Potential of endophytic fungi as biocontrol agents of Duponchelia fovealis ( Zeller ) ( Lepidoptera : Crambidae )

This study reports the first assessment of endophytic fungi isolated from strawberry leaves and selection of isolates for the control of Duponchelia fovealis, a new pest of strawberries. A total of 400 strawberry leaves of the cultivar ‘Albion’ were collected in four commercial farms. Leaves were disinfected, cut in fragments, and placed on Petri dishes containing potato dextrose agar media with tetracycline and incubated for 30 days. Following this time, 517 fungal colonies were isolated, and thirteen genera were identified: Cladosporium, Aspergillus, Nigrospora, Fusarium, Trichoderma, Chaetomium, Alternaria, Paecilomyces, Penicillium, Ulocladium, Bipolaris, Diaporthe, and Phoma. Eight isolates belonging to the genera Aspergillus, Diaporthe, Paecilomyces, and Cladosporium were selected for pathogenicity bioassays against third instar larvae of D. fovealis. Isolates of Paecilomyces induced the highest mortality rates.


Introduction
The European pepper moth, Duponchelia fovealis (Zeller, 1847), (Lepidoptera: Crambidae) is native to marshlands of southern Europe and the eastern Mediterranean region (CABI, 2016).This moth is a greenhouse pest of cut flowers, vegetables, and aquatic plants in northern Europe and Canada (CABI, 2016).It has also become a pest of strawberries grown commercially in Europe (Bonsignore and Vacante, 2009;Franco and Baptista, 2010;Efil et al., 2014) and South America (Zawadneak et al., 2016).Because this is an exotic species, arthropod natural enemies are absent or insufficiently effective to control it (CABI, 2016;Zawadneak et al., 2015a).In addition, chemical control may not be as effective as larvae inside stems are protected from treatment (CABI, 2016), requiring several applications.Since strawberries are mostly grown in greenhouses, the control based only on chemical pesticides would result in residues on fruits and impact on the environment (Bernardi et al., 2015).In these cases, microorganisms could be a sustainable alternative control method (Gonzalez et al., 2016) with benefits for consumers, farmers, and the environment (Bonsignore and Vacante, 2009;Lacey et al., 2015;Gonzalez et al., 2016).For these reasons, biological control based on microorganisms represents a complementary strategy for further development (Zawadneak et al., 2015a;Gonzalez et al., 2016); however, studies on this type of approach involving D. fovealis are nonexistent.
Fungi have several characteristics that make them suitable as biological control agents, as they can directly penetrate through the insect cuticle, and increase the potential for epizootics and mortality rates in the pest population (Hajek and Delalibera, 2010;Gonzalez et al., 2016).These fungi can colonize plant tissues as endophytes (Arnold, 2007), apparently remaining assymptomatic (Urrutia et al., 2002;Arnold and Lutzoni, 2007;Vega et al., 2009).Endophytes can have beneficial effects on host plants, e.g., promoting plant growth, reducing disease severity, inducing plant defense mechanisms, and producing anti-herbivore products (Arnold et al., 2003;Arnold and Lewis, 2005;Schulz and Boyle, 2005;Rudgers et al., 2007).These fungi have been detected in hundreds of plant species (Urrutia et al., 2002;Gonzalez et al., 2016).
This study was aimed at identifying endophytic fungi present in strawberry leaves and select isolates for the control of Duponchelia fovealis, a new pest of this crop.

Samples
Strawberry leaves were collected from four commercial farms in the state of Paraná, Brazil (Table 1), in March 2012, totaling 100 leaves randomly collected per area.Samples were taken to the laboratory in plastic bags and processed on the same day.

Isolation of endophytic fungi
Leaves were first washed in water to remove dust and dirt residues and then immersed in 70% ethanol for 30 seconds and 1% sodium hypochlorite solution for 3 minutes.
The material were then thoroughly rinsed twice in sterile distilled water.After this process, four foliar pieces (2×2cm) were placed in separate Petri dishes (90 × 15 mm) containing potato dextrose agar (PDA) with tetracycline (100 µL/ml).The adaxial part of the leaf was turned upward.Plates were incubated in a BOD chamber at 28 ± 0.5 °C for 30 days.Fungi grown from leaves were transferred to Petri dishes containing PDA media.
The frequency of isolation (FI= number of leaf fragments with fungus growth/ total number leaf fragments) was calculated (Azevedo, 1998).
To compare the frequency of isolation among farms, the data were transformed (log x+1) to normalize the distributions and analyzed with the ANOVA F test and the Tukey test (p <0.05) using the software Assistat 7.7.

