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.

Nicotiana benthamiana as a model plant host for Xylella fastidiosa: Control of infections by transient expression and endotherapy with a bifunctional peptide.

Transient expression of genes encoding peptides BP134 and BP178 by means of a Potato virus X (PVX) based-vector system, and treatment with synthetic peptides by endotherapy, were evaluated in the control of Xylella fastidiosa infections, in the model plant Nicotiana benthamiana. Transient production of BP178 significantly decreased disease severity compared to PVX and non-treated control (NTC) plants, without adverse effects. Plants treated with synthetic BP134 and BP178 showed consistently lower levels of disease than NTC plants. However, the coinfection with PVX-BP134 and X. fastidiosa caused detrimental effects resulting in plant death. The levels of X. fastidiosa in three zones sampled, upwards and downwards of the inoculation/treatment point, significantly decreased compared to the NTC plants, after the treatment with BP178, but not when BP178 was produced transiently. The effect of treatment and transient production of BP178 in the induction of defense-related genes was also studied. Synthetic BP178 applied by endotherapy induced the expression of ERF1, PR1a, PAL, PALII and WRKY25, while the transient expression of BP178 overexpressed the Cath, Cyc, PR4a, 9-LOX and Endochitinase B genes. Both treatments upregulated the expression of PR1, PR3, PR4 and CycT9299 genes compared to the NTC or PVX plants. It was concluded that the effect of BP178, either by endotherapy or by transient expression, on the control of the X. fastidiosa infections in N. benthamiana, was due in part to the induction of the plant defense system in addition to its bactericidal activity reported in previous studies. However, the protection observed when BP178 was transiently produced seems mainly mediated by the induction of plant defense, because the levels of X. fastidiosa were not significantly affected.

Virus classification based on in-depth sequence analyses and development of demarcation criteria using the Betaflexiviridae as a case study.

Currently, many viruses are classified based on their genome organization and nucleotide/amino acid sequence identities of their capsid and replication-associated proteins. Although biological traits such as vector specificities and host range are also considered, this later information is scarce for the majority of recently identified viruses, characterized only from genomic sequences. Accordingly, genomic sequences and derived information are being frequently used as the major, if not only, criteria for virus classification and this calls for a full review of the process. Herein, we critically addressed current issues concerning classification of viruses in the family Betaflexiviridae in the era of high-throughput sequencing and propose an updated set of demarcation criteria based on a process involving pairwise identity analyses and phylogenetics. The proposed framework has been designed to solve the majority of current conundrums in taxonomy and to facilitate future virus classification. Finally, the analyses performed herein, alongside the proposed approaches, could be used as a blueprint for virus classification at-large.

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.

Fast and Reliable Electronic Assay of a Xylella fastidiosa Single Bacterium in Infected Plants Sap.

Pathogens ultra-sensitive detection is vital for early diagnosis and provision of restraining actions and/or treatments. Among plant pathogens, Xylella fastidiosa is among the most threatening as it can infect hundreds of plant species worldwide with consequences on agriculture and the environment. An electrolyte-gated transistor is here demonstrated to detect X. fastidiosa at a limit-of-quantification (LOQ) of 2 ± 1 bacteria in 0.1 mL (20 colony-forming-unit per mL). The assay is carried out with a millimeter-wide gate functionalized with Xylella-capturing antibodies directly in saps recovered from naturally infected plants. The proposed platform is benchmarked against the quantitave polymerase chain reaction (qPCR) gold standard, whose LOQ turns out to be at least one order of magnitude higher. Furthermore, the assay selectivity is proven against the Paraburkholderia phytofirmans bacterium (negative-control experiment). The proposed label-free, fast (30 min), and precise (false-negatives, false-positives below 1%) electronic assay, lays the ground for an ultra-high performing immunometric point-of-care platform potentially enabling large-scale screening of asymptomatic plants.

Introduction and adaptation of an emerging pathogen to olive trees in Italy.

