Clavibacter Research Articles

A host shift as the origin of tomato bacterial canker caused by Clavibacter michiganensis.

The Actinomycetota (formerly Actinobacteria) genus Clavibacter includes phytopathogens with devasting effects in several crops. Clavibacter michiganensis, the causal agent of tomato bacterial canker, is the most notorious species of the genus. Yet, its origin and natural reservoirs remain elusive, and its populations show pathogenicity profiles with unpredictable plant disease outcomes. Here, we generate and analyse a decade-long genomic dataset of Clavibacter from wild and commercial tomato cultivars, providing evolutionary insights that directed phenotypic characterization. Our phylogeny situates the last common ancestor of C. michiganensis next to Clavibacter isolates from grasses rather than to the sole strain we could isolate from wild tomatoes. Pathogenicity profiling of C. michiganensis isolates, together with C. phaseoli and C. californiensis as sister taxa and the wild tomato strain, was found to be congruent with the proposed phylogenetic relationships. We then identified gene enrichment after the evolutionary event, leading to the appearance of the C. michiganesis clade, including known pathogenicity factors but also hitherto unnoticed genes with the ability to encode adaptive traits for a pathogenic lifestyle. The holistic perspective provided by our evolutionary analyses hints towards a host shift event as the origin of C. michiganensis as a tomato pathogen and the existence of pathogenic genes that remain to be characterized.

Production of bacteriocin-like inhibitory substance (BLIS) by Staphylococcus spp. isolates from dogs.

In the present study, 39 canine isolates of Staphylococcus spp. were tested for antimicrobial substance (AMS) production. Seven AMS producers were identified, whose products exhibited a non-acidic character and a proteinaceous nature, therefore being considered bacteriocin-like inhibitory substances (BLIS). The producer strains of BLIS P1, P16 and I3 showed a broad spectrum of antimicrobial activity. Human, veterinary and plant pathogens, such as Listeria monocytogenes, Klebsiella pneumoniae, Staphylococcus spp. and Clavibacter michiganensis, were among the inhibited micro-organisms, suggesting the potential biotechnological application of these peptides. MALDI-TOF mass spectrometry and 16S rDNA sequencing identified the producer strains of BLIS P1, P16 and I3 as Staphylococcus pseudintermedius P1, Staphylococcus schleiferi P16 and Staphylococcus pseudintermedius I3. The plasmid profile of these strains suggests that the BLIS production is linked to biosynthetic genes located on plasmids. PCR analyses revealed that BLIS P1, P16 and I3 are different from 11 staphylococcins already described in the literature and that their genomic DNAs do not carry the most prevalent staphylococcal enterotoxin genes. The highest levels of BLIS production were achieved after 18-24h of growth of the producer strains in TSB medium. Moreover, BLIS P1 and I3 exhibited high resistance to temperature and pH variations, and BLIS P16 maintained 100% of its activity in almost all conditions tested. The characteristics associated with BLIS P1, P16 and I3 described in this work encourage further investigation of these substances, in addition to this study being the first report of BLIS production by a strain of S. schleiferi.

Advancing sustainable agriculture: Metal-doped urea–hydroxyapatite hybrid nanofertilizer for agro-industry

Abstract Nanotechnology holds excessive potential for addressing agricultural challenges such as soil deprivation, nutrient deficiencies, low harvests, and nutrient leaching. Nanofertilizers enable more efficient nutrient absorption by plants due to their enlarged surface area, bestowing viable solutions. Urea–hydroxyapatite hybrid (urea–HA hybrid) was successfully synthesized via a coprecipitation approach by doping nanohydroxyapatite with copper and zinc along with urea. The synthesized nanohybrids were analyzed by applying various techniques such as Fourier transform infrared spectroscopy, energy-dispersive spectroscopy (EDS), scanning electron microscopy, and X-ray powder diffraction (XRD). The evidence for the crystalline structure of HA was confirmed by peaks present in XRD analysis at 25.89°, 28.77°, and 32.11°, while urea was validated at 39.29°. The nanosized HA hexagonal nanorods were approximately 16 ± 1.5 nm, with the incorporation of urea, Cu, and Zn. The components of urea–HA hybrid (Ca, P, C, O, and N) were confirmed by EDS analysis with traces of Si. Antibacterial and antifungal activities were investigated against phytopathogenic microbes. The nanohybrid significantly inhibits the growth of Clavibacter michiganensis, Xanthomonas campestris, Macrophomina phaseolina, and Sclerotium rolfsii. A fertilization trial using urea–HA hybrid on Citrus limon has demonstrated a growth of 30 cm within 8 weeks of treatment, accompanied by brighter-colored leaves. Thus, the synthesized urea–HA hybrid enabled the slow release of nutrients, which had a significant impact on plant growth and will also effectively manage disease control against phytopathogens. Thus, this innovative approach addresses agricultural challenges regarding nutrient delivery and disease control more effectively.

