NMR profiling of sour cherry in screening for leaf spot resistance Martin Jensen, Morten Rahr Clausen, Hanne Bertram, Hanne Lindhard Pedersen GUDP Danish Ministry of Food, Agriculture and Fisheries EU COST sour cherry meeting Dresden 13-16 July 2015
Project overview Funding: GUDP (Ministry of Food, Agriculture and Fisheries) Project period: January 2014 - december 2016 Funding theme: Development of cultivars for organic fruit production Partners: AU FOOD Science Plants-fruits-disease: Martin Jensen NMR analysis: Morten Rahr Clausen (Hanne Bertram). Horticultural Advisor: Hanne Lindhard Pedersen Fruit tree nursery: Westerskovgaard Nursery Organic sour cherry grower: Hedegaard Orchard Conventional sour cherry grower: Frederiksdal Estate
Objective and purpose Identification and development of sour cherry cultivars for organic production in DK. Hypothesis: A cocktail effect of a number of antifungal compounds together provide resistance to attack by leaf spot fungal disease Blumeriella jaapi and or Monilia laxa. Investigating if NMR metabolomics profiles of sour cherry leaves can predict resistance to cherry leaf spot disease and which compounds are involved. (using multivariat PLS analysis) Danish breeding pool of almost 3000 crosses in sour cherry Include invited samples from EU COST network Cherry: Leaf samples from most leaf spot resistant cultivars and breeding material from Germany, Hungary, Romania and Serbia
Year 1. AARHUS UNIVERSITET Project tasks Identifying clones with different resistance/tolerance to cherry leaf spot and Monilia laxa Develop method to quantify leaf spot attack on leaves with digital picture analysis Year 2. Year 3. Develop methods to ensure correct resistance evaluation field/lab methods Develop multivariate model that predict resistance from specific NMR profile metabolites. Validate model on new samples Propagate selected clones by grafting in nursery company Analyse leaf (spring and autumn) and flower samples with NMR Identify compounds that may corellate with resistance/ tolerance. Use model to identify and check resistance of most interesting clones for organic production Establish clones in organic trials at 2 growers, to evaluate them over the coming years.
Population Field 25 Population Field 26 UNIKT løbenr AARHUS UNIVERSITET Prøvenr indeholder Træid nr. 1 25_1 2501134 2 25_2 250118 3 25_3 250124 4 25_4 250146 5 25_5 250148 6 25_6 250161 7 25_7 250166 8 25_8 250188 9 25_9 250190 10 25_10 2501100 11 25_11 2501110 12 25_12 2501136 13 25_13 2501178 14 25_14 2501165 15 25_15 2502136 16 25_16 2501122 17 25_17 2502112 18 25_18 2501139 19 25_19 2501103 20 25_20 250233 21 25_21 250354 22 25_22 250375 23 25_23 2503130 24 25_24 2504151 25 25_25 250467 26 25_26 250351 27 25_27 250401 28 25_28 250536 29 25_29 2505118 30 25_30 2504150 31 25_31 2505143 32 25_32 2505101 33 25_33 250533 34 26_1 260155 35 26_2 260180 36 26_3 260191 37 26_4 2601148 38 26_5 2601168 39 26_6 2601177 40 26_7 2601224 41 26_8 2601240 42 29_9 2601239 43 26_10 2601221 44 26_11 2601205 45 26_12 260196 46 26_13 260106 47 26_14 260203 48 26_15 260211 49 26_16 260250 50 26_17 260254 51 26_18 2602104 52 26_19 2602132 53 26_20 2602142 54 26_21 2602161 55 26_22 2602178 56 26_23 2602188 57 26_24 2602224 58 26_25 2602226 59 26_26 2602228 60 26_27 mangler 61 26_28 2602215 62 26_29 2602206 63 26_30 2602185 64 26_31 2602126 65 26_32 2602122 66 26_33 260264 67 26_34 260220 68 26_35 260210 69 26_36 260347 70 26_37 260383 71 26_38 2603191 72 26_39 2603207 73 26_40 2602236 74 26_41 2603244 75 26_42 2603232 76 26_43 260453 77 26_44 260302 78 26_45 260420 79 26_46 260496 80 26_47 2604111 81 26_48 2604167 82 26_49 2604199 83 26_50 2604215 Strong attack Birgitte Viki Nefris Sumadinka Fanal European cultivars /clones Romania HV 45/40 HV 43/32 HV 16/18 Feketicka Spanska Futoska Fruskogors ka Reference cultivars/species P.a. Kordia Prunus incisa Resistant or almost Prunus subhirtella Prunus serrulata 'Shirotae' VI.P. 5/41-44 VI.H. 7/67-68 VI.P. 2/41-44 VI.P. 5/37-40 Serbia Hungary M221 x Csengődi Érdi bőtermő x Csengődi Pándy 279 x Csengődi Érdi bőtermő x Csengődi VI.H. 7/141 Érdi bőtermő x Csengődi VI.H. 9/91 Érdi bőtermő x Csengődi Germany
