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.

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.

Solanum lycopersicum heme-binding protein 2 as a potent antimicrobial weapon against plant pathogens.

The rise in antibiotic-resistant bacteria caused by the excessive use of antibiotics has led to the urgent exploration of alternative antimicrobial solutions. Among these alternatives, antimicrobial proteins, and peptides (Apps) have garnered attention due to their wide-ranging antimicrobial effects. This study focuses on evaluating the antimicrobial properties of Solanum lycopersicum heme-binding protein 2 (SlHBP2), an apoplastic protein extracted from tomato plants treated with 1-Methyl tryptophan (1-MT), against Pseudomonas syringae pv. tomato DC3000 (Pst). Computational studies indicate that SlHBP2 is annotated as a SOUL heme-binding family protein. Remarkably, recombinant SlHBP2 demonstrated significant efficacy in inhibiting the growth of Pst within a concentration range of 3-25 µg/mL. Moreover, SlHBP2 exhibited potent antimicrobial effects against other microorganisms, including Xanthomonas vesicatoria (Xv), Clavibacter michiganensis subsp. michiganensis (Cmm), and Botrytis cinerea. To understand the mechanism of action employed by SlHBP2 against Pst, various techniques such as microscopy and fluorescence assays were employed. The results revealed that SlHBP2 disrupts the bacterial cell wall and causes leakage of intracellular contents. To summarize, the findings suggest that SlHBP2 has significant antimicrobial properties, making it a potential antimicrobial agent against a wide range of pathogens. Although further studies are warranted to explore the full potential of SlHBP2 and its suitability in various applications.

Interactive effects of Pseudomonas entomophila strain 23S and Clavibacter michiganensis subsp. michiganensis on proteome and anti-Cmm compound production.

Pseudomonas entomophila strain 23S is an effective biocontrol bacterium for tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm); it produces an inhibitory compound affecting the growth of Cmm. In this study, the interactions between pure cultures of P. entomophila 23S and Cmm were investigated. First, the population dynamics of each bacterium during the interaction was determined using the selective media. Second, the amount of anti-Cmm compound produced by P. entomophila 23S in the presence of Cmm was quantified using HPLC. Lastly, a label-free shotgun proteomics study of P. entomophila 23S, Cmm, and a co-culture was conducted to understand the effects of the interaction of each bacterium at the proteomic level. Compared with the pure culture grown, the total number of proteins decreased in the interaction for both bacteria. P. entomophila 23S secreted stress-related proteins, such as chaperonins, peptidases, ABC-transporters and elongation factors. The bacterium also produced more proteins related with purine, pyrimidine, carbon and nitrogen metabolisms in the presence of Cmm. The population enumeration study revealed that the Cmm population declined dramatically during the interaction, while the population of P. entomophila 23S maintained. The quantification of anti-Cmm compound indicated that P. entomophila 23S produced significantly higher amount of anti-Cmm compound when it was cultured with Cmm. Overall, the study suggested that P. entomophila 23S, although is cidal to Cmm, was also negatively affected by the presence of Cmm, while trying to adapt to the stress condition, and that such an environment favored increased production of the anti-Cmm compound by P. entomophila 23S. SIGNIFICANCE: Pseudomonas entomophila strain 23S is an effective biocontrol bacterium for tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm); it produces an inhibitory compound affecting the growth of Cmm. In this study, secreted proteome of pure cultures of P. entomophila 23S and Cmm, and also of a co-culture was first time identified. Furthermore, the study found that P. entomophila strain 23S produced significantly higher amount of anti-Cmm compound when the bacterium was grown together with Cmm. Co-culture enhancing anti-Cmm compound production by P. entomophila 23S is useful information, particularly from a commercial point of view of biocontrol application, and for scale-up of anti-Cmm compound production.

Clavibacter lycopersici sp. nov.: a peach-colored actinobacterium isolated from symptomless tomato plant.

