The aim of this study was to evaluate the effects of commercially available bioinsecticides based on Metarhizium anisopliae (Metschnikoff) Sorokin and Beauveria bassiana (Balsamo) Vuillemin, i.e., Biometha WP Plus® (M. anisopliae), Biovéria G® (B bassiana), Boverril WP® (B. bassiana), Metarril WP® (M. anisopliae), and Metiê WP® (M. anisopliae) on the pupae and adults of Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) at concentrations of 1 × 109, 5 × 109, and 10 × 109 conidia mL-1. This braconid is released to control the sugarcane borer, Diatraea saccharalis. In the completely randomized first experiment with each commercial product, 10 C. flavipes female adults were held individually in disposable cups, which contained a 9-cm2 sugarcane leaf that had been treated with the one of the entomopathogenic fungal products. The mortality of C. flavipes females was assessed at 24, 48, 72, 96, and 120 h after treatment. In the second experiment, the same treatments were applied to C. flavipes pupae, because the latter can be exposed when the fungal products are applied to sugarcane to control various pests. In the second experiment we assessed the emergence of adults from treated pupae, the capacity of these adults to parasitize Diatraea saccharalis caterpillars, numbers of progeny of these C. flavipes, longevity of C. flavipes males and females, total adults emerged, and the percent emergence and longevity of males and females of the F1 generation. The mortality levels of C. flavipes pupae and adults were not affected by the 2 Entomopathogenic fungi. Therefore the use of Beauveria bassiana and M. anisopliae to protect sugarcane is compatible with the use of C. flavipes to suppress D. saccharalis.
Brazil is the world's largest sugarcane producer, and the major pests of this crop are Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae) and Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae) (Tiago et al. 2011; Tiago et al. 2012; Vacari et al. 2012; Simões et al. 2012). Caterpillars of D. saccharalis can cause direct losses in the cane stem by inducing biomass losses and the death of apical buds (White et al. 2008; Rossato et al. 2013). Chemical insecticides have low efficiency against this pest, because its third instar remains hidden inside the sugarcane stalk (Cruz et al. 2011; Rodrigues et al. 2013).
Microbial control agents such as entomopathogenic fungus can regulate insect populations through inundative and inoculative applications (Kurtti & Keyhani 2008; Mahdavi et al. 2013). These fungi can cause disease in up to 80% of the insects of a population, and they offer advantages of high genetic variability, infection at different development stages of the host, penetration through the integument, and high capacities of dispersal in the field (Destéfano et al. 2004).
Diatraea saccharalis is susceptible to Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) and Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae), and contact with these entomopathogenic fungi can affect the biological characteristics of this insect (Williams et al. 2013). In Brazil, the entomopathogenic fungus M. anisopliae is produced on rice and then applied to the sugarcane crop to reduce populations of M. fimbriolata (Loureiro et al. 2005).
Entomopathogenic fungi and Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) are used in sugarcane crops, and M. anisopliae may impact beneficial parasitoids and other non-target insects (Roy & Cottrell 2008) synergistically or antagonistically (Fuentes-Contreras & Niemeyer 2000; Stolz et al. 2002; Delpuech & Delahaye 2013). Thus host-parasitoid-entomopathogen interactions in agricultural systems can be harmful to populations of beneficial arthropods and ecological communities (Meyling et al. 2009; Meyling et al. 2011). Clearly, it is important to know the mortality patterns and interactions between fungi and natural enemies involved in integrated pest management programs (Santos Jr. et al. 2006).
In sugarcane fields in Brazil, M. anisopliae is applied to suppress Mahanarva fimbriolata , and B. bassiana is applied against termites, Metamasius hemipterus (Coleoptera: Curculionidae) and Sphenophorus levis (Coleoptera: Curculionidae). These treatments typically occur after C. flavipes has been released to suppress D. saccharalis. The parasitoid searches for and parasitizes D. saccharalis caterpillars inside and outside of the sugarcane stalks. After the parasitoid larva emerges from the caterpillar, they pupate. Because C. flavipes pupae are exposed, they may come in contact with M. anisopliae and B. bassiana.
