Optimisation of sampling and testing for asymptomatic olive trees infected by Xylella fastidiosa in Apulia region, Italy

Early detection of Xylella fastidiosa outbreaks in Apulian olive groves is crucial, especially in buffer zones and pathogen-free areas where olive trees are asymptomatic. Three studies were conducted. Two were on the spatial and temporal progression of X. fastidiosa infections in tree canopies of asymptomatic or mildly symptomatic olive trees of tolerant ('Leccino') and susceptible ('Cellina di Nardò' and 'Ogliarola salentina') cultivars. Despite different trends in pathogen infection rates and concentrations between 'Leccino' and susceptible olive cultivars over the study period, results showed that sampling was most effective in the mid-upper part of tree canopies throughout the year, excluding the warmest and coldest periods. Stem xylem tissues were the most appropriate for detecting the pathogen compared to lower parts of mature leaves with petioles, using serological and molecular assays. Based on these results, a third study was conducted to compare molecular and serological tests (qPCR, real-time LAMP, DAS-ELISA, DTBIA) for detection of X. fastidiosa in the mid-upper part of asymptomatic branches of infected 'Leccino' trees that were sampled in an appropriate collection time, using stem xylem tissue as the most appropriate matrix for testing. The molecular methods showed the greatest sensitivity, with no undetermined results, while among the serological assays, DTBIA was more sensitive than DAS-ELISA. An improved protocol for monitoring asymptomatic olive trees is recommended

A real time loop-mediated isothermal amplification (RealAmp) assay for rapid detection of Pleurostoma richardsiae in declining olive plants

Pleurostoma richardsiae is associated with host trunk diseases, known to cause dieback, cankers and wilting of woody trees, and human infections. This fungus was isolated from wood tissues of declining olive trees and grapevines showing esca disease symptoms, in the Apulia region of Italy. Fungus detection has been based on morphological and molecular features, which are time-consuming to identify and require well-trained personnel. Improvement of Pl. richardsiae detection in olive was achieved through development of real time loop-mediated isothermal amplification targeting the intergenic spacer (IGS) region of the fungus. Specificity of the assay was confirmed using ten Pl. richardsiae strains and 36 other fungus strains of species usually isolated from declining olive trees. The achieved limit of detection was 7.5 × 10-2 ng μL-1 of Pl. richardsiae genomic DNA. A preliminary validation of RealAmp was also performed using material from infected olive plants artificially inoculated in a greenhouse

Infection Cycle of Artichoke Italian Latent Virus in Tobacco Plants : Meristem Invasion and Recovery from Disease Symptoms

Peer reviewe

Scheme of a Samsun tobacco plant showing position of leaves used in this study.

<p>Scheme of a Samsun tobacco plant showing position of leaves used in this study.</p

Mixed infections of PVY-SON41 and AILV-V in tobacco exacerbate disease symptoms.

<p>In <b>A,</b> mild mosaic and moderate leaf blade malformation induced in tobacco at 30 dpi with PVY-SON41. In <b>B,</b> Chlorotic/necrotic ringspots, severe reduction of leaf lamina and plant growth induced at 30 dpi by a mixed infection of PVY-SON41 and AILV-V.</p

AILV-V RNA-2 accumulation varies in the same leaf, progressively decreasing moving to the successive leaf.

<p>Load of viral RNA (lines) was estimated by quantitative dot blot hybridization. RNA data are expressed as means of two independent experiments, were derived from spot intensity values of the target RNAs and were calculated on the basis of a standard curve generated by serial dilutions of a plasmid preparation containing the fragment of the RNA-2 of AILV-V targeted by the probe. Samples were collected from the 3^rd, 4^th, 5^th, and 6^th leaves at 24 h intervals from 10 to 23 dpi and then at 28 and 60 dpi. Each point in the line chart represents average of three biological replicates for each of the two experiments and error bars on lines represent the standard error among replicates. Figure shows also the relative quantity (RQ) of <i>RDR6</i> and <i>DCL4</i> transcripts (columns chart) in samples of tobacco plants collected at selected time points between 10 and 60 dpi with AILV-V. The values were first normalized on the accumulation level of the <i>GAPDH</i> mRNA (Δ cycle threshold [Ct] = Ct_GAPDH–Ct_target RNA) and then used to determine the relative quantification of each target RNA with a calibrator, according to the formula ΔΔCt = ΔCt_calibrator–ΔCt_target RNA. Each target mRNA in an individual mock-inoculated plant served as calibrator (RQ set to 1) for the respective gene. RQ for <i>RDR6</i> and <i>DCL4</i> transcripts was deduced by the formula expression 2^−ΔΔCt. Columns represent mean RQ values from three biological replicates for each of the two experiments and different letters represent statistically significant differences values according to separate one-way ANOVA analysis for each target mRNA, using Tukey's test (P<0 05). Vertical bars on columns represent standard deviations among replicates.</p

Small interfering RNAs (siRNA) produced in response to AILV-V infection remain below a detectable level.

