Research on infection by S.subterranea and host resistance to powdery scab Alison Lees
PMTV/ S. subterranea infection experiment Jennie Brierley, Alison Roberts, Alison Lees
PMTV/Spongospora Zoospores of S.s. acquire PMTV from an infected root or tuber and transmit the virus to new host plants. The mechanism is not known It is not known whether PMTV muliplies within Ss zoospores Plant viruses don t move to neighbouring cells through the plant cell wall; they encode movement proteins that enable passage of the viral genome through intercellular communication channels (plasmodesmata) PMTV can spread from seed tubers to foliage and progeny tubers Davey et al (2013) showed that direct infection of progeny tubers with PMTV from soil inoculum is more important than vertical transmission to progeny tubers
Detection of Ss and PMTV in roots and tubers Know from our previous work that: There is no effect of cultivar (Agria and Nicola) on the amount of Ss or PMTV detected in roots. More PMTV detectable in tubers of Nicola compared with Agria Confirms that PMTV is able to multiply independently in tubers Can PMTV move from root infections to foliage?
Aim To determine whether root infections caused by viruliferous S.s. are confined to root/stolons in contact with inoculum or whether the vector and/or virus are then transmitted throughout the plant
Experimental set-up 3 x cultivars (Cara, Nicola, Gladiator) 5 x reps 2 x blocks (Cabinets) 30 plants in total
Hydroponic potato culture Spongospora maintained in a constantly circulating (increasing) system. Inoculum added Stolons and tubers form above barrier and are sandwiched between plastic to keep dark Nutrient-Film Technique (NFT) Hydroponics
Roots, stolons and tubers in hydroponics
Sampling For each plant Above barrier all tissue samples removed and placed in labelled bags Leaves bulked, Stolons bulked, Tubers individual Below barrier all tissue samples rinsed thoroughly in running water before blotting dry and bagging Roots bulked, Stolons bulked, Tubers individual All material tested for the presence of S.s. and PMTV using a combined NA extraction method and real-time PCR assays.
Preliminary Results PMTV detected in roots of all 30 plants 24/29 plants with stolons below the barrier had detectable PMTV No significant difference in the amount of detectable PMTV between cultivars. 23/30 plants produced stolons above the barrier, 5/23 had detectable PMTV (4 Cara, 1 Nicola).
Preliminary Results Ss detected in roots of 30/30 plants Significantly more Ss detected in Cara than in Gladiator. 29/29 plants which produced stolons below the barrier had detectable Ss. More Ss detected in Cara than in Nicola or Gladiator but not significant. 16/23 plants which produced stolons above the barrier had detectable levels of Ss - amount was much lower than in below barrier stolons no significant difference between cultivars.
Current Experiment is being repeated to see whether results are consistent or could be due to leakage in the system An absorbent layer added to try to minimise chance of contamination
Powdery scab resistance Alison Lees Brierley, J.L.; Sullivan, L.; Hilton, A.J.; Wale, S.J.; Hilton, A.J.; Kiezebrink, D.T.; Lees, A.K. (2013) Relationship between Spongospora subterranea f.sp. subterranea soil inoculum level, host resistance and powdery scab on potato tubers in the field. Plant Pathology, 62, 413-420.
Root and tuber infection in S.phureja Know from previous work that diploid S. phureja clones very resistant to powdery scab Never able to demonstrate heritability in crosses with S. phureja parents - too many resistant clones Tested a range of S. phureja clones for resistance Have a well characterised diploid population (06H1) Prashar et al (2014). Dense SNP-based linkage map. Use linkage map in combination with phenotypic assessments to perform QTL analysis of traits Marker assisted selection?
