Combat phytopathogenic bacteria employing Argirium-SUNCs: limits and perspectives.

Bacterial plant diseases are difficult to control as the durability of deployed control measures is thwarted by continuous and rapid changing of bacterial populations. Although application of copper compounds to plants is the most widespread and inexpensive control measure, it is often partially efficacious for the frequent appearance of copper-resistant bacterial strains and it is raising concerns for the harmful effects of copper on environment and human health. Consequently, European Community included copper compounds in the list of substances candidates for substitution. Nanotechnologies and the application of nanoparticles seem to respond to the need to find new very effective and durable measures. We believe that Argirium-SUNCs®, silver ultra nanoclusters with an average size of 1.79 nm and characterized by rare oxidative states (Ag2+/3+), represent a valid candidate as a nano-bactericide in the control of plant bacterial diseases. Respect to the many silver nanoparticles described in the literature, Argirium-SUNCs have many strengths due to the reproducibility of the synthesis method, the purity and the stability of the preparation, the very strong (less than 1 ppm) antimicrobial, and anti-biofilm activities. In this mini-review, we provide information on this nanomaterial and on the possible application in agriculture. KEY POINTS: • Argirium-SUNCs have strong antimicrobial activities against phytopathogenic bacteria. • Argirium-SUNCs are a possible plant protection product. • Argirium-SUNCs protect tomato plants against bacterial speck disease.

Chitosan-Coated Fosetyl-Al Nanocrystals efficacy on Nicotiana tabacum colonized by Xylella fastidiosa.

Xylella fastidiosa (Xf) is a quarantine plant pathogen capable of colonizing the xylem of a wide range of hosts. Currently, there is no cure able to eliminate the pathogen from a diseased plant, whereas several integrated strategies have been implemented for containing the spread of Xf. Nanotechnology represents an innovative strategy based on the possibility of maximizing the potential antibacterial activity by increasing the surface-to-volume ratio of nanoscale formulations. Nanoparticles based on Chitosan and/or Fosetyl-Al have shown different in vitro antibacterial efficacy against Xf subspecies fastidiosa (Xff) and pauca (Xfp). This work demonstrated the uptake of Chitosan-Coated Fosetyl-Al nanocrystals (CH-nanoFos) by roots and their localization in the stems and leaves of olea europaea plants. Additionally, the antibacterial activity of Fosetyl-Al, nano-Fosetyl, nano-chitosan, and Chitosan-Coated Fosetyl-Al nanocrystals (CH-nanoFos) was tested on Nicotiana tabacum cv. SR1 (Petite Havana) inoculated with Xff, Xfp, or Xf subsp. multiplex (Xfm). The bacterial load was evaluated with qPCR, and the results showed that CH-nanoFos was the only treatment able of reducing the colonization of Xff, Xfm, and Xfp in tobacco plants. Additionally, the Area Under Disease Progress Curve (AUDPC), used to assess symptoms development in tobacco plants inoculated with Xff, Xfm, and Xfp and treated with CH-nanoFos, showed a reduction in symptom development. Furthermore, the twitching assay and bacterial growth under microfluidic conditions confirmed the antibacterial activity of CH-nanoFos.

Silver nanoclusters with Ag2+/3+ oxidative states are a new highly effective tool against phytopathogenic bacteria.

