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AFTER-Cu LIFE (2014-2016)

AFTER-Cu LIFE (2014-2016). Project LIFE12 ENV/IT/000336 “Anti-infective environmental friendly molecules against plant pathogenic bacteria for reducing Cu" (AFTER-Cu ). Carlos García Izquierdo. CEBAS-CSIC. FAENZA, JANUARY 2015 PARTNER CSIC (CEBAS-CSIC). CEBAS. OUR INSTITUT: CEBAS-CSIC.

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AFTER-Cu LIFE (2014-2016)

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  1. AFTER-Cu LIFE (2014-2016) Project LIFE12 ENV/IT/000336 “Anti-infective environmental friendly molecules against plant pathogenic bacteria for reducing Cu" (AFTER-Cu) Carlos García Izquierdo. CEBAS-CSIC FAENZA, JANUARY 2015 PARTNER CSIC (CEBAS-CSIC)

  2. CEBAS OUR INSTITUT: CEBAS-CSIC Centro de Edafología y BiologíaAplicada del Segura CEBAS-CSIC (Murcia, Spain) CEBAS CAMPUS UNIVERSITARIO CSIC

  3. AFTER-Cu (LIFE) CEBAS CEBAS-CSIC (AFTER-Cu) Dr. Carlos Garcia Dra. Teresa Hernández Dr. J. Moreno Lda. Mª Dolores Coll Ing. Carmen Chocano EXPERIMENTAL GREENHOUSES (SANTOMERA-ABARÁN

  4. AFTER-Cu (LIFE) CEBAS WORKING-GROUP (AFTER-Cu) “EFFECT OF CuSO4 ON SOIL-PLANT SYSTEM”

  5. PHOTODEGRADAtION VOLATILIZACIÓN ABSORCION Y EXUDACION RUNOFF INFILTRACION ADSORPTION DESORPTION CHEMICAL DEGRADATION BIODEGRADATION LIXIVIACION CAPILARIDAD WATER TABLE TRANSFER ADSORTION-DESORPTION (Cu) LEACHING VOLATILISATION RUNOF ABSORTION BY PLANTS AND ORGANISMS (Cu) TRANSFORMATI0N CHEMICAL (Cu) PHOTOCHEMICAL BIOLOGICAL (Cu) DYNAMIC OF CONTAMINANTS IN SOIL-WATER HEAVY METALS “Cu” Agriculture Industrial Domestic Wastes

  6. AFTER-Cu (LIFE) CEBAS-CSIC EXPERIMENT (AFTER-Cu Project) “EFFECT OF COPPER ON SOIL-PLANT SYSTEMS AT FIELD LEVEL (DIFFERENT CROPS: kiwi, lemon, olive) OBJECTIVE: to demonstrate the environmentally negative impact of COOPER on SOIL-PLANT SYSTEMS. It will be demonstrated at field level and on different crops (kiwi, lemon and olive)

  7. AFTER –Cu PROJECT CEBAS-CSIC EXPERIMENT Greenhouse (kiwi, olive, lemon) Bacteria inoculation (pseudomona) 1) Cu addition (environmentalproblem) 2) Peptides use (cleanagriculture)

  8. CEBAS-CSIC: PLANT EXPERIMENT WITH CITRUS, OLIVE AND KIWI (BACTERIA INOCULATION: pseudomona) CEBAS

  9. AFTER-Cu (LIFE) “In vitro” EFFECT OF BACTERIA INOCULATION Kiwi leavesinfectedby bacteria

  10. AFTER-Cu (LIFE) GREENHOUSE PLANTS EXPERIMENT K= kiwifruit Ps+= infected plant with pseudomone L= lemon Ps-= not infected plant with pseudomone O= Olive CuSO4+= plant/soil treated with CuSO4+ at 12k/Ha/year Control= distilled water CuSO4-= plant/soil not treated with CuSO4+ at 12k/Ha/year

  11. AFTER-Cu (LIFE) Concentrations of macro- and micronutrients and heavy metals in kiwi soils (dwt) Kiwifruit_ T6 months Riskusing CuSO4 Soilsshowed > Cu concentrationwhen CuSO4wasused as bactericide. Itcould be negativeonsoilenzyme, reducing the production of enzymes through its toxic effect on soil microflora

  12. AFTER-Cu (LIFE) Concentrations of macro- and micronutrients and heavy metals in kiwi leaves (dwt) Riskusing CuSO4. Plantswhere CuSO4wasused as bactericideshowedhigh quantity of Cu in leaves. Itcould be negativeforfruits and also for human health

