04 Vannini Presentazione Trabzon

Transcription

COST Action FP1401 Global Warning Trabzon Turkey 24-25/3/15
01/04/2015
Sentinel Trees as a Tool to
Forecast Invasions of Alien
Plant Pathogens
Approach 1: PlosOne in Press
AnnaMaria Vettraino 1, Alain Roques 2, Annie Yart 2,
Jian-ting Fan3 , Jiang-hua Sun 4, Andrea Vannini1
1. DIBAF, University of Tuscia, Viterbo, Italy
2. INRA- UR633, Zoologie Forestière, Centre de recherche d'Orléans, Orléans,
France
3. School of Forestry and Bio-technology, Zhejiang A & F University, Lin'an , China
4. State key laboratory of Integrated Management of pest Insects and Rodents,
Institute of Zoology, Chinese Academy of Sciences, Beijing , China
The increasing globalisation of trade in plants and plant material, together
with the impacts of climate change, has led to an increase in the introduction
and spread of new and damaging plant pathogens
Number of Invasive Forest Pathogens in Europe the past 200
years
Most of these species have
been first described when
already escaped from their
centers of origin and begun
causing damage in a
susceptible plant community
through host jump or
hybridization
From Santini et al., 2012
1
01/04/2015
Why do we fail in preventing
introductions?
Most of the National Plant Protection Organizations (NPPOs) regulate the
inspections of living plants and commodities
based on lists of known organisms described as invasive elsewhere (Lists of
regulated quarantine and no-quarantine harmful organisms (e.g. the EPPO A1
and A2 list). These lists consider pathogens only after they have escaped
from its geographic center of origin and begun causing damage in a
susceptible plant community
PRA protocols are developed on already escaped organisms and lack of data
on their ecology, potential host range and adaptation
Only a tiny proportion of imports is inspected (APHIS ~2%)
Phytosanitary inspections in all EU countries don’t adhere to the same
standard
Why we fail in preventing
introductions?
Because most of the diagnostic protocols and inspections measures are
directed to specific taxa (eg A1 and A2 listed organisms)
As a consequence they may cover only a minority of the organisms which
pose a threat………..around 10%? Basically they do not, or barely, consider
the unknowns to science
They did not allow for the existence of large numbers of high risk ‘unknown
pathogens’ that, due to Darwinian co-evolution with their hosts, were
causing little damage in their centres of origin
Unknowns to science might enter unnoticed with their native hosts or in
associated materials (eg soil, potting mix) with non-hosts
2
Increase of invasions by IFP’s in
the past 2 centuries
01/04/2015
Phytosanitary regulations and
measures are flawed
New strategies need to be employed in order to identify organisms harmful to
European forests before their introduction
Sentinel trees Network in its broad
The concept of
sense fully responds to this need
In fact it responds to:
• Approach 1 (pests & pathogens). The need of
identify plant pathogens aggressive to EU
species by establishing and monitoring
sentinel plantations of selected (selection
based on? WG1) EU plants in target exotic
environments.
