Field and Landscape Risk Factors Impacting Flavescence Dorée Infection: Insights from Spatial Bayesian Modeling in the Bordeaux Vineyards.

Flavescence dorée (FD) is a quarantine disease threatening European vineyards. Its management is based on mandatory insecticide treatments and the uprooting of infected plants identified during annual surveys. Field surveys are currently not optimized because the drivers affecting FD spread in vineyard landscapes remain poorly understood. We collated a georeferenced dataset of FD detection, collected from 34,581 vineyard plots over 5 years in the South West France wine region. Spatial models fitted with integrated nested Laplace approximation were used to identify local and landscape factors affecting FD detection and infection. Our analysis highlights the importance of sampling period on FD detection and of local practices and landscape context on FD infection. At field scale, altitude and cultivar choice were the main factors affecting FD infection. In particular, the odds ratio of FD infection in fields planted with the susceptible Cabernet Sauvignon, Cabernet Franc, or Muscadelle varieties were approximately twice those in fields planted with the less susceptible Merlot. Field infection was also affected by the field's immediate surroundings (within a circle with a radius of 150 to 200 m), corresponding to landscapes of 7 to 12 ha. In particular, the probability of FD infection increased with the proportions of forest and urban land and with the proportion of susceptible cultivars, demonstrating that the cultivar composition impacts FD epidemiology at landscape scale. The satisfactory predictive performance of the model for identifying districts with a prevalence of FD detection >10% of the fields suggests that it could be used to target areas in which future surveys would be most valuable.

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.

'Candidatus Phytoplasma asteris' subgroups display distinct disease progression dynamics during the carrot growing season.

Aster Yellows phytoplasma (AYp; 'Candidatus Phytoplasma asteris') is an obligate bacterial pathogen that is the causative agent of multiple diseases in herbaceous plants. While this phytoplasma has been examined in depth for its disease characteristics, knowledge about the spatial and temporal dynamics of pathogen spread is lacking. The phytoplasma is found in plant's phloem and is vectored by leafhoppers (Cicadellidae: Hemiptera), including the aster leafhopper, Macrosteles quadrilineatus Forbes. The aster leafhopper is a migratory insect pest that overwinters in the southern United States, and historical data suggest these insects migrate from southern overwintering locations to northern latitudes annually, transmitting and driving phytoplasma infection rates as they migrate. A more in-depth understanding of the spatial, temporal and genetic determinants of Aster Yellows disease progress will lead to better integrated pest management strategies for Aster Yellows disease control. Carrot, Daucus carota L., plots were established at two planting densities in central Wisconsin and monitored during the 2018 growing season for Aster Yellows disease progression. Symptomatic carrots were sampled and assayed for the presence of the Aster Yellows phytoplasma. Aster Yellows disease progression was determined to be significantly associated with calendar date, crop density, location within the field, and phytoplasma subgroup.

Silencing of ATP synthase ß reduces phytoplasma multiplication in a leafhopper vector.

The leafhopper Euscelidius variegatus is a natural vector of the chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of the Flavescence dorée phytoplasma (FDp). Previous studies indicated a crucial role for insect ATP synthase α and ß subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase ß was obtained by injection of specific dsRNAs in E. variegatus. Here we present the long-lasting nature of such silencing, its effects on the small RNA profile, the significant reduction of the corresponding protein expression, and the impact on phytoplasma acquisition capability. The specific transcript expression was silenced at least up to 37 days post injection with an average reduction of 100 times in insects injected with dsRNAs targeting ATP synthase ß (dsATP) compared with those injected with dsRNAs targeting green fluorescent protein (dsGFP), used as negative controls. Specific silencing of this gene was also confirmed at protein level at 15 days after the injection. Total sRNA reads mapping to dsATP and dsGFP sequences in analysed libraries showed in both cases a peak of 21 nt, a length consistent with the generation of dsRNA-derived siRNAs by RNAi pathway. Reads mapped exclusively to the fragment corresponding to the injected dsATPs, probably indicating the absence of a secondary machinery for siRNA synthesis. Insects injected either with dsATP or dsGFP successfully acquired CYp and FDp during feeding on infected plants. However, the average phytoplasma amount in dsATP insects was significantly lower than that measured in dsGFP specimens, indicating a probable reduction of the pathogen multiplication when ATP synthase ß was silenced. The role of the insect ATP synthase ß during phytoplasma infection process is discussed.

Plant health status effects on arbuscular mycorrhizal fungi associated with Lavandula angustifolia and Lavandula intermedia infected by Phytoplasma in France.