Morphological characterization
The morphological identification of endophytic fungi was based on macro and micromorphology (Kern and Blevins, 1999).Monosporic colonies were cultivated in Sabouraud media and incubated for 14 days at 28 ± 0.5 °C.Non-sporulated isolates were classified as Mycelia sterilia.
Isolates were maintained at the CMRP (Microbiological Collections of the Parana Network -TAXON line).

DNA sequencing
DNA sequencing was performed with 1-3 μL of the purified PCR product, 0.5 μL of buffer, 0.5 μL of each primer, 0.5 μL of BigDye  , and ultra-pure water, making up a final volume of 10 μL.Amplification conditions were: 96 °C for 1 min, and 35 cycles at 96 °C for 10s, 50 °C for 5s and 60 °C for 4 minutes.Sequencing was carried out with a DNA Sequencer Auto Applied BioSystem 3500.
The sequences obtained were aligned and edited with the package Staden Package version 1.6 and the software MEGA version 4.0.Subsequently they were compared with other sequences from the NCBI database with the software BLAST.

Insects
Larvae used in the experiments were obtained from the rearing facility at the Professor Angelo Moreira da Costa Lima Laboratory of Entomology/ UFPR, fed an artificial diet and kept at 70 ± 10% RH, 25 ± 2 °C, and 14-h photoperiod (Zawadneak et al., 2017).

Fungal preparations
In order to mass produce conidia, isolates were inoculated on malt and incubated at 28 ± 1 °C for 14 days in the dark.Colonies were then scraped and added to 100 mL of sterilized distilled water and Tween 80  (0.001%).The suspension was agitated for ten minutes at room temperature on an orbital shaking incubator at 150 and filtered through a sterilized cheesecloth.Conidia concentration was determined using a hemocytometer and adjusted to a final concentration of 1×10 9 conidia/mL.Conidial viability was assessed before the suspension preparation, and germination rates were higher than 95% in 24 hours at 28 ± 0.5 °C.

Pathogenicity bioassay
Eight endophytic fungi were evaluated in bioassays against D. fovealis.
Artificial diet was place in twenty-four well culture plates.D. fovealis larvae were transferred individually to the wells with the aid of a fine tip brush (#2).This assay consisted of three replicates per treatment and a negative control, totaling 96 larvae per treatment.
In the plate containing 24 larvae, 1mL of conidial suspension at the concentration of 1×10 9 conidia/mL was sprayed with a SAGYMA  Airbrush.A 1mL solution of 0.1% Tween 80 was used as control.Plates were incubated at 25 ± 1 °C, 14-h photoperiod, and 60 ± 10% RH for eight days.
Mortality criteria were based on insect's response to touch, color, and texture.Dead larvae were placed in Petri dishes with moistened sterile cotton for emergence of fungi from the host.The experimental design was completely randomized.
Fungal efficacy was based on means and mortality data (%) (Abbott, 1925) and compared with the ANOVA F test and the Tukey test (p < 0.05) with softwares GraphPad Prism 5.03 and Assistat 7.7.
The procedures were the same as those used in the first phase, consisted of D. fovealis larvae in twenty-four well culture plates sprayed with a conidial suspension at a given concentration, with five replicates per treatment.Mortality was assessed using the same criteria described previously.
The experimental design was completely randomized and the results were compared with the Probit analsyis) with Polo-PC -Leora Software, 1987.

Prevalence of isolates
A total of 1600 foliar fragments were collected from the four areas and 517 endophytic colonies were isolated.The prevalence of isolates was 32% (Table 2), varying among the areas evaluated, with values ranging from low to average prevalence (24.3 -45.8%).Area A showed the highest number of isolates ( 183), followed by area B with 123 isolates, while areas C and D had 114 and 97 (F: 13.2939, df: 3; 396; p < 0.05) isolates, respectively.

