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.

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.

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.

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.

Herbicide safener isoxadifen-ethyl associated with increased Goss's wilt severity in corn (Zea mays).

BACKGROUND: Goss's bacterial wilt and leaf blight (Goss's wilt), caused by the bacterium Clavibacter nebraskensis, is a corn disease that has been a top ten yield-reducing disease in North America in the past 15 years. Isoxadifen-ethyl is an herbicide safener that effectively increases cytochrome P450 activity in corn which enhances a plant's metabolism of herbicide molecules. Recent research found a potential link between isoxadifen-ethyl and increased Goss's wilt severity. RESULTS: The application of isoxadifen-ethyl increased (P = 0.014-0.046) area under disease progress curve (AUDPC) by 19%, 7%, and 9% at three environments, independent of accompanying herbicide or herbicide application timing. However, no significant differences in incidence of systemic wilt or corn grain yield occurred among treatments at any environment. CONCLUSION: These data provide evidence for an association between isoxadifen-ethyl safener and Goss's wilt in corn. The reason for this association is unknown, but the safener may affect plant or pathogen physiological mechanisms. While the increased disease severity did not result in decreased grain yield in these experiments, an increase in pathogen inoculum due to higher disease severity could influence Goss' wilt epidemics in future years. © 2024 Society of Chemical Industry.

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.

Uracil hydrazones: design, synthesis, antimicrobial activities, and putative mode of action.

BACKGROUND: Crop diseases caused by plant pathogenic fungi and bacteria have led to substantial losses in global food production. Chemical pesticides have been widely used as a primary means to mitigate these issues. Nevertheless, the persistent and excessive use of pesticides has resulted in the emergence of microbial resistance. Moreover, the improper application and excessive utilization of pesticides can contribute to environmental pollution and the persistence of pesticide residues. Consequently, the development of novel and highly effective bactericides and fungicides to combat plant pathogens holds immense practical importance. RESULTS: A series of uracil hydrazones IV-B was deliberately designed and evaluated for their antimicrobial efficacy. The results of bioassays indicated that most IV-B exhibited >80% inhibition against the fungal species Monilia fructigena and Sclerotium rolfsii, as well as the bacterial species Clavibacter michiganensis subsp. michiganensis, Xanthomonas oryzae pv. oryzae, and Ralstonia solanacearum, at 50 µg/mL in vitro. In vivo, IV-B20 showed 89.9% of curative and 71.8% of protective activities against C. michiganensis subsp. michiganensis at 100 µg/mL superior to thiodiazole copper and copper hydroxide. IV-B20 also showed excellent protective activity against M. fructigena (96.3% at 200 µg/mL) and S. rolfsii (80.4% at 1000 µg/mL), which were greater than chlorothalonil and equivalent to thifluzamide. Mechanistic studies revealed that IV-B20 induced oxidative damage in pathogenic bacteria and promoted the leakage of cellular contents. CONCLUSION: This study suggests that IV-B20 with uracil hydrazone skeleton has great potential as an antimicrobial candidate. These findings lay a foundation for practical application in agriculture. © 2023 Society of Chemical Industry.

Unlocking a Mystery: Characterizing the First Appearance of Clavibacter nebraskensis in Mexican Cornfields.

The Goss's wilt and leaf blight is a disease of maize (Zea mays) caused by Clavibacter nebraskensis, which was widespread in the last several years throughout the Midwest in the United States, south in Texas, and north to Canada. The bacterium is included within the high-risk list of quarantine pathogens by many plant protection organizations and countries including Mexico. Severe blight symptoms on maize plants were found in different provinces from Coahuila and Tlaxcala, Mexico, in 2012 and 2021, respectively. Twenty bacterial isolates with morphology similar to C. nebraskensis were obtained from the diseased maize leaves. The isolates were confirmed by phenotypic tests and 16S rRNA and gyrB sequencing. Two strains were tested for pathogenicity tests on seven hybrid sweet corn cultivars available in Mexico, and the most sensitive cultivar was tested for all the strains to fulfill Koch's postulates. The phylogenetic reconstruction based on two single loci reveals a remarkable clustering of Mexican strains to American strains reported approximately 50 years ago. The presence of this pathogen represents a risk and a significant challenge for plant protection strategies in Mexico and maize diversity.

A Putative Apoplastic Effector of Clavibacter capsici, ChpGCc as Hypersensitive Response and Virulence (Hrv) Protein in Plants.

Clavibacter bacteria use secreted apoplastic effectors, such as putative serine proteases, for virulence in host plants and for hypersensitive response (HR) induction in nonhost plants. Previously, we have shown that Clavibacter capsici ChpGCc is important for the necrosis development in pepper (Capsicum annuum) leaves. Here, we determine the function of ChpGCc, along with three paralogous proteins, for HR induction in the apoplastic space of a nonhost plant, Nicotiana tabacum. The full-length and signal peptide-deleted (ΔSP) mature forms of all proteins fused with the tobacco PR1b signal sequence were generated. The full-length and ΔSP forms of ChpGCc and only the ΔSP forms of ChpECc and Pat-1Cc, but none of the ChpCCc, triggered HR. Based on the predicted protein structures, ChpGCc carries amino acids for a catalytic triad and a disulfide bridge in positions like Pat-1Cm. Substituting these amino acids of ChpGCc with alanine abolished or reduced HR-inducing activity. To determine whether these residues are important for necrosis development in pepper, alanine-substituted chpGCc genes were transformed into the C. capsici PF008ΔpCM1 strain, which lacks the intact chpGCc gene. The strain with any variants failed to restore the necrosis-causing ability. These results suggest that ChpGCc has a dual function as a virulence factor in host plants and an HR elicitor in nonhost plants. Based on our findings and previous results, we propose Clavibacter apoplastic effectors, such as ChpGCc, Pat-1Cm, Chp-7Cs, and ChpGCm, as hypersensitive response and virulence (Hrv) proteins that display phenotypic similarities to the hypersensitive response and pathogenicity (Hrp) proteins found in gram-negative bacteria. [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.