Molecular variability and host distribution of ‘candidatus phytoplasma solani’ strains from different geographic origins

The knowledge of phytoplasma genetic variability is a tool to study their epidemiology and to implement an effective monitoring and management of their associated diseases. ‘Candidatus Phytoplasma solani’ is associated with “bois noir” disease in grapevines, and yellowing and decline symptoms in many plant species, causing serious damages during the epidemic outbreaks. The epidemiology of the diseases associated with this phytoplasma is complex and related to numerous factors, such as interactions of the host plant and insect vectors and spreading through infected plant propagation material. The genetic variability of ‘Ca. P. solani’ strains in different host species and in different geographic areas during the last two decades was studied by RFLP analyses coupled with sequencing on vmp1, stamp, and tuf genes. A total of 119 strains were examined, 25 molecular variants were identified, and the variability of the studied genes was linked to both geographic distribution and year of infection. The crucial question in ‘Ca. P. solani’ epidemiology is to trace back the epidemic cycle of the infections. This study presents some relevant features about differential strain distribution useful for disease monitoring and forecasting, illustrating and comparing the phytoplasma molecular variants identified in various regions, host species, and time periods

Molecular characterization of ‘Candidatus Phytoplasma mali’ strains in outbreaks of apple proliferation in north eastern Italy, Hungary, and Serbia

During 2005-2008 apple plants of different varieties showing proliferation symptoms were observed in diverse areas of north eastern Italy, Hungary and Serbia. PCR/RFLP analyses showed that all the samples were infected with ‘Candidatus Phytoplasma mali’. In the 16S plus spacer region two phytoplasma profiles (P-I and P-II) were distinguished. P-I profile was detected in reference strains AP, AT1, AT2, in samples from Serbia, and in the majority of samples from Trentino; the P-II profile was prevalent in samples from Veneto; both profiles were identified in samples from Hungary, in some cases together in single samples. The analyses of rpl22-s3 genes allow the identification, in all the samples showing a P-I profile, the presence of phytoplasmas belonging to rpX-A subgroup, while in the samples showing a P-II profile it was possible to distinguish the other three reported rpX subgroups. In the majority of samples from the Veneto region phytoplasmas belonging to rpX-D subgroup were identified, while rpX-B and rpX-C subgroups were identified only in a few samples from Trentino and Veneto regions, respectively. Further RFLP analyses on AP13/AP10 amplicons differentiate among strains belonging to the rpX-A subgroup: the samples from Serbia show AP profiles, while those from Trentino show AT-2 profiles. In the samples from Hungary the presence of AT1, AT2, and AP profiles was identified.Keywords: Apple, ‘Candidatus Phytoplasma mali’, phytoplasma strains, PCR/RFLP analyses, epidemiolog

Rubbery taproot disease of sugar beet in Serbia associated with 'Candidatus phytoplasma solani'

Rubbery taproot disease (RTD) of sugar beet was observed in Serbia for the first time in the 1960s. The disease was already described in neighboring Bulgaria and Romania at the time but it was associated with abiotic factors. In this study on RTD of sugar beet in its main growing area of Serbia, we provide evidence of the association between 'Candidatus Phytoplasma solani' (stolbur phytoplasma) infection and the occurrence of typical RTD symptomatology. 'Ca. P. solani' was identified by PCR and the sequence analyses of 16S ribosomal RNA, tuf, secY, and stamp genes. In contrast, the causative agent of the syndrome “basses richesses” of sugar beet-namely, 'Ca. Arsenophonus phytopathogenicus'-was not detected. Sequence analysis of the stolbur strain's tuf gene confirmed a previously reported and a new, distinct tuf stolbur genotype (named 'tuf d') that is prevalent in sugar beet. The sequence signatures of the tuf gene as well as the one of stamp both correlate with the epidemiological cycle and reservoir plant host. This study provides knowledge that, for the first time, enables the differentiation of stolbur strains associated with RTD of sugar beet from closely related strains, thereby providing necessary information for further epidemiological work seeking to identify insect vectors and reservoir plant hosts. The results of this study indicate that there are differences in hybrid susceptibility. Clarifying the etiology of RTD as a long-known and economically important disease is certainly the first step toward disease management in Serbia and neighboring countries.This is the peer reviewed version of the following article: Ćurčić Ž., Stepanović J., Zübert C., Taški-Ajduković K., Kosovac A., Rekanović E., Kube M., Duduk B. Rubbery taproot disease of sugar beet in Serbia associated with 'Candidatus phytoplasma solani'. Plant Disease 2021, 105 (2), 255 – 263. [https://doi.org/10.1094/PDIS-07-20-1602-RE]

Identification and ecology of alternative insect vectors of ‘Candidatus Phytoplasma solani’ to grapevine

Bois noir, a disease of the grapevine yellows complex, is associated with 'Candidatus Phytoplasma solani' and transmitted to grapevines in open fields by the cixiids Hyalesthes obsoletus and Reptalus panzeri. In vine-growing areas where the population density of these vectors is low within the vineyard, the occurrence of bois noir implies the existence of alternative vectors. The aim of this study was to identify alternative vectors through screening of the Auchenorrhyncha community, phytoplasma typing by stamp gene sequence analyses, and transmission trials. During field activities, conducted in Northern Italy in a vineyard where the bois noir incidence was extremely high, nine potential alternative insect vectors were identified according to high abundance in the vineyard agro-ecosystem, high infection rate, and harbouring phytoplasma strains characterized by stamp gene sequence variants found also in symptomatic grapevines. Transmission trials coupled with molecular analyses showed that at least eight species (Aphrodes makarovi, Dicranotropis hamata, Dictyophara europaea, Euscelis incisus, Euscelidius variegatus, Laodelphax striatella, Philaenus spumarius, and Psammotettix alienus/confinis) are alternative vectors of 'Candidatus Phytoplasma solani' to grapevines. These novel findings highlight that bois noir epidemiology in vineyard agro-ecosystems is more complex than previously known, opening up new perspectives in the disease management

Multi-gene analysis for differentiation of aster yellows phytoplasmas infecting carrots in Serbia. Annals of Applied Biology, 154(2): 219-229.

During a survey of large carrot fields in Serbia, plants showing leaf reddening and/or yellowing, adventitious shoot production and reduction in taproot size and quality were observed in a low percentage of plants. To verify phytoplasma association with the described symptoms and to carry out pathogen differentiation, PCR assays followed by restriction fragment length polymorphism (RFLP) analyses and/or sequencing of phytoplasma 16Sr DNA and ribosomal protein genes l22 and s3, tuf, putative aa kinase plus ribosomal recycling factor genes and DNA helicase gene were carried out. Phytoplasmas belonging to 16SrI-A and 16SrI-B ribosomal subgroups and to rpI-A and rpI-B ribosomal protein subgroups, respectively, were identified by RFLP analyses in 13 of 15 symptomatic plants tested. No amplification was obtained with non-symptomatic carrot samples. The identification was confirmed by sequence analyses of the phytoplasma genes studied. In two carrot samples, presence of interoperon sequence heterogeneity was detected and phytoplasma strains were identified as belonging to 16SrI group but were not assigned to any 16S rRNA or ribosomal protein subgroup. This research allowed the first molecular identification of phytoplasmas infecting carrot in Serbia using several molecular markers, and it indicates that under field conditions in non-epidemic outbreaks a certain amount of genetic mutation may occur in conserved genes of these prokaryotes