The focus of this research was the development and evaluation of different complex liquid and solid media for the isolation and growth of phytoplasma strains infecting grapevine plants. Previously ...reported media supporting phytoplasma isolation are commercial and not easy to modify in order to improve performance and selectivity towards obtaining pure cultures of ‘Candidatus Phytoplasma’ species. Three media (Piv®, CB and MB) were therefore evaluated for phytoplasma isolation and colony formation under microaerophilic growing conditions, using grapevine canes from plants showing yellows symptoms, and infected by “flavescence dorée”, “bois noir” and aster yellows phytoplasmas as sources. The newly developed methodology was applied for two years at three sample collection times. Broad applicability and a good repeatability in supporting phytoplasma colony formation were obtained in Pivs® and CBs media. While the MB medium did not support phytoplasma isolation and growth, the CB media support a phytoplasma growth comparable to the one obtained in the previously reported media. This medium has the advantage of a formulation that allow its modification to implement specificity towards selective phytoplasma growth. Moreover preliminary trials on serial dilutions and tetracycline addition confirmed some phytoplasma growth behaviours.
•Phytoplasmas were isolated from infected field-collected grapevine samples.•Complex media for phytoplasma isolation and growth were tested.•Unreported microaerophilic conditions are settled for phytoplasma plate colony growth.•“Flavescence dorée”, “bois noir” and aster yellows phytoplasma colonies are obtained from field infected materials.•Phytoplasma presence in colonies was detected by nested-PCR and sequencing on two genes.
Bois noir is the most widespread phytoplasma grapevine disease in Europe. It is associated with '
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 '
. 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.
As the causal agent of the grapevine yellows disease Bois noir, '
Phytoplasma solani' has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the ...very complex epidemiological cycles that involve the multiple "
. P. solani" host plants and insect vectors, of which
is the most important. In the present study, multiple genotyping of the
,
, and
genes was performed. This involved archived grapevine samples that were collected during an official survey of grapevine yellows throughout the wine-growing regions of Slovenia (from 2003 to 2016), plus samples from Austrian grapevines, stinging nettle, field bindweed, and insect samples (collected from 2012 to 2019). The data show that the tuf-b2 type of the
gene has been present in eastern Slovenia since at least 2003. The hypotheses that the occurrence of the haplotypes varies due to the geographical position of Slovenia on the Italian-Slovenian Karst divide and that the haplotypes are similar between Slovenian and Austrian Styria were confirmed. The data also show haplotype changes for host plants and
.
associated with '
. P. solani,' which might be linked to new epidemiological cycles of this phytoplasma that involve not just new plant sources and new insect vectors, but also climate and land-use changes.
Phytoplasmas are insect-vectored, difficult-to-culture bacterial pathogens that infect a wide variety of crop and non-crop plants, and are associated with diseases that can lead to significant yield ...losses in agricultural production worldwide. Phytoplasmas are currently grouped in the provisional genus ‘
Candidatus
Phytoplasma’, which includes 49
‘Candidatus’
species. Further differentiation of phytoplasmas into ribosomal groups is based on the restriction fragment length polymorphism (RFLP) pattern of the 16S rRNA-encoding operon, with more than 36 ribosomal groups (16Sr) and over 100 subgroups reported. Since disease symptoms on plants are not associated with phytoplasma identity, accurate diagnostics is of critical importance to manage disease associated with these microorganisms. Phytoplasmas are typically detected from plant and insect tissue using PCR-based methods targeting universal taxonomic markers. Although these methods are relatively sensitive, specific and are widely used, they have limitations, since they provide limited resolution of phytoplasma strains, thus necessitating further assessment of biological properties and delaying implementation of mitigation measures. Moreover, the design of PCR primers that can target multiple loci from phytoplasmas that differ at the sequence level can be a significant challenge. To overcome these limitations, a PCR-independent, multilocus sequence typing (MLST) assay to characterize an array of phytoplasmas was developed. Hybridization probe s targeting
cpn60
,
tuf
,
secA
,
secY
, and
nusA
genes, as well as 16S and
rp
operons, were designed and used to enrich DNA extracts from phytoplasma-infected samples for DNA fragments corresponding to these markers prior to Illumina sequencing. This method was tested using different phytoplasmas including ‘
Ca
. P. asteris’ (16SrI-B), ‘
Ca
. P. pruni’ (16SrIII-A),‘
Ca
. P. prunorum’ (16SrX-B), ‘
Ca
. P. pyri’ (16SrX-C), ‘
Ca
. P. mali’ (16SrX-A), and ‘
Ca
. P. solani’ (16SrXII-A). Thousands of reads were obtained for each gene with multiple overlapping fragments, which were assembled to generate full-length (typically >2 kb), high-quality sequences. Phytoplasma groups and subgroups were accurately determined based on 16S ribosomal RNA and
cpn60
gene sequences. Hybridization-based MLST facilitates the enrichment of target genes of phytoplasmas and allows the simultaneous determination of sequences corresponding to seven different markers. In this proof-of-concept study, hybridization-based MLST was demonstrated to be an efficient way to generate data regarding ‘
Ca
. Phytoplasma’ species/strain differentiation.
