insects Article Susceptibility of Duponchelia fovealis Zeller (Lepidoptera: Crambidae) to Soil-Borne Entomopathogenic Fungi Rafaela F. Amatuzzi 1,* ID , Carolina G. Poitevin 1, Alex S. Poltronieri 2 ID , Maria A. C. Zawadneak 1 ID and Ida C. Pimentel 1 1 Departamento de Patologia Básica, Universidade Federal do Paraná, Curitiba 81531-980, Paraná, Brazil; [email protected] (C.G.P.); [email protected] (M.A.C.Z.); [email protected] (I.C.P.) 2 Departamento de Fitotecnia, Universidade Federal de Santa Catarina, Florianópolis 88034-000, Santa Catarina, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +55-41-3361-1700 Received: 1 May 2018; Accepted: 11 June 2018; Published: 19 June 2018 Abstract: Duponchelia fovealis (Lepidoptera: Crambidae) is an invasive species that has had a large impact on strawberry crops in Brazil. Pesticides have had limited effectiveness and the use of biological control agents to improve its management is the most appropriate approach. The aim of this study was to evaluate the pathogenicity and virulence of entomopathogenic fungi—isolated from soil—against Duponchelia fovealis larvae under laboratory and greenhouse conditions. Pathogenicity screenings were performed for twenty isolates from Beauveria bassiana, Beauveria caledonica, Isaria javanica, Metarhizium anisopliae, and Lecanicillium sp. against third instar 9 −1 larvae of D. fovealis at the concentration of 10 conidia·mL . Lethal concentration (LC50) and lethal time (LT50) were determined for the most pathogenic isolates and for one commercial mycoinsecticide. Mortality rates varied from 10 to 89%. The isolates B. bassiana Bea1, Bea110, Bea111 and I. javanica Isa340 were the most pathogenic. The most virulent isolates were B. bassiana Bea111 and I. javanica 6 5 −1 Isa340 with LC50 values of 2.33 × 10 and 9.69 × 10 conidia·mL , respectively. Under greenhouse conditions, the efficacy of LC50 of the isolates I. javanica Isa340 and B. bassiana Bea111 were 45% and 52%, respectively. Our results indicate that these isolates are strong candidates for application in the control of D. fovealis. This study is the first evaluation of soil-borne entomopathogenic fungi against D. fovealis. Keywords: Duponchelia fovealis; strawberry; Crambidae; Beauveria bassiana; Isaria javanica; biological control; entomopathogenic isolates; IPM 1. Introduction Duponchelia fovealis Zeller (Lepidoptera: Crambidae) is a native species from the marshlands of southern Europe and has become one of the most destructive pests of greenhouse and strawberry crops in several countries, including Portugal [1], Italy [2], Turkey [3] and Brazil [4]. The larvae cause economic damage by feeding on the leaves, flowers, and crown, which results in wilting, collapse, and death of the plants and consequently reducing crop quality and yield [4]. Due to the need for daily picking of strawberry fruit and with the increasing awareness of problems associated with pesticide use, development of alternative control strategies have become the main goal in the integrated pest management (IPM) of pests such as D. fovealis [5–7]. Biological control is one of the most promising options [4,6,8], although there have been few studies on its application against D. fovealis. Entomopathogenic fungi (EPF) have attributes that make them strong candidates for Insects 2018, 9, 70; doi:10.3390/insects9020070 www.mdpi.com/journal/insects Insects 2018, 9, 70 2 of 9 use against this pest [9]. They are widespread in terrestrial environments, especially in the soil, which is considered a reservoir [10], and are important natural regulators of insect populations through infection by contact, consequently increasing the potential for epizootics and mortality rates in the pest population [11]. Fungi of the genera Beauveria, Metarhizium, and Isaria are widely known as biological control agents [12,13] and they are safe for farmers, consumers, and the environment [14–16]. Thus, preventive methods through integrated pest management can be used as an alternative to the chemical control of D. fovealis. Traits such as virulence and pathogenicity are considered important properties of entomopathogens used in pest control [17]. Although entomopathogenic fungi have been widely used against a number of insect pests, no data are available on the efficacy of fungi against D. fovealis. This study aims to contribute to the implementation of an improved IPM program for this pest, with the first report of tested EPF isolated from native forest and crop systems soils against D. fovealis larvae under laboratory and greenhouse conditions. 