DB.378 (1) Gladiator DB.271 (39) DB.161 (10) DB.270 (43) HB.171 (13) DB.441 (2) DB.168 (11) DB.375 (1) 71.T.6 71.P.10 DB.337 (37) DB.358 (23) DB.207 (35) PHU.951 (901) DB.377 (4) DB.244 (37) DB.354 (901) 842.P.75 851.T.8 ExMS86(13) DB.226 (70) DB.358 (30) 81.S.66 DB.333 (16) 80.CP.23 DB.323 (3) 71.T.46 DB.199 (10) HB.165 (1) DB.358(24) DB.170 (35) DB.299(39) DB.175 (5) DB.257 (28) PHU.950 (412) DB.375 (2) 99 FT 1B5 Nicola Agria Mean Composite Disease Score (Incidence x severity) Resistance screening of S. phureja clones for p.scab 350 300 250 200 150 100 50 0
06H1 Population Mayan Gold Susceptible parent Resistant parent
Testing 06H1 population Disease nursery 100 06H1 clones + parents + grandparents + Agria/Gladiator Scored disease at harvest
06H1 powdery scab scores Resistant parent Gladiator Susceptible parent Agria
06H1 QTL? Unfortunately no QTLs identified Many very resistant S. phureja clones Long day adapted S. phureja as parents for breeding for powdery scab resistance? Same clones have resistance to other diseases
Infection v disease in S.phureja and S.tuberosum Assess whether root and tuber infection is same in P.scab resistant S.phureja (diploid grandparents of 06H1) DB337(37) = Mayan Gold DB226(70) S. phureja x S. tuberosum parents of 06H1 with differing resistance HB171(13) = Resistant 06H1a83 (replacing 99FT1b5) = susceptible P. scab resistant (Gladiator) and susceptible S. tuberosum (Agria). Artificially inoculated land, 4 reps, 5 tuber plots 1 plant sample from each plot at tuber Initiation + 2 weeks assessed for root galling Remaining plants at harvest (visual symptoms testing roots and tubers with RT PCR)
06H1 Population Mayan Gold
Mean Root Gall Score (0-4 scale) 13 th July Root galling 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Gladiator 06H1a83 HB171(13) DB337(37) - Mayan Gold DB226(70) Agria
ng DNA/gfwt Root DNA Ss 45000.0 S.s. DNA detected in roots 40000.0 35000.0 30000.0 25000.0 20000.0 15000.0 10000.0 5000.0 0.0 Gladiator 06H1a83 HB171(13) DB337(37) DB226(70) Agria Large STDEV
ng DNA/gfwt Tuber DNA Ss 25000000.0 S.s DNA detected from tubers 20000000.0 15000000.0 10000000.0 5000000.0 0.0 Gladiator 06H1a83 HB171(13) DB337(37) DB226(70) Agria Large STDEV
Other projects: Poptical Aim to detect early disease symptoms To identify pathogen hotspots for soil-borne pathogens Powdery scab is included but is not a major target Plant Plot 1 Agria Plot 2 M. Piper 12 Agria M.Piper 11 10 279 B250 273 273 288 286 340 308 297 6.02 3.19 4.94 4.38 23.15 0.91 5.41 7 9 8 7 264 303 B164 269 287 277 310 253 270 0.92 2.37 3.24 0.25 1.02 2.22 0.72 5.44 6 5 4 252 280 288 272 286 272 273 B263 265 4.65 2.04 7.17 4.13 1.56 31.95 1.04 6.25 3 2 1 253 265 278 250 206 246 265 289 1.84 2.49 1.78 1.27 3.83 0.77 1 2.3
The Centre for Sustainable Cropping (CSC): The James Hutton Institute
Balruddery rotation & field layout Potato Winter Barley S S C C S Road field Mid East Den South C Winter OSR Winter wheat Field Beans Spring Barley S C S C C S Pylon Kennels Estate
Crop cultivars and sustainable treatment Reduced inorganic fertilizer Reduced herbicide application Reduced fungicide/pesticide application
Data collection On-site meteorological station providing continuous measurements of air temperature, precipitation, wind speed and direction, and solar radiation. Soil temperature, moisture content, microbial and invertebrate activity, nutrient and water uptake by plants and plant growth are measured alongside nutrient leaching and GHG emissions Crop yield & quality Nutrient budgets Soil structure Biodiversity Met station
Seed inoculum Disease assessed visually, symptomless infections identified with real-time PCR. Soil inoculum Inoculum levels in soil are determined pre-planting and post-harvest. The persistence of inoculum in the soil is being tracked through the rotation.
Lady Balfour Mayan Gold Vales Sovereign Cabaret Maris Piper Balruddery rotation & field layout Potato Spring Barley S S C C S Road field Mid East Den South C Field Beans Winter wheat Winter OSR Winter barley S C S C C S Pylon Kennels Estate
Incidence of silver scurf on progeny (%) Incidence of common scab on progeny 100 80 60 40 20 0 MP MP MP 0 20 40 60 80 100 Incidence of common scab on seed Seasonal and cultivar differences 2011 2012 2013 2014 2015 100 80 60 40 20 0 2011 2012 2013 2014 2015 0 20 40 60 80 100 Incidence of silver scurf on seed (%) No soil inoculum detected, seasonal effect
Acknowledgements Jennie Brierley Louise Sullivan James Lynott