Diagnosis, detection and identification of Pseudomonas syringae pv. actinidiae

Success story of the Euphresco project 'Pseudomonas syringae pv. actinidiae (PSA): diagnosis, detection, identification and study of epidemiological aspects

Lipid profile of Xylella fastidiosa Subsp. pauca associated with the olive quick decline syndrome

Lipids, components of the plasma and intracellular membranes as well as of droplets, provide different biological functions related to energy, carbon storage, and stress responses. Bacterial species display diverse membrane composition that changes in response to the different environmental conditions. During plant-pathogen interactions, lipids might have roles in several aspects such as recognition, signal transduction, and downstream responses. Among lipid entities, free fatty acids (FFAs) and their oxidized form, the oxylipins, represent an important class of signaling molecules in host-pathogen perception, especially related to virulence and defense. In bacteria, FFAs (e.g., diffusible signaling factors) and oxylipins have a crucial role in modulating motility, biofilm formation, and virulence. In this study, we explore by LC-TOF and LC-MS/MS the lipid composition of Xylella fastidiosa subsp. pauca strain De Donno in pure culture; some specific lipids (e.g., ornithine lipids and the oxylipin 7,10-diHOME), characteristic of other pathogenic bacteria, were revealed. Nicotiana tabacum was used for testing the ability of this pathogen in producing such lipids in the host. Different lipid compounds present a clear distribution pattern within the infected plant tissues compared to the uninfected ones

Sonication-Assisted Production of Fosetyl-Al Nanocrystals: Investigation of Human Toxicity and In Vitro Antibacterial Efficacy against Xylella Fastidiosa

none10Recently, there is a growing demand in sustainable phytopathogens control research. Nanotechnology provides several tools such as new pesticides formulations, antibacterial nanomaterials and smart delivery systems. Metal nano-oxides and different biopolymers have been exploited in order to develop nanopesticides which can offer a targeted solution minimizing side effects on environment and human health. This work proposed a nanotechnological approach to obtain a new formulation of systemic fungicide fosetyl-Al employing ultrasonication assisted production of water dispersible nanocrystals. Moreover, chitosan was applicated as a coating agent aiming a synergistic antimicrobial effect between biopolymer and fungicide. Fosetyl-Al nanocrystals have been characterized by morphological and physical-chemical analysis. Nanotoxicological investigation was carried out on human keratinocytes cells through cells viability test and ultrastructural analysis. In vitro planktonic growth, biofilm production and agar dilution assays have been conducted on twoXylella fastidiosasubspecies. Fosetyl-Al nanocrystals resulted very stable over time and less toxic respect to conventional formulation. Finally, chitosan-based fosetyl-Al nanocrystals showed an interesting antibacterial activity againstXylella fastidiosasubsp.paucaandXylella fastidiosasubsp.fastidiosa.Regione Puglia, Deliberazione della Giunta Regionale n. 1410 del 12/06/2015 “Linee guida per il parco della ricerca e sperimentazione finalizzata alla prevenzione e al contenimento del complesso del disseccamento rapido dell’olivo (CODIRO)” Project FATA- “Trattamenti fitoterapici innovativi a base di vettori di chitosano” MIPAAFT, Project Oli.Di.X.I.It-“OLIvicoltura e Difesa da Xylella fastidiosa e da Insetti vettori in Italia”, D.M. 23773 del 6/09/2017.openFrancesca Baldassarre; Giuseppe Tatulli; Viviana Vergaro; Stefania Mariano; Valeria Scala; Concetta Nobile; Nicoletta Pucci; Luciana Dini; Stefania Loreti; Giuseppe CiccarellaBaldassarre, Francesca; Tatulli, Giuseppe; Vergaro, Viviana; Mariano, Stefania; Scala, Valeria; Nobile, Concetta; Pucci, Nicoletta; Dini, Luciana; Loreti, Stefania; Ciccarella, Giusepp

Detection of Pseudomonas syringae pv. actinidiae, causal agent of bacterial canker of kiwifruit, from symptomless fruits and twigs, and from pollen