The main measure worldwide adopted to manage plant bacterial diseases is based on the application of copper compounds, which are often partially efficacious for the frequent appearance of copper-resistant bacterial strains and have raised concerns for their toxicity to the environment and humans. Therefore, there is an increasing need to develop new environmentally friendly, efficient, and reliable strategies for controlling plant bacterial diseases, and among them, the use of nanoparticles seems promising. The present study aimed to evaluate the feasibility of protecting plants against attacks of gram-negative and gram-positive phytopathogenic bacteria by using electrochemically synthesized silver ultra nanoclusters (ARGIRIUM­SUNCs®) with an average size of 1.79 nm and characterized by rare oxidative states (Ag2+/3+). ARGIRIUM­SUNCs strongly inhibited the in vitro growth (effective concentration, EC50, less than 1 ppm) and biofilm formation of Pseudomonas syringae pv. tomato and of quarantine bacteria Xanthomonas vesicatoria, Xylella fastidiosa subsp. pauca, and Clavibacter michiganensis subsp. michiganensis. In addition, treatments with ARGIRIUM­SUNCs also provoked the eradication of biofilm for P. syringae pv. tomato, X. vesicatoria, and C. michiganensis subsp. michiganensis. Treatment of tomato plants via root absorption with ARGIRIUM­SUNCs (10 ppm) is not phytotoxic and protected (80%) the plants against P. syringae pv. tomato attacks. ARGIRIUM­SUNCs at low doses induced hormetic effects on P. syringae pv. tomato, X. vesicatoria, and C. michiganensis subsp. michiganensis as well as on tomato root growth. The use of ARGIRIUM­SUNCs in protecting plants against phytopathogenic bacteria is a possible alternative control measure. KEY POINTS: • ARGIRIUM­SUNC has strong antimicrobial activities against phytopathogenic bacteria; • ARGIRIUM­SUNC inhibits biofilm formation at low doses; • ARGIRIUM­SUNC protects tomato plants against bacterial speck disease.

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.

Fungal and bacterial oxylipins are signals for intra- and inter-cellular communication within plant disease.

Lipids are central at various stages of host-pathogen interactions in determining virulence and modulating plant defense. Free fatty acids may act as substrates for oxidizing enzymes [e.g., lipoxygenases (LOXs) and dioxygenases (DOXs)] that synthesize oxylipins. Fatty acids and oxylipins function as modulators of several pathways in cell-to-cell communication; their structural similarity among plant, fungal, and bacterial taxa suggests potential in cross-kingdom communication. We provide a prospect of the known role of fatty acids and oxylipins in fungi and bacteria during plant-pathogen interactions. In the pathogens, oxylipin-mediated signaling pathways are crucial both in development and host infection. Here, we report on case studies suggesting that oxylipins derived from oleic, linoleic, and linolenic acids are crucial in modulating the pathogenic lifestyle in the host plant. Intriguingly, overlapping (fungi-plant/bacteria-plant) results suggest that different inter-kingdom pathosystems use similar lipid signals to reshape the lifestyle of the contenders and occasionally determine the outcome of the challenge.

Intra-Laboratory Evaluation of DNA Extraction Methods and Assessment of a Droplet Digital PCR for the Detection of Xanthomonas citri pv. citri on Different Citrus Species

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca), causal agents of citrus bacterial canker, are both regulated by the European Union to prevent their introduction. Xcc is responsible for severe outbreaks of citrus production worldwide, therefore, a prompt and reliable detection is advisable for the early detection of this bacterium either in symptomatic or asymptomatic plant material. The current EPPO (European and Mediterranean Plant Protection Organization) diagnostic protocol, PM 7/44(1), includes several diagnostic tests even if new assays have been developed in the latter years for which validation data are needed. Recently, a test performance study was organized within the Valitest EU Project to validate Xcc diagnostic methods and provide evidence on the most reliable assays; however, the influence of DNA extraction methods (DEM) on the reliability of the detection has never been assessed. In this study we evaluate four different DEM, by following two different approaches: (i) a comparison by real-time PCR standard curves of bacterial DNA versus bacterial DNA added to plant DNA (lemon, leaves and fruit; orange fruit); and (ii) the evaluation of performance criteria of spiked samples (plant extract added with ten-fold diluted bacterial suspensions at known concentrations). Droplet digital PCR is developed and compared with real-time PCR, as the detection method.

Mass Spectrometry-Based Targeted Lipidomics and Supervised Machine Learning Algorithms in Detecting Disease, Cultivar, and Treatment Biomarkers in Xylella fastidiosa subsp. pauca-Infected Olive Trees.

