sixième conférence internationale

AFPP – 3rd CONFERENCE ON MAINTENANCE OF AMENITIES AREA
TOULOUSE – 15, 16 AND 17 OCTOBER 2013
CONTROL OF PHYTOPHTHORA SPREAD BY EARLY DETECTION ON ORNAMENTAL
AND WOODY PLANTS IN NURSERY
D. MIGLIORINI
(1, 2)
(1) .
(1)
, E. TONDINI , N. LUCHI , L. GHELARDINI
P. CAPRETTI (2), A. SANTINI (1)
(1)
,
1
IPP-CNR -Via Madonna del Piano, 10 – 50019 Sesto Fiorentino, FI, Italy
[email protected]
2
Dept. Agriculture, Food and Environmental Science, DISPAA
– Piazzale delle Cascine, 28 – 50144 Firenze, Italy
ABSTRACT
The international trade of ornamental plant for planting is a primary pathway of diffusion of
invasive plant pathogen species. Nurseries play a primary role in pathogens spread within
the whole Europe, since the infected plants imported from abroad are kept for long periods.
One of the main European nursery pole for ornamental plants (80% exported to foreign
countries; mainly woody plants in pot) is located in Pistoia, Tuscany, Italy, where nurseries
are spread over more than 5300 hectares. Species of the pathogenic genus Phytophthora
(Oomycetes) were isolated from potted plants in two nurseries of the area. Eight different
Phytophthora species were isolated both from symptomatic and symptomless plant species.
These informations are particurlarly usefull for those work sectors involved in forest and
green urban planning, to employ plant’s material certified by Pathogens bio-security
protocols.
Keywords: Phytophthora infections, ornamental plant disease, international plant trade,
invasive species pathway, Bio-security protocols.
RÉSUMÉ
CONTROLE DE LA PROPAGATION DU PHYTOPHTHORA PAR DETECTION
PRELIMINAIRE EN PEPINIERE SUR LES PLANTES ORNAMENTALES ET
FORESTIERES
Le commerce international est la principale voie de propagation des espèces pathogènes
envahissantes. La vente des plantes répresente une voie importante d'introduction. Les
pépinières ont un rôle central dans la diffusion des agents pathogènes à travers l'Europe
lorsque les plantes infectées importées de l'étranger sont gardées durant de longue période.
L'un des centres les plus importants de pépinières de plantes ornamentales se trouve à
Pistoia, en Toscane, Italie (pépinières réparties sur plus de 5300 hectares, dont 80% de la
production des plantes en pot, principalement destinée aux pays étrangers). Des espèces de
l'agent pathogène Phytophthora (Oomycetes) ont été isolées à partir de plantes en pot
échantillonées dans deux pépinières de la région, fournissant des données sur la diversité
taxonomique et le degré de diffusion (huit espèces de Phytophthora différents ont été isolés)
On peut donc envisager la nécessité d'utiliser, dans le secteur forestier et des espaces verts
urbains, des plants végétaux certifiés grâce à l'utilisation de protocoles phytosanitaires de
biosécurité.
Mots-clés : Infections de Phytophthora, maladies de plantes ornamentales, commerce
intarnational de plantes, voies de propagation des espèces envahissantes, protocoles de
biosécurité.
INTRODUCTION
Worldwide human activity and changes in climatic conditions as increase of atmospheric
temperatures and modifications in rainfall dynamics occurred in the past decades are the
most influent and studied topics according to the alien species occurrence. The research
activity of this phenomenon is strongly increased during the last years on an international
scale. European Union, according to an estimate damage of 12.5 billions of Euro per year,
financed research programs focused on the problem.
Pysek et al. (2010) prove that country richness and human density factors, when valuated
with climate, geography and vegetation’s soil cover, are statistical significant indexes for the
largest part of biological invasion forecast models. Economic and demographic variables, as
indexes of human activity grade, increase the effect of factors like propagulus pressure,
pathways, eutrophication and human disturbing intensity, directly involved in the outcome of
invasion events.
Nursery’s commercial trades, due to continuous and wide movements of products, are one
of the most important introductions and spread pathways for exotic pathogens and pests.
Inside nurseries constant irrigation and greenhouse climate conditions create optimal
environments for the establishment and growth of new introduced microorganisms. Once
plants got diseased, financial losses occur, due to the impossibility to sell them and the need
of frequent chemical treatments. Fungicides and fungi-statics just silence the disease
outcome on the plants leading the persistence of the pathogen and, providing the market by
apparently healthy material with cryptic infections, its diffusion in the field.
