Microb Ecol (2012) 63:964–974
DOI 10.1007/s00248-011-9955-z
HOST MICROBE INTERACTIONS
Fungal Diversity Associated to the Olive Moth, Prays Oleae
Bernard: A Survey for Potential Entomopathogenic Fungi
Ivo Oliveira & José A. Pereira & Teresa Lino-Neto &
Albino Bento & Paula Baptista
Received: 28 May 2011 / Accepted: 5 August 2011 / Published online: 13 October 2011
# Springer Science+Business Media, LLC 2011
Abstract Olive production is one of the main agricultural
activities in Portugal. In the region of Trás-os-Montes, this
crop has been considerably affected by Prays oleae. In
order to evaluate the diversity of fungi on Prays oleae
population of Trás-os-Montes olive orchards, larvae and
pupae of the three annual generations (phyllophagous,
antophagous and carpophagous) were collected and evaluated for fungal growth on their surface. From the 3,828
larvae and pupae, a high percentage of individuals
exhibited growth of a fungal agent (40.6%), particularly
those from the phyllophagous generation. From all the
moth generations, a total of 43 species from 24 genera were
identified, but the diversity and abundance of fungal species
differed between the three generations. Higher diversity
was found in the carpophagous generation, followed by the
antophagous and phyllophagous generations. The presence
of fungi displaying entomopathogenic features was highest
in the phyllophagous larvae and pupae, with Beauveria
bassiana as the most abundant taxa. The first report of
Beauveria bassiana presence on Prays oleae could open
new strategies for the biocontrol of this major pest in olive
groves since the use of an already adapted species increases
the guarantee of success of a biocontrol approach. The
identification of antagonistic fungi able to control agents
that cause major olive diseases, such as Verticillium
I. Oliveira : J. A. Pereira : A. Bento : P. Baptista (*)
CIMO/School of Agriculture, Polytechnic Institute of Bragança,
Campus de Santa Apolónia, Apartado 1172,
5301-854 Bragança, Portugal
e-mail: pbaptista@ipb.pt
T. Lino-Neto
Centre for Biodiversity Functional and Integrative Genomics
(BioFIG), Plant Functional Biology Centre, University of Minho,
Campus de Gualtar,
4710-057 Braga, Portugal
dahliae, will benefit future biological control approaches
for limiting this increasingly spreading pathogen.
Introduction
The olive tree is an important crop for Mediterranean basin
countries, including Portugal. Extensive areas occupied by
olive groves in Trás-os-Montes region (Northeast of
Portugal) not only have a significant economic impact but
also exhibit a social, environmental and landscape significance. The olive moth, Prays oleae Bern., is one of the
major pests on these olive orchards, being responsible for
high losses in the olive yield as much as 40% [33]. This
lepidopteran presents three generations per year that
damage the olive tree in different organs. The antophagous
generation, occurring from April to June, causes damages
to the olive tree flowers; the carpophagous generation,
which usually appears from July to September, attacks the
fruits, leading to their premature fall; and the phyllophagous generation, present from October to March of the
following year, damages the leaves [33]. Due to the
growing awareness of detrimental effects of pesticides to
the ecosystems [6], agricultural practices in these orchards
have been changing to biological agriculture. Therefore, the
search for methods to control pests and diseases has
acquired a new motivation. One of the promising methods
to control pests can be the use of entomopathogenic fungi,
which are known by their ability to infect and kill several
insect species [25].
Entomopathogenic fungi comprise a large group of
pathogens that includes approximately 700 species in
almost 85 genera [5]. Due to their large host range,
entomopathogenic fungi could be potentially useful as
control agents against different insect orders. When
Fungal Associated to Prays oleae
compared to conventional chemical pesticides, the use of
insect pathogens presents many advantages, such as the
safety for humans and other non-target organisms, environmental reduction of pesticide residues and a smaller effect
on the natural biodiversity [15]. However, pest management
using entomopathogenic fungi has been difficult to achieve.
The ecological and environmental variations within agroecosystems turn the formulation and application of this kind
of biocontrol agent difficult to manage [42]. The isolation
of native fungi could provide a collection of isolates for the
development of potential control agents already adapted
and suited to a particular habitat. In this work, the fungal
diversity encountered on Prays oleae population was
evaluated in olive groves from Trás-os-Montes, and it will
be discussed by taking into account the olive moth
generation where it appears. From this survey, potential
entomopathogenic fungi for future biocontrol strategies will
be selected.
