Molecular Variability and Host Distribution of ‘Candidatus Phytoplasma solani’ Strains from Different Geographic Origins

The knowledge of phytoplasma genetic variability is a tool to study their epidemiology and to implement an effective monitoring and management of their associated diseases. ‘Candidatus Phytoplasma solani’ is associated with “bois noir” disease in grapevines, and yellowing and decline symptoms in many plant species, causing serious damages during the epidemic outbreaks. The epidemiology of the diseases associated with this phytoplasma is complex and related to numerous factors, such as interactions of the host plant and insect vectors and spreading through infected plant propagation material. The genetic variability of ‘Ca. P. solani’ strains in different host species and in different geographic areas during the last two decades was studied by RFLP analyses coupled with sequencing on vmp1, stamp, and tuf genes. A total of 119 strains were examined, 25 molecular variants were identified, and the variability of the studied genes was linked to both geographic distribution and year of infection. The crucial question in ‘Ca. P. solani’ epidemiology is to trace back the epidemic cycle of the infections. This study presents some relevant features about differential strain distribution useful for disease monitoring and forecasting, illustrating and comparing the phytoplasma molecular variants identified in various regions, host species, and time periods.

Differential Response of Grapevine to Infection with ‘Candidatus Phytoplasma solani’ in Early and Late Growing Season Through Complex Regulation of mRNA and Small RNA Transcriptomes

Bois noir is the most widespread phytoplasma grapevine disease in Europe. It is associated with ‘Candidatus Phytoplasma solani’, but molecular interactions between the causal pathogen and its host plant are not well understood. In this work, we combined the analysis of high-throughput RNA-Seq and sRNA-Seq data with interaction network analysis for finding new cross-talks among pathways involved in infection of grapevine cv. Zweigelt with ‘Ca. P. solani’ in early and late growing seasons. While the early growing season was very dynamic at the transcriptional level in asymptomatic grapevines, the regulation at the level of small RNAs was more pronounced later in the season when symptoms developed in infected grapevines. Most differentially expressed small RNAs were associated with biotic stress. Our study also exposes the less-studied role of hormones in disease development and shows that hormonal balance was already perturbed before symptoms development in infected grapevines. Analysis at the level of communities of genes and mRNA-microRNA interaction networks revealed several new genes (e.g., expansins and cryptdin) that have not been associated with phytoplasma pathogenicity previously. These novel actors may present a new reference framework for research and diagnostics of phytoplasma diseases of grapevine.

New Cross-Talks between Pathways Involved in Grapevine Infection with ‘Candidatus Phytoplasma solani’ Revealed by Temporal Network Modelling

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.

Symptomatology, (Co)occurrence and Differential Diagnostic PCR Identification of ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma convolvuli’ in Field Bindweed

Field bindweed (Convolvulus arvensis) is one of the major natural plant hosts and reservoirs of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), the causal agent of plant diseases in diverse agricultural crops, including Bois noir (BN) disease of grapevine. Phylogenetically, the most closely related phytoplasma to ‘Ca. P. solani’, the ‘Ca. P. convolvuli’, induces disease in field bindweed that is known by its symptoms as bindweed yellows (BY). The occurrence, coinfection and symptoms association of the two phytoplasmas in shared host plants were the subject of this study. Specific primers for the amplification of the elongation factor Tu gene (tuf) were developed for the identification of ‘Ca. P. convolvuli’ (by conventional nested PCR), as well as primers for simultaneous detection of ‘Ca. P. solani’ and ‘Ca. P. convolvuli’ by duplex SYBR Green real-time PCR. Among symptomatic bindweed plants, 25 and 41% were infected with a single phytoplasma species, ‘Ca. P. solani’ and ‘Ca. P. convolvuli’, respectively, while 34% were infected with both phytoplasmas. None of the non-symptomatic control plants carried phytoplasma, while non-symptomatic plants from our previous epidemiological studies in BN-affected vineyards were confirmed to be infected solely with ‘Ca. P. solani’. Stamp gene typing revealed Rqg50 and Rqg31 ‘Ca. P. solani’ genotypes in plants coinfected with ‘Ca. P. convolvuli’, while three diverse genotypes (Rqg50, GGY and Rpm35) were identified in a single locality with symptomatic bindweeds infected solely with ‘Ca. P. solani’. Variations in symptoms and their association with each of the phytoplasmas are described and documented. The symptom of bushy appearance could be single out as specific for ‘Ca. P. convolvuli’ infection, while occurrence of ‘Ca. P. solani’ could not be unequivocally associated with specific alterations in infected bindweeds. The results are discussed in the context of the epidemiological and ecological complexity of ‘Ca. P. solani’-induced diseases and the relationship between the two phytoplasma relatives in shared host plant.

