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Flavescence dorée phytoplasma (FDp) is a great threat to grapevine cultivation in Europe. Severe symptoms induced by FDp in grapevine plants, along with side effects of insecticide treatments used to control disease spread, bring considerable economic and environmental toll and challenge the sustainability of European viticulture. Previous research of map gene sequences identified three genetic clusters of FDp – Map-FD1, -FD2 and -FD3. Each cluster is affiliated with different geographical distribution and characterized by different genetic variability and epidemiological properties. Those clusters are identified through laborious and time consuming nested PCR-RFLP or sequencing and phylogenetic analysis. Here, a multiplex real-time PCR assay was developed for rapid and sensitive detection of the three FDp genetic clusters by using a single primer pair and three distinct BHQ probes labelled with different fluorescent dyes. The assay was evaluated using FDp reference strains, phytoplasma strains belonging to different phytoplasma groups and field collected FDp isolates. The assay was found to be highly sensitive and reliably detected and discriminated all three Map-FD genetic clusters within the 16Sr-V phytoplasma group. Early detection of Map-FD genetic clusters and thus phytoplasma genotypes which are less likely to cause epidemic outbreaks due to different transmission pathways, can significantly accelerate FDp epidemiological studies and facilitate the efforts to control the FD disease. [ABSTRACT FROM AUTHOR]

Production from crops of pome, stone fruit, nut, berry fruit, citrus, grapevine, and olive is increasingly threatened by fungal trunk diseases (FTD). These diseases and the consequent production losses are major problems. Many fungi (including Botryosphaeriaceae, Calosphaeriaceae, Diaporthaceae, Diatrypaceae, Nectriaceae, Phaeomoniellaceae, Pleosporaceae, Togniniaceae, Valsaceae) infect host wood, mainly through wounds and subsequent colonization of woody tissues, causing symptoms such as cankers, gummosis, wood rotting, blight and dieback. Propagative plant material, seedlings and fruit play a significant role in pathogen spread. Several abiotic factors (e.g. shifts in cultural practices and climate change) are involved in the disease development. This paper reviews recent literature on FTD of fruit crops, particularly focusing on the European status of pathogen occurrence. Case studies are described related to diseases of apple, citrus, grapevine, berry, nut and stone fruit, and olive trees. Aspects related to epidemiology and the increase in disease incidence along with the future perspectives on the FTD research are also discussed. [ABSTRACT FROM AUTHOR]

Here, published crop disease risk simulations are summarized, which were driven by climate change scenarios on a timescale terminating between 2001 and 2100. Thirty different agricultural and horticultural crops were specifically considered. Wheat diseases (mainly leaf rust) were most often simulated, followed by rice diseases (mainly leaf blast), grapevine diseases (mainly downy mildew) and potato diseases (mainly late blight). Most simulations suggest that within the projection period simulated, crop disease risk will more often increase (86 simulations) than decrease (45 simulations) or remain similar (12 simulations). The majority of crop disease risk simulations focus on Europe and Brazil. For example, there is agreement across crop disease risk simulations that in Europe the risk of leaf rust of wheat will increase. In other cases, there is disagreement across crop disease risk simulations, for example, due to contemporary climatic differences of locations. Therefore, it is risky to extrapolate results across locations. Mitigation and adaptation methods should be more often integrated in the crop disease risk simulations in order to inform about potential methods to reduce disease risk in future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The European grape berry moth, Eupoecilia ambiguella (Hübner) (Lepidoptera: Tortricidae), since its first identification in 1796, was defined as a key pest for European viticulture despite its polyphagy. Although between the late 1800s and early 1900s its presence and spread in Europe was of concern, to date its populations are low and limited to cooler and wetter areas, leading to a decline in its importance. In the present work, we reviewed its global distribution as well as its morphology, biology, and ecology. Considering the monitoring and management of this pest, the present review summarised insecticidal, agronomic, and cultural control strategies. Moreover, given the need to reduce the impact of agriculture on the environment, humans, and non-target species, we focused on control strategies relying on pheromones and biological control agents (e.g. predators and parasitoids) involved in conservation biological control. • The European grape berry moth, Eupoecilia ambiguella , has a long history as grape pest. • We reviewed knowledge on its morphology, bio-ecology, and geographical distribution. • Current monitoring and management tools were discussed. • Potential biocontrol agents were extensively reviewed. • Challenges for E. ambiguella IPM and biological control were formulated. [ABSTRACT FROM AUTHOR]