The invasive plant pathogen Xylella fastidiosa currently threatens European flora through the loss of economically and culturally important host plants. This emerging vector-borne bacterium, native to the Americas, causes several important diseases in a wide range of plants including crops, ornamentals, and trees. Previously absent from Europe, and considered a quarantine pathogen, X. fastidiosa was first detected in Apulia, Italy in 2013 associated with a devastating disease of olive trees (Olive Quick Decline Syndrome, OQDS). OQDS has led to significant economic, environmental, cultural, as well as political crises. Although the biology of X. fastidiosa diseases have been studied for over a century, there is still no information on the determinants of specificity between bacterial genotypes and host plant species, which is particularly relevant today as X. fastidiosa is expanding in the naive European landscape. We analysed the genomes of 79 X. fastidiosa samples from diseased olive trees across the affected area in Italy as well as genomes of the most genetically closely related strains from Central America. We provided insights into the ecological and evolutionary emergence of this pathogen in Italy. We first showed that the outbreak in Apulia is due to a single introduction from Central America that we estimated to have occurred in 2008 [95 % HPD: 1930-2016]. By using a combination of population genomic approaches and evolutionary genomics methods, we further identified a short list of genes that could play a major role in the adaptation of X. fastidiosa to this new environment. We finally provided experimental evidence for the adaptation of the strain to this new environment.

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.

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.

Predominance and Diversity of GLRaV-3 in Native Vines of Mediterranean Croatia.

Sixteen grapevine cultivars from Mediterranean Croatia were surveyed for the presence of 10 of the most economically important grapevine viruses. The presence of Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine leafroll associated virus-1, -2, and -3 (GLRaV-1; GLRaV-2 and GLRaV-3), Grapevine virus A (GVA) and B (GVB), Grapevine fleck virus (GFkV), Grapevine rupestris stem pitting associated virus (GRSPaV), and Grapevine Pinot gris virus (GPGV) were tested by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). All 71 analyzed clones were positive for the presence of one or more viruses. The most abundant one, detected in almost 95% of samples was GLRaV-3. In most of cases it was reported in mixed infections with GVA, GRSPaV, and GPGV. Virus genomes of GLRaV-3 infected vines were further characterized molecularly in order to determine their genetic diversity. Different genomic variants of heat shock 70 protein homologue (HSP70h) were identified by single-strand conformation polymorphism (SSCP) and sequenced. Sequence analysis confirmed their clustering into phylogenetic group I and/or phylogenetic group II. This study emphasizes the wide virus heterogenicity in Mediterranean vines and the predominant presence of GLRaV-3 phylogenetic groups I and II, either individually or in combination.

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.

Antagonistic activity of olive endophytic bacteria and of Bacillus spp. strains against Xylella fastidiosa.

Strains of Xylella fastidiosa subsp. pauca characterized by a specific genotype, the so called sequence type "ST53", have been associated with a severe disease named Olive Quick Decline Syndrome (OQDS). Despite the relevant research efforts devoted to control the disease caused by X. fastidiosa, so far there are no therapeutic means able to cure the infected host plants. As such, the aim of this study was the identification of antagonistic bacteria potentially deployable as bio-control agents against X. fastidiosa. To this end, two approaches were used, i.e. the evaluation of the antagonistic activity of: i) endophytic bacteria isolated from olive trees located in an infected area but showing mild or no symptoms, and ii) Bacillus strains, as they are already known as bio-control agents. Characterization of endophytic bacterial isolates revealed that the majority belonged to different species of the genera Sphingomonas, Methylobacterium, Micrococcus and Curtobacterium. However, when they were tested in vitro against X. fastidiosa ST53 none of them showed antagonistic activity. On the contrary, when strains belonging to different species of the genus Bacillus were included in these tests, remarkable antagonistic activities were recorded. Some B. velezensis strains also produced culture filtrates with inhibitory activity against X. fastidiosa ST53. Taking also into account that two of these B. velezensis strains (namely strains D747 and QST713) are already registered and commercially available as bio-control agents, our results pave the way for further studies aimed at the development of a sustainable bio-control strategy of the OQDS.

Emergence of a Plant Pathogen in Europe Associated with Multiple Intercontinental Introductions.