In 2015, Gram-positive peach-coloured actinobacterial strains were isolated from symptomless tomato phyllosphere in Iran. Biochemical and physiological characteristics, as well as 16S rRNA phylogeny showed that the strains belong to Clavibacter sp., while they were non-pathogenic on the host of isolation, and morphologically distinct from the tomato pathogen C. michiganensis and other plant-associated bacteria. Multilocus sequence analysis of five housekeeping genes showed that the two peach-coloured strains CFBP 8615T (Tom532T) and CFBP 8616 (Tom495) were phylogenetically distinct from all validly described Clavibacter species. Whole genome sequence-based indices, i.e. average nucleotide identity (orthoANI) and digital DNA-DNA hybridization (dDDH), showed that the two peach-colored strains share nearly 100 % orthoANI value with one another, while they differ from all validly described Clavibacter species with the orthoANI/dDDH values <93 % and <50 %, respectively. Thus, based on both phenotypic features and orthoANI/dDDH indices the peach-coloured strains could belong to a new species within Clavibacter. In this study, we provide a formal species description for the peach-coloured tomato-associated Clavibacter strains. Clavibacter lycopersici sp. nov. is proposed for the new species with Tom532T = CFBP 8615T = ICMP 22100T as type strain.

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

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

A highly branched novel galactofuranan in the cell wall of Clavibacter tesselarius VKM Ac-1406T.

The structures of two cell wall glycopolymers were studied in the plant pathogenic bacterium Clavibacter tesselarius VKM Ac-1406T (family Microbacteriaceae, order Micrococcales, class Actinomycetes). The predominant polymer was a novel (1 â†’ 6)-linked ß-d-galactofuranan with a highly branched repeating unit, α-L-Rhap-(1 â†’ 3)-α-D-Galp-(1 â†’ 2)-[α-L-Rhap-(1 â†’ 3)]-α-D-Fucp-(1 →, at O-2 on every second galactofuranose residue. The second polymer present in small amounts was acidic with the repeating unit, →3)-α-D-Galp-(1 â†’ 3)-α-D-[4,6-S-Pyr]-Manp-(1 â†’ 3)-α-D-Manp-[2OAc]0.2-(1→, and was reported in all Clavibacter species investigated to date. The presented results expand our knowledges of structural diversity of phosphate-free cell wall glycopolymers and provide evidence in support of their taxonomic specificity for bacterial species and genera.

A novel cell wall galactofuranan in Clavibacter phaseoli VKM Ac-2641T.

A glycopolymer of novel structure was found in the cell wall of plant pathogen Clavibacter phaseoli VKM Ac-2641T (family Microbacteriaceae, class Actinomycetes). The glycopolymer was (1 â†’ 6)-linked ß-d-galactofuranan with side branched trisaccharide, α-D-Manp-(1 â†’ 2)-[α-D-Manp-(1 â†’ 3)]-α-D-Ribf-(1→ at O-2 on every second galactofuranose residue. The galactofuranan structure was established by chemical and NMR spectroscopic methods using one- and two-dimensional techniques 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC. The results of this study provide new data on diversity of bacterial glycopolymers, may prove useful for bacterial taxonomy and contribute to the understanding of the host plant-microbiota interaction mechanisms.

CRISPRa-mediated transcriptional activation of the SlPR-1 gene in edited tomato plants.

With the continuous deterioration of arable land due to an ever-growing population, improvement of crops and crop protection have a fundamental role in maintaining and increasing crop productivity. Alternatives to the use of pesticides encompass the use of biological control agents, generation of new resistant crop cultivars, the application of plant activator agrochemicals to enhance plant defenses, and the use of gene editing techniques, like the CRISPR-Cas system. Here, we test the hypothesis that epigenome editing, via CRISPR activation (CRISPRa), activate tomato plant defense genes to confer resistance against pathogen attack. We provide evidence that edited tomato plants for the PATHOGENESIS-RELATED GENE 1 gene (SlPR-1) show enhanced disease resistance to Clavibacter michiganensis subsp. michiganensis infection. Resistance was assessed by evaluating disease progression and symptom appearance, pathogen accumulation, and changes in SlPR-1 gene expression at different time points. We determined that CRISPRa-edited plants develop enhanced disease-resistant to the pathogen without altering their agronomic characteristics and, above all, preventing the advancement of disease symptoms, stem canker, and plant death.