The European grapevine moth (Lobesia botrana) is currently the most damaging pest in many viticultural regions of South and Central Europe and is highly responsible for the development of secondary infections by Botrytis cinerea to both table and wine grapes. Entomopathogenic fungi (EPF) are considered as substantial biological control agents of insect pests. However, they are able to infect their hosts only under a narrow spectrum of environmental conditions. In this study, the effects of temperature on pathogenicity of EPF for the control of L. botrana are reported. Among a collection of 33 EPF isolates, six virulent isolates were selected for the experiments. The bioassays took place at controlled conditions, under the temperature regimes of 20, 22.5, 25, 27.5, 30, and 32.5 °C. A conidial concentration of 1 × 108 conidia/ml was prepared for each isolate, and fungi were applied by spraying them on third-instar larvae (L3) of L. botrana. Both factors, fungal isolate and temperature, significantly affected mortality of L. botrana. Most fungal isolates exhibited their highest larval mortality rates seven days post-treatment at the temperatures of 25 and 27.5 °C. Some isolates caused considerably high larval mortality that exceeded 90% under specific temperature conditions. Overall, EPF showed great potential as bio-control agents for controlling larval populations of L. botrana. [Display omitted] • Entomogenous fungi showed great potential as biocontrol agents of Lobesia botrana. • Temperature significantly influenced all fungi on larval mortality of L. botrana. • An isolate from a cool region (Isaria fumosorosea EBAC01) was the most virulent. • Most fungal isolates demonstrated their highest efficacy at 25 and 27.5 °C. [ABSTRACT FROM AUTHOR]

Flavescence dorée (FD) is one of the most devastating diseases threatening grapevine cultivation in Europe. The causal agents are phytoplasmas, which reside in phloem vessels of infected plants and in the body of leafhopper vectors, the most important being Scaphoideus titanus. Current control mainly relies on vector management, which is often insufficient to prevent from disease outbreaks. Suitable methods to interfere with phytoplasmas in planta are constantly sought to support insecticide sprays. We tested a biocomplex containing trace elements and soluble sugars, with potential systemic spread and antimicrobial activity, with the aim to verify its capability to penetrate plant phloem and to interfere with phytoplasma survival in vector-inoculated plants, using two FD isolates (FD-C and FD-D). The penetration into the plant phloem, as well as its acquisition by phloem-feeding vectors, were tested using an iridium-marked biocomplex. Phytoplasma infection was detected by real Time PCR and compared in plants exposed to biocomplex treatment before inoculation (short-time and mid-time preventive application), and to treatment after inoculation (curative application). We also assessed if the biocomplex interferes with the vector competence of S. titanus, by conducting phytoplasma transmission trials with sequential inoculation steps, where untreated and biocomplex-treated grapevine plantlets were sequentially offered to infectious leafhoppers. Foliar treatment with the biocomplex supported the penetration of phloem tissues, and the product was successfully acquired by the experimental phytoplasma vector Euscelidius variegatus after feeding on the phloem of treated plants. When the biocomplex was applied on grapevines, it induced a significant reduction of phytoplasma infection regardless of FD isolate, both as a preventive and curative treatment, indicating an antibacterial activity in the plant. Moreover, sequential inoculation experiments showed a decrease of S. titanus inoculation rates after vector exposure to a biocomplex-treated plant, suggesting a reduction of transmission efficiency that was higher using the FD-C rather than FD-D isolate. Our results indicate the tested biocomplex as a promising tool for the creation of anti-phytoplasma formulations to support vector management in FD containment. • An experimental biocomplex was tested against grapevine Flavescence dorée phytoplasma. • The biocomplex effectively penetrated phloem vessels after foliar spray. • Leafhopper vectors acquired the biocomplex through phloem feeding. • Lower phytoplasma infection was found in vines after preventive and curative treatment. • Insect inoculation efficiency was reduced after exposure to sprayed plants. [ABSTRACT FROM AUTHOR]