Methods
Study Area
Larvae (mostly in the fourth and fifth instars of development) and pupae of Prays oleae were collected in six olive
groves located in Mirandela—Bragança region, Northeast
of Portugal (GPS coordinates: 41°34′03.77″N, 7°05′39.21″
W; 41°33′53.29 N, 7°05′40.23″W; 41°33′52.51″N, 7°05′
30.59 W; 41°33′33.11 N, 7°05′35.62 W; 41°33′08.02″N, 7°
07′24.87″W; 41°32′35.20″N, 7°07′26.27″W). This region
comprises an olive tree growing area around 65.000 ha,
corresponding to 22% of the olive tree covered area in
Portugal. It contains 20% of Portuguese olive trees, which
altogether are responsible for 39% of national olive oil
production [31]. The topography of this region is mountainous, with altitudes ranging between 300 and 500 m. The
prevailing climate is Mediterranean, with cold and rainy
winters and long, hot and dry summers. The average annual
rainfall ranged from 600 to 800 mm, occurring mainly
between October and February, and the annual mean
temperature ranged from 9°C to 20°C. For this study, the
selected orchards have been managed through organic [11]
or integrated production guidelines [18] and display high
levels of olive moth infestations, as observed by delta traps
baited with pheromone. Although two different management systems are referred, the studied olive groves are very
similar, with low-external inputs. The grove area ranged
from 0.4 to 1.5 ha. Trees are of medium size, with a
planting density of 7×7 m. Pruning was made every 2–
3 years. No irrigation was done, only copper-based
products were used in the beginning of autumn for fungal
diseases control, and no other pesticides were used in the
965
last 5 years. The differences between the two management
systems are related to the products allowed in soil
fertilization. The most important cultivars were Cobrançosa
and Verdeal Transmontana.
Experimental Design and Collection of Plant Material
The collection of larvae and pupae was performed in 15
randomly chosen trees in each of the selected orchards for
the three annual generations of Prays oleae. Sampling dates
were variable according to the life stages of the pest. For
the phyllophagous generation, the collection was conducted
between 17 and 27 March 2009; for the antophagous
generation, it was conducted between 24 May and 5 June;
and for the carpophagous generation, it was conducted
between 1 and 3 September of the same year. For the first
two generations, sampling of leaves (phyllophagous generation) and flower clusters (antophagous generation), in
which larvae and pupae were present, was performed. For
the carpophagous generation, the collection of whole fruits
was randomly performed due to the presence of larvae and
pupae within olives. The plant material (leaves and flower
clusters) and olives were collected individually to sterile
bags and processed within a few hours after sampling.
Isolation of Fungi from Mycosed Larvae and Pupae
of Prays Oleae
In the laboratory, the plant material was further examined
for the presence of olive moth larvae and pupae. Since
carpophagous larvae develop inside the olive stone, the
stone was smoothly broken to expose larvae. Whenever
present, the larvae and pupae were individually placed into
sterile tubes containing a food source (leaves for the
phyllophagous generation, flower buds for the antophagous
generation and olive stones for the carpophagous generation). The tubes were sealed and maintained at 25±2°C,
under 16/8 h light/dark regime, for an average period of
10 days. Larvae and pupae were observed daily in order to
detect their death or evaluate the insect emergence. Each
time a fungal agent was growing on the surface of dead
larvae or pupae, the fungal specimen was isolated by
inoculating Potato Dextrose Agar (PDA) medium, supplemented with 0.01% (w/v) chloramphenicol (Oxoid). Pure
cultures of each isolate were deposited in the culture
collection of the Polytechnic Institute of Bragança (School
of Agriculture).
Extraction of Fungal DNA
Fungal isolates were inoculated onto PDA medium and
maintained at 25±2°C in the dark for 1–2 weeks. The
colony morphology, spore size and shape were used for the
966
first identification and to group strains. Spores were
collected from each isolate and used for DNA extraction.
Isolation of genomic DNA was performed by transferring
spores into a microtube containing 500 μL of Lysis buffer
(200-mM Tris-HCl pH 8.0, 250-mM NaCl, 25-mM EDTA
pH 8.0 and 0.5% SDS) and sterile glass spheres. Tubes
were vortexed for 5 min to disrupt cells. After addition of
250 μL of cold 3-M NaOAc pH 5.5, the mixture was gently
homogenized by inversion and incubated for 10 min at
−20°C. Following centrifugation at 10.500 rpm (4°C) for
10 min, the supernatant was collected to another microtube,
and one volume of isopropanol (−20°C) was added. This
mixture was slowly homogenized and incubated at −20°C
for 1 h. The DNA precipitate was collected by centrifugation at 10.500 rpm (4°C) for 10 min, and the pellet was
washed with cold 70% ethanol. The DNA pellet was airdried for 20 min at room temperature and re-suspended in
50 μL of ultra-pure water. DNA was stored at −20°C until
use.
Molecular Identification of Fungal Iisolates
Molecular identification was achieved by amplification of
the internal transcribed spacer region (ITS), using the
universal primers ITS1 and ITS4 [44]. PCR reactions
(50 μl) comprised 50 ng of genomic DNA, 0.2 μM of
each primer (ITS1 and ITS4), 1× GoTaq® Flexi buffer
(Promega), 2 mM MgCl2 (Promega), 0.2 μM dNTP Mix
(Fermentas) and 1 U GoTaq® DNA polymerase (Promega).
Amplifications were carried out in the thermocycler Biometra UNO II (Thermoblock, Biotron) using a temperature
gradient protocol as follows: initial denaturation at 94°C for
3 min, followed by 35 cycles of 0.5 min at 94°C, 0.5 min at
55°C, 1 min at 72°C and a final 10-min extension at 72°C.
