Xylella phage MATE 2: a novel bacteriophage with potent lytic activity against Xylella fastidiosa subsp. pauca.

Xylella fastidiosa (Xf) is a major phytosanitary threat to global agricultural production. The complexity and difficulty of controlling Xf underscore the pressing need for novel antibacterial agents, i.e., bacteriophages, which are natural predators of bacteria. In this study, a novel lytic bacteriophage of Xf subsp. pauca, namely Xylella phage MATE 2 (MATE 2), was isolated from sewage water in southern Italy. Biological characterization showed that MATE 2 possessed a broad-spectrum of antibacterial activity against various phytobacteria within the family Xanthomonadaceae, a rapid adsorption time (10 min), and high resistance to a broad range of pH (4-10) and temperatures (4-60°C). Most importantly, MATE 2 was able to suppress the growth of Xf subsp. pauca cells in liquid culture for 7 days, demonstrating its potential as an effective antibacterial agent against Xf. The genomic and electron microscopy analyses revealed that MATE 2 is a new species tentatively belonging to the genus Carpasinavirus within the class Caudoviricetes, with an isometric capsid head of 60 ± 5 nm along with a contractile tail of 120 ± 7.5 nm. Furthermore, the high-throughput sequencing and de novo assembly generated a single contig of 63,695 nucleotides in length; representing a complete genome composed of 95 Open Reading Frames. Bioinformatics analysis performed on MATE 2 genome revealed the absence of lysogenic mediated genes, and genes encoding virulence factors, antibiotic resistance, and toxins. This study adds a new phage to the very short list of Xf-infecting lytic phages, whose in-vitro antibacterial activity has been ascertained, while its efficacy on Xf-infected olive trees in the field has yet to be determined.

Nisin-based therapy: a realistic and eco-friendly biocontrol strategy to contrast Xylella fastidiosa subsp. pauca infections in planta.

The lack of sustainable strategies for combating Xylella fastidiosa (Xf) highlights the pressing need for novel practical antibacterial tools. In this study, Lactococcus lactis subsp. lactis strain ATCC 11454 (L. lactis), known for its production of nisin A, was in vitro tested against Xf subsp. pauca. Preliminary investigations showed that nisin A was involved in a strong antagonistic activity exhibited by L. lactis against Xf. Thus, the efficacy of nisin A was comprehensively assessed through a combination of in vitro and in planta experiments. In vitro investigations employing viable-quantitative PCR, spot assay, turbidity reduction assay, fluorescence microscopy, and transmission electron microscopy demonstrated nisin's robust bactericidal effect on Xf at a minimal lethal concentration of 0.6 mg/mL. Moreover, results from fluorescence and transmission electron microscopies indicated that nisin directly and rapidly interacts with the membranes of Xf cells, leading to the destruction of bacterial cells in few minutes. In in planta tests, nisin also demonstrated the ability to tackle Xf infections within Nicotiana benthamiana plants that remained asymptomatic 74 days post inoculation. Furthermore, RPLC-ESI-MS/MS analyses showed that nisin translocated to all parts of the plants and remains intact for up to 9 days. For the first time, this study underscores the nisin-based strategy as a realistic and eco-friendly approach to be further investigated against Xf infections in the field.

Zinc Oxide Nanoparticles Biosynthesized by Eriobotrya japonica Leaf Extract: Characterization, Insecticidal and Antibacterial Properties.

Zinc oxide nanoparticles (ZnO-NPs) have gained significant attention in nanotechnology due to their unique properties and potential applications in various fields, including insecticidal and antibacterial activities. The ZnO-NPs were biosynthesized by Eriobotrya japonica leaf extract and characterized by various techniques such as UV-visible (UV-vis) spectrophotometer, X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and zeta potential analysis. The results of SEM revealed that NPs were irregular and spherical-shaped, with a diameter between 5 and 27 nm. Meanwhile, DLS supported that the measured size distributions were 202.8 and 94.7 nm at 11.1° and 90.0°, respectively, which supported the polydisperse nature of NPs, and the corresponding zeta potential was -20.4 mV. The insecticidal activity of the produced ZnO-NPs was determined against the adult stage of coleopteran pests, Sitophilus oryzae (Linnaeus) (Curculionidae) and Tribolium castaneum (Herbst) (Tenebrionidae). The LC50 values of ZnO-NPs against adults of S. oryzae and T. castaneum at 24 h of exposure were 7125.35 and 5642.65 µg/mL, respectively, whereas the LC90 values were 121,824.56 and 66,825.76 µg/mL, respectively. Moreover, the biosynthesized nanoparticles exhibited antibacterial activity against three potato bacterial pathogens, and the size of the inhibition zone was concentration-dependent. The data showed that the inhibition zone size increased with an increase in the concentration of nanoparticles for all bacterial isolates tested. The highest inhibition zone was observed for Ralstonia solanacearum at a concentration of 5 µg/mL, followed by Pectobacterium atrosepticum and P. carotovorum. Eventually, ZnO-NPs could be successfully used as an influential agent in pest management programs against stored-product pests and potato bacterial diseases.