Grapevine trunk diseases (GTDs) are increasing in vineyards in many grape production regions. Among the pathogens causing these diseases, Neofusicoccum parvum, is one of the most frequent and virulent. To control GTDs, biocontrol is being developed using plant beneficial microorganisms. Strains of the oomycete Pythium oligandrum have been shown to naturally colonize grapevine roots in vineyards in several countries in Europe. This study examined the ability of the root-coloniser P. oligandrum to induce grapevine resistance against N. parvum, by deciphering the gene expression changes in a set of 62 genes involved in different grapevine defence pathways. Two greenhouse assays showed that the wood necrosis of vine cuttings caused by N. parvum was reduced by 65% when P. oligandrum colonized root systems of the plants. The relative expression levels of selected genes in the host trunks were studied by real-time PCR. Plant responses were assessed after inoculation by P. oligandrum and/or N. parvum, at three different sampling time points (0, 14 and 150 d after N. parvum inoculation). Sampling time influenced gene expressions for the different inoculation treatments. At each sampling time, specific host responses to the different treatments were also detected, for controls, and for inoculations with P. oligandrum, N. parvum or P. oligandrum + N. parvum. When P. oligandrum colonized grapevine root systems, inoculation with the pathogen was associated with increased up-regulation and over-expression of particular genes, including those regulating Pathogen-Related proteins, cell wall reinforcement proteins and hormone signalling pathways. A priming effect of the grapevine defence system was induced in roots colonized by P. oligandrum. [ABSTRACT FROM AUTHOR]

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To study the role of wild areas around the vineyards in the epidemiology of flavescence dorée (FD) and track the origin of new foci, two phytoplasma genetic markers, dnaK and malG, were developed for FD phytoplasma (FDp) characterization. The two genes and the vmpA locus were used to genetically characterize FDp populations at seven agroecosystems of a wine-growing Italian region. Vitis vinifera, "gone-wild" V. vinifera and rootstocks, Clematis spp., and Scaphoideus titanus adults were sampled within and outside the vineyards. A range of genotypes infecting the different hosts of the FDp epidemiological cycle was found. Type FD-C isolates were fairly homogeneous compared to type FD-D ones. Most of the FD-D variability was correlated with the malG sequence, and a duplication of this locus was demonstrated for this strain. Coinfection with FD-C and FD-D strains was rare, suggesting possible competition between the two. Similar levels of FDp genetic variation recorded for grapevines or leafhoppers of cultivated and wild areas and co-occurrence of many FDp genotypes inside and outside the vineyards supported the idea of the importance of wild or abandoned Vitis plants and associated S. titanus insects in the epidemiology of the disease. Genetic profiles of FDp found in Clematis were never found in the other hosts, indicating that this species does not take part in the disease cycle in the area. Due to the robustness of analyses using dnaK for discriminating between FD-C and FD-D strains and the high variability of malG sequences, these are efficient markers to study FDp populations and epidemiology at a small geographical scale. IMPORTANCE Flavescence dorée, a threatening disease of grapevine caused by FD phytoplasma (FDp), is distributed within the most important wine-producing areas of Europe and has severe effects on both vineyard productivity and landscape management. FDp is a quarantine pest in Europe, and despite the efforts to contain the pathogen, the disease is still spreading. In this work, new genetic markers for the fine genetic characterization of FDp at local scale are presented. Our findings improve the knowledge of FDp epidemiological cycle and offer the possibility of tracking the route of the FDp infection. In particular, due to its high genetic variability, one of the newly developed markers could be sufficient to track the origin of new infection foci, either from the wild areas or from nurseries. [ABSTRACT FROM AUTHOR]

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the agents responsible for leafroll disease of cultivated grapevines. To secure information on the molecular variability of GLRaV-3 isolates from central Europe, a 3,477 nucleotide long genome fragment coding for the end of a 55kDa protein, the complete capsid protein (CP), the minor CP (CPm), a 21kDa protein, and the N-terminal part of the p20A protein was sequenced from an atypical Slovak viral isolate, inducing no leafroll symptoms on the original Vitis vinifera host. In the sequenced ca. 3.5 kb region, the nucleotide identities with previously characterized isolates from different parts of the world ranged from 89.4 (Group 3 isolates) to 99.5% (Group 1 isolates). The Slovak SK04 isolate clustered within the largest Group 1, although in a slightly separated branch. Vegetative propagation by infected cuttings did not lead to changes of the dominant GLRaV-3 sequence, determined from the mother plant, when compared with the sequences obtained from the newly established 5-year-old self-rooted plants, indicating the high genetic stability of the SK04 viral population. [ABSTRACT FROM AUTHOR]

