Olive quick decline and Xylella fastidiosa in Southern Italy: the state of the art

The identification in 2013 of an outbreak of Xylella fastidiosa (Xf) in olive groves in the Salento peninsula (southern Italy) resulted in a plant health emergency of unprecedented proportions for the EU. Infected olive trees show extensive desiccation of the canopy and severe quick decline symptoms. In the outbreak area, the bacterium was found to be efficiently spread by the meadow spittlebug Philaenus spumarius, abundant on the olive canopies during the dry season. The initial demarcated foci rapidly expanded over the past 4 years, establishing a new demarcation line distant 80 km from the first reported outbreak; while few species were found infected in 2013 the currently known susceptible hosts reached the number of ca. 30 different plant species. Phytosanitary measures to combat the spread and mitigate the impact of the bacterial infections, included restrictions for the new plantations, for the movement of propagating materials and removal of infected trees. The severe damage suffered by the infected olive trees combined with the imposed phytosanitary restrictions determined severe economic and social impacts in the local community, raising major concerns against the application of the containment measures and determining the failure to implement timely, effective and coordinated preventive measures. Due to the novelty of the Xylella‐associated disease in olives and in general the fact that Xf is conquering new geographical area, like the EU territories, the EU Commission mobilized dedicated resources to build EU research actions to fulfil research gaps for this emerging pathogen threatening the entire EU territory. Between 2015 and 2016, two relevant research projects in the framework of the H2020 programs have been funded: the project "Pest Organisms Threatening Europe" (POnTE) and the project "Xylella Fastidiosa Active Containment Through a multidisciplinary‐Oriented Research Strategy" (XF‐ACTORS) the latter targeting exclusively Xf. From the intense research activity developed in the past three years some major results have been already achieved, providing data on the genetic and biological properties of the population of the bacterium, the range of hosts, the identification and biology of the vector, the identification of olive cultivars with promising traits of resistance

Tomato spotted wilt virus associated with lettuce dieback in Bekaa Valley, Lebanon

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The endophytic microbiome of X. fastidiosa susceptible and resistant olives

A multi-factorial strategy is required to co-exist with X. fastidiosa infections, which are devastating olive trees in the southern area of Apulia (Italy). Observations in the outbreak area can provide information on potential approaches for containment. Olive cvs Leccino and FS17 show lessened symptoms and host lower bacterial populations (1,2) than cvs Ogliarola salentina, Cellina di Nardò and Kalamata. We are evaluating whether microbial communities inhabiting the xylem vessels of olive cvs showing different susceptibilities to X. fastidiosa -infection play a role in resistance. To explore these endophytic microbiomes, a whole-metagenome shotgun analysis is currently ongoing. X. fastidiosa -infected and healthy olive plants of the cultivars FS17, Leccino and Kalamata, were selected from the same plot to limit the influence of diverse soil composition and crop management. Shotgun sequencing of DNA extracted from the xylem tissues will be used to investigate the microbiome community by bio-informatic analysis. Moreover, efforts to isolate culturable microorganisms to be used in antagonistic assays against X. fastidiosa, will be performed. Concurrently, the X. fastidiosa-biocontrol potency of Paraburkholderia phytofirmans PsJN strain, whose beneficial effects in the reduction of symptoms in Pierce’s Disease (3) have been recently described, are under evaluation. We are testing the ability of P. phytofirmans to colonise xylem vessels and interact with X. fastidiosa in tobacco and olive

First Report of ‘CandidatusLiberibacter asiaticus’ Associated with Huanglongbing in Sweet Orange in Ethiopia

Huanglongbing (HLB) is a serious disease of citrus worldwide. Three different ‘Candidatus Liberibacter’ species are associated with HLB: ‘Ca. Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ (1). ‘Ca. L. africanus’ and its vector, Trioza erytreae, are both heat sensitive, and when present, occur in citrus when temperatures remain below 30 to 32°C. In Africa, ‘Ca. L. africanus’ and T. erytreae have been reported in South Africa, Zimbabwe, Malawi, Burundi, Kenya, Somalia, Ethiopia, Cameroon, and Madagascar (1). Inspection of citrus trees in orchards and budwood sources in nurseries located in the warmer citrus-growing areas of Tigray and North Wollo in northern Ethiopia revealed nearly 100 trees with symptoms of leaf yellowing with a blotchy mottle pattern, dead branches, and decreased fruit quality and yield. Two symptomatic sweet orange budwood trees and three symptomatic orchard plants were sampled in April 2009, along with three healthy-looking sweet orange plants. DNA was extracted from 200 mg of desiccated leaf midribs using the CTAB method (4) and subjected to conventional PCR using the primer pairs A2/J5 (2) and OI2/23S1 (3) that amplify the ribosomal protein gene in the rplKAJL-rpoBC operon and the 16S/23S ribosomal intergenic regions, respectively, of ‘Ca. L. africanus’ and ‘Ca. L. asiaticus’. Positive PCR reactions were obtained for all five symptomatic samples with both primer pairs. PCR amplicons of 703 bp (A2/J5) and 892 bp (OI2/23S) recovered from two of these samples were purified, cloned, and sequenced. BLAST analysis revealed that the nucleotide sequences we obtained for the ribosomal protein (GenBank Accessions Nos. GQ890155 and GQ890156) shared 100% identity with each other and 99% identity with sequences of ‘Ca. L. asiaticus’ from Brazil (DQ471904), Indonesia (AB480161), China (DQ157277), and Florida (CP001677). Similarly, the 16S/23S ribosomal intergenic sequences (GU296538 and GU296539) shared 100% identity with each other and 99% identity with homologous ‘Ca. L. asiaticus’ sequences from Brazil (DQ471903), Indonesia (AB480102), China (DQ778016), and Florida (CP001677) and contained two tRNA genes as occurs in ‘Ca. L. asiaticus’ but not in ‘Ca. L. africanus’ (3). To our knowledge, this is the first report of ‘Ca. L. asiaticus’ in Africa. The presence of ‘Ca. L. asiaticus’ is a threat for warmer citrus-growing areas of Africa that are less favorable for ‘Ca. L. africanus’ and T. erytreae. In areas where ‘Ca. L. asiaticus’ was confirmed, symptomatic trees must be promptly eradicated and surveys to determine spread of the disease and its vectors are necessary

