Giuseppe CICCARELLA 1,2 1 Dipartimento di Scienze e Tecnologie Biologiche e Ambientali e Centro di...

Giuseppe CICCARELLA1,2

1Dipartimento di Scienze e Tecnologie Biologiche e Ambientali e Centro di Ricerche per la Salute dell’Uomo e dell’AmbienteUniversità del Salento

2CNR-NANOTEC , Istituto di Nanotecnologia del CNREmail: [email protected]

Nanotecnologie per salvare gli ulivi da Xylella fastidiosa

mailto:[email protected]

Olive Quick Decline Syndrome

Different factors affecting the olive trees are actually under evalutation together with their combination:

InsectZeuzera pyrina

Pathogenic Fungi Phaeoacremonium parasiticum

BacteriaXylella fastidiosa

The necrosis starts fromZeuzera galleries


Galleries in the trunk


Vascular system of plants

Transpiration

Xylematic liquid

xylem

phloem

Leaves and sprouts are feeded by the xylematic flow of water drained by roots.


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


Transmission• Xylella is transmitted by xylem-feeding insects (Philenus spumarius )• The identity of insects that vector QDS in olive trees is however

under study


Hosts

Almond Oleander Cherry tree

Components for disease development

• Economically important host (such as olive trees)• Vector (xylem-feeding insect)• Alternative host vegetation

Alternative host

vegetation

fruit tree

Vector movement between host types

?Vector movement within host canopy


Olive Quick Decline SyndromeDiffusion

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.


1.Development of an early diagnostic methodology“Know your enemy”

MetabolomicsAll 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 metabolomicsSimultaneous 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


1. Development of an early diagnostic methodology

…in 3000 m3

HPLC/ESI-Q-TOFHigh detection limit (10 nmol/L)

For a chemical with MW 100 g/mol this means

1 g / 1000 m3

3 g



….Useful to find an impressive number of metabolytes

Infectedculture 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.



Multivariate Analysis

Not infected culture medium

Xylella fastidiosa culture medium



Samples

preparation

HPLC-ESI-QTOF

analysis

Sick leaves

Healthy leaves

Elaboration of datasets

xylem



1. Development of an advanced diagnostic methodology

Healthy leavesSick leaves






Interactive heatmap

XCMS online metanalysisBy 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


Structure elucidation


1. Development of an advanced 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 leavesSick leaves


2. Development of nanoagrochemical drugs“Then destroy him”

Why nanodrgus?The bacteria are in the xylem so the drug has to be introduced INSIDE

1. Controlled release of drugsPlants 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 bacteriaWe 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).


2. Development of nanoagrochemical drugs

Are possible two scenarios

1



Nanocarriers adhere to the membrane and release the biocide.

Therfore:bacteria die.

Nanocarriers penetrate inside the cell and release the biocide

Therfore:bacteria die.



2

Are possible two scenarios

Nanocarrier requirements for large scale applications



Biocompatible

Unexpensive

Industrializable

V. Vergaro, G. Ciccarella : Synthesis of nano-sized CaCO3 particles by spray dryer. European Patent EP13425061.2 - (2013).

nano CaCO3

Nanocarrier requirements for large scale applications



Three main issues need to be addressed:

1. Can nanoCaCO3 be safe for humans and plants?

2. Can nanoCaCO3 move inside the xylems?

3. Can nanoCaCO3 be selective for Xylella fastidiosa?



Nano-CaCO3

MTT Test


Cell viability is not compromised



The germination rate is not influenced by the nanocarrierTests were carried out on:Wheat: T.dur. Triticum durum, cv Croesus 2002Tomato: L.esc. Lycopersicum esculentum, cv MicrotomTobacco: N.tab. Nicotiana tabacum, cv SR1

Phyto-toxicity tests

nanoCaCO3 conc.




No interference on root growth

Wheat

Ctrl Nano-CaCO3 100mg/L




Wheat

Ctrl Nano-CaCO3 100mg/L


nanoCaCO3 can be reasonably considered safe for plants and humans

Nanocarriers: 100 nmXylem diameter : 20 m

Ratio = 1: 200

10 m




xylem magnification 1:10.000

xylem magnification 1:10.000xylem magnification 1:10.000



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




Nanocarriers can be introduced inside the tree.The xylematic flux can distribute the nanoparticles along the trunck reaching capllarly branches and leaves


therefore nanovectors move within the vessels and…when they encounter bacteria hit the target

Analysis by confocal microscopy of xylems before the introduction of nanoCaCO3




Experimental tests

Le strategie oggetto della ricerca all’Università del Salento

Lower part of the stem

Upper part of the stem

fluorescentnanoCaCO3

Analysis by confocal microscopy of xylems after the introduction of nanoCaCO3


Experimental tests

Le strategie oggetto della ricerca all’Università del Salento

Lower part of the stem

Upper part of the stem

fluorescentnanoCaCO3


nanoCaCO3 has a diffusion gradient and then spreads into the plant




Due to its z-potential (-15-20 mV) nanoCaCO3 has a strong affinity for various sugar-based polymers such as:

ChitosanDextran



Therefore we expect that nanoCaCO3 could be also able to bind bacterial exopolysaccharides


There are two possible scenarios

1



Nanocarriers adhere to the membrane.

Therfore:- No aggregation in colonies (with

the formation of a cap responsible of leaves drying);

- Membrane destabilization?- Bacteria die?


Nanocarriers penetrate inside the cell

Therfore:- Membrane destabilization?- Bacteria die?



2


There are two possible scenarios



3. Can nanoCaCo3 be selective for Xylella fastidiosa?

Endosomes (permeation)

Adhesion?

A certain selectivity with the ability to

perforate the membrane seems

possible



In conclusion

Can nanoCaCO3 be safe for humans and plants?

nanoCaCO3 can be reasonably considered safe for plants and humans

Can nanoCaCO3 move inside the xylems?

nanoCaCO3 has a diffusion gradient and then spreads into the plant

Can nanoCaCO3 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

XYLELLA RESEARCH GROUP @ Unisalento Collaborations:

Università di BariProf. Giovanni MartelliProf. Vito Nicola SavinoProf. Giuliana LoconsoleProf. Oriana Potere

CNR IPSPIstituto per la Protezione Sostenibile delle PianteDr. Donato Boscia Dr. Maria SaponariDr. 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 DIAGNOSTICSProf. Giuseppe Cannazza (UniMORE)Dr. Cinzia Citti, Ph.D.NANOCARRIERSDr. Francesca Baldassarre, Ph.D.Dr. Viviana Vergaro, Ph.D.MICROSCOPYProf. Luciana DIniDr. Elisabetta Carata, Ph.D.Dr. Francesco Mura Ph. D. PLANT PHYSIOLOGYProf. Gian Pietro Di Sansebastiano, Ph.D

Thank you for your attention


Xylella fastidiosa

Thank you for your attention


Xylella “irosa”

Giuseppe CICCARELLA 1,2 1 Dipartimento di Scienze e Tecnologie Biologiche e Ambientali e Centro di...

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