Composition of the endophytic assemblages
The 278 endophytic fungi isolated in this study were categorized into 15 taxa (Table 3) based on morphological characteristics, but 239 non-sporulating fungi could not be identified.
The genera Aspergillus spp., Paecilomyces spp., Alternaria spp., Penicillium spp.and Cladosporium spp.were separated into two or more morphospecies based on the size of the colony and conidial characteristics.As shown in Figure 1, Cladosporium sp. had the highest frequency of isolates.Alternaria sp. was the second most frequent endophytic taxon followed by Diaporthe sp., with frequencies of 19, 12, and 9.31% respectively.

Estimation of the mean lethal concentration (LC50)
The values of LC50 of two P. lilacinus isolates (G41 and G42) were similar (Table 5).The low slope (0.524 ± 0.081 and 0.422 ± 0.050) and χ2 values (0.444 and 0.880), respectively indicate the Probit model was a good fit to the data.The effective lethal concentration was 3.6×10 7 conidia/mL for G41 and 5.4×10 7 conidia/mL for G42.

Discussion
The highest number of fungal endophytes isolates was obtained from strawberry leaves collected in area A, which has been an organic farm for 12 years.Although area B is also characterized as an organic grower, the period of time converting from the conventional system to organic production was only three years and the biodiversity found was similar to those of conventional areas.The model of conventional agricultural is characterized by single crops, large dependence on chemical products, and high yields (Feiden et al., 2002).A considerable biodiversity is lost   et al., 2005).Thus, information on the biodiversity is an important strategy in the ecological management of the populations inhabiting in the agroecosystem.Biodiversity creates a balance among species that, when it is restored, numerous and complex interactions are established among soil, plants, and animals (Altieri et al., 2005).
C. cladosporioides and C. sphaerospermum were pathogenic for D. fovealis larvae.Endophytic species of the genera Cladosporium are used in the biocontrol of pests and diseases (Veja et al., 2008).Bahar et al.
(2011) tested a strain of Cladosporium against larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) and reported increased mortality rates of larvae.Similar results were obtained in this study, where C. cladosporioides and C. sphaerospermum were pathogenic to D. fovealis larvae.
Among the tested fungi, the isolates of P. lilacinus induced a high mortality rate of D. fovealis larvae.P. lilacinus is widely used in the biological control of diseases in plants cultivated under field conditions (Veja et al., 2008).Other studies performed with this species reported mortality in nymphs of Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) (Fiedler and Sosnowska, 2007) and showed a potential in the control of Nasonovia ribisnigri (Mosley, 1841) (Hemiptera, Aphididae) a pest of lettuce (Lactuca sativa L.) in Brazil (Zawadneak et., al. 2015b) and its applicability in the control of nematodes of the genus Meloidogyne sp.(Fiedler and Sosnowska, 2007).
In the present study, the isolate D. endophytica induced a mortality rate of 68% in D. fovealis.This endophytic species has not been reported as entomopathogenic in the literature.This is the first study to report this genus as a potential biological control agent of insects.
The isolates G41 and G42 had LC50 values considered good compared to those reported in the literature.Other studies have found significant results for isolates of this genus, as observed in our study.Hussein et al. (2013) treated third instar larvae of Spodoptera littoralis (Noctuide: Lepidoptera) with a new lineage of Paecilomyces sp (CCM 8367) in a suspension at a concentration of 5.0 × 10 7 conidia/mL and reported a mortality rate above 90%.
In conclusion, G41 and G42 isolates induced the highest mortality rates in D. fovealis larvae with the lowest LC50.Our findings showed the pathogenic and virulence potential of Paecilomyces lilacinus isolates as biological control agents against D. fovealis in laboratory as an alternative to currently employed traditional chemical insecticides.This is the first study on endophytic fungi isolated from strawberry tested against D. fovealis.

Table 1 .
Location of the sampling areas of strawberry leaves.

Table 2 .
Prevalence of isolates and number of taxa of fungal endophytes isolated from strawberry leaves at each site.Comparison of means using the Tukey test at 0.05 probability.Same lowercase letters do not differ at 95% level; ** Isolate prevalence (%) = (total number of leaf pieces yielding more than one isolate)/(total number of leaf pieces in that trial) x100. *

Table 3 .
Prevalence of endophytic fungal genus isolated from strawberry leaves.

Table 4 .
Molecular identification of species used in the mortality bioassay against D. fovealis.

Table 5 .
Probit analysis of mortality and log-concentration of conidia using in the bioassay with two isolates against third instar larvae of D. fovealis.LC50 values and their 95% confidence levels are expressed in conidia per milliliter. a