Mehle, Nataša

ABSTRACT ‘ Candidatus Phytoplasma solani’ is a widespread and epidemiologically important phytoplasma in Europe with a broad host range, including sugar beet, potato and grapevine. In sugar beet, the recently emerging 16SrXII‐P subgroup of ‘ Ca . P. solani' frequently co‐occurs with the phloem‐associated bacterium ‘ Candidatus Arsenophonus phytopathogenicus’, which is considered the primary causal agent of the ‘syndrome des basses richesses’ (SBR), a disease that significantly affects yield and sugar content in central Europe. Currently, diagnosis and differentiation of ‘ Ca . P. solani' rely on real‐time quantitative PCR (qPCR) and nested PCR, which are labour‐intensive, costly and require complex instrumentation. Therefore, we developed a loop‐mediated isothermal amplification (LAMP) assay for specific detection of 16SrXII‐P phytoplasma that is suitable for rapid laboratory use and potential on‐site testing using crude plant and insect extracts. The new LAMP assay targets the stamp gene of 16SrXII‐P and was evaluated together with a published LAMP assay targeting the secA gene of ‘ Ca . P. solani' 16SrXII‐A. This combination enabled reliable detection and discrimination of 16SrXII‐P and 16SrXII‐A phytoplasma in sugar beet tissues and in the vector Pentastiridius leporinus . The 16SrXII‐P‐specific LAMP assay showed high analytical specificity and showed comparable performance to a TaqMan qPCR assay in the reliable detection of positive samples. In combination with simple NaOH‐Tris and phosphate‐buffered saline (PBS)‐based crude extraction protocols, the assay provided clear positive or negative results within 30 min, making it a practical tool for surveillance of 16SrXII‐P phytoplasma and rapid differentiation between the two ‘ Ca . P. solani' subgroups infecting sugar beet.

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.

Phytoplasmas of the 16SrIII group are wide spread, and have a broad plant host range. Among these, ‘Candidatus phytoplasma pruni’ (‘Ca. P. pruni’; phytoplasmas of 16SrIII subgroup A) can cause serious diseases in Prunus species and ‘Ca. P. pruni’-related strains can infect other plant species, including grapevines. In this study, a new real-time PCR detection system was developed for ‘Ca. P. pruni’ using TaqMan chemistry. This test was designed to detect ‘Ca. P. pruni’, by amplifying the species-specific secY gene. In addition, a test to amplify the group-specific 16S rRNA gene region was also developed. The performances of both tests were evaluated. The test that amplifies the secY gene provided reliable and quick detection of ‘Ca. P. pruni’. Using the newly developed and validated test, ‘Ca. P. pruni’ was not found in any of the 434 field samples collected from different plants species grown in different regions of Slovenia.

AbstractIn July 2017, a survey was conducted in a fig collection plot at Locorotondo (south of Italy) to investigate the possible presence of phytoplasmas in plants showing yellowing, deformed leaves, short internodes, mottling and mosaic. Samples were collected from symptomatic plants and tested by nested PCR assays using universal and specific primers to amplify the 16S rDNA of these prokaryotes. PCR results detected the presence of phytoplasma sequences in twenty plant samples that resulted clustering two phylogenetically distinct phytoplasmas, i.e., “Candidatus Phytoplasma asteris” and “Candidatus Phytoplasma solani” affiliated to 16SrI and 16SrXII ribosomal groups, respectively. The presence of phytoplasmas belonging to both ribosomal groups was confirmed with group specific quantitative PCR and RFLP assays on 16S ribosomal amplicons. Results of this study indicate for the first time the occurrence of phytoplasmas in fig; however, more work should be carried out to verify their association with the symptoms observed on diseased fig plants.