Induction of systemic defense responses in tomato against Clavibacter michiganensis by application of aerated compost tea

Induction of systemic defense responses in tomato against Clavibacter michiganensis by application of aerated compost tea

Выявление патогенов картофеля с использованием различных методов диагностики

Abstract. Potato diseases have far reaching implications for the agricultural industry as they not only reduce productivity and quality of potato crop but also negatively affect the livelihood of farmers. The purpose of the studies detection and identification of potato phytopathogens by visual assessment, real time polymerase chain reaction (PCR) and molecular genetic screening for phytopathogen resistance genes. Methods. Visual evaluation of each potato variety for major fungal, viral and bacterial diseases was carried out during the period of full sprouting, at plant height of 15–20 cm and in the phase of full flowering. The percentage of plants affected by diseases in relation to the total number of inspected plants was calculated. Diagnosis of resistance of potato samples to diseases was carried out on the basis of PCR. DNA markers of phytopathogen resistance genes YES3-3A, RYSC3, Ry186, TG 689, Gro1-4-1, Gpa2-2, PVX were used for molecular screening of potato. Infection of samples by PCR-RV (real-time) was determined using reagent kits of Syntol LLC Potato Virus X and Potato Virus Y, Potato spindle tuber viroid-PB, Clavibacter michiganensis subsp. sepedonicus-PB, Dickeya spp-PB. Results. According to the results of visual evaluation, the absence of symptoms of wrinkle mosaic in 20% of hybrids, mottle mosaic in 40%, mosaic leaf curl in 96 %, leaf curl in 72 hybrids, black leg in 100%, phytophthorosis in 88% of hybrids, alternaria in 84 %, rhizoctoniosis in 96 % of hybrids were observed. According to the results of PCR-RV analysis in 21 samples detected virus Y. The scientific novelty consists in the diagnosis of potato viruses using traditional visual assessment with the use of molecular markers that allow to identify correlation relationship between trhis methods.

Phytochemical Study of Ethanol Extract of Gnaphalium uliginosum L. and Evaluation of Its Antimicrobial Activity.

This study evaluates the antibacterial and antifungal effects of ethanol extracts from Gnaphalium uliginosum L. derived from freshly harvested plant biomass, including stems, leaves, flowers, and roots. The extract was analyzed using gas chromatography-mass spectrometry (GC-MS) to determine its antimicrobial activity against phytopathogenic bacteria and fungi. Two methods were used in the experiments: agar well diffusion and double serial dilution. Extraction was carried out using the maceration method with different temperature regimes (25 °C, 45 °C, and 75 °C) and the ultrasonic method at various powers (63-352 W) for different durations (5 and 10 min). It was found that the 70% ethanol extract obtained through the ultrasonic experiment at 189 W power for 10 min and at 252 W power for 5 min had the highest antimicrobial activity compared to the maceration method. The most sensitive components of the extracts were the Gram-positive phytopathogenic bacteria Clavibacter michiganensis and the Gram-negative phytopathogenic bacteria Erwinia carotovora spp., with MIC values of 156 μg/mL. Among the fungi, the most sensitive were Rhizoctonia solani and Alternaria solani (MIC values in the range of 78-156 µg/mL). The evaluation of the antimicrobial activity of extracts using the diffusion method established the presence of a growth suppression zone in the case of C. michiganensis (15-17 mm for flowers, leaves, and total biomass), which corresponds to the average level of antimicrobial activity. These findings suggest that G. uliginosum has potential as a source of biologically active compounds for agricultural use, particularly for developing novel biopesticides.