<p>In <b>A</b> and <b>B</b>, panels represent total RNA preparations enriched in siRNA obtained from samples collected from apical (Ap), rub-inoculated (I) and 3rd (3rd) leaf at 7, 14 and 21 dpi with AILV-V. RNA preparations were first separated in by polyacrylamide gel electrophoresis and stained with ethidium bromide (EtBr)), then transferred to nylon membrane by electroblotting and hybridized with an AILV-V -specific RNA probe (AILV-V) for the last 760 3′-terminal sequences, labeled with digoxigenin and hydrolyzed by alkaline treatment. H =  total RNA extracted from an healthy tobacco leaf. P = 23 bp primer. Arrows point the position expected for the 23 bp primer, after hybridization. In <b>C</b>, detection of siRNAs in samples collected from leaves of a tobacco plant at 10 dpi with PVY-SON41. Panels show ethidium bromide staining (EtBr) after PAGE analysis and signals produced after hybridization with a PVY-specific RNA probe (PVY) labeled with digoxigenin and hydrolyzed by alkaline treatment. Arrows point position of 21 and 23 bp primers used as size markers.</p

Effect of suppressors of RNA silencing (VSR) derived from different RNA and DNA viruses and expressed as transgenes in lines of <i>N. tabacum</i> cv Xanthi on the development of symptoms and induction of the recovery phenotype upon infection of AILV-V.

<p>*Transgenic plant line expressing the following VSR: P1 from <i>Cocksfoot mottle virus</i> (P1 CfMV) and from <i>Rice yellow mottle virus</i> (P1 RYMV), Hc-Pro from Potato virus Y (PVY), AC2 of <i>African cassava mosaic</i> (ACMV), P25 from <i>Potato virus X</i> (PVX), 2b from <i>Cucumber mosaic virus</i> (CMV) and P19 from <i>Tomato bushy stunt virus</i> (TBSV). Symptoms were recorded between 3 and 50 dpi with AILV-V. VC =  vein clearing; LR =  local ringspots; RS = systemic ringspots; M =  mosaic; B =  leaf blistering R =  recovery. WT =  untransformed <i>N. tabacum</i> cv Xanthi; pBin61 =  empty vector.</p

AILV-V is unable to revert GFP silencing while interferes with cell-to-cell movement of silencing signal.

<p>In <b>A,</b> progression of GFP silencing (indicated by dark red areas along the major veins) in a plant of <i>N. benthamiana</i>, line 16c, at 14 dpi with the TMV-GFP vector. Silenced areas were inoculated with AILV-V but no desilencing effects were observed at 30 dpi with AILV-V; rather the silenced areas expanded (in <b>B</b>) following the spread of TMV-GFP infection. In <b>C</b> Free GFP was expressed transiently in 16c <i>N. benthamiana</i> from the binary vector pBIN-mGFP4 carried by <i>A. tumefaciens</i>. Prior to agroinfiltration, leaves were mock-inoculated with buffer (Mock) or with AILV-V (AILV), PVY-SON41 (PVY), AILV-V and PVY- SON41 (AILV+PVY) and PVA-B11 (PVA). Upon ectopic expression of GFP, a thin border of dark red tissue was visible at 14 dpi in plants mock-inoculated indicating short-range movement of GFP silencing. This border was not produced in leaves of plants inoculated with AILV-V, suggesting a viral interference with cell-to-cell movement of the silencing signal. Green fluorescent areas visible in AILV+PVY, PVY and PVA infected plants indicate suppression of silencing driven by VSR coded by PVY-SON41 and PVA-B11.</p

AILV-V would not replicate in leaves recovered from disease symptoms but retains infectivity.

<p>In <b>A.</b> local and systemic symptoms consisting, respectively, in chlorotic spots and line patterns and, chlorotic/necrotic ringspots surrounding the veins and peripheral vein clearing induced by AILV-V in tobacco at 12 dpi with sap extracted from tobacco leaves, which had recovered from disease symptoms at 40 dpi. In <b>B.</b> Northern blot hybridization for detection of (+)RNA (1) and (−)RNA (2) on RNA preparations extracted from the following sources: A =  apical leaves at 40 dpi; 5thR and 5thL =  samples collected from opposite sites (Right and Left) from the 5th leaf of two tobacco plants at 40 dpi with recovery phenotype; 4th =  sample collected from the 4th leaf of a tobacco plant with severe symptoms of systemic infection; V =  RNA from a purified preparation of AILV-V particles, used as positive control; H =  sample collected from an healthy tobacco plant, used as negative control. The picture shows the presence of a weak signal of hybridization with the plus-sense RNA probe (which detects the replication specific minus-sense RNA) only in correspondence of the sample collected from the 4th leaf with severe symptoms of systemic infection. In <b>C.</b> Accumulation of AILV-V RNA2 determined by dot blot hybridization in leaf samples collected from two recovered plants (P1 and P2) at 28 dpi before and 14 days after secondary inoculation. + and – indicate positive (pAILV769 plasmid DNA) and negative (mock-inoculated plant leaf) controls, respectively.</p