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Reducing Copper's Impact on Soil-Plant Systems: Effects on Microbial Populations

This study investigates the environmental impact of copper on soil-plant systems, specifically on microbial populations. The experiment demonstrates the negative effects of copper on soil enzymes, soil respiration, and the Bacteria/Fungi ratio. The study concludes that alternatives to using copper as a fungicide and bactericide should be considered.

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Reducing Copper's Impact on Soil-Plant Systems: Effects on Microbial Populations

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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 FLORENCE, JUNE 2015 PARTNER CSIC (CEBAS-CSIC)

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

  3. AFTER-Cu (LIFE) CEBAS-CSIC EXPERIMENT (AFTER-Cu Project) “EFFECT OF COPPER ON SOIL-PLANT SYSTEMS (DIFFERENT CROPS: kiwi, lemon, olive) January to June 2014 (sixmonths) OBJECTIVE: to demonstrate the environmentally impact of COOPER on SOIL-PLANT SYSTEMS. It will be demonstrated at field level and on different crops (kiwi, lemon and olive) B1: Demostration of the use of Cu for the control of bcterial diseases of plants important for the EU C1: Monitoring on the environmental impact of Cu for the crop defence against bacterial phytopathogens

  4. AFTER-Cu (LIFE) ACTIVITIES B1 and C1 (at lablevel: microcosm) ON SOIL BIOLOGICAL AND BIOCHEMICAL PROPERTIES, CU ADDITION TO THE SOIL SHOWED A NEGATIVE EFFECT: -- Soil enzymes, particularly hydrolases implied in the N, C and P cycles (urease, b-glucosidase and phosphatase activities) showed a negative effect when Cu SO4 is added to the soil. This negative effect is observed only in the first days of experiment (15 days of application). It could be indicative that Cu can influence on microbial population which can synthetized the enzyme; but when Cu is long-term in the soil, this heavy metal could be complexed by organic or mineral colloid and it decreases your toxicity. For the dehydrogenase activity (endocellular enzyme), the negative effect on this enzyme by Cu is more weekly that on hydrolases. At initial point of our experiment, can be observed a little negative effect of Cu on soil dehydrogenase activity. -- Soil respiration: at T15 a negative effect of Cu on soil microorganisms is observed particularly at higher doses; however, at the end of the experiment (T60), all Cu doses showed an increase on soil microbial respiration. It can be due to the possible complex formed in the soil with Cu, and there is a decrease of toxicity. It was already observed with some soil enzymes. -- PLFAs analysis showed that at initial time (T0), the Bacteria/Fungi ratio decreased on control soil. This effect was not showed at the end of experiment (T60). It is indicative that the Cu changes your toxicity with the time. And we can indicate that Cu As main Conclusion in this study we can say that the use of Cu in the soil should be avoid; a negative effect of Cu on soil microbial populations has been showed. In addition, Cu is accumulative in the soil, and is possible that the quantity of Cu can increase with the time. For this reason, new alternatives for not using CuSO4 as fungicide and bactericide should be proposed. AFTER-Cu Project is in this way.

  5. 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 ACTIVITIES B1 and C1

  6. 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 ACTIVITIES B1 and C1

  7. 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 ACTIVITIES B1 and C1

  8. AFTER-Cu (LIFE) T_6 months (27/10/2014) KIWI Kiwi cropwasaffectedby bacteria inoculation. CuSO4 seems to have a possitiveeffect . Survival kiwi plants ACTIVITIES B1 and C1

  9. AFTER-Cu (LIFE) CONCLUSIONS TO THE EXPERIMENT (Activities B1 and C1) 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 wherethe use of CuSO4 seems be more effective , January to June 2014 (sixmonths)

  10. AFTER-Cu (LIFE) CEBAS-CSIC EXPERIMENT (AFTER-Cu Project) “EFFECT OF PEPTIDES ON SOIL-PLANT SYSTEMS (DIFFERENT CROPS: kiwi, lemon, olive) C2 and B5/C3/B2: April2014-April 2015 OBJECTIVE: C2. Monitoring of the in vitro peptides at laboratory levelon different crops (kiwi, lemon and olive) B5/C3: Collaboration. Demostration and Monitoring of the absence of side effects for the PEPTIDES on soil microorganisms B2: (collaboration): to demonstrate the changes induced by PESTIDES

  11. AFTER-Cu (LIFE) ACTIVITIES C2; collaboration C3 and B5) Peptide assays 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:

  12. 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)

  13. AFTER-Cu (LIFE) LEMON AND KIWI TREE (Peptideassay) ) ACTIVITIES C2

  14. AFTER-Cu (LIFE) ICP-OES EQUIPMENT Micro and macronutrients was analyzed by ICP-OES (Inductively coupled plasma-optical emision spectrometer) cuantitative determination, is an elemental analysis technic (IONOMIC SERVICE). The “Ionoma” of soil is important to know the equilibrium of the elements in the soil. It can be indicative of the influence of peptides on some macro and micro elements found in soils. ACTIVITIES C2-C3/B5

