Seasonal Dynamics and Distribution of Xylella fastidiosa in Infected Almond Trees.

This research focused on studying the dynamics of the bacterial pathogen Xylella fastidiosa in almond trees across different developmental stages. The objective was to understand the seasonal distribution and concentration of X. fastidiosa within almond trees. Different tree organs, including leaves, shoots, branches, fruits, flowers, and roots, from 10 X. fastidiosa-infected almond trees were sampled over 2 years. The incidence and concentration of X. fastidiosa were determined using qPCR and isolation. Throughout the study, X. fastidiosa was consistently absent from fruits, flowers, and roots, whereas it was detected in leaves as well as in shoots and branches. We demonstrate that the absence of X. fastidiosa in the roots is likely linked to the inability of this isolate to infect the peach-almond hybrid rootstock GF677. X. fastidiosa incidence in shoots and branches remained consistent throughout the year, whereas in leaf petioles, it varied across developmental stages, with lower detection during the early and late stages of the season. Similarly, viable X. fastidiosa cells were isolated from shoots and branches at all developmental stages, but no successful isolations were achieved from leaf petioles during the vegetative and nut growth stage. Studying the progression of almond leaf scorch symptoms in trees with initial infections showed that once symptoms emerged on one branch, symptomless branches were likely already infected by the bacterium. Therefore, selectively pruning symptomatic branches is unlikely to cure the tree. This study enhances our understanding of X. fastidiosa dynamics in almond trees and may have practical applications for its detection and control.

Assessing Carrot Accessions Susceptibility to the Bacterial Pathogen 'Candidatus Liberibacter solanacearum' and Its Associated Symptoms.

'Candidatus Liberibacter solanacearum' is an insect-transmitted bacterium associated with several plant diseases. In the Mediterranean Basin, 'Ca. L. solanacearum' haplotype D is vectored by Bactericera trigonica and can severely infect carrot plants leading to abnormal growth phenotypes and significant yield losses. Insecticide applications are insufficient to suppress disease spread and damage, and additional means for disease control are needed. In the current study, we evaluated the resistance of 97 carrot accessions to the bacterial pathogen 'Ca. L. solanacearum' and its associated symptoms. Accessions (Western and Asian types) were first screened in two commercial carrot fields. We found that Western type accessions were less prone to develop disease symptoms in both fields and were less frequently visited by the insect vector in one field. Overall, 22 Asian and five Western accessions with significantly lower disease incidence compared with the commercial cultivar were found. These accessions were then inoculated with 'Ca. L. solanacearum' under controlled conditions and were assessed for disease incidence, insect oviposition, and bacterial relative titer. Five accessions (three Asian and two Western) had significantly lower disease incidence compared with the reference cultivar. Interestingly, disease incidence was not necessarily in line with insect oviposition or in planta bacterial titer, which may indicate that other, perhaps physiological, differences among the accessions may govern the susceptibility of plants to the disease. The resistant accessions found in this study could be used in future resistance breeding programs and to better understand the underlying mechanisms of resistance to 'Ca. L. solanacearum'.

Effect of Plant Age, Temperature, and Vector Load on 'Candidatus Liberibacter solanacearum' in Planta Titer and Shoot Proliferation Symptoms in Carrot.

