Flavescence dorée phytoplasma (FDp) is a quarantine pathogen associated with a severe and epidemic grapevine yellows disease representing a great threat for grapevine cultivation in Europe. An ...increase in disease spread prompted efforts to identify FDp strains in Croatia. Over 800 samples of grapevine together with presumed reservoir plants and almost 400 samples of Scaphoideus titanus and other potential vectors were collected countrywide and analysed. FDp isolates were characterized by multilocus sequence typing (MLST) of map, secY and uvrB‐degV genes in order to determine genetic diversity and structure of FDp populations, and to trace transmission pathways. FD‐related phytoplasmas were found in Croatia for the first time in alder, the invasive tree species Ailanthus altissima and leafhopper Phlogotettix cyclops. Phylogenetic analysis revealed the presence of three mapFD strain clusters: mapFD1, mapFD2 and mapFD3, and for the first time in Croatia a case of Palatinate grapevine yellows strain A (PGY‐A). In total, 7 different map, 10 secY and 11 uvrB‐degV genotypes were detected. The identification of 15 comprehensive FDp genotypes based on MLST suggests separate routes for disease introduction and propagation origins in Croatia. Moreover, high genetic variability of Croatian isolates indicates a complex ecological cycle of FDp involving various hosts.
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•Feeding behavior of Scaphoideus titanus was studied using electropenetreography.•Males and females had different feeding behaviors.•Scaphoideus titanus fed both in phloem and ...xylem.•This significant mixed diet questions on S. titanus role in pathogen transmission.
In phytophagous piercing-sucking insects, salivary sheath-feeding species are often described as xylem- or phloem-sap feeding specialists. Because these two food sources have very different characteristics, two feeding tactics are often associated with this supposed specialization. Studying the feeding behavior of insects provides substantial information on their biology, ecology, and evolution. Furthermore, study of feeding behavior is of primary importance to elucidate the transmission ability of insects that act as vectors of plant pathogens. In this study, we compared the durations of ingestion performed in xylem versus phloem by a leafhopper species, Scaphoideus titanus Ball, 1932. This was done by characterizing and statistically analyzing electrical signals recorded using the electropenetrography technique, derived from the feeding behaviors of males and females. We identified three groups of S. titanus based on their feeding behavior: 1) a group that reached the phloem quickly and probed for a longer time in phloem tissue than the other groups, 2) a group that reached the xylem quickly and probed for a longer time in xylem tissue than the other groups, and 3) a group where individuals did not ingest much sap. In addition, the numbers and durations of waveforms representing ingestion of xylem and phloem saps differed significantly depending on the sex of the leafhopper, indicating that the two sexes exhibit different feeding behaviors. Males had longer phloem ingestion events than did females, which indicates that males are greater phloem feeders than females. These differences are discussed, specifically in relation to hypotheses about evolution of sap feeding and phytoplasma transmission from plant to plant.
Analyses of space-time statistical features of a flavescence dorée (FD) epidemic in
plants are presented. FD spread was surveyed from 2011 to 2015 in a vineyard of 17,500 m
surface area in the ...Piemonte region, Italy; count and position of symptomatic plants were used to test the hypothesis of epidemic Complete Spatial Randomness and isotropicity in the space-time static (year-by-year) point pattern measure. Space-time dynamic (year-to-year) point pattern analyses were applied to newly infected and recovered plants to highlight statistics of FD progression and regression over time. Results highlighted point patterns ranging from disperse (at small scales) to aggregated (at large scales) over the years, suggesting that the FD epidemic is characterized by multiscale properties that may depend on infection incidence, vector population, and flight behavior. Dynamic analyses showed moderate preferential progression and regression along rows. Nearly uniform distributions of direction and negative exponential distributions of distance of newly symptomatic and recovered plants relative to existing symptomatic plants highlighted features of vector mobility similar to Brownian motion. These evidences indicate that space-time epidemics modeling should include environmental setting (e.g., vineyard geometry and topography) to capture anisotropicity as well as statistical features of vector flight behavior, plant recovery and susceptibility, and plant mortality.
Digital PCR-based methods, such as droplet digital PCR, are one of the best tools for determination of absolute nucleic-acid copy numbers. These techniques avoid the need for reference materials with ...known target concentrations. Compared to real-time PCR, they provide higher accuracy of quantification at low target concentrations, and have higher resilience to inhibitors. In this Chapter, we describe the droplet digital PCR workflow for the detection and quantification of flavescence dorée phytoplasma.