The objective of this study was to measure mortality of C. flavipes after exposure to commercial products containing M. anisopliae and B. bassiana that are used for controlling pests in sugarcane fields.
Materials and Methods
The experiments were performed in the Laboratories of Entomology /Biological Control (LECOBIOL) of the Faculdade de Ciências Agrárias (FCA), Microbiology and Entomology of the Faculdade de Ciências Biológicas e Ambientais (FCBA) of Universidade Federal da Grande Dourados (UFGD), Dourados, Mato Grosso do Sul State, Brazil, as detailed below.
Obtaining Commercial Formulations Based on Metarhizium anisopliae and Beauveria bassiana
Commercial formulations used included Biometha WP Plus® (M. anisopliae), Biovéria G® (B. bassiana), Metarril WP®(M. anisopliae), Boverril WP® (B. bassiana), and Metiê WP® (M. anisopliae) provided by the companies Biotech Controle Biológico Ltda., Itaforte Bioprodutos, and Ballagro Agro Tecnologia, respectively. All commercial formulations showed over 95% viable spores.
Rearing of Diatraea saccharalis
Eggs of D. saccharalis were obtained by rearing this species in the LECOBIOL. Eggs were placed in glass jars (8.5-cm diam × 13-cm high) with artificial diet based on wheat germ, soybean, and the phagostimulant, sugarcane yeast (Saccharomyces cerevisiae Meyen ex E.C. Hansen; Saccaromycetales: Saccaromycetaceae) to provide food for neonates and 2nd, and 3rd instars. Fourth instars were transferred to disposable Petri dishes (6.5-cm diam × 2.5-cm high) and fed the same diet until they reached the pupal stage. Pupae were selected depending on their morphological characteristics and each held individually in a plastic pot covered with a screen until they reached the adult stage. The adults were separated in groups of 20 males and 30 females per cage of polyvinyl chloride (PVC) tubes (10-cm diam × 22-cm high). These cages were closed with bond paper and elastic and internally lined with paper sheets to aid oviposition. Eggs of D. saccharalis were collected daily, washed with a solution of copper sulfate, and then stored in a climatic chamber at 25 ± 2 °C, 70 ± 10% RH, and 14:10 h L:D, i.e., methodology adapted from Parra (2007).
Rearing the Parasitoid Cotesia flavipes
Fourth instar D. saccharalis caterpillars were individually exposed to a mated 24-h-old C. flavipes female. After being parasitized, the fourth instars were placed in disposable Petri dishes (6.5-cm diam × 2.5-cm high) and provided with artificial diet. These disposable Petri dishes were placed at 25 ± 2 °C, 70 ± 10% RH, and 14:10 h L:D until C. flavipes pupae formed. Pupae were held individually in disposable cups with lids (100 mL) using a drop of honey to feed the adults at 25 ± 2 °C, 70 ± 10% RH, and 14:10 h L:D until emergence of parasitoids (Garcia et al. 2009).
Experiment I, Quantification of Mortalities of C. flavipes Female Adults Exposed to Various Doses of M. anisopliae and B. bassiana
The objective of Experiment I was to quantify the mortality of adult females of C. flavipes when exposed to various doses of M. anisopliae and B. bassiana. Newly emerged C. flavipes females were exposed to commercial formulations of M. anisopliae and B. bassiana at the concentrations of 1 × 109,5 × 109, and 10 × 109 conidia mL-1. These concentrations of M. anisopliae are recommended for the control of M. fimbriolata. Doses of B. bassiana were identical. Ten C. flavipes females were enclosed per 100 mL disposable cup with a lid and with one droplet of honey inside. The surface of the sugarcane leaf was disinfected with a hypochlorite solution (0.02%). A disinfected sugarcane leaf (9 cm2; 3-cm long × 3-cm wide) was introduced into each cup. The surface contacting the insect was treated with 1 mL of each standardized suspension of the biopesticide in a Neubauer® chamber (Alves & Leucona 1998). The contact surfaces were treated with the aid of a micropipette and placed on paper towels to dry (Cardoso et al. 2007). The experiment was developed in a completely randomized design (CRD) with 16 treatments and 5 replications each using 10 parasitoid females, and totaling to 50 females per treatment.