A decade ago, shoot proliferation symptoms (i.e., witches' broom) in carrots were believed to be the cause of 'Candidatus Phytoplasma' and Spiroplasma infection, yet in recent years this association appeared to have weakened, and a closer association was found with the yet-unculturable, psyllid-transmitted Gram-negative bacterium 'Candidatus Liberibacter solanacearum'. In Israel, carrots are grown throughout the year, yet shoot proliferation symptoms tend to appear only in mature plants and mostly in late spring to early summer. We hypothesized that factors such as plant age, temperature, and vector load, which vary during the year, have a critical effect on symptom development and examined these factors under controlled conditions. Here we show that young carrot seedlings are as prone as older plants to develop shoot proliferation symptoms after 'Ca. L. solanacearum' inoculation. Surprisingly, we found that the local 'Ca. L. solanacearum' haplotype was extremely sensitive to constant temperature of 30°C, which led to a significant reduction in bacterial growth and symptom development compared with 18°C, which was very conducive to symptom development. We have also found that inoculations with 10 or 20 psyllids per plant results in faster symptom development compared with inoculations with two psyllids per plant; however, the difference between vector loads in disease progress rate was not significant. These data provide important insights to the effects of plant age, growth temperature, and vector load on 'Ca. L. solanacearum' and its associated symptoms and further strengthen the notion that 'Ca. L. solanacearum' is the main responsible agent for carrot witches' broom in Israel.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Xylella fastidiosa Outbreak in Israel: Population Genetics, Host Range, and Temporal and Spatial Distribution Analysis.

Diseases caused by the insect-transmitted bacterium Xylella fastidiosa have been reported in the Americas since the 19th century, causing diseases such as Pierce's disease of grapevine, almond leaf scorch (ALS), and citrus variegated chlorosis. In the last decade X. fastidiosa was reported from different parts of the world, most notably from southern Italy, infecting olives. In 2017, X. fastidiosa was reported to be associated with ALS symptoms in Israel. Here, we investigated the causal agent of ALS in Israel, its genetic diversity, and host range, and we characterized the temporal and spatial distribution of the disease. X. fastidiosa subsp. fastidiosa sequence type 1 was isolated from symptomatic almond trees and was used to infect almond and grapevine by mechanical inoculation. The pathogen, however, did not infect olive, peach, cherry, plum, nectarine, clementine, and grapefruit plants. Genomic analysis of local isolates revealed that the local population is derived from a single introduction and that they are closely related to X. fastidiosa strains from grapevines in California. Distribution analyses revealed that ALS did not expand from 2017 to 2019; however, since 2020, newly symptomatic trees appeared in the tested orchards. Symptomatic trees were located primarily in clusters, and symptoms tended to spread within rows. Our study confirms that X. fastidiosa is the causal agent of ALS in Israel and describes its genetic and host range characteristics. Although there is no clear evidence yet for the identity of the vectors in Israel, ALS spread continues to threat the almond and grapevine industries in Israel.

Biosynthesis and secretion of the microbial sulfated peptide RaxX and binding to the rice XA21 immune receptor.

The rice immune receptor XA21 is activated by the sulfated microbial peptide required for activation of XA21-mediated immunity X (RaxX) produced by Xanthomonas oryzae pv. oryzae (Xoo). Mutational studies and targeted proteomics revealed that the RaxX precursor peptide (proRaxX) is processed and secreted by the protease/transporter RaxB, the function of which can be partially fulfilled by a noncognate peptidase-containing transporter component B (PctB). proRaxX is cleaved at a Gly-Gly motif, yielding a mature peptide that retains the necessary elements for RaxX function as an immunogen and host peptide hormone mimic. These results indicate that RaxX is a prokaryotic member of a previously unclassified and understudied group of eukaryotic tyrosine sulfated ribosomally synthesized, posttranslationally modified peptides (RiPPs). We further demonstrate that sulfated RaxX directly binds XA21 with high affinity. This work reveals a complete, previously uncharacterized biological process: bacterial RiPP biosynthesis, secretion, binding to a eukaryotic receptor, and triggering of a robust host immune response.

Frateuria defendens sp. nov., bacterium isolated from the yellows grapevine's disease vector Hyalesthes obsoletus.