First report of bacterial wilt caused by Clavibacter michiganensis subsp. michiganensis affecting tomato in Iğdır

Tomato wilt disease caused by Clavibacter mihiganensis subsp. michiganensis (Cmm) is one of the most destructive tomato diseases and causes significant crop loss in both greenhouse and field tomato production areas worldwide. In this study, the presence of the causal agent of bacterial wilt disease in tomato plants was investigated in Aras Valley. Isolation was made from diseased plant samples and it was determined whether the strains were pathogenic by cellulase activity and HR test. The virulence, morphological and biochemical characteristics of the strains were determined. Strains that fatty acid methyl ester extraction, isolation and purification were performed were identified at species and subspecies level with % similarity index using gas chromatography system. The diagnosis was confirmed with the Biolog Gen III System and all strains were identified at the subspecies level with a % similarity index. As a result of this study, 57 strains were obtained in the isolation, and 39 of the strains were determined not to be pathogenic. Strain 18 was determined as the pathogen causing the most damage to tomato plants with 100 % disease severity. Strains were identified as Cmm at subspecies level with a similarity index of 71-87 % using gas chromatography system and 54-75 % similarity index with Biolog Gen III System. According to the heat map created, it was determined that the strains consisted of two main clusters. The presence of pathogen in Aras Valley was proved for the first time by this study.

Nanobiological synthesis of silver oxide-doped titanium oxide bionanocomposite targeting foodborne and phytopathogenic bacteria

Fungi possess remarkable capabilities for metal speciation, dissolution, and mineral formation, which contribute to the production of mycogenic nanostructures. This study explores a green chemistry approach for synthesizing silver oxide-doped titanium oxide (Ag2O-doped TiO2) bionanocomposite utilizing Trichoderma virens. The light yellowish fungal filtrate transformed into a dark brownish colloidal suspension after reacting with silver nitrate and rutile titanium (IV) oxide. X-ray diffractometry (XRD) unveiled the crystalline structure of Ag2O-doped TiO2 bionanocomposite (22.15 nm), showing the coexistence of cubic and rutile tetragonal phases of Ag2O and TiO2, respectively. Fourier-transform infrared spectroscopy (FTIR) showed the presence of functional groups of alcohols, phenols nitro compounds, and aromatic amines derived from the cultural filtrate of T. virens. Raman analysis revealed vibrational modes corresponding to Ag2O and TiO2 nanoparticles. Distinct sharp emission peaks characteristic to Ti, Ag, and O were depicted using energy dispersive X-ray analysis (EDX) analysis. X-ray photoelectron spectroscopy (XPS) confirmed the presence of elemental valence states and binding energies of Ag, Ti, and O in the mycogenic nanocomposite. Field emission scanning electron microscopy (FESEM) revealed aggregation of polydispersed Ag2O-doped TiO2 bionanocomposite, displaying spherical- and cuboctahedron-shaped nanostructures with rough surfaces. High-resolution transmission electron microscopy (HRTEM) showed the presence of circular-, semi-spherical-, hexagonal-, and polygonal-shaped monodispersed Ag2O NPs, with defined boundaries. The Ag2O NPs were obviously deposited on the sheet-like TiO2 NPs. Selected area electron diffraction pattern implied the polycrystallinity of the as-synthesized bionanocomposite. A broad antibacterial spectrum of the prepared bionanocomposite was attained against foodborne pathogenic bacteria; Escherichia coli (12.05 mm), Salmonella enterica (11.26 mm), and Staphylococcus aureus (11.44 mm) and phytopathogenic bacteria; Clavibacter michiganensis subsp. michiganensis (15.72 mm), C. michiganensis subsp. capsici (10.80 mm), streptomycin-sensitive and -resistant Xanthomonas citri pv. citri (14.11 and 14.53 mm, respectively), and streptomycin-sensitive and -resistant Pectobacterium carotovorum subsp. carotovorum (11.36 and 11.07 mm, respectively) using in vitro Kirby-Bauer method. Minimum inhibitory and minimum bactericidal concentrations were determined via a broth micro-dilution assay. FESEM revealed significant morphological alterations in bacterial cells upon treatment with the Ag2O-doped TiO2 bionanocomposite, including deformed shape, rough surface, cell thinning, wrinkled cell wall, protrusions, cavitations, and cracks. These findings signify the successful mycosynthesis of Ag2O-doped TiO2 bionanocomposite, which acted a potent antibacterial agent against a variety of foodborne and phytopathogenic bacteria, which could be employed for environmental, biomedical, agricultural, food bio-processing applications.

Allelic variations in the chpG effector gene within Clavibacter michiganensis populations determine pathogen host range.

