1. Giuseppe CICCARELLA 1,2 1 Dipartimento di Scienze e Tecnologie Biologiche e Ambientali e Centro di Ricerche per la Salute dell’Uomo e dell’Ambiente Università del Salento 2 CNR-NANOTEC, Istituto di Nanotecnologia del CNR Email: giuseppe.ciccarella@unisalento.itgiuseppe.ciccarella@unisalento.it Nanotecnologie per salvare gli ulivi da Xylella fastidiosa

2. Olive Quick Decline Syndrome

4. Different factors affecting the olive trees are actually under evalutation together with their combination: Insect Zeuzera pyrina Pathogenic Fungi Phaeoacremonium parasiticum Bacteria Xylella fastidiosa Insect Zeuzera pyrina Pathogenic Fungi Phaeoacremonium parasiticum Bacteria Xylella fastidiosa The necrosis starts from Zeuzera galleries Olive Quick Decline Syndrome Galleries in the trunk

5. Olive Quick Decline Syndrome Vascular system of plants Transpiration Xylematic liquid xylem phloem Leaves and sprouts are feeded by the xylematic flow of water drained by roots.

6. Olive Quick Decline Syndrome Xylella fastidiosa Xylematic liquid Scorching is caused by a low-level moisture stress that occurs as xylem vessels in leaf veins become blocked Transpiration blocking Pathogen lives in the xylem vessels of host plant. Bacteria form biofilms blocking the xylematic vessel

7. Olive Quick Decline Syndrome Transmission Xylella is transmitted by xylem-feeding insects (Philenus spumarius ) The identity of insects that vector QDS in olive trees is however under study

8. Olive Quick Decline Syndrome Hosts Almond Oleander Cherry tree

9. Components for disease development Economically important host (such as olive trees) Vector (xylem-feeding insect) Alternative host vegetation fruit tree Vector movement between host types ? Vector movement within host canopy Olive Quick Decline Syndrome

10. Diffusion

11. Strategies for sustainable protection 1.Development of an early diagnostic methodology “Know your enemy” 2. Development of nanoagrochemical drugs “Then destroy him” So….why sustainable protection? Because actually the solution proposed is to cut sick olive trees and to spread pesticides to kill bugs.

12. Strategies for sustainable protection 1.Development of an early diagnostic methodology “Know your enemy”

13. Metabolomics All the living organisms leave a trace of their vital activity. Metabolomics looks for small molecules and their profile. Two analytical approach are possible to perform the investigation: 1.Targeted metabolomics This approach is used with known molecules (e.g. drugs) 2. Untargeted metabolomics Simultaneous measure of many metabolites as possible from biological samples. The differences are used to identify specific metabolites as potential diagnostic agent. As the presence of Xylella fastidiosa bacteria inside the olive tree modifies the metabolome of the plant we investigate the variations of metabolites occurring in infected trees by using HPLC-MS Strategies for sustainable protection 1.Development of an early diagnostic methodology

14. …in 3000 m 3 HPLC/ESI-Q-TOF High detection limit (10 nmol/L) For a chemical with MW 100 g/mol this means 1 g / 1000 m 3 3 g Strategies for sustainable protection 1.Development of an early diagnostic methodology ….Useful to find an impressive number of metabolytes

15. Infected culture medium Not infected culture medium Samples preparation HPLC-ESI-QTOF analysis Elaboration of datasets *METLIN is a metabolomics database, it serves as a data management system to assist in metabolite research and metabolite identification by providing public access to its repository of comprehensive MS/MS metabolite data. Strategies for sustainable protection 1.Development of an early diagnostic methodology

16. Multivariate Analysis Not infected culture medium Xylella fastidiosa culture medium Strategies for sustainable protection 1.Development of an early diagnostic methodology

17. Samples preparation HPLC-ESI-QTOF analysis Sick leaves Healthy leaves Elaboration of datasets xylem Strategies for sustainable protection 1.Development of an early diagnostic methodology

18. 1.Development of an advanced diagnostic methodology Healthy leaves Sick leaves Multivariate Analysis Strategies for sustainable protection 1.Development of an early diagnostic methodology

19. Strategies for sustainable protection 1.Development of an early diagnostic methodology Interactive heatmap

20. XCMS online metanalysis By comparing the metabolytes of sick olive trees and Xylella fastidiosa culture media we found two metabolytes adducible to bacteria. Two compounds showed significant difference (p<0.01)in both pairwise comparisons: Xylella fastidiosa culture media and olive tree leaves Strategies for sustainable protection Structure elucidation 1.Development of an early diagnostic methodology

