Orthology-Based Estimate of the Contribution of Horizontal Gene Transfer from Distantly Related Bacteria to the Intraspecific Diversity and Differentiation of Xylella fastidiosa

Xylella fastidiosa is a xylem-limited bacterium phylogenetically related to the xanthomonads, with an unusually large and diversified range of plant hosts. To ascertain the origin of its peculiarities, its pan-genome was scanned to identify the genes that are not coherent with its phylogenetic position within the order Xanthomonadales. The results of the analysis revealed that a large fraction of the genes of the Xylella pan-genome have no ortholog or close paralog in the order Xanthomonadales. For a significant part of the genes, the closest homologue was found in bacteria belonging to distantly related taxonomic groups, most frequently in the Betaproteobacteria. Other species, such as Xanthomonas vasicola and Xanthomonas albilineans which were investigated for comparison, did not show a similar genetic contribution from distant branches of the prokaryotic tree of life. This finding indicates that the process of acquisition of DNA from the environment is still a relevant component of Xylella fastidiosa evolution. Although the ability of Xylella fastidiosa strains to recombine among themselves is well known, the results of the pan-genome analyses stressed the additional relevance of environmental DNA in shaping their genomes, with potential consequences on their phytopathological features

Convergent evolution of phytopathogenic pseudomonads onto hazelnut

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Xylella fastidiosa subsp. pauca, Neofusicoccum spp. and the decline of olive trees in salento (Apulia, Italy). Comparison of symptoms, possible interactions, certainties and doubts

Xylella fastidiosa subsp. pauca (XFP), Neofusicoccum mediterraneum, N. stellenboschiana and other fungi have been found in olive groves of Salento (Apulia, Italy) that show symptoms of severe decline. XFP is well known to be the cause of olive quick decline syndrome (OQDS). It has also been assessed that Neofusicoccum spp. causes a distinct disease syndrome, namely, branch and twig dieback (BTD). All these phytopathogens incite severe symptoms that can compromise the viability of large canopy sectors or the whole tree. However, their specific symptoms are not easily distinguished, especially during the final stages of the disease when branches are definitively desiccated. By contrast, they can be differentiated during the initial phases of the infection when some facets of the diseases are typical, especially wood discoloration, incited solely by fungi. Here, we describe the typical symptomatological features of OQDS and BTD that can be observed in the field and that have been confirmed by Koch postulate experiments. Similar symptoms, caused by some abiotic adverse conditions and even by additional biotic factors, are also described. Thus, this review aims at: (i) raising the awareness that declining olive trees in Salento do not have to be linked a priori to XFP; (ii) defining the guidelines for a correct symptomatic diagnosis to orient proper laboratory analyses, which is crucial for the application of effective control measures. The possibility that bacterium and fungi could act as a polyspecies and in conjunction with predisposing abiotic stresses is also widely discussed

1H-NMR Metabolomics Study after Foliar and Endo-Therapy Treatments of Xylella fastidiosa subsp. pauca Infected Olive Trees: Medium Time Monitoring of Field Experiments

Here we report the medium-term effects of foliar spray and endo-therapy treatments with different doses of a Cu/Zn citric acid biocomplex (Dentamet®) in Xylella fastidiosa infected olive trees of Salento, Apulia region (South-east Italy). Leaf extract samples from field-treated 150 years old olive trees cvs Ogliarola salentina and Cellina di Nardò were studied by 1H NMR-based metabolomics. The result of different applications of Dentamet® endo-therapy after 60, 120 and 180 days in comparison with traditional foliar spray treatment and water injection as a control have been investigated. The metabolic profile analyses, performed by 1H NMR-based metabolomic approach, indicated plant metabolites variations connected to the disease progression such as mannitol, quinic acid, and oleuropein related compounds. The best results, in terms of discrimination of the metabolic profiles with respect to water injection, were found for monthly endo-therapy treatments. Dentamet® foliar application demonstrated more specific time related progressive effectiveness with respect to intravascular treatments. Therefore, besides a possible more effective performance of endo-therapy with respect to foliar treatments, the need of further doses/frequencies trimming to obtain longterm results was also assessed. The present field studies confirmed the indication of Dentamet® effectiveness in metabolic variation induction, potentially linked with reducing the X. fastidiosa subspecies pauca related Olive Quick Decline Syndrome (OQDS) symptoms development

Identification and characterization of Neofusicoccum stellenboschiana in branch and twig dieback-affected olive trees in Italy and comparative pathogenicity with N. mediterraneum

