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.

Cellulose Nanocrystal-Based Emulsion of Thyme Essential Oil: Preparation and Characterisation as Sustainable Crop Protection Tool.

Essential oil-based pesticides, which contain antimicrobial and antioxidant molecules, have potential for use in sustainable agriculture. However, these compounds have limitations such as volatility, poor water solubility, and phytotoxicity. Nanoencapsulation, through processes like micro- and nanoemulsions, can enhance the stability and bioactivity of essential oils. In this study, thyme essential oil from supercritical carbon dioxide extraction was selected as a sustainable antimicrobial tool and nanoencapsulated in an oil-in-water emulsion system. The investigated protocol provided high-speed homogenisation in the presence of cellulose nanocrystals as stabilisers and calcium chloride as an ionic crosslinking agent. Thyme essential oil was characterised via GC-MS and UV-vis analysis, indicating rich content in phenols. The cellulose nanocrystal/essential oil ratio and calcium chloride concentration were varied to tune the nanoemulsions' physical-chemical stability, which was investigated via UV-vis, direct observation, dynamic light scattering, and Turbiscan analysis. Transmission electron microscopy confirmed the nanosized droplet formation. The nanoemulsion resulting from the addition of crosslinked nanocrystals was very stable over time at room temperature. It was evaluated for the first time on Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease. In vitro tests showed a synergistic effect of the formulation components, and in vivo tests on olive seedlings demonstrated reduced bacterial colonies without any phytotoxic effect. These findings suggest that crosslinked cellulose nanocrystal emulsions can enhance the stability and bioactivity of thyme essential oil, providing a new tool for crop protection.

Thymol-Nanoparticles as Effective Biocides against the Quarantine Pathogen Xylella fastidiosa.

Quarantine pathogens require the investigation of new tools for effective plant protection. In particular, research on sustainable agrochemicals is the actual challenge. Plant extracts, essential oils, and gels are natural sources of efficient biocides, such as aromatic secondary metabolites. Thymol is the major phenolic constituent of thyme and oregano essential oils, and it can inhibit many pathogenic microbes. Thymol nanoparticles were obtained through adsorption on CaCO3 nanocrystals, exploiting their carrier action. High loading efficiency and capability were reached as verified through UV and TGA measurements. We report the first study of thymol effect on Xylella fastidiosa, conducing both fluorometric assay and in vitro inhibition assay. The first test confirmed the great antibacterial effect of this compound. Finally, an in vitro test revealed an interesting synergistic action of thymol and nanocarriers, suggesting the potential application of thymol-nanoparticles as effective biocides to control Xylella fastidiosa infection.

Boosting Immunity and Management against Wheat Fusarium Diseases by a Sustainable, Circular Nanostructured Delivery Platform.

Fusarium head blight (FHB) and Fusarium crown rot (FCR) are managed by the application of imidazole fungicides, which will be strictly limited by 2030, as stated by the European Green Deal. Here, a novel and eco-sustainable nanostructured particle formulation (NPF) is presented by following the principles of the circular economy. Cellulose nanocrystals (CNC) and resistant starch were obtained from the bran of a high amylose (HA) bread wheat and employed as carrier and excipient, while chitosan and gallic acid were functionalized as antifungal and elicitor active principles. The NPF inhibited conidia germination and mycelium growth, and mechanically interacted with conidia. The NPF optimally reduced FHB and FCR symptoms in susceptible bread wheat genotypes while being biocompatible on plants. The expression level of 21 genes involved in the induction of innate immunity was investigated in Sumai3 (FHB resistant) Cadenza (susceptible) and Cadenza SBEIIa (a mutant characterized by high-amylose starch content) and most of them were up-regulated in Cadenza SBEIIa spikes treated with the NPF, indicating that this genotype may possess an interesting genomic background particularly responsive to elicitor-like molecules. Quantification of fungal biomass revealed that the NPF controlled FHB spread, while Cadenza SBEIIa was resistant to FCR fungal spread. The present research work highlights that the NPF is a powerful weapon for FHB sustainable management, while the genome of Cadenza SBEIIa should be investigated deeply as particularly responsive to elicitor-like molecules and resistant to FCR fungal spread.

Complete genome assembly of the levan-positive strain PVFi1 of Pseudomonas savastanoi pv. savastanoi isolated from olive knots in Central Italy.

Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease, is a fluorescent Gram-negative bacterium classified, according to the specific LOPAT profile, as Ib. However, during the 90s, a number of atypical non-fluorescent levan-positive strains of Pseudomonas savastanoi pv. savastanoi have been unexpectedly isolated from olive knots in Central Italy. Since its first report, several studies were conducted on this species variant, but its genome sequence has never been reported. The complete genome sequence and two additional plasmids of PVFi1, a representative strain, were here obtained using a hybrid sequencing approach with both Oxford Nanopore Technology and Illumina sequencing. A thorough genomic analysis unravelled several genetic features of this peculiar strain, showing a transposase insertion downstream a fragmented copy of the levansucrase gene. The same features were previously reported on levan-negative Pseudomonas savastanoi pv. savastanoi strains. In addition, a second copy of the levansucrase gene fully equipped for a gene expression and comparable to the levan-positive Pseudomonas savastanoi pv. glycinea, may explain the levan-positive test. This result provides a solid genetic demonstration that the bacterial species Pseudomonas savastanoi contains either levan-positive or levan-negative strains, providing insights for an update of the related LOPAT classification.

A Green Nanostructured Pesticide to Control Tomato Bacterial Speck Disease.

Bacterial speck disease, caused by Pseudomonas syringae pv. tomato (Pst), is one of the most pervasive biological adversities in tomato cultivation, in both industrial and in table varieties. In this work synthesis, biochemical and antibacterial properties of a novel organic nanostructured pesticide composed of chitosan hydrochloride (CH) as active ingredient, cellulose nanocrystals (CNC) as nanocarriers and starch as excipient were evaluated. In order to study the possibility of delivering CH, the effects of two different types of starches, extracted from a high amylose bread wheat (high amylose starch-HA Starch) and from a control genotype (standard starch-St Starch), were investigated. Nanostructured microparticles (NMP) were obtained through the spray-drying technique, revealing a CH loading capacity proximal to 50%, with a CH release of 30% for CH-CNC-St Starch NMP and 50% for CH-CNC-HA Starch NMP after 24 h. Both NMP were able to inhibit bacterial growth in vitro when used at 1% w/v. Moreover, no negative effects on vegetative growth were recorded when NMP were foliar applied on tomato plants. Proposed nanostructured pesticides showed the capability of diminishing Pst epiphytical survival during time, decreasing disease incidence and severity (from 45% to 49%), with results comparable to one of the most used cupric salt (hydroxide), pointing out the potential use of CH-CNC-Starch NMP as a sustainable and innovative ally in Pst control strategies.

UAV-Based Thermal, RGB Imaging and Gene Expression Analysis Allowed Detection of Fusarium Head Blight and Gave New Insights Into the Physiological Responses to the Disease in Durum Wheat.

Wheat is one of the world's most economically important cereal crop, grown on 220 million hectares. Fusarium head blight (FHB) disease is considered a major threat to durum (Triticum turgidum subsp. durum (Desfontaines) Husnache) and bread wheat (T. aestivum L.) cultivars and is mainly managed by the application of fungicides at anthesis. However, fungicides are applied when FHB symptoms are clearly visible and the spikes are almost entirely bleached (% of diseased spikelets > 80%), by when it is too late to control FHB disease. For this reason, farmers often react by performing repeated fungicide treatments that, however, due to the advanced state of the infection, cause a waste of money and pose significant risks to the environment and non-target organisms. In the present study, we used unmanned aerial vehicle (UAV)-based thermal infrared (TIR) and red-green-blue (RGB) imaging for FHB detection in T. turgidum (cv. Marco Aurelio) under natural field conditions. TIR and RGB data coupled with ground-based measurements such as spike's temperature, photosynthetic efficiency and molecular identification of FHB pathogens, detected FHB at anthesis half-way (Zadoks stage 65, ZS 65), when the percentage (%) of diseased spikelets ranged between 20% and 60%. Moreover, in greenhouse experiments the transcripts of the key genes involved in stomatal closure were mostly up-regulated in F. graminearum-inoculated plants, demonstrating that the physiological mechanism behind the spike's temperature increase and photosynthetic efficiency decrease could be attributed to the closure of the guard cells in response to F. graminearum. In addition, preliminary analysis revealed that there is differential regulation of genes between drought-stressed and F. graminearum-inoculated plants, suggesting that there might be a possibility to discriminate between water stress and FHB infection. This study shows the potential of UAV-based TIR and RGB imaging for field phenotyping of wheat and other cereal crop species in response to environmental stresses. This is anticipated to have enormous promise for the detection of FHB disease and tremendous implications for optimizing the application of fungicides, since global food crop demand is to be met with minimal environmental impacts.

Chitosan Hydrochloride Decreases Fusarium graminearum Growth and Virulence and Boosts Growth, Development and Systemic Acquired Resistance in Two Durum Wheat Genotypes.

