCR (ddPCR), gave similar results in infected samples. Finally, a loop-mediated isothermal amplification (LAMP) assay provided rapid detection of SbGP/MPV phytoplasma DNA. Application of these assays revealed that SbGP/MPV phytoplasma is widely distributed in Central Mexico, with positive samples identified from eleven localities within three states separated by hundreds of kilometres. These results also provide tools for determining the presence and geographic distribution of this pathogen in plant and insect samples in other localities.

Periwinkle proliferation disease associated with 16SrI-B phytoplasma in Mexico

Catharantus roseus, known as periwinkle, is highly susceptible to phytoplasma infection. Periwinkle plants showing proliferation symptoms were detected during 2013–2014 in four geographically distant states in Mexico. The presence of phytoplasmas was confirmed through the amplification of 16S F2nR2 and cpn60 UT sequences from symptomatic plants. Sequencing, phylogenetic analysis and in vitro RFLP revealed that the isolates were ‘Candidatus Phytoplasma asteris’-related strains and members of the 16SrI-B subgroup, confirming the association of this phytoplasma group with periwinkle proliferation disease in Mexico. We also demonstrated that the use of the approximately 550 pb cpn60 universal target sequences allow the differentiation of two 16SrI-B strains, designated here as MePP-Centre, and MePP-South.

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.