G. P. Martelli

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).

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.

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.

Subcellular localization and immunodetection of movement proteins of olive latent virus 1

Summary.Polyclonal sera raised to Escherichia coli-expressed movement proteins encoded by ORF 3 (p8K) and ORF 4 (p6K) of olive latent virus 1, were used for their immunodetection in infected Nicotiana benthamiana plants. In subfractionated locally infected tissues 4 days post inoculation (d.p.i.) that were analysed by Western blot, p8K was found in the fast-sedimenting fractions P1 and P30 containing membranous material and/or cell organelles and, likely, the fibrous structures mentioned below, but not in the soluble protein-containing supernatant. No p6K could be detected in these extracts. In locally inoculated leaves p8K began to accumulate from 2 d.p.i onwards reaching its peak at 4 d.p.i. Intracellular immunogold labelling of cells from locally and systemically infected tissues localized p8K primarily in fibrous inclusions made up of thin filaments with a helical structure present in the cytoplasm of locally and systemically infected cells. In systemic infections a light and scattered labelling was oberved in the cytoplasm and near the cell wall. The specific serum to p6K did not label the fibrous structures and failed to recognize its antigen in systemically and locally infected tissues except at 4 d.p.i., when scattered labelling was observed in the cytoplasm and near plasmodesmata.

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.

FIG TREE VIRUSES IN NEW ZEALAND

In late March 2012, cuttings were collected from 11 fig trees growing in Anaura Beach and in a private garden in the vicinity of Gisborne (Bay of Poverty, New Zealand). Each tree apparently belonged to a different cultivar, whose name was unknown to the owners. Due to the late season, symptoms on the leaves were not outstanding. When visible, they consisted of various patterns of mottling resembling very much the symptoms characterizing fig mosaic (Martelli, 2011). Total RNA was extracted from cortical scrapings and subjected to RT-PCR, which was conducted accord- ing to the protocols and using the virus-specific primers routinely utilized in our laboratory (Martelli, 2011 and references therein) for the detection of the following viruses: Fig mosaic virus (FMV), Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-asso- ciated virus 2 (FLMaV-2), Fig mild mottle virus (FMMV), and Fig latent virus 1 (FLV-1). PCR was also carried out for the presence of Fig badnavirus 1 (FBaV-1), using the primers P1s: 5’-GCT GAT CAC AAG AGG CAT GA-3’ and P1as: 5’-TCC TTG TTT CCA CGT TCC TT-3’ designed on the sequence of the 5’ termi- nal portion of the polyprotein encoded by the viral ORF3 (Gen- Bank accession No. JF411989). Amplification products were ob- tained from all samples, most of which proved to be infected by two (e.g. FMV, FBaV-1) or more (e.g. FMV, FBaV-1, FLMaV-1, FMMV) different viruses. FLV-1 was not detected, whereas the most widespread viruses were FMV and FBaV-1. Interestingly, the latter virus was detected in the totality of the samples. Fig mosaic is known to occur in New Zealand (Li and Procter, 1944; Cairns, 2006) but, to the best of our knowledge, there are no records of viruses associated with the disease.

A SURVEY FOR FIG-INFECTING VIRUSES IN PALESTINE

SUMMARY A virus disease of fig (Ficus carica) known as fig mosaic (FM) is widely spread in Palestine, where its severity varies according to the cultivar and the growing area. At least 10 viruses and three viroids have been detected so far in fig trees. This study reports the results of a preliminary survey carried out in Palestine to secure information on the viruses associated with mosaic-infected figs. Samples were collected from scattered trees, fig orchards and nurseries of different areas of central Palestine, where figs are traditionally grown, and tested for the presence of Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1), Fig badnavirus-1 (FBV-1), Fig leaf mottle-associated virus 1 (FLMaV-1) and 2 (FLMaV-2), and Fig cryptic virus (FCV) in addition to Apple dimple fruit viroid (ADFVd). The following viruses. FMV, FBV-1, FLV-1 and FLMaV-2, were detected by RT-PCR. FBV-1 was the most widespread followed by FMV. The genetic diversity of FMV was assessed by sequencing a fragment from the viral p4 protein, revealing a low divergence from the homologous sequences from GenBank.