First report of Ceratocystis ficicola causing wilt disease on common fig (Ficus carica) in Italy.

Common fig (Ficus carica L.) in is one of the most important crops in the Mediterranean area. In Italy, it is grown on a total area of 2118 ha. In Apulia (South-eastern Italy), the annual production of fig exceeds 3200 tons annually and together with olive and grapevine, they characterize the Apulian agricultural panorama. In September 2021, symptoms of a vascular wilt disease and, in severe cases, decline tree mortality were observed in Salento area (Apulia). Symptomatic Affected plants showed symptoms of leaf wilt and different stages of disease expression, which begins with leaf chlorosis on shoots, followed by wilting, extensive defoliation and twig dieback. On the main branches andlower part of the trunk and in some cases on lateral branches, bark cracks and cankers were observed and extended wood discoloration was detected in cross sections. In two orchards located in Salice Salentino (Site 1) and Squinzano (Site 2), where disease incidence exceeded 80%, 3-5 wood discs per tree were gathered from affected tissues from two (Site 1) and four (Site 2) trees. Isolations were performed on malt extract agar 2% amended with 0.5 g L-1 streptomycin sulfate. A Ceratocystis species was recovered from all samples trees with high frequencies (83.3%). Two-week-old colonies on potato dextrose agar showed black ascomata with 300-600 µm wide bases and 1100-2250 µm long necks. On the tips of the necks, ascospores (5-6x4-5 µm) exuded in creamy white sticky masses. Endoconidia (5-9x4.5-7 µm) were abundant, cylindrical, aseptate, and produced in chains. Two monoconidial representative strains CRSFA.Cer.033 (Site 1) and CRSFA.Cer.035 (Site 2) were deposited in the DISSPA Di.S.S.P.A. collection of the University of Bari. Species identification was done through sequence analyses of rDNA internal transcribed spacer region (ITS) using ITS5/ITS4 primers (White et al. 1990), elongation factor 1 alpha gene (TEF) using EF1/EF2 (O'Donnell et al. 1998) and RNA polymerase II gene (RPB2) gene using RPB2-5F/FRPB2fRPB2-7cR (Liu et al. 1999). Sequences were deposited in NCBI GenBank (accession numbers: OQ329983-OQ335969 (ITS), OQ352265-OQ352266 (TEF), OQ352268-OQ352267 (RPB2)). The sequences of both Apulian isolates were identical. BLAST searches revealed high similarity to the sequences of two isolates of Ceratocystis ficicola Kajitani and Masuya from Japan: ex-type CMW38543 and CMW38544, specifically 98.41% identity matching with KY685076 (ITS), 100% with KY685079 (TEF), and 99.87% with KY685083 and KY685082 (RPB2). Pathogenicity tests were conducted, on six common fig 'Dottato' seedlings by inoculating one year-old twigs with mycelium plugs (Bolboli et al. 2022). Control plants were inoculated with PDA plugs without mycelium. After one month, all inoculated twigs showed symptoms of wilt. Forty days post-inoculation, the bark of inoculated twigs was removed, and longitudinal and transverse sections revealed wood discolorations extending above and below the inoculation point. Ceratocystis ficicola was consistently reisolated from symptomatic tissues and identified as described above, thus fulfilling Koch's postulates. Neither symptoms nor positive isolations were observed in control seedlings. The pathogen was first described in 2011 in Japan (Kajitani and Masuya, 2011) where it currently affects all fig-producing areas. In 2018, it was reported for the first time in the EPPO region in Greece (Tsopelas et al., 2021), and to our knowledge, this is the first report on its detection in Italy. Since February 2022, C. ficicola has been included in the EPPO alert list because of its potential to cause tree mortality and the difficulty of its eradication. Regional national surveys are therefore urgently needed to determine its distribution in the fig growing areas of Italy and limit its spread. References Bolboli, Z., et al. 2022. Mycol. Prog. 21:89. doi: 10.1007/s11557-022-01834-9 Kajitani, Y., and Masuya, H., 2011. Mycoscience 52:349. doi: 10.1007/s10267-011-0116-5 Liu, Y. J., et al. 1999. Mol. Biol. and Evol. 16:1799. doi: 10.1093/oxfordjournals.molbev.a026092 O'Donnell, K., et al. 1998. Proc Natl Acad Sci USA 95:2044. doi: 10.1073/pnas.95.5.2044 Tsopelas, P., et al. 2021. Phytopathol. Mediterr. 60:337. doi: 10.36253/phyto-12794 White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. doi: 10.1016/0307-4412(91)90165-5.

