Coding-Complete Genome Sequence of a Black Queen Cell Virus Isolate from Honey Bees (Apis mellifera) in Italy

In this study, we documented the complete coding genome sequence of a Black queen cell virus (BQCV) isolate from honey bees in Italy. This genome sequence illustrates a high similarity with other BQCV isolates reported worldwide and could provide insights into BQCV genome phylogeny and divergence

Xylella fastidiosa in Olive in Apulia: Where We Stand

A dramatic outbreak of Xylella fastidiosa decimating olive was discovered in 2013 in Apulia, Southern Italy. This pathogen is a quarantine bacterium in the European Union (EU) and created unprecedented turmoil for the local economy and posed critical challenges for its management. With the new emerging threat to susceptible crops in the EU, efforts were devoted to gain basic knowledge on the pathogen biology, host, and environmental interactions (e.g., bacterial strain(s) and pathogenicity, hosts, vector(s), and fundamental drivers of its epidemics) in order to find means to control or mitigate the impacts of the infections. Field surveys, greenhouse tests, and laboratory analyses proved that a single bacterial introduction occurred in the area, with a single genotype, belonging to the subspecies pauca, associated with the epidemic. Infections caused by isolates of this genotype turned to be extremely aggressive on the local olive cultivars, causing a new disease termed olive quick decline syndrome. Due to the initial extension of the foci and the rapid spread of the infections, eradication measures (i.e., pathogen elimination from the area) were soon replaced by containment measures including intense border surveys of the contaminated area, removal of infected trees, and mandatory vector control. However, implementation of containment measures encountered serious difficulties, including public reluctance to accept control measures, poor stakeholder cooperation, misinformation from some media outlets, and lack of robust responses by some governmental authorities. This scenario delayed and limited containment efforts and allowed the bacterium to continue its rapid dissemination over more areas in the region, as shown by the continuous expansion of the official borders of the infected area. At the research level, the European Commission and regional authorities are now supporting several programs aimed to find effective methods to mitigate and contain the impact of X. fastidiosa on olives, the predominant host affected in this epidemic. Preliminary evidence of the presence of resistance in some olive cultivars represents a promising approach currently under investigation for long-term management strategies. The present review describes the current status of the epidemic and major research achievements since 2013

Spatial Bayesian Modeling Applied to the Surveys of Xylella fastidiosa in Alicante (Spain) and Apulia (Italy)

The plant-pathogenic bacterium Xylella fastidiosa was first reported in Europe in 2013, in the province of Lecce, Italy, where extensive areas were affected by the olive quick decline syndrome, caused by the subsp. pauca. In Alicante, Spain, almond leaf scorch, caused by X. fastidiosa subsp. multiplex, was detected in 2017. The effects of climatic and spatial factors on the geographic distribution of X. fastidiosa in these two infested regions in Europe were studied. The presence/absence data of X. fastidiosa in the official surveys were analyzed using Bayesian hierarchical models through the integrated nested Laplace approximation (INLA) methodology. Climatic covariates were obtained from the WorldClim v.2 database. A categorical variable was also included according to Purcell’s minimum winter temperature thresholds for the risk of occurrence of Pierce’s disease of grapevine, caused by X. fastidiosa subsp. fastidiosa. In Alicante, data were presented aggregated on a 1 km grid (lattice data), where the spatial effect was included in the model through a conditional autoregressive structure. In Lecce, data were observed at continuous locations occurring within a defined spatial domain (geostatistical data). Therefore, the spatial effect was included via the stochastic partial differential equation approach. In Alicante, the pathogen was detected in all four of Purcell’s categories, illustrating the environmental plasticity of the subsp. multiplex. Here, none of the climatic covariates were retained in the selected model. Only two of Purcell’s categories were represented in Lecce. The mean diurnal range (bio2) and the mean temperature of the wettest quarter (bio8) were retained in the selected model, with a negative relationship with the presence of the pathogen. However, this may be due to the heterogeneous sampling distribution having a confounding effect with the climatic covariates. In both regions, the spatial structure had a strong influence on the models, but not the climatic covariates. Therefore, pathogen distribution was largely defined by the spatial relationship between geographic locations

Isolation and pathogenicity of Xylella fastidiosa associated to the olive quick decline syndrome in southern Italy

In autumn 2013, the presence of Xylella fastidiosa, a xylem-limited Gram-negative bacterium, was detected in olive stands of an area of the Ionian coast of the Salento peninsula (Apulia, southern Italy), that were severely affected by a disease denoted olive quick decline syndrome (OQDS). Studies were carried out for determining the involvement of this bacterium in the genesis of OQDS and of the leaf scorching shown by a number of naturally infected plants other than olive. Isolation in axenic culture was attempted and assays were carried out for determining its pathogenicity to olive, oleander and myrtle-leaf milkwort. The bacterium was readily detected by quantitative polymerase chain reaction (qPCR) in all diseased olive trees sampled in different and geographically separated infection foci, and culturing of 51 isolates, each from a distinct OQDS focus, was accomplished. Needle-inoculation experiments under different environmental conditions proved that the Salentinian isolate De Donno belonging to the subspecies pauca is able to multiply and systemically invade artificially inoculated hosts, reproducing symptoms observed in the field. Bacterial colonization occurred in prick-inoculated olives of all tested cultivars. However, the severity of and timing of symptoms appearance differed with the cultivar, confirming their differential reaction

