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. 970 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. References 1. Alagesaboopathi C (1994) Biological control of damping-off disease of cotton seedling. Curr Sci 66:864–868 2. Amóra S, Bevilaqua C, Feijó F, Silva M, Pereira R, Silva S, Alves N, Freire F, Oliveira D (2009) Evaluation of the fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes), a potential biologi- 973 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. cal control agent of Lutzomyia longipalpis (Diptera, Psychodidae). Biol Control 50:329–335 Aquino de Muro M, Elliot S, Moore D, Parker B, Skinner M, Reid W, El Bouhssni M (2005) Molecular characterisation of Beauveria bassiana isolates obtained from overwintering sites of sunn pests (Eurygaster and Aelia species). Mycol Res 109:294–306 Bourbos V, Skoundridakis M, Metzidakis I (1999) Alternaria alternata: a new disease of leafy cuttings of olive shoots. In: Metzidakis I, Voyiatzis D (eds), Proceedings 3rd International ISHS Symposium on Olive Growing. Acta Horticulturae 474:585–587 Charnley A, Collins S (2007) Entomopathogenic fungi and their role in pest control. In: Kubicek C, Druzhinina I (eds) The Mycota, Volume 4—Environmental and Microbial Relationships 2nd edn. Springer-Verlag Berlin Heidelberg, pp 159–187. Cuthbertson A, Walters K (2005) Pathogenicity of the entomopathogenic fungus, Lecanicillium muscarium, against the sweetpotato whitefly Bemisia tabaci under laboratory and glasshouse conditions. Mycopathologia 160:315–319 Dalzoto P, Glienke-Blanco C, Kava-Cordeiro V, Ribeiro J, Kitajima E, Azevedo J (2006) Horizontal transfer and hypovirulence associated with double-stranded RNA in Beauveria bassiana. Mycol Res 110:1475–1481 De Lucca A (2007) Harmful fungi in both agriculture and medicine. Rev Iberoamer Micol 24:3–13 Devi K, Sridevi V, Mohan C, Padmavathi J (2005) Effect of high temperature and water stress on in vitro germination and growth in isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin. J Invertebr Pathol 88:181–189 Eken C, Hayat R (2009) Preliminary evaluation of Cladosporium cladosporioides (Fresen.) de Vries in laboratory conditions, as a potential candidate for biocontrol of Tetranychus urticae Koch. World J Microbiol Biotechnol 25:489–492 European Union. Council Regulation (EC) No 834/2007 of 28 June 2007 Fravel D, Olivain C, Alabouvette C (2002) Fusarium oxysporum and its biocontrol. New Phytol 157:493–502 Fuguet R, Vey A (2004) Comparative analysis of the production of insecticidal and melanizing macromolecules by strains of Beauveria spp.: in vivo studies. J Invertebr Pathol 85:152–167 Konstantopoulou M, Mazomenos B (2005) Evaluation of Beauveria bassiana and B. brongniartii strains and four wild-type fungal species against adults of Bactrocera oleae and Ceratitis capitata. BioControl 50:293–305 Lacey L, Frutos R, Kaya H, Vail P (2001) Insect pathogens as biological control agents: do they have a future? Biol Control 21:230–248 Logrieco A, Bottalico A, Mulé G, Moretti M, Perrone G (2003) Epidemiology of toxigenic fungi and their associated mycotoxins for some Mediterranean crops. Eur J Plant Pathol 109:645–667 Mahmoud M (2009) Pathogenicity of three commercial products of entomopathogenic fungi, Beauveria bassiana, Metarhizum anisopilae and Lecanicillium lecanii against adults of olive fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae) in the laboratory. Plant Protect Sci 45:98–102 Malavolta C, Delrio G, Boller EF (2002) Guidelines for integrated production of olives. IOBC Technical Guideline III, 1st Edition, 25 (4), pp. 1–8. Mander U, Mikk M, Külvik M (1999) Ecological and low intensity agriculture as contributors to landscape and biological diversity. Landsc Urban Plann 46:169–177 Marannino P, Santiago-Álvarez C, Lillo E, Quesada-Moraga E (2006) A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera; Buprestidae). J Invertebr Pathol 93:210–213 974 21. Maroco J (2003) Análise Estatística, com utilização do SPSS. Edições Sílabo, Lisboa, 822 pp 22. Maurer P, Couteaudieri Y, Girard P, Bridge P, Riba G (1997) Genetic diversity of Beauveria bassiana and relatedness to host insect range. Mycol Res 101:159–164 23. Medrela-Kuder E (2010) Seasonal variations in the