cThe Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial wilt and canker of tomato, is an economically devastating pathogen that inflicts considerable damage throughout all major tomato-producing regions. Annual outbreaks continue to occur in New York, where C. michiganensis subsp. michiganensis spreads via infected transplants, trellising stakes, tools, and/or soil. Globally, new outbreaks can be accompanied by the introduction of contaminated seed stock; however, the route of seed infection, especially the role of fruit lesions, remains undefined. In order to investigate the modes of seed infection, New York C. michiganensis subsp. michiganensis field strains were stably transformed with a gene encoding enhanced green fluorescent protein (eGFP). A constitutively eGFP-expressing virulent C. michiganensis subsp. michiganensis isolate, GCMM-22, was used to demonstrate that C. michiganensis subsp. michiganensis could not only access seeds systemically through the xylem but also externally through tomato fruit lesions, which harbored high intra-and intercellular populations. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed, once the fruits began to ripen. These results highlight the ability of C. michiganensis subsp. michiganensis to invade tomato fruits and seeds through multiple entry routes.

New York Clavibacter michiganensis subsp. michiganensis isolates, collected from disparate bacterial canker of tomato outbreaks over the past 11 years, were characterized with a multilocus sequence analysis (MLSA) scheme that differentiated the 51 isolates into 21 haplotypes with a discriminatory power of 0.944. The MLSA scheme consisted of five housekeeping genes (kdpA, sdhA, dnaA, ligA, and gyrB) and three putative pathogenicity genes (celA, tomA, and nagA). Repetitive polymerase chain reaction (PCR), with the BOX-A1R primer, confirmed the high diversity of C. michiganensis subsp. michiganensis isolates in New York by demonstrating that all six PCR patterns (A, B, 13C, 65C, 81C, and D) were present, with PCR patterns C and A being the most common. The MLSA scheme provided higher resolving power than the current repetitive-PCR approach. The plasmid profiles of New York isolates were diverse and differed from reference strain NCPPB382. PCR analysis indicated that the presence of putative pathogenicity genes varied between isolates and highlighted the ephemeral nature of pathogenicity genes in field populations of C. michiganensis subsp. michiganensis. Analysis of molecular variance between Serbian and New York C. michiganensis subsp. michiganensis isolates demonstrated that the two populations were not significantly different, with 98% genetic variation within each population and only 2% genetic variation between populations.

Expansin proteins, which loosen plant cell walls, play critical roles in normal plant growth and development. The horizontal acquisition of functional plant-like expansin genes in numerous xylem-colonizing phytopathogenic bacteria suggests that bacterial expansins may also contribute to virulence. To investigate the role of bacterial expansins in plant diseases, we mutated the non-chimeric expansin genes (CmEXLX2 and RsEXLX) of two xylem-inhabiting bacterial pathogens, the Actinobacterium Clavibacter michiganensis ssp. michiganensis (Cmm) and the β-proteobacterium Ralstonia solanacearum (Rs), respectively. The Cmm ΔCmEXLX2 mutant caused increased symptom development on tomato, which was characterized by more rapid wilting, greater vascular necrosis and abundant atypical lesions on distant petioles. This increased disease severity correlated with larger in planta populations of the ΔCmEXLX2 mutant, even though the strains grew as well as the wild-type in vitro. Similarly, when inoculated onto tomato fruit, ΔCmEXLX2 caused significantly larger lesions with larger necrotic centres. In contrast, the Rs ΔRsEXLX mutant showed reduced virulence on tomato following root inoculation, but not following direct petiole inoculation, suggesting that the RsEXLX expansin contributes to early virulence at the root infection stage. Consistent with this finding, ΔRsEXLX attached to tomato seedling roots better than the wild-type Rs, which may prevent mutants from invading the plant's vasculature. These contrasting results demonstrate the diverse roles of non-chimeric bacterial expansins and highlight their importance in plant-bacterial interactions.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

A corroded lead service line was removed from a drinking water distribution system, and the microbial community was profiled using 16S rRNA gene techniques. This is the first report of the characterization of a biofilm on the surface of a corroded lead drinking water service line. The majority of phylotypes have been linked to heavy-metal-contaminated environments.

Bacterial wilt caused by Ralstonia solanacearum is considered among the most damaging diseases of potato in Sub-Saharan Africa and the most significant biotic constraint of potato production alongside late blight. Unlike late blight, which can be managed by chemical means, R. solanacearum can only be managed through cultural methods and clean seed. Laboratory testing to certify seed before planting is required to confirm the absence of the pathogen in Kenya. A loop-mediated isothermal amplification (LAMP) assay was developed using the UDP-(3-O-acyl)-N-acetylglucosamine deacetylase gene (IpxC) to screen seed potato for R. solanacearum strains. The assay was assessed using DNA extracted from R. solanacearum and other soil and potato pathogens to demonstrate specificity and sensitivity. The LAMP assay was validated using field samples from different potato growing regions of Kenya collected over two growing seasons and compared with established nucleic acid and protein-based assays. The IpxC LAMP assay was found to be specific and sensitive to R. solanacearum, detecting as low as 2.5 pg/µl of R. solanacearum DNA. Of the 47 potentially infected field samples collected, both IpxC LAMP and quantitative polymerase chain reaction (PCR) detected R. solanacearum DNA in 90% of the samples, followed by conventional PCR (86%) and ELISA (75%). This IpxC LAMP assay is a promising diagnostic tool to rapidly screen for R. solanacearum in seed potato with high sensitivity in Kenya. [Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

The Gram-positive actinobacterium Clavibacter michiganensis is the causal agent of bacterial canker of tomato, an economically impactful disease with a worldwide distribution. This seedborne pathogen systemically colonizes tomato xylem leading to unilateral leaflet wilt, marginal leaf necrosis, stem and petiole cankers, and plant death. Additionally, splash dispersal of the bacterium onto fruit exteriors causes bird’s-eye lesions, which are characterized as necrotic centers surrounded by white halos. The pathogen can colonize developing seeds systemically through xylem and through penetration of fruit tissues from the exterior. There are currently no commercially available resistant cultivars, and bactericidal sprays have limited efficacy for managing the disease once the pathogen is in the vascular system. In this review we summarize research on epidemiology, host colonization, the bacterial genetics underlying virulence, and management of bacterial canker. Finally, we highlight important areas of research into this pathosystem that have the potential to generate new strategies for prevention and mitigation of bacterial canker.

To assess the diversity of Xanthomonas campestris spp. infecting crucifers in New York, 154 isolates were collected over 10 years across the state. The goal was to determine if isolates of the pathogen were overwintering in New York and serving as primary inoculum in subsequent years, or if novel isolates were entering the state each year. Pure cultures of isolates were characterized using multilocus sequence analysis (MLSA), a greenhouse pathogenicity assay, repetitive element-polymerase chain reaction (Rep-PCR) using the BOX-A1R primer, and enzyme-linked immunosorbent assay. The MLSA scheme proved to be more efficient than Rep-PCR for a large sample population and for comparison with global isolates. X. campestris isolated from crucifers in New York comprised of X. campestris pv. campestris and X. campestris pv. raphani, with X. campestris pv. raphani being predominately isolated from transplants. Evidence for unique haplotypes persisting on the same farm for several years due to improper seedbed rotations was documented in addition to novel haplotypes being spread throughout states through infected transplants and seed. Rep-PCR confirmed the high diversity of X. campestris and was used to generate 15 unique fingerprint patterns from isolates collected in the first 5 years. A worldwide comparison of isolates suggests that the X. campestris pv. campestris population appears to be very homogenous with dominant haplotypes persisting for extended periods and being globally disseminated.