Grapevine is largely cultivated in several parts of the world, and a spurt in its cultivation has occurred in the last two decades in grapevine cultivated areas of South and East Asia, mainly in ...China, India, Japan, Korea, Thailand, and Indonesia. Grapevine yellows (GY) represent one of the most important diseases in viticultural areas of the world, and they have been assigned to five different groups: aster yellows AY (16SrI), peanut witches’ broom PnWB (16SrII), X-disease (16SrIII), elm yellows EY (16SrV), and Stolbur (16SrXII). This study provides a comprehensive overview of the presence of phytoplasma strains and their vectors associated with GY complex, and their potential impact on viticulture of the South and East Asia. In general, both AY and EY were reported on several herbaceous plants and/or cultivated plants in South and East Asia, along with its vectors that were largely reported in China and sporadically in Japan. Interestingly, AY and EY are yet not found in South and East Asia grapevine regions; however, their presence on different plant species suggests the potential spread of the pathogens that may occur in grapevine regions in the near future. Additionally, a few reports also suggest the presence of Stolbur group in Asian countries, along with one study that found a Stolbur-related strain in China on
Vitis vinifera
. Similarly, PnWB was also frequently reported in India and China on several plant species, but not in grapes. Conversely, sporadic detections of phytoplasma strains related to X-disease in Thailand, South Korea, and China indicate that their potential influence in viticulture is rather negligible. Our review suggests that monitoring and control strategies against GY are essential in order to prevent epidemic phytoplasma spread, especially in vine-allocated areas in Asia.
Grapevine “bois noir”, related to the presence of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), represents a serious threat in several vine-growing areas worldwide. In surveys conducted over two ...years, mild and/or moderate symptoms and lower pathogen titer were mainly associated with ‘Ca. P. solani’ strains harboring a secY gene sequence variant (secY52), whereas severe symptoms and higher titer were mainly observed in grapevines infected by phytoplasma strains carrying any one of another four variants. A comparison of amino acid sequences of the protein SecY of ‘Ca. P. solani’ strains revealed the presence of conservative and semi-conservative substitutions. The deduced three-dimensional (3D) protein analysis unveiled that one semi-conservative substitution identified in the sequence variant secY52 is responsible for a structural disordered region that probably confers a flexibility for binding to distinct molecular complexes. In fact, the other analyzed variants show an organized structure and the 3D in silico prediction allowed the identification of β-sheets. Thus, differences in symptom severity and pathogen concentration observed in grapevines infected by ‘Ca. P. solani’ strains carrying distinct secY gene sequence variants suggest a possible relationship between SecY protein structure and phytoplasma strain virulence.
