Identification and characterization of an isolate of apple green crinkle associated virus involved in a severe disease of quince (Cydonia oblonga, Mill.).

A quince tree showing severe symptoms of a previously undescribed viral disease occurring in northern Apulia (Italy) was analysed using high-throughput sequencing of small RNA libraries, leading to the identification of a new strain of apple green crinkle associated virus (isolate AGCaV-CYD) showing peculiar traits. RT-PCR with specific primers detected AGCaV-CYD in consistent association with symptoms in the surveyed orchards. Molecular characterization of the reconstructed genome, together with phylogenetic analysis, showed it to be closely related to an AGCaV strain causing green crinkle disease in apple (AGCaV-AUR) and divergent from the type strain of apple stem pitting virus (ASPV-PA66).

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

BACKGROUND: The 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. RESULTS: A 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. CONCLUSIONS: Collectively 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.

Unusual genomic features of a badnavirus infecting mulberry.

Mulberry badnavirus 1 (MBV1) has been characterized as the aetiological agent of a disease observed on a mulberry tree in Lebanon (accession L34). A small RNA next-generation sequencing library was prepared and analysed from L34 extract, and these data together with genome walking experiments have been used to obtain the full-length virus sequence. Uniquely among badnaviruses, the MBV1 sequence encodes a single ORF containing all the conserved pararetrovirus motifs. Two genome sizes (6 kb and 7 kb) were found to be encapsidated in infected plants, the shortest of which shares 98.95 % sequence identity with the full L34 genome. In the less-than-full-length deleted genome, the translational frame for the replication domains was conserved, but the particle morphology, observed under electron microscopy, was somehow altered. Southern blot hybridization confirmed the coexistence of the two genomic forms in the original L34 accession, as well as the absence of cointegration in the plant genome. Both long and deleted genomes were cloned and proved to be infectious in mulberry. Differently from other similar nuclear-replicating viruses or viroids, the characterization of the MBV1-derived small RNAs showed a reduced amount of the 24-mer class size.

Transmission of grapevine Pinot gris virus by Colomerus vitis (Acari: Eriophyidae) to grapevine.

Grapevine Pinot gris virus (GPGV) is a new virus reported in Europe and several other grape-growing countries. In an attempt to identify a vector for GPGV, samples of the eriophyid mite Colomerus vitis collected from buds and erinea in GPGV-infected vines were analysed by RT-PCR, using specific primers. Molecular analysis revealed the presence of GPGV in C. vitis. Transmission trials were conducted using C. vitis collected from GPGV-infected vines. Mites were able to transmit GPGV to healthy grapevines, suggesting that C. vitis is a potential vector of this virus.

Detection and molecular characterization of a Grapevine Roditis leaf discoloration-associated virus (GRLDaV) variant in an autochthonous grape from Apulia (Italy).

The complete nucleotide sequence and genome organization of a new Badnavirus isolated from the autochthonous grapevine variety "Bombino nero" from Apulia (Italy) was determined. The genome of this virus consists of 7097 nt and has four open reading frames (ORFs). Analysis of putative proteins encoded by each ORF revealed greatest sequence similarity to Grapevine Roditis leaf discoloration-associated virus w4 (GRLDaV; NC_027131). In a pairwise alignment with GLRDaV w4 genome sequence, the "Bombino Nero" sequence was 109 nt longer with a major 57 nt insertion between positions 2405 and 2413. Furthermore, its putative ORF4 is located after the ORF3, while in the GLRDaV w4 sequence, the putative ORF4 completely overlapped ORF3. Nucleotide analysis classifies this new Badnavirus as a GLRDaV strain, which was named GRLDaV-BN. Multi-year field observations showed that the GLRDaV-BN-infected vine was symptomless.

Update of the Xylella spp. host plant database - systematic literature search up to 30 June 2022.

This scientific report provides an update of the Xylella spp. host plant database, aiming to provide information and scientific support to risk assessors, risk managers and researchers dealing with Xylella spp. Upon a mandate of the European Commission, EFSA created and regularly updates a database of host plant species of Xylella spp. The current mandate covers the period 2021-2026. This report is related to the seventh version of the database published in Zenodo in the EFSA Knowledge Junction community, covering literature published from 1 January 2022 up to 30 June 2022, and recent Europhyt outbreak notifications. Informative data have been extracted from 30 selected publications. Fifteen new host plants were identified and added to the database. Those plant species were reported from Brazil, France, Italy, Portugal and Spain, and infected by subsp. multiplex, pauca or unknown (i.e. not reported). No additional data were retrieved for X. taiwanensis. Two new STs (namely ST88 and ST89) belonging to subspecies multiplex were identified in host plants in natural conditions, and new information on the tolerant/resistant response of plant species to X. fastidiosa infection were added to the database. The overall number of Xylella spp. host plants determined with at least two different detection methods or positive with one method (between sequencing and pure culture isolation) reaches now 423 plant species, 194 genera and 68 families. Such numbers rise to 679 plant species, 304 genera and 88 families if considered regardless of the detection methods applied.