Nanopore Technology Applied to Targeted Detection of Tomato Brown Rugose Fruit Virus Allows Sequencing of Related Viruses and the Diagnosis of Mixed Infections.

Tomato (Solanum lycopersicum) plants from a commercial glasshouse were identified with symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection. Reverse transcription-PCR and quantitative PCR confirmed the presence of ToBRFV. Subsequently, the same RNA sample and a second from tomato plants infected with a similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT). For the targeted detection of ToBRFV, the two libraries were synthesized by using six ToBRFV sequence-specific primers in the reverse transcription step. This innovative target enrichment technology enabled deep coverage sequencing of ToBRFV, with 30% of the total reads mapping to the target virus genome and 57% mapping to the host genome. The same set of primers applied to the ToMMV library generated 5% of the total reads mapping to the latter virus, indicating that sequencing of similar, non-target viral sequences was also allowed. Further, the complete genome of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus suggesting that, even using multiple sequence-specific primers, a low rate of off-target sequencing can usefully provide additional information on unexpected viral species coinfecting the same samples in an individual assay. These results demonstrate that targeted nanopore sequencing can specifically identify viral agents and has sufficient sensitivity towards non-target organisms to provide evidence of mixed virus infections.

Exploring the xylem-sap to unravel biological features of Xylella fastidiosa subspecies pauca ST53 in immune, resistant and susceptible crop species through metabolomics and in vitro studies.

Xylella fastidiosa subsp. pauca ST53 (Xfp) is a pathogenic bacterium causing one of the most severe plant diseases currently threatening the olive-growing areas of the Mediterranean, the Olive Quick Decline Syndrome (OQDS). The majority of the olive cultivars upon infections more or less rapidly develop severe desiccation phenomena, while few are resistant (e.g. Leccino and FS17), being less impacted by the infections. The present study contributes to elucidating the basis of the resistance phenomenon by investigating the influence of the composition of the xylem sap of plant species on the rate of bacterial multiplication. Xylem saps from Xfp host and non-host species were used for growing the bacterium in vitro, monitoring bacterial growth, biofilm formation, and the expression of specific genes. Moreover, species-specific metabolites, such as mannitol, quinic acid, tartaric acid, and choline were identified by non-targeted NMR-based metabolomic analysis in olive, grapevine, and citrus. In general, the xylem saps of immune species, including grapevine and citrus, were richer in amino acids, organic acids, and glucose. The results showed greater bacterial growth in the olive cultivar notoriously susceptible to Xfp (Cellina di Nardò), compared to that recorded in the resistant cultivar Leccino. Conversely, higher biofilm formation occurred in Leccino compared to Cellina di Nardò. Using the xylem saps of two Xfp-immune species (citrus and grapevine), a divergent bacterial behavior was recorded: low planktonic growth and biofilm production were detected in citrus compared to the grapevine. A parallel evaluation of the expression of 15 genes showed that Xfp directs its molecular functions mainly to virulence. Overall, the results gained through this multidisciplinary study contribute to extending the knowledge on the host-pathogen interaction, while confirming that the host response and resistance mechanism have a multifactorial basis, most likely with a cumulative effect on the phenotype.

Susceptible and resistant olive cultivars show differential physiological response to Xylella fastidiosa infections.

