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

Principali patogeni e difesa

Olive as other woody perennial crops can be affected by several systemic pathogens, including bacteria, fungi and several vector-borne viruses. The most important olive diseases, caused by different pathogens, are described in this text. Pathogen biology, diagnostic technique and control strategies are reported for each disease. The main and widespread bacterial disease, caused by Pseudomonas savastanoi pv. savastanoi, is the olive knot. As concerns the most important and widespread fungal diseases, in this text are described the olive leaf spot, Verticillum wilt, cercosporiosis, anthracnose, fruit rot, parasitic brusca, root rot, sooty mold and wood rot. Infections caused by viruses are generally symptomless and do not result in diseased plants. Although, viruses do not cause detrimental disease on olive varieties, the infected olive trees represent a reservoir of virus inoculum for other crops, where these viruses are known to cause severe disease. Detection of these viruses represent an important critical step in the sanitary improvement of this crop. Recently, molecularbased assays have been effectively implemented to detect at least 8 of the 15 viruses known to infect this crop. Sanitation program using in vitro culture of shoot tip and thermotherapy have been recently described to recover virus-free plantlets.Realizzato nell'ambito del progetto "Ricerca ed Innovazione per l'Olivicoltura Meridionale", finanziato dal MiPAAFMiPAAF - Ministero delle politiche agricole alimentari e forestal

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

Presence of viruses in croatian olive groves

Tijekom 2005. i 2006. godine, prvi puta u Hrvatskoj je provedeno testiranje biljnog materijala masline na prisutnost virusa. Uzorci za testiranje su sakupljani na tradicionalnim maslinarskim područjima iz komercijalnih nasada masline, na dvadeset i pet lokaliteta. Uzorci su testirani na prisutnost osam vrsta virusa: ArMV, CMV, CLRV, OLV-1, OLV-2, OLRV, OLYaV i SLRV. Rezultati analiza provedenih primjenom RT-PCR metode, potvrdili su nazočnost virusa na oko 25 % uzoraka, a ukupno je potvrđena nazočnost četiri vrste virusa: OLYaV - Olive leaf yellowing-associated virus, CMV - Cucumber mosaic virus, OLV-2 - Olive latent virus 2 i SLRV – Strawberry latent ringspot virus. Nazočnost virusa CMV je utvrđena na uzorcima sorata Istarska bjelica i Lastovka, SLRV na sortama Levantinka i Oblica, OLV-2 na sorti Lastovka, te OLYaV na sortama Bjelica, Istarska bjelica, Lastovka i Levantinka. Uzorci sorata Buža, Perišićeva mastrinka i Rosulja bili su negativni na nazočnost virusa.During the years 2005 and 2006, testing of the olive plant material on the presence of olive viruses was conducted for the first time in Croatia. The samples for testing were collected from twenty five locations in commercial olive orchards, from traditional olive growing areas. All samples were teseted on the presence of eight virus types: ArMV, CMV, CLRV, OLV-1, OLV-2, OLRV, OLYaV and SLRV. The results of analyses conducted by using the RT-PCR method, confirmed the presence of viruses in 25% of samples, and four virus types were confirmed: OLYaV - Olive leaf yellowing-associated virus, CMV - Cucumber mosaic virus, OLV-2 - Olive latent virus 2 and SLRV – Strawberry latent ringspot virus. Presence of CMV was confirmed on Istarska bjelica and Lastovka samples, SLRV was confirmed on Levantinka and Oblica samples, OLV-2 was confirmed on Lastovka samples, OLYaV was confirmed on Bjelica, Istarska bjelica, Lastovka and Levantinka samples. Samples of Buža, Perišićeva mastrinka and Rosulja cultivars were free from tested viruses