In 2013, Xylella fastidiosa (Xf) was detected for the first time in Apulia and, subsequently, recognized as the causal agent of the olive quick decline syndrome (OQDS). To contain the disease, the olive germplasm was evaluated for resistance to Xf, identifying cultivars with different susceptibility to the pathogen. Regarding this, the resistant cultivar Leccino has generally a lower bacterial titer compared with the susceptible cultivar Ogliarola salentina. Among biomolecules, lipids could have a pivotal role in the interaction of Xf with its host. In the grapevine Pierce's disease, fatty acid molecules, the diffusible signaling factors (DSFs), act as regulators of Xf lifestyle and are crucial for its virulence. Other lipid compounds derived from fatty acid oxidation, namely, oxylipins, can affect, in vitro, biofilm formation in Xf subsp. pauca (Xfp) strain De Donno, that is, the strain causing OQDS. In this study, we combined high-performance liquid chromatography-mass spectrometry-MS-based targeted lipidomics with supervised learning algorithms (random forest, support vector machine, and neural networks) to classify olive tree samples from Salento. The dataset included samples from either OQDS-positive or OQDS-negative olive trees belonging either to cultivar Ogliarola salentina or Leccino treated or not with the zinc-copper-citric acid biocomplex Dentamet®. We built classifiers using the relative differences in lipid species able to discriminate olive tree samples, namely, (1) infected and non-infected, (2) belonging to different cultivars, and (3) treated or untreated with Dentamet®. Lipid entities emerging as predictors of the thesis are free fatty acids (C16:1, C18:1, C18:2, C18:3); the LOX-derived oxylipins 9- and 13-HPOD/TrE; the DOX-derived oxylipin 10-HPOME; and diacylglyceride DAG36:4(18:1/18:3).

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.

Further In Vitro Assessment and Mid-Term Evaluation of Control Strategy of Xylella fastidiosa subsp. pauca in Olive Groves of Salento (Apulia, Italy).

During recent years; Xylella fastidiosa subsp. pauca (Xfp) has spread in Salento causing relevant damage to the olive groves. Measures to contain the spreading of the pathogen include the monitoring of the areas bordering the so-called "infected" zone and the tree eradication in case of positive detection. In order to provide a control strategy aimed to maintain the tree productivity in the infected areas, we further evaluated the in vitro and in planta mid-term effectiveness of a zinc-copper-citric acid biocomplex. The compound showed an in vitro bactericidal activity and inhibited the biofilm formation in representative strains of X. fastidiosa subspecies, including Xfp isolated in Apulia from olive trees. The field mid-term evaluation of the control strategy assessed by quantitative real-time PCR in 41 trees of two olive groves of the "infected" area revealed a low concentration of Xfp over the seasons upon the regular spraying of the biocomplex over 3 or 4 consecutive years. In particular, the bacterial concentration lowered in July and October with respect to March, after six consecutive treatments. The trend was not affected by the cultivar and it was similar either in the Xfp-sensitive cultivars Ogliarola salentina and Cellina di Nardò or in the Xfp-resistant Leccino. Moreover, the scoring of the number of wilted twigs over the seasons confirmed the trend. The efficacy of the treatment in the management of olive groves subjected to a high pathogen pressure is highlighted by the yielded a good oil production.

Antibacterial Activity of Essential Oils and Trametes versicolor Extract against Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum for Seed Treatment and Development of a Rapid In Vivo Assay.