A Dyella-like bacterium was previously isolated from the planthopper Hyalesthes obsoletus (Hemiptera). Based on its 16S rRNA gene sequence, strain DHoT was assigned to the family Rhodanobacteraceae with Dyella and Frateuria as its closest relatives. The closest 16S rRNA gene sequences were Frateuria aurantia DSM 6220T (98.2 %), Dyella thiooxydans ATSB10T (98 %), Dyella terrae JS14-6T (97.8 %) and Dyella marensis CS5-B2T (97.8 %). Strain DHoT is a Gram-negative, aerobic, motile, yellow-pigmented, rod-shaped bacterium. Strain DHoT cells grew well at 28-30 °C and at pH 6.5-7.5 on a nutrient agar plate. DNA-DNA hybridization showed that the relatedness between strain DHoT and D. jiangningensis strain SBZ3-12T, and F. aurantia DSM 6220T was 42.7 and 42.6 %, respectively. Ubiquinone Q-8 was the predominant respiratory quinone, and the major fatty acids (>10 %) were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 0. In silico analysis based on phylogenetics and sequence identity at the nucleotide and protein levels suggests that Frateuria is the closest known relative of strain DHoT. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain DHoT was designated as a novel species of the genus Frateuria, for which the name Frateuria defendens sp. nov. is proposed. The type strain is DHoT (=NCCB 100648T; =DLBT=DSM 106169T).

Correction: Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses.

[This corrects the article DOI: 10.1371/journal.ppat.1004809.].

Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand-dependent activation of defense responses.

Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components.

Bacterial Outer Membrane Vesicles Induce Plant Immune Responses.

Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions.

Transgenic expression of the rice Xa21 pattern-recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum.

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern-recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21-mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar 'Gonja manjaya' (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.

Methods for Assessing Plant Immune System Activation by Bacterial Extracellular Vesicles and Other Elicitors.

Bacterial extracellular vesicles (BEVs) are released from the surface of bacterial cells and contain a diverse molecular cargo. Studies conducted primarily with bacterial pathogens of mammals have shown that BEVs are involved in multiple processes such as cell-cell communication, the delivery of RNA, DNA, and proteins to target cells, protection from stresses, manipulation of host immunity, and other functions. Until a decade ago, the roles of BEVs in plant-bacteria interactions were barely investigated. However, recent studies have shown that BEVs of plant pathogens possess similar functions as their mammalian pathogen counterparts, and more research is now devoted to study their roles and interactions with plants. In the following methods chapter, we provide five well-validated assays to examine the interaction of BEVs with the plant immune system. These assays rely on different markers or immune outputs, which indicate the activation of plant immunity (defense marker gene expression, reactive oxygen species burst, seedling inhibition). Furthermore, we offer assays that directly evaluate the priming of the immune system following BEV challenge and the effectiveness of its response to subsequent local or systemic infection. Altogether, these assays provide a thorough examination to the interactions of BEVs and the plant immune system.

Bacterial outer membrane vesicles induce a transcriptional shift in arabidopsis towards immune system activation leading to suppression of pathogen growth in planta.

Gram-negative bacteria form spherical blebs on their cell periphery, which later dissociate from the bacterial cell wall to form extracellular vesicles. These nano scale structures, known as outer membrane vesicles (OMVs), have been shown to promote infection and disease and can induce typical immune outputs in both mammal and plant hosts. To better understand the broad transcriptional change plants undergo following exposure to OMVs, we treated Arabidopsis thaliana (Arabidopsis) seedlings with OMVs purified from the Gram-negative plant pathogenic bacterium Xanthomonas campestris pv. campestris and performed RNA-seq analysis on OMV- and mock-treated plants at 2, 6 and 24 h post challenge. The most pronounced transcriptional shift occurred at the first two time points tested, as reflected by the number of differentially expressed genes and the average fold change. OMVs induce a major transcriptional shift towards immune system activation, upregulating a multitude of immune-related pathways including a variety of immune receptors. Comparing the response of Arabidopsis to OMVs and to purified elicitors, revealed that OMVs induce a similar suite of genes and pathways as single elicitors, however, pathways activated by OMVs and not by other elicitors were detected. Pretreating Arabidopsis plants with OMVs and subsequently infecting with a bacterial pathogen led to a significant reduction in pathogen growth. Mutations in the plant elongation factor receptor (EFR), flagellin receptor (FLS2), or the brassinosteroid-insensitive 1-associated kinase (BAK1) co-receptor, did not significantly affect the immune priming effect of OMVs. All together these results show that OMVs induce a broad transcriptional shift in Arabidopsis leading to upregulation of multiple immune pathways, and that this transcriptional change may facilitate resistance to bacterial infection.