Entomopathogenic nematodes and fungi to control Hyalesthes obsoletus (Hemiptera: Auchenorrhyncha: Cixiidae)

Hyalesthes obsoletus Signoret (Hemiptera: Auchenorrhyncha: Cixiidae) is a univoltine, polyphagous planthopper that completes its life cycle, including the subterranean nymph cryptic stage, on herbaceous weeds. In vineyards, it can transmit ‘Candidatus Phytoplasma solani’, an obligate parasitic bacterium associated with bois noir (BN) disease of grapevine, from its host plants to grapevine when occasionally feeding on the latter. The main disease management strategies are based on vector(s) control. Insecticide treatments on grapevine canopy are completely inefficient on H. obsoletus, due to its life cycle. Consequently, control of this planthopper focuses on the nymphs living on the roots of their host plants. Such practices, based on herbicide application and/or weed management, can reduce vector density in the vineyard but can impact the environment or may not be applicable, highlighting the necessity for alternative strategies. In this study, the efficacy of entomopathogenic nematodes (EPNs; Steinernema carpocapsae, S. feltiae, Heterorhabditis bacteriophora) and fungi (EPFs; Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Lecanicillium muscarium) against H. obsoletus nymphs (EPNs) and adults (EPNs and EPFs) was assessed under laboratory and greenhouse conditions. The majority of examined EPNs and EPFs were able to kill H. obsoletus exhibiting a range of effectiveness. S. carpocapsae (among EPNs) and I. fumosorosea (among EPFs) were found to be the most effective biocontrol agents in all trials carried out. Advantages and limitations of such promising biocontrol agents were discussed. Ecological competency and conditions that can impede or enhance the EPNs and EPFs performance should be investigated to optimize their performance under field conditions.

Hyalesthes obsoletus is an active vector of Wood blackening in the Republic of Moldova

Hyalesthes obsoletus (Signoret) is an important vector of Wood Blackening in the Republic of Moldova. To identify the causes of the epidemiology of this disease in vineyards, the period of flight and the preference of the host plant H. obsoletus in the field were studied. Monitoring of vineyards showed that this species was found on the Field Bindweed (Convolvulus arvensis L.), and during the period of mass flight, the leafhopper was observed on Xanthium strumarium and Gorets bindweed (Fallopia convolvulus L.). The captured leafhoppers were diagnosed with the presence of Bois Noir stolbur phytoplasm (STOL). The adults of H. obsoletus were also caught from other herbaceous plants: Povoy fence (Calystegia sepium L.), Garden quinoa (Ariplex hortensis L.), Sow thistle (Sonchus oleraceus L.), Medicinal dandelion (Taraxacum officinale L.), as well as c Stinging nettle (Urtica dioica L.). Changes in climatic conditions in the region, causing premature drying of the grass cover, forces cicadas - vectors to switch to actively growing plants during this period, including grapes, which contributes to the widespread of the disease.

AGROTECHNICAL METHOD TO PREVENT “BOIS NOIR”SPREAD

In the Republic of Moldova the defeat of bushes with a disease of phytoplasmic eti-ology – Bois noir occurs everywhere. The article discusses and analyzes the factors contributing to the spread of this disease on grapevine plantations. This process is facilitated by plants-reserves of phyto-plasm, leafhoppers-carriers and changes in climatic conditions. As a result of the stud-ies carried out, plants reserves of phyto-plasma Bois noir were identified: Convol-vulus arvensis L., Fallópia convólvulus L., Chenopodium bonus-henricus L., Rosa L., Ulmus L. Long-term studies have shown that the destruction of weeds on the plan-tation leads to the removal of infection res-ervoir plants and the larvae of the leafhop-per-vector feeding on weeds on the roots. Therefore, systematic soil cultivation in the fight against weeds is an agricultural tech-nique that prevents the spread of Bois noir.

Detection of Two Different Grapevine Yellows in Vitis vinifera Using Hyperspectral Imaging

Grapevine yellows (GY) are serious phytoplasma-caused diseases affecting viticultural areas worldwide. At present, two principal agents of GY are known to infest grapevines in Germany: Bois noir (BN) and Palatinate grapevine yellows (PGY). Disease management is mostly based on prophylactic measures as there are no curative in-field treatments available. In this context, sensor-based disease detection could be a useful tool for winegrowers. Therefore, hyperspectral imaging (400–2500 nm) was applied to identify phytoplasma-infected greenhouse plants and shoots collected in the field. Disease detection models (Radial-Basis Function Network) have successfully been developed for greenhouse plants of two white grapevine varieties infected with BN and PGY. Differentiation of symptomatic and healthy plants was possible reaching satisfying classification accuracies of up to 96%. However, identification of BN-infected but symptomless vines was difficult and needs further investigation. Regarding shoots collected in the field from different red and white varieties, correct classifications of up to 100% could be reached using a Multi-Layer Perceptron Network for analysis. Thus, hyperspectral imaging seems to be a promising approach for the detection of different GY. Moreover, the 10 most important wavelengths were identified for each disease detection approach, many of which could be found between 400 and 700 nm and in the short-wave infrared region (1585, 2135, and 2300 nm). These wavelengths could be used further to develop multispectral systems.