21. 1.Development of an advanced diagnostic methodology Multivariate Analysis Strategies for sustainable protection 1.Development of an early diagnostic methodology Infected trees present a different metabolomic pattern, two metabolytes attributable to Xylella have been observed. We can monitor the helth of olive trees by a ‘single leaf’ analysis. Healthy leaves Sick leaves

22. Strategies for sustainable protection 2. Development of nanoagrochemical drugs “Then destroy him”

23. Why nanodrgus? The bacteria are in the xylem so the drug has to be introduced INSIDE 1. Controlled release of drugs Plants are devoid of the active immune systems therefore the longer is the release of the biocide the higher is the protection towards new inoculations of bacteria due to the bugs. 2. Specificity towards bacteria We have to avoid the diffusion of drugs inside the fruit (olives) this leads to a minor usage of drugs and to costs containment (it is estimated that about 1 million of olive trees over 11 millions are infected). Strategies for sustainable protection 2. Development of nanoagrochemical drugs

24. Are possible two scenarios 1 Strategies for sustainable protection 2. Development of nanoagrochemical drugs Nanocarriers adhere to the membrane and release the biocide. Therfore: bacteria die.

25. Nanocarriers penetrate inside the cell and release the biocide Therfore: bacteria die. Strategies for sustainable protection 2. Development of nanoagrochemical drugs 2 Are possible two scenarios

26. Nanocarrier requirements for large scale applications Strategies for sustainable protection 2. Development of nanoagrochemical drugs Biocompatible Unexpensive Industrializable V. Vergaro, G. Ciccarella : Synthesis of nano-sized CaCO 3 particles by spray dryer. European Patent EP13425061.2 - (2013). nano CaCO 3

27. Nanocarrier requirements for large scale applications Strategies for sustainable protection 2. Development of nanoagrochemical drugs Three main issues need to be addressed: 1.Can nanoCaCO 3 be safe for humans and plants? 2.Can nanoCaCO 3 move inside the xylems? 3.Can nanoCaCO 3 be selective for Xylella fastidiosa?

28. Strategies for sustainable protection 2. Development of nanoagrochemical drugs Nano-CaCO 3 MTT Test 1.Can nanoCaCO 3 be safe for humans and plants? Cell viability is not compromised

29. Strategies for sustainable protection 2. Development of nanoagrochemical drugs The germination rate is not influenced by the nanocarrier Tests were carried out on: Wheat: T.dur. Triticum durum, cv Croesus 2002 Tomato: L.esc. Lycopersicum esculentum, cv Microtom Tobacco: N.tab. Nicotiana tabacum, cv SR1 Phyto-toxicity tests nanoCaCO3 conc. 1.Can nanoCaCO 3 be safe for humans and plants?

30. Strategies for sustainable protection 2. Development of nanoagrochemical drugs No interference on root growth Wheat Ctrl Nano-CaCO3 100mg/L 1.Can nanoCaCO 3 be safe for humans and plants?

31. Strategies for sustainable protection 2. Development of nanoagrochemical drugs Wheat Ctrl Nano-CaCO3 100mg/L 1.Can nanoCaCO 3 be safe for humans and plants? nanoCaCO 3 can be reasonably considered safe for plants and humans

32. Nanocarriers: 100 nm Xylem diameter :20  m Ratio = 1: 200 10  m Strategies for sustainable protection 2. Development of nanoagrochemical drugs 2. Can nanoCaCO 3 move inside the xylems? xylem magnification 1:10.000

33. Strategies for sustainable protection 2. Development of nanoagrochemical drugs This can be possible if the plant do not have interactions with the nanocarriers Nanocarriers can be introduced inside the tree. The xylematic flux can distribute the nanoparticles along the trunck reaching capllarly branches and leaves 2. Can nanoCaCO 3 move inside the xylems?

