Exploring endophytic microbial communities to identify potential biocontrol agents against Xylella fastidiosa strain 'De Donno' - EFSA
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Exploring endophytic microbial communities to identify potential biocontrol agents against Xylella fastidiosa strain ‘De Donno’ Morelli M.1 , Giampetruzzi A.1 , Baptista P.2 , Sisto A.3 , Abou Kubaa R.1 , Altamura G.1 , Boscia D.1 , Cameirão C.2 , Costa D.4 , D’Attoma G.1, De Bellis P.3 , Lino Neto T.4 , Masiello M.3 , Pereira J. A.2 , Saponari M.1 , Zicca S.1 , Saldarelli P.1 1C onsiglio Nazionale delle R icerche, Istituto per la Protezione Sostenibile delle Piante, Se de Secondaria di Bari, Bari, Italy 2Ce ntro de Investigação de Montanha (CIMO), Campus de Santa Apolónia, Instituto Politécnico de Bragança, Bragança, Portugal 3C onsiglio Nazionale delle R icerche, Istituto di Scienze delle Produzioni Alimentari, Bari, Italy 4Biosystems & Integrative Sciences Institute (BioISI), Plant Functional Biology Center (CBFP), Campus de Gualtar, Unive rsity of Minho, Braga, Portugal
Objectives
◼ Investigate changes in the olive
xylem microbiome associated to
Xylella fastidiosa infection
◼ Assess correlations between xylem
microbiome and cultivar responses
to Xylella fastidiosa infections
◼ Identify microorganisms associated
with the resistant phenotypes and
potential biocontrol agents
2
Samples used in this study
Kalamata, November 2018
◼ 6 olive trees cultivar Kalamata
◼ 6 olive trees cultivar FS17
◼ Same age (15 yrs old)
◼ Same management practices (irrigation and yearly
pruning)
◼ First sampling, Spring 2017 FS17®, November 2018
Low or no Xylella fastidiosa infection levels
detected by qPCR
◼ Second sampling, Autumn 2018
qPCR-detectable Xylella fastidiosa population
levels in both cultivars
Manifest desiccations in Kalamata, very mild or no
symptoms in FS17
Giampetruzzi et al., Pathogens 2020, 9, 723. https://doi.org/10.3390/pathogens9090723 3
Sequencing approaches
Whole Metagenome Shotgun Pros and Cons
Sequencing (WMSS)
◼ DNA extraction from xylem shavings + Increased sequencing depth
◼ Library preparation: Illumina DNA + Higher data recovery
PCR-Free Library Prep Kit
- Increased sequencing costs
◼ Sequencing: Illumina TruSeq 2 x 150
bp
16S rRNA and ITS Sequencing
◼ DNA extraction from xylem shavings
+ Consolidated pipeline
◼ Bacteria: amplification of V3-V4
region 16S rRNA gene + More cost-effective
◼ Fungi: amplification of ITS1 region - Analysis on a single gene
◼ Library preparation
◼ Sequencing: Illumina MiSeq paired-
end (2 x 250 bp)
4
General comparative description
WMSS 16S rRNA/ITS
12 phyla 3 phyla
23 classes 10 classes
62 orders 13 orders
115 families 19 families
225 genera 29 genera
◼ Bacteria ◼ Bacteria
◼ Fungi ◼ Fungi
◼ Archaea
◼ Viruses
5
Parameters considered for the analysis Season Spring 2017 vs Autumn 2018 Cultivar Resistant (FS17 ®) vs Susceptible (Kalamata) Xylella fastidiosa relative abundance (Xylella reads/Bacteria reads): High (>5%) vs Low (
Season was the main factor shaping microbial communities
WMSS
Bacteria Fungi
◼ PCA and ANOSIM clearly separated ◼ Significant differences in fungal
olive trees sampled in Spring 2017 microbiomes were found related to
from those sampled in Autumn 2018 the sampling period
Giampetruzzi et al., Pathogens 2020, 9, 723. https://doi.org/10.3390/pathogens9090723 716S rRNA/ITS
Nonmetric multidimensional scaling (NMDS) PERMANOVA analysis: variability in
plots followed by ANOSIM test: bacterial bacterial and fungal composition is mainly
and fungal communities differ significantly explained by season and Xylella fastidiosa
between seasons abundance
Bacteria Fungi
30 ** **
% Total variation