  13. AFTER-Cu (LIFE) Concentrations of macro- and micronutrients and heavy metals in lemon soils (dwt) Lemon_T6 months Riskusing CuSO4 Ourresultsobtainedonlemonsoilsweresimilars to kiwi soil. When CuSO4wasused, highquantity of Cu in thesoilwasfound. Anincrease of soil Ca contentwasobserved. Itcould be due to the bacteria inoculation

  14. AFTER-Cu (LIFE) Concentrations of macro- and micronutrients and heavy metals in lemon leaves(dwt) Riskusing CuSO4 When CuSO4wasused as bactericideonlemoncrop, Cu increasedonlemonleaves. Cr and Ni alsoincreased in leaves inoculatedwith bacteria. Heavy metal could be a risk to fruits and the human health

  15. AFTER-Cu (LIFE) Olive_T6 months Concentrations of macro- and micronutrients and heavy metals in olives soils (dwt) Riskusing CuSO4 Onsoil olive crop, ourresultsweresimilars to kiwi and lemoncrops. When CuSO4wasused, > Cu in soilwasfound. Also Ca and Ni increaseddue to the bacteria inoculation.

  16. AFTER-Cu (LIFE) Concentrations of macro- and micronutrients and heavy metals in olive leaves(dwt) Riskusing CuSO4 When CuSO4wasused as bactericideon olive crop, > Cu in Leaveswasobserved. Cr and Ni alsoincreased in leaves inoculatedwith bacteria. Heavy metal could affect to fruits and to the human health

  17. AFTER-Cu (LIFE) WATER SOLUBLE C WATER SOLUBLE N Water soluble C and N is a labilfraction. Forthisreason, theseparameterschangealongtheexperiment. Water soluble C and N are influencedbyorganicmattermineralzationThereisnottoodifferencesbetweenthedifferenttreatments. Onlyfor kiwi, the CuSO4addition and bacteria inoculationincreased soluble N in soil.

  18. AFTER-Cu (LIFE) Thedegradation of organicmatteris a property of allheterotrophs, and itsrateiscommonlyused to indicatethelevel of microbialactivity After 6 months of theexperiment, no negativeeffectwasobservedonsoilmicrobialrespirationwhen Cu wasadded to thesoilforlemon and kiwi. For olive, soilrespirationincreased in control soil. A majornegativeeffectcouldprobably be observedwith > Cu in soil

  19. Microbialbiomass: PhosphoLipidFattyAcids (PLFAs) Microbialbiomass C and N (Powlson et al., 1987) ATP, Ergosterol, etc. PhosphoLipidFattyAcids (PLFAs) Bligh & Dyer, 1959 Frostegard et al., 1993

  20. AFTER-Cu (LIFE) PLFAs ON SOILS Bacterial. fungal. Gram+. Gram-. satured and monosatured PLFAs concentration in kiwi soils T6 months The total PLFA and bacteria/fungi. Gram+/Gram- and monounsatured/satured ratios in kiwi soils T6 months. Differencesbetween control soil and soilwith CuSO4additionweredetectedforgram- bacteria and monounsaturated PLFA . Bacteria, fungi and actinobacteriadidnot show differences

  21. AFTER-Cu (LIFE) T_6 months (27/10/2014) KIWI Kiwi cropwasaffectedby bacteria inoculation. CuSO4 seemsto havea possitiveeffect. Survival kiwi plants

  22. AFTER-Cu (LIFE) T_6 months (27/10/2014) LEMON Lemoncropwasnotaffectedby bacteria inoculation. CuSO4notseems to haveanyeffectonlemoncrop. Survival lemon plants Lemons haven’t symptoms.

  23. AFTER-Cu (LIFE) T_6 months (27/10/2014) OLIVE Olive cropwasaffectedby bacteria inoculation. CuSO4 havenot a clearly possitiveeffect. Survival olive plants

  24. AFTER-Cu (LIFE) CONCLUSIONS TO THE EXPERIMENT 1) Cu is a heavy metal which can cause someproblemsonsoilquality (soilenzymeactivities, microbialpopulation, soilcontamination,…) Soilswhere CuSO4isused, Cu accumulationwasfound. 2)Ourstudyindicatesthat Cu couldmovefromsoil to plant (leaves). 3) The use of CuSO4 as bactericideshould be considered as a risk to theenvironment 4)Resultsobtained in the AFTER-Cu Project showedthat kiwi is thecrop more affectedby bacteria, and wheretheuse of CuSO4 seemsbe more effective ,

  25. Organic product Fusarium sp. NEW PRODUCTS FOR A CLEAN AGRICULTURE: BIOPESTICIDES HIGH QUALITY ORGANIC PRODUCTS (peptides, aminoacids) Biopesticide effect • Antibiotics induction by organic products • Competitive action between microorganisms • Enzyme productions with biopesticide activity • Systemic resistance induction for plants Without organic product Fungal supression to avoid root rooting and necrosis Pythium sp.