The cucurbit pathogenic bacterium Acidovorax citrulli requires a polar flagellum for full virulence before and after host-tissue penetration.

Acidovorax citrulli causes seedling blight and bacterial fruit blotch of cucurbits. Previous reports demonstrated the contribution of type IV pili (T4P) to A. citrulli virulence and to systemic infection of melon seedlings. Microfluidic flow-chamber assays demonstrated the involvement of T4P in surface adhesion and biofilm formation, whereas polar flagella did not appear to contribute to either of these features. On the other hand, a transposon mutant impaired in the biosynthesis of polar flagella was identified in screens for reduced virulence of an A. citrulli mutant library. Further characterization of polar flagellum mutants confirmed that A. citrulli requires a polar flagellum for full virulence on melon plants. Foliage and stem inoculation experiments revealed that polar flagella contribute to A. citrulli virulence and growth in planta at both pre- and post-host-tissue penetration. Interestingly, light microscope observations revealed that almost all A. citrulli wild-type cells extracted from the xylem sap of stem-inoculated melon seedlings remained motile, supporting the importance of this organelle in virulence and colonization of the host vascular system. We also report a negative effect of polar flagellum impairment on T4P-mediated twitching motility of A. citrulli and discuss a possible co-regulation of these two motility machineries in this bacterium.

Antimicrobial Activity of Metabolites Secreted by the Endophytic Bacterium Frateuria defendens.

Candidatus Phytoplasma, the causative agent of yellows disease, inflicts substantial damage on several hundred plant species including perennials and annual plants. The endophytic bacterium Frateuria defendens reduces the symptoms of yellows disease in a number of agricultural crops. One possible mode of action is that the bacterium secretes antimicrobial metabolites. To test this hypothesis, the substances secreted by the endophyte during 10 days of growth in an artificial medium were identified by GC-MS (gas chromatography-mass spectrometry). Synthetic analogues to these substances were then used on periwinkle, a nurse culture plant infected by phytoplasma. Phytoplasma quantities were evaluated by quantitative PCR, and disease symptoms were monitored and recorded. It was found that specific compounds identified by the biochemical analysis caused a significant reduction in both the titer of phytoplasma and the disease symptoms in periwinkle when compared to untreated infected plants. Further research is required to examine the potential of these compounds as an effective treatment against yellows disease.

Type IV Pili are required for virulence, twitching motility, and biofilm formation of acidovorax avenae subsp. Citrulli.

Acidovorax avenae subsp. citrulli is the causal agent of bacterial fruit blotch (BFB), a threatening disease of watermelon, melon, and other cucurbits. Despite the economic importance of BFB, relatively little is known about basic aspects of the pathogen's biology and the molecular basis of its interaction with host plants. To identify A. avenae subsp. citrulli genes associated with pathogenicity, we generated a transposon (Tn5) mutant library on the background of strain M6, a group I strain of A. avenae subsp. citrulli, and screened it for reduced virulence by seed-transmission assays with melon. Here, we report the identification of a Tn5 mutant with reduced virulence that is impaired in pilM, which encodes a protein involved in assembly of type IV pili (TFP). Further characterization of this mutant revealed that A. avenae subsp. citrulli requires TFP for twitching motility and wild-type levels of biofilm formation. Significant reductions in virulence and biofilm formation as well as abolishment of twitching were also observed in insertional mutants affected in other TFP genes. We also provide the first evidence that group I strains of A. avenae subsp. citrulli can colonize and move through host xylem vessels.

Liberibacter crescens biofilm formation in vitro: establishment of a model system for pathogenic 'Candidatus Liberibacter spp.'