Members of the genus ‘Candidatus Phytoplasma’ are obligate intracellular bacteria restricted to phloem sieve elements and are able to colonize several tissues and the hemolymph in their insect ...vectors. The current unfeasibility of axenic culture and the low complexity of genomic sequences are obstacles in assembling complete chromosomes. Here, a method combining pathogen DNA enrichment from infected insects and dual deep-sequencing technologies was used to obtain the complete genome of a phytoplasma causing Grapevine Flavescence dorée. The de novo assembly generated a circular chromosome of 654,223 bp containing 506 protein-coding genes. Quality assessment of the draft showed a high degree of completeness. Comparative analysis with other phytoplasmas revealed the absence of potential mobile units and a reduced amount of putative phage-derived segments, suggesting a low genome plasticity. Phylogenetic analyses identified Candidatus Phytoplasma ziziphi as the closest fully sequenced relative. The “Flavescence dorée” phytoplasma strain CH genome also encoded for several putative effector proteins potentially playing a role in pathogen virulence. The availability of this genome provides the basis for the study of the pathogenicity mechanisms and evolution of the Flavescence dorée phytoplasma.
“Flavescence dorée” (FD) is a grape vine disease caused by the bacterial agent “Candidatus Phytoplasma vitis” and spread by the leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae). The ...disease is very closely monitored in Europe, as it reduces vine productivity and causes vine death and is also highly transmissible. Currently, the control method used against this disease is a two-pronged approach: i) the spraying of insecticide on a regular basis to kill the vector, and ii) a survey of each row in a vineyard by experts in this disease. Unfortunately, these experts are not able to carry out such a task every year on every vineyard and need an aid for planning their survey.In this study, we propose and evaluate an original automatic method for the detection of FD based on computer vision and artificial intelligence algorithms applied to images acquired by proximal sensing. A two-step approach was used, mimicking an expert’s scouting in the vine rows: (i) the three known isolated symptoms (red or yellow leaves depending on variety, together with a lack of shoot lignification and the presence of desiccated bunches) were detected, (ii) isolated detections were combined to make a diagnosis at image scale; i.e., vine scale. A detection network was used to detect and classify non-healthy leaves into three classes: ‘FD symptomatic leaf', 'Esca leaf' and 'Confounding leaf'; while a segmentation network was used for the retrieval of FD symptomatic shoots and bunches. Finally, the association of detected symptoms was performed by a RandomForest classifier for diagnosis at the image scale. The experimental evaluation was conducted on more than 1000 images collected from 14 blocks planted with five different grape varieties. The detection of the isolated symptoms achieved a precision of between 0.67 and 0.82 and a recall of between 0.39 and 0.59. The classification at the image scale obtained very good results when applied to images acquired under the same conditions, with the same grape varieties as the training images (precision and recall of more than 0.89). The results of the tests on the other grape varieties show the importance of having some of them in the training base in these AI-based approaches.
Endophytic bacteria benefit host plant directly or indirectly, e.g. by biocontrol of the pathogens. Up to now, their interactions with the host and with other microorganisms are poorly understood. ...Consequently, a crucial step for improving the knowledge of those relationships is to determine if pathogens or plant growing season influence endophytic bacterial diversity and dynamic.
Four healthy, four phytoplasma diseased and four recovered (symptomatic plants that spontaneously regain a healthy condition) grapevine plants were sampled monthly from June to October 2010 in a vineyard in north-western Italy. Metagenomic DNA was extracted from sterilized leaves and the endophytic bacterial community dynamic and diversity were analyzed by taxon specific real-time PCR, Length-Heterogeneity PCR and genus-specific PCR. These analyses revealed that both sampling date and phytoplasma infection influenced the endophytic bacterial composition. Interestingly, in June, when the plants are symptomless and the pathogen is undetectable (i) the endophytic bacterial community associated with diseased grapevines was different from those in the other sampling dates, when the phytoplasmas are detectable inside samples; (ii) the microbial community associated with recovered plants differs from that living inside healthy and diseased plants. Interestingly, LH-PCR database identified bacteria previously reported as biocontrol agents in the examined grapevines. Of these, Burkholderia, Methylobacterium and Pantoea dynamic was influenced by the phytoplasma infection process and seasonality.
Results indicated that endophytic bacterial community composition in grapevine is correlated to both phytoplasma infection and sampling date. For the first time, data underlined that, in diseased plants, the pathogen infection process can decrease the impact of seasonality on community dynamic. Moreover, based on experimental evidences, it was reasonable to hypothesize that after recovery the restructured microbial community could maintain the main structure between seasons.
The leafhopper
is the primary vector of Flavescence dorée phytoplasma (FDp) in European vineyards. Flavescence dorée is one of the most severely damaging diseases of
and, consequently, a major threat ...to grape and wine production in several European countries. Control measures are compulsory, but they mainly involve large-scale insecticide treatments, with detrimental impacts on the environment. One possible solution is to exploit the largely unexplored genetic diversity of viruses infecting
as highly specific and environmentally benign tools for biological control.
