Progress 10/01/14 to 09/30/18
Outputs
Target Audience:The target audience for this project is primarily other researchers seeking to understand and control brown rot (bacterial wilt) of potato caused by Ralstonia solanacearum. A secondary taget audience are federal quarantine authorities (USDA-APHIS-PPQ working to prevent introduction of select agent Race 3 biovar 2 strains of R. solanacearum. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate student Alicia N. Truchon successfully obtained her MS degree in Plant Pathology in August 2017. She is now enrolled in the UW-Madison Microbiology Doctoral Training Program. She continues working in my lab, focusing on the biology of latent infection and tuber transmission in Race 3 biovar 2 strains of R. solanacearum in contrast to the biologically distinct potato brown rot strains from Madagascar. Research Intern Madeline Hayes also contributed to research project. This experience strengthened her applications to graduate school; she has been accepted to several Ph.D. programs for Fall 201,9 but has not yet made a choice. How have the results been disseminated to communities of interest?In addition to the publications listed under "Products", results from this project were presented last year as invited talks to the following groups: "How the bacterial wilt pathogen Ralstonia solanacearum deceives and exploits its host plants" Invited keynote talk at PNIA Symposium on Phytophthora infestans and Ralstonia solanacearum, major threats to potato. International Potato Center (CIP) in Lima, Peru. March 13, 2018. "Hungry and stickyorfat and vicious? Strategic switches inthe bacterial plant pathogen Ralstonia solanacearum". Invited seminar to University of Tennessee-Knoxville Dept of Microbiology April 2, 2018. "Life beyond the crop: exploring the roles of non-agricultural habitats in epidemiology and plant health. Phytoviews Symposium Moderator, International Congress of Plant Pathology & American Phytopathological Society Annual Meeting, Boston, MA, July 29-August 3, 2018. "How Ralstonia solanacearum manipulates and exploits the flowing plant xylem environment" Invited keynote talk to XanthoMeeting International, Sao Paulo, Brazil, September 5-6 2018. "How Ralstonia solanacearum manipulates and exploits the flowing plant xylem environment". Invited seminar to University of Illinois-Urbana Department of Crop Sciences. October 17, 2018. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
American strain K60 makes several proteins, called bacteriocins, that inhibit other R. solanacearum strains, including a strain called Race 3, which causes potato brown rot and geranium wilt. Race 3 is listed as a federal Select Agent quarantine pathogen because it poses a serious threat to U.S. agriculture. Strain K60 outcompetes Race 3 in both stems and rhizospheres of tomato plants. This project looked at how to better understand the role of bacteriocins in competitive fitness and exclusion of this major pathogen. We accomplished Aims 1 and 2 as described in previous reports. For aim 2, we used two defined mutant strains lacking these bacteriocins to show that the two RHS-family proteins are essential for the competitive advantage of native (endemic) American strain K60 in plants. Mutants had lost their ability to outcompete Race 3 biovar 2 strains in roots or stems of tomato plants. As explain in the the 2017 report, we could not overcome technical obstacles to overexpressing bacteriocins because these toxins were acutely toxic when expressed even in other bacteria such as E. coli. We therefore pivoted to a related emerging problem, the introduction and rapid expansion of R. solanacearum R3bv2 into Madagascar in the past 10 years. Local reports show thatfor decades endemic R. solanacearum strains have been causing low levels of crop losses to potato brown rot in Madagascar highlands, where potatoes are an important food security crop. However, brown rot incidence increased dramatically beginning around 2006 and this disease is now the most destructive disease of potatoes in the highlands. We analyzed strains collected in a survey by collaborator Philippe Prior (CIRAD La Reunion, France). We found both African (phylotype IIII ) strains of R. solanacearum and typical R3bv2 strains, which originated in the Andes along with the potato. We initially hypothesized that African strains were directly out-competed by R3bv2 strains, possibly via a bacteriocin-mediated exclusionb. Direct competition experiments and inhibition assays in vitro did not support this hypothesis. We then hypothesized that the African brown rot strains were less able to cause latent infections, especially to infect potato seed tubers. Phenotypic analysis under growth chamber conditions showed that the African brown rot strains rarely form latent infections of tubers. This could explain their slow spread and moderate impact relative to the aggressively-spreading R3bv2 strains. Comparative genomic analysis of African and R3bv2 strains of R. solanacearum are currently underway. These may help us identify pathogen genes that correlate with the ability to form latent infections and be tyransmitted in seed tubers.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Dalsing BL*, AN Truchon*, D Khokhani, T Lowe-Power, A MacIntyre, B McDonald, F Ailloud, R Anex, J Klassen, ET Gonzalez-Orta, C Currie, P Prior, and C Allen. 201x. Subgroups in the Ralstonia solanacearum species complex use divergent respiratory strategies to grow in tomato xylem. Applied and Environmental Microbiology, accepted pending revision.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
How the bacterial wilt pathogen Ralstonia solanacearum deceives and exploits its host plants Invited keynote talk at PNIA Symposium on Phytophthora infestans and Ralstonia solanacearum, major threats to potato. International Potato Center (CIP) in Lima, Peru. March 13, 2018