Embryo Culture, In Vitro Propagation, and Molecular Identification for Advanced Olive Breeding Programs

The high biodiversity of the olive tree is an important opportunity to develop sustainable plans to control Xylella fastidiosa (X) through breeding programs. Olive tree breeding activities have been limited due to various features of this species including the long time required for seed germination caused by the inhibition effect of the woody endocarp, the seed integument, and the endosperm. Starting from F1 seeds by cross-breeding, the embryo culture was compared with traditional seed germination, evaluating the effectiveness of in vitro multiplication of the plantlets for large-scale production. The isolated embryos were established on a new medium based on Rugini ‘84 macroelements, Murashige & Skoog ‘62 microelements, with Nitsch J. P. & Nitsch C. ‘69 vitamine and subcultured on Leva MSM modified. The results obtained confirmed that in vitro culture of olive embryos is a valid tool for increasing the percentage and speed of germination, helping to reduce the time of the olive breeding programs, offering the possibility to effectively propagate plantlets for further experiments

A non-targeted metabolomics study on Xylella fastidiosa infected olive plants grown under controlled conditions

In the last decade, the bacterial pathogen Xylella fastidiosa has devastated olive trees throughout Apulia region (Southern Italy) in the form of the disease called “Olive Quick Decline Syndrome” (OQDS). This study describes changes in the metabolic profile due to the infection by X. fastidiosa subsp. pauca ST53 in artificially inoculated young olive plants of the susceptible variety Cellina di Nardò. The test plants, grown in a thermo-conditioned greenhouse, were also co-inoculated with some xylem-inhabiting fungi known to largely occur in OQDS-affected trees, in order to partially reproduce field conditions in terms of biotic stress. The investigations were performed by combining NMR spectroscopy and MS spectrometry with a non-targeted approach for the analysis of leaf extracts. Statistical analysis revealed that Xylella-infected plants were characterized by higher amounts of malic acid, formic acid, mannitol, and sucrose than in Xylella-non-infected ones, whereas it revealed slightly lower amounts of oleuropein. Attention was paid to mannitol which may play a central role in sustaining the survival of the olive tree against bacterial infection. This study contributes to describe a set of metabolites playing a possible role as markers in the infections by X. fastidiosa in olive

First international proficiency testing for laboratory performance on Xylella fastidiosa detection

A proficiency test (PT) to evaluate the performance of laboratories involved in molecular and serological detection of X. fastidiosa was carried out in early 2017; 35 laboratories from EU/non- EU Countries tested 4 different methods to purify DNA, conventional and qPCR assays, and 2 ELISA tests. The number of resultant positive agreement/negative agreement/positive deviation/negative deviation was used to determine the laboratory performance (i.e. accuracy 100%). The overall results showed that all laboratories were able to correctly diagnose X. fastidiosa in the blind samples containing the highest X. fastidiosa concentrations, whereas the performance of several laboratories was negatively affected by the lack of detection in the samples with the lowest concentrations, both through molecular and serological tests. Accuracy level of 100% (laboratory conformed to the PT) was successfully recovered in the majority of the laboratories performing qPCR and PCR assays on DNA purified using at least 2 of the 4 tested protocols. The use of automated platform ensured higher laboratory performance. As expected, results of the ELISA tests generated lower performance values in the majority of the laboratories, due to the lack of detection of positive samples containing the lowest the bacterial concentration. This study provides a good overview on the laboratory performance for the diagnostics currently used in the EPPO countries and indicate useful improvements that laboratories can adopt to achieve a better performance

Next-generation sequencing and metagenomic analysis advances plant virus diagnosis and discovery

The advent of next generation sequencing (NGS) technologies dramatically advanced our ability to comprehensively investigate diseases of unknown etiology and expedited the entire process of virus discovery, identification, viral genome sequencing and, subsequently, the development of routine assays for new viral pathogens. Unlike traditional techniques, these novel approaches require no preliminary knowledge of the suspected virus(es). Currently, the RNA-Seq approach has been widely used to identify new viruses in infected plants, by analyzing virus-derived small interfering RNA populations, single- and double-stranded RNA (dsRNA) molecules extracted from infected plants. The method generates sequence in an unbiased fashion, likely allowing to detect all viruses that are present in a sample. We applied the Illumina NGS, coupled with metagenomic analysis, to generate large sequence dataset in different woody crops affected by diseases of unknown origin or infected with uncharacterized viruses or new strains. This approach allowed the identification of five novel viral species and, in addition, the sequencing of the whole genome of several viruses and viroids infecting Citrus spp., Prunus spp., grapes, fig, hazelnut, olive, persimmon and mulberry. Combined analysis of the datasets generated by using either siRNA fractions and dsRNA templates, enhanced the characterization of the whole virus-derived sequences in the infected tissues. Furthermore, profiling small RNAs from virus-infected plants led to a better understanding of host-plant response to virus and viroid infections in perennial plants. A general bioinformatic pipeline and an experimental validation strategy were developed and its application illustrated

Draft Genome Sequence of Xylella fastidiosa subsp. Fastidiosa Strain IVIA5235, Isolated from Prunus avium in Mallorca Island, Spain

We report the complete annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. fastidiosa strain IVIA5235. This strain was recovered from a cherry tree in Mallorca, Spain