Identification and GroEL gene characterization of green petal phytoplasma infecting strawberry in Italy

none5The presence of phytoplasmas in strawberry showing malformation of the fruits together with the typical green petals symptoms was detected in some North Western Italy cultivations. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rDNA and GroEL sequences specific for phytoplasmas. Bands of 1.2 kb were obtained in both cases after nested-PCR assays and RFLP analyses allowed to classify the detected phytoplasmas in the aster yellows subgroup 16SrI-C, the GroELI grouping confirm all the strains from strawberry to be identical to each other and to GroELI-VI group. This is the first multigene molecular identification of strawberry green petals phytoplasmas in Italy.openContaldo N.; J.F. Mejia; S. Paltrinieri; A. Calari; A. BertacciniContaldo N.; J.F. Mejia; S. Paltrinieri; A. Calari; A. Bertaccin

Axenic culture of plant pathogenic phytoplasmas

Phytoplasmas are microorganisms associated with severe plant diseases affecting diverse agrarian activites worldwide. Since their discovery, conclusive proof of their pathogenicity is lacking due to failure to culture these organisms. This has also hindered studies on their biology, biochemistry and physiology, although significant taxonomic progress has been achieved from study of the 16S ribosomal gene and full genomic sequencing of four phytoplasma strains. The inability to fulfil Koch’s postulates severely restricts the understanding of the roles of these organisms in plant diseases and in plant/insect/phytoplasma interactions. Here we show that specific commercial media support axenic growth of phytoplasmas under defined conditions; the identity of the organisms was confirmed by PCR/RFLP analyses and sequencing of phytoplasma-specific genes. We have demonstrated for the first time that phytoplasmas, similarly to mycoplasmas, can grow independently from their host(s). This should assist reduction of the socio-economic impact of phytoplasma diseases worldwide through improved pathogen detection and consequently better management of the diseases they cause. Knowledge of mechanisms underlying the autonomous life of phytoplasmas, that are among the smallest living organisms, should also provide important information about basic mechanisms of life

Simultaneous detection of mixed ‘Candidatus Phytoplasma asteris’ and ‘Ca. Liberibacter solanacearum’ infection in carrot

Symptoms of shoot and root malformation were observed in carrot plants in fields located in the North of Gran Canaria Island (Spain), during surveys carried out in spring 2015 and 2016. Total DNA extraction from the leaves of symptomatic plants was performed and PCR assays were carried out to detect the agents possibly associated to the observed symptoms. While ‘Candidatus Liberibacter solanacearum’ was detected in the majority of tested symptomatic samples, phytoplasmas belonging to the ribosomal group 16SrI were detected only in two samples. Phylogenetic analyses and sequencing, together with virtual RFLP, confirmed that ‘Ca. L. solanacearum’ strains detected in carrot samples belong to haplotype D

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

Studio dei meccanismi molecolari coinvolti nel determinismo sintomatologico di piante infette da virus e virus-satelliti

The aim of our work was to study the molecular mechanisms involved in symptoms appearance of plants inoculated either with a virus or with a virus-satellite complex. In the first case, we tried to set up a reliable method for an early identification of PVYNTN strains present in Italy and causing potato tuber necrosis. This, to prevent their spread in the field and to avoid severe yield losses, especially in seed potato production. We tried to localize the particular genomic region responsible for tuber necrosis. To this purpose, we carried out RT-PCR experiments using various primer combinations, covering PVY genomic regions larger than those previously used by other authors. As the previous researchers, though, we were not able to differentiate all NTN from others PVY strains. This probably because of the frequent virus variability, due to both genomic mutations and possible recombination events among different strains. In the second case, we studied the influence of Y-sat (CaRNA5 satellite) on symptoms of CMV (Cucumber mosaic virus) in Nicotiana benthamiana plants: strong yellowing appearance instead of simple mosaic. Wang et al (2004), inoculating the same infectious complex on tobacco plants transformed with a viral suppressor of plant silencing (HC-PRO), did not experience the occurrence of yellowing anymore and, therefore, hypotesized that changes in symptoms were due to plant post transcriptional gene silencing (PTGS) mechanism. In our case, inoculation of N. benthamiana plants transformed with another PTGS viral suppressor (p19), and other plants defective for RNA polymerase 6 (involved in systemic silencing), still resulted in yellowing appearance. This, to our opinion, suggests that in our system another possible mechanism is involved

