M. Saponari

IDENTIFICATION OF DNA SEQUENCES RELATED TO XYLELLA FASTIDIOSA IN OLEANDER, ALMOND AND OLIVE TREES EXHIBITING LEAF SCORCH SYMPTOMS IN APULIA (SOUTHERN ITALY)

Xylella fastidiosa is an important pathogen of commercial crops, landscape trees and ornamentals in North and South America. In Europe, symptoms resembling those caused by X. fastidiosa have occasionally been observed, but the presence of this EPPO quarantinable pathogen has never been confirmed. Recently, a rapidly spreading decline of aged olive trees has taken place in a large area of the Salento peninsula (Apulia, southern Italy). PCR assays on extracts from leaf veins and petioles of diseased trees gave positive reactions using X. fastidiosa gene-specific primers. In particular, PCR amplicons were generated by primers targeting the conserved hypothetical HL protein (Francis et al., 2006), the RNA polymerase sigma-70 factor, and the 16S rDNA genes (Rodrigues et al., 2003). Furthermore, molecular tests extended to almond and oleander trees with leaf scorching symptoms, growing next to diseased olive orchards, were also positive for X. fastidiosa. PCR products amplified from diseased olive trees were sequenced in duplicate and the sequences (EMBL-EBI provisional accession Nos HX2000034932- HX2000035003) showed 95 to 99% identity with the homologous genomic regions of X. fastidiosa. Tests for ascertaining the presence of X. fastidiosa by DAS-ELISA using two commercial kits (Agadia, USA and Bio-Rad, USA) were also positive, thus confirming molecular tests. Studies aimed at isolating the bacterium, determining the strain, evaluating its pathogenicity, and identifying the putative local vector(s) are currently in progress. X. fastidiosa has an extensive natural host range, including olive, from which the bacterial genotype A, pathogenic to oleander and almond, but not to grapevine, has been isolated in California (Krugner et al., 2010).

DETECTION OF XYLELLA FASTIDIOSA IN OLIVE TREES BY MOLECULAR AND SEROLOGICAL METHODS

SUMMARY Xylella fastidiosa has recently been identified in the Apulian province of Lecce (south-eastern Italy) in olive trees affected by a devastating disease denoted Olive Quick Decline Syndrome (OQDS), that appeared suddenly in 2010. Symptoms of OQDS consist of withering and desiccation of scattered terminal shoots, which rapidly expands to the rest of the canopy, and results in the collapse and death of the tree. The identification of X. fastidiosa in OQDS-affected trees represents the first confirmed detection of this bacterium in the European Union (EU), but its exact role in the aetiology of this disease is yet to be determined. Since X. fastidiosa is a regulated quarantine pathogen in the EU, upon request of the Apulian Plant Protection Service, surveys were initiated in order to delineate the contaminated area. To this effect, diagnostic protocols based on ELISA and conventional PCR for X. fastidiosa detection in olive samples were compared and validated via an interlaboratory ring-test in which three accredited laboratories, all located in Italy, participated. Both procedures proved to be equally effective but, due to the simplicity of sample preparation, ELISA was chosen for the large-scale X. fastidiosa monitoring programme now in progress.

Identification and Characterization of Citrus yellow vein clearing virus, A Putative New Member of the Genus Mandarivirus

Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing disease of lemon trees, reported in Pakistan, India, and more recently in Turkey and China. CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus-induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants. A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses. Contigs assembled from the Illumina sequenced short reads were used to construct the whole genome of Citrus yellow vein clearing virus (CYVCV), consisting in a positive-sense RNA of 7,529 nucleotides and containing six predicted open reading frames. The CYVCV genome organization and size resembled that of flexiviruses, and search for sequence homologies revealed that Indian citrus ringspot virus (ICRSV) (Mandarivirus, Alphaflexiviridae) is the most closely related virus. However, CYVCV had an overall nucleotide sequence identity of ≈74% with ICRSV. Although the two viruses were similar with regard to genome organization, viral particles, and herbaceous host range, CYVCV caused different symptoms in citrus and was serologically distinct from ICRSV. Primer pairs were designed and used to detect the virus by conventional and quantitative reverse transcription-polymerase chain reaction on yellow vein clearing symptomatic field trees as well as graft- and mechanically inoculated host plants. Collectively, these data suggest that CYVCV is the causal agent of yellow vein clearing disease and represents a new species in the genus Mandarivirus.