Exploring Active Peptides with Antimicrobial Activity In Planta against Xylella fastidiosa.

Xylella fastidiosa (Xf) is a xylem-limited quarantine plant bacterium and one of the most harmful agricultural pathogens across the world. Despite significant research efforts, neither a direct treatment nor an efficient strategy has yet been developed for combatting Xylella-associated diseases. Antimicrobial peptides (AMPs) have been gaining interest as a promising sustainable tool to control pathogens due to their unique mechanism of action, broad spectrum of activity, and low environmental impact. In this study, we disclose the bioactivity of nine AMPs reported in the literature to be efficient against human and plant pathogen bacteria, i.e., Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, against Xf, through in vitro and in vivo experiments. Based on viable-quantitative PCR (v-qPCR), fluorescence microscopy (FM), optical density (OD), and transmission electron microscopy (TEM) assays, peptides Ascaphin-8 (GF19), DASamP1 (FF13), and DASamP2 (IL14) demonstrated the highest bactericidal and antibiofilm activities and were more efficient than the peptide PB178 (KL29), reported as one of the most potent AMPs against Xf at present. Furthermore, these AMPs showed low to no toxicity when tested on eukaryotic cells. In in planta tests, no Xf disease symptoms were noticed in Nicotiana tabacum plants treated with the AMPs 40 days post inoculation. This study highlighted the high antagonistic activity of newly tested AMP candidates against Xf, which could lead to the development of promising eco-friendly management of Xf-related diseases.

Identification and Characterization of Erwinia Phage IT22: A New Bacteriophage-Based Biocontrol against Erwinia amylovora.

Erwinia amylovora is a quarantine phytopathogenic bacterium that is the causal agent of fire blight, a destructive disease responsible for killing millions of fruit-bearing plants worldwide, including apple, pear, quince, and raspberry. Efficient and sustainable control strategies for this serious bacterial disease are still lacking, and traditional methods are limited to the use of antibiotics and some basic agricultural practices. This study aimed to contribute to the development of a sustainable control strategy through the identification, characterization, and application of bacteriophages (phages) able to control fire blight on pears. Phages isolated from wastewater collected in the Apulia region (southern Italy) were characterized and evaluated as antibacterial agents to treat experimental fire blight caused by E. amylovora. Transmission electron microscopy (TEM) conducted on purified phages (named EP-IT22 for Erwinia phage IT22) showed particles with icosahedral heads of ca. 90 ± 5 nm in length and long contractile tails of 100 ± 10 nm, typical of the Myoviridae family. Whole genome sequencing (WGS), assembly, and analysis of the phage DNA generated a single contig of 174.346 bp representing a complete circular genome composed of 310 open reading frames (ORFs). EP-IT22 was found to be 98.48% identical to the Straboviridae Erwinia phage Cronus (EPC) (GenBank Acc. n° NC_055743) at the nucleotide level. EP-IT22 was found to be resistant to high temperatures (up to 60 °C) and pH values between 4 and 11, and was able to accomplish a complete lytic cycle within one hour. Furthermore, the viability-qPCR and turbidity assays showed that EP-IT22 (MOI = 1) lysed 94% of E. amylovora cells in 20 h. The antibacterial activity of EP-IT22 in planta was evaluated in E. amylovora-inoculated pear plants that remained asymptomatic 40 days post inoculation, similarly to those treated with streptomycin sulphate. This is the first description of the morphological, biological, and molecular features of EP-IT22, highlighting its promising potential for biocontrol of E. amylovora against fire blight disease.

Exploring Antimicrobial Peptides Efficacy against Fire Blight (Erwinia amylovora).

Antimicrobial peptides (AMPs) are a various group of molecules found in a wide range of organisms and act as a defense mechanism against different kinds of infectious pathogens (bacteria, viruses, and fungi, etc.). This study explored the antibacterial activity of nine candidates reported in the literature for their effect on human and animal bacteria, (i.e., Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) against Erwinia amylovora (E. amylovora), the causal agent of fire blight disease on pome fruits. The antibacterial activity of these peptides against E. amylovora was evaluated in vitro using viable-quantitative PCR (v-qPCR), fluorescence microscopy (FM), optical density (OD), and transmission electron microscopy (TEM), while the in vivo control efficacy was evaluated in treating experimental fire blight on pear fruits. With a view to their safe and ecofriendly field use in the future, the study also used animal and plant eukaryotic cells to evaluate the possible toxicity of these AMPs. Results in vitro showed that KL29 was the most potent peptide in inhibiting E. amylovora cell proliferation. In addition, the results of v-qPCR, FM, and TEM showed that KL29 has a bifunctional mechanism of action (lytic and non-lytic) when used at different concentrations against E. amylovora. KL29 reduced fire blight symptoms by 85% when applied experimentally in vivo. Furthermore, it had no impact on animal or plant cells, thus demonstrating its potential for safe use as an antibacterial agent. This study sheds light on a new and potent antibacterial peptide for E. amylovora and its modes of action, which could be exploited to develop sustainable treatments for fire blight.