• Approach 2 (pathways). The need of making
invasion pathways secure by establishing
sentinel nurseries of the most traded exotic
plants to EU at their area of origin, and
to monitor them for pathogens infection
3
01/04/2015
Sentinel trees network- Approach 1
Example 1: monitoring (how? Common protocols? WG2) the impact of native
pathogens to European tree species planted in China
Sentinel trees network- Approach 2
Example 2. monitoring (how? Common protocols? WG2) the presence of
native organisms in Chinese tree species commonly exported to Europe, and
potentially harmful to European plant communities
4
01/04/2015
Sentinel trees network
Approach
1
Approach
2
Approach
1
1°
2°
• symptoms and signs are expected (lack of co-evolution)
• symptoms assessment, biological and molecular detection
ARE NECESSARY
• molecular analysis of resident microbial community is
relevant
• symptoms and sign are NOT necessarily expected (coevolution)
• biological and molecular detection is relevant
• Molecular analysis of resident microbial community IS
NECESSARY
European tree species
planted in China
• Plantation of European tree species in China
(which propagation material? According to
which agreement? WG1 &3)
Q. suber
• Survey of diseases (WG1&2 exp. design)
Q. petraea
3°
•Pathogens detection (how? Unique shared
protocol? WG2)
Q. ilex
5
Approach
1
01/04/2015
planted in China
Fuyang-Zhejiang
Approach
1
planted in China
In 2008 bare-rooted plants were shipped to China
(feasible as standard procedure? WG1 & 3)
A phytosanitary certificate declared their healthy
status (useful? WG3)
The plants received a fungicide treatment:
UNKNOWN
The bare-rooted plants were kept 3 weeks in
quarantine at the airport before being planted
No symptoms were detectable after 3 weeks of
quarantine
6
Approach
1
01/04/2015
Fungal detection
Fungal isolation: morphological analysis
Fungal isolation: molecular analysis (how to
barcode? which markers? WG2) :
Q. suber
rDNA Internal Transcribed
Spacer (ITS)
glyceraldehyde-3-phosphate
dehydrogenase (gpd) gene
Q. petraea
beta-tubulin gene
Q.ilex
Approach
1
454-NGS
DNA extraction/amplification
ITS1F-ITS2 (appropriate marker? WG2)
Q. suber
Pyrosequencing (other useful
platforms? WG2)
Reads: filtering and trimming
Q. petraea
Blastclust/Phylogenetic analysis (shared
software & protocol WG2)
Identification (which reference database?
WG2)
Q.ilex
7
01/04/2015
Symptoms assessment
Approach
1
Signs and perythecia of Leptosphaeria
cankers on Q. petraea
Spots on Quercus spp.
Conidia of Pestalopsis sp. and
Alternaria sp. from Quercus spp.
Signs of Erysiphe on Q. petraea
Approach
1
A total of 9 OTUs have
been identified of possible
Asiatic origin
8
Approach
1
01/04/2015
OTU 102 refers to a
Chinese
Leptosphaeria sp
The genus Leptosphaeria includes a large number of species mainly
considered saprobic or necrothrophic on stems and leaves. Nevertheless
the genus comprises also highly pathogenic species
Approach
1
The OTUs 38 and 88 refer to
the Mycosphaerella
complex
Mycosphaerella spp. are
among the most common
and destructive plant
pathogens known, causing
considerable economic
losses on a wide variety of
host plants worldwide,
including economically
important horticultural and
ornamental crops
9
Approach
1
01/04/2015
OUT 4 refers to Erysiphe quercicola
2006- Takamatsu et al. proposed the
Asian origin
2012 - E. quercicola has been detected
in France and associated to flagshoot symptoms of Q. robur and
Q. petraea
2008- Mougou et al. suggested that E.
quercicola oak powdery mildew
might originate from host shifts
of tropical Erysiphe species
introduced to Europe through
infected exotic host plants
2014- Kirshner and Liu reported
anamorphic E. quercicola on 2
tropical hosts commonly traded
to Europe as ornamentals
Approach
1
Discussion
Our study reports for the first time the colonization of a selected collection of EU
trees by native fungi while growing in China, and the potential of sentinel
plantations to detect taxa pathogenic to EU trees that might pose a biosecurity
risk for Europe. The sentinel plantation of Fuyang represents an improvement of
the ISPN hypothesis based on the use of existing botanical gardens worldwide.
Infact, in this study, the EU trees were selected according to ecological criteria
(WG1).
They were planted in China in sites comparable in climate and plant community
composition to those of their area of origin in Europe (WG1). Presence of native
species belonging to the same family or genus (e.g. the Fagaceae family in the
Fuyang plot) and having similar ecological requirements enhances the probability
of exposure of sentinel trees to natural inoculum pressure. The placing of the
European trees in these specific sites was thought to favours “host jump” by
native pathogens facilitating the evaluation of their aggressiveness.
