Flavescence dorée phytoplasma (FDP) titre in two red grapevine cultivars, Barbera and Nebbiolo, was measured over the vegetative seasons of two consecutive years in two vineyards of the Piemonte ...Region (northwestern Italy), with a double absolute quantification of FDP cells and grapevine DNA in real‐time PCR. The relationships of pathogen concentration to cultivar susceptibility and symptom severity were investigated. FDP titre was always higher in cv. Barbera than in cv. Nebbiolo infected vines, and this difference was significant at early and late summer samplings of 2008 and at early summer sampling of 2009. A seasonal trend in FDP concentration (low in spring, high in early summer and intermediate in late summer) was conserved for cvs Barbera and Nebbiolo in both years and vineyards. Considering both cultivars and years from both vineyards, a significant positive correlation between FDP concentration and symptom severity was found in the spring samples. Regarding the FDP strains (‐C or ‐D), no differences in pathogen titres were detected for either cultivar. Similarly, the presence of another grapevine yellows phytoplasma, bois noir, a subgroup 16SrXII‐A phytoplasma, in mixed infection with FDP strains had no effect on FDP concentration. These results demonstrate for the first time that grapevine cultivars with different susceptibility to FDP support different pathogen titres.
(FD) is a grapevine disease caused by phytoplasmas and transmitted by leafhoppers that has been spreading in European vineyards despite significant efforts to control it. In this study, we aim to ...develop a model for the automatic detection of FD-like symptoms (which encompass other grapevine yellows symptoms). The concept is to detect likely FD-affected grapevines so that samples can be removed for FD laboratory identification, followed by uprooting if they test positive, all to be conducted quickly and without omission, thus avoiding further contamination in the fields. Developing FD-like symptoms detection models is not simple, as it requires dealing with the complexity of field conditions and FD symptoms' expression. To address these challenges, we use deep learning, which has already been proven effective in similar contexts. More specifically, we train a Convolutional Neural Network on image patches, and convert it into a Fully Convolutional Network to perform inference. As a result, we obtain a coarse segmentation of the likely FD-affected areas while having only trained a classifier, which is less demanding in terms of annotations. We evaluate the performance of our model trained on a white grape variety, Chardonnay, across five other grape varieties with varying FD symptoms expressions. Of the two largest test datasets, the true positive rate for Chardonnay reaches 98.48% whereas for Ugni-Blanc it drops to 8.3%, underlining the need for a multi-varietal training dataset to capture the diversity of FD symptoms. To obtain more transparent results and to better understand the model's sensitivity, we investigate its behavior using two visualization techniques, Guided Gradient-weighted Class Activation Mapping and the Uniform Manifold Approximation and Projection. Such techniques lead to a more comprehensive analysis with greater reliability, which is essential for in-field applications, and more broadly, for all applications impacting humans and the environment.
Quantitative PCR protocols for phytoplasma detection were used to monitor grapevine yellows (GY) in 373 vineyards located in nine Tuscan districts. Among more than 70,000 plants visually monitored, ...1.867 plants were sampled and “flavescence dorée” phytoplasmas (FD) were detected in 122 plants and mainly identified as trains belonging to 16SrV-C subgroup. The “bois noir” (BN) phytoplasma was found in 734 samples, with prevalence of tufB type-b strains. The 2013–2015 monitoring program was strongly influenced by the first survey (2012) in which FD was found consistently in the North West (15 samples), whereas only a few cases were observed in the East territory (2 samples). Both areas were thoroughly monitored in the following years: few foci were found in the East (2 in 2014, 1 in 2015), while several infected areas were found in the North West (6, 10 and 22 foci in 2013, 2014 and 2015, respectively). Definitely, the novel FD foci detected in the survey (17, 6, 12 and 23 in each year of survey) and the widespread of BN, suggest a dangerous distribution of GY in Tuscany.
Laboratory trials were carried out under controlled conditions (24 °C, 75% RH, 16L:8D) to evaluate the effect of six selected insecticides on egg hatching of Scaphoideus titanus Ball (Hemiptera ...Cicadellidae). Standardized cuttings of two-year-old Isabella (Vitis vinifera × Vitis labrusca) canes were used as test plant material. A newly developed test cage allowed the treatment of infested canes in a vertical position and a standardized assessment of the number of S. titanus nymphs on yellow sticky traps over the whole hatching period. The six test substances etofenprox, spirotetramat, paraffin oil, azadirachtin, spirodiclofen and aluminium silicate (kaolinite) resulted in a reduction of egg hatching of S. titanus of 100%, 99%, 83%, 72%, 64% and 47% in relation to the control (water) respectively. The present study suggests that plant protection treatments against the immobile stage of S. titanus at an earlier application date (BBCH-scale 13-17) than the usually recommended one in Austria (BBCH 65-77) could effectively reduce S. titanus population at the start of the growing season and lead to an improved suppression of the vector-assisted spread of Flavescence dorée phytoplasma.