Pathogen introductions have led to numerous disease outbreaks in naive regions of the globe. The plant pathogen Xylella fastidiosa has been associated with various recent epidemics in Europe affecting agricultural crops, such as almond, grapevine, and olive, but also endemic species occurring in natural forest landscapes and ornamental plants. We compared whole-genome sequences of X. fastidiosa subspecies multiplex from America and strains associated with recent outbreaks in southern Europe to infer their likely origins and paths of introduction within and between the two continents. Phylogenetic analyses indicated multiple introductions of X. fastidiosa subspecies multiplex into Italy, Spain, and France, most of which emerged from a clade with limited genetic diversity with a likely origin in California, USA. The limited genetic diversity observed in X. fastidiosa subspecies multiplex strains originating from California is likely due to the clade itself being an introduction from X. fastidiosa subspecies multiplex populations in the southeastern United States, where this subspecies is most likely endemic. Despite the genetic diversity found in some areas in Europe, there was no clear evidence of recombination occurring among introduced X. fastidiosa strains in Europe. Sequence type taxonomy, based on multilocus sequence typing (MLST), was shown, at least in one case, to not lead to monophyletic clades of this pathogen; whole-genome sequence data were more informative in resolving the history of introductions than MLST data. Although additional data are necessary to carefully tease out the paths of these recent dispersal events, our results indicate that whole-genome sequence data should be considered when developing management strategies for X. fastidiosa outbreaks.IMPORTANCEXylella fastidiosa is an economically important plant-pathogenic bacterium that has emerged as a pathogen of global importance associated with a devastating epidemic in olive trees in Italy associated with X. fastidiosa subspecies pauca and other outbreaks in Europe, such as X. fastidiosa subspecies fastidiosa and X. fastidiosa subspecies multiplex in Spain and X. fastidiosa subspecies multiplex in France. We present evidence of multiple introductions of X. fastidiosa subspecies multiplex, likely from the United States, into Spain, Italy, and France. These introductions illustrate the risks associated with the commercial trade of plant material at global scales and the need to develop effective policy to limit the likelihood of pathogen pollution into naive regions. Our study demonstrates the need to utilize whole-genome sequence data to study X. fastidiosa introductions at outbreak stages, since a limited number of genetic markers does not provide sufficient phylogenetic resolution to determine dispersal paths or relationships among strains that are of biological and quarantine relevance.

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.

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.

Draft Genome Sequence Resources of Three Strains (TOS4, TOS5, and TOS14) of Xylella fastidiosa Infecting Different Host Plants in the Newly Discovered Outbreak in Tuscany, Italy.

An outbreak of Xylella fastidiosa was discovered in late 2018 in northern Italy affecting several plant species. Multilocus sequence typing analyses detected the presence of strains clustering in X. fastidiosa subsp. multiplex and harboring a hitherto uncharacterized sequence type, ST87. Three cultured strains (TOS4, TOS5, and TOS14) were subjected to high-throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated that they belong to the subspecies multiplex. The genetic information generated for these newly discovered strains further supports the evidence that sequence types are associated with the emergence of X. fastidiosa in Europe, posing major challenges for predicting the main threatened European and Mediterranean crops and plant species.

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.

Draft Genome Resources of Two Strains ("ESVL" and "IVIA5901") of Xylella fastidiosa Associated with Almond Leaf Scorch Disease in Alicante, Spain.

An outbreak of Xylella fastidiosa subsp. multiplex sequence type ST6 was discovered in 2017 in mainland Spain affecting almond trees. Two cultured almond strains, "ESVL" and "IVIA5901," were subjected to high throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated they belong to the subspecies multiplex, and pairwise comparisons of the chromosomal genomes showed an average nucleotide identity higher than 99%. Interestingly, the two strains differ for the presence of the plasmids pXF64-Hb_ESVL and pUCLA-ESVL detected only in the ESVL strain. The availability of these draft genomes contribute to extend the European genomic sequence dataset, a first step toward setting new research to elucidate the pathway of introduction and spread of the numerous strains of this subspecies so far detected in Europe.

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

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.