For about a decade, olive groves in Apulia (Southern Italy) have been progressively destroyed by Olive Quick Decline Syndrome (OQDS), a disease caused by the bacterium Xylella fastidiosa subsp. pauca (Xfp). Recently, we described an additional wilting syndrome affecting olive trees in that area. The botryosphaeriaceous fungus Neofusicoccum mediterraneum was found associated with the diseased trees, and its high virulence toward olive trees was demonstrated. Given the common features with Branch and Twig Dieback (BTD) of olive tree, occurring in Spain and California, we suggested that the observed syndrome was BTD. During our first survey, we also found a botryosphaeriaceous species other than N. mediterraneum. In the present article, we report the morphological and molecular characterization of this fungal species which we identified as Neofusicoccum stellenboschiana. In the study, we also included for comparison additional N. stellenboschiana isolates obtained from olive trees in Latium and Tuscany region (Central Italy). The occurrence of N. stellenboschiana in olive trees is reported here for the first time in the northern hemisphere. The pathogenicity and virulence were tested in nine inoculation trials, where the Apulian N. stellenboschiana isolate was compared with the isolate from Latium and with the Apulian isolate of N. mediterraneum. Both isolates of N. stellenboschiana proved pathogenic to olive trees. They caused evident bark canker and wood discolouration when inoculated at the base of the stem of two/three-year-old trees and on one-year-old twigs. However, virulence of N. stellenboschiana was significantly lower, though still remarkable, compared with N. mediterraneum in term of necrosis rogression in the bark and the wood and capacity of wilting the twigs. Virulence of N. stellenboschiana and N. mediterraneum did not substantially change when noculations were performed in spring/summer and in autumn, suggesting that these fungal species have the potential to infect and damage olive trees in all seasons. The high thermotolerance of N. stellenboschiana was also revealed with in vitro growth and survival tests. The high virulence of these otryosphaeriaceae species highlights their contribution in BTD aetiology and the necessity to investigate right away their diffusion and, possibly, the role of additional factors other than Xfp in the general decline of olive groves in Apulia. Hence the importance of assessing the degree of overlap of BTD/Botryosphariaceae with OQDS/Xfp is discussed

Extensive remodeling of the Pseudomonas syringae pv. avellanae type III secretome associated with two independent host shifts onto hazelnut

BACKGROUND: Hazelnut (Corylus avellana) decline disease in Greece and Italy is caused by the convergent evolution of two distantly related lineages of Pseudomonas syringae pv. avellanae (Pav). We sequenced the genomes of three Pav isolates to determine if their convergent virulence phenotype had a common genetic basis due to either genetic exchange between lineages or parallel evolution. RESULTS: We found little evidence for horizontal transfer (recombination) of genes between Pav lineages, but two large genomic islands (GIs) have been recently acquired by one of the lineages. Evolutionary analyses of the genes encoding type III secreted effectors (T3SEs) that are translocated into host cells and are important for both suppressing and eliciting defense responses show that the two Pav lineages have dramatically different T3SE profiles, with only two shared putatively functional T3SEs. One Pav lineage has undergone unprecedented secretome remodeling, including the acquisition of eleven new T3SEs and the loss or pseudogenization of 15, including five of the six core T3SE families that are present in the other Pav lineage. Molecular dating indicates that divergence within both of the Pav lineages predates their observation in the field. This suggest that both Pav lineages have been cryptically infecting hazelnut trees or wild relatives for many years, and that the emergence of hazelnut decline in the 1970s may have been due to changes in agricultural practice. CONCLUSIONS: These data show that divergent lineages of P. syringae can converge on identical disease etiology on the same host plant using different virulence mechanisms and that dramatic shifts in the arsenal of T3SEs can accompany disease emergence

Net blotch (Pyrenophora teres Drechsler): An increasingly significant threat to barley production

Pyrenophora teres is a pathogen causing a net blotch disease in cultivated barley, which is present worldwide and can thus significantly reduce barley yields. This fungus also infects wild barley and other plants of the Hordeum genus, as well as barley grass, wheat, oats and plants from various genera, including Agropyron, Bromus, Elymus, Hordelymus and Stipa. Based on the symptoms it causes on the infected barley plants, the pathogen can be divided into two forms: P. teres f. teres, which causes net-like symptoms, and P. teres f. maculata, which causes blotchy symptoms. Infected seeds, stubble and plant debris, and volunteer and weed plants represent primary sources of pathogen inoculum. During the growing season, the pathogen enters a sexual stage, developing pseudothecia with asci and ascospores. This is followed by an asexual stage, during which conidiophores with conidia are formed. The conidial (anamorphic) stage is much more common, whereby conidia is a source of inoculum for secondary infection during the barley growing season. The first symptoms appear at the end of winter and the beginning of spring, often during the tilling phase. The most characteristic symptoms form on barley leaves. Frequently, symptoms of the net form can be mistaken for other diseases occurring on barley, making molecular analysis essential for accurate detection of P. teres, its forms, mating types and hybrids. Current net blotch control measures are based on the combined application of cultural, chemical and biological control methods and the selection of resistant varieties