occurrence of culturable airborne fungi in outdoor and indoor air in Craców. Int Biodeterior Biodegrad 52:203–205 24. Mercado-Blanco J, Rodríguez-Jurado D, Hervás A, Jiménez-Díaz R (2004) Suppression of Verticillium wilt in olive planting stocks by root-associated fluorescent Pseudomonas spp. Biol Control 30:474–486 25. Meyling N, Eilenberg J (2007) Ecology of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae in temperate agroecosystems: potential for conservation biological control. Biol Control 43:145–155 26. Murad A, Laumann R, Mehta A, Noronha E, Franco O (2007) Screening and secretomic analysis of entomopathogenic Beauveria bassiana isolates in response to cowpea weevil (Callosobruchus maculatus) exoskeleton. Comp Biochem Physiol C 145:333–338 27. Ordentlich A, Nachmias I (1990) Integrated control of Verticillium dahliae in potato by Trichoderma harzianum and captan. Crop Prot 9:363–366 28. Pandey R, Arora D, Dubey R (1993) Antagonistic interactions between fungal pathogens and phylloplane fungi of guava. Mycopathologia 124:31–39 29. Pathan A, Devi K, Vogel H, Reineke A (2007) Analysis of differential gene expression in the generalist entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin grown on different insect cuticular extracts and synthetic medium through cDNAAFLPs. Fungal Genet Biol 44:1231–1241 30. Paterson R (2008) Cordyceps—A traditional Chinese medicine and another fungal therapeutic biofactory? Phytochemistry 69:1469–1495 31. Pereira J, Bento A, Cabanas E, Torres L, Herz A, Hassan A (2004) Ants as predators of the egg parasitoid Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae) applied for biological control of the olive moth, Prays oleae (Lepidoptera: Plutellidae) in Portugal. Biocontrol Sci Technol 14:653–664 32. Quesada-Moraga E, Maranhao E, Valverde-García P, SantiagoÁlvarez C (2006) Selection of Beauveria bassiana isolates for control of the whiteflies Bemisia tabaci and Trialeurodes vapor- I. Oliveira et al. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. ariorum on the basis of their virulence, thermal requirements, and toxicogenic activity. Biol Control 36:274–287 Ramos P, Campos M, Ramos J (1998) Long-term study on the evaluation of yield and economic losses caused by Prays oleae Bern. in the olive crop of Granada (southern Spain). Crop Prot 17:645–647 Rencher AC (1995) Methods of multivariate analysis. John Willey, New York, 708 pp Roussos S, Zaouia N, Salih G, Tantaoui-Elaraki A, Lamrani K, Cheheb M, Hassouni H, Verh F, Perraud-Gaime I, Augur C, Ismaili-Alaoui M (2006) Characterization of filamentous fungi isolated from Moroccan olive and olive cake: toxinogenic potential of Aspergillus strains. Mol Nutr Food Res 50:500–506 Santoro P, Neves P, Alexandre T, Sartori D, Alves L, Fungaro M (2008) Selection of Beauveria bassiana isolates to control Alphitobius diaperinus. J Invertebr Pathol 97:83–90 Silva V, Barros R, Marques E, Torres J (2003) Suscetibilidade de Plutella xylostella (L.) (Lepidoptera: Plutellidae) aos Fungos Beauveria bassiana (Bals.) Vuill. e Metarhizium anisopliae (Metsch.) Sorok. Neotrop Entomol 32:653–658 Sun B, Yu H, Chen A, Liu X (2008) Insect-associated fungi in soils of field crops and orchards. Crop Prot 27:1421–1426 Topbaş M, Tosun U, Çan G, Kaklikkaya N, Aydin F (2006) Identification and seasonal distribution of airborne fungi in urban outdoor air in an Eastern Black Sea Turkish town. Turk J Med Sci 31:31–36 Torres-Barragán A, Anaya A, Alatorre R, Toriello C (2004) Entomopathogenic fungi from ‘El Eden’ Ecological Reserve, Quintana Roo, Mexico. Mycopathologia 158:61–71 Vänninen I, Hokkanen H (1997) Efficacy of entomopathogenic fungi and nematodes against Argyrestthia conjugella (Lep: Yponomeutidae). Entomophaga 42:377–385 Vega F, Goettel M, Blackwell M, Chandler D, Jackson M, Keller S, Koike M, Maniania N, Monzón A, Ownley B, Pell J, Rangel D, Roy H (2009) Fungal entomopathogens: new insights on their ecology. Fungal Ecol 2:149–159 Verma M, Brar S, Tyagi R, Surampalli R, Valéro J (2007) Antagonistic fungi, Trichoderma spp.: panoply of biological control. Biochem Eng J 37:1–20 Tj W, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Shinsky J, White Tj (eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, pp 315–322