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The discovery of new mechanisms of resistance and natural bioactive molecules could be two of the possible ways to reduce fungicide use in vineyard and assure an acceptable and sustainable protection against Plasmopara viticola, the grapevine downy mildew agent. Emission of volatile organic compounds (VOCs), such as terpenes, norisoprenoids, alcohols and aldehydes, is frequently induced in plants in response to attack by pathogens, such as P. viticola, that is known to cause a VOCs increment in cultivars harboring American resistance traits. In this study, the role of leaf VOCs in the resistance mechanism of two resistant cultivars (Mgaloblishvili, a pure Vitis vinifera cultivar, and Bianca, an interspecific hybrid) and the direct antimicrobial activity of four selected VOCs have been investigated. The leaf VOCs profiles, analyzed through solid-phase microextraction gas chromatography-mass spectrometry analysis, as well as the expression of six terpene synthases (TPSs), were determined upon pathogen inoculation. In both cultivars, the expression pattern of six TPSs increased soon after pathogen inoculation and an increment of nine VOCs has been detected. While in Mgaloblishvili VOCs were synthesized early after P. viticola inoculation, they constituted a late response to pathogen in Bianca. All the four terpenes (farnesene, nerolidol, ocimene and valencene), chosen according to the VOC profiles and gene expression analysis, caused a significant reduction (53-100%) in P. viticola sporulation. These results support the role of VOCs into defense mechanisms of both cultivars and suggest their potential role as a natural and eco-friendly solution to protect grapevine from P. viticola., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Mgaloblishvili, a Vitis vinifera cultivar, exhibits unique resistance traits against Plasmopara viticola , the downy mildew agent. This offers the unique opportunity of exploring the molecular responses in compatible and incompatible plant-pathogen interaction. In this study, whole transcriptomes of Mgaloblishvili, Pinot noir (a V. vinifera susceptible cultivar), and Bianca (a resistant hybrid) leaves, inoculated and non-inoculated with the pathogen , were used to identify P. viticola effector-encoding genes and plant susceptibility/resistance genes. Multiple effector-encoding genes were identified in P. viticola transcriptome, with remarkable expression differences in relation to the inoculated grapevine cultivar. Intriguingly, five apoplastic effectors specifically associated with resistance in V. vinifera . Gene coexpression network analysis identified specific modules and metabolic changes occurring during infection in the three grapevine cultivars. Analysis of these data allowed, for the first time, the detection in V. vinifera of a putative P. viticola susceptibility gene, encoding a LOB domain-containing protein. Finally, the de novo assembly of Mgaloblishvili, Pinot noir, and Bianca transcriptomes and their comparison highlighted novel candidate genes that might be at the basis of the resistant phenotype. These results open the way to functional analysis studies and to new perspectives in molecular breeding of grapevine for resistance to P. viticola ., Competing Interests: The authors declare no conflict of interest.

Citrus Decline Disease was recently reported to affect several citrus species in Iran when grafted on a local rootstock variety, Bakraee. Preliminary studies found “ Candidatus Phytoplasma aurantifoliae” and “ Candidatus Liberibacter asiaticus” as putative etiological agents, but were not ultimately able to determine which one, or if an association of both, were causing the disease. The current study has the aim of characterizing the microbiota of citrus plants that are either asymptomatic, showing early symptoms, or showing late symptoms through amplification of the V1–V3 region of 16S rRNA gene using an Illumina sequencer in order to (i) clarify the etiology of the disease, and (ii) describe the microbiota associated to different symptom stages. Our results suggest that liberibacter may be the main pathogen causing Citrus Decline Disease, but cannot rule out the possibility of phytoplasma being involved as well. The characterization of microbiota shows that the leaves show only two kinds of communities, either symptomatic or asymptomatic, while roots show clear distinction between early and late symptoms. These results could lead to the identification of bacteria that are related to successful plant defense response and, therefore, to immunity to the Citrus Decline Disease.