PCR amplification products were analysed by electrophoresis, and those reactions that amplified a single PCR
product were selected for purification using the JETquick
PCR product purification kit (Genomed). Amplified fragments were sequenced using both ITS1 and ITS4 primers at
the sequencing services of STAB Vida (Oeiras, Portugal).
DNA sequences were analysed with DNASTAR v.2.58
software, and fungal identification was performed using the
NCBI database (http://www.ncbi.nlm.nih.gov) and BLAST
algorithm.
The ecological classification of identified species was
only based on the impact they can have on olive
orchards regarding their potential role on pest and disease
control. Even though many of the identified fungi are
also considered general saprophytes, they were only
classified into three ecological roles: phytopathogenic,
antagonistic and entomopathogenic. The classification
given to each fungal species was based on previously
described characteristics.
I. Oliveira et al.
Data Analysis
For each Prays oleae generation, the species richness,
Simpson (D) and Shannon–Wiener (H) diversity indexes,
total and relative abundances were estimated. Calculations
of Simpson and Shannon–Wiener diversity indexes were
done using the software Species Diversity and Richness (v.
3.0). Total abundance (N) was estimated as the number of
isolates per fungal taxa, whereas the proportion of isolates
from each fungal taxa in relation to the total number of
fungal isolates was considered as the relative abundance
of a certain taxa. Principal component analysis (PCA)
was applied to access the relationship between fungal
taxa and the three Prays oleae generations. PCA was
performed using the SPSS software, version 17.0 (SPSS,
Inc.). It was applied as an unsupervised approach for
reducing the number of variables (43, corresponding to the
number of identified fungal species) to a smaller number
of new derived variables (principal component or factors)
that adequately summarize the original information. This
analysis will define which fungal species are correlated
with each olive moth generation. PCA analysis also
allowed the recognition of patterns in the data by plotting
them in a multidimensional space using the newly derived
variables as dimensions (factor scores). The aim of the
PCA is to produce components suitable to be used as
predictors or response variables in subsequent analysis.
The number of factors to keep in data treatment was
evaluated by the Scree plot, taking into account the
eigenvalues and the internal consistency by means of
αCronbach’s value [21, 34].
Results
Diversity and Abundance of Fungi Encountered
in Prays Oleae
From the 3,828 larvae and pupae (2,552 larvae and 1,276
pupae) collected in all generations of Prays oleae, an insect
emergence percentage of 38.6% was observed (Table 1). Of
the total number of dead larvae and pupae (1,477), 40.6%
(599) exhibited the growth of a fungal agent on their
surface (Table 1). From those, the isolation of fungi allowed
the identification of 43 species, belonging to 24 genera and
14 families (Table 2). The families comprising more
diversity were Pleosporaceae and Quambalariaceae (eight
species each) and Mucoraceae and Nectriaceae (five species
each). These families accounted for as much as 62% of the
total identified species. The greatest number of taxa
belonged to the genera Penicillium (7), Arthrinium, Mucor,
Fusarium and Alternaria (all with four taxa). Concerning
abundance, 166 different isolates were obtained in this
Fungal Associated to Prays oleae
967
Table 1 Larvae and pupae collected from each generation of Prays
oleae. The insect emergence percentage was determined for each moth
generation. The percentage of cadavers exhibiting fungal growth on
their surface was determined in relation to the total of dead larvae and
pupae
Number of
collected larvae
and pupae
Insect
emergence
Cadavers
presenting surface
fungal growth
Phyllophagous
1,246
30.1%
61.5%
Antophagous
Carpophagous
Total
1,745
837
3,828
53.9%
19.5%
38.6%
32.7%
23.0%
40.6%
Generation
work (Table 2). The most common taxa were Beauveria
bassiana (N=50) and Fusarium oxysporum (N=25), representing together 45.2% of the total identified isolates.
The number of fungal taxa identified in the present study
varied between the three generations of Prays oleae
(Table 2). In the phyllophagous generation, where a total
of 70 fungal isolates were obtained, 16 different species
were identified, belonging to 13 genera and 9 families. The
most representative family was Quambalariaceae, comprising 25% of the identified species in this generation, with the
genus Penicillium as the most represented (3 species). In
the antophagous generation, 52 isolates were obtained,
belonging to 13 species, 8 genera and 8 families. The
families Nectriaceae and Apiosporaceae include the majority of the identified taxa (54%). The genus that included
more taxa (4) was Fusarium (Nectriaceae), which contained
31% of the fungal species surveyed in this generation.
Concerning the carpophagous generation, 44 fungal isolates
were obtained, with 21 identified species, belonging to 14
genera and 10 families. Pleosporaceae was the family
representing more species (33%), and the genera Alternaria
(Pleosporaceae) and Penicillium (Quambalariaceae), both
with four species each, comprised 38% of the total fungal
taxa found in this generation of Prays oleae.