Plant pathogenic bacteria often have a narrow host range, which can vary among different isolates within a population. Here, we investigated the host range of the tomato pathogen Clavibacter michiganensis (Cm). We determined the genome sequences of 40 tomato Cm isolates and screened them for pathogenicity on tomato and eggplant. Our screen revealed that out of the tested isolates, five were unable to cause disease on any of the hosts, 33 were exclusively pathogenic on tomato, and two were capable of infecting both tomato and eggplant. Through comparative genomic analyses, we identified that the five non-pathogenic isolates lacked the chp/tomA pathogenicity island, which has previously been associated with virulence in tomato. In addition, we found that the two eggplant-pathogenic isolates encode a unique allelic variant of the putative serine hydrolase chpG (chpGC), an effector that is recognized in eggplant. Introduction of chpGC into a chpG inactivation mutant in the eggplant-non-pathogenic strain Cm101, failed to complement the mutant, which retained its ability to cause disease in eggplant and failed to elicit hypersensitive response (HR). Conversely, introduction of the chpG variant from Cm101 into an eggplant pathogenic Cm isolate (C48), eliminated its pathogenicity on eggplant, and enabled C48 to elicit HR. Our study demonstrates that allelic variation in the chpG effector gene is a key determinant of host range plasticity within Cm populations.

A New Multiplex TaqMan qPCR for Precise Detection and Quantification of Clavibacter michiganensis in Seeds and Plant Tissue.

Bacterial canker of tomato caused by Clavibacter michiganensis (Cm) is one of the most devastating bacterial diseases affecting the tomato industry worldwide. As the result of Cm colonization of the xylem, the susceptible host shows typical symptoms of wilt, marginal leaf necrosis, stem cankers, and ultimately plant death. However, what makes Cm an even more dangerous pathogen is its ability to infect seeds and plants without causing symptoms. Unfortunately, there are no resistant cultivars or effective chemical or biological control methods available to growers against Cm. Its control relies heavily on prevention. The implementation of a rapid and accurate detection tool is imperative to monitor the presence of Cm and prevent its spread. In this study, we developed a specific and sensitive multiplex TaqMan qPCR assay to detect Cm and distinguish it from related bacterial species that affect tomato plants. Two Cm chromosomal virulence-related genes, rhuM and tomA, were used as specific targets. The plant internal control tubulin alpha-3 was included in each of the multiplexes to improve the reliability of the assay. Specificity was evaluated with 37 bacterial strains including other Clavibacter spp. and related and unrelated bacterial pathogens from different geographic locations affecting a wide variety of hosts. Results showed that the assay is able to discriminate Cm strains from other related bacteria. The assay was validated on tissue and seed samples following artificial infection, and all tested samples accurately detected the presence of Cm. The tool described here is highly specific, sensitive, and reliable for the detection of Cm and allows the quantification of Cm in seeds, roots, stems, and leaves. The diagnostic assay can also be adapted for multiple purposes such as seed certification programs, surveillance, biosafety, the effectiveness of control methods, border protection, and epidemiological studies.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Whole-genome sequence of a novel lytic bacteriophage infecting Clavibacter michiganensis subsp. michiganensis from Turkey.

Clavibacter michiganensis subsp. michiganensis (Cmm) is an important plant-pathogenic bacterium that causes canker and wilt diseases. Biological control of the disease with bacteriophages is an alternative to conventional methods. In this study, Phage33 infecting Cmm was characterized based on morphological and genomic properties. Morphological characteristics such as shape and size were investigated using electron microscopy. The whole genome was sequenced using the Illumina Novaseq 6000 platform and the sequence was assembled and annotated. VICTOR and VIRIDIC were used for determining the phylogeny and comparing viral genomes, respectively. Electron microscopy showed that Phage33 has an icosahedral head with a diameter of ~55 nm and a long, thin, non-contractile tail ~169 nm in length. The genome of Phage33 is 56 324 bp in size, has a GC content of 62.49 % and encodes 67 open reading frames. Thirty-seven ORFs showed high homology to functionally annotated bacteriophage proteins in the NCBI database. The remaining 30 ORFs were identified as hypothetical with unknown functions. The genome contains no antimicrobial resistance, no lysogenicity and no virulence signatures, suggesting that it is a suitable candidate for biocontrol agents. The results of a blastn search showed similarity to the previously reported Xylella phage Sano, with an average nucleotide sequence identity of 92.37 % and query coverage of 91 %. This result was verified using VICTOR and VIRIDIC analysis, and suggests that Phage33 is a new member of the genus Sanovirus under the class Caudoviricetes.