Axenic culture of plant pathogenic phytoplasmas

open5siPhytoplasmas are microorganisms associated with severe plant diseases affecting diverse agrarian activites worldwide. Since their discovery, conclusive proof of their pathogenicity is lacking due to failure to culture these organisms. This has also hindered studies on their biology, biochemistry and physiology, although significant taxonomic progress has been achieved from study of the 16S ribosomal gene and full genomic sequencing of four phytoplasma strains. The inability to fulfil Koch’s postulates severely restricts the understanding of the roles of these organisms in plant diseases and in plant/insect/phytoplasma interactions. Here we show that specific commercial media support axenic growth of phytoplasmas under defined conditions; the identity of the organisms was confirmed by PCR/RFLP analyses and sequencing of phytoplasma-specific genes. We have demonstrated for the first time that phytoplasmas, similarly to mycoplasmas, can grow independently from their host(s). This should assist reduction of the socio-economic impact of phytoplasma diseases worldwide through improved pathogen detection and consequently better management of the diseases they cause. Knowledge of mechanisms underlying the autonomous life of phytoplasmas, that are among the smallest living organisms, should also provide important information about basic mechanisms of life.openContaldo N.; A. Bertaccini; S. Paltrinieri; H.M. Windsor; D.G. WindsorContaldo N.; A. Bertaccini; S. Paltrinieri; H.M. Windsor; D.G. Windso

TROPICSAFE: WP1: Grapevine yellows Italy

This dataset contains the underlying data of the following publication: Y. Zambon, A. Canel, A. Bertaccini, N. Contaldo, 2018. Molecular diversity of phytoplasmas associated with grapevine yellows disease in North-Eastern Italy. Phytopathology, 108:2, 206-214. DOI: 10.1094/PHYTO-07-17-0253-R In particular, the results obtained from a 3-year survey in different vineyards located in Treviso province (Italy) were presented in tables and figures. The tables 1-3 contain the number of grapevine and insect samples positive to phytoplasma presence and the molecular characterization of these prokaryotes on different genes. Lineages corresponding to different restriction profiles are shown, supporting the findings of a number of variants of different phytoplasmas among both, plants and insects. Table 4 contains the list of GenBank accession number of the sequences used in phylogenetic analyses in comparison with sequences obtained in the survey and listed in table 2

Biological and molecular proof of phytoplasma seed transmission in corn

Phytoplasma seed transmission is still a poorly investigated topic inspite of growing experimental evidences. Corn seeds deriving from 5 symptomatic and phytoplasma-infected cobs were sown and the germinated plants were analyzed to verify the presence of infected progenies by nested PCR on 16S ribosomal DNA, and by attempts of phytoplasma cultivation in chemically defined media to verify their viability. After sterilization, seeds were in vitro germinated and the plantlets were transferred in sterile soil in insect-proof cages and tested when they were 30 to 100 days old. Preliminary trials were carried out on 9 seedlings, 6 of which, positive to aster yellows and “stolbur” phytoplasmas, were used as plant sources for isolation trials in PivL® medium. From three plants the isolation allowed to detect aster yellows and “stolbur” phytoplasma DNAs after chloroform/phenol extraction from 1 ml of liquid medium from isolation tubes and from tubes obtained after serial dilution that have shown color change. Further tests on 79 seedlings resulted in 17 of them positive for aster yellows and “stolbur” phytoplasmas. Isolation from three of these plants after 30 days from germination resulted positive for phytoplasma DNA in tubes deriving after 2 to 10 serial dilution in fresh medium that have all shown colour change.Reisolation carried out at 90 days from germination confirmed these results only from one plants. Plating carried out together with DNAs extraction from liquid medium produced colonies of different size and shapes. The same type of colonies were obtained from plating tubes maintained for seven month at 25°C. Single colonies were picked and transferred in broth for purification steps at several times and small colonies were obtained resulting to consistently contain aster yellows DNAs; moreover this type of colony growth was observed for at least 3 subsequent passages liquid/solid media carried out about every 5 days. These preliminary results are indicating the viability of phytoplasmas isolated in corn seedlings grown in insect-proof environment