Many studies, due to the primary need of recognizing and stopping a disease when is still inside the
nursery, are focused on develop rapid detection methods for pathogens introduced with commercial
plant trades. Phytophthora, Oomycetes group, counts more than 70 species diffused all over the
world and it’s one of the most damaging genus for the ornamental and forestry wood plants market
production. Species are mainly root rot agents and the infection occurs via rot system. A few
Phytophthora species are agents of stem canker or crown disease, in these case the infection is
aerial. Thanks to a large taxonomical diversity and several reproduction system, they are present in
all temperate and tropical environments demonstrating efficient adaptability to different conditions
from sub-tropical to oceanic climates.
The aim of this work is to evaluate the occurrence rate of Phytophthora in nursery using
traditional techniques and molecular markers analysis.
An adequate panel of the studied species distribution will allow to evaluate significant
correlation between pathogen’s taxonomy and health state of the host plant in order to
estimate the potential risk of infection inside and outside the nursery.
MATERIAL AND METHODS
Area of study
The survey was achieved in two nurseries in Pistoia (Italy). This area is the most important
European nursery pole for woody ornamental plant production: 1.732 companies in the year
2007; import and export 2011 of 18.350.231 € and 210.102.283 €.
The material sampled consists of plants growing in pot showing three different health’s state:
a) “symptomatic plants” (visible problems on the crown); b) “asymptomatic plants” (plants
with healthy crown but necrotic roots);c) “healthy plants”(Image I).
Image I Plant samples of the three different health state: a) “symptomatic plants”
(visible problems on the crown); b) “asymptomatic plants” (plants with healthy crown but
necrotic roots);c) “healthy plants”
(Des échantillons de plantes présentant les
trois différents états sanitaires: a) «plantes avec symptômes» (problèmes visibles sur la
couronne), b) "végétaux sans symptômes» (plantes avec couronne en bonne santé mais
des racines nécrosées), c) "plantes saines")
a
b
c
Isolation
Isolations were made on potting media and roots of all the plants. Root fragments of 2-3 mm
were directly isolated on PARP-CME substrate (Erwin et al.,1996) and then transferred on
potato dextrose agar media (PDA).
Pot substrate was also assayed for Phytophthora presence by apple baits. Baiting was
performed according to Jung et al., 1999.
DNA extraction and ITS sequencing
Phytophthora isolates were grown on Petri dishes with cellophane; the micelia were
harvested and DNA extraction was realized using the EZNA Plant DNA Kit (Omega Bio-tek)
following the Plant DNA Short Protocol. Amplification of the ribosomial DNA ITS region was
carried out with primers ITS6 and ITS4 that are currently indicate for taxonomical
investigation within the Phytophthora genus (White et al.,1990; Grunwald et al., 2012).
The morphological identification of the isolates was confirmed by sequences alignment on
NCBI BLAST database.
RESULTS
ITS analysis revealed a total of eight Phytophthora species. Taxons obtained icludes
common species of ornamental nurseries in Europe such as Phytophthora syringae,
Phytophthora citrophthora, Phytophthora cactorum, Phytophthora nicotianae, Phytophthora
cryptogea, Phytophthora cinnamomi, Phytophthora citricola and less common or rare
species such as Phytophthora palmivora (Tab 1).
Table I Oomycetes and other fungal pathogens isolated on each plant species
included in the study.
(Oomycetes et autres agents pathogènes fongiques isolés à partir des
espèces végétales étudiées.)
Plant species
Arbutus unedo L.
Oomycetes and others fungi detected
P.cinnamomi, P.citrophthora, P.nicotianae, P.palmivora,
Pythium
helicandrum,
Pythium
vexans,
Morteriella
sp.,Grogronella sp.
Buxus sempervirens P.citrophthora, P.nicotianae, P.palmivora, Pythium irregolare,
L.
Pythium arrhenomanes
Ceanothus thyrsiflorus P.citrophthora, P.nicotianae, P.palmivora,
E.
Cupressocyparis
Pythium vexans, Mortierella sp.
leylandii
Cupressus
P.palmivora, Pythium vexans, Pythium undulatum, Mortierella sp
sempervirens L.
Euonymus sp.
P.citrophthora, P.nicotianae, Pythium chamaihypho, Pythium
dissotocum, Pythium vexans, Phytopythium litorale, Mortierella
sp., Grongronella sp.
Hibiscus sp.
Pythium cylindrosporum
Laurus nobilis L.
P.cinnamomi, Pythium perplexum
Myrtus communis L.
P.cinnamomi, P.nicotianae, Pythium vexans, Mortierella,
Zygorhynchus sp, Fusarium sp.
Nerium oleander L.
Ceratobasidion sp.