  15. AFTER-Cu (LIFE) Concentrations of macro- and micronutrients and heavy metals in kiwi crop soil (dwt): Initial analysis KIWI Bacteria + peptideinoculation. Ourresultsshowed no influence of peptides ON IONOMA SOIL. Onlyforsomeelements (Ca) wecouldindicateanincreasewithpeptidesaddition; Itcould be due to a possitiveeffectonelementabsorption ACTIVITIES C2-C3/B5

  16. AFTER-Cu (LIFE) Concentrations of macro- and micronutrients and heavy metals in kiwi crop soil (dwt): final analysis KIWI No differencesonIONOMA soilweredetected at end of theexperiment ACTIVITIES C2

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

  18. AFTER-Cu (LIFE) PLFAs EXTRACTION AND DETERMINATION PLFAs are only found in intact cells and certain groups of microorganisms have different signature fatty acids, changes in the PLFAs pattern represent changes in the soil microbial community structure. The PLFA profile can give an overall picture of the community structure, and it has been used to evaluate soil environmental quality

  19. AFTER-Cu (LIFE) PLFAs ACTIVITIES C2-C3/B5 Bacterial. fungi. 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) Changeswereshowedontotal microbialbiomass (bacteria and fungi)

  20. 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. At theend of theexperiment, alsothemicrobialbiomasswasaffectedby bacteria and peptides. Thesoilbiodiversitywaschangedregarding to the control soil. Itisindicative thatpeptides are actives in thesoils. ACTIVITIES C2-C3/B5

  21. AFTER-Cu (LIFE) SOIL BIODIVERSITY: FUNCTIONALITY SOIL RESPIRATION Thedegradation of organicmatteris a property of allheterotrophs, and itsrateiscommonlyused to indicatethelevel of microbialactivity ACTIVITIES C2-C3/B5

  22. AFTER-Cu (LIFE) Thedegradation of organicmatteris a property of allheterotrophs, and itsrateiscommonlyused to indicatethelevel of microbialactivity Theintroduction of peptides In thesoilhaveaneffecton soilrespiration (metabolic biota activity); itdecreased whenpeptides are used ACTIVITIES C2-C3/B5

  23. AFTER-Cu (LIFE) Peptide AP17 assay two months later Peptide Li 21 assay two months later Kiwi treesshowed a negativeeffectonplantwith bacteria inoculo; peptide (particularlyLi21) wasshoweduseful to eliminatethisnegativeeffect Kiwi leavesinfectedby bacteria ACTIVITIES C2-C3/B5

  24. AFTER-Cu (LIFE) A NEW EXPERIMENT FROM CEBAS-CSIC “Effect of peptidesadditiononsomepathogenmicroorganisms in soils”. EFFECT ON SOIL BIODIVERSITY SOIL + Pathogen SOIL + PEPTIDES + Pathogen CONTROL SOIL ACTIVITIES C2-C3/B5

  25. 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. ACTIVITIES C2-C3/B5

  26. AFTER-Cu (LIFE) Enzyme activities Indicator of specific 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-

  27. AFTER-Cu (LIFE) Glucosidase compost (µmoles PNF /g,h) Glucosidase (µmoles PNF /g,h) B-Glucosidaseis a hydrolaseincluded in the C cycle. Itisimportantforsoilquality. When compost isadded, theenzymeincreasesthevalue in thesoil. Thisenzyme no changedwithpeptides ACTIVITIES C2-C3/B5

  28. AFTER-Cu (LIFE) Phosphatase Compost (µmoles PNF /g,h) Phosphatase (µmoles PNF /g,h) Phosphataseis a hydrolase of P cycle. Itincreasedwhencompost was added to thesoil. No changeswereshowed at theend of theexperiment ACTIVITIES C2-C3/B5

  29. AFTER-Cu (LIFE) Urease Compost (µmoles PNF /g,h) Urease (µmoles PNF /g,h) Ureaseisanenzyme of the N cycle. Thishydrolaseshowedsomediffrences at theend of theexperiment ACTIVITIES C2-C3/B5

  30. AFTER-Cu (LIFE) Bacterial. fungal. Gram+. Gram-. satured and monosatured, actinobacteria PLFAs concentration in peptide microcoms T0.. The total PLFA and bacteria/fungi. Gram+/Gram- and monounsatured/satured ratios in in peptide microcoms T0. Peptides and soilbiodiversity. The use of peptidesshowed someinfuenceonsoilbiodiversity ACTIVITIES C2-C3/B5

  31. AFTER-Cu (LIFE) Bacterial. fungal. Gram+. Gram-. satured and monosatured, actinobacteria PLFAs concentration in peptide microcoms T60.. The total PLFA and bacteria/fungi. Gram+/Gram- and monounsatured/satured ratios in in peptide microcoms T60. Peptides and soilbiodiversity. Bacteria and fungi are influencedbypeptides Addition at theend of theexperiment. Itwould be considered in thefuture. ACTIVITIES C2-C3/B5