10
Approach
1
01/04/2015
Discussion
A total of 9 OTUs have been identified of possible Asiatic origin, based on clustering in
NJ trees (WG2) within groups of sequences of taxa mostly reported from Asia.
Four out of the nine taxa of Asiatic origin, are related to plant pathogenic genera and were
associated with symptoms and signs typical of infection on the sentinel trees (WG2
prerequisite?).
In conclusion, the sentinel plantations represent an appropriate answer to the growing
need of preventive identification and characterization of plant pathogens at risk of
introduction and invasion in Europe (and elsewhere). However the preliminary experience
carried out in the Fuyang plantation highlighted the logistic and technical difficulties in
utilizing sentinel trees for pathogens detection. Efforts should be made to solve regulatory
and logistic bottlenecks associated with biosecurity issues (WG3) and the possibility of
full characterization of biological samples, in order to make possible the routinary and
reciprocal utilization of this approach between countries (WG3).
Approach
2
monitoring the presence of
native microbes in Chinese
tree species commonly
exported to Europe
11
Approach
2
01/04/2015
Why Approach 2 is
important?
Darwinian evolution predicts that, being adapted to and co-evolved with
their hosts, many of these “unknown to science” are unlikely to do
noticeable damage in their native ecosystems, and so are less likely to be
detected even when introduced in a new environment with their native hosts
We do not expect necessarily symptoms on native hosts caused by native
pathogens
Approach
2
Location of the sentinel tree
plot in China
Beijing
Acer palmatum, Fraxinus chinensis , Ilex conrnuta , Zelkova schneideriana , Buxus microphylla
12
Approach
2
01/04/2015
Chinese tree species
commonly exported to
Europe (WG1&3)
1°
• Plantation of Chinese
species in China (WG1-3)
2°
• Survey of diseases (WG1
& 2 exp. design)
3°
• Pathogens detection
(WG2)
Approach
2
Samplings and diagnostic activities
Acer palmatum, Fraxinus chinensis , Ilex conrnuta , Zelkova schneideriana , Buxus
microphylla
13
Approach
2
01/04/2015
Biological detection
Acer palmatum
Fungal isolation: morphological analysis
(protocols?)
Zelkova schneideriana
Fungal solation: molecular analysis (WG2 issue;
appropriate barcoding?):
Buxus microphylla
rRNA gene internal transcribed
spacer (ITS)
glyceraldehyde-3-phosphate
dehydrogenase (gpd) gene
Ilex conrnuta
beta-tubulin gene
Fraxinus chinensis
Approach
2
Symptoms assessment
Deep canker Sup canker
Shoot blight
Fraxinus chinensis
Ilex cornuta
Acer palmatum
14
01/04/2015
Tricothecium roseum
Approach
2
Biological
detection
Botryosphaeria dothidea
Alternaria alternata
Colletotrichum gleosporioides
Phomopsis sp.
Pestalotiopsis sp.
Phoma sp.
Approach
2
monitoring the presence of native microbes
in Chinese tree species commonly exported
to Europe, and potentially harmful to
European plant communities
Chinese plants developed symptoms associated to
worldwide diffused plant pathogens and to some taxa
apparently not described (unknowns to science?)
These undescribed taxa belong to genera that host a
large number of plant pathogens
For none of them has been possible to recognize a
“chinese origin” (invasives?)
However their pathogenicity to European ‘relatives’
hosts has to be studied (risk of host jump?)
microphylla
15
Approach
2
01/04/2015
454-NGS
Acer palmatum
DNA extraction/amplification
ITS1F-ITS2
Pyrosequencing
Buxus microphylla
Reads: filtering and trimming
Blastclust/ Phylogenetic analysis
Ilex conrnuta
Identification
Fraxinus chinensis
Approach
2
NGS statistics
35.839 reads after trimming
Minimum 93, maximum 6.549 per sample
A total of 148 Operational Taxonomic Units
Botryosphaeriales
Chaetothyriales
Dothideales
Eurotiales
Helotiales
Lichenostigmatales
Myriangiales
Pleosporales
Taphrinales
Agaricales
Erythrobasidiales
Leucosporidiales
Sporidiobolales
Capnodiales
Xylariales
Dothideomycetes
Glomerellales
Hypocreales
Microascales
nd
Saccharomycetales
Trichosphaeriales
Corticiales
Filobasidiales
Polyporales
Tremellales
25 fungal orders
16
01/04/2015
Symptoms
Acer palmatus
Plant
Stock
SB1
Buxus microphylla
Sentinel
Nursery
Taxa
sp
.