Complete Genome Sequence Resource for a Recently Isolated Potato Ring Rot Pathogen, Clavibacter sepedonicus K496.

Potato ring rot caused by Clavibacter sepedonicus has been a devastating disease in the U.S. since 1930. In this study, we isolated a recent C. sepedonicus strain, K496, from potato tubers showing discolorations of the vascular cylinder or pith tissues. We de novo assembled the genome sequence of K496 with 1,924,544,313 bp of Nanopore reads (N50 = 13,785 bp) using Flye v2.9 and polished it with 2 × 150 bp paired-end Illumina reads (855,788,703 bp in total). The resulting genome of K496 consists of a single circular chromosome 3,266,016 bp long and a linear plasmid of 135,489 bp. Using the NCBI PGAP v5.3, this genome was predicted to have 3,301 genes, encompassing 3,247 protein-coding genes, 90 pseudogenes, two 5S rRNA-coding, two 16S rRNA-coding, two 23S rRNA-coding sequences, 45 tRNAs, and three noncoding RNAs. The chromosome and plasmid sequences have been deposited at the NCBI GenBank database under the accession numbers CP088266 and CP088267, respectively.

Clavibacter nebraskensis causing Goss's wilt of maize: Five decades of detaining the enemy in the New World.

Goss's bacterial wilt and leaf blight of maize (Zea mays) caused by the gram-positive coryneform bacterium Clavibacter nebraskensis is an economically important disease in North America. C. nebraskensis is included within the high-risk list of quarantine pathogens by several plant protection organizations (EPPO code: CORBMI), hence it is under strict quarantine control around the world. The causal agent was reported for the first time on maize in Nebraska (USA) in 1969. After an outbreak during the 1970s, prevalence of the disease decreased in the 1980s to the early 2000s, before the disease resurged causing a serious threat to maize production in North America. The re-emergence of Goss's wilt in the corn belt of the United States led to several novel achievements in understanding the pathogen biology and disease control. In this review, we provide an updated overview of the pathogen taxonomy, biology, and epidemiology as well as management strategies of Goss's wilt disease. First, a taxonomic history of the pathogen is provided followed by symptomology and host range, genetic diversity, and pathogenicity mechanisms of the bacterium. Then, utility of high-throughput molecular approaches in the precise detection and identification of the pathogen and the management strategies of the disease are explained. Finally, we highlight the role of integrated pest management strategies to combat the risk of Goss's wilt in the 21st century maize industry. DISEASE SYMPTOMS: Large (2-15 cm) tan to grey elongated oval lesions with wavy, irregular water-soaked margins on the leaves. The lesions often start at the leaf tip or are associated with wounding caused by hail or wind damage. Small (1 mm in diameter), dark, discontinuous water-soaked spots, known as "freckles", can be observed in the periphery of lesions. When backlit, the freckles appear translucent. Early infection (prior to growth stage V6) may become systemic and cause seedlings to wilt, wither, and die. Coalescence of lesions results in leaf blighting. HOST RANGE: Maize (Zea mays) is the only economic host of the pathogen. A number of Poaceae species are reported to act as secondary hosts for C. nebraskensis. TAXONOMIC STATUS OF THE PATHOGEN: Class: Actinobacteria; Order: Micrococcales; Family: Microbacteriaceae; Genus: Clavibacter; Species: Clavibacter nebraskensis. SYNONYMS: Corynebacterium nebraskense (Schuster, 1970) Vidaver & Mandel 1974; Corynebacterium michiganense pv. nebraskense (Vidaver & Mandel 1974) Dye & Kemp 1977; Corynebacterium michiganense subsp. nebraskense (Vidaver & Mandel 1974) Carlson & Vidaver 1982; Clavibacter michiganense subsp. nebraskense (Vidaver & Mandel 1974) Davis et al. 1984; Clavibacter michiganensis subsp. nebraskensis (Vidaver & Mandel 1974) Davis et al. 1984. TYPE MATERIALS: ATCC 27794T ; CFBP 2405T ; ICMP 3298T ; LMG 3700T ; NCPPB 2581T . MICROBIOLOGICAL PROPERTIES: Cells are gram-positive, orange-pigmented, pleomorphic club- or rod-shaped, nonspore-forming, nonmotile, and without flagella, approximately 0.5 × 1-2.0 µm. DISTRIBUTION: The pathogen is restricted to Canada and the United States. PHYTOSANITARY CATEGORIZATION: EPPO code CORBNE.