The mortality of C. flavipes female adults was assessed after 24, 48, 72, 96, and 120 h of contact with the fungal suspensions. Each dead female was transferred to a graduated Eppendorf® microtube (1.5-mL volumetric capacity) and capped with moistened cotton wool with sterile distilled water. These microtubes were kept in a climatic chamber at 25 ± 2 °C, 70 ± 10% RH, and 14:10 h L:D to observe the growth of the fungus and to confirm the death of the insect.
Cumulative mortality of C. flavipes data were subjected to analysis of variance at 5% probability and to regression analysis. The choice of the equation that best fit the data was obtained with the linear model, based on the coefficient of determination (R2) and the significance of the regression by F test (up to 5% probability).
Experiment II, Quantification of Mortalities and Other Effects When C. flavipes Pupae Were Exposed to Various Doses of M. anisopliae and B. bassiana
The objective of Experiment II was to determine the effects of various doses of M. anisopliae and B. bassiana, when applied on C. flavipes pupae. Masses (size between 2.35 ± 0.83) of newly formed C. flavipes pupae were treated with 1 mL suspension of the insecticides based on M. anisopliae and B. bassiana at the concentrations of 1 × 109 conidia mL-1, 5 × 109 conidia mL-1, and 1 × 109 conidia mL-1 using an automatic pipette. These masses were then placed on absorbent paper to dry and each mass was held individually in 100 mL disposable cups with lids. These cups were placed in chambers at 25 ± 2 °C, 70 ± 10% RH, and 14:10 h L:D until the emergence of the adult parasitoids) Folegatti et al. 1990).
The experiment was developed in a CRD with 16 treatments and 10 replications, each replication included a mass of pupae with the potential for 50 C. flavipes adults to emerge. The data were subjected to analysis of variance (F test) and the means were compared by the Scott-Knott test at 5% probability.
Twenty females and 20 males of newly emerged C. flavipes were used per 1.5 mL Eppendorf® microtube, fed a droplet of honey, and capped with cotton wool to evaluate their longevity (days). The microtubes with insects were placed in a climatic chamber temperature at 25 ± 2 °C, 70 ± 10% RH, and 14:10 h L:D.
The percentage of parasitism (based on the number of parasitized larvae per treatment, discounting the natural mortality of the host) was evaluated with 5 D. saccharalis fourth-instars each exposed to five 24-h-old C. flavipes females. The experiment was performed with 10 replications, each replication included 5 parasitized caterpillars, totaling 50 caterpillars per treatment. Each caterpillar was parasitized by a female C. flavipes immediately when she found it. The caterpillars were placed in disposable Petri dishes (6-cm diam), fed with the above-mentioned artificial diet, and then transferred to an air conditioned room at 25 ± 2 °C, 70 ± 10% RH, and 14:10 h L:D.
The percentage of C. flavipes that emerged from parasitized larvae (filial generation - F1), the progeny (total individuals emerged), and the longevity (days) of this parasitoid were evaluated. The data were subjected to analysis of variance (F test) and the means were compared by Scott-Knott test at 5% probability.
Results
Experiment I, Quantification of Mortalities of C. flavipes Female Adults Exposed to Various Doses of M. anisopliae and B. bassiana.
Cumulative mortality of C. flavipes exposed to M. anisopliae commercial products was similar to the control at 24 h of exposure (HAE) (Table 1). Mortality at 24, 48, and 72 HAE was zero with Metarril WP® at a concentration of 1 × 109 conidia mL-1 (Table 1). Linear regression analysis showing mortality as a function of time was 80% at 96 HAE with Metarril WP® at 1 × 109 conidia mL-1 (Fig. 1B). Metiê WP® at 5 × 109conidia mL-1 and 10 × 109 conidia mL-1 at 96 HAE caused cumulative mortalities of this parasitoid of 42% and 48%, respectively. Biovéria G® (10 × 109 conidia mL-1) caused the least cumulative mortality (12%) after 24, 48, and 72 HAE (Fig. 1C). The cumulative mortalities of C. flavipes adults exposed to M. anisopliae products were similarly great among treatments after 96 and 120 h of the exposure to the fungi (Table 1).