Olive quick decline syndrome (OQDS) is a severe disease, first described in Italy in late 2013, caused by strains of Xylella fastidiosa subsp. pauca (Xfp) in susceptible olive cultivars. Conversely, resistant olive cultivars do not develop OQDS but present scattered branch dieback, which generally does not evolve to severe canopy decline. In the present study, we assessed the physiological responses of Xfp-infected olive trees of susceptible and resistant cultivars. Periodic measurements of stomatal conductance (gs) and stem water potential (Ψstem) were performed using a set of healthy and Xfp-infected plants of the susceptible "Cellina di Nardò" and resistant "Leccino" and "FS17" cultivars. Strong differences in Δgs and ΔΨstem among Xfp-infected trees of these cultivars were found, with higher values in Cellina di Nardò than in Leccino and FS17, while no differences were found among healthy plants of the different cultivars. Both resistant olive cultivars showed lower water stress upon Xfp infections, compared to the susceptible one, suggesting that measurements of gs and Ψstem may represent discriminating parameters to be exploited in screening programs of olive genotypes for resistance to X. fastidiosa.

Transmission of Xylella fastidiosa subsp. pauca ST53 by the Sharpshooter Cicadella viridis From Different Source Plants and Artificial Diets.

The sharpshooter Cicadella viridis L. (Hemiptera: Cicadellidae) is the most common sharpshooter in Europe and, given its xylem feeding behavior, is considered a potential vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae). We tested X. fastidiosa subsp. pauca ST53 (Xfp) transmission capabilities of C. viridis adults, namely 1) acquisition efficiency from four host plant species-periwinkle, milkwort, lavender, alfalfa-and from two artificial diets (PD3 and Xfm), 2) inoculation efficiency to periwinkle at different times post acquisition from different plant and artificial diet sources. The main European vector species-Philaenus spumarius L. (Hemiptera: Aphrophoridae)-was used as a control. C. viridis was able to acquire Xfp from periwinkle, milkwort, and lavender, although with low efficiency (3-16%) and from artificial diets (23-25%). Successful inoculation on periwinkle was extremely rare, being observed only three times, following feeding on milkwort plant and PD3 artificial diet sources. Our study shows that C. viridis is not a relevant vector of Xfp, given the very low transmission rate in controlled conditions, and the inability to feed on olive. The low efficiency reported here correlates with ecological constraints of the vector (mainly monocots host plants, humid environments) that make it difficult to forecast a relevant role in dispersing X. fastidiosa, at least within the present distribution of the exotic bacterium in Europe. However, a possible role of this species in spreading Xf in other agroecosystems, e.g., vineyard and stone fruits grown in humid areas, cannot be excluded.

Metabolomics Approach to Characterize Green Olive Leaf Extracts Classified Based on Variety and Season.

The huge interest in the health-related properties of plant polyphenols to be applied in food and health-related sectors has brought about the development of sensitive analytical methods for metabolomic characterization. Olive leaves constitute a valuable waste rich in polyphenols with functional properties. A (HR)LC-ESI-ORBITRAP-MS analysis with a multivariate statistical analysis approach using PCA and/or PLS-DA projection methods were applied to identify polyphenols in olive leaf extracts of five varieties from the Apulia region (Italy) in two different seasonal times. A total of 26 metabolites were identified, further finding that although metabolites are common among the different cultivars, they differ in the relative intensity of each peak and within each cultivar in the two seasonal periods taken into consideration. The results of the total phenol contents showed the highest content in November for Bambina and Cima di Mola varieties (1816 and 1788 mg/100 g, respectively), followed by Coratina, Leccino, and Cima di Melfi; a similar trend was found for the antioxidant activity and RapidOxy evaluations by reaching in Bambina values of 45 mmol TE/100 g and 85 min of induction time.

Olea Europaea Geminivirus: A Novel Bipartite Geminivirid Infecting Olive Trees.