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker of kiwifruit, was monitored in symptomless fruits, twigs and pollen of the host using bacterial isolation and DNA-extraction followed by two PCR-assays (direct-PCRs). A procedure for Psa detection from symptomless twigs was established. Out of 16 symptomless twigs samples, Psa was detected in 12 samples by isolation and 13 samples by direct-PCR. Thirteen pollen samples were treated using two different procedures; Psa was detected in eight samples by isolation and ten samples by direct-PCR. By washing 108 samples of fruits, Psa was detected by isolation in only two samples, collected from severely affected orchards. However, one of these samples contained wilted fruits, whereas for the other, only one colony was isolate. From 60 bulk-samples of fruits, endophytic Psa was detected in six samples by isolation and ten samples by direct-PCRs. A Psa-positive bulk-sample of fruits was analyzed separately as individual fruits: there was a faint signal in five or seven fruits out of 50 depending on the PCR assay used. Isolation was negative for these samples. Presence of the pathogen on bulk-fruit samples could be due to low amounts of inoculum distributed over many fruits: as a consequence, there is a negligible risk of introducing the pathogen into countries free of bacterial canker. This integrated approach (isolation plus PCR) is proposed as a tool for the analysis of symptomless kiwifruit material for the presence of Psa.

The Italian inter-laboratory study on the detection of Pseudomonas syringae pv. actinide

A severe form of bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), has been detected in all the main areas of cultivation of kiwifruit (Actinidia deliciosa and A. chinensis). Since 2010 several research groups have been assessing methods and procedures to detect and identify Psa, both from symptomatic and symptomless host material. In 2011, a study to compare Psa diagnostic methods was performed with reference to Psa strains and related pathovars, and with plant extracts or DNA obtained from healthy and naturally infected leaves, pollen or wood. The study revealed the strengths and the weaknesses of the assessed methods. The procedure included screening tests for Psa detection and for identification of Psa colonies. The methods assessed were bacterial isolation on generic and semi-selective media, PCR analysis (single, duplex and rep-PCR assay, the latter for identification only). The results highlighted the best performance of semi-selective with respect the generic media; the usefulness of the direct-PCR as screening tests for Psa detection; and the greater specificity of duplex-PCR and sensitivity of simple-PCR. The use of semi-selective medium for isolation and of two PCR-based methods - in parallel - for Psa detection are suggested. Both rep-PCR and duplex-PCR, were found to be specific, and are recommended as an identification test for this pathogen

Analysis of Italian isolates of Pantoea stewartii subsp. stewartii and development of a real-time PCR-based diagnostic method

Pantoea stewartii subsp. stewartii (Pss) causes Stewart’s vascular wilt of maize, and it is responsible for serious crop losses. Pss is indigenous to North America and spreads with maize seeds. The presence of Pss has been notified in Italy since 2015. The risk assessment of the entry of Pss in the EU from the United States through seed trade is in the order of magnitude of hundred introductions per year. Several molecular or serological tests were developed for the detection of Pss and used as official analysis for the certification of commercial seeds. However, some of these tests lack adequate specificity, not allowing to correctly discriminate Pss from P. stewartii subsp. indologenes (Psi). Psi is occasionally present in maize seeds and is avirulent for maize. In this study, several Italian isolates of Pss recovered in 2015 and 2018 have been characterized by molecular, biochemical, and pathogenicity tests; moreover, their genomes have been assembled through MinION and Illumina–sequencing procedures. Genomic analysis reveals multiple introgression events. Exploiting these results, a new primer combination has been defined and verified by real-time PCR, allowing the development of a specific molecular test able to detect the presence of Pss down to the concentration of 103  CFU/ml in spiked samples of maize seed extracts. Due to the high analytical sensitivity and specificity achieved with this test, the detection of Pss has been improved disentangling the inconclusive results in Pss maize seed diagnosis, overcoming its misidentification in place of Psi. Altogether, this test addresses the critical issue associated with maize seeds imported from regions where Stewart’s disease is endemic

