INTRODUCTION

Among the main challenges facing the poultry industry, the control of Alphitobius diaperinus (A. diaperinus) (^{Crippen et al., 2018}) stands out, since this beetle is a reservoir of numerous pathogens in birds and humans (^{do Carmo et al., 2019}). In addition, although the main habitat of A. diaperinus, is manure or garbage (^{Govorushko, 2019}), its larvae can climb walls (^{Santoro et al., 2008}) and support posts in bird facilities (^{Rice and Lambkin, 2009}). Causing damage to insulation materials during their pupation processes (^{Hazeleger et al., 2008}; ^{Wales et al.,2010}; ^{Crippen et al., 2018}). The control of this insect is generally carried out with insecticides (^{Arena et al., 2019}). Highlighting the i) fenitrotion, ii) cyßuthrin and iii) spinosad (^{Tomberlin et al., 2014}; ^{Hickmann et al., 2018}). However, many of these chemicals have no effect on the adult stage of the beetle (^{do Prado et al., 2013}).

In addition, ^{Lambkin et al. (2010}) reported a greater resistance of A. diaperinus to fenitrotion, in 11 poultry farms for fattening. Especially southeast of Queensland, where numerous applications of fenitrotion have been carried out constantly. A control alternative for A. diaperinus is the entomopathogenic fungus Beauveria bassiana (B. bassiana) (^{Martins et al., 2016}).

This fungus is used as a biological insecticide or biopesticide controlling a large number of parasites such as caterpillars, termites, whiteflies, aphids and beetles (^{Daniel et al., 2019}), causing them mycosis or white muscardine disease (^{Santoro et al., 2008}; ^{Mascarin and Jaronski, 2016}). In Mexico, as a result of a previous investigation carried out by (^{Prado et al., 2014}), the pathogenicity of five strains of the Beauveria bassiana fungus in adults of Alphitobius diaperinus beetle was evaluated under laboratory conditions, obtaining good results. Therefore, from this study, the need arose to carry out research with New Mexican strains of the Beauveria bassiana fungus. Therefore, the objective of the present study was to evaluate the effect of different strains of B. bassiana (Deuteromycotina: Hyphomycetes) against A. diaperinus (Coleoptera: Tenebrionidae) of poultry farms in Colima state.

MATERIAL AND METHODS

Experimental location.

The bioassays were performed at the DES Biological Control Laboratory: Agricultural Sciences of the University of Colima. Laboratory conditions were 25±1 °C temperature, 80±4% of relative humidity (HR), and 12:12 h light/dark (L/O).

Origin of A. diaperinus.

A total of 360 A. diaperinus beetles (adults) were collected from poultry farms in Colima state, during the 7^th week of production (September and October 2016). The capture was carried out in various sites of the bird's bed, with the use of entomological clamps. diaperinus were transported in bottles 6 cm in diameter x 7 cm high, to the laboratory for taxonomic identification according to (^{Chernaki and Almeida, 2001}; ^{Dunford and Kaufman, 2006}).

Origin of B. bassiana strains.

Five strains of B. bassiana (Bb3, Bb4, Bb5, Bb6 and Bb17) from the entomopathogenic fungi collection of the DES Biological Control Laboratory: Agricultural Sciences of the University of Colima and the Laboratory of Parasitology and Biological Control of the Department of Veterinary and Zootechnics, Life Sciences Division of the University of Guanajuato.

Experimental stage (Phase 1). Multiplication, viability and concentration of B. bassiana strains.

The five strains of B. bassiana were multiplied in solid medium solid dextrose sabouraud agar (ADS) and incubated for 21 d. One day before performing the bioassay, the conidia were harvested with a bacteriological handle and placed in a 2x8 cm glass jar with a capacity of 25 mL, containing 10 mL of sterile distilled water with Tween 80 (00102; Sigma Chemical CO., Saint Louis, MO) at 0.01%. The mixture was homogenized (stirring) for 5 min and the viability of the conidia of each strain was determined by microculture (^{Santoro et al., 2008}; ^{Mascarin and Jaronski, 2016}). It was considered as a viable strain, one where more than 90% of the germinated conidia were observed (^{Geden and Steinkraus, 2003}; ^{Daniel et al., 2019}). To determine the concentration of conidia, a Neubawer chamber was used. The five treatments of B. bassiana (Bb3, Bb4, Bb5, Bb6 and Bb17) had a concentration of 1x10⁸ conidia/mL.

Experiment (Phase 2). Pathogenicity.

