Brader, Günter

AbstractRecent outbreaks of ‘Candidatus Phytoplasma solani’ resulted in severe losses in potatoes, vegetable crops and grapevines in certain regions of Austria and constituted a major challenge for seed potato production. Therefore, the effects of various insecticides and insect deterrents on pathogen spread were studied both in laboratory and field experiments from 2018 to 2021. In laboratory transmission experiments, field captured Hyalesthes obsoletus were caged on differently treated Catharanthus roseus for five days. The insecticides lambda-cyhalothrin, deltamethrin, esfenvalerate, acetamiprid and chlorpyriphos showed the most rapid impact on insect survival and fully prevented phytoplasma transmission. The particle film forming products kaolin and diatomaceous earth had some effect. A transfer of the promising laboratory results to potato fields, however, was achieved to a limited extent only. Treatments with pyrethroids and acetamiprid every 8–10 days over the flight period of H. obsoletus roughly halved the number of symptomatic plants and tubers in case of moderately susceptible varieties and moderate infection pressure. In the event of susceptible varieties and high disease pressure, treatment effects were hardy discernible. In practical terms, the experiments indicate that insecticide applications alone are not sufficient to mitigate the disease. Spraying of diatomaceous earth and mineral oil did not affect disease incidence in the field.

The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including ‘Candidiatus Phytoplasma solani’ are unknown. Six putative pathogenicity factors/effectors from six different strains of ‘Ca. P. solani’ were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate–glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate–glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.

As the causal agent of the grapevine yellows disease Bois noir, ‘Candidatus 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 “Ca. P. solani” host plants and insect vectors, of which Hyalesthes obsoletus is the most important. In the present study, multiple genotyping of the tuf, secY, stamp, and vmp1 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 tuf 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 H. obsoletus associated with ‘Ca. 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.

AbstractPear decline, induced by the phytoplasma 'Candidatus Phytoplasma pyri', transmitted by pear psyllids, is one of the most devastating diseases on Pyrus communis in Europe and North America. Investigations of pear psyllids in 4 pear orchards in lower Austria showed the presence of Cacopsylla pyri, C. pyricola and C. pyrisuga at all locations. PCR analyses revealed overall phytoplasma infection rates for C. pyri of 5.4%, for C. pyricola, of 4.6%, for C. pyrisuga remigrants of 9.6% and for C. pyrisuga emigrants of 0%. The rates of PCR-positive C. pyri and C. pyricola individuals varied greatly in the course of the year, and the highest infection rates were observed in late summer, autumn and in late winter. In transmission experiments with healthy pear seedlings, winterform individuals of C. pyri and C. pyricola transmitted the pathogen to 19.2% (5 out of 26) and 4.8% (2 out of 41) of the test plants, respectively. The vectoring ability of C. pyrisuga was experimentally proven for the first time, and in transmission experiments with remigrants, 9.5% (2 out of 21) of the pear seedlings were infected. Our data indicate a significant risk of pathogen transmission in pear orchards during the greater part of the year, especially in late winter, early spring and autumn. Multilocus sequence analysis by aid of the genes aceF and imp allowed the discrimination between 15 phytoplasma types. Three so far undescribed aceF genotypes and four undescribed imp genotypes were identified.

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.

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.