The accelerated evolution and spread of pathogens are threats to host species. Agrobacteria require an oncogenic Ti or Ri plasmid to transfer genes into plants and cause disease. We developed a strategy to characterize virulence plasmids and applied it to analyze hundreds of strains collected between 1927 and 2017, on six continents and from more than 50 host species. In consideration of prior evidence for prolific recombination, it was surprising that oncogenic plasmids are descended from a few conserved lineages. Characterization of a hierarchy of features that promote or constrain plasticity allowed inference of the evolutionary history across the plasmid lineages. We uncovered epidemiological patterns that highlight the importance of plasmid transmission in pathogen diversification as well as in long-term persistence and the global spread of disease.

bThe improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.

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Field experiments were conducted in silty-clay loam in Corvallis, OR during the summers of 1995 and 1996 to study the effects of green manure cover crops (Sudan grass, rape, and barley), soil solarization, soil fumigation, and combinations of those treatments on population densities of soil pathogens Verticillium dahliae, Phytophthora cinnamomi, Pratylenchus penetrans, and Agrobacterium rhizogenes. Nylon mesh bags containing soil infested with V. dahliae and Phytophthora cinnamomiwere buried 5, 10, 20, and 30 cm deep. Soil solarization was performed over a 54- to 59-day period using a 0.6-mil clear polyethylene film. Maximum soil temperatures recorded at depths of 5, 10, 20, and 30 cm were 53, 48, 39, and 34°C in solarized soil, respectively; these temperatures were 8 to 16°C higher than in corresponding nonsolarized plots. Soil samples were collected before, during, and after solarization to quantify pathogen populations at those four depths. Pot or field studies were conducted subsequent to treatments to determine the effects of treatments on susceptible plants. Soil solarization, cover crops plus solarization, or fumigation with metam sodium resulted in a significant decrease (P< 0.05) in density of P. cinnamomi populations at all four depths and reduced (P< 0.05) V. dahliae at 5 and 10 cm. In greenhouse assays of solarized soils, disease severity was reduced (P< 0.05) for Verticillium spp. on eggplant and Phytophthora spp. on snapdragons. Cover crops alone were not effective in reducing P. cinnamomi and V. dahliae populations. Agrobacterium spp. population densities declined within solarized plots and incidence of crown gall on ‘Mazzard’ cherry rootstock planted in solarized plots was reduced significantly. Population densities of Pratylenchus penetranswere reduced in the upper 30-cm soil profile by solarization.Solarization for an 8-week period during the warmest months of summer could provide an additional management alternative for several important soilborne pathogens in western Oregon.

The Ti plasmid in Agrobacterium tumefaciens strain 15955 carries two alleles of traR that regulate conjugative transfer. The first is a functional allele, called traR, that is transcriptionally induced by the opine octopine. The second, trlR, is a nonfunctional, dominant-negative mutant located in an operon that is inducible by the opine mannopine (MOP). Based on these findings, we predicted that there exist wild-type agrobacterial strains harboring plasmids in which MOP induces a functional traR and, hence, conjugation. We analyzed 11 MOP-utilizing field isolates and found five where MOP induced transfer of the MOP-catabolic element and increased production of the acyl-homoserine lactone (acyl-HSL) quormone. The transmissible elements in these five strains represent a set of highly related plasmids. Sequence analysis of one such plasmid, pAoF64/95, revealed that the 176-kb element is not a Ti plasmid but carries genes for catabolism of MOP, mannopinic acid (MOA), agropinic acid (AGA), and the agrocinopines. The plasmid additionally carries all of the genes required for conjugative transfer, including the regulatory genes traR, traI, and traM. The traR gene, however, is not located in the MOP catabolism region. The gene, instead, is monocistronic and located within the tra-trb-rep gene cluster. A traR mutant failed to transfer the plasmid and produced little to no quormone even when grown with MOP, indicating that TraR pAoF64/95 is the activator of the tra regulon. A traM mutant was constitutive for transfer and acyl-HSL production, indicating that the anti-activator function of TraM is conserved.