25
20
**
15 **
10
5 *
0
Bacteria Fungi
Season X. fastidiosa Cultivar
Giampetruzzi et al., Pathogens 2020, 9, 723. https://doi.org/10.3390/pathogens9090723 8WMSS
Kalamata FS17
Spring 2017
Effect of Xylella fastidiosa abundance
Autumn 2018
◼ Xylella tends to occupy xylem niche
negatively affecting bacterial 0 20 40 60
community
% Xylella/Bacteria reads
16S rRNA
◼ Xylella represented 72.14 % of the
bacterial microbiome in WMSS
Kalamata FS17
Spring 2017
Autumn 2018
0 20 40 60
% Xylella/Bacteria reads 9Changes in Xylella fastidiosa population size
◼ Kalamata : Xylella fastidiosa
population size increased between
Spring 2017 and Autumn 2018
◼ FS17 ®: Not significant changes
◼ Xylella fastidiosa colonization was
significantly more extensive in
Kalamata
Giampetruzzi et al., Pathogens 2020, 9, 723. https://doi.org/10.3390/pathogens9090723 10Effects on microbiome diversity
Xylella Cultivar Season
Bacteria
◼ Alpha-diversity of Bacteria,
Fungi and Archaea was
higher in FS17®
◼ A higher diversity was
found in Spring 2017 and
Fungi
in presence of low Xylella
fastidiosa populations
Archaea
Giampetruzzi et al., Pathogens 2020, 9, 723. https://doi.org/10.3390/pathogens9090723 11Further objectives Selection of olive trees
◼ Isolation, characterization and ◼ Resistant vs Susceptible cultivars
identification of endophytic bacterial
strains 4 olive trees cultivar Leccino
3 olive trees cultivar FS17
4 olive trees cultivar Kalamata
◼ Evaluation of antagonistic activity
against Xylella fastidiosa strain De
Donno ◼ Mild or no symptoms of OQDS
Leccino and FS17 ®: symptomless
◼ Evaluation of Bacillus spp. strains
Kalamata: few scattered desiccations
known to produce antimicrobial
substances
◼ Low infection levels
qPCR: Xylella fastidiosa undetectable
or at low population size
12◼ A low number of culturable endophytic
bacteria was isolated from olive tissues
Kalamata: Sphingomonas, Curtobacterium
and Methylobacterium
FS17 ®: Sphingomonas, Methylobacterium
Leccino: Micrococcus, Methylobacterium and
Frondihabitans
◼ It was not possible identify bacterial species
specifically associated to one cultivar or
correlated with resistant phenotype or Xylella
fastidiosa infection levels
◼ A total of 120 representative endophytic
bacteria were tested against Xylella fastidiosa
strain De Donno
◼ None showed antagonistic activity
Zicca et al., Microbiol Res. 2020 Jun;236:126467. doi: 10.1016/j.micres.2020.126467 13◼ Strains of Bacillus velenzensis and ◼ Culture filtrates of Bacillus
Bacillus amyloliquefaciens showed the amyloliquefaciens N3.2, D747 and
highest antagonistic activity Bacillus velenzensis QST713 showed
antimicrobial activity, increasing over
time
Zicca et al., Microbiol Res. 2020 Jun;236:126467. doi: 10.1016/j.micres.2020.126467 1415
Acknowledgements
◼ Consiglio Nazionale delle Ricerche, ◼ Centro de Investigação de Montanha
Istituto per la Protezione Sostenibile (CIMO), Campus de Santa Apolónia,
delle Piante, Sede Secondaria di Bari, Instituto Politécnico de Bragança,
Bari, Italy Bragança, Portugal
Giampetruzzi Annalisa Baptista Paula
Zicca Stefania Cameirão Cristina
Abou Kubaa Raied Pereira José Alberto
Altamura Giuseppe
D’Attoma Giusy ◼ Biosystems & Integrative Sciences
Boscia Donato Institute (BioISI), Plant Functional
Saponari Maria Biology Center (CBFP), Campus de
Saldarelli Pasquale Gualtar, University of Minho, Braga,
Portugal
◼ Consiglio Nazionale delle Ricerche,
Istituto di Scienze delle Produzioni Lino Neto Teresa
Alimentari, Bari, Italy Costa Daniela
Sisto Angelo ◼ Apulia Region Research Program
De Bellis Palmira EZIOCONTROL
Masiello Mario ◼ European Union Horizon 2020
POnTE
◼ European Union Horizon 2020
XF-ACTORS
16You can also read