Elevation of Clavibacter michiganensis subsp. californiensis to species level as Clavibacter californiensis sp. nov., merging and re-classification of Clavibacter michiganensis subsp. chilensis and Clavibacter michiganensis subsp. phaseoli as Clavibacter phaseoli sp. nov. based on complete genome in silico analyses.

The Gram-positive genus Clavibacter is currently divided into seven species (Clavibacter michiganensis, Clavibacter nebraskensis, Clavibacter capsici, Clavibacter sepedonicus, Clavibacter tessellarius, Clavibacter insidiosus and Clavibacter zhangzhiyongii) and three subspecies (C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli). Recent studies have indicated that the taxonomic rank of the subspecies must be re-evaluated. In this research, we assessed the taxonomic position of the three C. michiganensis subspecies and clarified the taxonomic nomenclature of other 75 Clavibacter strains. The complete genomes of the type strains of the three Clavibacter subspecies, the type strain of C. tessellarius and C. nebraskensis A6096 were sequenced using PacBio RSII technology. Application of whole-genome-based computational approaches such as average nucleotide identity (ANI), digital DNA-DNA hybridization, multi-locus sequence analysis of seven housekeeping genes (acnA, atpD, bipA, icdA, mtlD, recA and rpoB), a phylogenomic tree reconstructed from 1 028 core genes, and ANI-based phylogeny provided sufficient justification for raising C. michiganensis subsp. californiensis to the species level. These results led us to propose the establishment of Clavibacter californiensis sp. nov. as a species with its type strain C55T (=CFBP 8216T=ATCC BAA-2691T). Moreover, the orthologous and in silico dot plot analyses, along with the above described bioinformatic strategies, revealed a high degree of similarity between C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli. Based on these analyses, we propose that both subspecies be combined into a single taxon and elevated to the species level as Clavibacter phaseoli sp. nov., with LPPA 982T (= CECT 8144T= LMG 27667T) as the type strain.

Epiphytic and endophytic microorganisms associated to different cultivar of tomato fruits in greenhouse environment and characterization of beneficial bacterial strains for the control of post-harvest tomato pathogens.

Several bacterial and fungal diseases affect greenhouse-grown tomato crops, causing severe annual yield losses worldwide. The need to reduce chemical compound applications has encouraged the search of alternative approach for the control of tomato diseases, including the use of biological control agents. The presence of total and beneficial microbial populations was investigated on the surface and in the pulp of seven cultivars of tomato fruit coming from eleven greenhouses in the Pachino district (south-east of Sicily), recognized by the European Community with the "Protected Geographical Indication" label. Principal component analysis (PCA) showed that epiphytic and endophytic microbial populations clustered into groups according to the areas of origin. Approximately 240 tomato fruit-associated bacterial isolates were selected and a high percentage of them showed antagonistic activity against Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, Xanthomonas euvesicatoria pv. perforans, Botrytis cinerea and Alternaria alternata. Analysis of the 16S rRNA gene sequences revealed a predominance of bacteria in Bacillus and Pseudomonas genera, followed by Citrobacter and Enterobacter. The presence of these genera differed according to the geographical areas of tomato samples, whereas their antagonistic capabilities varied according to the five tomato pathogens. The in vitro effectiveness of eight representative bacterial strains belonging to Pseudomonas, Bacillus and Enterobacter genera was confirmed in in vivo assays, carried out on tomato fruit artificially inoculated with A. alternata and B. cinerea. Altogether, these results revealed differences in population density of native microbiota of "Pomodoro di Pachino" fruits and allowed the selection of antagonistic bacterial strains that can be applied in field and in postharvest conditions as alternatives to chemical compounds.