Experiment II, Quantification of Mortalities and Other Effects When C. flavipes Pupae Were Exposed to Various Doses of M. anisopliae and B. bassiana
Parental Generation. The number of C. flavipes progeny (males and females) that emerged was similar among treatments with both entomopathogens (Table 2). Longevities (days) of females and males that emerged from pupae treated with Biometha WP Plus® (10 × 109conidia mL-1), Metiê WP® (5 × 109conidia mL-1) and Metarril WP® (1 × 109 conidia mL-1) were not significantly different from the control. The levels of parasitism induced in D. saccharalis by C. flavipes females that had emerged from pupae treated with M. anisopliae-based commercial products did not differ among treatments (Table 2).
The number of progeny (males and females) of C. flavipes that emerged from pupae treated with B. bassiana-based commercial products was similar among treatments (Table 2). The longevities of both female and male C. flavipes that emerged from pupae treated with all B. bassiana formulations were significantly shorter than the control. The longevities of females and males treated with Boverril WP® at 1 × 109 con.mL-1 and 10 × 109 con.mL-1 were also significantly shorter than the control (Table 2). The levels of parasitism induced in D. saccharalis by C. flavipes females that had emerged from pupae treated with B. bassiana-based products did not differ significantly among treatments and the control with the exception of Boverril WP® at the 1 × 109 conidia mL-1 treatment (Table 2).
F1 Generation. The emergence of F1 adults was not significantly different from the control in the following M. anisopliae treatments: Biometha WP Plus® at 1 × 109 con.mL-1 and 5 × 109 con.mL-1, Metiê WP® at 10 × 109 con.mL-1) and Metarril WP® at 1 × 109 con.mL-1. However in the remaining M. anisopliae treatments the emergence of F1 adults was significantly lower than in the control; and the lowest rate of emergence occurred with the Metarril WP® treatment at 5 × 109 con.mL-1 and 10 × 109 con.mL-1 (Fig. 2A).
The longevity of Fl C. flavipes female adults did not differ significantly from the control for the following M. anisopliae treatments: Biometha WP Plus® at 5 × 109 con.mL-1 and 10 × 109 con. mL-1 and Metiê WP® at 1 × 109 con.mL-1), but female longevity was significantly reduced in all remaining M. anisopliae treatments (Table 3). The longevity of males did not differ significantly from that of the control except it was lower with Metiê WP® 10 × 109con.mL-1(1.65 days) (Table 3).
The percent emergence of F1 adults from D. saccharalis larvae parasitized by C. flavipes females in treatments with B. bassiana was not significantly different from the control in the following treatments: Biovéria G® at 1 × 109 con.mL-1, Boverril WP® at 1 × 109 con.mL-1 and Boverril WP® at 10 × 109 con.mL-1. However the percent emergence of F1 adults was significantly lower than the control in the following treatments: Biovéria G® at 5 × 109 con.mL-1, Biovéria G® at 10 × 109con.mL-1 and Boverril WP® at 5 × 109con. mL-1 (Fig. 2B ).
The longevity of C. flavipes F1 females was not significantly different from the control (3.20 days) for the treatments Biovéria G® at 1 × 109 con.mL-1 (3.05 days), Biovéria G® at 5 × 109con. mL-1 (3.00 days) and Boverril WP® at 1 × 109con. mL-1 (2.70 days). However the longevity of F1 females emerged was significantly shorter than the control for the following treatments: Biovéria G® at 10 × 109con.mL-1 (2.00 days), Boverril WP® at 5 × 109 con.mL-1 (2.15 days) and Boverril WP® at 10 × 109 con.mL-1 (2.40 days). The longevity of F1 males was significantly different from the control only for the following 2 treatments: Biovéria G® at 10 × 109con.mL-1 (1.90 days) and Boverril WP® at 5 × 109con.mL-1 (1.80 days).