Pittosporum
tobira P.palmivora
(Tumb.) W.T.Aiton
Prunus laurocerasus P.criptogea,
P.palmivora,
Pythium
chamaihyphon,
L.
Phytopythium litorale, Pythium vexans, Mortierella sp.,
Ceratobasidion sp
Prunus lusitanica L.
P.palmivora , P.nicotianae
Thuja occidentalis L.
P.cinnamomi, P.nicotianae, P.syringae, Pythium litorale,
Pythium vezans, Pythium elicoides, Fusarium oxysporum
Viburnum tinus L.
P.cactorum, P.citricola, Mortierella sp., Ceratobasidion sp.,
Conidiobolus sp.
A preliminary elaboration of the data has shown higher isolation frequencies for P.
nicotianae, P. cinnamomi, and P. palmivora (Tab.II).
Table II molecular analysis.
Isolation frequency of Phytophthora species detected in the study by
(Fréquence d'isolement des espèces de Phytophthora obtenues dans
l'étude en utilisant des techniques moléculaires.)
Phytophthora species
P. citricola
P. cactorum
P. cryptogea
P. nicotianae
P. palmivora
P. cinnamomi
P. citrophtora
P. syringae
Taxon frequency
<5%
<5%
10%
27%
25%
25%
5%
<5%
The highest number of Phytophthora species is been obtained on Arbutus unedo (Fig.1).
High level of susceptibility is also observed for Buxus sempervirens, Ceanothus thyrsiflorus
and Thuja occidentalis, each one hosting three species of the pathogen.
Figure 1-
Number of Phytophthora species per host species.
(Nombre d'espèces de Phytophthora par espèce hôte.)
Phytophthora taxons distribution per host health’s class is showed in the diagrams of figure
2. “Symptomatic plants” class contains all the eight Phytophthora species detected in this
work, while in both “Asymptomatic Plants” and “Healthy plants” classes the number decrease
to four .
Figure 2 Number of Phytophthora species isolated on symptomatic plants
(above), plants with no crown’s symptoms and healthy palnats (midle and below).
(Nombre d'espèces de Phytophthora isolées sur les plantes présentant
des symptômes (ci-dessus), plantes à feuillage sans symptôme et plantes saines (au milieu
et en bas)).
DISCUSSION
The presence of Phytophthora species in two nursery of Tuscany has been demonstred in
this work. The main species found in this survey are P. cinnamomi and P. nicotianae
considered as main agents of damage in nurseries and in natural ecosystems. P.
cinnamomi, originally from either Papua New Guinea or Sumatra, has long been introduced
in the agricultural and horticultural environments from which, thanks to its broad host range,
it has repeatedly escaped on different wild hosts around the globe (Garbelotto, 2008).
P.nicotianae is able to cross-hybridization with P. cactorum as demonstrated by Bonants et
al. (2000) who detected the new line generated pathogen in many host plants. As mentioned
in th introduction paragraph, nurseries and green house environments offers optimal
conditions for the cross-breeding due to a continuous flux of infected plants and constant
presence of water.
Arbutus unedo L. has the largest amount of pathogen species detected in the present work.
That plant, as many others not included in the study, is both diffused as an ornamental
product in Mediterranenan area and in natural ecosystems, showing an ecological value in
temperate regions. Potential level of damage for this host is amplified if we consider its high
grade of susceptibility in relation to the spreading of the host.
In this study Phytophthora species were isolated from soil and roots collected from
symptomatic plants, but also from symptomless hosts.
Samples collected from “Symptomatic plants” revealed to be a reserve of Phytophthora
inoculum where the pathogen, due to its diffusion capacity throw nursery’s irrigation water
sistems, reaches healthy roots plant, starting new infections. Interestlingly, the presence of
Phytophthora has been found also from “healthy plants”, where the pathogen can easily hide
its presence spreading outside the nursery, occasionally causing large damages on natural
stends or diffuseing its inoculum in nurseryes of different continent, as an alien species.
CONCLUSION
The commercial trade of potted plants enhance the spreading of several pathogens such as
the Phytophthora species. For these reasons the development of early detection methods,
based on molecular tools, such as real time PCR assay, could help to detect these
pathogens before symptoms occurs in the hosts, and monitoring with adequate
phytosanitary protocols for control the outbreak of disease inside and outside the nursery.
An adequate knowledge of these aspects are of a primary importance supporting the urban
green area planning and reforestation, in order to minimized the risk of damages in natural
stands and losses of plant material, with economical consequences.
ACKNOWLEDGEMENTS
The authors are grateful to the two nursery companies to collect plant material for the
investigations.
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