The most abundant species also differed between
generations. For the phyllophagous generation, the most
abundant one was Beauveria bassiana (N=49), which
represented 70% of the relative abundance in this generation. For the antophagous generation, F. oxysporum was the
most abundant (N=23), corresponding to a relative abundance of 44.2%. In the carpophagous generation, several
taxa presented comparable relative abundances, with
Penicillium pinophilum as the most frequent (N = 7,
corresponding to a relative abundance of 15.9%), followed
by Alternaria sp.1 and Aspergillus ustus, both with N=6
corresponding to 13.6% of relative abundance.
In order to provide more information about fungal
community composition in each Prays oleae generation,
the Simpson (D) and Shannon–Wiener (H) diversity indexes
were determined (Table 3). In the present work, both
diversity indexes differed between the three Prays oleae
generations. As verified for species richness, the highest
fungal diversity was verified in the carpophagous generation (D=0.09 and H=2.70). In fact, when using larvae or
pupae from this generation, the highest number of unique
species (17) was obtained. The number of exclusive species
identified from phyllophagous and antophagous generations
was only 11 and 9, respectively. The lowest value of
species diversity (D=0.5 and H=1.39) was detected in the
phyllophagous generation.
When performing a principal component analysis (PCA)
using the total fungal taxa, according to the moth
generation from which they were isolated, it was possible
to separate the samples in eight groups (Fig. 1). The
phyllophagous generation is clearly related to groups 4 and
7, the antophagous generation is linked to groups 3 and 6,
and the carpophagous generation is associated to groups 1
and 2. The two remaining groups (5 and 8) do not appear
associated to a specific generation. Group 5 is mainly
correlated to the antophagous generation, although presenting some relation to the phyllophagous generation. Group
8 is equally correlated to both phyllophagous and carpophagous generations.
Fungal Ecological Roles
By taking into account the potential use for the biological
control of pests and diseases, 45% of the isolates found in
this work have been described in the literature as presenting
the useful features of antagonism and/or entomopathogenecity (Fig. 2a). The remaining 55% of the isolates
presented phytopathogenic features, displaying also in large
extent (41%) antagonistic and/or entomopathogenic characteristics. When comparing the number of identified
species, the majority (75%) has been described as phytopathogenic fungi (Fig. 2b). While the mentioned attribute
was present alone in 28% of the identified species, the
remaining 47% also displayed antagonistic and/or entomopathogenic characteristics. From those species described as
non-phytopathogenic (23%), the majority are defined as
antagonistic (17%).
The ecological roles of the identified fungi differed
between generations. In the phyllophagous generation, the
majority (87%) of the fungal isolates have been described
as antagonistic and entomopathogenic (Fig. 2a). Being
included in the phytopathogenic fungi (23%), 13% of
fungal isolates also display antagonistic and/or entomopathogenic characteristics. This relation is reversed when
considering the number of identified fungal species. The
number of phytopathogenic taxa was higher (77%) than
those displaying only antagonistic and/or entomopathogenic features (23%) (Fig. 2b).
968
I. Oliveira et al.
Table 2 Total abundance (N) and relative percentage (%) of each
fungal taxa isolated from dead Prays oleae larvae and pupae in
relation to the total number of identified fungi. Values are presented
for all the three moth generations. The ecological role (ER) of each
fungus is also presented (P phytopathogenic, A antagonistic, E
entomopathogenic)