Insect vectors transmit viruses and bacteria that can cause severe diseases in plants and economic losses due to a decrease in crop production. Insect vectors, like all other organisms, are colonized by a community of various microorganisms, which can influence their physiology, ecology, evolution, and also their competence as vectors. The important ecological meaning of bacteriophages in various ecosystems and their role in microbial communities has emerged in the past decade. However, only a few phages have been described so far in insect microbiomes. The leafhopper Euscelidius variegatus is a laboratory vector of the phytoplasma causing Flavescence dorée, a severe grapevine disease that threatens viticulture in Europe. Here, the presence of a temperate bacteriophage in E. variegatus (named Euscelidius variegatus phage 1, EVP-1) was revealed through both insect transcriptome analyses and electron microscopic observations. The bacterial host was isolated in axenic culture and identified as the bacterial endosymbiont of E. variegatus (BEV), recently assigned to the genus Candidatus Symbiopectobacterium. BEV harbors multiple prophages that become active in culture, suggesting that different environments can trigger different mechanisms, finely regulating the interactions among phages. Understanding the complex relationships within insect vector microbiomes may help in revealing possible microbe influences on pathogen transmission, and it is a crucial step toward innovative sustainable strategies for disease management in agriculture. [ABSTRACT FROM AUTHOR]

Understanding temporal biological phenomena is a challenging task that can be approached using network analysis. Here, we explored whether network reconstruction can be used to better understand the temporal dynamics of bois noir, which is associated with 'Candidatus Phytoplasma solani', and is one of the most widespread phytoplasma diseases of grapevine in Europe. We proposed a methodology that explores the temporal network dynamics at the community level, i.e., densely connected subnetworks. The methodology offers both insights into the functional dynamics via enrichment analysis at the community level, and analyses of the community dissipation, as a measure that accounts for community degradation. We validated this methodology with cases on experimental temporal expression data of uninfected grapevines and grapevines infected with 'Ca. P. solani'. These data confirm some known gene communities involved in this infection. They also reveal several new gene communities and their potential regulatory networks that have not been linked to 'Ca. P. solani' to date. To confirm the capabilities of the proposed method, selected predictions were empirically evaluated. [ABSTRACT FROM AUTHOR]

Background: Flavescence dorée is the most serious grapevine yellows disease in Europe. It is caused by phytoplasmas which are transmitted from grapevine to grapevine by the leafhopper Scaphoideus titanus. Differences in susceptibility among grapevine varieties suggest the existence of specific genetic features associated with resistance to the phytoplasma and/or possibly with its vector. In this work, RNA-Seq was used to compare early transcriptional changes occurring during the three-trophic interaction between the phytoplasma, its vector and the grapevine, represented by two different cultivars, one very susceptible to the disease and the other scarcely susceptible. Results: The comparative analysis of the constitutive transcriptomic profiles suggests the existence of passive defense strategies against the insect and/or the phytoplasma in the scarcely-susceptible cultivar. Moreover, the attack by the infective vector on the scarcely-susceptible variety prompted immediate and substantial transcriptomic changes that led to the rapid erection of further active defenses. On the other hand, in the most susceptible variety the response was delayed and mainly consisted of the induction of phytoalexin synthesis. Surprisingly, the jasmonic acid- and ethylene-mediated defense reactions, activated by the susceptible cultivar following FD-free insect feeding, were not detected in the presence of the phytoplasma-infected vector. Conclusions: The comparison of the transcriptomic response in two grapevine varieties with different levels of susceptibility to Flavescence dorèe highlighted both passive and active defense mechanisms against the vector and/or the pathogen in the scarcely-susceptible variety, as well as the capacity of the phytoplasmas to repress the defense reaction against the insect in the susceptible variety. [ABSTRACT FROM AUTHOR]