We investigated root communities of arbuscular mycorrhizal fungi (AMF) in relation to lavender (Lavandula angustifolia) and lavandin (Lavandula intermedia) health status from organic and conventional fields affected by Phytoplasma infection. The intensity of root mycorrhizal colonization was significantly different between diseased and healthy plants and was higher in the latter regardless of agricultural practice. This difference was more pronounced in lavender. The root AMF diversity was influenced by the plant health status solely in lavender and only under the conventional practice resulting in an increase in the AMF abundance and richness. The plant health status did not influence the distribution of root AMF communities in lavandin unlike its strong impact in lavender in both agricultural practices. Finally, among the most abundant molecular operational taxonomic units (MOTUs), four different MOTUs for each plant species were significantly abundant in the roots of healthy lavender and lavandin in either agricultural practice. Our study demonstrated that the plant health status influences root colonization and can influence the diversity and distribution of root AMF communities. Its effects vary according to plant species, can be modified by agricultural practices and allow plants to establish symbiosis with specific AMF species.

The glutathione peroxidase family of Theobroma cacao: Involvement in the oxidative stress during witches' broom disease.

The glutathione peroxidases (GPXs) are enzymes which are part of the cell antioxidant system inhibiting the ROS-induced damages of membranes and proteins. In cacao (Theobroma cacao L.) genome, five GPX genes were identified. Cysteine insertion codons (UGU) were found in TcPHGPX, TcGPX2, TcGPX4, TcGPX6 and tryptophan insertion codon (UGG) in TcGPX8. Multiple alignments revealed conserved domains between TcGPXs and other plants and human GPXs. Homology modeling was performed using the Populus trichocarpa GPX5 structure as template, and the molecular modeling showed that TcGPXs have affinity with selenometionine in their active site. In silico analysis of the TcGPXs promoter region revealed the presence of conserved cis-elements related to biotic stresses and hormone responsiveness. The expression analysis of TcGPXs in cacao plantlet meristems infected by M. perniciosa showed that TcGPXs are most expressed in susceptible variety than in resistant one, mainly in disease stages in which oxidative stress and programmed cell death occurred. This data, associated with phylogenetic and location analysis suggested that TcGPXs may play a role in protecting cells from oxidative stress as a try of disease progression reduction. To our knowledge, this is the first study of the overall GPX family from T. cacao.

Haplaxius crudus (Hemiptera: Cixiidae) Transmits the Lethal Yellowing Phytoplasmas, 16SrIV, to Pritchardia pacifica Seem. & H.Wendl (Arecaceae) in Yucatan, Mexico.

Lethal yellowing (LY) affects several palm species in the Americas. It is caused by 16SrIV group phytoplasmas. In Florida (USA), LY was shown to be transmitted by the planthopper Haplaxius crudus ( Van Duzee ) (Hemiptera, Cixiidae) to different palm species, including Pritchardia pacifica Seem . & H. Wendl . (Arecaceae) in insect-proof cage experiments in the 1980s, a result that had never been reproduced later. LY has destroyed many coconut plantations as well as other palm species in the Caribbean and Mexico. In order to evaluate if H. crudus is a vector of LY phytoplasmas in Mexico, experiments were carried out in Yucatan (Mexico). Several H. crudus from palms infected by LY in the field were introduced into cages containing young P. pacifica palms. These insects were able to transmit 16SrIV group phytoplasmas to P. pacifica palms. According to DNA sequences comparative analysis, virtual restriction fragment length polymorphism, and phylogenetic analysis, the phytoplasmas detected in these infected P. pacifica were of subgroups A and D. All of ten P. pacifica palms infected with the subgroup D phytoplasmas developed symptoms of LY and died, whereas only one of two palms infected with subgroup A developed LY symptoms and died. This is the first time, more than 30 years later, that the role of H. crudus as a vector of LY is confirmed.

Functional variation in phyllogen, a phyllody-inducing phytoplasma effector family, attributable to a single amino acid polymorphism.