  32. Cs Bac_1 Thedynamic of labile C and N no showedanyvariation withpeptides ACTIVITIES C2-C3/B5

  33. AFTER-Cu (LIFE) ACTIVITIES C2-C3/B5

  34. AFTER-Cu (LIFE) Soilrespirationwasinfluencedbypeptides use. Thisparameterdecreasedregardingsoil control. ACTIVITIES C2-C3/B5

  35. AFTER-Cu (LIFE) • CONCLUSIONS • Peptidescouldalter soilbiodiversity • Peptides can influenceontheactivity of soilmicroorganisms (soilrespiration) • Peptidescouldchangethebiodiverdity at functionallevel (biota size) • Peptidesnotshowedanyeffectonbiogeochemicalcycles of C, P and N

  36. AFTER-Cu (LIFE) 2015. NEW ASSAY WITH PEPTIDES ACTIVITIES B5 and C3: Effects of some PEPTIDES onPhytotoxicityand Ecotoxicity

  37. AFTER-Cu (LIFE) Peptidesplaced directlyonPetri dish againstphatogens bacteria showed a litle biopesticideeffect. ACTIVITIES B3/C5

  38. AFTER-Cu (LIFE) PHYTOTOXICITY. Germination experiment (Biostimulant and Phytotoxic effect) The objective of this assay will be determined if the obtained digestates contained some phytotoxic compound that could make them inadvisable for agronomic use. To this, germination tests using seeds of rye grass (Lolliumperenne) or barley (Hordeum vulgaris) will be carried out. Rye-grass seeds will be used due to its high sensitivity to phytotoxic compounds and salinity, and barley seeds due to its greater resistance to these compounds. This study is carried out in laboratory growth chambers with control of temperature, humidity, and light conditions. ACTIVITIES B3/C5

  39. AFTER-Cu (LIFE) Somebiostimulanteffectshas beenobserved in Petri dishswithpeptides. A possiblephytohormonaleffectcould be responsible of thisbehaoviur. ACTIVITIES B3/C5

  40. AFTER-Cu (LIFE) Ecotoxicity assay A toxicity test was carried out using luminescent bacteria (Microtox), in which the inhibition of the luminescence of Photobacteriumphosphoreum was measured using a luminometer (Kapanen and Itävaara, 2001) after adding extracts of the samples. This assay uses a suspension of luminescent bacteria (Photobacteriumphosphoreum) as bioassay organism for measuring acute toxicity in aqueous extracts (Bulich, 1979; Matthews and Hastings, 1987). Lyophilized bacteria were used after rehydration in the commercial solution. All assays were carried out at 15 ºC with 15 min and 30 min contact periods between 0.5 ml of bacterial suspension and compost suspension. Compost suspension was prepared by mixing 1g sample with 10ml of 2 % NaCl (w/w) solution Ensayo de Ecotoxicidad Los ensayos de Ecotoxicidad se realizaron sobre los tres péptidos objeto de estudio a una concentración de 60 µM (concentración de trabajo en planta). Resultados de ecotoxicidad de los péptidos Según BOE 10 de noviembre de 1989, número 270/1989 se considera que una sustancia es tóxica si los lixiviados presentan una EC50 (quince minutos, 15ºC) inferior o igual a 3000 mg/l. Según esto, ninguno de los péptidos presentan toxicidad. ACTIVITIES B3/C5

  41. AFTER-Cu (LIFE) • CONCLUSIONS • Peptidesshoweda bioestimulanteffectonplants • Ecotoxicitymeasuredonpeptidesdidnot show anyproblem • Peptides show a litlebiopesticideeffecton Petri dishassays

  42. AFTER-Cu (LIFE) 2015-- NEW ASSAY WITH PLANTS Greenhouse and fieldexperiments ACTIVITIES B6 AND C5 Demostration and monitoring of theeffect of PEPTIDES onplantssystem B6 and C5: October2014-December 2015

  43. AFTER-Cu (LIFE) KIWI TREES IN MURCIA (SPAIN) ACTIVITIES B6/C5

  44. AFTER-Cu (LIFE) KIWI TREES IN MURCIA (SPAIN) Kiwi treesinoculation ACTIVITIES B6/C5

  45. AFTER-Cu (LIFE) KIWI TREES IN MURCIA (SPAIN) Possible diseasesymtoms ACTIVITIES B6/C5

  46. AFTER-Cu (LIFE) LEMON TREES IN MURCIA (SPAIN) Lemontreesinoculation ACTIVITIES B6/C5

  47. AFTER-Cu (LIFE) LEMON CROP IN MURCIA (SPAIN) LeavezonewithPs. Syr. Syringae. Lemonleavewithsomediseasesymtoms ACTIVITIES B6/C5

  48. AFTER-Cu (LIFE) LEMON CROP IN MURCIA (SPAIN) Lemonleaveswithsomediseasesymtoms ACTIVITIES B6/C5

  49. AFTER-Cu (LIFE) OLIVE TREES IN MURCIA (SPAIN) Olive plantsinoculationprocess. ACTIVITIES B6/C5

  50. AFTER-Cu (LIFE) OLIVE TREES IN MURCIA (SPAIN) Olive treesinoculation. Diseasesymtomsof theseplantsshould be noted aftersomeweeks. ACTIVITIES B6/C5

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