Molecular characterisation of bois noir phytoplasmas from Bosnia and Herzegovina

Bois noir (BN) phytoplasmas were associated with grapevine yellows diseases in Bosnia and Herzegovina (Delic and Lolic, 2010). The disease is widely to moderate distributed in vineyards with both domestic and imported grapevine cultivars. Molecular characterisation of selected strains of BN phytoplasmas detected in heavily BN-infected vineyards in was done to verify the presence of genetic variability. Thirty-five DNA samples of grapevines that were shown to be positive by PCR assays on the 16S rDNA were selected for the study. Polymorphisms were studied in the 16S rDNA, Tuf and ribosomal protein genes of the selected samples. Tuf1f/r, TufAYf/r, and TufINT1f/TufINT4r primer pairs were used in nested PCR for amplification of Tuf genes; RFLP analyses with HpaII showed the presence of the tuf type-b (VK-II) in all the tested samples. The BN infected samples were further amplified with rp/STOL primers and PCR products were digested with TaiI and SspI restriction enzymes. Finally 16S rDNA genes were amplified with P1/P7 primers in direct PCR and with R16F2n/R16R2 primers in nested PCR assays: both types of amplicons obtained were restricted with MboII and RsaI, MboII, Hpy188I enzymes, respectively. Restriction profiles obtained after RFLP analyses on 16S rDNA and ribosomal protein amplicons weren't uniform. Some samples showed profiles comparable with some of those recently published (Contaldo et al., 2009), while some of the profiles of other samples were totally different from any of those reported in literature. Therefore, polymorphism of these regions should be further studied to better understand BN epidemiology in Bosnia and Herzegovina

Molecular diversity of phytoplasmas associated with grapevine yellows disease in north-eastern Italy

A 3-year survey was conducted in Northern Italy to verify the presence and diversity of phytoplasmas in selected vineyards showing symptoms of severe yellows. Symptomatic and asymptomatic grapevines were sampled, and insects were collected using yellow sticky traps. The phytoplasmas detected in grapevine samples were different according to the years: "flavescence dor\ue9e" (16SrV-C/D) was detected together with other phytoplasmas such as 16SrXII-A ('Candidatus Phytoplasma solani'- related, bois noir), 16SrI-B ('Ca. P. asteris'-related, aster yellows), 16SrX-B ('Ca. P. prunorum'-related, European stone fruit yellows), and 16SrV-A ('Ca. P. ulmi'-related, elm yellows). Moreover, phytoplasmas belonging to 16SrVII-A ('Ca. P. fraxini'-related) and 16SrVI ('Ca. P. Trifolii'-related) subgroups were also identified. Identification of phytoplasmas was also carried out from insects and showed the presence of some of these phytoplasmas in Scaphoideus titanus and Orientus ishidae: 16SrXII-A, 16SrVII, and 16SrVI phytoplasmas were detected in specimens of both species, while 16SrXII-A and 16SrI-B phytoplasma strains were identified in Orientus ishidae and Hyalesthes obsoletus, and 16SrX-B in S. Titanus. Direct sequencing of selected amplicons obtained from 16S rRNA, rp, and tuf genes from grapevine and insect samples confirmed the phytoplasma identification. The 16SrVII-A and 16SrVI phytoplasmas were never detected before in grapevine, S. Titanus and Orientus ishidae in Europe and their epidemiological importance is being monitored