Fusarium head blight (FHB) is a devastating disease for cereals. FHB is managed by fungicides at anthesis, but their efficacy is variable. Conventional fungicides accumulate in the soil and are dangerous for animal and human health. This study assayed the antifungal ability of chitosan hydrochloride against Fusarium graminearum. Chitosan reduced F. graminearum growth and downregulated the transcript of the major genes involved in the cell growth, respiration, virulence, and trichothecenes biosynthesis. Chitosan promoted the germination rate, the root and coleoptile development, and the nitrogen balance index in two durum wheat genotypes, Marco Aurelio (FHB-susceptible) and DBC480 (FHB-resistant). Chitosan reduced FHB severity when applied on spikes or on the flag leaves. FHB severity in DBC480 was of 6% at 21 dpi after chitosan treatments compared to F. graminearum inoculated control (20%). The elicitor-like property of chitosan was confirmed by the up-regulation of TaPAL, TaPR1 and TaPR2 (around 3-fold). Chitosan decreased the fungal spread and mycotoxins accumulation. This study demonstrated that the non-toxic chitosan is a powerful molecule with the potential to replace the conventional fungicides. The combination of a moderately resistant genotype (DBC480) with a sustainable compound (chitosan) will open new frontiers for the reduction of conventional compounds in agriculture.

A new inclusive MLVA assay to investigate genetic variability of Xylella fastidiosa with a specific focus on the Apulian outbreak in Italy.

The Olive Quick Decline Syndrome by Xylella fastidiosa subspecies pauca is among the most severe phytopathological emergencies nowadays. In few years, the outbreak devastated olive groves in Apulia (Italy), potentially endangering the entire Mediterranean basin. This research aimed to develop a multiple locus VNTR analysis assay, a molecular tool to differentiate between populations of the pathogen. It has already been successfully applied to different X. fastidiosa subspecies from various plant hosts. The previously published TR loci, together with a set of new design, have been tested in silico on the genome of the Apulian De Donno strain. The resulting selection of 37 TR loci was amplified on the genomic DNAs of the Apulian strains AND from representatives of X. fastidiosa subspecies, and directly on DNA extracted from infected plants. The assay clearly discerned among subspecies or even sequence types (ST), but also pointed out variants within the same ST so as to provide more detailed information on the dynamics and pathogen diffusion pathways. Its effective application even on total DNAs extracted from infected tissues of different host plants makes it particularly useful for large-scale screening of infection and for the strengthening of containment measures.

The modulation of stomatal conductance and photosynthetic parameters is involved in Fusarium head blight resistance in wheat.

Fusarium head blight (FHB) is one of the most devastating fungal diseases affecting grain crops and Fusarium graminearum is the most aggressive causal species. Several evidences shown that stomatal closure is involved in the first line of defence against plant pathogens. However, there is very little evidence to show that photosynthetic parameters change in inoculated plants. The aim of the present study was to study the role of stomatal regulation in wheat after F. graminearum inoculation and explore its possible involvement in FHB resistance. RT-qPCR revealed that genes involved in stomatal regulation are induced in the resistant Sumai3 cultivar but not in the susceptible Rebelde cultivar. Seven genes involved in the positive regulation of stomatal closure were up-regulated in Sumai3, but it is most likely, that two genes, TaBG and TaCYP450, involved in the negative regulation of stomatal closure, were strongly induced, suggesting that FHB response is linked to cross-talk between the genes promoting and inhibiting stomatal closure. Increasing temperature of spikes in the wheat genotypes and a decrease in photosynthetic efficiency in Rebelde but not in Sumai3, were observed, confirming the hypothesis that photosynthetic parameters are related to FHB resistance.

Cellulose nanocrystals from Actinidia deliciosa pruning residues combined with carvacrol in PVA_CH films with antioxidant/antimicrobial properties for packaging applications.

Kiwi Actinidia deliciosa pruning residues were here used for the first time as precursors for the extraction of high performing cellulose nanocrystals (CNC) by applying a bleaching treatment followed by an acidic hydrolysis. The resultant cellulosic nanostructures, obtained by an optimize extraction procedure (0.7% wt/v two times of sodium chlorite NaClO2) followed by an hydrolysis step, were then used as reinforcements phases in poly(vinyl alcohol) (PVA) blended with natural chitosan (CH) based films and also combined, for the first time, with carvacrol used here as active agent. Morphological and optical characteristics, mechanical response, thermal and migration properties, moisture content and antioxidant and antimicrobial assays were conducted. The morphological, optical and colorimetric results underlined that no particular alterations were induced on the transparency and color of PVA and PVA_CH blend by the presence of CNC and carvacrol, while they were able to modulate the mechanical responses, to induce antioxidant activities maintaining the migration levels below the permitted limits and suggesting the possible application in industrial sectors. Finally, inhibitions on bacterial development were detected for multifunctional systems, suggesting their protective function against microorganisms contamination.