Family, genera and species
ER
Phyllophagous
Antophagous
Carpophagous
Total
N
N
N
N
%
%
%
%
Amphisphaeriaceae
Truncatella
T. angustata (Pers.) S. Hughes
P
0
0.0
0
0.0
1
2.3
1
0.6
A. phaeospermum (Corda) M.B. Ellis
A
1
1.4
0
0.0
0
0.0
1
0.6
Arthrinium sp.1
A
0
0.0
1
1.9
0
0.0
1
0.6
Arthrinium sp.2
A
0
0.0
1
1.9
0
0.0
1
0.6
Arthrinium sp.3
A
0
0.0
1
1.9
0
0.0
1
0.6
P, A
2
2.9
0
0.0
0
0.0
2
1.2
P
0
0.0
1
1.9
0
0.0
1
0.6
P, A
0
0.0
0
0.0
1
2.3
1
0.6
P, A
0
0.0
0
0.0
1
2.3
1
0.6
A, E
49
70.0
0
0.0
1
2.3
50
30.1
E
1
1.4
0
0.0
0
0.0
1
0.6
P, A, E
1
1.4
0
0.0
1
2.3
2
1.2
A
0
0.0
8
15.4
0
0.0
8
4.8
P
0
0.0
0
0.0
1
2.3
1
0.6
A
1
1.4
2
3.8
0
0.0
3
1.8
–
0
0.0
0
0.0
1
2.3
1
0.6
Apiosporaceae
Arthrinium
Bionectriaceae
Bionectria
B. ochroleuca (Schwein.) Schroers & Samuels
Botryosphaeriaceae
Botryosphaeria
B. dothidea (Moug.) Ces. & De Not.
Microdiplodia
M. hawaiiensis Crous
Chaetomiaceae
Chaetomium
C. funicola Cooke
Cordycipitaceae
Beauveria
B. bassiana (Bals.-Criv.) Vuill
Cordyceps
C. sinensis (Berk.) Sacc.
Davidiellaceae
Cladosporium
C. cladosporioides (Fresen.) G.A. de Vries
Hypocreaceae
Trichoderma
T. gamsii Samuels & Druzhin.
Incertae sedis
Septogloeum
S. mori (Lév.) Briosi & Cavara
Trichothecium
T. roseum (Pers.) Link
Mucoraceae
Lichtheimia
L. ramosa (Zopf) Vuill.
Mucor
M. circinelloides Tiegh.
P, A, E
0
0.0
0
0.0
3
6.8
3
1.8
M. fragilis Bainier
P
1
1.4
0
0.0
0
0.0
1
0.6
M. hiemalis Wehmer
P, E
0
0.0
1
1.9
0
0.0
1
0.6
M. racemosus Bull.
P
1
1.4
0
0.0
0
0.0
1
0.6
P, A,
0
0.0
8
15.4
0
0.0
8
4.8
Nectriaceae
Fusarium
F. equiseti (Corda) Sacc.
Fungal Associated to Prays oleae
969
Table 2 (continued)
Family, genera and species
ER
Phyllophagous
Antophagous
Carpophagous
Total
N
N
N
N
%
%
%
%
F. oxysporum Schltdl.
P, A, E
2
2.9
23
44.2
0
0.0
25
F. solani (Mart.) Sacc.
P, E
0
0.0
1
1.9
0
0.0
1
15.1
0.6
Fusarium sp.1
P, A, E
0
0.0
1
1.9
0
0.0
1
0.6
P
1
1.4
0
0.0
0
0.0
1
0.6
Alternaria arborescens E. G. Simmons
P
0
0.0
0
0.0
1
2.3
1
0.6
Alternaria sp.1
P, A
3
4.3
3
5.8
6
13.6
12
7.2
Alternaria sp.2
P, A
0
0.0
0
0.0
2
4.5
2
1.2
Alternaria tenuissima (Kunze) Wiltshire
P
0
0.0
0
0.0
5
11.4
5
3.0
P
0
0.0
0
0.0
1
2.3
1
0.6
P, A
1
1.4
0
0.0
0
0.0
1
0.6
Gibberella
Gibberella sp.1
Pleosporaceae
Alternaria
Embellisia
Embellisia sp.
Epicoccum
E. nigrum Link
Stemphylium
S. solani G. F. Weber
P
0
0.0
0
0.0
1
2.3
1
0.6
S. vesicarium (Wallr.) E.G. Simmons
P
0
0.0
0
0.0
1
2.3
1
0.6
P
3
4.3
0
0.0
0
0.0
3
1.8
Quambalariaceae
Quambalaria
Q. cyanescens (de Hoog & G.A. de Vries) Z.W. Beer, Begerow & R. Bauer
Penicillium
P. biourgeianum K.M. Zalessky
P
1
1.4
0
0.0
0
0.0
1
0.6
P. commune Thom
P
1
1.4
0
0.0
0
0.0
1
0.6
P. echinulatum Fassat.
P
1
1.4
0
0.0
0
0.0
1
0.6
P. italicum Wehmer
P
0
0.0
0
0.0
1
2.3
1
0.6
P. pinophilum Thom
A
0
0.0
1
1.9
7
15.9
8
4.8
Penicillium sp. 1
P, A, E
0
0.0
0
0.0
1
2.3
1
0.6
Penicillium sp. 2
P, A, E
0
0.0
0
0.0
1
2.3
1
0.6
P, A, E
0
0.0
0
0.0
6
13.6
6
3.6
Trichocomaceae
Aspergillus
A. ustus (Bainier) Thom & Church
Talaromyces
T. flavus (Klöcker) Stolk & Samson
Total
In the antophagous generation, a large fraction of
isolates (73%) has been described as displaying phytopathogenic characteristics, of which 71% also displays antagonistic and/or entomopathogenic features (Fig. 2a). The
remaining 27% has been described as antagonistic fungi.
Concerning the carpophagous generation, the higher fraction of fungal isolates displays phytopathogenic characteristics (77%) (Fig. 2a). Included in these, 50% of fungal
isolates also exhibit antagonistic and/or entomopathogenic
features. Fungal isolates displaying only antagonistic and/or
entomopathogenic features were also found but in a lower
A
0
0.0
0
0.0
1
2.3
1
0.6
70
100.0
52
100.0
44
100.0
166
100.0
proportion (20%). When considering the number of
identified fungal taxa, the same trend was observed for
the antophagous and carpophagous generations. In both, the
phytopathogenic fungi comprised the majority of identified
taxa, but most of them also present antagonistic and/or
entomopathogenic features. It was in the carpophagous
generation that the lowest amount of non-phytopathogenic
species with antagonistic and/or entomopathogenic properties were found (12%). This was the only generation where
a fungal taxon (Lichtheimia ramosa) with no described
ecological role was isolated.
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I. Oliveira et al.