Flower malformation represented by phyllody is a common symptom of phytoplasma infection induced by a novel family of phytoplasma effectors called phyllogens. Despite the accumulation of functional and structural phyllogen information, the molecular mechanisms of phyllody have not yet been integrated with their evolutionary aspects due to the limited data on their homologs across diverse phytoplasma lineages. Here, we developed a novel universal PCR-based approach to identify 25 phytoplasma phyllogens related to nine "Candidatus Phytoplasma" species, including four species whose phyllogens have not yet been identified. Phylogenetic analyses showed that the phyllogen family consists of four groups (phyl-A, -B, -C, and -D) and that the evolutionary relationships of phyllogens were significantly distinct from those of phytoplasmas, suggesting that phyllogens were transferred horizontally among phytoplasma strains and species. Although phyllogens belonging to the phyl-A, -C, and -D groups induced phyllody, the phyl-B group lacked the ability to induce phyllody. Comparative functional analyses of phyllogens revealed that a single amino acid polymorphism in phyl-B group phyllogens prevented interactions between phyllogens and A- and E-class MADS domain transcription factors (MTFs), resulting in the inability to degrade several MTFs and induce phyllody. Our finding of natural variation in the function of phytoplasma effectors provides new insights into molecular mechanisms underlying the aetiology of phytoplasma diseases.

Genome-wide DNA methylation analysis of paulownia with phytoplasma infection.

DNA methylation, an important epigenetic modification, regulates a wide range of biological processes. Previous MSAP results showed that the occurrence of PaWB related to changes of DNA methylation level; however, the relationship between DNA methylation and gene expression remains obscure in paulownia. Therefore, in the present study, we applied WGBS and RNA-seq techniques to investigate the DNA methylation and gene expression changes between healthy Paulownia fortunei seedlings and the phytoplasma-infected ones. A map of methylated cytosines at the single base pair resolution of paulownia was constructed. Compared to the healthy seedlings, the DNA methylation level increased after phytoplasma infection, and the change of mCHH was the main methylation pattern. DMR analysis showed that 422,662 DMRs in the genome were identified, in which, 27,871 DMR-associated genes were differentially expressed. Finally, 436 genes with significant differences in their methylation levels and mRNA expression profiles were identified through integrated analysis of the DNA methylomic and transcriptomic. KEGG pathway analysis revealed that these genes are mainly involved in plant hormone signal transduction, carbon metabolism, and starch and sucrose metabolism pathways. Two of DMR-associated genes were verified by BS- PCR. Finally, we selected TRP 1 and R2R3-MYB protein were closely related to the occurrence of PaWB. Our findings provide valuable insight into the mechanism of PaWB at the epigenetic level.

Multi-scale spatial genetic structure of the vector-borne pathogen 'Candidatus Phytoplasma prunorum' in orchards and in wild habitats.

Inferring the dispersal processes of vector-borne plant pathogens is a great challenge because the plausible epidemiological scenarios often involve complex spread patterns at multiple scales. The spatial genetic structure of 'Candidatus Phytoplasma prunorum', responsible for European stone fruit yellows disease, was investigated by the application of a combination of statistical approaches to genotype data of the pathogen sampled from cultivated and wild compartments in three French Prunus-growing regions. This work revealed that the prevalence of the different genotypes is highly uneven both between regions and compartments. In addition, we identified a significant clustering of similar genotypes within a radius of 50 km or less, but not between nearby wild and cultivated Prunus. We also provide evidence that infected plants are transferred between production areas, and that both species of the Cacopsylla pruni complex can spread the pathogen. Altogether, this work supports a main epidemiological scenario where 'Ca. P. prunorum' is endemic in - and generally acquired from - wild Prunus by its immature psyllid vectors. The latter then migrate to shelter plants that epidemiologically connect sites less than 50 km apart by later providing infectious mature psyllids to their "migration basins". Such multi-scale studies could be useful for other pathosystems.

Phytoplasma SAP11 effector destabilization of TCP transcription factors differentially impact development and defence of Arabidopsis versus maize.

Phytoplasmas are insect-transmitted bacterial pathogens that colonize a wide range of plant species, including vegetable and cereal crops, and herbaceous and woody ornamentals. Phytoplasma-infected plants often show dramatic symptoms, including proliferation of shoots (witch's brooms), changes in leaf shapes and production of green sterile flowers (phyllody). Aster Yellows phytoplasma Witches' Broom (AY-WB) infects dicots and its effector, secreted AYWB protein 11 (SAP11), was shown to be responsible for the induction of shoot proliferation and leaf shape changes of plants. SAP11 acts by destabilizing TEOSINTE BRANCHED 1-CYCLOIDEA-PROLIFERATING CELL FACTOR (TCP) transcription factors, particularly the class II TCPs of the CYCLOIDEA/TEOSINTE BRANCHED 1 (CYC/TB1) and CINCINNATA (CIN)-TCP clades. SAP11 homologs are also present in phytoplasmas that cause economic yield losses in monocot crops, such as maize, wheat and coconut. Here we show that a SAP11 homolog of Maize Bushy Stunt Phytoplasma (MBSP), which has a range primarily restricted to maize, destabilizes specifically TB1/CYC TCPs. SAP11MBSP and SAP11AYWB both induce axillary branching and SAP11AYWB also alters leaf development of Arabidopsis thaliana and maize. However, only in maize, SAP11MBSP prevents female inflorescence development, phenocopying maize tb1 lines, whereas SAP11AYWB prevents male inflorescence development and induces feminization of tassels. SAP11AYWB promotes fecundity of the AY-WB leafhopper vector on A. thaliana and modulates the expression of A. thaliana leaf defence response genes that are induced by this leafhopper, in contrast to SAP11MBSP. Neither of the SAP11 effectors promote fecundity of AY-WB and MBSP leafhopper vectors on maize. These data provide evidence that class II TCPs have overlapping but also distinct roles in regulating development and defence in a dicot and a monocot plant species that is likely to shape SAP11 effector evolution depending on the phytoplasma host range.