Immune recognition of the secreted serine protease ChpG restricts the host range of Clavibacter michiganensis from eggplant varieties.

Bacterial wilt and canker caused by Clavibacter michiganensis (Cm) inflict considerable damage in tomato-growing regions around the world. Cm has a narrow host range and can cause disease in tomato but not in many eggplant varieties. The pathogenicity of Cm is dependent on secreted serine proteases, encoded by the chp/tomA pathogenicity island (PI), and the pCM2 plasmid. Screening combinations of PI deletion mutants and plasmid-cured strains found that Cm-mediated hypersensitive response (HR) in the Cm-resistant eggplant variety Black Queen is dependent on the chp/tomA PI. Singular reintroduction of PI-encoded serine proteases into Cm∆PI identified that the HR is elicited by the protease ChpG. Eggplant leaves infiltrated with a chpG marker exchange mutant (CmΩchpG) did not display an HR, and infiltration of purified ChpG protein elicited immune responses in eggplant but not in Cm-susceptible tomato. Virulence assays found that while wild-type Cm and the CmΩchpG complemented strain were nonpathogenic on eggplant, CmΩchpG caused wilt and canker symptoms. Additionally, bacterial populations in CmΩchpG-inoculated eggplant stem tissues were c.1000-fold higher than wild-type and CmΩchpG-complemented Cm strains. Pathogenicity tests conducted in multiple Cm-resistance eggplant varieties demonstrated that immunity to Cm is dependent on ChpG in all tested varieties, indicating that ChpG-recognition is conserved in eggplant. ChpG-mediated avirulence interactions were disabled by alanine substitution of serine231 of the serine protease catalytic triad, suggesting that protease activity is required for immune recognition of ChpG. Our study identified ChpG as a novel avirulence protein that is recognized in resistant eggplant varieties and restricts the host range of Cm.

RNA-Seq Analysis Discovers the Critical Role of Rel in ppGpp Synthesis, Pathogenicity, and the VBNC State of Clavibacter michiganensis.

The viable but nonculturable (VBNC) state is a unique survival strategy of bacteria in response to stress conditions. It was confirmed that Clavibacter michiganensis, the causal agent of bacterial canker in tomato, could be induced into the VBNC state by exposure to CuSO4 in an oligotrophic solution. RNA-sequencing analysis was used to monitor the mechanisms of the VBNC state during CuSO4 induction in C. michiganensis. The results identified that numerous genes involved in stringent response, copper resistance, and stress resistance were upregulated, and some involved in cell division were downregulated significantly. The study investigated the importance of Rel, which is an essential enzyme in the synthesis of the molecular alarmone ppGpp, via the generation of a Δrel mutant and its complementation strain. Biological characterization revealed that deficiency of rel reduced the bacterial growth, production of exopolysaccharides, and pathogenicity as well as ppGpp production. The Δrel mutant increased the sensitivity to environmental stress, exhibiting reduced growth on minimal media and a propensity to enter the VBNC state in response to CuSO4. These findings have important implications for the understanding of survival mechanism and management of C. michiganensis and other phytopathogenic bacteria.