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Target audiences are: The community of R. solanacearum researchers and regulatory agency scientists:This year, we communicated projectfindings of complete genome sequences as a peer-reviewed journal article (Hayes et al 2017). Ornamental growers who contacted me informally for diagnostic testingand disease management advice. Changes/Problems:As described above, we encountered technical difficulties expressing R. solanacearum bacteriocin proteins in E. coli. They appear to be highly toxic. This, combined with the interesting field data from Madagascar, caused us to shift the project focus slightly towards understanding inter-strain competition in this more practical context. What opportunities for training and professional development has the project provided?Graduate student Alicia N. Truchon, who is supported by the project, successfully defended her MS in Microbiology in August 2017. In September 2017, Alicia enrolled in the highly competitive Microbiology Doctoral Training Program (MDTP) here at UW-Madison. She is continuing to work on this project. Research intern Madeline Hayes (not directly supported by the project) was also active in the genome sequencing efforts. She will be applying to graduate school next year. How have the results been disseminated to communities of interest?By publication in the peer-reviewed literature: Hayes, M. M., A. M. MacIntyre, and C. Allen. 2017. Complete genome sequences of the plant pathogensRalstonia solanacearumtype strain K60 andR. solanacearumRace 3 biovar 2 strain UW551. Genome Announcements 5: e01088-17 What do you plan to do during the next reporting period to accomplish the goals?Our goals for the next year are: 1) to characterize the virulence and etiology of the Madagascar (phyl III) potato brown rot strains of R. solanacearum. Specifically, we want to determine their competitive fitness and transmissibility relative to R3bv2 (phyl II) Andean potato brown rot strains. 2) To write up and submit a paper describing the genetic basis of bacteriocin production by highly competitive R. solanacearum strains.
Impacts
What was accomplished under these goals?
Despite repeated efforts to express the strain K60 bacteriocins in E. coli, we have not been successful. The most likely explanation is that these proteins are highly toxic to E. coli. We have decided to write up and publish the results to date. Closed R. solanacearum genome sequences. Genetic analysis of bacterial wilt virulence and detection has been hampered by the poor-quality genome sequences generated with previous-generation technologies. Research intern Madeline Hayes therefore used single molecule read technology (SMRT, PacBio) to generate complete closed genome sequences for R3bv2 Select Agent strain UW551 and for K60, the R. solanacearum type strain that was isolated in 1952 by Arthur Kelman in North Carolina. These will save time and effort for the Ralstonia research and regulatory communities. Metabolic diversity and competitive fitness. This year, we expanded our analyses of differences among the diverse strains of Ralstonia solanacearum in order to better understand the traits associated with their competitive fitnessand persistence in agricultural environments. Genomic data suggest that the R. solanacearum species complex (RSSC) falls into four distinct phylotypes that correspond to geographic origins. Among other differences, the four phylotypes appeared to have strikingly distinct capacities for anaerobic respiration using nitrate as an alternative electron acceptor. Nitrate is abundant in some agricultural soils and is also present in xylem sap, the niche ofthis pathogen inside its host plants.To determine the biological relevance of this behavior, we conducted a comparative analysis of inorganic nitrogen metabolism in the four phylotypes of the RSSC usingarepresentative strain fromeach phylotype that can all infectashared host, the economicallyimportant crop plant tomato. We found that strains in RSSC phylotypes II and IV (from the Americas and Indonesia, respectively) cannot complete denitrifying respiration.Interestingly, this correlates perfectly with their behavior in culture, where they grow poorly in nitrogen-rich but low-oxygen environments and move activelyup an oxygen gradient. Further, scanning electron microscopy of tomato plants infected with phylotype II andIV strains revealed that they tend to swim freely in host xylem sap or form single-cell layers on xylem walls. In contrast, strains from phylotypes I and III (from Asia and Africa), which can complete denitrifying respiration and are not attracted to higher oxygen environments in culture, form thickaggregates inside host xylem. The interiors of these aggregates are presumably hypoxic or anoxic. We are excited about these findings because they suggest that closely-related pathogen strains may exploit physically distinct micro-niches inside a common host.This work will besubmitted for publication in 2018. Strain competition and displacement in Madagascar. In parallel, we have initiated a study in response to aninteresting real-world observation. Potato bacterial wilt has long been a minor problem in the highland potato-growing regions of Madagascar. The disease there is caused by phylotype III strains of R. solanacearum, which havebeen present on the island for decades or longer. However, around 2006, an R3bv2 strain was introduced and it has rapidly spread and appears to be replacing the native African strains.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Hayes, M. M., A. M. MacIntyre, and C. Allen 2017 Complete genome sequences of the plant pathogens Ralstonia solanacearum type strain K60 and R. solanacearum Race 3 biovar 2 strain UW551. Genome Announcements 5: e01088-17
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One student (Alicia Truchon) will obtain her Masters degree in microbiology, with specific expertise in agricultural microbes and microbial ecology. She is expected to defend her thesis in summer, 2017. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the next year we plan to: 1) Complete the last experiments to demonstrate that RHS proteins specifically confer competitive fitness of certain R. solanacearum strains during co-infection of host plants. 2) Submit this research for publication in a peer-reviewed science journal. 3) Continue efforts to overexpress functional bacteriocin proteins in E. coli and/or plant hosts of R. solanacearum, such as potato.