Tr
ro ich
se o
um th
ec
iu
Co
m
lle
to
tri
ch
um
sp
Bo
.
do t r
th yo
id sp
ea h
ae
ria
Ph
om
op
si
s
sp
.
Ph
om
a
sp
.
Pe
st
al
ot
io
ps
is
sp
.
Source
Al
te
rn
ar
ia
Hosts
Plant
Stock
Fraxinus chinensis
Ilex cornuta
Sentinel
Nursery
Plant
Stock
Plant
Stock
Sentinel
Nursery
6
LN1
DC1
DC2
DC1
DC2
SB
SC1
SC2
SC3
SC4
SC5
LN1
LN2
LN1
SN1
SN2
SN3
1
1
9
8
3
21
10
2
6
1
2
1
2
7
2
4
2
2
3
3
2
2
1
1
1
4
2
9
3
4
2
SN4
3
Taphrina sp.
17
01/04/2015
Buxus microphylla
Elsinoe sp.
Max 89% identity on NCBI…possible new species
Buxus microphylla
Volutella buxi
Pathogen on Buxus
18
01/04/2015
Buxus microphylla
Colletotrichum yunnanense
Endophyte on Buxus in China
Liu, Xiaoying, Xiangming Xie & Junxing Duan. Colletotrichum
yunnanense sp. nov., a new endophytic species from Buxus
sp. Mycotaxon 100: 137–144. 2007.
Buxus microphylla
Phyllosticta citrichinaensis
Foliar pathogen of Citrus in China
19
01/04/2015
Fraxinus chinensis
Phyllactinia populi
Powdery mildew of poplar in China and Korea (CABI)
Blumeria graminis
Powdery mildew of wheat: Japanese and Chinese
genotypes?
20
01/04/2015
Fraxinus chinensis
Strelitziana mali
Zhang, Rong, Hanli Yang, Guangyu Sun, Huanyu Li,
Jieli Zhuang, Xiaoru Zhai & Mark L. Gleason.
Strelitziana mali, a new species causing sooty blotch on
apple fruit. Mycotaxon 110: 477–485. 2009.
Zhang, Rong, Hanli Yang, Guangyu Sun, Huanyu Li, Jieli Zhuang, Xiaoru
Zhai & Mark L. Gleason. Strelitziana mali, a new species causing sooty
blotch on apple fruit. Mycotaxon 110: 477–485. 2009.
Approach
2
NGS analysis revealed a rich fungal community
associated to the 5 Chinese trees species
Among them many common endophytes and
epiphytes taxa
A number of ‘known’ native fungal pathogens and
additional ‘candidates’ have been identified whose
impact to European plant community deserve to be
investigated
Some of these taxa were known as pathogens on plant
hosts other than the 5 tree species considered in this
study (possibility of unnoticed introduction)
microphylla
21
Approach
2
01/04/2015
NGS seems to be a convenient technique to be used to
scan the microbial community associated to native
plants to be shipped overseas (yes it is but needs to be
used in a shared protocol WG2)
Obviously NGS results need confirmation through
biological detection especially for those organisms
potentially candidates to be aggressive to exotic hosts
(very important but not necessarily feasible WG2)
Furthermore the technique offers the opportunity to
individuate undescribed taxa (unknown to science)
microphylla
Kauri trees threatened by
Phytophthora taxon Agathis in New
Zealand
22

04 Vannini Presentazione Trabzon

Transcription

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