A Novel Effector Protein of Apple Proliferation Phytoplasma Disrupts Cell Integrity of Nicotiana spp. Protoplasts.

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by 'Candidatus Phytoplasma mali' (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called "Protein in Malus Expressed 2" (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.

Structural insights into the interaction between phytoplasmal effector causing phyllody 1 and MADS transcription factors.

Phytoplasmas are bacterial plant pathogens which can induce severe symptoms including dwarfism, phyllody and virescence in an infected plant. Because phytoplasmas infect many important crops such as peanut and papaya they have caused serious agricultural losses. The phytoplasmal effector causing phyllody 1 (PHYL1) is an important phytoplasmal pathogenic factor which affects the biological function of MADS transcription factors by interacting with their K (keratin-like) domain, thus resulting in abnormal plant developments such as phyllody. Until now, lack of information on the structure of PHYL1 has prevented a detailed understanding of the binding mechanism between PHYL1 and the MADS transcription factors. Here, we present the crystal structure of PHYL1 from peanut witches'-broom phytoplasma (PHYL1PnWB ). This protein was found to fold into a unique α-helical hairpin with exposed hydrophobic residues on its surface that may play an important role in its biological function. Using proteomics approaches, we propose a binding mode of PHYL1PnWB with the K domain of the MADS transcription factor SEPALLATA3 (SEP3_K) and identify the residues of PHYL1PnWB that are important for this interaction. Furthermore, using surface plasmon resonance we measure the binding strength of PHYL1PnWB proteins to SEP3_K. Lastly, based on confocal images, we found that α-helix 2 of PHYL1PnWB plays an important role in PHYL1-mediated degradation of SEP3. Taken together, these results provide a structural understanding of the specific binding mechanism between PHYL1PnWB and SEP3_K.

Molecular and biological characterization of phytoplasmas from coconut palms affected by the lethal yellowing disease in Africa.

Côte d'Ivoire lethal yellowing (CILY) is a devastating disease associated with phytoplasmas and has recently rapidly spread to several coconut-growing areas in the Country. Phytoplasmas are phloem-restricted bacteria that affect plant species worldwide. These bacteria are transmitted by plant sap-feeding insects, and their cultivation was recently achieved in complex artificial media. In this study, phytoplasmas were isolated for the first time from coconut palm trunk borings in both solid and liquid media from CILY symptom-bearing and symptomless coconut palms. The colony morphology, PCR and sequencing analyses indicated the presence of phytoplasmas from different ribosomal groups. This study reports the first biochemical characterization of two of these phytoplasma isolates. Moreover, a disc-diffusion antibiotic susceptibility assay revealed that these bacteria exhibit tobramycin susceptibility and cephalexin hydrate and rifampicin resistance. Urea and arginine hydrolysis, and glucose fermentation tests that were performed on colonies of phytoplasmas and Acholeplasma laidlawii indicated that both phytoplasmas tested were negative for urea and positive for glucose and arginine, whereas A. laidlawii was positive for glucose and negative for urea and arginine. The growth of coconut phytoplasmas in both solid and liquid artificial media and the biological characterization of these isolates are novel and important advancements in the field of disease management and containment measures for the CILY disease. The characterization of isolated phytoplasmas will allow for more efficient management strategies in both the prevention of a coconut phytoplasma epidemics and the reduction of the economic impact of the disease in the affected areas.

Nested and TaqMan® probe based quantitative PCR for the diagnosis of Ca. Phytoplasma in coconut palms.