Leaf samples from Dresden, Germany No. Genotype Ploidy Parentage Rootstock Accession-No. Type Place Leaf spot Monilia blossom blight 1 P. canescens 2x Piku 1 KZ-PRU0037 species DK1,A,35 resistant tolerant DK1,A,36 2 F5,18,167 2x P. avium M53 x P. canaescens P. avium KZP86-1 F1 clone DK1,1,51 resistant DK1,1,52 3 DK1,2,25 2x Namati x F5,18,167 own root DK1,2,25 F2 clone DK1,2,25 resistant 4 DK1,2,4 2x Namati x F5,18,167 own root F2 clone cultivar DK1,2,4 resistant 5 Achat 4x Köröser x B7,2,40 (Fanal x P. avium KZC82-1 cultivar DK1,4,35 susceptible tolerant Kelleriis 16) DK1,4,36 6 Coralin 4x Kelleriis 16 x (Köröser x P. avium KZC83-1 cultivar DK1,4,70 tolerant susceptible Schattenmorelle) DK1,4,71 7 P. maackii 4x Piku 1 KZP02-2 species DK1,A,37 resistant resistant DK1,A,38 8 DK2,3,105 4x Schattenmorelle x P. maackii P. avium DK2,3,105 F1 clone DK1,5,282 resistant susceptible DK1,5,283 9 DK2,3,106 4x Schattenmorelle x P. maackii P. avium DK2,3,106 F1 clone DK1,5,284 resistant susceptible DK1,5,285 10 DK1,5,11 4x DK2,3,105 x Fanal own root DK1,5,11 F2 clone DK1,5,11 resistant resistant 11 DK1,5,56 4x DK2,3,105 x Fanal own root DK1,5,56 F2 clone DK1,5,56 resistant susceptible 12 Prunus serotina 4x own root KZP04-2 species DK1,3,26 resistant resistant 13 DK1,8,12 4x P. serotina x F14,7,15 (Köröser x own root Vowi) DK1,8,12 F1 clone DK1,8,12 resistant resistant (?) Thanks to all for valuable contribution!
Sampling (from strongly infected field populations) Leaves: (two times, same clones each time, compare) Autumn 2014 (6-9 October) Attacked old leaves. Purpose to identify clones that will keep the leaves for longest time, be most tolerant to attack and still with large variation in attack by leaf spot. OBS: Increasing resistance with leaf age OBS: Possible induction of resistance compounds after infection (SAR), possible metabolites from fungus. Early summer 2015 (start July) Healthy young leaves Purpose to get young fresh leaves without any infection, get a pure plant chemical signal at the time of late primary infection by ascospores/season conidia spore infection. Flowers (Early June 2015, Monilia) (sampling once, same clones as leaf samples (Clones characterised for attack of Monilia laxa) Could the same compounds give resistance to both Blumeriella jaapi and to Monilia laxa?
Autumn 2014 sampling (6-9 October) NMR chemical analysis Fresh leaves Stored -25 C Freeze dried Ground Stored -80 C WinRHIZO picture analysis or visual analysis Fresh leaves dried in paper press 3-4 days at 30 C Scanned with WinRHIZO 8
Samples for NMR 9
WinRHIZO 20 leaves per plant RÉGENT scanner WinRHIZO software Colour analysis Setup Batch analysis 10
Photoshop CS5 - preparing picture Raw picture After image editing Automatic actions: Adjustment layer Change luminous intensity from 0 to 70 % Select and delete background 11
Photoshop CS5 (ZOOM 200) After Automatic actions: Select the brown necrotic tissue damage - 10% tolerance (one click) Fill with Red colour (one click) Save Ready for analysis 12
WinRHIZO Colour analysis method: Select 3 colour groups (Red Green -White) Select scanning areas and analysis method Save Configuration and Data file 13
WinRHIZO (data file) Data file contains 1700 analysed scanned pictures 266 columns 180298 rows Results % Area of leaf spot attack of whole leaf Area of single spots and number of spots (ImageJ) 14
Visual assessment of attacked area on damaged leaves not adaptable to scanning based on examples of scanned leaves 15
Variation in % area of attack of leaf spot necrosis in autumn leaf samples. Blue dots are mean of 20 leaves pr plant and red dots are max attack for the leaf with highest degree of attack in the same plant.
Scanned mean (of 20 leaves) compared to max area (the leaf with max damage out of 20 examined). Does the max area better describe the potential infection given worst case conditions (high inoculum and optimum weather conditions)
Correlation between field visual evaluation of degree of attack on the tree (necrosis area per leaf and cover in the entire tree crown, 0= no infection, 10= most infected (very strong) and the scanning results for same clones.
Sumadinka Nefris Fanal Birgitte Viki P. subhirtella P. incisa, serrulata, P.a. Kordia Correlation between field evaluation of degree of attack and the % of remaining leaves on the tree (beginning of October). Some plants can keep leaves very late even if they are quite heavily attacked by leaf spot disease. Red spots are selected reference cultivars/other species
Procedure for NMR on ground sour cherry leaves: 3 extraction repeatings of each sample. 1. Weigh 50 mg freezedry finely ground material into 1½ ml eppendorf tube. 2. Add 500 ul 90mM phosphate buffer ph 6,0 3. Add 500 ul MeOH-d 4. Whirlmix thorughly for ca. 1 min. 5. Ultrasound bath for 15 min. 6. Centrifuge for 10 min. v. 15.000 g at room temperature. 7. Transfer 600 ul to NMR tube and analyze at room temperature Tubes can be stored at 0-4 C for few days before measurement, acclimatise at room temperature for half an hour before measuring. Production of buffer: 100 ml 90 mm phosphate buffer ph=6,0: 1,232 g KH2PO4 (R5) 10 mg TSP (NMR4) 400 ul 1M NaOD (40 ul 40% NaOD in 400 ul D2O) Fill up to 100 ml with D2O A 1:1 relation between deuteriated water and deuteriated methanol buffer is known to give a good evaluation of both phenolic compounds and more hydrophilic compounds like carbohydrates and amino acids. 20
NMR analysis 21
Carbohydrates (and other compounds) Fanal Sein Uninfected Infected Sumadinka Uninfected Infected Nefris Uninfected Infected
Phenolics and amino acids Fanal Sein Uninfected Infected Sumadinka Uninfected Infected Nefris Uninfected Infected
Scores on PC 2 (27.87%) Scores on PC 2 (27.87%) PCA of NMR full spectra of selected samples (preliminary data) The same plot (PC1 and PC2) but with different color codes, 3 samples of each cv. Reference cultivar leaves were harvested with both high and low degree of infection Birgitte 9 Samples/Scores Plot of ref_samples Samples/Scores Plot of ref_samples FanalSein 9 0.8 0.6 0 10 5 7 8 95% Confidence Level 0.8 0.6 Incisa 9 Kordia 9 Nefris 9 Serrulata 9 0.4 0.4 Subhirtella 9 Sumadinka 9 0.2 0 0.2 0 Viki 9 95% Confidence Level -0.2-0.2-0.4-0.4-0.6-0.6-0.8-0.8-1 -0.8-0.6-0.4-0.2 0 0.2 0.4 0.6 0.8 1 Scores on PC 1 (31.93%) -1-0.8-0.6-0.4-0.2 0 0.2 0.4 0.6 0.8 1 Scores on PC 1 (31.93%) Coloured according to the degree of field infection (1-10). Within the reference cultivars (left part of the plot) it was possible to see difference between infected and uninfected/low infected samples per cultivar. Normalized, mean centered and pareto-scaled. Colour according to cultivar or species. Along PC1 samples are separated according to cultivar/species, with the sour cherry reference cultivars on the left hand side and the other cherry species on the right hand side.
Scores on PC 4 (9.00%) AARHUS UNIVERSITET Same plot but showing PC2 and PC4 0.5 0.4 Samples/Scores Plot of ref_samples 0 10 5 7 8 95% Confidence Level 0.3 0.2 0.1 0-0.1-0.2-0.3-0.4-0.5-0.8-0.6-0.4-0.2 0 0.2 0.4 0.6 0.8 Scores on PC 2 (27.87%) It is possible to find a direction in data that describes differences between infected and uninfected samples irrespective of the cultivar/species. Reference cultivars (± infected) and other species samples (no. 101 114). Normalized, mean centered and paretoscaled. Coloured according to the degree of infection in field evaluation.
Scores on PC 2 (8.96%) 40 30 20 Samples/Scores Plot of x 100 111 200 201 207 210 25 26 95% Confidence Level 10 0-10 Groupings related to field, cultivar and country (preliminary analysis only). All samples autoscaled. Coloured according to classes (groups): (3 repl./cultivar). -20-30 -40-40 -20 0 20 40 60 80 100 Scores on PC 1 (34.84%) Autumn 2014 leaf data 25: open pollinated crosses in field 25 planted 2006-7 26: controlled crosses in field 26 planted 2008 100: reference cultivars (Nefris, Fanal, Sumadinka, Birgitte, Viki) 111: other species; (P. avium Kordia, subhirtella, serrulata, incisa) 200+201: Hungarian samples 207: Romanian samples 210: Serbian samples
Further analysis coming up NMR analysis with scan data degree of necrosis /compare to field evalution of attack Analyse for specific compound differences (loading scores) especially phenolics. (make sure they are not produced by the fungi). Use pure reference compounds as reference for identity and quantity. What changes comes up with infection (SAR)? What compounds are at higher frequency in resistant/tolerant clones? Combined effect of compounds (multivariat, cocktail). Evaluate infection effect on carbohydrate profile : Percentage of leaf necrosis should corelate with photosynthetic performance and net assimilation and thus carbohydrate status in leaves. How much damage is incurred by infection? Do tolerant cultivars incur less damage maintain better carbohydrate status? Analyse spring/early summer leaves with no or minimum infection to get clean plant metabolite profile. Correlate spring and autumn metabolite profile and compare rank and differences over a season. Analyse flower samples and correlate with NMR results and degree of field attack with Monilia laxa.