In 2014, high-throughput sequencing of libraries of total DNA from olive trees allowed the identification of two geminivirus-like contigs. After conventional resequencing of the two genomic DNAs, their analysis revealed they belonged to the same viral entity, for which the provisional name of Olea europaea geminivirus (OEGV) was proposed. Although DNA-A showed a genome organization similar to that of New World begomoviruses, DNA-B had a peculiar ORF arrangement, consisting of a movement protein (MP) in the virion sense and a protein with unknown function on the complementary sense. Phylogenetic analysis performed either on full-length genome or on coat protein, replication associated protein (Rep), and MP sequences did not endorse the inclusion of this virus in any of the established genera in the family Geminiviridae. A survey of 55 plants revealed that the virus is widespread in Apulia (Italy) with 91% of the samples testing positive, although no correlation of OEGV with a disease or specific symptoms was encountered. Southern blot assay suggested that the virus is not integrated in the olive genome. The study of OEGV-derived siRNA obtained from small RNA libraries of leaves and fruits of three different cultivars, showed that the accumulation of the two genomic components is influenced by the plant genotype while virus-derived-siRNA profile is in line with other geminivirids reported in literature. Single-nucleotide polymorphism (SNP) analysis unveiled a low intra-specific variability.

Differences in the Endophytic Microbiome of Olive Cultivars Infected by Xylella fastidiosa across Seasons.

The dynamics of Xylella fastidiosa infections in the context of the endophytic microbiome was studied in field-grown plants of the susceptible and resistant olive cultivars Kalamata and FS17. Whole metagenome shotgun sequencing (WMSS) coupled with 16S/ITS rRNA gene sequencing was carried out on the same trees at two different stages of the infections: In Spring 2017 when plants were almost symptomless and in Autumn 2018 when the trees of the susceptible cultivar clearly showed desiccations. The progression of the infections detected in both cultivars clearly unraveled that Xylella tends to occupy the whole ecological niche and suppresses the diversity of the endophytic microbiome. However, this trend was mitigated in the resistant cultivar FS17, harboring lower population sizes and therefore lower Xylella average abundance ratio over total bacteria, and a higher α-diversity. Host cultivar had a negligible effect on the community composition and no clear associations of a single taxon or microbial consortia with the resistance cultivar were found with both sequencing approaches, suggesting that the mechanisms of resistance likely reside on factors that are independent of the microbiome structure. Overall, Proteobacteria, Actinobacteria, Firmicutes, and Bacteriodetes dominated the bacterial microbiome while Ascomycota and Basidiomycota those of Fungi.

Spittlebugs of Mediterranean Olive Groves: Host-Plant Exploitation throughout the Year.

Spittlebugs are the vectors of the bacterium Xylella fastidiosa Wells in Europe, the causal agent of olive dieback epidemic in Apulia, Italy. Selection and distribution of different spittlebug species on host-plants were investigated during field surveys in 2016-2018 in four olive orchards of Apulia and Liguria Regions of Italy. The nymphal population in the herbaceous cover was estimated using quadrat samplings. Adults were collected by sweeping net on three different vegetational components: herbaceous cover, olive canopy, and wild woody plants. Three species of spittlebugs were collected: Philaenus spumarius L., Neophilaenus campestris (Fallén), and Aphrophora alni (L.) (Hemiptera: Aphrophoridae). Philaenus spumarius was the predominant species both in Apulia and Liguria olive groves. Nymphal stages are highly polyphagous, selecting preferentially Asteraceae Fabaceae plant families, in particular some genera, e.g., Picris, Crepis, Sonchus, Bellis, Cichorium, and Medicago. Host-plant preference of nymphs varies according to the Region and through time and nymphal instar. In the monitored sites, adults peak on olive trees earlier in Apulia (i.e., during inflorescence emergence) than in Liguria (i.e., during flowering and beginning of fruit development). Principal alternative woody hosts are Quercus spp. and Pistacia spp. Knowledge concerning plant selection and ecological traits of spittlebugs in different Mediterranean olive production areas is needed to design effective and precise control strategies against X. fastidiosa vectors in olive groves, such as ground cover modifications to reduce populations of spittlebug vectors.

Re.Ger.O.P.: An Integrated Project for the Recovery of Ancient and Rare Olive Germplasm.