Exopolysaccharides of lactic acid bacteria as protective agents against bacterial and viral plant pathogens

In this study, 25 exopolysaccharides produced by lactic acid bacteria (LAB) were screened for their effect on plant pathogens. The molecular masses of EPS were found to be 3,8-5,0 × 104 Da. The GC–MS analysis revealed that EPSs were majorly composed of glucose (85.85–97.98 %). The FT-IR spectra of EPSs were in agreement with the typical absorption peaks of polysaccharides. EPSs showed a hydroxyl radical scavenging ability. The scavenging rate of EPS ranged from 20 to 50 % at a concentration of 5.0 mg/mL. Significant growth delay of phytopathogenic bacteria was observed after 3–6 h of cultivation. Optical density values of indicator cultures growing in the medium with EPS (1 mg/mL) were lower compared to the control by 24–100 % for Pseudomonas fluorescens, 9–46 % for P. syringae, 47–79 % for Pectobacterium carotovorum, 14–90 % for Clavibacter michiganensis, 9–100 % for Xantomonas campestris, and 45–100 % for X. vesicatorium. EPS retained their inhibitory effect on the growth of X. campestris, X. vesicatorium and C. michiganensis strains after 24–48 h of cultivation, but stimulating effect on the growth of some strains also was observed. LAB EPS showed antibiofilm activity against P. carotovorum, P. syringae, and P. fluorescent, decreasing their biofilm formation by 16–50 %, 14–39 %, and 29–59 %, respectively. Also, stimulation of biofilm formation by X. campestris (by 8–29 %), X. vesicatorium (by 3–32 %) and C. michiganensis (by 31–41 %) strains was observed. EPSs showed antiviral activity against tobacco mosaic virus (TMV). At a concentration of 100 μg/mL, they decreased the infective ability of TMV by 61–92 %. This is the first study demonstrating that LAB EPS exhibited in vitro antibacterial and antibiofilm activity against phytopathogenic bacteria and anti-viral activity against TMV. Thus, LAB EPSs could have great potential for plant protection strategies.

Management of Tomato Bacterial Canker Disease by the Green Fabricated Silver Nanoparticles

Bacterial canker disease caused by Clavibacter michiganensis is a substantial threat to the cultivation of tomatoes, leading to considerable economic losses and global food insecurity. Infection is characterized by white raised lesions on leaves, stem, and fruits with yellow to tan patches between veins, and marginal necrosis. Several agrochemical substances have been reported in previous studies to manage this disease but these were not ecofriendly. Thus present study was designed to control the bacterial canker disease in tomato using green fabricated silver nanoparticles (AgNps). Nanosilver particles (AgNPs) were synthesized utilizing Moringa oleifera leaf extract as a reducing and stabilizing agent. Synthesized AgNPs were characterized using UV–visible spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), energy-dispersive X-ray (EDX), and Fourier transform infrared spectrometry (FTIR). FTIR showed presence of bioactive compounds in green fabricated AgNPs and UV-visible spectroscopy confirmed the surface plasmon resonance (SPR) band in the range of 350 nm to 355 nm. SEM showed the rectangular segments fused together, and XRD confirmed the crystalline nature of the synthesized AgNPs. The presence of metallic silver ions was confirmed by an EDX detector. Different concentrations (10, 20, 30, and 40 ppm) of the green fabricated AgNPs were exogenously applied on tomato before applying an inoculum of Clavibacter michigensis to record the bacterial canker disease incidence at different day intervals. The optimal concentration of AgNPs was found to be 30 µg/mg that exhibited the most favorable impact on morphological (shoot length, root length, plant fresh and dry weights, root fresh and dry weights) and physiological parameters (chlorophyll contents, membrane stability index, and relative water content) as well as biochemical parameters (proline, total soluble sugar and catalase activity). These findings indicated a noteworthy reduction in biotic stress through the increase of both enzymatic and non-enzymatic activities by the green fabricated AgNPs. This study marks a first biocompatible approach in assessing the potential of green fabricated AgNPs in enhancing the well-being of tomato plants that affected with bacterial canker and establishing an effective management strategy against Clavibacter michiganensis. This is the first study suggests that low concentration of green fabricated nanosilvers (AgNPs) from leaf extract of Moringa oleifera against Clavibacter michiganensis is a promisingly efficient and eco-friendly alternative approach for management of bacterial canker disease in tomato crop.