34. Strategies for sustainable protection 2. Development of nanoagrochemical drugs Nanocarriers can be introduced inside the tree. The xylematic flux can distribute the nanoparticles along the trunck reaching capllarly branches and leaves 2. Can nanoCaCO 3 move inside the xylems? therefore nanovectors move within the vessels and… when they encounter bacteria hit the target

35. Analysis by confocal microscopy of xylems before the introduction of nanoCaCO 3 Strategies for sustainable protection 2. Development of nanoagrochemical drugs 2. Can nanoCaCO 3 move inside the xylems? Experimental tests

36. Le strategie oggetto della ricerca all’Università del Salento Lower part of the stem Upper part of the stem fluorescent nanoCaCO 3 Analysis by confocal microscopy of xylems after the introduction of nanoCaCO 3 2. Can nanoCaCO 3 move inside the xylems? Experimental tests

37. Le strategie oggetto della ricerca all’Università del Salento Lower part of the stem Upper part of the stem fluorescent nanoCaCO 3 2. Can nanoCaCO 3 move inside the xylems? nanoCaCO3 has a diffusion gradient and then spreads into the plant

38. Strategies for sustainable protection 2. Development of nanoagrochemical drugs 3. Can nanoCaCO 3 be selective for Xylella fastidiosa? Due to its z-potential (-15-20 mV) nanoCaCO 3 has a strong affinity for various sugar-based polymers such as: Chitosan Dextran

39. Strategies for sustainable protection 2. Development of nanoagrochemical drugs Therefore we expect that nanoCaCO 3 could be also able to bind bacterial exopolysaccharides 3. Can nanoCaCO 3 be selective for Xylella fastidiosa?

40. There are two possible scenarios 1 Strategies for sustainable protection 2. Development of nanoagrochemical drugs Nanocarriers adhere to the membrane. Therfore: - No aggregation in colonies (with the formation of a cap responsible of leaves drying); - Membrane destabilization? - Bacteria die? 3. Can nanoCaCO 3 be selective for Xylella fastidiosa?

41. Nanocarriers penetrate inside the cell Therfore: - Membrane destabilization? - Bacteria die? Strategies for sustainable protection 2. Development of nanoagrochemical drugs 2 3. Can nanoCaCO 3 be selective for Xylella fastidiosa? There are two possible scenarios

42. Strategies for sustainable protection 2. Development of nanoagrochemical drugs 3. Can nanoCaCO 3 be selective for Xylella fastidiosa?

43. Strategies for sustainable protection 2. Development of nanoagrochemical drugs 3. Can nanoCaCo 3 be selective for Xylella fastidiosa? Endosomes (permeation) Adhesion? A certain selectivity with the ability to perforate the membrane seems possible

44. Strategies for sustainable protection 2. Development of nanoagrochemical drugs In conclusion Can nanoCaCO 3 be safe for humans and plants? nanoCaCO 3 can be reasonably considered safe for plants and humans Can nanoCaCO 3 move inside the xylems? nanoCaCO 3 has a diffusion gradient and then spreads into the plant Can nanoCaCO 3 be selective for Xylella fastidiosa? A certain selectivity with the ability to perforate the membrane seems possible, experiments are still in progress Early detection Infected trees present a different metabolomic pattern, two metabolytes attributable to Xylella have been observed

45. XYLELLA RESEARCH GROUP @ Unisalento Collaborations: Università di Bari Prof. Giovanni Martelli Prof. Vito Nicola Savino Prof. Giuliana Loconsole Prof. Oriana Potere CNR IPSP Istituto per la Protezione Sostenibile delle Piante Dr. Donato Boscia Dr. Maria Saponari Dr. Angelo De Stradis Fundings: PON 254/Ric. Potenziamento del “CENTRO RICERCHE PER LA SALUTE DELL'UOMO E DELL'AMBIENTE” Cod. PONa3_00334. PRIN 2010-2011 (D.M. 1152/ric del 27/12/2011) Nanotecnologie molecolari per il rilascio controllato di farmaci Tecnologie Abilitanti per Produzioni Agroalimentari Sicure e Sostenibili (T.A.P.A.S.S) “AIUTI A SOSTEGNO DEI CLUSTER TECNOLOGICI REGIONALI PER L’INNOVAZIONE” Regione Puglia Acknowledgements ADVANCED DIAGNOSTICS Prof. Giuseppe Cannazza (UniMORE) Dr. Cinzia Citti, Ph.D. NANOCARRIERS Dr. Francesca Baldassarre, Ph.D. Dr. Viviana Vergaro, Ph.D. MICROSCOPY Prof. Luciana DIni Dr. Elisabetta Carata, Ph.D. Dr. Francesco Mura Ph. D. PLANT PHYSIOLOGY Prof. Gian Pietro Di Sansebastiano, Ph.D

46. Thank you for your attention Strategies for sustainable protection Xylella fastidiosa

47. Thank you for your attention Strategies for sustainable protection Xylella “irosa”