Field experiments were conducted on a silty clay loam in Corvallis, OR during the summers of 1995 and 1996 to study the effects of soil solarization, spring-planted green manure crops, fumigation with metham, and combinations of these treatments on annual bluegrass seed survival. Annual bluegrass seeds were incorporated into the soil as a bioassay species and soil samples extracted to a depth of 15 cm to determine effects on seed survival. Soil solarization was applied over a 53- or 59-d period using a 0.6-mil clear polyethylene film. Soil samples were collected from four depths after the solarization period in both solarized and nonsolarized plots and surviving seeds germinated in a greenhouse. Maximum soil temperatures recorded at 5-, 10-, and 20-cm depths were 52, 47, and 33 C in solarized soil, respectively. Solarization reduced annual bluegrass seed survival from 89 to 100% in the upper 5 cm of soil, but did not reduce survival below 5 cm. Solarization may have enhanced seed survival below 5 cm. Cover crops of barley, rapeseed, and sudangrass generally increased survival of annual bluegrass seeds buried 2.5 to 15 cm deep in the soil. Green manure cover crops plus solarization did not improve the efficacy of solarization alone and in some cases diminished the effectiveness of solarization. Solarization significantly improved the efficacy of one-quarter rates of metham (230 L/ha) in the top 5 cm of soil, reducing overall annual bluegrass seed survival in the soil by 40% compared with metham alone (230 L/ha) but only 30% compared with solarization alone. The conventional rate of metham (930 L/ha) was the most effective and consistent treatment across all depths.

Members of the agrobacteria–rhizobia complex (ARC) have multiple and diverse plasmids. The extent to which these plasmids are shared and the consequences of their interactions are not well understood. We extracted over 4000 plasmid sequences from 1251 genome sequences and constructed a network to reveal interactions that have shaped the evolutionary histories of oncogenic virulence plasmids. One newly discovered type of oncogenic plasmid is a mosaic with three incomplete, but complementary and partially redundant virulence loci. Some types of oncogenic plasmids recombined with accessory plasmids or acquired large regions not known to be associated with pathogenicity. We also identified two classes of partial virulence plasmids. One class is potentially capable of transforming plants, but not inciting disease symptoms. Another class is inferred to be incomplete and non-functional but can be found as coresidents of the same strain and together are predicted to confer pathogenicity. The modularity and capacity for some plasmids to be transmitted broadly allow them to diversify, convergently evolve adaptive plasmids and shape the evolution of genomes across much of the ARC. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Rhodococcus fascians is a phytopathogenic actinobacterium which causes leafy galls and other plant distortions that result in economically significant losses to nurseries producing ornamental plants. Traditional assays for detection and identification are time-consuming and laborious. We developed a rapid polymerase chain reaction (PCR) diagnostic assay based on two primer pairs, p450 and fas, which target the fasA and fasD genes, respectively, that are essential for pathogenicity. We also developed a faster, more convenient, loop-mediated isothermal amplification (LAMP) assay targeting the fasR gene, which regulates expression of virulence genes. Both assays were evaluated for sensitivity and specificity in vitro and in planta. The p450 and fas primers amplified DNA only from pure cultures of pathogenic reference isolates of R. fascians. Nonpathogenic isolates and 51 other plant-associated bacteria were not amplified. The PCR primers correctly detected pathogenic R. fascians from 73 of 75 (97%) bacterial strains isolated from naturally infected plants. The PCR assay correctly discriminated between pathogenic R. fascians and other bacteria in 132 of 139 (95%) naturally infected plants, and in 34 of 34 (100%) artificially inoculated plants. The fas primers were slightly more accurate than the p450 primers. The LAMP assay accurately detected pathogenic R. fascians in 26 of 28 (93%) naturally infected plants and did not react with 23 asymptomatic plants. The LAMP primers also amplified product for DNA extracts of 40 of 41 bacterial strains isolated from plants with leafy galls. The detection limit of both the PCR and LAMP assays was approximately 103 CFU/30-μl reaction. These new tools allow fast, reliable, and accurate detection of R. fascians in vitro and in planta. The LAMP assay in particular is a significant advancement in rapid R. fascians diagnostics, and enables those with limited laboratory facilities to confirm the presence of this pathogen in infected plants.