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 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.

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.

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.