Download New pathogens of Solanum elaeagnifolium investigated as

3rd International Symposium on Weeds and Invasive Plants
October 2-7, 2011 in Ascona, Switzerland
New pathogens of Solanum elaeagnifolium investigated as possible biological
control agents of the weed in Greece
Javid Kashefi1,* and Anastasia L. Lagopodi2
ARS European Biological Control Laboratory, Thessaloniki, Greece
2 Plant Pathology Laboratory, School of Agriculture, Aristotle University, Thessaloniki, Greece
1 USDA
ABSTRACT: Solanum elaeagnifolium (Silverleaf nightshade) is an alien invasive weed in
Greece, parts of southern Europe and many North African countries with Mediterranean climate. Since
its establishment in Greece in early 1900’s, it has become rapidly one the most dangerous and aggressive weeds in the country, especially in north and central parts. Climate change and the rise of
temperature in the Mediterranean basin increase the possibility of spread and establishment of the
weed in new regions, since it is extremely resistant to drought. Recently two new fungi were discovered attacking the weed in Greece and the goal is to evaluate the impact of their presence, and the
possibility of using endemic pathogens for biological control of S. elaeagnifolium. The first fungus has
been identified as Verticillum dahliae. However, V. dahliae is a serious pathogen and most of its
strains are pathogenic to numerous hosts, so it is not appropriate as a biological control agent. On the
other hand the presence of this fungus on a widespread weed is a threat to the cultivated plants, since
diseased S. elaeagnifolium would facilitate the maintenance and proliferation of the fungus’s propagules in the soils. The second fungus is Erysiphe sp. Chances of Erysiphe sp. to be specific to S.
elaeagnifolium are very high, since most powdery mildew pathogens are host specific, but this needs
further justification. This is the first world record of both fungi attacking silverleaf nightshade. The use
of endemic natural enemies of the invasive, alien weeds in Europe would bring the challenge of their
control to a new level.
KEYWORDS: Solanum elaeagnifolium, Verticillum dahliae, Erysiphe sp., biological control
1
INTRODUCTION
Solanum elaeagnifolium is a perennial plant
20 cm to 1.2 m in height. The stems are covered
with short spines, ranging from very few on
some plants to very dense on others. Leaves
and stems are covered with downy hairs that lie
against and hide the surface, giving a silvery or
greyish appearance. The leaves are up to 10 cm
long and 0.5 to 2.5 cm wide, with shallowly
waved edges. The flowers, appearing from April
to August, have five petals united to form a star,
ranging from blue to pale lavender or white; five
yellow stamens and a pistil form a projecting
centre. The plant produces yellow or orange
berries that last all winter and may turn brown as
they dry. According to recent studies, (MarieClaude Bon, personal communication), the
origin of the Greek population is Texas, USA
and probably it has been introduced into Greece
by contaminated seeds around 1930. The epicenter of the weed in Greece is central Macedonia with the heaviest infestations. However, it
has been reported from all the other regions of
the country. Studies concerning the economic
impacts of the weed in Greece have not been
conducted yet. Control of the weed is extremely
difficult and confounded by its ability to produce
thousands of viable seeds from a single plant,
Corresponding author address: Javid Kashefi,
USDA ARS European Biological Control Laboratories, Thessaloniki, Greece;
Tel.: +30 2310 473252; fax: +30 2310 473272;
e-mail: [email protected]
as well as a deep and well developed root system, which gives to the plant the ability to propagate vegetatively from creeping lateral roots,
root fragments and rhizomes. The weeds’ resistance to drought and harsh environmental
conditions is well known and documented. The
most usual used methods of control are ploughing and the use of strong herbicides which
brings short time relief but no permanent solution. Intensive mowing all year around can help
to control smaller infestations in farms and gardens.
Some of the classical biological control
methods use natural enemies of the target organism in order to minimize its population to a
tolerant level. The method has several advantages as compared to chemical control. Most
of all it is environmentally friendly, sometimes it
is the only control method available and can be
more easily accepted by the public. Fungal
pathogens over other plant pathogens are preferably used as biological control agents because they are easy to reproduce, easy to maintain and require no vectors for their application.