Clavibacter michiganensis subsp. michiganensis (Smith) Davis et al. (Cmm) and Ralstonia solanacearum Yabuuchi et al. (Smith) (Rs) are important seed-borne bacterial pathogens of tomato (Solanum lycopersicum) listed as A2 pests in the EPPO (European and Mediterranean Plant Protection Organization) region. At present, there are few strategies to control these pathogens, and seed control with eco-compatible approaches is widely encouraged. In this work, the essential oils (EOs) of oregano (Origanum vulgare), garlic (Allium sativum), basil (Ocimum basilicum), cinnamon (Cinnamomum zeylanicum), clove buds (Syzygium aromaticum), thyme (Thymus vulgaris), and Trametes versicolor extract (Tve) were tested in vitro for their antimicrobial activity against Cmm and Rs (broth microdilution method). The tested EOs and the Tve extract caused a significant inhibition of bacterial growth, with very promising MBC (minimum bactericidal concentration) and MIC90 (minimum inhibitory concentration causing a 90% growth inhibition) values. Moreover, an in vivo germination test showed no major reduction in seed germination when the substances were applied as seed treatment. A rapid molecular screening method has been developed, through real-time PCR, for the specific quantification of Cmm in the presence of a vegetable matrix to test in vivo the antimicrobial efficacy of oregano and cinnamon oil on seed treatment without resorting to whole plant essays, which are time- and space-consuming.

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

Recently, 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 two Xylella fastidiosa subspecies. 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 against Xylella fastidiosa subsp. pauca and Xylella fastidiosa subsp. fastidiosa.

Xylella fastidiosa subsp. pauca and olive produced lipids moderate the switch adhesive versus non-adhesive state and viceversa.

Global trade and climate change are re-shaping the distribution map of pandemic pathogens. One major emerging concern is Xylella fastidiosa, a tropical bacterium recently introduced into Europe from America. In last decades, X. fastidiosa was detected in several European countries. X. fastidiosa is an insect vector-transmitted bacterial plant pathogen associated with severe diseases in a wide range of hosts. X. fastidiosa through a tight coordination of the adherent biofilm and the planktonic states, invades the host systemically. The planktonic phase is correlated to low cell density and vessel colonization. Increase in cell density triggers a quorum sensing system based on mixture of cis 2-enoic fatty acids-diffusible signalling factors (DSF) that promote stickiness and biofilm. The lipidome profile of Olea europaea L. (cv. Ogliarola salentina) samples, collected in groves located in infected zones and uninfected zones was performed. The untargeted analysis of the lipid profiles of Olive Quick Decline Syndrome (OQDS) positive (+) and negative (-) plants showed a clustering of OQDS+ plants apart from OQDS-. The targeted lipids profile of plants OQDS+ and OQDS- identified a shortlist of 10 lipids that increase their amount in OQDS+ and X. fastidiosa positive olive trees. These lipid entities, provided to X. fastidiosa subsp. pauca pure culture, impact on the dual phase, e.g. planktonic ↔ biofilm. This study provides novel insights on OQDS lipid hallmarks and on molecules that might modulate biofilm phase in X. fastidiosa subsp. pauca.

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.

The diagnosis of plant pathogenic bacteria: a state of art.

Plant protection plays an important role in agriculture for the food quality and quantity. The diagnosis of plant diseases and the identification of the pathogens are essential prerequisites for their understanding and control. Among the plant pests, the bacterial pathogens have devastating effects on plant productivity and yield. Different techniques (microscopy, serology, biochemical, physiological, molecular tools and culture propagation) are currently used to detect and identify bacterial pathogens. Detection and identification are critical steps for the appropriate application of phytosanitary measures. The "harmonization of phytosanitary regulations and all other areas of official plant protection action" mean the good practices for plant protection and plant material certification. The prevention of diseases progression and spread by early detection are a valuable strategy for proper pest management and disease control. For this purpose, innovative methods aim achieving results within a shorter time and higher performance, to provide rapidly, accurately and reliably diagnosis. In this review, we focus on the techniques for plant bacterial diagnosis and on the regulations for harmonizing plant protection issue.

Pseudomonas syringae pv. coryli, the Causal Agent of Bacterial Twig Dieback of Corylus avellana.

ABSTRACT Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.