A metatranscriptomic approach was adopted to identify viruses that may infect individuals caught in the wild in both its native (United States) and invasive (Europe) areas. Reverse transcription PCR was used to confirm their presence in RNA pools and explore their prevalence.
We described nine new RNA viruses, including members of "Picorna-Calici", "Permutotetra", "Bunya-Arena", "Reo", "Partiti-Picobirna", "Luteo-Sobemo" and "Toti-Chryso" clades. A marked difference in the diversity and abundance of the viral species was observed between the US population and the European ones.
This work represents the first survey to assess the viral community of a phytoplasma insect vector. The possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of
could be the answer to increasing demand for a more sustainable viticulture.
The leafhopper Scaphoideus titanus is able to transmit 16SrV phytoplasmas agents of grapevine's flavescence dorée (FD) within 30–45 days, following an acquisition access period (AAP) of a few days ...feeding on infected plants as a nymph, a latency period (LP) of 3–5 weeks becoming meanwhile an adult, and an inoculation access period (IAP) of a few days on healthy plants. However, several aspects of FD epidemiology suggest how the whole transmission process may take less time, and may start directly with adults of the insect vector. Transmission experiments have been set up under lab condition. Phytoplasma‐free S. titanus adults were placed on broad bean (BB) plants (Vicia faba) infected by FD‐C (16SrV‐C) phytoplasmas for an AAP = 7 days. Afterwards, they were immediately moved onto healthy BB for IAP, which were changed every 7 days, obtaining three timings of inoculation: IAP 1, IAP 2 and IAP 3, lasting 7, 14 and 21 days from the end of AAP, respectively. DNA was extracted from plants and insects, and PCR tests were performed to identify FD phytoplasmas. Insects were dissected and fluorescence in situ hybridisation was made to detect the presence of phytoplasmas in midguts and salivary glands. The rate of infection in insects ranged 46–68% without significant differences among IAPs. Inoculation in plants succeeded in all IAPs, at a rate of 16–23% (no significant differences). Phytoplasma load was significantly higher in IAP 3 than IAP 1–2 for both plants and insects. Phytoplasmas were identified both in midgut and salivary glands of S. titanus at all IAP times. The possible implications of these results in the epidemiology of flavescence dorée are discussed.
The phytoplasmal agent of Flavescence dorée (FD) is transmitted to grapevine by the leafhopper Scaphoideus titanus. Even though 30‐45 days are reported to be required from acquisition (by nymphs) to inoculation (by adults), successful transmission could be achieved even after a shorter period, and it could start directly from adults. FD phytoplasma transmission experiments with S. titanus adults revealed that as soon as 14 days from acquisition the leafhoppers were able to transmit the pathogen to healthy broad beans. The results of this work open to new epidemiologic scenarios, which should be taken into account for FD management.
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•Feeding waveforms were characterized and their biological meanings defined.•Four typical waveform phases and four families within those phases were characterized.•Sustained ingestion ...by a deltocephaline leafhopper can occur from both xylem and phloem vascular cells.•EPG-recorded X wave was very similar to the model X wave of sharpshooters.
The leafhopper Scaphoideus titanus (Hemiptera: Cicadomorpha: Cicadellidae), an invasive deltocephaline species introduced into Europe from North America, is the vector of the most important phytoplasma disease in European viticulture, flavescence dorée. In this first electropenetrography (EPG) study of S. titanus, we characterized its feeding waveforms and defined their biological meanings. Four typical waveform phases (pathway, X wave, sustained ingestion, and interruption) and four families within those phases (A, B, C, and N) were characterized using DC EPG technology. We proposed biological meanings for these waveforms based on excreta pH-ingestion correlations, presence of X waves, and comparison with previous AC, DC, and AC-DC EPG waveforms conducted on Cicadomorpha. We observed that sustained (i.e., >10min) ingestion by a deltocephaline leafhopper can occur from both xylem and phloem vascular cells. Waveform C2x represented ingestion of xylem fluid, and two waveforms represented behaviors when stylets were inserted into phloem sieve elements: C2p variant 1 (C2p-1), which may represent salivation (perhaps simultaneous with ingestion), and C2p variant 2 (C2p-2), which represented active ingestion. Furthermore, we found that the EPG-recorded X wave has a dual meaning by occurring prior to sustained ingestion from either phloem or xylem. This X wave was very similar in appearance to the model X wave of sharpshooters, an entirely different leafhopper subfamily, Cicadellinae. All cicadellines are obligate xylem-ingesters. Such a “dual-meaning X wave” will provide insights into how the feeding tactics of S. titanus relate to other sheath-feeding hemipterans, and will provide support for future research to clarify the role of this leafhopper as a vector of plant pathogens.
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