ISOLATION OF A XYLELLA FASTIDIOSA STRAIN INFECTING OLIVE AND OLEANDER IN APULIA, ITALY

SUMMARY The isolation in pure culture of the Xylella fastidiosa strain associated with the quick decline syndrome of olive, recently observed in Apulia (Salento peninsula, southern Italy) was attempted from symptomatic, naturally infected olive and oleander plants, and a periwinkle seedling that had been exposed to, and was infected by Xylella-positive spittlebugs. Prior to isolation, the presence of Xylella was ascertained in all donor hosts by PCR, indirect immunofluorescence and electron microscopy. Isolations from olive failed because of the heavy contamination by bacteria other than Xylella. By contrast, pure bacterial cultures were obtained from oleander and periwinkle extracts plated in periwinkle wilt gelrite (PWG) and buffered cysteine-yeast extract (BCYE) media. In both media, colonies were slow-growing, small-sized (less than 1 mm 25 days from plating), non pigmented, opalescent and exhibited the same morphology, except for the margin that was entire in BCYE and somewhat irregular in PWG. Bacterial cells were rod-shaped with rounded ends, had a thick and rippled cell wall, an average width of 0.35 µm, and a maximum length of ca. 5 µm. They gave a positive reaction in immunofluorence assays and were clearly decorated by colloidal gold in immunogold labelling tests. Sequenced PCR products amplified from periwinkle and oleander colonies shared 97-99% sequence identity with known X. fastidiosa strains from database and were 100% identical to one another and to comparable sequences obtained from infected olive trees. These sequences grouped in a distinct cluster of a branch comprising X. fastidiosa isolates belonging to the subspecies pauca.

Draft Genome Sequence of the Xylella fastidiosa CoDiRO Strain

ABSTRACT We determined the draft genome sequence of the Xylella fastidiosa CoDiRO strain, which has been isolated from olive plants in southern Italy (Apulia). It is associated with olive quick decline syndrome (OQDS) and characterized by extensive scorching and desiccation of leaves and twigs.

Spittlebugs as vectors of Xylella fastidiosa in olive orchards in Italy

The recent introduction of Xylella fastidiosa in Europe and its involvement in the Olive Quick Decline Syndrome (OQDS) in Apulia (Salento, Lecce district, South Italy) led us to investigate the biology and transmission ability of the meadow spittlebug, Philaenus spumarius, which was recently demonstrated to transmit X. fastidiosa to periwinkle plants. Four xylem-sap-feeding insect species were found within and bordering olive orchards across Salento during a survey carried out from October 2013 to December 2014: P. spumarius was the most abundant species on non-olive vegetation in olive orchards as well as on olive foliage and was the only species that consistently tested positive for the presence of X. fastidiosa using real-time PCR. P. spumarius, whose nymphs develop within spittle on weeds during the spring, are likely to move from weeds beneath olive trees to olive canopy during the dry period (May to October 2014). The first X. fastidiosa-infective P. spumarius were collected in May from olive canopy: all the individuals previously collected on weeds tested negative for the bacterium. Experiments demonstrated that P. spumarius transmitted X. fastidiosa from infected to uninfected olive plants. Moreover, P. spumarius acquired X. fastidiosa from several host plant species in the field, with the highest acquisition rate from olive, polygala and acacia. Scanning electron microscopy (SEM) revealed bacterial cells resembling X. fastidiosa in the foreguts of adult P. spumarius. The data presented here are essential to plan an effective IPM strategy and limit further spread of the fastidious bacterium.