Update of the Xylella spp. host plant database - systematic literature search up to 30 June 2021.

Following a request from the European Commission, EFSA was asked to create and regularly update a database of host plant species of Xylella spp. The mandate now covers the period 2021-2026 and EFSA is requested to release an update of the database twice per year. The aim of the database is to provide information and scientific support to risk assessors, risk managers and researchers dealing with Xylella spp. This report is related to the fifth version of the database published in Zenodo in the EFSA Knowledge Junction community, covering literature published from 1 January 2021 up to 30 June 2021, and recent Europhyt outbreak notifications. Informative data have been extracted from 41 selected publications. Nineteen new host plants were identified and added to the database since the previous update published in June 2021. Those plant species were reported naturally infected by subsp. multiplex or unknown (i.e. not reported in the publication) of X. fastidiosa in the UE (France, Spain and Portugal). No additional data were retrieved for X. taiwanensis. New information on the tolerant/resistant response of plant species to X. fastidiosa infection were added, while no new STs have been identified worldwide compared to the previous update published in May 2021. The overall number of Xylella spp. host plants determined with at least two different detection methods or positive with one method (between: sequencing, pure culture isolation) now reaches 407 plant species, 185 genera and 68 families. Such numbers raise to 655 plant species, 293 genera and 88 families if considered regardless of the detection method applied.

Update of the Xylella spp. host plant database - systematic literature search up to 31 December 2021.

This Scientific report provides an update of the Xylella spp. host plant database, aiming to provide information and scientific support to risk assessors, risk managers and researchers dealing with Xylella spp. Upon a mandate of the European Commission, EFSA created and regularly updated a database of host plant species of Xylella spp. The current mandate covers the period 2021-2026. This report is related to the sixth version of the database published in Zenodo in the EFSA Knowledge Junction community, covering literature published from 1 July 2021 up to 31 December 2021, and recent Europhyt outbreak notifications. Informative data have been extracted from 29 selected publications. Eleven new host plants were identified and added to the database: six plant species naturally infected by subsp. multiplex of X. fastidiosa in the EU (France, Italy and Portugal) and five plant species artificially infected by different X. fastidiosa subspecies (multiplex, pauca, fastidiosa and sandyi). No additional data were retrieved for X. taiwanensis. New information on the tolerant/resistant response of plant species to X. fastidiosa infection were added, while no new STs have been identified worldwide compared to the previous update published in January 2022. The overall number of Xylella spp. host plants determined with at least two different detection methods or positive with one method (between: sequencing, pure culture isolation) reaches now 412 plant species, 190 genera and 68 families. Such numbers rise to 664 plant species, 299 genera and 88 families if considered regardless of the detection methods applied.

Update of the Xylella spp. host plant database - systematic literature search up to 31 December 2020.

Following a request from the European Commission, EFSA was asked to create and regularly update a database of host plant species of Xylella spp. Complying with an extension of the previous mandate, which now covers the period 2021-2026, the current version of Xylella spp. host plant database updates the previous release dated April 2020. Informative data have been extracted from 86 recent publications retrieved through an extensive literature search. This report is related to the fourth version of the database published in Zenodo in the EFSA Knowledge Junction community, covering articles selected from: a systematic literature review conducted up to 31 December 2020, Europhyt outbreak notifications up to 18 March 2021 and communications from research groups and national authorities. Forty-three new host plant species of X. fastidiosa, identified through the data extracted from the selected publications, have been added to the database. Those plant species were reported as naturally or artificially infected by subsp. fastidiosa, multiplex, pauca or unknown (i.e. not reported in the publication) subspecies of X. fastidiosa. New information on the tolerant/resistant response of plant species or varieties to X. fastidiosa infection is also reported. No additional data were retrieved for X. taiwanensis. This new version of the database includes no update on the number of Sequence Types (STs) identified so far, which remains unchanged. The overall number of Xylella spp. host plants determined with at least two different detection methods or positive with one method (between: sequencing, pure culture isolation) reaches now 385 plant species, 179 genera and 67 families. Such numbers rise to 638 plant species, 289 genera and 87 families if considered regardless of the detection method applied. The database will be issued twice per year, with the aim to provide information and scientific support to risk assessors, risk managers and researchers dealing with Xylella spp.

Piriformospora indica Primes Onion Response against Stemphylium Leaf Blight Disease.