Establishment of an experimental field to explore the differential olive cultivar response to Xylella fastidiosa infection

While different sources of natural resistance to Xylella fastidiosa (Xf) have been described in grapevines and citrus, lack of consolidated information exists on the wide panel of cultivars characterizing the vast olive germplasm. Preliminary observations on few cultivars, support the evidence that differential cultivar responses to Xf infections may exist. To explore the response of a larger panel of cultivars, in April 2015, an experimental olive plot, located within the Xf-heavily affected olive groves, was established in the Apulia Region (Italy). Twenty-four trees for each of the ten different cultivars were planted in randomized blocks. Each tree was caged with 15-20 specimens of Philaenus spumarius collected from the neighboring infected olive groves. Upon removing the cages, the trees are then continuously exposed to the natural vector populations occurring in the area. Nine and 12-months after planting, the trees were sampled, tested for Xf and inspected for symptoms. The first data confirmed the infectivity of the vector populations occurring in the Apulian contaminated area and the Xf susceptibility of the olive cultivars tested. Almost 50% of the trees tested positive, with an infection incidence ranging from 25% (Leccino) to 78% (Koroneiki). Symptoms of shoot dieback started to appear 1-year after planting, limitedly on few replicates of Cellina di Nardò. In April 2016, the number of cultivars has been increased up to 30. Periodical surveys for symptoms and quantitative analyses to monitor the differential bacterial titer and expression of target genes involved in the host response, are underway

Complete Genome Sequence of the Olive-Infecting Strain Xylella fastidiosa subsp. pauca De Donno

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

Characterization of Citrus tristeza virus isolates recovered in Syria and Apulia (southern Italy) using different molecular tools

Citrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Syria in citrus groves from two Governorates (Lattakia and Tartous) and several CTV outbreaks have been reported in Apulia (southern Italy) since 2003. To molecularly characterize the CTV populations spreading in Syria and Italy, a number of isolates from each region was selected and examined by different molecular approaches including: Multiple Molecular Markers analysis (MMM), real time RT-(q)PCR, single strand conformation polymorphism (SSCP) of the major coat protein (CP) gene (P25), and sequence analysis of the CP (P25), P18, P20 and RdRp genes. SSCP analysis of CP25 yielded two distinct simple patterns among the Syrian isolates and three different patterns in the Italian isolates. Based on MMM analysis, all Syrian CTV isolates were categorized as VT-like genotype, whereas the Italian isolates reacted only with the markers specific for the T30 genotype. These findings were also confirmed by RT-qPCR and by sequencing analysis of four genomic regions. The Italian isolates had nucleotide identities which varied: from 99.5 to 99.8 for the CP gene; from 97.4% to 98.3% for the P18 gene; from 98.6% to 99.8% for the P20 and from 97.8% to 99.1% for the partial RdRp sequenced. High sequence identity was found for all genomic regions analyzed between the Syrian isolates (from 98.9% to 99.6%). These results show that the CTV populations spreading in Apulia and Syria are associated with different genotypes, indicating different potential impacts on the citrus trees in the field. Since in both areas the introduction of the virus is relatively recent, infected plants resulted to contain a single and common genotype, suggesting that CTV is spreading from the first outbreaks by aphids or local movement of autochthonous infected plant material

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

A Negative-Stranded RNA Virus Infecting Citrus Trees: The Second Member of a New Genus Within the Order Bunyavirales

A new RNA virus has been identified from a sweet orange tree in southern Italy. This virus, tentatively named citrus virus A (CiVA), has a bipartite genome composed of (i) a negative-stranded (ns) RNA1, encoding the viral RNA-dependent RNA polymerase (RdRp), and (ii) an ambisense RNA2, coding for the putative movement protein (MP) and nucleocapsid protein (NP), with the two open reading frames separated by a long AU-rich intergenic region (IR) adopting a hairpin conformation. CiVA genomic RNAs and the encoded proteins resemble those of the recently discovered citrus concave gum-associated virus (CCGaV). This CCGaV, a nsRNA virus associated with the ancient citrus concave gum disease, has been proposed as the representative member of a new genus tentatively named Coguvirus. Molecular and phylogenetic analyses presented here support the classification of CiVA, and likely of other two recently described nsRNA viruses infecting plants, in this new genus. By showing that the evolutionary origin of the MP of all the putative coguviruses likely differs from that of their respective RdRp and NP, this study also provides evidence of a likely modular genome evolution for these viruses. Moreover, phylogenetic data support the proposal that, during the evolutionary history of nsRNA viruses, the plant-infecting viruses most likely emerged from an invertebrate-infecting ancestor several times as independent events. CiVA was identified in a field sweet orange tree not showing any obvious symptom and was graft-transmitted to sweet orange, grapefruit, rough lemon and Dweet tangor indicator plants that did not developed symptoms. The capacity of infecting citrus hosts of several species was also confirmed by a preliminary survey that identified orange, mandarin, clementine and lemon trees as natural hosts of CiVA in several fields of southern Italy, again without any obvious association with specific symptoms