•A viroid sharing 85% of nucleotide identity with apple dimple fruit viroid is present in fig.•Deep sequencing of a small RNA library from the fig accession produced a complete coverage of the viroid ...sequence.•A first characterization of small RNAs from an ADFVd fig variant was done.•A nested RT-PCR was developed allowing viroid detection in several fig accessions.
Viroids are small (246–401nt) circular and non coding RNAs infecting higher plants. They are targeted by host Dicer-like enzymes (DCLs) that generate small RNAs of 21–24nt (sRNAs), which are involved in the host RNA silencing pathways. The accumulation in plant tissues of such viroid-derived small RNAs (vd-sRNAs) is a clear sign of an ongoing viroid infection. In this study, next generation sequencing of a sRNAs library and assembling of the sequenced vd-sRNAs were instrumental for the identification of a viroid resembling apple dimple fruit viroid (ADFVd) in a fig accession. After confirming by molecular methods the presence of this viroid in the fig tree, its population was characterized, showing that the ADFVd master sequence from fig diverges from that of the ADFVd reference variant from apple. Moreover, since this viroid accumulates at a low level in fig, a semi-nested RT-PCR assay was developed for detecting it in other fig accessions. ADFVd seems to have a wider host range than thought before and this poses questions about its epidemiology. A further characterization of ADFVd-sRNAs showed similar accumulation of (+) or (−) vd-sRNAs that mapped on the viroid genome generating hotspot profiles. Moreover, similarly to other nuclear-replicating viroids, vd-sRNAs of 21, 22 and 24nt in size prevailed in the distribution profiles. Altogether, these data support the involvement of double-stranded RNAs and different DCLs, targeting the same restricted viroid regions, in the genesis of ADFVd-sRNAs.
The detection of the four grapevine viruses (GLRaV-1, GLRaV-3, GFLV and ArMV) regulated in European Union plant material certification, requires sensitive and specific diagnostic tools. A strategy of ...simultaneous detection in a real-time single tube amplification was developed, based on the EvaGreen binding dye. The melting curve analysis (MCA) of the amplicons allows a qualitative detection of the four different virus targets in multiplex analysis. A plasmid dilution assay calculated an analytical sensitivity with an amplification threshold up to 100 copies of the target sequences. A small cohort of field grapevine samples, with a known status of infection by mixtures of the target viruses or free of them, respectively, was successfully tested for the evaluation of the amplicons Tm.
High‐throughput sequencing (HTS) technologies have revolutionized plant pest research and are now raising interest for plant pest diagnostics, with plant virus diagnostics at the forefront of ...development. However, the application of HTS in plant pest diagnostics raises important challenges that plant health regulators will have to address. Adapted infrastructures, technical guidelines and training are pivotal for further use and adoption of the HTS technologies in the phytosanitary framework.
Technologies de séquençage génétique à haut débit pour le diagnostic des organismes nuisibles aux végétaux: défis et perspectives
Les technologies de séquençage génétique à haut débit ont révolutionné la recherche des organismes nuisibles aux végétaux et suscitent maintenant de l'intérêt dans le contexte de leur diagnostic, avec au premier plan celui des virus des végétaux. Cependant, l'application de ces technologies au diagnostic des organismes nuisibles aux végétaux fait émerger de nombreux challenges auxquels les personnes développant la réglementation en santé des végétaux devront faire face. Des infrastructures adaptées, des lignes directrices techniques ainsi que la formation sont des éléments clefs pour l'utilisation future et l'adoption de ces technologies dans le contexte phytosanitaire.
Пpимeнeниe тexнoлoгий выcoкoпpoизвoдитeльнoгo ceквeниpoвaния для диaгнocтики вpeдныx для pacтeний opгaнизмoв: cyщecтвyющиe пpoблeмы и oткpывaющиecя вoзмoжнocти
Texнoлoгии пиpoceквeниpoвaния (HTS) пoдняли нa кaчecтвeннo нoвый ypoвeнь иccлeдoвaния вpeдныx для pacтeний opгaнизмoв, и в нacтoящee вpeмя oни пoвышaют интepec к вoпpocaм диaгнocтики вpeдныx opгaнизмoв. Пpи этoм диaгнocтикa виpycoв pacтeний явлeтcя oднoй из пpиopитeтныx зaдaч. Oднaкo пpимeнeниe HTS для диaгнocтики вpeдныx для pacтeний opгaнизмoв coпpяжeнo c цeлым pядoм вaжныx пpoблeм, нa кoтopыe дoлжнo быть oбpaщeнo внимaниe нopмaтивныx opгaнoв фитocaнитapии. Boт пoчeмy для дaльнeйшeгo иcпoльзoвaния и пpинятия нa вoopyжeниe тexнoлoгий пиpoceквeниpoвaния (HTS) в фитocaнитapныx cтpyктypax нeoбxoдимa иx aдaптaция, coглacoвaнныe pyкoвoдящиe пpинципы и тpeнинг.