Challenges of Prevention for a Sustainable Personalized Medicine

The development and implementation of the approaches of personalized medicine for disease prevention are still at infancy, although preventive activities in healthcare represent a key pillar to guarantee health system sustainability. There is an increasing interest in finding informative markers that indicate the disease risk before the manifestation of the disease (primary prevention) or for early disease detection (secondary prevention). Recently, the systematic collection and study of clinical phenotypes and biomarkers consented to the advance of Rehabilomics in tertiary prevention. It consents to identify relevant molecular and physiological factors that can be linked to plasticity, treatment response, and natural recovery. Implementation of these approaches would open avenues to identify people at high risk and enable new preventive lifestyle interventions or early treatments targeted to their individual genomic profile, personalizing prevention and rehabilitation. The integration of personalized medicine into prevention may benefit citizens, patients, healthcare professionals, healthcare authorities, and industry, and ultimately will seek to contribute to better health and quality of life for Europe\u2019s citizens

Progress towards Sustainable Control of Xylella fastidiosa subsp. pauca in Olive Groves of Salento (Apulia, Italy)

Xylella fastidiosa subsp. pauca is the causal agent of "olive quick decline syndrome" in Salento (Apulia, Italy). On April 2015, we started interdisciplinary studies to provide a sustainable control strategy for this pathogen that threatens the multi-millennial olive agroecosystem of Salento. Confocal laser scanning microscopy and fluorescence quantification showed that a zinc-copper-citric acid biocomplex-Dentamet®-reached the olive xylem tissue either after the spraying of the canopy or injection into the trunk, demonstrating its effective systemicity. The biocomplex showed in vitro bactericidal activity towards all X. fastidiosa subspecies. A mid-term evaluation of the control strategy performed in some olive groves of Salento indicated that this biocomplex significantly reduced both the symptoms and X. f. subsp. pauca cell concentration within the leaves of the local cultivars Ogliarola salentina and Cellina di Nardò. The treated trees started again to yield. A 1H-NMR metabolomic approach revealed, upon the treatments, a consistent increase in malic acid and γ-aminobutyrate for Ogliarola salentina and Cellina di Nardò trees, respectively. A novel endotherapy technique allowed injection of Dentamet® at low pressure directly into the vascular system of the tree and is currently under study for the promotion of resprouting in severely attacked trees. There are currently more than 700 ha of olive groves in Salento where this strategy is being applied to control X. f. subsp. pauca. These results collectively demonstrate an efficient, simple, low-cost, and environmentally sustainable strategy to control this pathogen in Salento

A zinc, copper and citric acid biocomplex shows promise for control of Xylella fastidiosa subsp. pauca in olive trees in Apulia region (southern Italy)

The bacterium Xylella fastidiosa subsp. pauca is associated with the “olive quick decline syndrome” in the Apulia region of southern Italy. To investigate control of this phytopathogen, a compound containing zinc and copper complexed with citric-acid hydracids (Dentamet®) was evaluated for in vitro and in planta bactericidal activity. Confocal laser scanning microscopy, fluorescent quantification and atomic emission spectroscopy were then used to determine if the compound reached the xylem networks of leaves, twigs and branches of olive, to release zinc and copper within the xylem. A 3-year field trial in an olive orchard containing mature Cellina di Nardò and Ogliarola salentina olive trees, and officially declared infected by X. fastidiosa subsp. pauca,was also carried out o to determine if the compound affected severity of the disease. Each year, from early April to October (excluding July and August), six spray treatments of 0.5% (v:v) Dentamet® were applied on the olive tree crowns. The compound reduced severity of symptoms in both cultivars. Most untreated trees died by the end of the trial, whereas all treated trees survived with good vegetative status as assessed by a normalized difference vegetation index. Quantitative real-time PCR was performed from June 2016 to September 2017, following the official procedures established by the European and Mediterranean Plant Protection Organization. The analysis revealed a statistically significant reduction of X. fastidiosa cell densities within the leaves of treated trees. These promising results suggest that integrated management to reduce severity of X. fastidiosa that includes regular pruning and soil harrowing with spring and summer spray treatments with Dentamet®, is likely to effectively control the disease.