  26. AFTER-Cu (LIFE) Peptide assays T0 We have performed a co-inoculation assay (Pseudomonespp.+peptide) in kiwi, lemon and olive plants in order to verify the antimicrobial capacity of two different peptides known that P1: AP17 and P2: Li 27 at two concentrations (30 and 100 µM). For this, were prepared the bacterial solutions in sterile physiological solution and were added the peptide in the corresponding quantity to the final concentration in plant 30 and 100 µM. Each plant was infected with the corresponding Pseudomonesp: Kiwi: P. syringaepv. actinidae Lemon: P. syringaepv. syringae Olive: P. savastanoipv. Nerii This final solution (bacteria+peptide) was sprayed on kiwi and lemon plants and in the olive case was incorporated to plant through stems wounds. The different references are showed in the following table:

  27. AFTER-Cu (LIFE) Bacteria culture: Kiwi: P. syringaepv. Actinidae. Lemon: P. syringaepv. syringae Olive: P. savastanoipv. Nerii Peptides: P1: AP17 and P2: Li 27 , produced by Florence University (Dr. S.Tegli)

  28. AFTER-Cu (LIFE) LEMON AND KIWI TREE (Peptideassay) T1 (22/12/2014)

  29. AFTER-Cu (LIFE) Concentrations of macro- and micronutrients and heavy metals in kiwi crop soil (dwt) KIWI Bacteria + peptideinoculation: > Ca in soil. Itcould be due to a possitive effectonelementabsorption

  30. AFTER-Cu (LIFE) Concentrations of macro- and micronutrients and heavy metals in lemon crop soil (dwt) LEMON Bacteria + peptideinoculationnotshowedanydifferenceon control soil (lemoncrop).

  31. AFTER-Cu (LIFE) LEMON KIWI

  32. AFTER-Cu (LIFE) PLFAs Bacterial. fungal. Gram+. Gram-. satured and monosatured PLFAs concentration in kiwi soils T0 before treatments. The total PLFA and fungi/bacteria. Gram+/Gram- and satured/monounsatured ratios in kiwi soils T0 before treatments. Microbialbiomasswasaffectedby bacteria and peptides (alsobypeptide doses)

  33. AFTER-Cu (LIFE) Peptide AP17 assay two months later Peptide Li 21 assay two months later Kiwi cropshowed a negativeeffectwith bacteria inoculo; peptide (particularly Li21) couldavoidthiseffect Kiwi leavesinfectedby bacteria

  34. AFTER-Cu (LIFE) LEMON CROP. Lemonleaves: plantsnotshowedinfectionsintomsby bacteria

  35. AFTER-Cu (LIFE) OLIVE CROP. Olive cropwith bacteria and peptides inoculation. At thismoment, notnegative effects has beenshowed

  36. AFTER-Cu (LIFE) A NEW EXPERIMENT FROM CEBAS “Effect of peptidesadditiononsomepathogenmicroorganisms in soils”. EFFECT ON SOIL BIODIVERSITY SOIL + PEPTIDES SOIL + Pathogen SOIL + PEPTIDES + Pathogen CONTROL SOIL

  37. AFTER-Cu (LIFE) PEPTIDE MICROCOMS ASSAY Table 1. Sample description Sampling: T0, T15, T30 and T60 days Bac 1: P. syringaepv. actinidae Bac 2: P. syringaepv. syringae Bac:3 P. savastanoi pv. nerii Bac (1,2,3) P1 30: Pseudomone+ antimicrobial peptide P1 (AP17) 30 µM Bac (1,2,3) P1 100: Pseudomone+ antimicrobial peptide P1 (Ap17) 100 µM Bac (1,2,3) P2 30: Pseudomone+ antimicrobial peptide P2 (Li 27) 30 µM Bac (1,2,3) P2 100: Pseudomone+ antimicrobial peptide P2 (Li 27)100 µM Sample (soil and soil 10% compost) were disposed in 100 c.c. Several containers, each one with one hundred g of soil or soli amendment with 10% compost , were treated as show in Table 1, and placed in a growth chamber.Each treatment was replicated three times and placed in a random design into the growth chamber set at 16h photoperiod with a day/night temperature regime of 24/15ºC.

  38. CEBAS WORKING GROUP THANK YOU FOR YOUR ATTENTION

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