The olive tree is one of the most important economic, cultural, and environmental resources for Italy, in particular for the Apulian region, where it shows a wide diversity. The increasing attention to the continuous loss of plant genetic diversity due to social, economic and climatic changes, has favored a renewed interest in strategies aimed at the recovery and conservation of these genetic resources. In the frame of a project for the valorization of the olive Apulian biodiversity (Re.Ger.O.P. project), 177 minor genotypes were recovered in different territories of the region. They were submitted to morphological, molecular, technological and phytosanitary status analysis in comparison with reference cultivars, then they were propagated and transferred in an ex situ field. All the available information was stored in an internal regional database including photographic documentation and geographic position. The work allowed obtaining information about the genetic diversity of Apulian germplasm, to clarify cases of homonymy and synonymy, to check the sanitary status, and to identify candidate genotypes useful both to set up breeding programs and to enrich the panel of olive cultivars available to farmers for commercial exploitation.

Fantoni translaryngeal tracheostomy versus ciaglia blue rhino percutaneous tracheostomy: a retrospective comparison.

PURPOSE: To compare the operative technique and complications of the Fantoni tracheostomy (TLT) with those of the Ciaglia Blue Rhino tracheostomy (CBR). We also compared the costs of mini-invasive tracheostomy with those of surgical tracheostomy (ST). METHODS: Between January 1998 and January 2006, 530 patients needed emergency intubation and protracted assisted ventilation in our department. We performed 470 mini-invasive tracheostomies: as TLT in 350 and as CBR in 120. The time between intubation and tracheostomy was 4 +/- 1 days. Interventions were carried out in our intensive care unit (ICU). RESULTS: One hundred and nine patients died within 20 +/- 5 days of intervention, but 361 are still alive after 100 +/- 3 months. TLT and CBR complications were independent of the operative technique (P = 0.74, r = 0.285 vs P = 0.61, r = 0.271) or procedure time (P = 0.95, r = 0.297 vs P = 0.92, r = 0.295). Ciaglia Blue Rhino tracheostomy was noted to have a cost-benefit advantage over TLT and ST (P = 0.0002, P = 0.009, P = 0.22, respectively). The average time until decannulation was 20 +/- 1 days. CONCLUSIONS: Mini-invasive tracheostomies are easy, safe, and fast. Ciaglia Blue Rhino tracheostomy took less time to perform and had fewer complications than TLT, because the technique was simpler.

Infections of the Xylella fastidiosa subsp. pauca Strain "De Donno" in Alfalfa (Medicago sativa) Elicits an Overactive Immune Response.

Diseases caused by Xylella fastidiosa are among the most destructive for several agricultural productions. A deadly disease of olive, termed olive quick decline syndrome, is one of the most recent examples of the severe impacts caused by the introduction and spread of this bacterium in new ecosystems with favorable epidemiological conditions. Deciphering the cascade of events leading to the development of severe alterations in the susceptible host plants is a priority of several research programs investigating strategies to mitigate the detrimental impacts of the infections. However, in the case of olives, the long latent period (>1 year) makes this pathosystem not amenable for such studies. We have inoculated alfalfa (Medicago sativa) with the olive-infecting strain "De Donno" isolated from a symptomatic olive in Apulia (Italy), and we demonstrated that this highly pathogenic strain causes an overactive reaction that ends up with the necrosis of the inoculated stem, a reaction that differs from the notoriously Alfalfa Dwarf disease, caused by X. fastidiosa strains isolated from grapes and almonds. RNASeq analysis showed that major plant immunity pathways are activated, in particular, several calcium transmembrane transporters and enzymes responsible for the production of reactive oxygen species (ROS). Signs of the necrotic reaction are anticipated by the upregulation of genes responsible for plant cell death and the hypersensitive reaction. Overall the whole infection process takes four months in alfalfa, which makes this pathosystem suitable for studies involving either the plant response to the infection or the role of Xylella genes in the expression of symptoms.

Molecular characterisation of a novel gemycircularvirus associated with olive trees in Italy.