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.

Development of an FTP-LAMP assay based on TaqMan real-time PCR and LAMP for the specific detection of Xylella fastidiosa De Donno and mulberry strains in both plants and insect vectors.

We developed two real-time detection assays, TaqMan real-time PCR and LAMP, using primers and probe designed based on a sequence annotated to code for a Haemagglutinin-related protein (Hg) of Xylella fastidiosa (Xf), a gene uniquely present in the Italian olive (De Donno of olive) and American mulberry strains, for specific detection of the target Xf strains. These assays were validated with DNA samples extracted from Xf-infected plant samples and from two species of insect vectors (Philaenus spumarius, Ps; and Neophilaenus campestris, Nc). Both techniques were proven to be highly sensitive (100 fg of Xf-genomic DNA) and specific to the Italian De Donno and American mulberry strains of Xf. When our LAMP was utilized in a duplex manner by combining with previously published universal primers and probe for detection of all Xf-subspecies and strains, the duplex LAMP showed high versatility in the simultaneous detection and differentiation of the Italian De Donno and American mulberry stains form other subspecies/strains. Furthermore, the Hg gene-specific LAMP primers and TaqMan probe were exploited to develop a new approach; henceforth referred to as the Fluorescence of TaqMan Probe upon Dequenching - Loop mediated Isothermal Amplification (FTP-LAMP). In the FTP-LAMP, the Xf-Hg specific fluorophore-quenched probe was added to a standard LAMP reaction and fluoresces only when bound to its target, allowing for a sequence-specific detection of the Xf-Italian De Donno and American mulberry strains in a LAMP context. Our FTP-LAMP assay showed to be highly sensitive detecting down to 100 fg genomic DNA of Xf, when tested on Xf-genomic DNA extracted from infected plants, DAS-ELISA-crude saps and insect vectors. Furthermore, the assay showed high specificity (98.7% vs 89% for LAMP) when applied on DNA templates from insect vectors. With the addition of an extra target sequence-specific probe acting as a direct Xf-specific dye, the FTP-LAMP has gained more specificity and reduced one of the main problems of the LAMP assay (false positives) when used for detecting of Xf in insect vectors. To the best of our knowledge, this study reports the development of the first LAMP assay and the first novel FTP-LAMP method for specific detection of the Italian De Donno and the American mulberry strains of Xf. Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP could represent a useful tool not only for the specific detection of the olive-associated strain in Italy, but also to differentiate the De Donno strain from other strains of Xf already reported in Italy and Europe (Germany, France, Spain and Portugal).

Seasonal Fluctuations of Sap-Feeding Insect Species Infected by Xylella fastidiosa in Apulian Olive Groves of Southern Italy.

A study on seasonal abundance of Auchenorrhyncha species and their infectivity by Xylella fastidiosa in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were monitored monthly over a 2-yr period from five olive groves. A total of 15 species were captured, identified, and tested for presence of X. fastidiosa by polymerase chain reaction (PCR). For three species, Philaenus spumarius L., Neophilaenus campestris (Fallèn), and Euscelis lineolatus Brullé, positive reactions to X. fastidiosa were obtained, on average, in 16.3, 15.9 and 18.4% of adult insects, respectively. Philaneous spumarius was the dominant species (39.8% of total Auchenorrhyncha captured) with the highest adult abundance in summer months. Adult P. spumarius and N. campestris were first detected between March and May in both years, and all insects tested during these periods (year 1: n = 42, year 2: n = 132) gave negative reactions to X. fastidiosa by PCR. Similarly, first adults of E. lineolatus that appeared from October to November (year 1: n = 20, year 2: n = 15) tested negative for presence of X. fastidiosa Given the lack of transstadial and transovarial transmission of X. fastidiosa and considering that P. spumarius is univoltine, control measures against nymphal stages of P. spumarius should be investigated as means of population suppression to reduce spread of X. fastidiosa in olive groves.

Identification of pigmented Serratia marcescens symbiotically associated with Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae).

To characterize red pigment-producing bacteria (RPPB) regularly released during oviposition by red palm weevil (RPW), RPPB were recovered from eggs deposited in apples supplied as substrate for oviposition. The presence of RPPB was also detected from gut, the reproductive apparatus of dissected adult and virgin insects and from pupal cases collected within infested palms. RPPB were also identified all along the tissue of these palms. Analysis of the 16S rDNA, gyrB, rpoB, recA, and groEL sequences assigned RPPB to the species Serratia marcescens. RPPB exhibited an antimicrobial activity assessed by the agar well diffusion method against a number of gram-positive and gram-negative bacteria. In this study, we first report the identification of a red pigment-producing S. marcescens as extracellular symbiont of RPW. Route of transmission, detection within different organs, and a wide spread along the infested palm tissue, suggested S. marcescens is present as extracellular symbiont in different developmental stages of the RPW. Additionally, the antimicrobial activity exhibited versus Bacillus spp., Paenibacillus spp., and Lysinibacillus spp., reported as insect pathogens and potential candidates for biocontrol agents, could ascribe for S. marcescens a potential protective role.