Impacts
What was accomplished under these goals?
During this reporting period, we focused primarily on Major Goal 2(Determine the role of bacteriocins in the competitive fitness ofR. solanacearum). Screening a transposon mutant library of endemic native American R. solanacearum strain K60 identified five mutants that had lost the ability to inhibit other R. solanacearum strains. Four of the five contained insertions in two adjacent RHS-family genes that typically encode bacteriocins and toxins in other microbes. We used two defined mutant strains lacking these bacteriocinsto show that the two RHS-family proteins are essential for the competitive advantage of native (endemic) American strain K60 in plants. Mutants had lost their ability to outcompete Race 3 biovar 2 strains in roots or stems of tomato plants. To explore the feasibility of using bacteriocins to control potato brown rot and exclude Race 3 from imported geranium cuttings, by Constructing and testing bacteriocin-expressing potato and geranium lines (Major Goal 3).Alicia has been working to clone and over-express active bacteriocins in the lab bacterium Escherichia coli. This has been challenging. She has expressed the cloned proteins in E. coli, but they do not have inhibitory activity like they do in the native R. solanacearum background.We are working on alternative strategies to produce active, functional proteins in a heterologous background.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:We published one peer-reviewed paper and presented an oral report of progress on this project to the target audience of researchers in the area of plant pathology and applied environmental microbiology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One graduate student, Alejandra I. Huerta, completed her Ph.D. with support from this project. Dr. Huerta is currently doing postdoctoral research in the lab of Dr. Jan Leach at Colordao State University, with support from an NSF Postdoctoral Fellowship. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the coming year, we hope to complete experients to accomplish Major Goal 2, Determine the role of bacteriocins in the competitive fitness of R. solanacearum on host plants, using defined mutant strains. To explore the feasibility of using bacteriocins to control potato brown rot and exclude Race 3 from imported geranium cuttings, we will initiate Major Goal 3, Construct and test bacteriocin-expressing potato and geranium lines. This funding will support a new MS student, Ms Alicia Truchon, who is continuing this project. She is currently working to express solanacin proteins in E. coli to determine if the purified proteins can inhibit growth of R. solanacearum strain R3bv2. In addition, she is developing techniques for potato transformation with technical support from our collaborator, USDA researcher Dr. Dennis Halterman.
Impacts
What was accomplished under these goals?
During this first year of the project, we made significant progress on the first two goals. We essentially completed Major Goal 1 (Characterize the biological functions of K60's bacteriocins). These findings were disseminated in a paper published in the peer-reviewed journal Applied and Environmental Microbiology. A brief summary of the motivation, findings, and implications of this research follows: Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands. We initiated research on Major Goal 2 (Determine the role of bacteriocins in the competitive fitness of R. solanacearum on host plants, using defined mutant strains). To determine if solanacins can explain differences in strain fitness, we screened a K60 mutant library for loss of inhibitory ability. This screen identified a locus encoding two secreted Rhs-repeat domain proteins, named SinA and SinB. Rhs proteins mediate intercellular competition in microbes. In addition to losing the ability to inhibit other R. solanacearum strains in culture, sinA and sinB mutants also could no longer outcompete R3bv2 strain UW551 in tomato plants. Complementation of these mutants restored inhibition in culture and competitive fitness in plants. These results indicate that solanacins are the mechanism of K60's high interstrain competitive fitness and may explain why R3bv2 strains have not become established in the lowland tropics. Expression of sinA and sinB was positively controlled by the quorum sensing-mediated virulence regulators PhcA and VsrAD. Bioinformatic analyses revealed that the genomes of 27 diverse R. solanacearum strains each encode three or more Rhs proteins. Parallel functional assays showed that 20 of 27 tested strains inhibited growth of at least one other strain in culture. This suggests that diversifying selection has generated distinct inhibitor repertoires in related strains that compete to occupy the same niche.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Huerta, A. I., F. Ailloud, and C. Allen. 2015. Identification and characacterization of interstrain chemical weapons of diverse Ralstonia solanacearum pathogens. Phytopathology 105(Suppl. 4):S4.162
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Huerta, A. I., A. S. Milling, and C. Allen. 2015. Tropical strains of Ralstonia solanacearum outcompete Race 3 biovar 2 strains at lowland tropical temperatures. Applied and Environmental Microbiology 81:3542-3551
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