Biologically inspired nanoformulations for the control of bacterial canker pathogens Clavibacter michiganensis subsp. michiganensis and subsp. capsici

Clavibacter michiganensis subsp. michiganensis (Cmm) and C. michiganensis subsp. capsici (Cmc) are phytopathogenic bacteria that cause bacterial canker disease in tomatoes and peppers, respectively. Bacterial canker disease poses serious challenges to solanaceous crops, causing significant yield losses and economic costs. Effective management necessitates the development of sustainable control strategies employing nanobiotechnology. In this study, the antibacterial effects of four Aspergillus sojae-mediated nanoformulations, including cobalt oxide nanoparticles (Co3O4 NPs), zinc oxide nanoparticles (ZnO NPs), cobalt ferrite nanoparticles (CoFe2O4 NPs), and CoFe2O4/functionalized multi-walled carbon nanotube (fMWCNT) bionanocomposite, were evaluated against Cmm and Cmc. The diameters of the zone of inhibition of A. sojae-mediated Co3O4 NPs, ZnO NPs, CoFe2O4 NPs, and CoFe2O4/fMWCNT bionanocomposite against Cmm and Cmc were 23.60 mm, 22.09 mm, 27.65 mm, 22.51 mm, and 19.33 mm, 17.66 mm, 21.64 mm, 18.77 mm, respectively. The broth microdilution assay was conducted to determine the minimal inhibitory and bactericidal concentrations. The MICs of Co3O4 NPs, ZnO NPs, CoFe2O4 NPs, and CoFe2O4/fMWCNT bionanocomposite against Cmm were 2.50 mg/mL, 1.25 mg/mL, 2.50 mg/mL, and 2.50 mg/mL, respectively. While, their respective MBCs against Cmm were 5.00 mg/mL, 2.50 mg/mL, 5.00 mg/mL, and 5.00 mg/mL. The respective MICs of Co3O4 NPs, ZnO NPs, CoFe2O4 NPs, and CoFe2O4/fMWCNT bionanocomposite against Cmc were 2.50 mg/mL, 1.25 mg/mL, 5.00 mg/mL, and 5.00 mg/mL. While, their respective MBCs against Cmc were 5.00 mg/mL, 2.50 mg/mL, 10.00 mg/mL, and 10.00 mg/mL. The morphological and ultrastructural changes of Cmm and Cmc cells were observed using field-emission scanning and transmission electron microscopy before and after treatment with sub-minimal inhibitory concentrations of the nanoformulations. Nanoformulation-treated bacterial cells became deformed and disrupted, displaying pits, deep cavities, and groove-like structures. The cell membrane detached from the bacterial cell wall, electron-dense particles accumulated in the cytoplasm, cellular components disintegrated, and the cells were lysed. Direct physical interactions between the prepared nanoformulations with Cmm and Cmc cells might be the major mechanism for their antibacterial potency. Further research is required for the in vivo application of the mycosynthesized nanoformulations as countermeasures to combat bacterial phytopathogens.

Delving into Chemical Control Options for Bacterial Canker (Clavibacter michiganensis subsp. michiganensis) in Tomatoes: An In-vitro Study

The 2022/2023 tomato crop witnessed the emergence of Clavibacter michiganensis subsp. michiganensis in a tomato field, leading to symptoms resembling bacterial canker. Identification of the suspected bacterium, C. michiganensis subsp. michiganensis, utilized specific primers (CMM5 and CMM6) for PCR reaction, resulting in a 614 bp fragment. Several fungicides and bactericides were tested for their ability to control bacterial growth in Petri dishes. Fungicides and bactericides that completely inhibit the bacterial growth in Petri dishes included benzalkonium chloride (250 mg a.i./L), copper oxychloride (1680 mg a.i./L with 1000 mg metallic copper/L), copper hydroxide (2764 mg a.i./L with 1800 mg metallic copper/L), fluazinam (500 µL a.i./L), difenoconazole + pidiflumetofen (200 + 120 µL a.i./L), cuprous oxide (1344 mg a.i./L with 1200 mg metallic copper /L), mancozeb + famoxadone (1000 + 100 mg a.i./L), mancozeb (4000 mg a.i./L) and metiram + pyraclostrobin (2200 + 200 mg a.i./L). The packaged dose of casugamycin (60 µL a.i./L) failed to completely inhibit C. michiganensis subsp. michiganensis growth, necessitating doses exceeding 140 µL a.i./L for complete inhibition. Only at a dosage of 140 µL a.i./L was there no observable growth on the Petri dish containing YDC. Label doses of casugamycin did not prevent the growth of any bacteria, albeit partially controlling Clavibacter and Pectobacterium populations. At the dose of 140 µL a.i./L, the sole bacterium that proliferated was Xanthomonas hortorum pv. gardneri. The other bacteria were included in this study focusing on Clavibacter solely to understand the effect of certain products on other important bacteria in tomato cultivation. The active ingredients, difenoconazole + pidiflumetofen (200 + 120 µL/L active ingredient) and fluazinam (500 µL/L active ingredient) effectively suppressed C. michiganensis subsp. michiganensis growth. The study indicates that various tested fungicides and bactericides were effective in curbing C. michiganensis subsp. michiganensis growth under laboratory conditions. Nonetheless, efficacy may fluctuate based on dose and specific product used. Further research, including field trials, is imperative to evaluate product efficacy under real-world conditions and devise comprehensive management strategies for tomato bacterial canker control.

