Federica Terlizzi

A new grapevine virus discovered by deep sequencing of virus- and viroid-derived small RNAs in Cv Pinot gris.

Field symptoms of chlorotic mottling and leaf deformations were observed on the cv Pinot gris (PG) in the Trentino region (Italy). Extensive assays excluded the presence of widely distributed nepo-, ampelo- and vitiviruses. An analysis of small RNA populations from two PG grapevines showing or not symptoms was carried out by Illumina high throughput sequencing. The study disclosed the virus and viroids contents of the two vines that was composed by Grapevine rupestris stem pitting-associated virus (GRSPaV), two viroids Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1), the marafiviruses Grapevine rupestris vein feathering virus (GRVFV) and Grapevine Syrah virus 1 (GSyV-1), and a hitherto unrecorded virus. This virus had a genome organization identical to that of Grapevine berry inner necrosis virus (GINV), a trichovirus reported only from Japan, with which it grouped in phylogenetic trees constructed with sequences of the RdRp domain and the coat protein gene. However, molecular differences with GINV are wide enough to warrant classification of the virus in question as a new species, for which the provisional name of Grapevine Pinot gris virus (GPGV) is proposed. A limited field survey for the presence of GPGV in diseased and symptomless plants from three different cultivars did not allow to clearly associating the virus to the observed symptoms.

Independent origins of vectored plant pathogenic bacteria from arthropod-associated Arsenophonus endosymbionts.

The genus Arsenophonus (Gammaproteobacteria) is comprised of intracellular symbiotic bacteria that are widespread across the arthropods. These bacteria can significantly influence the ecology and life history of their hosts. For instance, Arsenophonus nasoniae causes an excess of females in the progeny of parasitoid wasps by selectively killing the male embryos. Other Arsenophonus bacteria have been suspected to protect insect hosts from parasitoid wasps or to expand the host plant range of phytophagous sap-sucking insects. In addition, a few reports have also documented some Arsenophonus bacteria as plant pathogens. The adaptation to a plant pathogenic lifestyle seems to be promoted by the infection of sap-sucking insects in the family Cixiidae, which then transmit these bacteria to plants during the feeding process. In this study, we define the specific localization of an Arsenophonus bacterium pathogenic to sugar beet and strawberry plants within the plant hosts and the insect vector, Pentastiridius leporinus (Hemiptera: Cixiidae), using fluorescence in situ hybridization assays. Phylogenetic analysis on 16S rRNA and nucleotide coding sequences, using both maximum likelihood and Bayesian criteria, revealed that this bacterium is not a sister taxon to “Candidatus Phlomobacter fragariae,” a previously characterized Arsenophonus bacterium pathogenic to strawberry plants in France and Japan. Ancestral state reconstruction analysis indicated that the adaptation to a plant pathogenic lifestyle likely evolved from an arthropod-associated lifestyle and showed that within the genus Arsenophonus, the plant pathogenic lifestyle arose independently at least twice. We also propose a novel Candidatus status, “Candidatus Arsenophonus phytopathogenicus” novel species, for the bacterium associated with sugar beet and strawberry diseases and transmitted by the planthopper P. leporinus.

A Rapid Protocol of Crude RNA/DNA Extraction for RT-qPCR Detection and Quantification of 'Candidatus Phytoplasma prunorum'

Many efforts have been made to develop a rapid and sensitive method for phytoplasma and virus detection. Taking our cue from previous works, different rapid sample preparation methods have been tested and applied to Candidatus Phytoplasma prunorum (‘Ca. P. prunorum’) detection by RT-qPCR. A duplex RT-qPCR has been optimized using the crude sap as a template to simultaneously amplify a fragment of 16S rRNA of the pathogen and 18S rRNA of the host plant. The specific plant 18S rRNA internal control allows comparison and relative quantification of samples. A comparison between DNA and RNA contribution to qPCR detection is provided, showing higher contribution of the latter. The method presented here has been validated on more than a hundred samples of apricot, plum and peach trees. Since 2013, this method has been successfully applied to monitor ‘Ca. P. prunorum’ infections in field and nursery. A triplex RT-qPCR assay has also been optimized to simultaneously detect ‘Ca. P. prunorum’ and Plum pox virus (PPV) in Prunus.

Mycoviruses Infecting True Truffles

The study of viruses able to infect fungi (mycoviruses) is a relative new branch of virology that is expected to increase drastically in the next future due to the next-generation sequencing technology contribution. Mycoviruses are often latent or symptomless in many fungi but can also cause various effects on the hosts such as hypovirulence, lack of sporulation and also hypervirulence. Several viral agents have been identified as the etiologic agents of economically important diseases of the mushrooms Agaricus bisporus and Pleurotus ostreatus, while many others have been isolated from asymptomatic edible fungi. In the last years, few reports confirmed that mycoviruses also infect both arbuscular and ectomycorrhizal fungi such as Rhizophagus clarus, Hebeloma circinans, or Pisolithus microcarpus. Up to now just several viruses have been reported infecting the true truffles Tuber aestivum and Tuber excavatum, apparently not associated to alterations on morphology of the colonised fruiting bodies or mycelia. We recently obtained evidences that new viral species within the genus Endornavirus infect symptomatic fruiting bodies of Tuber magnatum. Considering the lack of information about mycoviruses infecting the genus Tuber and the devastating effect of some viral diseases of mushrooms, we should not underestimate the potential risk of viral infection in truffles. We therefore need to improve knowledge regarding the viral population of true truffle worldwide, in particular considering the high economic impact of this crop.

Genetic variability of the movement and coat protein genes of Grapevine fanleaf virus isolates from Italy

AbstractGrapevine fanleaf virus (GFLV), a nematode-transmitted virus belonging to the genus Nepovirus, is the major pathogen responsible for fanleaf degeneration, one of the most widespread and damaging viral diseases of grapevine. The virus is characterized by a genome constituted by two single positive sense RNAs (RNA1 and RNA2), coding for two polyproteins. Here we investigated the genetic variability of the movement and coat protein genes (2BMP and 2CCP) of Italian GFLV isolates by sequencing analyses. The presence of high molecular heterogeneity between our isolates suggests that GFLV comprises a family of sequence variants.