Deep Sequencing Analysis of RNAs from Citrus Plants Grown in a Citrus Sudden Death-Affected Area Reveals Diverse Known and Putative Novel Viruses.

Citrus sudden death (CSD) has caused the death of approximately four million orange trees in a very important citrus region in Brazil. Although its etiology is still not completely clear, symptoms and distribution of affected plants indicate a viral disease. In a search for viruses associated with CSD, we have performed a comparative high-throughput sequencing analysis of the transcriptome and small RNAs from CSD-symptomatic and -asymptomatic plants using the Illumina platform. The data revealed mixed infections that included Citrus tristeza virus (CTV) as the most predominant virus, followed by the Citrus sudden death-associated virus (CSDaV), Citrus endogenous pararetrovirus (CitPRV) and two putative novel viruses tentatively named Citrus jingmen-like virus (CJLV), and Citrus virga-like virus (CVLV). The deep sequencing analyses were sensitive enough to differentiate two genotypes of both viruses previously associated with CSD-affected plants: CTV and CSDaV. Our data also showed a putative association of the CSD-symptomatic plants with a specific CSDaV genotype and a likely association with CitPRV as well, whereas the two putative novel viruses showed to be more associated with CSD-asymptomatic plants. This is the first high-throughput sequencing-based study of the viral sequences present in CSD-affected citrus plants, and generated valuable information for further CSD studies

A high-throughput analysis of high-resolution X-ray CT images of stems of olive and citrus plants resistant and susceptible to Xylella fastidiosa

The bacterial plant pathogen Xylella fastidiosa causes disease in several globally important crops. However, some cultivars harbour reduced bacterial loads and express few symptoms. Evidence considering plant species in isolation suggests xylem structure influences cultivar susceptibility to X. fastidiosa. We test this theory more broadly by analysing high-resolution synchrotron X-ray computed tomography of healthy and infected plant vasculature from two taxonomic groups containing susceptible and resistant varieties: two citrus cultivars (sweet orange cv. Pera, tangor cv. Murcott) and two olive cultivars (Koroneiki, Leccino). Results found the susceptible plants had more vessels than resistant ones, which could promote within-host pathogen spread. However, features associated with resistance were not shared by citrus and olive. While xylem vessels in resistant citrus stems had comparable diameters to those in susceptible plants, resistant olives had narrower vessels that could limit biofilm spread. And while differences among olive cultivars were not detected, results suggest greater vascular connectivity in resistant compared to susceptible citrus plants. We hypothesize that this provides alternate flow paths for sustaining hydraulic functionality under infection. In summary, this work elucidates different physiological resistance mechanisms between two taxonomic groups, while supporting the existence of an intertaxonomical metric that could speed up the identification of candidate-resistant plants.</p

Parallel host shifts in a bacterial plant pathogen suggest independent genetic solutions

Beyond Xylella, Integrated Management Strategies for Mitigating Xylella fastidiosa Impact in Europe (BeXyl) (Grant Agreement 101060593). Partner/Coordinador principal: Blanca B. Landa del Castillo, Investigadora Científica del Instituto de Agricultura Sostenible (IAS-CSIC).While there are documented host shifts in many bacterial plant pathogens, the genetic foundation of host shifts is largely unknown. Xylella fastidiosa is a bacterial pathogen found in over 600 host plant species. Two parallel host shifts occurred-in Brazil and Italy-in which X. fastidiosa adapted to infect olive trees, whereas related strains infected coffee. Using 10 novel whole-genome sequences from an olive-infecting population in Brazil, we investigated whether these olive-infecting strains diverged from closely related coffee-infecting strains. Several single-nucleotide polymorphisms, many derived from recombination events, and gene gain and loss events separated olive-infecting strains from coffee-infecting strains in this clade. The olive-specific variation suggests that this event was a host jump with genetic isolation between coffee- and olive-infecting X. fastidiosa populations. Next, we investigated the hypothesis of genetic convergence in the host shift from coffee to olive in both populations (Brazil and Italy). Each clade had multiple mutations and gene gain and loss events unique to olive, yet no overlap between clades. Using a genome-wide association study technique, we did not find any plausible candidates for convergence. Overall, this work suggests that the two populations adapted to infect olive trees through independent genetic solutions.This work was partially funded by the California Department of Food and Agriculture Pierce's Disease Control Program. Additional funding was from Horizon 2020 (XF Actors project number 727987), BeXyl Project (HORIZON, id: 101060593), and FAPESP (São Paulo Research Foundation 2016/02176-7). H.D.C.F. received CNPq research fellowships (proc. no. 313676/2017-8).Peer reviewe

Temporal and Spatial Scaling of the Genetic Structure of a Vector-Borne Plant Pathogen