Bacteriostatic Activity of Janthinobacterium lividum and Purified Violacein Fraction against Clavibacter michiganensis

Clavibacter michiganensis causes plant diseases and is included in the list of microorganisms subject to export control. Janthinobacterium lividum is capable of synthesizing a pigment with antagonistic potential. The purpose of the study was to evaluate the activity of J. lividum VKM B-3705D and the pigment fraction against C. michiganensis VKM Ac-1402. The results of spectrophotometric and nuclear magnetic resonance analysis showed that the pigment synthesized by the J. lividum VKM B-3705D corresponds to violacein. The J. lividum strain demonstrated potential bacteriostatic activity against C. michiganensis VKM Ac-1402 when both strains were co-cultured. Compared to the control (DMSO), the violacein solution suppressed the specific growth of Clavibacter by 57.7%. The mechanism of suppression of the growth of Clavibacter is discussed. One of the ways to suppress the growth of C. michiganensis may be the inhibition of key enzymes. Violacein inhibited the activity of adenosine triphosphatase (ATPase, EC 3.6.1.3) compared to the control (DMSO) by 23.2%. Thus, the current study of the bacteriostatic effect may be a decisive step towards the development of a plant protection product.

Mycofabrication of bimetal oxide nanoparticles (CuO/TiO2) using the endophytic fungus Trichoderma virens: Material properties and microbiocidal effects against bacterial pathogens

Bimetallic oxide nanoparticles (NPs) have gained the interest of researchers owing to the synergistic mechanism of action by the elements present. Mycosynthesis of nanostructures is a promising, facile, and an environmentally friendly approach. In the present study, CuO/TiO2 NPs were produced using the cell-free filtrate of the endophytic fungus Trichoderma virens. The reaction of the fungal filtrate with the two metal salts, CuSO4.5H2O and TiO2, resulted in the mycosynthesis of the CuO/TiO2 NPs. The prepared bimetal oxide NPs were characterized using XRD, FTIR spectroscopy, EDX, DLS, zeta potential analysis, XPS, Raman scattering, FESEM, and HRTEM. The mycosynthesized CuO/TiO2 NPs exhibited diffraction peaks characteristic of the monoclinic CuO and tetragonal rutile TiO2 crystal planes. Homogeneity and stability were observed as a result of the biological moieties present in the fungal filtrate, which capped the CuO/TiO2 NPs. The prepared CuO/TiO2 NPs were distributed as flake/plate-like and quasi-spherical/nanorod CuO and TiO2 NPs, respectively. A broad-spectrum antibacterial effect was recorded, with zones of inhibition as follows: foodborne pathogens Escherichia coli (14.34 mm), Salmonella enterica serovar Typhimurium (11.32 mm), and Staphylococcus aureus (9.63 mm); and phytopathogenic bacteria involving Clavibacter michiganensis subsp. michiganensis (Cmm, 12.16 mm), C. michiganensis subsp. capsici (Cmc, 11.26 mm), wild type and streptomycin-resistant Pectobacterium carotovorum subsp. carotovorum (Pcc, 11.06 and 12.37 mm, respectively), and wild type and streptomycin-resistant Xanthomonas citri pv citri (Xcc, 12.48 and 12.69 mm, respectively). The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of the CuO/TiO2 NPs were determined using the broth microdilution assay. NP-treated bacterial cells at sub-MICs had rough surfaces with membrane defects and exhibited shrinkage, reduction of cell edges, cracks, and fractures on their surfaces. This study provides insights into the potential of the mycosynthesized CuO/TiO2 NPs as alternative nano-antimicrobial agents, which could be employed for future biomedical, agricultural, and pharmaceutical applications.