A principle of biological control using fungi is
that they should be host specific on the target
host and should not cause diseases to other
plants (Gnanamanikam et al., 2002). As S.
elaeagnifolium is a relatively new species for
Greece, there is no information about its natural
pathogens in this country. The goal of this research is to investigate areas with dense popu-
3rd International Symposium on Weeds and Invasive Plants
October 2-7, 2011 in Ascona, Switzerland
lations of S. elaeagnifolium, report the occurrence of new fungal diseases, and further on to
investigate the possibility that these pathogens
are used as potential biological control agents of
this weed.
2
MATERIALS AND METHODS
2.1 Area and time of research
Field surveys were initiated in late summer
2009 in fields near Thermi and Triadi, areas in
Thessaloniki prefecture northern Greece. Fields
with S. elaeagnifolium were periodically visited
from spring, a time when new plants growing
from old rhizomes are blooming, until late summer-autumn, when the plant’s fruit is approaching maturity.
2.2 Occurrence of diseases, description of
symptoms and identification of pathogens
A high incidence of a disease with plants
showing severe wilt symptoms was observed
over large areas around Thermi, in late summer
2009. In some cases plants showed wilting
symptoms on one side. Diseased plants were
retarded in growth, dried out prematurely, and
most of them produced few or no fruit. Examination of internal tissues revealed extensive dark
discoloration of the vascular system. A fungus
producing slowly growing white colonies, which
eventually turned to black, was repeatedly isolated from the infected tissues. Microscopic examination of the colonies showed that the fungus produced spores on conidiophores characteristic for Verticillium dahliae Kleb. (Goud et al.,
2003). Hyphae gave rise to microsclerotia which
turned the colour of the colony white culture to
black. The fungus was molecularly identified
after amplification of the internal transcribed
spacer 1 (ITS1) and ITS2 regions, including the
ribosomal 5.8S RNA gene (White et al. 1990).
During summer 2010 another disease was
observed on silverleaf nightshade over large
areas in Triadi. Leaves exhibited yellowish areas
covered with a fine whitish mycelium, typical of a
powdery mildew infection, and dried out prematurely. The mycelial masses expanded over the
leaf, covering much of its surface, turning to
whitish-grey. Infected plants died prematurely.
Pin-point, black round structures were observed,
scattered in the mycelial mass, when leaves
started to dry out. Microscopic examination of
the infected leaves showed numerous barrelshaped conidia. The tiny, dot-like black structures were cleistothecia that lacked characteristic appendages, and in late autumn contained
numerous asci with ascospores. Based on the
presence and morphology of cleistothecia and
the number of asci the sexual stage of the fungus was recognized as an Erysiphe species,
and the barrel-shaped conidia were considered
as spores of its anamorph (Hanlin, 1990).
2.3. Pathogenicity tests and investigation of host
range
First, pathogenicity of V. dahliae isolated
from S. elaeagnifolium was tested on the weed
itself in order to fulfil Koch’s postulates. After
proving pathogenicity on its original host, the
fungus was used to inoculate paprika (Capsicum
annuum) plants. Additionally, two virulent isolates of V. dahliae, one from eggplant, and another from olive tree were tested for pathogenicity on S. elaeagnifolium. Fungal inocula were
prepared by growing each fungus in 250ml
Czapek-Dox broth, in 500ml Erlenmeyer flasks,
at 23oC, under continuous shaking, for 1 week.
The cultures were filtered through 4 layers of
cheesecloth, and the collected number of conidia was adjusted to a concentration of 106
spores/ml, by the use of a haematocytometer.
S. elaeagnifolium seedlings grew in Jiffy peat
pellets for 6 weeks prior to inoculation. The outer fine net of each Jiffy peat pellet was removed,
so tiny wounds would be produced on the roots
and the whole content of the Jiffy, containing the
seedling, was dipped in V. dahliae inoculum for
10 min. Twenty S. elaeagnifolium seedlings
were inoculated with each of the three V. dahliae isolates, and 20 seedlings dipped in deionized water served as control.
Paprika seedlings grew in a nursery for 4
weeks prior to inoculation and were dipped with
naked roots in V. dahliae inoculum. A slight trim
of the paprika roots, in order to produce tiny
wounds, took place prior to inoculation. Twenty
plants were inoculated with V. dahliae isolated
from S. elaeagnifolium, and another 20 plants
dipped in de-ionized water served as control.
The inoculated plants grew for 6 weeks in a
greenhouse. They were visually examined for
wilt symptoms, and the disease progress was
followed for up to 6 weeks post inoculation. At
the same time isolations from surface disinfected tissues were made, from root and stem of the
inoculated plants, exhibiting severe wilt symptoms, in order to confirm the success of the artificial inoculations. The experiment was repeated
twice.