The ecology of plant pathogens of perennial crops is affected by the long-lived nature of their immobile hosts. In addition, changes to the genetic structure of pathogen populations may affect disease epidemiology and management practices; examples include local adaptation of more fit genotypes or introduction of novel genotypes from geographically distant areas via human movement of infected plant material or insect vectors. We studied the genetic structure of Xylella fastidiosa populations causing disease in sweet orange plants in Brazil at multiple scales using fast-evolving molecular markers (simple-sequence DNA repeats). Results show that populations of X. fastidiosa were regionally isolated, and that isolation was maintained for populations analyzed a decade apart from each other. However, despite such geographic isolation, local populations present in year 2000 were largely replaced by novel genotypes in 2009 but not as a result of migration. At a smaller spatial scale (individual trees), results suggest that isolates within plants originated from a shared common ancestor. In summary, new insights on the ecology of this economically important plant pathogen were obtained by sampling populations at different spatial scales and two different time points.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Transmission and distribution of Xylella fastidiosa subsp. pauca in olive trees as a parameter for managing olive quick decline syndrome

Systemic bacteria such as Xylella fastidiosa can be vertically transmitted by using diseased propagative material during nursery tree production. We tested the hypothesis that X. fastidiosa is not transmitted by symptomless olive tree sprouts. In addition, we investigated the distribution of bacteria in olive plants (different parts of canopy and root system) with initial leaf scorch symptoms and with symptoms spread throughout the canopy. In both studies, the presence of bacteria was tested by quantitative real-time PCR (qPCR). For the first hypothesis, sprouts from a symptomless olive tree were rooted and checked for X. fastidiosa infection 32 months later. A total of 57.7% (41 of 71) of rooted seedlings were positive for X. fastidiosa (Ct from 24.25 to 31.82) but only one plant had scorched leaves. Regarding the distribution of the bacteria in the diseased plants, results based on qPCR showed that X. fastidiosa was systemically distributed through the olive plant canopy (30% to 77% of sampled tissues, with Ct values from 19.56 to 31.59). Samples from trees showing symptoms of olive quick decline syndrome (OQDS), taken 3 m above the lateral branch with symptoms, were positive for the presence of X. fastidiosa, as was the root system. The results demonstrate that vegetative material from olive plants used for rooting must be carefully selected for the absence of X. fastidiosa. Also, based on the qPCR-positive results, pruning may have a limited effect on eliminating the bacteria from diseased plants, even from the ones showing few OQDS symptoms.This work was funded by Horizon 2020 (XF Actors grant 727987) and FAPESP (São Paulo Research Foundation grant 2016/02176-7). The authors are very grateful to Cristina Maria Vicentine for supporting the sampling of the olive plants. H.D.C.F. thanks the National Council for Scientific and Technological Development (CNPq) for the research fellowship (project 308164/2021-0). Manuel Anguita-Maeso acknowledges the research stay fellowship from SEGIB - Carolina Foundation.Peer reviewe

Culture and metagenomic approaches for the identification of olive xylem microbial communities as a biological control tool to cope against Xylella fastidiosa infection

Trabajo presentado en la 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021.The xylem-inhabiting plant pathogenic bacterium Xylella fastidiosa (Xf) represents one of the major phytopathological threats to olive crop worldwide, due to its devastating effects on agricultural yields losses and high tree mortality that causes profound socioeconomic and environmental impacts. Endophytes play an essential role on plant growth and its physiological status, but they can also act as an innate natural defense to cope against infection by xylem-inhabiting pathogenic organisms. Today, vast majority of microorganisms residing in olive xylem are unknown; therefore this work pursues the characterization of the olive microbiome through culture-dependent and independent (NGS) techniques as a tool for identifying potential biological control agents for this pathogen. Hence, four cultivated olive genotypes (Arbequina, Arbosana, Koroneiki and Grappolo) located in Sao Paulo state (Brazil) showing visual Xf symptoms or asymptomatic-non-infected were selected. Xf infection was verified by qPCR. For the culture-dependent approach, chips extracts of xylem tissue from branches and roots were plated in two solid media (R2A and R2A supplemented with plant extract). For culture independent approach, total DNA extracted from xylem tissue was analyzed by metagenomic analysis of 16S and ITS region to characterize the xylem-inhabiting bacterial and fungal communities. Preliminary culture results indicated differences in the frequency of microbial communities depending on the olive genotype and the type of plant material analyzed, as well as, the presence or absence of Xf symptoms on the sampled trees that correlated with Xf infection. These results will help to expand our knowledge on the olive xylem microbiome community composition and understand its driving factors when Xf infection occurs and more importantly to identify xylem-inhabiting microorganisms with potential to combat this harmful bacterium.Study supported by Projects 727987 XF-ACTORS (EU-H2020) and AGL2016-75606-R (MEIC Spain and FEDER-EU) and SEGIB – Carolina Foundation

Impacts of local population history and ecology on the evolution of a globally dispersed pathogen.

