Giuliana Loconsole

Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family Geminiviridae

In the attempt to identify the causal agent of Citrus chlorotic dwarf disease (CCDD), a virus-like disorder of citrus, the small RNA fraction and total DNA from symptomatic citrus plants were subjected to high-throughput sequencing. DNA fragments deriving from an apparently new geminivirus-like agent were found and assembled by NGS to re-construct the entire viral genome. The newly identified virus has a circular single-stranded DNA genome comprising five open reading frames (ORFs) with sequence homologies with those encoded by geminiviruses. PCR and qPCR assays were successfully used for determining its presence in the CCDD-affected plants obtained by graft propagation. The larger genome size (3.64 vs. 2.5-3.0 kb) and a number of differences in its structural organization, identified this virus as a highly divergent member of the family Geminiviridae, to which the provisional name of Citrus chlorotic dwarf-associated virus (CCDaV) is assigned.

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.

Infectivity and Transmission of Xylella fastidiosa by Philaenus spumarius (Hemiptera: Aphrophoridae) in Apulia, Italy

ABSTRACT Discovery of Xylella fastidiosa from olive trees with “Olive quick decline syndrome” in October 2013 on the west coast of the Salento Peninsula prompted an immediate search for insect vectors of the bacterium. The dominant xylem-fluid feeding hemipteran collected in olive orchards during a 3-mo survey was the meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Aphrophoridae). Adult P. spumarius, collected in November 2013 from ground vegetation in X. fastidiosa-infected olive orchards, were 67% (40 out of 60) positive for X. fastidiosa by polymerase chain reaction (PCR) assays. Euscelis lineolatus Brullé were also collected but tested negative for the pathogen. Transmission tests with P. spumarius collected from the Salento area were, therefore, conducted. After a 96-h inoculation access period with 8 to 10 insects per plant and a 30-d incubation period, PCR results showed P. spumarius transmitted X. fastidiosa to two of five periwinkle plants but not to the seven olive plants. Sequences of PCR products from infected periwinkle were identical with those from X. fastidiosa-infected field trees. These data showed P. spumarius as a vector of X. fastidiosa strain infecting olives trees in the Salento Peninsula, Italy.

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.

Development of real-time PCR based assays for simultaneous and improved detection of citrus viruses

Citrus, one of the most economically important crops, is susceptible to a number of arthropod- and graft-transmissible pathogens. Rapid and reliable methods for detecting multiple pathogens are important for routine diagnosis by reducing time, labour and costs. To this end, primers and TaqMan probes for Citrus psorosis virus (CPsV) and Citrus variegation virus (CVV) detection by singleplex real-time (q) reverse transcription (RT)- PCR were initially designed. Further optimizations included the development of a multiplex (m) RT-qPCR assay to detect simultaneously CPsV, CVV, and Citrus tristeza virus (CTV) in a single reaction. When 10-fold serial dilutions prepared using total RNAs from CPsV- and CVV-infected plants were tested, RT-qPCR assays proved to be 100 and 1000 times more sensitive than conventional RT-PCR, respectively. The target viruses were effectively identified by mRT-qPCR in field-infected clementine and sweet orange trees. The optimized multiplex assay proved to be as sensitive as the singleplex tests, thus providing a valuable alternative tool for detection of these citrus viruses.

Transcriptome profiling of two olive cultivars in response to infection by the CoDiRO strain of Xylella fastidiosa subsp. pauca

BackgroundThe recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show much milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To determine whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated.ResultsA global quantitative transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection.ConclusionsCollectively these findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response.

Genome-Wide Analysis Provides Evidence on the Genetic Relatedness of the Emergent Xylella fastidiosa Genotype in Italy to Isolates from Central America

Xylella fastidiosa is a plant-pathogenic bacterium recently introduced in Europe that is causing decline in olive trees in the South of Italy. Genetic studies have consistently shown that the bacterial genotype recovered from infected olive trees belongs to the sequence type ST53 within subspecies pauca. This genotype, ST53, has also been reported to occur in Costa Rica. The ancestry of ST53 was recently clarified, showing it contains alleles that are monophyletic with those of subsp. pauca in South America. To more robustly determine the phylogenetic placement of ST53 within X. fastidiosa, we performed a comparative analysis based on single nucleotide polymorphisms (SNPs) and the study of the pan-genome of the 27 currently public available whole genome sequences of X. fastidiosa. The resulting maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pan-genome analysis are consistent with previously described X. fastidiosa taxonomy, distinguishing the subsp. fastidiosa, multiplex, pauca, sandyi, and morus. Within the subsp. pauca, the Italian and three Costa Rican isolates, all belonging to ST53, formed a compact phylotype in a clade divergent from the South American pauca isolates, also distinct from the recently described coffee isolate CFBP8072 imported into Europe from Ecuador. These findings were also supported by the gene characterization of a conjugative plasmid shared by all the four ST53 isolates. Furthermore, isolates of the ST53 clade possess an exclusive locus encoding a putative ATP-binding protein belonging to the family of histidine kinase-like ATPase gene, which is not present in isolates from the subspecies multiplex, sandyi, and pauca, but was detected in ST21 isolates of the subspecies fastidiosa from Costa Rica. The clustering and distinctiveness of the ST53 isolates supports the hypothesis of their common origin, and the limited genetic diversity among these isolates suggests this is an emerging clade within subsp. pauca.

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.

SURVEY FOR THE PRESENCE OF XYLELLA FASTIDIOSA subsp. PAUCA (STRAIN CoDiRO) IN SOME FORESTRY AND ORNAMENTAL SPECIES IN THE SALENTO PENINSULA

Xylella fastidiosa (Xf) is a xylem-inhabiting, vector-transmitted bacterium and the agent of a variety of diseases in a broad range of plant hosts. A destructive Xf outbreak has recently emerged in some olive groves of the province of Lecce (south-eastern Italy), caused by a strain (called CoDiRO from the Italian for Rapid desiccation complex of olive) of Xf subsp. pauca, a pathogen known to infect coffee and citrus in South America. Taking into account that this is the first confirmed record of the presence of Xf in the European Union (EU) territory and fairing its possible spread from the affected site, the EU has issued the Commission Implementing Decision L 45/29- 31 (February 24, 2014), which prohibits the transfer from the province of Lecce of all plant genera and species not listed in the Annex I of the cited Decision. A survey was therefore initiated to verify the health status of a number of ornamental and forestry plants growing mostly in nurseries, but also in public and private gardens, which are or can be naturally exposed to high inoculum pressure. Samples collected from 207 conifers, 105 members of the family Arecaceae, Musaceae and Cycadaceae, and 208 succulent plants (totalling 520) were comparatively examined by serological (DAS-ELISA) and molecular (PCR) methods. None of the tested plant species proved to be infected, providing a strong indication that they may not be susceptible to field infection by the Xf subsp. pauca strain currently present in the province of Lecce.

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.