The root (wilt) disease caused by phytoplasma (Ca. Phytoplasma) is one of the major and destructive occurs in coconut gardens of Southern India. As this organism could not be cultured in vitro, the early detection in the palm is very much challenging. Hence, proper early diagnosis and inoculum assessment relay mostly on the molecular techniques namely nested and quantitative PCR (qPCR). So, the present study qPCR assay conjugated with TaqMan® probe was developed which is a rapid, sensitive method to detect the phytoplasma. For the study, samples from different parts of infected coconut palms viz., spindle leaflets, roots and the insect vector-leaf hopper (Proutista moesta) were collected and assessed by targeting 16S rRNA gene. Further, nested PCR has been carried out using p1/p7 and fU5/rU3 primers and resulted in the amplification product size of 890 bp. From this amplified product, specifically a target of 69 bp from the 16S rRNA gene region has been detected through primers conjugated with Taqman probe in a step one instrument. The results indicated that the concentration of phytoplasma was more in spindle leaflets (8.9 × 105 g of tissue) followed by roots (7.4 × 105 g of tissue). Thus, a qPCR approach for detection and quantification of coconut phytoplasma was more advantageous than other PCR methods in terms of sensitivity and also reduced risk of cross contamination in the samples. Early diagnosis and quantification will pave way for the healthy coconut saplings selection and management under field conditions.

Phytoplasmas: An Introduction.

Phytoplasmas are among the most recently discovered plant pathogens. They are wall-less prokaryotes restricted to phloem tissue, associated with diseases affecting several hundred plant species. The impact of phytoplasma diseases on agriculture is impressive and, at the present day, no effective curative strategy has been developed. The availability of rapid and sensitive techniques for phytoplasma detection as well as the possibility to study their relationship with the host plants is a prerequisite for the management of phytoplasma-associated diseases.

Micro-Tom Tomato Grafting for Stolbur-Phytoplasma Transmission: Different Grafting Techniques.

Tomato plant, being a model system in scientific research, is widely used to study plant-phytoplasma interaction. Grafting is the faster and most effective method to obtain infected plants. This chapter describes the greenhouse culture of tomato, cv. Micro-Tom, and different herbaceous grafting techniques for efficient stolbur-phytoplasma transmission.

Phytoplasma Transmission: Insect Rearing and Infection Protocols.

Phytoplasmas are obligate pathogens and thus they can be studied only in association with their plants or insect hosts. In this chapter, we present protocols for rearing some phytoplasma insect vectors, to obtain infected insects and plants under controlled environmental conditions. We focus on Euscelidius variegatus and Macrosteles quadripunctulatus that can infect Arabidopsis thaliana, and Hyalesthes obsoletus and Scaphoideus titanus, that can infect grapevine.

Symptoms of Phytoplasma Diseases.

Phytoplasmas are associated with diseases in several hundreds of cultivated herbaceous and woody plants. Their impact in agriculture and the periodical outbreak of worrying epidemics make very important, besides precise laboratory-based diagnosis, the direct in-field recognition of phytoplasma disease symptoms. Even if some symptoms are typical of this kind of pathogens, in-field diagnosis requires the knowledge of the host plant, strong field experience, and awareness of the symptom variability of the various organs of the plant during different seasons and under various environmental conditions. It is therefore very important to be familiar with factors like environmental conditions, agronomical features, and disease progression that influence symptom expression. Therefore, a satisfactory diagnosis should be based on repeated and complete observations scored over the entire plant and across different times of the year. A more suitable diagnosis is possible if the observer is able to recognize and distinguish the symptoms of other biotic or abiotic diseases. A general rule is to observe three different symptoms, at least, and to seek input from the grower about the initial development, frequency, diffusion, and particular characteristics of the disease.After a short introduction the following symptoms are presented: the most common and representative symptoms caused by phytoplasmas; the most common symptoms of phytoplasma diseases occurring in particular plant organs, with some references to specific diseases; phytoplasma symptoms on the model plant periwinkle (Vinca rosea or Catharanthus roseus); the main factors influencing phytoplasma symptoms expression; and several practical procedures that should be followed for suitable diagnosis. A series of original photos have been included to illustrate typical symptoms.

Transcriptomic Analyses of Phytoplasmas.

Transcriptomic analyses addressed to study phytoplasma gene expression may present few difficulties due to the uncultivable nature of these intracellular, obligate pathogens. While RNA extraction from insect vectors does not imply any particular adaptation of the protocols used in most commercial kits, RNA isolation from phytoplasma-infected plants can be a challenging task, given the high levels of polyphenol contents and accumulation of sucrose and starch in the different plant tissues. Here, we describe two different transcriptomic approaches, one focused on RNA phytoplasma sequencing and the other on phytoplasma quantitative gene expression in relation to pathogen load.