Bois noir (BN) is the most widespread disease of the grapevine yellows complex in the Euro-Mediterranean area. BN is caused by 'Candidatus Phytoplasma solani' (BNp), transmitted from herbaceous plants to grapevine by polyphagous insect vectors. In this study, genetic diversity among BNp strains and their prevalence and possible association with grapevine symptom severity were investigated in a Sangiovese clone organic vineyard in the Chianti Classico area (Tuscany). Field surveys over 2 years revealed a range of symptom severity on grapevine and an increase of BN incidence. A TaqMan allelic discrimination assay detected only tufB type b among BNp strains, suggesting the prevalence of the bindweed-related ecology. Nucleotide sequence analyses of vmp1 and stamp genes identified 12 vmp1 and 16 stamp sequence variants, showing an overall positive selection for such genes. The prevalent genotype was Vm43/St10, reported for the first time in this study and closely related to strains identified only in the French Eastern Pyrenees. BNp strains identified in the examined vineyard and mostly grouped in separate bindweed-related phylogenetic clusters showed statistically significant differences in their distribution in grapevines exhibiting distinct symptom severity. These results suggest the possible occurrence of a range of virulence within BNp strain populations in the Chianti Classico area.

Background: Almond witches'-broom (AlmWB), a devastating disease of almond, peach and nectarine in Lebanon, is associated with 'Candidatus Phytoplasma phoenicium'. In the present study, we generated a draft genome sequence of 'Ca. P. phoenicium' strain SA213, representative of phytoplasma strain populations from different host plants, and determined the genetic diversity among phytoplasma strain populations by phylogenetic analyses of 16S rRNA, groEL, tufB and inmp gene sequences., Results: Sequence-based typing and phylogenetic analysis of the gene inmp, coding an integral membrane protein, distinguished AlmWB-associated phytoplasma strains originating from diverse host plants, whereas their 16S rRNA, tufB and groEL genes shared 100 % sequence identity. Moreover, dN/dS analysis indicated positive selection acting on inmp gene. Additionally, the analysis of 'Ca. P. phoenicium' draft genome revealed the presence of integral membrane proteins and effector-like proteins and potential candidates for interaction with hosts. One of the integral membrane proteins was predicted as BI-1, an inhibitor of apoptosis-promoting Bax factor. Bioinformatics analyses revealed the presence of putative BI-1 in draft and complete genomes of other 'Ca. Phytoplasma' species., Conclusion: The genetic diversity within 'Ca. P. phoenicium' strain populations in Lebanon suggested that AlmWB disease could be associated with phytoplasma strains derived from the adaptation of an original strain to diverse hosts. Moreover, the identification of a putative inhibitor of apoptosis-promoting Bax factor (BI-1) in 'Ca. P. phoenicium' draft genome and within genomes of other 'Ca. Phytoplasma' species suggested its potential role as a phytoplasma fitness-increasing factor by modification of the host-defense response.

Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including 'Candidatus Phytoplasma australiense', 'Candidatus Phytoplasma japonicum' and 'Candidatus Phytoplasma fragariae'. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis vinifera L.). Such latter strains have been informally proposed to represent a separate species, 'Candidatus Phytoplasma solani', but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 (STOL) was distinguished from reference strains of previously described species of the 'Candidatus Phytoplasma' genus based on 16S rRNA gene sequence similarity and a unique signature sequence in the 16S rRNA gene. Other stolbur- and bois noir-associated ('Ca. Phytoplasma solani') strains shared >99 % 16S rRNA gene sequence similarity with strain STOL11 and contained the signature sequence. 'Ca. Phytoplasma solani' is the only phytoplasma known to be transmitted by Hyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse 'Ca. Phytoplasma solani' strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA, tuf, secY and rplV-rpsC gene sequences supported this view and yielded congruent trees in which 'Ca. Phytoplasma solani' strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of 'Ca. Phytoplasma australiense'-related strains. Based on distinct molecular and biological properties, stolbur- and bois noir-associated strains are proposed to represent a novel species level taxon, 'Ca. Phytoplasma solani'; STOL11 is designated the reference strain.

'Candidatus Phytoplasma mali', the causal agent of apple proliferation (AP) disease, is a quarantine pathogen controlled by chemical treatments against insect vectors and eradication of diseased plants. In accordance with the European Community guidelines, novel strategies should be developed for sustainable management of plant diseases by using resistance inducers (e.g. endophytes). A basic point for the success of this approach is the study of endophytic bacteria associated with plants. In the present work, endophytic bacteria living in healthy and 'Ca. Phytoplasma mali'-infected apple trees were described by cultivation-dependent and independent methods. 16S rDNA sequence analysis showed the presence of the groups Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, and Firmicutes. In detail, library analyses underscored 24 and 17 operational taxonomic units (OTUs) in healthy and infected roots, respectively, with a dominance of Betaproteobacteria. Moreover, differences in OTUs number and in CFU/g suggested that phytoplasmas could modify the composition of endophytic bacterial communities associated with infected plants. Intriguingly, the combination of culturing methods and cloning analysis allowed the identification of endophytic bacteria (e.g. Bacillus, Pseudomonas, and Burkholderia) that have been reported as biocontrol agents. Future research will investigate the capability of these bacteria to control 'Ca. Phytoplasma mali' in order to develop sustainable approaches for managing AP.