Bacterial ring rot of potato caused by Clavibacter sepedonicus: A successful example of defeating the enemy under international regulations.

BACKGROUND: Bacterial ring rot of potato (Solanum tuberosum) caused by the gram-positive coryneform bacterium Clavibacter sepedonicus is an important quarantine disease threatening the potato industry around the globe. Since its original description in 1906 in Germany, management of ring rot has been a major problem due to the seedborne nature (via seed tubers not true seeds) of the pathogen allowing the bacterium to be transmitted long distances via infected tubers. DISEASE SYMPTOMS: On growing potato plants: interveinal chlorosis on leaflets leading to necrotic areas and systemic wilt. On infected tubers: vascular tissues become yellowish brown with a cheesy texture due to bacterial colonization and decay. HOST RANGE: Potato is the main host of the pathogen, but natural infection also occurs on eggplant, tomato, and sugar beet. TAXONOMIC STATUS OF THE PATHOGEN: Class: Actinobacteria; Order: Actinomycetales; Family: Microbacteriaceae; Genus: Clavibacter; Species: Clavibacter sepedonicus (Spieckermann and Kotthoff 1914) Li et al. 2018. SYNONYMS (NONPREFERRED SCIENTIFIC NAMES): Aplanobacter sepedonicus; Bacterium sepedonicum; Corynebacterium sepedonicum; Corynebacterium michiganense pv. sepedonicum; Clavibacter michiganensis subsp. sepedonicus. MICROBIOLOGICAL PROPERTIES: Gram-positive, club-shaped cells with creamy to yellowish-cream colonies for which the optimal growth temperature is 20-23°C. DISTRIBUTION: Asia (China, Japan, Kazakhstan, Nepal, North Korea, Pakistan, South Korea, Uzbekistan, the Asian part of Russia), Europe (Belarus, Bulgaria, Czech Republic, Estonia, Finland, Georgia, Germany, Greece, Hungary, Latvia, Lithuania, Norway, Poland, Romania, European part of Russia, Slovakia, Spain, Sweden, Turkey, Ukraine), and North America (Canada, Mexico, USA). PHYTOSANITARY CATEGORIZATION: CORBSE: EPPO A2 list no. 51. EU; Annex designation I/A2.

A novel glucofucobiose with potential prebiotic activity prepared from the exopolysaccharides of Clavibacter michiganensis M1.

Fucose and fucosylated oligosaccharides have important applications in various industries owing to their prebiotic, anti-inflammatory, anticoagulant, and antiviral activities. Here, we aimed to obtain fucosylated oligosaccharides using the acidolysis method to depolymerize exopolysaccharides extracted from Clavibacter michiganensis M1. Based on structural analysis, the prepared glucofucobiose was found to consist of d-glucose and l-fucose, with a molecular weight of 326 Da and a structure of d-Glcp-ß-(1→4)-l-Fucp. The prebiotic activity of glucofucobiose was compared with that of 2'-fucosyllactose (2'-FL), the most abundant oligosaccharide in human milk. According to the results, glucofucobiose could significantly promote the proliferation of six probiotic strains, and short-chain fatty acid production of five probiotic strains on glucofucobiose was substantially higher than that on 2'-FL at 48 h of fermentation. Overall, this study proposed a new technology for obtaining fucosylated oligosaccharides. The prepared glucofucobiose was found to exhibit potential prebiotic activity and should be further assessed.

Unmarked Gene Editing in Clavibacter michiganensis Using CRISPR/Cas9 and 5-Fluorocytosine Counterselection.

Plant-pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis, which couples the expression of cas9 and single-guide RNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site-directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the C. michiganensis rpsL gene to generate a streptomycin-resistant mutant, and complete gene knockout, in which the deletion of the C. michiganensis celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments, the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 5-fluorocytosine at 100 µg/ml, in which an unmarked katA mutant lacking the transformation vector was recovered. Compared with conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient because it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into C. michiganensis and other Clavibacter spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.