Clubroot Research Update: What have you done for me lately? Bruce D. Gossen AAFC, Saskatoon Research Centre Canola Industry Days, Saskatoon, December 2, 2015
Management Strategies Few management options available when clubroot first appeared. Long rotations out of Brassica crops. Equipment sanitization. These strategies were not popular with farmers and not widely adopted. R.J. Howard, AARD
2015
A B C D
Virulence Patterns on Differentials Genotype Clubroot reaction: (+) Susceptible, (-) Resistant Nine distinct virulence phenotypes detected Westar + + + + + + + + + 45H29 - - + + + + + + + Mendel - - - - + - + + - ECD 02 - - - - - - - - - ECD 05 + + + + + + + + + ECD 06 + + - - + + + + + ECD 08 + + + + + + + + + ECD 09 + + - - + + + + + ECD 10 - - - - - - - - - ECD 11 - - - - - - - + - ECD 13 + - - + + + + + - Laurentian + - - - + - + + + Williams 3 5 5 6 3 6 3 2 8 Some et al. P2 P2 P3 P3 P2 P2 P2 P2 P2
Map resistance genes, develop markers Genetic distance (cm) 2.53 1.80 3.64 1.21 A3 Marker ss2093 sr12302i sr6340i sj5076 sb4889b Mapping of clubroot resistance gene Rcr1 (Chu et al. 2013) 24.02 24.03 24.04 24.09 24.10 24.11 24.13 24.16 24.17 24.19 24.26 24.32 24.34 24.35 24.36 24.37 24.40 24.42 24.48 24.50 A3 A3S020 A3S030 Bra038790 Bra038789 Bra038788 Bra038778 Bra038777 Bra038776 Bra038774 Bra038771 Bra038768 Bra038766 Bra038753 Bra019413 Bra019412 Bra019411 Bra019410 Bra019409 Bra019407 Bra019405 Bra019401 Bra019398 Bra019394 Bra019391 sn8591 Resistance genes identified: Rcr1, Rcr2 A03 Rcr3 (B. rapa) A08 Rcr4 (B. nigra) B05 Rcr5 to 8 in progress q AAFC is forming a consortium with several major seed companies q Aim: To develop eight CR genes (Rcr1 Rcr8) for resistance breeding. q Several of these genes are resistant to (at least) some pathotype X collections.
Rotation reduces clubroot impact on CR canola cultivars Break (yr) spores/g soil 0 2.7 x 10 6 bc 1 2.9 x 10 6 c 2 5.7 x 10 4 a 3 2.1 x 10 5 ab 4 1.1 x 10 5 ab A >2-year break from canola lowers pathogen inoculum in soil and increases canola yield! Even the yield of resistant cultivars can be increased substantially
Spore conc. after Susceptible Canola Spores g -1 soil (log scale) 1.E+08 1.E+07 1.E+06 y =1E+07e -0.76x R² = 0.65 1.E+05 0 1 2 3 4 5 6 Break following a susceptible crop (years)
Spore conc., Resistant Canola Crop Spores g -1 soil (log scale) 1.E+09 1.E+08 1.E+07 1.E+06 Spring (pre-plant) Fall (post-harvest) 1.E+05 No break 1-yr break 2-yr break
Resting spore conc. and ph 100 90 5.5 6.0 6.5 7.0 7.5 Incidence (%) 80 70 60 50 40 30 20 10 0 1x10^3 1x10^5 1x10^7 Spores g -1 soil
Resting spore concentration, Flamborough, ON Spores g -1 (x10 3 )
Vertical distribution q Resting spores of P. brassicae were present at 30 100 cm depth at each of four established sites. Likely move downward (and back up?) in ground water. q Resting spores were found only near the surface at two sites where infestations were recent. q Do spores deep in the soil affect canola production? NO! q Are they viable? Studies underway. q Major implications for industries involved in excavation or movement of soil (e.g., oil & gas, highway construction).
Multiplex qpcr for detection and quantification of resting spores in soil Location & Sample Initial analysis Resting spores/g Analysis following dilution * CIPC C t Dilution CIPC C t Spores/g Corrected Quebec 23-1 No Amp. 1:10 1.2 Not detected - 23-4 NA 1:10 1.9 2.66 x 10 6 8.78 x 10 6 28-3 NA 1:10 2.6 1.23 x 10 6 6.15 x 10 6 29 NA 1:10 2.3 5.51 x 10 4 2.64 x 10 5 Drapeau NA 1:10 0.4 3.13 x 10 3 3.64 x 10 3 Edmonton Boron trial-1 NA 1:10 2.1 4.72 x 10 4 1.42 x 10 5 Boron trial-2 NA 1:10 2.3 7.64 x 10 4 3.06 x 10 5 MCRS 1 NA 1:10 2.4 2.3 x 10 4 1.17 x 10 5 2 NA 1:10 1.7 8.1 x 10 4 2.10 x 10 5
CIPC qpcr q One-step rapid TaqMan assay for detection and quantification of P. brassicae DNA in soil. q More accurate than standard method. q Sensitivity similar to standard (~800 spores/g) but more likely to detect false negatives. q No need for extra run - saves time & resources. q Suitable for wide range of soil types.
Fumigation trial, MCRS, 2015 0 3.3 1.1 3.3 3.3 11.1 3.3 7.8 7.8 60 48.9 56.7 21.1 26.7 31.1 10 35.6 48.9 3.3 12.2 10 15.6 71.1 28.9 15.6 45.6 67.8 20 64.4 74.4 36.7 25.6 20 18.9 66.7 48.9
Gossen s Recipe for Eradicating Clubroot Identify and mark infested area Symptomatic plants / spores in soil samples. Mark x2 affected area (at least!) in every direction. Exclude all traffic from marked area. Initial treatment Fumigate and cover, or incorporate lime to ph 7.5. Seed to sod-forming grass. When a strong sod is established, traffic allowed. Evaluation and termination Use soil sampling to monitor spore conc. When no longer detectable, break sod. Use only clubroot-resistant cultivars.
Mechanism of clubroot resistance Using transcriptomic, proteomic and metabolomic analyses to investigate the mechanisms of clubroot resistance genes. Identifying biological processes related to the modes of action of specific R genes. Pyramiding and rotating R genes with different action modes in canola breeding and production. Transcriptome analysis RNA sequencing
Research Priorities (Steering Group) 1. Develop multiple sources of resistance. 2. Develop reliable screening techniques to assess durability of new forms of resistance. 3. Identify and categorize new pathotypes. 4. Develop a differential set. 5. Molecular techniques for pathotyping. 6. Biology and physiology of clubroot. 7. Cultural control methods. 8. Surveys and mapping.