Understanding how bacteria affect plant health is crucial for developing sustainable crop production systems. We coupled ecological sampling and genome sequencing to characterize the population genetic history of Rhodococcus and the distribution patterns of virulence plasmids in isolates from nurseries. Analysis of chromosome sequences shows that plants host multiple lineages of Rhodococcus, and suggested that these bacteria are transmitted due to independent introductions, reservoir populations, and point source outbreaks. We demonstrate that isolates lacking virulence genes promote beneficial plant growth, and that the acquisition of a virulence plasmid is sufficient to transition beneficial symbionts to phytopathogens. This evolutionary transition, along with the distribution patterns of plasmids, reveals the impact of horizontal gene transfer in rapidly generating new pathogenic lineages and provides an alternative explanation for pathogen transmission patterns. Results also uncovered a misdiagnosed epidemic that implicated beneficial Rhodococcus bacteria as pathogens of pistachio. The misdiagnosis perpetuated the unnecessary removal of trees and exacerbated economic losses.

Agrobacterium is a genus of soilborne gram-negative bacteria. Members carrying oncogenic plasmids can cause crown gall disease, which has significant economic costs, especially for the orchard and nursery industries. Early and rapid detection of pathogenic Agrobacterium spp. is key to the management of crown gall disease. To this end, we designed oligonucleotide primers and probes to target virD2 for use in a molecular diagnostic tool that relies on isothermal amplification and lateral-flow-based detection. The oligonucleotide tools were tested in the assay and evaluated for detection limit and specificity in detecting alleles of virD2. One set of primers that successfully amplified virD2 when used with an isothermal recombinase was selected. Both tested probes had detection limits in picogram amounts of DNA. Probe 1 could detect all tested pathogenic isolates that represented most of the diversity of virD2. Finally, the coupling of lateral-flow detection to the use of these oligonucleotide primers in isothermal amplification helped to reduce the onerousness of the process, and alleviated reliance on specialized tools necessary for molecular diagnostics. The assay is an advancement for the rapid molecular detection of pathogenic Agrobacterium spp.

Rhodococcus is a genus of Gram-positive bacteria with species that can cause growth deformations to a large number of plant species. This ability to cause disease is hypothesized to be dependent on a cluster of three gene loci on an almost 200 kbsized linear plasmid. To reevaluate the roles of some of the genes in pathogenicity, we constructed and characterized deletion mutants of fasR and four fas genes. Findings confirmed that fasR, which encodes a putative transcriptional regulator, is necessary for pathogenesis. However, three of the fas genes, implicated in the metabolism of plant growth promoting cytokinins, are dispensable for the ability of the pathogen to cause disease. We also used long-read sequencing technology to generate high quality genome sequences for two phytopathogenic strains in which virulence genes are diverged in sequence and/or hypothesized to have recombined into the chromosome. Surprisingly, findings showed that the two strains carry extremely diverse virulence plasmids. Ortholog clustering identified only 12 genes present on all three virulence plasmids. Rhodococcus requires a small number of horizontally acquired traits to be pathogenic and the transmission of the corresponding genes, via recombination and conjugation, has the potential to rapidly diversify plasmids and bacterial populations.

A general mathematical model that describes the temporal behavior of a real‐time isothermal process used for nucleic acid amplification is derived. A monotonically‐increasing fluorescence signal s(t) generated and measured during the amplification reaction can be modeled in the form of a logistic function of time that is completely described by three parameters (k, t50, and Smax), which may be readily estimated from experimentally acquired s(t) data. Experimental data obtained from a real‐time loop‐mediated isothermal amplification (LAMP) assay for the infectious pathogen Erwinia amylovora (E. amylovora) are used to illustrate and validate the mathematical model. Implementation of such a modeling approach can allow for the extraction of quantitative information from real‐time LAMP data through parameter estimation techniques; this is demonstrated experimentally using real‐time amplification data acquired using the real‐time E. amylovora assay.

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Randall et al., 2018 and Vereecke, 2018 have raised concerns about a paper we published (Savory et al., 2017). Here, we respond to those concerns.

BACKGROUND: Global medical education is gradually moving toward more comprehensive implementations of a competency-based education (CBE) model. Elimination of standard time-based training and adoption of time-variable training (competency-based time-variable training [CB-TVT]) is one of the final stages of implementation of CBE. While CB-TVT has been implemented in some programs outside the United States, residency programs in the United States are still exploring this approach to training. The Accreditation Council for Graduate Medical Education (ACGME) and the American Board of Medical Specialties (ABMS) are encouraging member boards and residency review committees to consider innovative ways programs could implement CB-TVT. The goals of this study were to (1) identify potential problems with the implementation of CB-TVT in anesthesiology residency training, (2) rank the importance of the problems and the perceived difficulty of solving them, and (3) develop proposed solutions to the identified problems. METHODS: Study participants were recruited from key stakeholder groups in anesthesiology education, including current or former program directors, department chairs, residents, fellows, American Board of Anesthesiology (ABA) board members, ACGME residency review committee members or ACGME leaders, designated institutional officials, residency program coordinators, clinical operations directors, and leaders of large anesthesiology community practice groups. This study was conducted in 2 phases. In phase 1, survey questionnaires were iteratively distributed to participants to identify problems with the implementation of CB-TVT. Participants were also asked to rank the perceived importance and difficulty of each problem and to identify relevant stakeholder groups that would be responsible for solving each problem. In phase 2, surveys focused on identifying potential solutions for problems identified in phase 1. RESULTS: A total of 36 stakeholders identified 39 potential problems, grouped into 7 major categories, with the implementation of CB-TVT in anesthesiology residency training. Of the 39 problems, 19 (48.7%) were marked as important or very important on a 5-point scale and 12 of 19 (63.2%) of the important problems were marked as difficult or very difficult to solve on a 5-point scale. Stakeholders proposed 165 total solutions to the identified problems. CONCLUSIONS: CB-TVT is a promising educational model for anesthesiology residency, which potentially results in learner flexibility, individualization of curricula, and utilization of competencies to determine learner advancement. Because of the potential problems with the implementation of CB-TVT, it is important for future pilot implementations of CB-TVT to document realized problems, efficacy of solutions, and effects on educational outcomes to justify the burden of implementing CB-TVT.