Higher plants use post-transcriptional gene silencing (PTGS), an RNA-degradation system, as a defence mechanism against viral infections. To counteract this, plant viruses encode and express PTGS ...suppressor proteins. Four of the five proteins encoded by the Grapevine virus A (GVA) genome were screened using a green fluorescent protein (GFP)-based transient expression assay, and the expression product of ORF5 (protein p10) was identified as a suppressor of silencing. ORF5 p10 suppressed local and systemic silencing induced by a transiently expressed single-stranded sense RNA. This protein was active towards both a transgene and exogenous GFP mRNAs. Ectopic expression of GVA-ORF5 by a Potato virus X vector enhanced symptom severity. The findings that p10 markedly reduces the levels of small interfering RNAs (siRNAs) and that the recombinant protein is able to bind single-stranded and double-stranded forms of siRNAs and microRNAs, suggest the existence of a potential mechanism of suppression based on RNA sequestering.
Through the application of next generation sequencing, in synergy with conventional cloning of DOP-PCR fragments, two double-stranded RNA (dsRNA) molecules of about 1.5 kbp in size were isolated from ...leaf tissue of a Japanese persimmon (accession SSPI) from Apulia (southern Italy) showing veinlets necrosis. High-throughput sequencing allowed whole genome sequence assembly, yielding a 1,577 and a 1,491 bp contigs identified as dsRNA-1 and dsRNA-2 of a previously undescribed virus, provisionally named as Persimmon cryptic virus (PeCV). In silico analysis showed that both dsRNA fragments were monocistronic and comprised the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP) genes, respectively. Phylogenetic reconstruction revealed a close relationship of these dsRNAs with those of cryptoviruses described in woody and herbaceous hosts, recently gathered in genus
Deltapartitivirus
. Virus-specific primers for RT-PCR, designed in the CP cistron, detected viral RNAs also in symptomless persimmon trees sampled from the same geographical area of SSPI, thus proving that PeCV infection may be fairly common and presumably latent.
A study of the structural, optical and electrical properties of synthetic and natural melanin by means of X-ray diffraction, absorption and photocurrent techniques is reported. The model of the ...natural melanin film as a network of nano-aggregates of polymeric units based on the indolic structure is proposed to explain the X-ray diffraction results. The shape of the absorption spectra is similar to that of amorphous and disordered semiconductors, with a very strong, broad band UV and visible absorption and an optical gap value of about 0.5 eV. Photosensitivity to sun spectra has been demonstrated by photoconductivity measurements of synthetic melanin pellets under AM1 light source illumination.
Natural and synthetic melanin have been investigated by means of optical, electrical and photoelectronic measurements. Optical measurements evidence absorption curves which allow to estimate optical ...gap for synthetic melanin, by using Tauc’s method. Dark conductivity and photoconductivity measurements were performed as a function of temperature and for different duration of thermal treatments. It has been evidenced that both quantities are thermally activated and thermal treatments play a very important role as far as gap states are concerned.
A sensitive and reliable one step RT-PCR reaction with an internal control has been developed to detect and differentiate eight important viruses that affect stone fruit tress: Apple mosaic virus ...(ApMV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), American plum line pattern virus (APLPV), Plum pox virus (PPV), Apple chlorotic leaf spot virus (ACLSV), Apricot latent virus (ApLV) and Plum bark necrosis stem pitting associated virus (PBNSPaV). In addition, we investigated the detection limit and the efficiency of three different nucleic acid extraction methods that avoid the use of organic solvents, for both multiplex RT-PCR and dot-blot hybridisation assays. The primer cocktail was used to analyse 38 stone fruits originating from nine different countries and six species. A large number of virus combinations was detected and up to three different viruses were observed in five samples. A decrease in sensitivity was observed when the primer cocktail contained more than five different pair primers. However, comparative analyses showed that the multiplex RT-PCR containing the eight virus pair primers was even more sensitive than the ELISA or molecular hybridisation assays. The use of the multiplex RT-PCR technology in routine diagnosis of stone fruit tree viruses is discussed.
The family Closteroviridae revised Martelli, G P; Agranovsky, A A; Bar-Joseph, M ...
Archives of virology
147, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Recently obtained molecular and biological information has prompted the revision of the taxonomic structure of the family Closteroviridae. In particular, mealybug-transmitted species have been ...separated from the genus Closterovirus and accommodated in a new genus named Ampelovirus (from ampelos, Greek for grapevine). Thus, the family now comprises three genera. Their major properties are (i) Closterovirus: type species Beet yellows virus, genome monopartite, 15.5-19.3 kb in size, a 22-25 kDa major coat protein (CP), the gene encoding the divergent CP analogue (CPd) upstream of the CP cistron, transmission by aphids, a membership of 8 definitive and 4 tentative species; (ii) Ampelo-virus: type species Grapevine leafroll virus 3, genome monopartite 16.9-19.5 kb in size, a 35-37 kDa major CP, a CPd cistron generally located downstream of the CP gene, transmission by pseudococcid and coccid mealybugs, a membership of 6 definitive and 5 tentative species; (iii) Crinivirus: type species Lettuce infectious yellows virus, genome essentially bipartite 15.3-19 kb in size, a 28-33 kDa CP, a CPd cistron downstream of the CP gene, transmission by whiteflies (Bemisia, Trialeurodes), a membership of 7 definitive and 3 tentative species. There are five unassigned species in the family.
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