Suzbijanje zlatne žutice vinove loze obavezno je u područjima gdje je potvrđena njena pojava, a mjere suzbijanja su u većini vinogradarskih regija Europe regulirane pravnim aktima, odnosno naredbama ...o suzbijanju. Najučinkovitije mjere suzbijanja zlatne žutice uključuju suzbijanje američkog cvrčka primjenom insekticida, budući da je američki cvrčak primarni vektor zaraze te uklanjanje i uništavanje simptomatičnih trsova, koje treba ukloniti u što kraćem roku od prve pojave simptoma. Pored toga, kako bi se smanjio izvor nove zaraze, potrebno je krčiti zapuštene vinograde ili samoniklu lozu koja se nalazi u blizini proizvodnih vinograda. U svrhu uspješnog uklanjanja zaraženih trsova iznimno je važno pratiti pojavu simptoma unutar vinograda, što se provodi organizirano od strane stručnih službi na razini šireg vinogradarskog područja, ali i od strane proizvođača, koji trebaju pratiti pojavu simptoma u vlastitim vinogradima. U slučaju izostanka primjene mjera suzbijanja, zlatna žutica se može epidemijski širiti te se u svega nekoliko godina može zaraziti veći dio trsova u vinogradu. U ovom je radu dan prikaz mjera suzbijanja zlatne žutice s ciljem smanjenja šteta koje ona pričinjava i sprječavanja širenja zaraze.
Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine ...fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of "flavescence dorée" phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of "flavescence dorée", makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years.
Many aspects of plant diseases caused by phytoplasmas are still unknown, as these pathogens are phloem restricted, uncultivable wall-less bacteria and must be studied always in association with their ...host. Phytoplasma transcripts are strongly underrepresented within host tissues and this poses problems for gene expression analyses. In this study, a procedure was established to infect the model plant Arabidopsis thaliana with the phytoplasma Flavescence dorée, a serious threat to European viticulture. Rates of phytoplasma infective insects and transmission efficiency to A. thaliana as well as pathogen loads were measured in different tissues of infected A. thaliana plants, and modification of phloem cell ultrastructure was observed in infected plant tissues at microscopic level. Moreover, a protocol for the application of laser microdissection to analyze plant and phytoplasma gene expression profiles in the specific colonized tissue was designed. The procedure allowed a good preservation of the plant tissue anatomy. Results showed that the extracted RNA was suitable for qualitative and quantitative RT-PCR, since both plant and pathogen transcripts, either abundant or rare ones, could be detected without any pre-amplification step. The combined use of laser microdissection approach and A. thaliana to study phytoplasmas opens the way to exploit biological, molecular and bioinformatic tools available for the model plant and to elucidate key pathways of the infection mechanisms of these important plant pathogen.
The transmission of microbial pathogens by insect vectors can be affected by the insect’s microbial symbionts, which may compete in colonizing organs, express antagonistic factors or activate host ...immune response. Acetic acid bacteria of the genus
Asaia
are symbionts of the leafhopper
Scaphoideus titanus
, which transmits Flavescence dorée phytoplasma. These bacteria could be used as control agents against the disease. Here, we experimentally investigated the interaction between different strains of
Asaia
and phytoplasma transmission in the laboratory by using the model leafhopper
Euscelidius variegatus
and the plant host
Vicia faba
. We found that uncultivable and low concentrations of
Asaia
phylotypes were associated with
E. variegatus
. When we supplied different
Asaia
strains isolated from other insects and exhibiting different phenotypes to
E. variegatus
orally, the bacteria stably colonized the leafhopper, reached relatively higher densities and could then be isolated from the host. We conducted transmission trials of Flavescence dorée phytoplasma with individuals colonized with three exogenous
Asaia
strains. When the phytoplasma became established in the bodies of
E. variegatus
, leafhoppers were able to transmit it to broad beans, with transmission rates ranging from 33 to 76% in different experiments. However, leafhoppers that were colonized by one of the
Asaia
strains producing an air–liquid interface biofilm exhibited significantly reduced phytoplasma acquisition, with infection rates at 5–28%, whereas they were 25–77% in control insects. Although the mechanisms regulating this interference remain to be elucidated, our results provide evidence of the potential use of
Asaia
as a biocontrol agent.
We present a population model of the insect Scaphoideus titanus Ball, the leafhopper vector of Flavescence Dorée phytoplasma in Vitisvinifera L. The model accounted for the stage-dependent S. titanus ...life cycle rates and timing, and vineyard settings such as surface area, plant density, and sampling characteristics. The model parameters were estimated against 13 independent cases of population counting in both laboratory and field conditions, and returned a correlation coefficient in the range 86.4 to 99.1% with residuals in the range 3.5 to 26.3%. A statistical parametric analysis showed that the standard deviation of life cycle rates generally varied more than the one resulting from timing parameters. However, a stochastic sensitivity analysis showed that S. titanus dynamics were more susceptible to variations in timing than rate parameters. Analysis of scenarios of insecticide suppression efficiency and timing showed that S. titanus presence could be optimally controlled by a combination of suppression efficiency and timing. These results were instrumental to understand in which specific aspect of S. titanus life cycle could pest management operations be most effective to reduce S. titanus presence in vineyards, and possibly reduce the risk of Flavescence phytoplasma spread.
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