Discussion
The very low of mortality of C. flavipes females at 24 and 48 HAE with the products M. anisopliae and B. bassiana is important because the life period of the adult of this parasitoid is approximately 24 h in the laboratory at 24 ± 2 °C (Simões et al. 2012). The similar selectivity of B. bassiana and M. anisopliae to the parasitoid C. flavipes should not be generalized, because this natural enemy was susceptible to other isolates of M. anisopliae and B. bassiana (Folegatti et al. 1990).
Table 1.
Percent cumulative mortality of Cotesia flavipes (Hymenoptera: Braconidae) adults after continuous exposure to various commercial products based on Metarhizium Anisopliae (A) and beauveria bassiana (B) in the laboratory on sugarcane leaves for 24 to 120 hours (Hae) at 25 ± 2 °C, 70 ± 10% RH AND 14:10 H L:D.
Fig. 1.
Mortality of Cotesia flavipes (Hymenoptera: Braconidae) adults in the control (A) and after exposure to Metarril WP® (Metarhizium anisopliae) (B) at the 1 × 109conidia mL-1, and Biovéria G® (Beauveria bassiana) (C) at 10 × 109 conidia mL-1 inoculated on leaves of cane sugar at 25 ± 2 °C, 70 ± 10% RH and 14:10 h L:D.
Differences in the cumulative mortality of C. flavipes with the commercial products based on B. bassiana and M. anisopliae after 72 HAE and increased mortality in the control (38%) suggests the compatibility of this parasitoid with these fungi, as reported for the selectivity of B. bassiana to immature Habrobracon hebetor (Say) (Hymenoptera: Braconidae) (Mahdavi et al. 2013). The strain IPA 139E of M. anisopliae did not reduce egg parasitism of D. saccharalis by Trichogramma galloi Zucchi (Hymenoptera: Trichogrammatidae), which demonstrates the safety of this entomopathogenic fungus to this parasitoid (Broglio-Micheletti et al. 2006).
A similar longevity of C. flavipes females with the products based on M. anisopliae and B. bassiana corroborate the results with Oomyzus sokolowskii Kurdjumov (Hymenoptera: Eulophidae) and B. bassiana Esalq 447 and M. anisopliae E9 at the concentration of 107 conidia mL-1 These entomopathogenic fungi did not reduce the longevity, but B. bassiana caused higher mortality of this parasitoid (21%) than did M. anisopliae (9%) (Santos et al. 2006). However, B. bassiana is rarely used in sugarcane crops, which can contribute for the efficiency of C. flavipes in controlling D. saccharalis larvae.
The high mortality of C. flavipes at 72 HAE in the control and the treatments with the entomopathogens may not reduce parasitism by C. flavipes, whose adults start to parasitize their hosts approximately 24 h after emergence (Simões et al. 2012). This suggests that the 2 methods of biological control may act synergistically in the management of D. saccharalis in sugarcane crops.
Experiment II
Parent Generation. The emergence of similar numbers of individuals and similar proportions of males and females per parasitized C. flavipes pupae treated either with M. anisopliae or B. bassiana showed that these bioinsecticides did not affect the development of immature parasitoid. This may be because they are in the pupal stage, which is resistant to penetration and infection by entomopathogens (Armitage & Siva-Jothy 2005; Lemaitre & Hoffmann 2007; Mahdavi et al. 2013).
The shorter longevity of C. flavipes females with the bioinsecticides Metarril WP® (M. anisopliae) at 10 × 109 con.mL-1 (1.25 days) and Boverril WP® (B. bassiana) at 1 × 109con.mL-1 and 10 × 109 con.mL-1 by 1.10 days and 1.20 days, respectively, can be explained by the contact of C. flapives pupae with these entomopathogens. This result agrees with the premature death of other parasitoids species infected with these fungi (Rashki et al. 2009).
Table 2.
Progeny, number and longevity of females and males in the parental generation (G: P) of Cotesia flavipes that emerged from pupae treated with Metarhizium anisopliae (a) and Beauveria bassiana (B) and their capacities to parasitize Diatraea saccharalis at 25 ± 2 °C, 70 ± 10% RH and 14:10 H L:D.
Fig. 2.
Emergence (%) of adults in the F1 generation of Cotesia flavipes (Hymenoptera: Brachonidae) after exposure to the fungi Metarhizium anisopliae (A) and Beauveria bassiana (B) (Hypocreales: Clavicipitaceae) at 25 ± 2 °C, 70 ± 10% RH and 14:10 h L:D
Table 3.
Longevity of females (long, females) and males (long, males) of Cotesia flavipes (Hymenoptera: Brachonidae) in the F1 generation after exposure to Metarhizium anisopliae (a) and Beauveria bassiana (b) at 25 ± 2 °C, 70 ± 10% RH and 14:10 H L:D.
The reduction of parasitism by C. flavipes females that emerged from pupae treated with B. bassiana-based commercial products may indicate their weakening by this fungi (Emana 2007). On the other hand, similar parasitism in other treatments is due to the fact C. flavipes parasitizes its host almost immediately and even lives for a few days longer (Potting et al. 1997), which reinforces the safety of this fungus to its natural enemy.
The decrease in the longevity of C. flavipes males emerged from pupae treated with Boverril WP® (B. bassiana) at concentrations of 1 × 109 and 10 × 109 con.mL-1, 1.15 and 1.05 days, respectively, was half of the longevity of males in the control. However, this decrease differed from that obtained for Trichogramma pretiosum. Riley (Hymenoptera: Trichogrammatidae) that emerged from Anagasta kuehniella Zeller (Lepidoptera: Pyralidae) treated with B. bassiana and M. anisopliae (Potrich et al. 2009). The reduction in C. flavipes male longevity may be due to the infection by these fungi. These fungi may have only minor deleterious effects, because C. flavipes copulates in the first hour of its life and thus the reduction on its longevity may not affect this parameter (Sagarra et al. 2000a, 2000b; Chichera et al. 2012).
F1 generation. The lower emergence of C. flavipes adults in the F1 generation from D. saccharalis caterpillars after contact with M. anisopliae was also observed for Trichogramma galloi Zucchi with D. saccharalis eggs treated with isolated IPA 159E (M. anisopliae) (Broglio-Micheletti et al. 2006). However, the smallest percent emergence and longevity of C. flavipes males and females with B. bassiana differs from that reported for Trichogramma atopovirilia Oatman & Plainer (Hymenoptera: Trichogrammatidae) (Polanczyk et al. 2010) exposed to M. anisopliae and B. bassiana. The infection by entomopathogenics fungi on more advanced stages of immaturity may not reduce parasitoid emergence (Mesquita & Lacey 2001; Rashki et al. 2009), as observed in the parental generation. However, the exposure to this fungus may compromise other biological characteristics of C. flavipes females by producing offspring with lower parasitic capacity and development in D. saccharalis caterpillars.
Most formulations based on the entomopathogenic fungi B. bassiana and M. anisopliae reduced the longevity of C. flavipes males and females, but the percent emergence, number of progeny, and percent parasitism of D. saccharalis caterpillars were less affected. The contact with the entomopathogenic fungi with C. flavipes pupae did not affect parasitism by this parasitoid, and, thus, they are compatible.
Conclusion
The mortality of C. flavipes pupae and adults was not influenced by B. bassiana and M. anisopliae at the concentrations of 1 × 109, 5 × 109, and 10 × 109con.mL-1. Thus B. bassiana and M. anisopliae, at the concentrations of 1 × 109, 5 × 109, and 10 × 109con.mL-1, were compatible with the use of the parasitoid C. flavipes for biological control of sugarcane pests.
Acknowledgments
To the Brazilians institutions “Coordenação de Aperfeiçãamento de Pessoal de Nível Superior (CAPES)” for granting of the scholarship. To “Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq)” and “Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG).” Global Edico Services rewrote and edited this manuscript.