2. Materials and Methods 2.1. Fungal Isolates and Insects EPFs were isolated from soil samples collected between January and June 2015 from different geographical sites in the state of Parana, Brazil (Table1). The isolates are deposited at the Paran á State Microbiological Collection Network—TAXONline (CMRP), Department of Basic Pathology, Federal University of Paraná, Curitiba, Brazil. Tests were conducted on third instar larvae of D. fovealis obtained from a stock colony reared on an artificial diet and maintained under controlled conditions of 25 ± 2 ◦C, 70 ± 10% relative humidity (RH), and a 14-h photophase [18]. Table 1. Entomopathogenic fungi isolates tested against Duponchelia fovealis and the GenBank accession numbers for their respective ITS sequences. Species Isolate GenBank Soil Source Coordinates and Sites Bea1 KY471,648 Bea2 KY471,649 Conventional corn (Zea mays) 25◦53019000 S, 49◦43019500 W Beauveria bassiana Bea3 KY471,650 crop systems Bea4 KY471,651 Araucária Bea5 KY471,652 Conventional strawberry Beauveria caledonica Bea110 KY471,655 (Fragaria × ananassa) crop systems 25◦53037600 S, 49◦44065400 W Bea111 KY471,653 Conventional corn (Zea mays) Araucária Beauveria bassiana A2B KY471,654 crop systems Organic strawberry 25◦74001000 S, 49◦89042500 W Lecanicillium sp. In1 KY471,666 (Fragaria × ananassa) Lapa crop systems 104 KY471,656 Metarhizium anisopliae 25◦34015400 S, 48◦90008300 W 107 KY471,657 Native forest Morretes Isaria javanica Isa340 KY488,507 110B KY471,658 110C KY471,659 110D KY471,660 315 KY471,661 ◦ 0 00 ◦ 0 00 Metarhizium anisopliae Banana trees (Musa spp.) in 25 38 776 S, 48 86 026 W 381 KY471,662 native forest Morretes 399A KY471,663 399B KY471,664 399C KY471,665 Insects 2018, 9, 70 3 of 9 2.2. Production of Fungal Conidial Suspensions Each isolate (Table1) was cultured on Sabouraud dextrose agar (SDA) medium and incubated at 25 ◦C in complete darkness for 15 days until sporulation was abundant. Conidia from each isolate were harvested under sterile flow conditions using a sterile steel spatula to scrape them into 30 mL glass tubes with 20 mL of 0.3% (v/v) Tween® 80 solution. Conidial suspensions of each isolate were vortexed for 15 min at 120 rpm and then filtered, using sterile gauze (10 × 15 cm), into new 30 mL glass tubes. Suspensions were prepared independently for each assay and concentrations were adjusted by serial dilution to achieve a comparable number of conidia per milliliter in an aqueous 0.3% Tween® 80 solution. Spore suspensions were used immediately after preparation and concentrations were adjusted using a hemocytometer. Spore viability was determined by direct counting [19,20]. Viability was then determined using a microscope (400× magnification) by visualization of 200 spores; spores that presented germinative tube growth were considered as viable. This procedure was adopted for all isolates and viability ranged from 90 to 95%. 2.3. Pathogenicity Bioassay Strawberry leaves were disinfected superficially [21] and their petioles were wrapped in cotton and placed in a 20 mL glass tube with autoclaved sterile distilled water and then positioned in acrylic boxes (11 × 11 × 3.5 cm). The experimental design was completely randomized. Each treatment was conducted with five replications. Twenty-four third instar D. fovealis larvae were transferred to each box, so that each box was considered a replication, for a total of 120 larvae tested per treatment. A Sagyma SW776 airbrush (10 lb pol−1) was used to spray 1 mL of a conidial suspension at a concentration of 109 conidia·mL−1 and Tween® 80 at 0.3% over the leaves and larvae. The control treatment received 1 mL of sterile distilled water with 0.3% (v/v) Tween® 80. After spraying, boxes were kept under controlled conditions (25 ± 2 ◦C, 70 ± 10% RH, and 14-h photophase). Mortality was assessed after 7 days. Moribund individuals or those that did not respond to touch with a paintbrush were considered dead. These specimens were transferred to Petri dishes with moistened filter paper until fungal extrusion to check for postmortem sporulation. The experiment was carried out for 5 weeks. 2.4. Virulence Bioassay and Determination of LC50 Fungal strains that caused high mortality rates of third instar larvae D. fovealis were selected to determine lethal concentration (LC50) and lethal time (LT50). Five concentrations were tested: 1 × 104; 1 × 105; 1 × 106; 1 × 107, 1 × 108 conidia·mL−1. For each concentration, 120 larvae were used, divided into five replicates. The control treatment consisted of sterile distilled water added 0.3% (v/v) Tween® 80. A commercially available biopesticide (Bovemax®) was used for comparison, containing B. bassiana CG716 strain as the active ingredient. Spraying was conducted as described for the pathogenicity bioassay and the samples were monitored daily for 7 days. Mortality assessments were also conducted as described above. 2.5. Greenhouse Assay 5 6 −1 The fungi that presented