The Liberibacter genus comprises insect endosymbiont bacterial species that cause destructive plant diseases, including Huanglongbing in citrus and zebra chip in potato. To date, pathogenic 'Candidatus Liberibacter spp.' (CLs) remain uncultured, therefore the plant-associated Liberibacter crescens (Lcr), only cultured species of the genus, has been used as a biological model for in vitro studies. Biofilm formation by CLs has been observed on the outer midgut surface of insect vectors, but not in planta. However, the role of biofilm formation in the life cycle of these pathogens remains unclear. Here, a model system for studying CLs biofilms was developed using Lcr. By culture media modifications, bovine serum albumin (BSA) was identified as blocking initial cell-surface adhesion. Removal of BSA allowed for the first time observation of Lcr biofilms. After media optimization for biofilm formation, we demonstrated that Lcr attaches to surfaces, and form cell aggregates embedded in a polysaccharide matrix both in batch cultures and under flow conditions in microfluidic chambers. Biofilm structures may represent excellent adaptive advantages for CLs during insect vector colonization helping with host retention, immune system evasion, and transmission. Future studies using the Lcr model established here will help in the understanding of the biology of CLs.

Genome Analysis of Haplotype D of Candidatus Liberibacter Solanacearum.

Candidatus Liberibacter solanacearum (Lso) haplotype D (LsoD) is a suspected bacterial pathogen, spread by the phloem-feeding psyllid Bactericera trigonica Hodkinson and found to infect carrot plants throughout the Mediterranean. Haplotype D is one of six haplotypes of Lso that each have specific and overlapping host preferences, disease symptoms, and psyllid vectors. Genotyping of rRNA genes has allowed for tracking the haplotype diversity of Lso and genome sequencing of several haplotypes has been performed to advance a comprehensive understanding of Lso diseases and of the phylogenetic relationships among the haplotypes. To further pursue that aim we have sequenced the genome of LsoD from its psyllid vector and report here its draft genome. Genome-based single nucleotide polymorphism analysis indicates LsoD is most closely related to the A haplotype. Genomic features and the metabolic potential of LsoD are assessed in relation to Lso haplotypes A, B, and C, as well as the facultative strain Liberibacter crescens. We identify genes unique to haplotype D as well as putative secreted effectors that may play a role in disease characteristics specific to this haplotype of Lso.

Genome Sequence of "Candidatus Carsonella ruddii" Strain BT from the Psyllid Bactericera trigonica.

The genome of "Candidatus Carsonella ruddii" strain BT from Bactericera trigonica in Israel was sequenced. The full-length genome is 173,904 bp long and has a G+C content of 14.6%, with 224 predicted open reading frames (ORFs) and 30 RNAs.

Introduction of a putative biocontrol agent into a range of phytoplasma- and liberibacter-susceptible crop plants.

BACKGROUND: Phytoplasma, the causative agent of Bois Noir disease of grapevines, are vectored by the planthopper Hyalesthes obsoletus (Hemiptera: Cixiidae). A Dyella-like bacterium (DLB) isolated from H. obsoletus inhibits the growth of Spiroplasma melliferum, a cultivable relative of phytoplasma. Additional evidence suggests that DLB can reduce the symptoms of yellows disease in grapevine plantlets. The aim of this study was to test whether DLB could colonize a range of phytoplasma- and liberibacter-sensitive crop plants, and thus assess its potential agricultural use. RESULTS: Vitex agnus-castus, the preferred host plant of H. obsoletus was found to be a natural host of DLB, which was successfully introduced into a range of crop plants belonging to seven families. The most effective DLB application method was foliar spraying. Microscopy observation revealed that DLB aggregated on the leaf surface and around the stomata, suggesting that this is its route of entry. DLB was also present in the vascular tissues of plants, indicating that it moved systemically through the plant. CONCLUSIONS: DLB is a potential biocontrol agent and its broad spectrum of host plants indicates the possibility of its future use against a range of diseases caused by phloem-limited bacteria. © 2017 Society of Chemical Industry.