Length heterogeneity-PCR assays, combined with statistical analyses, highlighted that the endophytic bacterial community associated with healthy grapevines was characterized by a greater diversity than that present in diseased and recovered plants. The findings suggest that phytoplasmas can restructure the bacterial community by selecting endophytic strains that could elicit a plant defense response.

'Candidatus Liberibacter spp.' cause serious plant diseases. 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus' and 'Ca. L. africanus' are the aetiological agents of citrus greening (Huanglongbing) in Asia, America and Africa. 'Candidatus Liberibacter solanacearum' causes diseases in Solanaceae in America and New Zealand. All four species are vectored by psyllid insects of different genera. Here, we show that the pear psyllid pest Cacopsylla pyri (L.) hosts a novel liberibacter species that we named 'Ca. Liberibacter europaeus'. It can bloom to high titres in the psyllid host, with more than 10(9) 16S rRNA gene copies per individual. Fluorescent in situ hybridization experiments showed that 'Ca. L. europaeus' is present in the host midgut lumen, salivary glands and Malpighian tubules. 'Candidatus L. europaeus' has a relatively high prevalence (> 51%) in C. pyri from different areas in the Piedmont and Valle d'Aosta regions in Italy and can be transmitted to pear plants in experimental transmission trials. However, even though high titres of the bacterium (more than 10(8) 16S rRNA gene copies g(-1) of pear plant tissue) could be detected, in the pear tissues no specific disease symptoms could be observed in the infected plants over a 6-month period. Despite liberibacters representing potential quarantine organisms, 'Ca. L. europaeus', first described in Italy and Europe, apparently behaves as an endophyte rather than a pathogen.

During a survey on grapevine yellows disease complex in vineyards of Lombardy region (northern Italy), phytoplasmas associated with Flavescence dorée disease were identified in symptomatic grapevines. Polymerase chain reaction and restriction fragment length polymorphism (RFLP) analyses of 16S rDNA revealed the prevalence of phytoplasmal subgroup 16SrV-D. Bioinformatic analyses of nucleotide sequences of rplV and rpsC genes, amplified from 16SrV-D phytoplasma infected grapevines and cloned, underscored the presence of five confirmed rpsC single nucleotide polymorphism (SNP) lineages, determined by different combination of SNPs at nucleotide positions 29, 365, 680, and 720 of rpsC gene. Virtual and actual RFLP analyses with the enzyme TaqI validated the presence of these SNPs. Co-infections by up to four distinct rpsC SNP lineages of 16SrV-D phytoplasma were found in grapevines. These results could open new perspectives for the study of the ecology and the epidemiology of Flavescence dorée.

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, "Candidatus Phytoplasma vitis," which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the "Candidatus Cardinium hertigii" group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of "Ca. Cardinium hertigii." This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of "Ca. Phytoplasma vitis" were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and "Ca. Phytoplasma vitis" have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.

Flavescence dorée is a devastating disease of grapevine widespread in several countries in EU such as France, Italy and Spain. Genetic variability among 17 Italian and 3 French FD strains was investigated by RFLP analyses based on a fragment of the ribosomal protein operon and on the non-ribosomal DNA fragment FD9. RFLP analysis of the PCR amplified ribosomal protein fragment, coding for the 3' end of rpl22 and the entire rps3 genes, differentiated 4 rp-subgroups among the FD strains and 4 subgroups among the reference strains belonging to elm yellows group (16SrV). Sequencing and phylogenetic analysis of the same ribosomal protein DNA fragment validated the delineation of 4 distinct FD strain types derived by RFLP analyses. The results supported the differentiation based on analysis of the non-ribosomal DNA fragment FD9. The phylogenetic analysis further revealed relationships and a probable evolutionary trend among the FD strains and the other representatives of elm yellows group. All the FD strains together with the reference strains ALY, RuS and JWB formed a cluster very well distinct from the EY/ULW cluster. Moreover, ALY was shown to be more closely related to three FD strain types: the Lombardia/Piemonte, the French FD70, and the French FD88/Italian FD-D strain clusters.