Transmission trials were carried out using Reptalus quinquecostatus (Cixiidae), a potential vector of ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), to assess its ability to inoculate the ...phytoplasma to periwinkle (Catharanthus roseus), grapevine (Vitis vinifera), lavandin (Lavandula × intermedia) and tobacco (Nicotiana tabacum). Detection, genotyping and comparison of ‘Ca. P. solani’ strains carried by R. quinquecostatus showed that R. quinquecostatus carried a higher diversity of ‘Ca. P. solani’ than Hyalesthes obsoletus, major known vector of ‘Ca. P. solani’ strains. Molecular analyses also showed the presence of a new strain only in grapevines and R. quinquecostatus. ‘Ca. P. solani’ was successfully inoculated to periwinkles by R. quinquecostatus, but no transmission was achieved to the other tested plants. The ability to transmit ‘Ca. P. solani’ to plants and observations of adults feeding on grapevines in vineyards consolidate the hypothesis that R. quinquecostatus is a specific vector of ‘Ca. P. solani’ strains. Moreover, the discovery of a new genotype present in R. quinquecostatus and not in H. obsoletus, vector also present on grapes, suggests that R. quinquecostatus should have a direct role in ‘Ca. P. solani’ epidemiology. Overall, even if R. quinquecostatus has a minor or no role in ‘Ca. P. solani’ transmission from weeds to grapevines, it can have an indirect role in Bois Noir epidemiology. This planthopper can contribute to maintain an alternative ‘Ca. P. solani’ cycle in weeds even in the absence of H. obsoletus preferentially by maintaining pathogen reservoirs in wild compartments neighbouring susceptible crops.
Bois noir (BN) associated with ‘Candidatus Phytoplasma solani’ (Stolbur) is regularly found in Austrian vine growing regions. Investigations between 2003 and 2008 indicated sporadic presence of the ...confirmed disease vector Hyalesthes obsoletus and frequent infections of bindweed and grapevine. Infections of nettles were rare. In contrast present investigations revealed a mass occurrence of H. obsoletus almost exclusively on stinging nettle. The high population densities of H. obsoletus on Urtica dioica were accompanied by frequent occurrence of ‘Ca. P. solani’ in nettles and planthoppers. Sequence analysis of the molecular markers secY, stamp, tuf and vmp1 of stolbur revealed a single genotype named CPsM4_At1 in stinging nettles and more than 64 and 90 % abundance in grapevine and H. obsoletus, respectively. Interestingly, this genotype showed tuf b type restriction pattern previously attributed to bindweed associated ‘Ca. P. solani’ strains, but a different sequence assigned as tuf b2 compared to reference tuf b strains. All other marker genes of CPsM4_At1 clustered with tuf a and nettle derived genotypes verifying distinct nettle phytoplasma genotypes. Transmission experiments with H. obsoletus and Anaceratagallia ribauti resulted in successful transmission of five different strains including the major genotype to Catharanthus roseus and in transmission of the major genotype to U. dioica. Altogether, five nettle and nine bindweed associated genotypes were described. Bindweed types were verified in 34 % of grapevine samples, in few positive Reptalus panzeri, rarely in bindweeds and occasionally in Catharanthus roseus infected by H. obsoletus or A. ribauti. ‘Candidatus Phytoplasma convolvuli‘(bindweed yellows) was ascertained in nettle and bindweed samples.
A search for phytoplasma‐associated diseases was conducted for the first time in the main grapevine‐growing localities of the Dukagjini plain in Kosovo. A total of 144 samples were collected from ...grapevine cultivars displaying leaf yellowing, reddening, discolouration and irregular wood ripening, and analysed using nested and quantitative PCR assays. These assays showed that 35.4% of samples belonging to eight cultivars were positive to the presence of phytoplasmas in the 16SrXII group. The 16S rDNA phytoplasma sequences obtained from 15 samples shared identity greater than 99.5% with ‘Candidatus Phytoplasma solani’. Sequence analysis of the tuf gene showed that the strains found in Kosovar grapevines are in the tuf‐type b1 group, sharing 99.6% to 99.8% identity with ‘Ca. P. solani'‐related strains associated with the “bois noir” grapevine disease in many European countries; the secY gene sequences, on the other hand, shared 100% identity with ‘Ca. P. solani' strains from Bosnia and Herzegovina, Serbia, Croatia and Turkey. This study constitutes the first report on the presence and molecular characterization of phytoplasmas in Kosovar vineyards. Based on these results, it is recommended that testing for phytoplasma be included in the certification program for grapevine in Kosovo.
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