Phylogeographic Analysis of Soft-Rot-Causing Pectobacterium spp. Strains Obtained from Cabbage in Serbia

The aim of this study was to establish a link between genetic diversity and the geographic origin of Pectobacterium strains belonging to three species—P. carotovorum, P. versatile, and P. odoriferum—isolated from cabbage in Serbia by comparing their sequences with those of strains sourced from different hosts and countries in Europe, Asia, and North America. Phylogeographic relatedness was reconstructed using the Templeton, Crandall, and Sing’s (TCS) haplotype network based on concatenated sequences of the housekeeping genes dnaX, icdA, mdh, and proA, while pairwise genetic distances were computed by applying the p-distance model. The obtained TCS haplotype networks indicated the existence of high intra-species genetic diversity among strains of all three species, as reflected in the 0.2–2.3%, 0.2–2.5%, and 0.1–1.7% genetic distance ranges obtained for P. carotovorum, P. versatile, and P. odoriferum, respectively. Five new haplotypes (denoted as HPc1–HPc5) were detected among cabbage strains of P. carotovorum, while one new haplotype was identified for both P. versatile (HPv1) and P. odoriferum (HPo1). None of the TCS haplotype networks provided evidence of significant correlation between geographic origin and the determined haplotypes, i.e., the infection origin. However, as haplotype network results are affected by the availability of sequencing data in public databases for the used genes and the number of analyzed strains, these findings may also be influenced by small sample size

In-Depth Characterization of Crown Gall Disease of Tobacco in Serbia

In August 2020, the unusual appearance of crown gall symptoms was observed on the tobacco plants (hybrid PVH2310) grown in fields in the Golubinci (Srem district, Serbia) locality. The causal agent isolated from galls located on tobacco roots formed circular, convex, and glistening light blue colonies, and then dark to olive-green-colored bacterial colonies on a semi-selective D1 medium. Molecular analysis based on multiplex PCR and multi-locus sequence analysis (MLSA) using concatenated sequences of the atpD, dnaK, glnA, and rpoB genes as well as 16S rRNA identified Serbian tobacco isolates such as Agrobacterium tumefaciens (biovar 1). Two duplex PCR methods confirmed the presence of the virD2 and virC genes in tobacco isolates. Pathogenicity tests performed on carrot discs and squash fruits resulted in tumor/gall formation after 12 to 16 days post inoculation, respectively. Pathogenicity was also confirmed on tobacco plants, where isolates caused tumor development 21−25 days after inoculation. API 50 CH generated results regarding the biochemical features of the Serbian tobacco isolates. As A. tumefaciens (biovar 1) as a cause of tobacco crown gall has previously been documented solely in Japan, there is presently no data on its wider occurrence. Therefore, this first detailed investigation of A. tumefaciens isolated from naturally infected tobacco in Serbia will contribute to a better understanding of it at the global level

Assessment of dietary exposure to some persistent organic pollutants in the Republic of Karakalpakstan of Uzbekistan.

A 1999 study heightened long-standing concerns over persistent organic pollutant contamination in the Aral Sea area, detecting elevated levels in breast milk and cord blood of women in Karakalpakstan (western Uzbekistan). These findings prompted a collaborative research study aimed at linking such human findings with evidence of food chain contamination in the area. An international team carried out analyses of organochlorine and organophosphate pesticides, polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) on samples of 12 foods commonly produced and consumed in Karakalpakstan. Analysis consistently detected long-lasting organochlorine pesticides and their metabolites in all foods of animal origin and in some vegetables such as onions and carrots--two low-cost components of many traditional dishes. Levels of PCBs were relatively low in all samples except fish. Analyses revealed high levels of PCDDs and PCDFs (together often termed "dioxins") in sheep fat, dairy cream, eggs, and edible cottonseed oil, among other foodstuffs. These findings indicate that food traditionally grown, sold, and consumed in Karakalpakstan is a major route of human exposure to several persistent toxic contaminants, including the most toxic of dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). Intake estimations demonstrate that consumption of even small amounts of locally grown food may expose consumers to dioxin levels that considerably exceed the monthly tolerable dioxin intake levels set by the World Health Organization. Data presented in this study allow a first assessment of the risk associated with the consumption of certain food products in Karakalpakstan and highlight a critical public health situation