Table 3 Simpson (D) and Shannon–Wiener (H) diversity indexes of
fungal taxa identified in the three generations of Prays oleae
Generation
Diversity indexes
D
H
Phyllophagous
Antophagous
0.50
0.25
1.39
1.68
Carpophagous
0.09
2.70
Discussion
In the present work, the diversity of fungi isolated from
dead larvae and pupae of a major olive pest (Prays oleae)
was evaluated for the first time. Following molecular
identification, it was possible to identify 43 fungal species,
belonging to 24 genera and 14 families. The identification
of such a high number of fungal taxa described as
presenting antagonistic, entomopathogenic or phytopathogenic features was only possible because the studied olive
groves were maintained under organic or integrated
production guidelines. If sampling had been performed on
a conventional orchard, the expected fungal diversity would
have been lower. Organic and integrated production
management creates a healthier and safer environment with
higher biological diversity [19]. Similar diversity levels (46
fungal species and 27 genera) were obtained when studying
insect-associated fungi isolated from soil samples of
different field crops (wheat/maize, corn and soybean) and
orchards (peach and apple) [38].
Fungal Diversity and Abundance
Figure 1 Principal component analysis (PCA) of the identified fungal
taxa according to the moth generation from which they were isolated
(represented as closed circles; AG antophagous generation, PG
phyllophagous generation, CG carpophagous generation). Each
number corresponds to a fungal group defined by the coordinates
generated by SPSS. Fungal groups are defined as follows: 1
Chaetomium funicola, Stemphylium solani, Penicillium italicum,
Stemphylium vesicarium, Alternaria tenuíssima, Talaromyces flavus,
Alternaria sp.1, Penicillium sp.1, Microdiplodia hawaiiensis, Aspergillus ustus, Embellisia sp., Alternaria arborescens, Septogloeum
mori, Mucor circinelloides, Truncatella angustata, Lichtheimia
ramosa, Penicillium sp.2 and Alternaria sp.2; 2 Penicillium pinophilum; 3 Arthrinium sp.1, Fusarium sp.1, Trichoderma gamsii, F. solani,
Arthrinium sp.2, Mucor hiemalis, Botryosphaeria dothidea, F. equiseti
and Arthrinium. sp.3; 4 Mucor racemosus, Penicillium biourgeianum,
Arthrinium phaeospermum, Penicillium commune, Penicillium echinulatum, Gibberella sp., Epicoccum nigrum, Cordyceps sinensis,
Bionectria ochroleuca, Quambalaria cyanescens and Mucor fragilis;
5 Trichothecium roseum; 6 F. oxysporum; 7 Beauveria bassiana;
8 Cladosporium cladosporioides
Concerning genera diversity, three main patterns were
detected: (1) species-rich and highly abundant genera, e.g.
Fusarium, Alternaria and Penicillium (about 35% of the
taxonomic diversity and 42% of the total of isolates), (2)
species-rich genera, but displaying low abundance, e.g.
Mucor and Arthrinium (about 19% of the taxonomic
diversity and 5% of the total of isolates), and (3) speciespoor but highly abundant genera, e.g. Beauveria and
Trichoderma (about 5% of the taxonomic diversity and
35% of the total of isolates). Among the 43 species
recorded during the present study, the genera Alternaria,
Arthrinium, Fusarium, Mucor and Penicillium were the
most represented in terms of taxa number. These genera
include some of the most ubiquitous fungal species in
nature, which have been found in soils, plants and
agricultural communities. The wide presence of such
microorganisms has been related to the broad ecological
roles they play, either as saprophyts, phytopathogens or
biocontrol agents [8].
The genera that contributed most to species diversity
depended on which Prays oleae generation was used for
fungal isolation. While in phyllophagous generation Penicillium represented the genus with highest taxonomic
diversity (18%), in antophagous generation, that genus
was Fusarium (31%), and in carpophagous generation, they
were Alternaria and Penicillium (representing together
39%). The composition of fungal community was also
inferred by the Simpson (D) and Shannon–Wiener (H)
diversity indexes, which offer valuable information about
rarity and frequency of species in a community. Both
indexes provide more information than simply species
richness since they also take the relative abundances of
different species into account. While the carpophagous
generation presented the highest fungal diversity (display-
Fungal Associated to Prays oleae
Figure 2 Percentage of fungal isolates (a) and fungal species (b)
presenting different ecological roles. Isolates were obtained from dead
larvae and pupae of the three generations of Prays oleae (PG
phyllophagous generation, AG antophagous generation, CG carpophagous generation). After molecular identification, fungi were grouped
according to their described ecological role (A antagonistic, E
entomopathogenic, P phytopathogenic, U unknown)
ing the lowest D and highest H values), the phyllophagous
generation displayed the lowest diversity (presenting the
highest D and lowest H values). This difference between
moth generations could be related to two main factors:
climatic conditions during larvae and pupae collection and
the moth life cycle.