A new gemycircularvirus sequence was obtained from total DNAs extracted from olive leaves and twigs tissues. Its complete genome consists of a single-stranded circular DNA of 2,145 nt, whose sequence was obtained by rolling circle amplification (RCA). Genome analysis identified three open reading frames, sharing homologies with the coat and replication-associated proteins, these latter in the anti-parallel strand, of known gemycircularvirus species. Search for homologies showed that the circular ssDNA sequence is distantly related to other gemycircularviruses thus originating from a new virus species, for which the name olive-associated gemycircularvirus 1 (OaGV1) is proposed. A survey in two different olive-growing areas of the Apulian region (Southern Italy), showed a limited distribution of OaGV1.

Transmission of Xylella fastidiosa Subspecies Pauca Sequence Type 53 by Different Insect Species.

Diseases associated with Xylella fastidiosa have been described mostly in North and South America. However, during the last five years, widespread X. fastidiosa infections have been reported in a constrained area of the Apulia region (southern Italy), in olives trees suffering a severe disease, denoted as Olive Quick Decline Syndrome (OQDS). Because many xylem sap-feeding insects can function as vectors for the transmission of this exotic pathogen in EU, several research programs are ongoing to assess the role of candidate vectors in the spread of the infections. Initial investigations identified Philaenus spumarius (L.) as the predominant vector species in the olive orchards affected by the OQDS. Additional experiments have been carried out during 2016 and 2017 to assess the role of other species. More specifically, adults of the spittlebugs Philaenus italosignus Drosopolous and Remane, Neophilaenus campestris (Fallen) and of the planthopper Latilica tunetana (Matsumura) (Issidae) have been tested in transmission experiments to assess their ability to acquire the bacterium from infected olives and to infect different susceptible hosts (olives, almond, myrtle -leaf milkwort, periwinkle). Acquisition rates determined by testing individual insects in quantitative PCR assays, ranging from 5.6% in N. campestris to 22.2% in P. italosignus, whereas no acquisition was recorded for L. tunetana. Successful transmissions were detected in the recipient plants exposed to P. italosignus and N. campestris, whereas no trasmissions occurred with L. tunetana. The known vector Philaenus spumarius has been included in all the experiments for validation. The systematic surveys conducted in 2016 and 2017 provided further evidence on the population dynamics and seasonal abundance of the spittlebug populations in the olive groves.

Plant Selection and Population Trend of Spittlebug Immatures (Hemiptera: Aphrophoridae) in Olive Groves of the Apulia Region of Italy.

The xylem-limited bacterium Xylella fastidiosa Wells is the causal agent of severe diseases of several cultivated and wild plants. It is transmitted by xylem-sap feeder insects, such as spittlebugs (Hemiptera: Cercopoidea) and sharpshooters (Hemiptera: Cicadellinae). A dramatic epidemic of X. fastidiosa subspecies pauca sequence type 53 is currently affecting a large area of the Apulia Region of Italy, where it is spread by Philaenus spumarius L. adults within olives. In 2015 and 2016, field surveys were carried out in Apulian olive groves to investigate host plant selection of spittlebug nymphs, to identify the main plant species that can act as reservoirs of the vectors. Two different sampling methods were used: randomized plant sampling and quadrats sampling. Host plant selection by P. spumarius and Neophilaenus campestris (Fallén) nymphs was estimated using Manly's selection index. The botanic families presenting the highest number of plants infested by P. spumarius nymphs were Asteraceae, Fabaceae, and Apiaceae. Nymphs of P. spumarius were sampled on 72 plant genera, and among the most common 25 genera, Sonchus, Knautia, Glebionis, Urospermum (Asteraceae), Medicago, Vicia, Melilotus (Fabaceae), and Daucus (Apiaceae) were the ones selected preferentially, according to Manly's index results. Populations of P. spumarius nymphs peak in early April, with densities ranging between 10 and 40 nymph/m2, were about 10-fold those of N. campestris. Plant infestation rate by spittlebug nymphs in 2016 was significantly higher in olive groves located in Lecce province (infected area) than those situated in Bari province (noninfected area).