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

CITRUS TRISTEZA VIRUS RESISTANCE GENE LOCUS: SMALL RNA PROFILE AND PRELIMINARY EPIGENETIC STUDIES

Small interfering RNAs (siRNAs), play a vital role in epigenetics of plant virus-host plant interactions. It has been extensively studied at both the transcriptional and post-transcriptional levels. In plants, siRNAs initiate and manage gene silencing by directing DNA methylation and/or histone methylation. In Arabidopsis, the ~24 nt siRNAs directs DNA methylation (RNA-directed DNA methylation, RdDM) and chromatin remodeling at their target loci. Recent advances in highthroughput sequencing techniques has enabled thorough exploration of small RNAs populations and allow rapid analysis of massive datasets to assemble complete full-length genome sequence for different plant species. This large database of sequence information also allows identification of genome regions specifically matched by siRNAs that likely differ among tolerant, resistant or susceptible hosts and advance epigenetic studies on diseased plants. Resistance to Citrus tristeza virus (CTV), the most severe virus affecting Citrus spp., associated with a single dominant gene locus Ctv occurring in Poncirus trifoliata while all Citrus spp. are considered susceptible. This locus contains 22 putative genes, but their regulation and mechanism for resistance remains unknown. In our study, CTV was graft-inoculated on Carrizo citrange (Poncirus trifoliata x C. sinensis (I think) ) and C. aurantium (sour orange) seedlings, and the population of siRNA characterized by high-throughput sequencing using an ILLUMINA platform. The Ctv-derived siRNA (~2% of the total short reads) were dominated in both hosts by the 24-nt. However, CTV infection caused an increase in accumulation of 24-nt siRNA sequences homologous to the Ctv gene in Carrizo but it decreased in sour orange. Distribution of the 24nt along the Ctv gene locus (282Kb) had a clearly different distribution between the two host. The predominant hot spot of siRNA in Carrizo mapped in the putative gene Ctv-20, whereas in sour orange it associated to the intergenic region between the putative genes Ctv-11 and Ctv-12, where a Copia-like retrotransposon C is located. This distribution profile was conserved for each species between CTV-infected and uninfected plants but, as previously mentioned, the frequency of the 24nt siRNAs was altered by the presence of the virus. We supposed that the different profile of 24nt between the two host in the locus ctv is due to RdDM mechanisms. To demonstrate the methylation status of the resistance locus we performed a bisulfite treatment of DNA. in which unmethylated cytosine was converted to uracile, while methylated cytosine did not react. A methylcytosines mapping was carried out on Ctv-11 and Ctv-12 sequences. By specific software were found 5 different CpG islands in the Copia-likeretrotransposon sequence and 42 primer pair were designed. The PCR analyses have been carried out using MSP and BSP primers followed by combined bisulfite restriction analysis (COBRA)

Non-lethal effects of N-acetylcysteine on xylella fastidiosa strain De Donno biofilm formation and detachment

This study investigated in-vitro the non-lethal eects of N-acetylcysteine (NAC) on Xylella fastidiosa subspecies pauca strain De Donno (Xf-DD) biofilm. This strain was isolated fromthe olive trees aected by the olive quick decline syndrome in southern Italy. Xf-DD was first exposed to non-lethal concentrations of NAC from 0.05 to 1000 M. Cell surface adhesion was dramatically reduced at 500 M NAC (47%), hence, this concentration was selected for investigating the eects of pre-, postand co-treatments on biofilm physiology and structural development, oxidative homeostasis, and biofilm detachment. Even though 500 MNAC reduced bacterial attachment to surfaces, compared to the control samples, it promoted Xf-DD biofilm formation by increasing: (i) biofilm biomass by up to 78% in the co-treatment, (ii) matrix polysaccharides production by up to 72% in the pre-treatment, and (iii) reactive oxygen species levels by 3.5-fold in the co-treatment. Xf-DD biofilm detachment without and with NAC was also investigated. The NAC treatment did not increase biofilm detachment, compared to the control samples. All these findings suggested that, at 500 M, NAC diversified the phenotypes in Xf-DD biofilm, promoting biofilm formation (hyper-biofilm-forming phenotype) and discouraging biofilm detachment (hyper-attachment phenotype), while increasing oxidative stress level in the biofilm

A new variant of Xylella fastidiosa subspecies multiplex detected in different host plants in the recently emerged outbreak in the region of Tuscany, Italy

The vector-borne bacterial pathogen Xylella fastidiosa is widely distributed in the Americas; in the last decade it has emerged as a serious threat for agricultural crops, natural environment and landscape in Europe. Following the first EU outbreak in 2013 in southern Italy, associated with a severe disease in olive trees, annual mandatory surveys are now in place in the Member States, leading to the discovery of bacterial outbreaks in different countries. Among the latest findings, an outbreak has been reported in the Italian region of Tuscany, with infections identified in seven different plant species. In this work, we report the isolation and the genetic characterization of isolates associated with this newly discovered outbreak. Multilocus sequence typing approach revealed the occurrence of isolates harbouring a new sequence type, denoted ST87, genetically related to strains of subsp. multiplex, but different from the genotypes of this subspecies previously characterized in Europe. Five cultured strains were successfully recovered from four of the seven host plants, an important achievement for advancing the studies on genomics and pathogenicity of these isolates and thus assess their potential threat for European agriculture