Climate conditions, particularly relative humidity and
temperature, are known to affect both conidia dispersion
and germination [39]. The collection of larvae and pupae
from phyllophagous generation (March) matched with the
time of year where the temperature is low, decreasing the
ability of spore germination and thus reducing fungal
diversity. Furthermore, the moth life cycle also seems to
strongly affect the fungal diversity observed in this
generation as a large part of phyllophagous moth development occurs in the leaves as miner larvae. Therefore, the
reduced fungal diversity observed in this generation can be
associated to the low chances of larvae contact with fungal
spores. The high occurrence of the entomopathogenic
Beauveria bassiana in the phyllophagous generation (70%
of the isolates) could be related to an epizootic outbreak
that might have occurred during the time of larvae and
pupae sampling. These epizootics are described as being
dependent on host population dynamics, the number and
971
viability of infective stages in the pathogen population,
infection efficiency and development, in addition to a
complex set of environmental factors and timing [25].
Although dispersal of Beauveria bassiana conidia by larvae
could have been limited due to their small displacement in
trees, wind and rain may have caused the spread of those
infectious structures. Also, the high number of infected
Prays oleae individuals could have functioned as sources of
infective conidia, thus contributing for the abundance of
Beauveria bassiana in this generation.
In antophagous and carpophagous generations, the most
abundant taxa were F. oxysporum (44% of the total isolates)
and Penicillium pinophilum (15.9% of the total isolates),
respectively. As referred, these two taxa are very frequent in
nature, being present in almost all environments. Furthermore, the amount of spores of Fusarium species is known
to increase from April to July [39], and those from
Penicillium species rise between August and October [23].
These periods are coincident with the sampling dates of
larvae and pupae from antophagous (May–June) and
carpophagous (September) generations, explaining in some
degree the high abundance of Fusarium and Penicillium
genera in antophagous and carpophagous generations,
respectively.
The association of climate and life cycle negatively
affects the fungal diversity when using antophagous larvae
and pupae. In the region where the study was conducted,
temperature rapidly increases during May and June, leading
to an extremely fast larvae development. Therefore, larvae
and pupae stay a short period exposed to the surrounding
environment, reducing the possibility of fungal infection. In
contrast, carpophagous larvae are exposed to fungal spores
for a longer period. After ecloding from the egg, larvae
must travel from the oviposition site in the fruits to the
petiole and bore down into the stone, where they usually
feed for several weeks. After completing their development,
larvae re-emerge from the fruit. The chance of infection is
still enhanced by the extension of carpophagous generation
which, together with the favourable climatic conditions,
allows the development of more fungal taxa. Accordingly,
it was in this moth generation that higher species diversity
was found.
Fungal Ecological Roles
The relation between the amount of dead larvae and pupae
displaying fungal growth and the ecological role of the
identified fungal taxa provides interesting data. The higher
amount of infected dead larvae and pupae was observed in
the phyllophagous generation (61.5%). It was also from this
generation that the most isolates exhibiting entomopathogenic features (76%) were obtained. As the presence of
fungal growth on dead larvae and pupae decreases (from
972
61.5% to 32.7% and finally 23.0%, in the phyllophagous,
antophagous and carpophagous generations, respectively),
the abundance of fungi exhibiting entomopathogenic
characteristics also decreases (76%, 50% and 30%, respectively). These results seem to suggest that the infection with
entomopathogenic fungi could have led to larvae and pupae
death. Most of the fungal isolates (86%) and species (70%)
identified in this work have been described as antagonistic
and/or entomopathogenic, although some of them have also
been considered as displaying phytopathogenic features.
Because only the non-phytopathogenic fungi displaying
antagonistic and/or entomopathogenic features could be
explored for limiting fungal diseases and/or pests, the
percentage of identified fungal species that might have a
future application as biocontrol agents is reduced to 23%.
From the identified taxa in this work, Beauveria
bassiana seems to be the most conspicuous among the
entomopathogenic/antagonistic species. The natural occurrence of this fungus in over 700 insect hosts from almost all
taxonomic orders is well documented [25]. However, there
are still some reservations about the host range of this
fungus. Some authors claim that Beauveria bassiana is a
“species complex”, referring that different isolates have a
restricted host, while others point out that this fungus has
no host specificity [29]. Accordingly, several pests are
susceptible to the entomopathogenic aptitude of this fungus,
like Alphitobius diaperinus [36], whiteflies Bemisia tabaci
and Trialeurodes vaporariorum [32], Capnodis tenebrionis
[20], Lutzomyia longipalpis [2], Callosobruchus maculates
[26] and Tetranychus urticae [10]. Some studies also
indicate the presence of this fungus associated to several
lepidopterans [2, 3, 7, 13, 22, 32, 36]. In addition, natural
occurrence of mycoses caused by Beauveria bassiana is
reported in lepidopteran pests such as Helicoverpa armigera (Hubner), Spodoptera litura (Fab.) [9], Argyresthia
conjugella [41] and Plutella xylostella [37]. As far as we
know, this is the first report of Beauveria bassiana related
to Prays oleae, where a large amount of isolates were
obtained from larvae and pupae of the phyllophagous
generation. The presence of this fungus in olive grove soils
has already been reported [20, 32], as well as its ability to
control pests in this crop, such as Bactrocera oleae [14, 17].
