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Environmental Friendly Molecules for Reducing Plant Pathogenic Bacteria

Explore the effects of copper on soil microbiological community through enzyme activities in the AFTER Cu project. Understand the negative impact of copper on soil quality and soil enzymes.

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Environmental Friendly Molecules for Reducing Plant Pathogenic Bacteria

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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 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. Facilities (CEBAS-CSIC) CEBAS PILOT PLANT CULTURE CHAMBERS CEBAS LABORATORIES EXPERIMENTAL FIELD

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

  5. AFTER-Cu (LIFE) CEBAS WORKING-GROUP (AFTER-Cu) “EFFECT OF Cu ON SOIL BIOLOGICAL QUALITY”

  6. SOIL QUALITY Lithological material Topography Climate SOIL Natural resource Bioticcommunity -- Organics: pesticides -- Inorganics: Heavy metals Negativeeffectonsoilquality SOME TREATHS FOR SOILS: Climatechange, Management, Pollution, Salinization

  7. ContaminationbyDiffuse and Punctualsources 1 1 COMMERCIAL FERTILISER 2 Heavy metals Run-off AGROCHEMICALS HERBICIDES INSECTICIDES FUNGICIDES Atmospheric deposition N,P,K PHOTODECOMPOSITION 3 IRRIGATION 4 ACCIDENTAL SPILLS NITROGEN PHOSPHOROUS Run-off Run-off Erosion Root absorption Degradation Microorganisms BURIAL ANIMAL RESTS LEACHING Anaerobic degradation N,P Pesticides 5 Adsorption Desorption Saturated zone Sediments

  8. 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

  9. AFTER-Cu (LIFE) BIOLOGICAL INDICATORS OF SOIL • Soil structure formation • State of soil organic matter • Biogeochemical cycles (include N fixation): ENZYMES • Conditions of the vegetal development • Degradation of agrochemical and contaminants in soil Critical role in soil functions. They respond to soil management, contamination processes, changes in soil properties, ... SENSITIVES

  10. AFTER-Cu (LIFE) CEBAS-CSIC EXPERIMENT (B1 and C1, AFTER-Cu) “EFFECT OF COPPER ON SOIL MICRO-ORGANISMS ACTIVITY” OBJECTIVE: to demonstrate the environmentally negative impact of copper on soil microbiological community. The copper in soils can inhibit some enzyme activity through many pathways (Dick, 1997; Dussault et el., 2008): * Reducing the production of enzymes through its toxic effect on soil microflora. * Combining with the active protein groups of the enzyme. * Through complexation of the substrate. * Reacting with the enzyme-substrate complex.

  11. AFTER-Cu (LIFE) EXPERIMENT: SOIL + Cu (doses, leaching..) 60 days of experiment (sampling at 0, 15, 30 and 60 days)

  12. Cu in thesoil Cu: Ourexperimentshowedthatthe Cu was increasedlightly in thesoilwhenplants weretreatedwith CuSO4. Cu contentwillincrease in soilswiththe time becauseisused as continoustreatment. pH: No problemwith pH after Cu saltaddition. Itisindicative thatthesolubility of this metal isnotincreased in thesoil.

  13. AFTER-Cu (LIFE) SOIL ENZYMES versus SOIL POLLUTION (HEAVY METALS) • Enzymes are proteinswhichhave a main role in soilfunctionality. • They are implied in themaincycles of elementssuch as C, N, P and S. A lot of processes in soil are carriedoutbyenzymes (degradation of organicwastes, oxidation of organicmatter, …). • Soilenzymes can be bioindicators of thepotential of a soil to carryoutspecificbiochemicalreaction in order to maintainsoilfertility. Burns, 1982 E E E E E + S E E-S E+ P E E Enzyme-substractcomplex (soluble ornot soluble) Notproliferantcells Células vivas E E E E E E Deadcells and cellular wastes Arcillas y coloides húmicos E

  14. AFTER-Cu (LIFE) Enzyme activities Dheydrogenase enzymes catalyze the oxidation of organic compounds with the removal of two hydrogen atoms that are transferred to the molecule of NAD+ R-H2 + NAD+ NADH + R + H+ Indicator of global microbial metabolism b-Glucosidase enzymes catalyze the final limiting step of cellulose degradation Carbon cycle Phosphatase enzymes catalyze the hydrolysis of various organic phosphate esters Phosphorus cycle R-O-PO32- + H2O R-O-H + HO-PO32-

  15. AFTER-Cu (LIFE) Hidrolases: Extracellularenzymes Transformmacrosustratesintosmallercompounds Responsibles of importantscycles : C, N, P cycles) Degradationpesticides and otherxenobiotics Ureaseactivity: hydrolase of N cycle. Ureaseactivitydecreasedwhen Cu was added to thesoil (0 and 15 days). Cu showedaninitialnegativeeffect. No effect has beenobserveddue to thedifferentdose and leaching.