^{Rezende et al. (2009} and ^{Alves et al. (2015)} suggested using between 10 and 20 beetles. Therefore, 15 A. diaperinus beetles (adults) were used for the control and for each strain of B. bassiana (Bb3, Bb4, Bb5, Bb6 and Bb17), with four replicates/treatment. Each A. diaperinus was transferred to a sterile 250 mL beaker containing 10 mL of suspension (1x10⁸ conidia/mL) of each treatment and (only sterile distilled water with 0.01% Tween 80) for witness. After inoculation, A. diaperinus were placed in Petri dishes, with wet filter paper with sterile distilled water. Mortality was recorded every 12 h after inoculation for a period of 10 d. The dead beetles were individually placed in Petri dishes with wet filter paper with sterile distilled water and incubated for 21 d, to promote mycelial growth and thereby determine if the death was caused by B. bassiana.

Statistical analysis.

The statistical program (^{SAS, System, v. 9.0.2, Cary, NC}) was used to process the data obtained. To evaluate the design completely randomly, the following model was tested:

where:

γ_𝑖𝑗 = observation of the j-th u.e. of i-th treatment;

μ_𝑖 = mean of the ith treatment; and

ε_𝑖𝑗 = experimental error of the unit 𝑖𝑗.

The resulting data set was analyzed for descriptive statistics by (PROC UNIVARIATE; SAS, 2010). The comparison between treatments (control, Bb3, Bb4, Bb5, Bb6 and Bb17) was carried out by (PROC ANOVA; SAS, 2010). When significant effect was found by group (P <0.05), the Tukey multiple comparison test was performed. The data obtained for mycosis were transformed to the arcsine x-/100 * Sin-1 , expressed as a percentage and plotted.

RESULTS

Descriptive statistics for total deaths and total deaths from mycosis, caused by the five strains of B. bassiana on A. diaperinus beetles (adults) of poultry farms in Colima state. It can be seen that the strains tested in the total of dead beetles and the total of beetles killed by mycosis showed higher values than the control, where the Bb4 strain was the one that obtained the highest values, as shown in table 1.

The Bb4 strain of B. bassiana showed a difference between groups, with respect to mortality (5.25±0.95 dead A. diaperinus beetles). The lowest mortality among strains of bassiana was quantified in Bb3 (table 2). In dead beetles, mycosis due to mycelium growth of B. bassiana, presented the best results in strain Bb4 (2.50±1.29 beetles A. diaperinus with mycosis) equivalent to 16.66% (figure 1).

Figure 1 Mycosis by Beauveria bassiana in Alphitobius diaperinus of poultry farms in the state of Colima, n = 360 beetles. 

The Bb5 strain recorded (1.00±1.15 A. diaperinus beetles with mycosis) equivalent to 6.66% (figure 1). With the lowest value in strains Bb3 and Bb6 (3.33 and 1.66% of mycosis) respectively.

DISCUSSION

The results showed that all fungal isolates were pathogenic against A. diaperinus beetles, with a mycosis-induced mortality of 16.66% maximum and with significant differences between strains (Bb3, Bb4, Bb5, Bb6 and Bb17). These results are similar to the reports of (^{Rohde et al., 2006}; ^{Rezende et al., 2009}), who worked with the same beetle. It has been described that the first contact between B. bassiana and A. diaperinus, occurs when the spore (conidium) is deposited on the surface of the insect, hence the importance of the pathogenic dose (1 x 10⁸ conidia/mL) (^{Santoro et al., 2008}).

The conidium begins the development of its germination tube and its appressorium to fix itself to the beetle. For adequate germination, a HR of 92% and a temperature of between 23 and 25 °C (^{Vertyporokh et al., 2019}) are required. Then through enzymes such as proteases, lipases and chitinases, the entomopathogenic fungus enters A. diaperinus through its soft parts (^{Mascarin and Jaronski, 2016}).

Within the beetle, B. bassiana branches its structures and colonizes the host's cavities (^{Daniel et al., 2019}). With toxins p. eg, beauvericin, beauverolide, bassianolide and oxalic breaks the immune system of A. diaperinus, invading all its tissues and killing the beetle (^{Geden and Steinkraus, 2003}). Thus beginning the saprophytic phase of B. bassiana with its multiplication and growth (^{Mascarin and Jaronski, 2016}).

However, the highest expected mortality did not reach significant levels. ^{Daniel et al. (2019)} found that B. bassiana isolates are highly virulent and also indicated a wide variability in virulence among strains (from 2 to 70%). This indicates that, as in the present study, it is mandatory to evaluate as many strains as possible and thus be able to select the one with the greatest pathogenicity against A. diaperinus.

CONCLUSION

Beauveria bassiana proved useful as a biological control measure for Alphitobius diaperinus from poultry farms in Colima state. The potential to cause mycosis of strain Bb4, on strains Bb3, Bb5, Bb6 and Bb17 under laboratory conditions, is highlighted. The results found could be used in future trials to evaluate the Bb4 strain under normal conditions in poultry facilities.