All of these evidences may suggest that this fungus could
be effective in controlling Prays oleae in olive orchards,
although experiments to evaluate its infecting ability
towards Prays oleae have to be performed. The natural
occurrence of this species in the studied olive groves
guarantees an already adapted and suited strain to be used
as a control agent in this particular ecosystem.
Another potential entomopathogen identified in the
present study that could be able to control Prays oleae
larvae is Cordyceps sinensis. Although no literature is
available for its ability to infect Prays oleae, the capacity of
I. Oliveira et al.
infecting other lepidopterans larvae was already described,
such as those of Hepialus armoricanus [30]. Nevertheless,
these fungal taxa described as entomopathogenic cannot be
definitely linked to the cause of death of Prays oleae larvae
and pupae. Assays confirming the infection ability and
virulence of such fungi must be performed in order to
confirm their entomopathogenic potential.
The identification of fungi that could limit the growth of
other infectious fungi by their antagonistic properties may
also be important for designing future biocontrol strategies
for restricting fungal diseases in olive groves. The most
abundant taxa with antagonistic characteristics identified in
the present study were Penicillium pinophilum and Trichoderma gamsii. Penicillium pinophilum is one of the most
important antagonists of Rhizoctonia solani, a fungal
pathogen of tobacco [1]. Although scarce information is
available about Trichoderma gamsii, antagonistic properties
of Trichoderma species against a great number of fungal
species have already been described, including Verticillium
dahliae that causes one of the most severe diseases
affecting olive (Verticillium wilt) [27, 43]. The identification of several isolates with antagonistic features (44%),
some of them against fungi that cause olive diseases, opens
up the possibility of further research on those antagonistic
species for limiting the occurrence of such phytopathogenic
fungi in olive orchards.
In this work, besides the identification of potential
biocontrol agents for limiting pests and fungal diseases in
olive groves, many other fungi were identified, which could
play a role in olive grove ecosystems. One of the most
common genera found in this work was Alternaria, which
comprises species that have already been reported to cause
spoilage of olives [35] and cause a disease on olive shoots
grown under greenhouse conditions [4]. The most frequent
Alternaria species isolated in the present work was
Alternaria tenuissima, which has been associated to late
blight of pistachio and black point of small-grain cereals
[16], among other crops. One of the most abundant fungal
taxa identified in this study was F. Oxysporum, which
exhibits antagonistic, entomopathogenic and phytopathogenic properties. This species has been mainly described as
phytopathogenic, causing vascular wilts or rot and crown
rots in a large number of crops, including tree crops [12].
This species also presents antagonistic features against
Colletotrichum gloeosporioides and Pestalotia psidii [28],
and, most importantly, against V. dahliae [24]. Furthermore,
F. oxysporum has also been described as an opportunistic
insect pathogen [38]. Accordingly, it has been isolated from
several insect hosts, such as those from Homoptera and
Coleoptera orders, being able to act as parasite to the
greenhouse whitefly, Trialeurodes vaporariorum [40].
However, the identification of entomopathogenic fungi
from dead Prays oleae larvae and pupae cannot be
Fungal Associated to Prays oleae
unequivocally associated to their ability to infect this
lepidopteran since dead larvae and pupae could just become
increasingly susceptible to fungi.
Conclusion
As far as we know, the present work describes for the first
time the assessment of fungal diversity directly obtained
from mycosed dead Prays oleae larvae and pupae.
The strategy used for obtaining fungal isolates (collection of larvae and pupae from the field and isolation of
fungi from cadavers) allowed the identification of 43 fungal
species, displaying several ecological roles. The diversity
and abundance of fungal species differed when using larvae
or pupae from different moth generations. Higher fungal
diversity was found in the carpophagous generation,
followed by antophagous and phyllophagous generations.
Although the identified taxa could not be unequivocally
associated with the cause of moth death, almost 37% of
identified taxa presented entomopathogenic properties.
The identification of entomopathogenic and antagonist
fungi in olive orchards provided a pool of biocontrol agents
that could be used in the future for controlling pests and
fungal diseases. The first report of Beauveria bassiana
presence on Prays oleae could open new strategies for the
biocontrol of this major pest in olive groves. This fungus
was isolated with high incidence from phyllophagous
generation larvae and pupae. Future studies will be directed
to screening the entomopathogens isolates for virulence to
adult Prays oleae. As the environmental conditions influence the performance of a given strain of fungus, the use of
already adapted species to a particular ecosystem increases
the guarantee of success of a biocontrol approach. The
occurrence of antagonistic fungi able to control one of the
major fungus attacking olive trees (V. dahliae) may as well
be investigated in order to control this pathogen that is
spreading throughout olive orchards.
Acknowledgements This work was supported by the Science and
Technology Foundation (Fundação para a Ciência e Tecnologia—
FCT) project PTDC/AGR-AAM/102600/2008 “entomopathogenic
fungi associated to olive pests: isolation, characterization and selection
for biological control”. The first author is grateful to the Science and
Technology Foundation for the Ph.D. grant SFRH/BD/44265/2008.
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