  16. AFTER-Cu (LIFE) Phosphataseactivity: hydrolase of P cycle. It can hydrolizeorganic P to inorganic P, assimilablebyplants. Negativeeffect of Cu at 0 and 15 days has beenshowed. High doseshowed more negativeeffect at 15 days.

  17. AFTER-Cu (LIFE) B-Glucosidaseis a hydrolaseincluded in the C cycle. Itisimportantforsoilquality. Cu showed a negativeeffectonthisenzyme at theinitialpoint. Notcleardifferenceswereobservedbetweendifferent doses and leachates

  18. AFTER-Cu (LIFE) Fig 4. Changes in Dehydrogenase activity (µgr INTF/g dry soil h) in Control, D1, D1P, D2 and D2P treatments after 60 days of incubation in growth chamber. Same letter in each figure indicate no significant differences between treatment (Tuckey’s method). A negativeeffectonDehydrogenaseactivitywasobservedafter Cu addition (initialpoint and after 15 days in sometreatments). Total metabolicactivitywasinfluencedby Cu treatment.

  19. AFTER-Cu (LIFE) Thedegradation of organicmatteris a property of allheterotrophs, and itsrateiscommonlyused to indicatethelevel of microbialactivity After 30 days of theexperiment, no negativeeffectwasobservedonsoilmicrobialrespirationwhen Cu wasadded to thesoil. After 60 days, a positive effectwasshowed. A negativeeffectcouldprobably be observedwith a higuer Cu amount in soil

  20. AFTER-Cu (LIFE) ACCUMULATIVE SOIL RESPIRATION Accumulative micro-organismsrespiration. The cinetic of soilRespirationshowedanincreasewhen Cu wasadded to thesoil. Itcould be likelydue to a microbial stress.

  21. AFTER-Cu (LIFE) Water soluble C is a labil C fraction. Forthisreason, thisparameterchanges along of theexperiment. Water soluble C isinfluencedbyorganicmatter mineralzation

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

  23. AFTER-Cu (LIFE) PFLA Extraction Trace GC Ultra Lipidfractions

  24. CEBAS-CSIC GROUP: SOILS ANALYSIS Thisis ONLY an EXAMPLE!!! Bacteria (Gram+ and Gram-)

  25. AFTER-Cu (LIFE) WHAT about the Immobilized Enzymes and Cu ? Immobilized Enzyme Free Enzyme More resistant to denaturalization: ENVIRONMENTAL INTEREST (NEW EXPERIMENT) Could Cu affect to immobilized enzymes???

  26. AFTER –Cu PROJECT EXPERIMENTS WITH PLANTS Greenhouse; controlled culture chamber -- Bacteria inoculation (pseudomona…) -- Cu addition (environmentalproblem) -- Peptides use (cleanagriculture)

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

  28. AFTER-Cu (LIFE) PLANTS TREATED WITH Cu , AND INOCULATED (BACTERIA)

  29. AFTER-Cu (LIFE) CITRUS (+/- BACTERIA)

  30. AFTER-Cu (LIFE) EFFECT OF BACTERIA INOCULATION

  31. AFTER-Cu (LIFE) KIWI (+/- BACTERIA)

  32. AFTER-Cu (LIFE) OLIVE (+/- BACTERIA)

  33. 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.

  34. AFTER-Cu (LIFE) A NEW EXPERIMENT PROPOSED BY CEBAS?? “Effect of peptidesadditiononsomepathogenmicroorganisms in soils”. BIOSTIMULANT AND BIOPESTICIDE EFFECT SOIL + PEPTIDES SOIL + Pathogen SOIL + PEPTIDES + Pathogen CONTROL SOIL

  35. CEBAS WORKING GROUP THANK YOU FOR YOUR ATTENTION

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