BackgroundPathogens with a global distribution face diverse biotic and abiotic conditions across populations. Moreover, the ecological and evolutionary history of each population is unique. Xylella fastidiosa is a xylem-dwelling bacterium infecting multiple plant hosts, often with detrimental effects. As a group, X. fastidiosa is divided into distinct subspecies with allopatric historical distributions and patterns of multiple introductions from numerous source populations. The capacity of X. fastidiosa to successfully colonize and cause disease in naïve plant hosts varies among subspecies, and potentially, among populations. Within Central America (i.e. Costa Rica) two X. fastidiosa subspecies coexist: the native subsp. fastidiosa and the introduced subsp. pauca. Using whole genome sequences, the patterns of gene gain/loss, genomic introgression, and genetic diversity were characterized within Costa Rica and contrasted to other X. fastidiosa populations.ResultsWithin Costa Rica, accessory and core genome analyses showed a highly malleable genome with numerous intra- and inter-subspecific gain/loss events. Likewise, variable levels of inter-subspecific introgression were found within and between both coexisting subspecies; nonetheless, the direction of donor/recipient subspecies to the recombinant segments varied. Some strains appeared to recombine more frequently than others; however, no group of genes or gene functions were overrepresented within recombinant segments. Finally, the patterns of genetic diversity of subsp. fastidiosa in Costa Rica were consistent with those of other native populations (i.e. subsp. pauca in Brazil).ConclusionsOverall, this study shows the importance of characterizing local evolutionary and ecological history in the context of world-wide pathogen distribution

Mapping Xylella fastidiosa infection and xylem microbiome composition on olive tree branches

Trabajo presentado en la 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021.The plant-pathogenic bacterium Xylella fastidiosa, widely distributed in the Americas and with several recent outbreaks in EU, emerged as a serious threat to olive crop and the typical Mediterranean landscape. This xylem-limited plant pathogen spreads by xylem sap-feeding insects, causes symptoms of marginal leaf scorch, leaf chlorosis, defoliation, and a general decay in olive trees. Research on plant-associated microorganisms has gained importance in the last decade as a key component in plant health. However, the role of the xylem microbiome and its contribution to plant health is still scarce. Therefore, this work is focused on mapping X. fastidiosa infection and deciphering the changes in the composition of microbial communities on symptomatic and asymptomatic olive tree branches. Three olives trees of cultivar Grappolo growing at Sao Paulo state (Brazil) were selected and within each tree three main branches were sampled showing different severity of symptoms development; one branch presenting typical X. fastidiosa symptoms of pathogen infection in contrast to the other two branches that were asymptomatic. X. fastidiosa infection was verified by qPCR. Leaves and stem samples were collected in a gradual scale above and below the symptomatic branches. Chips extracts of xylem tissue from its stems and leaf veins and petioles were used for DNA extraction to undergo metagenomic analysis (NGS) for microbial composition estimation and to diagnose the presence of the pathogen by real-time PCR, respectively. These results will help to understand the occurrence of step-wise changes in olive xylem microbiome as a consequence of X. fastidiosa infection and determine which microorganisms respond faster or can be associated to a symptom progression on infected trees.Study supported by Projects 727987 XF-ACTORS (EU-H2020) and AGL2016-75606-R (MEIC Spain and FEDER-EU) and SEGIB – Carolina Foundation

Genomic Diversity and Recombination among Xylella fastidiosa Subspecies.

Xylella fastidiosa is an economically important bacterial plant pathogen. With insights gained from 72 genomes, this study investigated differences among the three main subspecies, which have allopatric origins: X. fastidiosa subsp. fastidiosa, multiplex, and pauca The origin of recombinogenic X. fastidiosa subsp. morus and sandyi was also assessed. The evolutionary rate of the 622 genes of the species core genome was estimated at the scale of an X. fastidiosa subsp. pauca subclade (7.62 × 10-7 substitutions per site per year), which was subsequently used to estimate divergence time for the subspecies and introduction events. The study characterized genes present in the accessory genome of each of the three subspecies and investigated the core genome to detect genes potentially under positive selection. Recombination is recognized to be the major driver of diversity in X. fastidiosa, potentially facilitating shifts to novel plant hosts. The relative effect of recombination in comparison to point mutation was calculated (r/m = 2.259). Evidence of recombination was uncovered in the core genome alignment; X. fastidiosa subsp. fastidiosa in the United States was less prone to recombination, with an average of 3.22 of the 622 core genes identified as recombining regions, whereas a specific clade of X. fastidiosa subsp. multiplex was found to have on average 9.60 recombining genes, 93.2% of which originated from X. fastidiosa subsp. fastidiosa Interestingly, for X. fastidiosa subsp. morus, which was initially thought to be the outcome of genome-wide recombination between X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex, intersubspecies homologous recombination levels reached 15.30% in the core genome. Finally, there is evidence of X. fastidiosa subsp. pauca strains from citrus containing genetic elements acquired from strains infecting coffee plants as well as genetic elements from both X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex In summary, our data provide new insights into the evolution and epidemiology of this plant pathogen.IMPORTANCEXylella fastidiosa is an important vector-borne plant pathogen. We used a set of 72 genomes that constitutes the largest assembled data set for this bacterial species so far to investigate genetic relationships and the impact of recombination on phylogenetic clades and to compare genome content at the subspecies level, and we used a molecular dating approach to infer the evolutionary rate of X. fastidiosa The results demonstrate that recombination is important in shaping the genomes of X. fastidiosa and that each of the main subspecies is under different selective pressures. We hope insights from this study will improve our understanding of X. fastidiosa evolution and biology