Michela Chiumenti

Draft Genome Sequence of the Xylella fastidiosa CoDiRO Strain

ABSTRACT We determined the draft genome sequence of the Xylella fastidiosa CoDiRO strain, which has been isolated from olive plants in southern Italy (Apulia). It is associated with olive quick decline syndrome (OQDS) and characterized by extensive scorching and desiccation of leaves and twigs.

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.

OCCURRENCE OF FIG BADNAVIRUS 1 IN FIG TREES FROM DIFFERENT COUNTRIES AND IN SYMPTOMLESS SEEDLINGS

The consistent identification of Fig badnavirus 1 (FBaV-1) in the totality of samples from mosaic-affected fig trees from New Zealand (Minafra et al., 2012) and Italy (unpublished informa- tion), prompted the examination of additional samples from adult fig trees with mosaic symptoms from Italy (10), France (1), Greece (3), Albania (1), Spain (1), Portugal (1), England (1), Hungary (3), Montenegro (1), Lebanon (19), Syria (11), Tunisia (15), Algeria (2), Turkey (4), South Africa (1), Mexico (1), Cuba (1), and Aus- tralia (1). Additional samples from Italy consisting of volunteer symptomless seedlings (20) and symptomless potted plants de- rived from explants of mosaic-affected trees subjected to heat therapy (4), brought to over 100 the total number of sources ana- lyzed. Total RNA was silica-extracted from cortical scrapings and subjected to RT-PCR 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 deposited in GenBank un- der the accession No. JF411989. A product with the expected size (214 bp) was amplified from all samples, regardless of the geo- graphical origin (18 countries) and the type of source (adult trees, seedlings, heat-treated plants). These results confirm what appears to be an amazing and unique association of FBaV-1 with Ficus car- ica. The extent of the association is such (100%) and involves trees from such a wide range of geographical origins, that makes it plausible, as in the case of other pararetroviruses (Staginnus and Richert-Poggeler, 2006), the suggestion that FBaV-1 may be inte- grated in the fig genome; a likelihood further supported by the virus presence in volunteer fig seedlings.

Identification and Characterization of Citrus tristeza virus Isolates Breaking Resistance in Trifoliate Orange in California

Most Citrus tristeza virus (CTV) isolates in California are biologically mild and symptomless in commercial cultivars on CTV tolerant rootstocks. However, to better define California CTV isolates showing divergent serological and genetic profiles, selected isolates were subjected to deep sequencing of small RNAs. Full-length sequences were assembled, annotated and trifoliate orange resistance-breaking (RB) isolates of CTV were identified. Phylogenetic relationships based on their full genomes placed three isolates in the RB clade: CA-RB-115, CA-RB-AT25, and CA-RB-AT35. The latter two isolates were obtained by aphid transmission from Murcott and Dekopon trees, respectively, containing CTV mixtures. The California RB isolates were further distinguished into two subclades. Group I included CA-RB-115 and CA-RB-AT25 with 99% nucleotide sequence identity with RB type strain NZRB-G90; and group II included CA-RB-AT35 with 99 and 96% sequence identity with Taiwan Pumelo/SP/T1 and HA18-9, respectively. The RB phenotype was confirmed by detecting CTV replication in graft-inoculated Poncirus trifoliata and transmission from P. trifoliata to sweet orange. The California RB isolates induced mild symptoms compared with severe isolates in greenhouse indexing tests. Further examination of 570 CTV accessions, acquired from approximately 1960 and maintained in planta at the Central California Tristeza Eradication Agency, revealed 16 RB positive isolates based on partial p65 sequences. Six isolates collected from 1992 to 2011 from Tulare and Kern counties were CA-RB-115-like; and 10 isolates collected from 1968 to 2010 from Riverside, Fresno, and Kern counties were CA-RB-AT35-like. The presence of the RB genotype is relevant because P. trifoliata and its hybrids are the most popular rootstocks in California.

Draft genome sequence of CO33, a coffee-infecting isolate of Xylella fastidiosa

ABSTRACT The draft genome sequence of Xylella fastidiosa CO33 isolate, retrieved from symptomatic leaves of coffee plant intercepted in northern Italy, is reported. The CO33 genome size is 2,681,926 bp with a GC content of 51.7%.

SANITATION TRIALS FOR THE PRODUCTION OF VIRUS-FREE FIG STOCKS

SUMMARY Heat therapy, meristem tip culture in vitro and a combination of both techniques were used for obtaining fig (Ficus carica) plants free from some viruses associated with fig mosaic, a disease with a worldwide distribution. Source plants were two field-grown adult fig accessions from a germplasm repository of the University of Bari (Italy) that showed severe and mild symptoms of mosaic, respectively, and two symptomless fig seedlings grown under screen. Adult plants were infected by Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1) and Fig badnavirus 1 (FBV-1). Seedlings were infected by FLV-1 and FBV-1. Progeny of explants subjected to meristem tip culture were still infected by FMV (93.8% elimination). This virus, however, was eradicated (100% sanitation) by shoot tip culture combined with heat therapy, or in vitro heat therapy. High sanitation rates from FLV-1 (81 to 100%) were also registered with all sanitation procedures employed, such as in vitro heat therapy alone (two cycles) or combined with tissue culture. By contrast, the DNA virus FBV-1 resisted all attempts of elimination, a behaviour that confirms indirectly its hypothesized integration in the fig genome.

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.

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.

IDENTIFICATION OF A BADNAVIRUS INFECTING MULBERRY

SUMMARY Electron microscope observation of leaf dips from a mulberry (Morus alba) tree from Lebanon showing leaf mottling and vein yellowing disclosed the presence of bacilliform particles ca. 150x30 nm in size, resembling those of members of the genus Badnavirus. BLAST analysis of the sequence of a DOP-PCR clone (810 bp) generated from DNA extracts from partially purified particle preparation disclosed a 67% (nucleotide) and 63% (amino acid) identity with the reverse transcriptase/RNase H gene (ORF3) of the badnavirus Cacao swollen shoot virus (CSSV). In a phylogenetic tree constructed with the amino acid sequence obtained, the putative mulberry badnavirus clustered close to CSSV and Citrus mosaic virus (CiMV) in a branch comprising also Fig badnavirus 1 (FBV-1) and Dioscorea bacilliform virus (DBALV). PCR analyses showed that the virus occurs with high infection rates in Lebanon (7 out of 13 trees examined), Turkey (2 out of 3) and Italy (29 out of 39). However, except for one, all PCR-positive trees were symptomless. Thus, since there is no clear-cut association of the virus in question with a specific disease, we propose for it the provisional name of Mulberry badnavirus 1 (MBV-1).

Small RNA Isolation from Tissues of Grapevine and Woody Plants

A protocol is described to purify small (s)RNA molecules from tissues of grapevine and other woody plants. The protocol has been specifically developed to analyze sRNA populations by high-throughput sequencing. It has been widely used on species of the genera Prunus and Vitis particularly rich in polyphenols and other enzyme-inhibiting compounds. The high quality of the sRNAs extracted from leaf or phloem tissues makes them suitable for all molecular biology reactions, in particular for next-generation sequencing library preparation.