NEW HOSTS OF XYLELLA FASTIDIOSA STRAIN CoDiRO IN APULIA

In the course of surveys carried out in June 2014 in the Salento (Apulia, southern Italy) area affected by an epidemic of a strain of Xylella fastidiosa subsp. pauca (Cariddi et al., 2014) denoted CoDiRO (abbreviation from the Italian name “Complesso del Disseccamento Rapido dell’Olivo”), the following symptomatic plants were observed: (i) cherry (Prunus avium), 13 trees showing scanty vegetation and bud failure, but no leaf scorching; (ii) myrtle-leaf milkwort (Polygala myrtifolia), three shrubs showing extensive desiccation of twigs and scorched leaves; (iii) coastal rosemary (Westringia fruticosa), one shrub with extensive chlorosis and desiccation of the leaves. Samples collected from all these hosts (except for two of the 13 cherry plants) were ELISA- and PCR-positive upon testing with the protocols described by Loconsole et al. (2014). Sequencing of the amplified products from five housekeeping genes (gyrB, 16S rRNA, dnaK, tonB, RNA polymerase sigma factor) and of the PCR products obtained using the X. fastidiosa strain- specific primers 272-1int/272-2int, showed that all these amplicons, regardless of the host of origin, had 100% sequence identity with the homologous products amplified from diseased olive trees (Cariddi et al., 2014). These results provide evidence that all the analyzed positive samples contain the same X. fastidiosa strain infecting olives in the same area. With the exception of cherry, for which there is a recorded infection by X. fastidiosa subsp. fastidiosa in California (Hernandez- Martinez et al., 2007), to the best of our knowledge P. myrtifolia and W. fruticosa are hitherto unreported hosts of this bacterium.

Draft genome sequence of CO33, a coffee-infecting isolate of Xylella fastidiosa

ABSTRACT The draft genome sequence of Xylella fastidiosa CO33 isolate, retrieved from symptomatic leaves of coffee plant intercepted in northern Italy, is reported. The CO33 genome size is 2,681,926 bp with a GC content of 51.7%.

Complete genome sequence of the olive-infecting strain Xylella fastidiosa subsp. pauca De Donno

ABSTRACT We report here the complete and annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. pauca strain De Donno. This strain was recovered from an olive tree severely affected by olive quick decline syndrome (OQDS), a devastating olive disease associated with X. fastidiosa infections in susceptible olive cultivars.

Molecular and biological characterization of a novel mild strain of citrus tristeza virus in California

Strain differentiating marker profiles of citrus tristeza virus (CTV) isolates from California have shown the presence of multiple genotypes. To better define the genetic diversity involved, full-length genome sequences from four California CTV isolates were determined by small-interfering RNA sequencing. Phylogenetic analysis and nucleotide sequence comparisons differentiated these isolates into the genotypes VT (CA-VT-AT39), T30 (CA-T30-AT4), and a new strain called S1 (CA-S1-L and CA-S1-L65). S1 isolates had three common recombination events within portions of genes from VT, T36 and RB strains and were transmissible by Aphis gossypii. Virus indexing showed that CA-VT-AT39 could be classified as a severe strain, whereas CA-T30-AT4, CA-S1-L and CA-S1-L65 were mild. CA-VT-AT39, CA-S1-L, and CA-S1-L65 reacted with monoclonal antibody MCA13, whereas CA-T30-AT4 did not. RT-PCR and RT-qPCR detection assays for the S1 strain were developed and used to screen MCA13-reactive isolates in a CTV collection from central California collected from 1968 to 2011. Forty-two isolates were found to contain the S1 strain, alone or in combinations with other genotypes. BLAST and phylogenetic analysis of the S1 p25 gene region with other extant CTV sequences from the NCBI database suggested that putative S1-like isolates might occur elsewhere (e.g., China, South Korea, Turkey, Bosnia and Croatia). This information is important for CTV evolution, detection of specific strains, and cross-protection.