The root-endophytic fungus Piriformospora indica (=Serendipita indica) has been revealed for its growth-promoting effects and its capacity to induce resistance in a broad spectrum of host plants. However, the bioefficacy of this fungus had not yet been tested against any pathogen affecting onion (Allium cepa). In this study, the biocontrol potency of P. indica against onion leaf blight, an impacting disease caused by the necrotrophic fungal pathogen Stemphylium vesicarium, was evaluated. First, it was proved that colonisation of onion roots by P. indica was beneficial for plant growth, as it increased leaf development and root biomass. Most relevantly, P. indica was also effective in reducing Stemphylium leaf blight (SLB) severity, as assessed under greenhouse conditions and confirmed in field trials in two consecutive years. These investigations could also provide some insight into the biochemical and molecular changes that treatment with P. indica induces in the main pathways associated with host defence response. It was possible to highlight the protective effect of P. indica colonisation against peroxidative damage, and its role in signalling oxidative stress, by assessing changes in malondialdehyde and H2O2 content. It was also showed that treatment with P. indica contributes to modulate the enzymatic activity of superoxide dismutase, catalase, phenylalanine ammonia-lyase and peroxidase, in the course of infection. qPCR-based expression analysis of defence-related genes AcLOX1, AcLOX2, AcPAL1, AcGST, AcCHI, AcWRKY1, and AcWRKY70 provided further indications on P. indica ability to induce onion systemic response. Based on the evidence gathered, this study aims to propose P. indica application as a sustainable tool for improving SLB control, which might not only enhance onion growth performance but also activate defence signalling mechanisms more effectively, involving different pathways.

Xylella fastidiosa in Olive: A Review of Control Attempts and Current Management.

Since 2013, Xylella fastidiosa Wells et al. has been reported to infect several hosts and to be present in different areas of Europe. The main damage has been inflicted on the olive orchards of southern Apulia (Italy), where a severe disease associated with X. fastidiosa subspecies pauca strain De Donno has led to the death of millions of trees. This dramatic and continuously evolving situation has led to European and national (Italian and Spanish) measures being implemented to reduce the spread of the pathogen and the associated olive quick decline syndrome (OQDS). Research has been also carried out to find solutions to better and directly fight the bacterium and its main insect vector, Philaenus spumarius L. In the course of this frantic effort, several treatments based on chemical or biological substances have been tested, in addition to plant breeding techniques and integrated pest management approaches. This review aims to summarize the attempts made so far and describe the prospects for better management of this serious threat, which poses alarming questions for the future of olive cultivation in the Mediterranean basin and beyond.

Phenotypic Characterization and Transformation Attempts Reveal Peculiar Traits of Xylella fastidiosa Subspecies pauca Strain De Donno.

Xylella fastidiosa subsp. pauca strain De Donno has been recently identified as the causal agent of a severe disease affecting olive trees in a wide area of the Apulia Region (Italy). While insights on the genetics and epidemiology of this virulent strain have been gained, its phenotypic and biological traits remained to be explored. We investigated in vitro behavior of the strain and compare its relevant biological features (growth rate, biofilm formation, cell-cell aggregation, and twitching motility) with those of the type strain Temecula1. The experiments clearly showed that the strain De Donno did not show fringe on the agar plates, produced larger amounts of biofilm and had a more aggregative behavior than the strain Temecula1. Repeated attempts to transform, by natural competence, the strain De Donno failed to produce a GFP-expressing and a knockout mutant for the rpfF gene. Computational prediction allowed us to identify potentially deleterious sequence variations most likely affecting the natural competence and the lack of fringe formation. GFP and rpfF- mutants were successfully obtained by co-electroporation in the presence of an inhibitor of the type I restriction-modification system. The availability of De Donno mutant strains will open for new explorations of its interactions with hosts and insect vectors.

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.

Ionomic Differences between Susceptible and Resistant Olive Cultivars Infected by Xylella fastidiosa in the Outbreak Area of Salento, Italy.

Olive quick decline syndrome (OQDS) is a devastating disease of olive trees in the Salento region, Italy. This disease is caused by the bacterium Xylella fastidiosa, which is widespread in the outbreak area; however, the "Leccino" variety of olives has proven to be resistant with fewer symptoms and lower bacterial populations than the "Ogliarola salentina" variety. We completed an empirical study to determine the mineral and trace element contents (viz; ionome) of leaves from infected trees comparing the two varieties, to develop hypotheses related to the resistance of Leccino trees to X. fastidiosa infection. All samples from both cultivars tested were infected by X. fastidiosa, even if leaves were asymptomatic at the time of collection, due to the high disease pressure in the outbreak area and the long incubation period of this disease. Leaves were binned for the analysis by variety, field location, and infected symptomatic and infected asymptomatic status by visual inspection. The ionome of leaf samples was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) and compared with each other. These analyses showed that Leccino variety consistently contained higher manganese (Mn) levels compared with Ogliarola salentina, and these levels were higher in both infected asymptomatic and infected symptomatic leaves. Infected asymptomatic and infected symptomatic leaves within a host genotype also showed differences in the ionome, particularly a higher concentration of calcium (Ca) and Mn levels in the Leccino cultivar, and sodium (Na) in both varieties. We hypothesize that the ionome differences in the two varieties contribute to protection against disease caused by X. fastidiosa infection.

Variation in Fumonisin and Ochratoxin Production Associated with Differences in Biosynthetic Gene Content in Aspergillus niger and A. welwitschiae Isolates from Multiple Crop and Geographic Origins.

The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that (i) isolates of both species differed in ability to produce the mycotoxins; (ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum) cluster; (iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and (iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas, a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin.