1. AFTER-Cu AFTER-Cu LIFE (2014-2015) Carlos García Izquierdo. CEBAS-CSIC FLORENCE, DECEMBER 2015 BENEFICIARY: CSIC (CEBAS-CSIC) Project LIFE12 ENV/IT/000336 “Anti-infective environmental friendly molecules against plant pathogenic bacteria for reducing Cu" (AFTER-Cu)

2. Dr. Carlos Garcia Dra. Teresa Hernández Dr. J. Moreno Lda. Mª Dolores Coll Ing. Carmen Chocano Carmen Montesinos Blanca Gonsalvez Francisco Tomás CEBAS-CSIC (AFTER-Cu) EXPERIMENTAL GREENHOUSES (SANTOMERA-ABARÁN

3. 2015-- ASSAY WITH PLANTS GREENHOUSE AND FIELD EXPERIMENTS ACTIVITIES B6 AND C5 Demostration and monitoring of the effect of PEPTIDES on plant system (and Soil) B6 and C5: From October 2014 to December 2015

4. ReferencesDescriptionControl Plant not treated Bac Plant treated with Pseudomone spp AP 17 Plants treated with Pseudomone+ antimicrobial peptide AP17 (lemon and olive) Li 27 Plants treated with Pseudomone+ antimicrobial peptide Li27 (lemon and olive) Psa 21 Plants treated with Pseudomone+ antimicrobial peptide Psa 21 (kiwi) Each plant was infected with the corresponding Pseudomone spp: Kiwi: P, syringae pv, actinidae Lemon: P, syringae pv, syringae Olive: P, savastanoi pv, nerii We have performed a co-inoculation assay (Pseudomone spp,+peptide) in kiwi, lemon and olive plants to verify the antimicrobial capacity of two different peptides known that P1: AP17 and P2: Li 27 at 60µM concentration in lemon and olive, and Psa 21 peptide specific for kiwi plants in 60µM concentration too. For this, were prepared the bacterial solutions (10 9 UFC/ml) in sterile physiological solution and were added the peptide in the corresponding quantity to the final concentration (60) µM in plant. Following, inoculation method are described so in pilot (greenhouse) as field assay. Plant parameters (Greenhouse and field assays) ClorophylHeight Trunk diameter Macro and micronutrients and Nt SYMTOMS Soil Parameters (field assay) ICP, Nt, COT, Ct Microrespiration C/ hidrosoluble PLFAs PREPARATION

5. GREENHOUSE EXPERIMENT

6. Greenhouse inoculation procedure Lemon and kiwi plants 1. Remove 2-3 leaves from each plant petiole included (100%wounds done), exposing the wound produced. 2. Co-inoculation: Inoculate a solution (10 µ l bacterial solution (10 9 UFC/ml) with peptide (60 µ M)) on the wound directly. 3. Let dry the co-inoculation liquid and protect the wound with parafilm. Remove the film on seventh day.

7. KIWI TREES IN MURCIA (SPAIN) B6/C5 ACTIVITIES

8. KIWI TREES IN MURCIA (SPAIN) Kiwi trees inoculation B6/C5 ACTIVITIES

9. KIWI TREES IN MURCIA (SPAIN) Possible symtoms on plant B6/C5 ACTIVITIES

10. LEMON TREES IN MURCIA (SPAIN) Lemon trees inoculation B6/C5 ACTIVITIES

11. Leave zone with Ps. Syr. Syringae. LEMON CROP IN MURCIA (SPAIN) Lemon leave with some disease symtoms B6/C5 ACTIVITIES

12. Planta pulverizada con peptido Li 27 Planta pulverizada con péptido AP 27 Planta control Planta pulverizada con peptido Li 27 Planta pulverizada con péptido AP 27 Planta control LEMON CROP IN MURCIA (SPAIN) Lemon leaves with some disease symtoms B6/C5 ACTIVITIES

13. OLIVE TREES IN MURCIA (SPAIN) Olive tree inoculation process. B6/C5 ACTIVITIES

14. OLIVE TREES IN MURCIA (SPAIN) Olive trees inoculation. Symtoms on these plants should be noted after some weeks. B6/C5 ACTIVITIES

15. OliveSurvivalSymptoms Control8000000000 Bac80101101 14 AP178000000000 Li 27 8000000000 LemonSurvivalSymptoms Control8100000001 Bac81100010 14 AP178100000001 8000000000 KiwiSurvivalSymptoms Control8100000001 Bac81100010 14 AP178100000001 8000000000 0 without symptoms 1 slight symptoms Some diseased leaves, although most of them are healthy 2 severe symptoms Most of the leaves are diseased. GREENHOUSE ASSAY

16. a. Clorophyl b. Height (cm) c. Trunk diameter (mm) Control 0,70 ab 93 a 9,22 a P. syr. syr. 0,68 ab 96 a 8,52 a Ap 17 0,64 a 94 a 9,87 a Li 27 0,74 b 113 a 9,54 a Lemon Clorophyl, height and trunk diameter in lemon plants 6 months old in greenhouse assay. Same letter in each parameter indicate no significantly differences between letters (Tuckey ’ s method, p<0.05) Fig 9, 10 and 11. Lemon plants, Clorophyl, Height and Trunk diameter results respectively. Some data should be confirmed by the field experiment

17. a. Clorophyl b. Height (cm) c. Trunk diameter (mm) Control 0,78 a 103 a 6,57 a AP 17 0,76 a 105 a 6,39 a Li 27 0,76 a 96 a 7,63 a P. sav. nerii 0,77 a 99 a 6,44 a Olive Clorophyl, height and trunk diameter in olive trees 6 months old in greenhouse assay. Same letter in each parameter indicate no significantly differences between letters (Tuckey ’ s method, p<0.05) Not significatives differences

18. P (g/100g) K (g/100g) Ca (g/100g) S (g/100g) Mg (g/100g) Na (g/100g) Control 0,16 a 1,01 a 0,28 a 0,16 a 0,073 a 0,48 b P. sav. nerii 0,16 a 1,15 a 0,26 a 0,16 a 0,069 a 0,36 ab Ap 17 0,17 a 1,28 a 0,33 a 0,13 a 0,082 a 0,27 a Li 27 0,17 a 1,06 a 0,36 a 0,16 a 0,088 a 0,41 ab Macro and micronutrients Macro and micronutrients in olive plants 6 month old greenhouse assay. Same letter in each parameter indicate no significantly differences between letters (Tuckey ’ s method, p<0.05) Fe (mg/Kg) Mn (mg/Kg) Control 75,65 a 26,42 a P. sav. nerii 73,34 a 17,32 a Ap 17 70,49 a 20,83 a Li 27 83,30 a 16,35 a Micronutrients in olive greenhouse assay. Same letter in each parameter indicate no significantly differences between letters (Tuckey ’ s method, p<0.05) Some data should be confirmed by the field experiment (Na)

19. FIELD EXPERIMENT

20. KIWI

21. LEMON

22. OLIVE CROP

23. LEMON CROP: SOME POSITIVE EFFECT WITH PEPTIDES LEMON CROP

24. KIWI CROP SOME POSITIVE EFFECT WITH PEPTIDES ON KIWI CROP

25. OLIVE CROP

26. KIWIOLIVELEMON 112000002 002000100 002011010 002000001 002000010 002000000 002000000 002101000 002000002 002000011 002010 002000 012000 001 000 0 without symptoms 1 slight symptoms Some diseased leaves, although most of them are healthy 2 severe symptoms Most of the leaves are diseased. Contro Control Pept Pept Pept Bac Bac Bac FIELD EXPERIMENT

27. P (g/100g) K (g/100g) Ca (g/100g) S (g/100g) Mg (g/100g) Na (g/100g) Control 0,14 a 2,49 a 1,24 a 0,17 a 0,16 a 0,010 a P. syr. syr. 0,16 a 2,87 a 1,42 a 0,21 a 0,23 b 0,016 a Ap 17 0,14 a 3,02 a 1,46 a 0,20 a 0,18 ab 0,012 a Li 27 0,16 a 3,08 a 1,47 a 0,23 a 0,19 ab 0,008 a MACRO AND MICRONUTRIENTS Macro and micronutrients in lemon leaves field assay. Same letter in each parameter indicate no significantly differences between letters (Tuckey ’ s method, p<0.05). Fe (mg/Kg) Mn (mg/Kg) Control 54,2 a 35,6 a P. syr. syr. 66,8 a 41,9 a Ap 17 79,9 a 48,9 a Li 27 73,4 a 45,2 a

28. Cu (mg/Kg) Cd (mg/Kg) Zn (mg/Kg) Pb (mg/Kg) Cr (mg/Kg) Ni (mg/Kg) Control 4,08 a <0,1 a 16,05 a 1,48 a 0,21 a 0,36 a P. syr. syr. 5,50 a <0,1 a 20,50 a 1,66 a 0,37 a Ap 17 3,78 a <0,1 a 19,60 a 2,14 a 0,22 a 0,44 a Li 27 4,95 a <0,1 a 19,89 a 1,80 a 0,28 a 0,44 a Heavy metals in lemon leaves field assay Heavy metals in lemon leaves field assay. Same letter in each parameter indicate no significantly differences between letters (Tuckey’s method, p<0.05).

29. SOIL ANALYSIS Biotic community Biotic community Climate Lithological material Topography SOIL Effects of peptides and bacteria on soil biological properties

30. SOIL RESPIRATION The degradation of organic matter is a property of all heterotrophs, and its rate is commonly used to indicate the level of microbial activity SOIL BIODIVERSITY: FUNCTIONALITY The metabolic activity of the sediments, measured as microbial respiration, showd the lowest values

31. Labile substrates for microorganisms: Soluble C and N Soil respiration: CO2 release

32. Microbial biomass: PhosphoLipid Fatty Acids (PLFAs) Lipid extractionFractionation Metil- esterificatión fatty acids Gass Chromatography Bligh & Dyer, 1959 Frostegard et al., 1993 Bligh & Dyer, 1959 Frostegard et al., 1993 PhosphoLipid Fatty Acids (PLFAs) Microbial biomass C and N (Powlson et al., 1987) ATP, Ergosterol, etc.

33. Anova analysis TreatmentFactor1Factor2 Control Controlba P,syr, actinidiae P,syr, actinidiaeba Psa 21 Psa 21ba Load factor for each variable Parameter Factor 1 Factor 2 c120,536,011 c130,072,711 C14-,238-,092 i15-,330,198 a15-,124,696 c15,712 Factor 2 (20,4) -,416 i16-,764,095 c16-,544-,203 c10me16-,197,948 c161w9-,218,968 ci17-,226-,508 cy17-,134-,472 c17,878-,131 c18,776-,228 c181n9t-,306,908 c10me18,877,025 c182n6t,133-,064 c181n9c,771-,412 c182n6c,861-,282 cy19,145,704 c19,627,134 c183w30,063-0,025 c200,875-0,168 c201w90,8740,006 c240,029-0,158 c241w9-0,2790,165 Kiwi crop: PLFAs measured on soil under kiwi

34. CEBAS WORKING GROUP THANK YOU FOR YOUR ATTENTION LIFE12 ENV/IT/000336