Pathogenicity of Erysiphe sp. isolated from
S. elaeagnifolium was tested on the weed itself
in order to fulfil Koch’s postulates. Release of
ascospores and artificial inoculation by these
spores was not successful. Inoculations by conidia were made by dusting the spores, with the
aid of a fine brush over the leaves of wet, 8
weeks old S. elaeagnifolium seedlings, in pots.
3rd International Symposium on Weeds and Invasive Plants
October 2-7, 2011 in Ascona, Switzerland
The leaves were then closed in plastic bags for
2 days. Ten seedlings were artificially inoculated
with conidia and another 10 seedlings with wet
leaves, closed in plastic bags, for 2 days, were
used as controls. Seedlings were examined for
symptoms of powdery mildew for the following
12 days.
3 RESULTS AND DISCUSSION
V. dahliae isolated from S. elaeagnifolium
was proved to be extremely virulent on its original host. All the artificially inoculated seedlings
of S. elaeagnifolium developed severe wilt
symptoms and all of them died within 3 weeks
post inoculation. The fungus was successfully
re-isolated from stems and roots of all inoculated plants. The V. dahliae isolate from S.
elaeagnifolium was also proved to be extremely
virulent on paprika. A hundred percent of artificially inoculated paprika seedlings developed
severe disease symptoms in 4 weeks post inoculation, and the fungus was successfully reisolated from stems and roots of all inoculated
paprikas.
At the same time, S. elaeagnifolium was
successfully inoculated from two other V. dahliae isolates originating from eggplant and olive
tree. All seedlings developed typical wilt symptoms 6 weeks post inoculation. The fungi were
re-isolated from roots and stems from artificially
inoculated S. elaeagnifolium.
V. dahliae is a serious pathogen worldwide,
causing great losses in many plant crops. It attacks many plant species causing wilt diseases.
Strains of this fungus usually are not host specific. The fungus once established in a field is
practically impossible to remove, since it can
survive for years forming microsclerotia. At the
same time it attacks several weeds, which by
this way help the pathogen to spread and form
more propagules in the soil. The results of the
present work demonstrate that S. elaeagnifolium
can be infected by V. dahliae strains isolated
from common horticultural crops. At the same
time the strain isolated from this weed can infect
cultivated plants such as paprika. These findings
indicate that S. elaeagnifolium exerts two serious threats for the cultivated plants in northern
Greece. Not only it is a weed difficult to control,
but at the same time it carries a very serious
pathogen, V. dahliae and contributes to its
maintenance in soil. For this reason it is extremely urgent that effective measures should
be implemented for the control of this weed. To
the best of our knowledge, this is the first world
record of V. dahliae attacking Silverleaf nightshade. At present, possible other hosts of the
strain isolated from S. elaeagnifolium are tested,
including tomato, eggplant, cucumber, tobacco
and olive tree.
S. elaeagnifolium plants inoculated with Erysiphe sp. developed the disease symptoms 8
days after inoculation. Forty percent of inoculated leaves were infected and eventually dried
out. A powdery mildew pathogen such as Erysiphe sp. could possibly act as a successful biological control agent due to its host specificity
and usually high virulence. To the best of our
knowledge, this is the first world record of Erysiphe sp. attacking silverleaf nightshade. At present the molecular identification of the fungus is
taking place. However the work presented here
can only be considered as a preliminary study.
Pathogenicity of the fungus was confirmed in
artificial inoculations, however possible other
hosts must be checked for susceptibility, since
in some cases, pathogenicity on more than one
host has been reported for some powdery mildew pathogens (Smith 1969, Rector et al.,
2005). Experiments should include other plants
especially in the Solanaceae family such as tomato, tobacco, eggplant and paprika, and possibly on members of other families, depending
on the species of the fungus. Conditions of artificial inoculations should be optimized, in order
to develop a high disease incidence.
Plant pathogens have successfully been
used as biological control agents due to their
host specificity (Charudatan, 2001). Erysiphe
spp. have been suggested for biological control
of weeds in the past (Ialongo, 1980). The use of
endemic natural enemies of the invasive, alien
weeds in Europe brings the challenge of their
control to a new level in the beginning of 21st
century Field surveys for new diseases of S.
elaeagnifolium are at present continued.
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3rd International Symposium on Weeds and Invasive Plants
October 2-7, 2011 in Ascona, Switzerland
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