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

Identification and characterization of privet leaf blotch-associated virus, a novel idaeovirus

A novel virus has been identified by next-generation sequencing (NGS) in privet (Ligustrum japonicum L.) affected by a graft-transmissible disease characterized by leaf blotch symptoms resembling infectious variegation, a virus-like privet disease with an unclear aetiology. This virus, which has been tentatively named ‘privet leaf blotch-associated virus’ (PrLBaV), was absent in non-symptomatic privet plants, as revealed by NGS and reverse transcription-polymerase chain reaction (RT-PCR). Molecular characterization of PrLBaV showed that it has a segmented genome composed of two positive single-stranded RNAs, one of which (RNA1) is monocistronic and codes for the viral replicase, whereas the other (RNA2) contains two open reading frames (ORFs), ORF2a and ORF2b, coding for the putative movement (p38) and coat (p30) proteins, respectively. ORF2b is very probably expressed through a subgenomic RNA starting with six nucleotides (AUAUCU) that closely resemble those found in the 5′-terminal end of genomic RNA1 and RNA2 (AUAUUU and AUAUAU, respectively). The molecular signatures identified in the PrLBaV RNAs and proteins resemble those of Raspberry bushy dwarf virus (RBDV), currently the only member of the genus Idaeovirus. These data, together with phylogenetic analyses, are consistent with the proposal of considering PrLBaV as a representative of the second species in the genus Idaeovirus. Transient expression of a recombinant PrLBaV p38 fused to green fluorescent protein in leaves of Nicotiana benthamiana, coupled with confocal laser scanning microscopy assays, showed that it localizes at cell plasmodesmata, strongly supporting its involvement in viral movement/trafficking and providing the first functional characterization of an idaeovirus encoded protein

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

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

Draft Genome Sequence of CO33, a Coffee-Infecting Isolate of Xylella fastidiosa

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%

Next-generation sequencing and metagenomic analysis advances plant virus diagnosis and discovery

The advent of next generation sequencing (NGS) technologies dramatically advanced our ability to comprehensively investigate diseases of unknown etiology and expedited the entire process of virus discovery, identification, viral genome sequencing and, subsequently, the development of routine assays for new viral pathogens. Unlike traditional techniques, these novel approaches require no preliminary knowledge of the suspected virus(es). Currently, the RNA-Seq approach has been widely used to identify new viruses in infected plants, by analyzing virus-derived small interfering RNA populations, single- and double-stranded RNA (dsRNA) molecules extracted from infected plants. The method generates sequence in an unbiased fashion, likely allowing to detect all viruses that are present in a sample. We applied the Illumina NGS, coupled with metagenomic analysis, to generate large sequence dataset in different woody crops affected by diseases of unknown origin or infected with uncharacterized viruses or new strains. This approach allowed the identification of five novel viral species and, in addition, the sequencing of the whole genome of several viruses and viroids infecting Citrus spp., Prunus spp., grapes, fig, hazelnut, olive, persimmon and mulberry. Combined analysis of the datasets generated by using either siRNA fractions and dsRNA templates, enhanced the characterization of the whole virus-derived sequences in the infected tissues. Furthermore, profiling small RNAs from virus-infected plants led to a better understanding of host-plant response to virus and viroid infections in perennial plants. A general bioinformatic pipeline and an experimental validation strategy were developed and its application illustrated

Conserved genetic defense response against X. fastidiosa subsp. pauca in olive and citrus

X. fastidiosa subsp. pauca causes diseases in citrus and olive plants. Fortunately, there are citrus species and olive varieties more tolerant to X. fastidiosa and therefore good genotypes to search for genetic sources of resistance. Following this approach, global gene expression analyses were recently achieved using Citrus reticulata cv. Ponkan and Olea europaea cv. Leccino allowing the identification of potential genes involved in plant defense response. Thus, the objective of this work was to identify key genes involved in common genetic defense responses that could be further explored to get resistant varieties. Overall we identified two main mechanisms for both plant species: i. Bacteria recognition and ii. Cell wall fortification. The former involve the expression of patternrecognition receptors, which recognize pathogen molecular patterns and trigger cell defense responses. Some of these receptors belong to the LRR-XII group which contains cell surface immune receptors. The latter involves downregulation of genes in tolerant host such as expansin, pectate lyases and polygalacturonases, related with cell wall expansion and degradation. This suggests that in tolerant hosts, plant cell recognizes X. fastidiosa and reprograms the cell wall development to impair its colonization through the xylem vessels. Therefore these genes represent good candidates to be explored aiming their use in breeding and/or genetic engineering program. Giampetruzzi A., Morelli M., Saponari M., Loconsole G., Chiumenti M., Boscia D., Savino V.N., Martelli G.P. & Saldarelli P. 2016. Transcriptome profiling of two olive cultivars in response to infection by the CoDiRO strain of Xylella fastidiosa subsp. pauca. BMC Genomics 17:475. Rodrigues C.M., De Souza A.A., Takita M.A., Kishi L.T. & Machado M.A. 2013. RNA-Seq analysis of Citrus reticulata in the early stages of Xylella fastidiosa infection reveals auxin-related genes as a defense response. BMC Genomics 14: 676