A novel virulence gene, cviA1 of Clavibacter michiganensis for necrosis development in the Nicotiana benthamiana plant

Clavibacter michiganensis is a Gram-positive bacterium that causes diverse disease symptoms in tomatoes and Nicotiana benthamiana, a surrogate host plant, including canker, blister lesions, and wilting. Previously, we reported that C. michiganensis also causes necrosis in N. benthamiana leaves. Here, to identify novel virulence genes of C. michiganensis required for necrosis development in N. benthamiana leaves, we screened 1,862 transposon-inserted mutants and identified a mutant strain that exhibited weak and delayed necrosis, whereas there was no discernible difference in blister lesions, canker, or wilting symptoms. Notably, this mutant caused canker similar to that of the wild-type strain, but caused mild wilting in tomato. This mutant carried a transposon in a chromosomal gene, called Clavibactervirulence gene A1 (cviA1). CviA1 encodes a 180-amino acid protein with a signal peptide (SP) at the N-terminus and two putative transmembrane domains (TMs) at the C-terminus. Interestingly, deletion of the SP or the C-terminus, including the two putative TMs, in CviA1 failed to restore full necrosis in the mutant, highlighting the importance of protein secretion and the putative TMs for necrosis. A paralog of cviA1, cviA2 is located on the large plasmid pCM2 of C. michiganensis. Despite its high similarity to cviA1, the introduction of cviA2 into the cviA1 mutant strain did not restore virulence. Similarly, the introduction of cviA1 into the Clavibacter capsici type strain PF008, which initially lacks cviA1, did not enhance necrosis symptoms. These results reveals that the chromosomal cviA1 gene in C. michiganensis plays an important role in necrosis development in N. benthamiana leaves.

Phosphate solubilizing Pseudomonas and Bacillus combined with rock phosphates promoting tomato growth and reducing bacterial canker disease.

Nowadays, sustainable agriculture approaches are based on the use of biofertilizers and biopesticides. Tomato (Solanum lycopersicum L.) rhizosphere could provide rhizobacteria with biofertilizing and biopesticide properties. In this study, bacteria from the rhizosphere of tomato were evaluated in vitro for plant growth promotion (PGP) properties. Five Pseudomonas isolates (PsT-04c, PsT-94s, PsT-116, PsT-124, and PsT-130) and one Bacillus isolate (BaT-68s), with the highest ability to solubilize tricalcium phosphate (TCP) were selected for further molecular identification and characterization. Isolates showed phosphate solubilization up to 195.42 μg mL-1. All isolates showed phosphate solubilization by organic acid production. The six isolates improved seed germination and showed effective root colonization when tomato seeds were coated with isolates at 106 cfu g-1 in axenic soil conditions. Furthermore, the selected isolates were tested for beneficial effects on tomato growth and nutrient status in greenhouse experiments with natural rock phosphate (RP). The results showed that inoculated tomato plants in the presence of RP have a higher shoot and root lengths and weights compared with the control. After 60 days, significant increases in plant Ca, Na, P, protein, and sugar contents were also observed in inoculated seedlings. In addition, inoculated tomato seedlings showed an increase in foliar chlorophyll a and b and total chlorophyll, while no significant changes were observed in chlorophyll fluorescence. In greenhouse, two Pseudomonas isolates, PsT-04c and PsT-130, showed ability to trigger induced systemic resistance in inoculated tomato seedlings when subsequently challenged by Clavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker. High protection rate (75%) was concomitant to an increase in the resistance indicators: total soluble phenolic compounds, phenylalanine-ammonia lyase, and H2O2. The results strongly demonstrated the effectiveness of phosphate-solubilizing bacteria adapted to rhizosphere as biofertilizers for tomato crops and biopesticides by inducing systemic resistance to the causal agent of tomato bacterial canker disease.

Insights into the action of phylogenetically diverse microbial expansins on the structure of cellulose microfibrils

BackgroundMicrobial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp.ResultsFive heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20–25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%.ConclusionThis direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.