Optimization of Factors Affecting Beauveria bassiana Fungus Ability in Control of Greater Wax Moth (Galleria mellonella L.) by Response Surface Method

Stored product pests are a major problem in the storage of agricultural products that cause damage from harvest until consumption. Greater Wax Moth (Galleria mellonella L.) is one of the most important pests of stored products and beehives. The most common method to control this pest in many countries is use of chemical compounds. However, these compounds have disadvantages such as pesticide residues in wax, the development of resistance in pest and irreversible effects on the environment and humans. The use of insect pathogenic fungi due to its low risk on mammals and natural enemies can be a good alternative to conventional chemical pesticides. Response surface methodology (RSM) is a statistical technique that is employed to optimize processes that are affected by several variables. This technique uses regression analysis to obtain optimal equations to estimate the values. Using this method, while maintaining the quality in the experiments, the number of those could be reduced. Therefore, this study was aimed to evaluate response surface methodology to determine the effect of optimum lethal level of concentration of B. bassiana conidia, temperature as well as humidity variables on the mortality of fifth instar larvae of greater wax moth.
Wax moth-eating insects were raised in plastic containers containing artificial food and old black wax at 30 ± 1 ° C and a relative humidity of 85 ± 1 % and photoperiod of 14:10 h (L: D). Isolation of insect pathogenic fungus B. bassiana was done by using Galleria Bait Method (GBM). For this purpose, after preparation of the fungus suspension from the infected larvae, 1 ml volume of the suspension was transferred to the water-agar 1.2% and then sealed petri dishes incubated at 30 ° C for three days. After identifying the single colony and formation of pure isolates, microscopic slides were prepared and eventually recovered isolates were recognized as B. bassiana. The bioassay was performed by determining the lethal concentrations of the B.bassiana that cause 20% to 80% casualties with a lot of concentration by immersion method for 10 seconds. Concentrations 1×106 and 1×108 conidia/ml were identified as high and low lethality ranges, respectively. In this study, the central composite design and response surface methodology with three independent variables including temperature (25-35°C), humidity (70-80 percent) and concentration (1×106-1×108 conidia/ml) and six replications in the central point of the design (to calculate the repeatability of the process) were used to evaluate the increase in mortality. The number of experiments was twenty and the dependent variable (response) was the mortality of the fifth instar larvae of greater wax moth. For each experiment, 10 last instar larvae were randomly selected and then 10 sterile petri dishes containing sterile wax to feed insect were prepared. Larvae were immersed for 10 seconds in a solution containing the fungus and then were placed in containers.
Analysis of variance (ANOVA) for the quadratic response surface model to factor mortality of the fifth instar larvae of greater wax moth showed that quadratic model is statistically significant (P≤0.001). Also high R2 (R2 = 0.9430) and coordination of adjusted R2 (Adj R2 = 0.9211) indicates the strength of the model to predict. According to tests, the optimal conditions for achieving maximum mortality of the fifth instar larvae of greater wax moth is 25 ° C temperature, 75% humidity and 1×108 conidia/ml concentration, respectively. Settings applied to the optimization process, was including maximum mortality. The effect of temperature on mortality of the fifth instar larvae of this insect showed that the mortality rate decreased with increasing temperature. Cause of mortality reduction as increasing the temperature is probably related to the characteristics of this fungus that could be affected by temperature, so that the impact of this fungus increases with decreasing temperature. The impact of the concentration on mortality rates showed that by gradually increasing of concentrations, the amount of mortality increases. This is because at the high concentrations of conidia, many more conidia could contact with the body of fifth instar larvae of this insect and could infect and destroy the larvae very quickly.
The results of current research indicate the efficiency of response surface method to optimize the use of insect pathogenic fungi. Among the conditions that were imposed on mortality, it was found that the increase in mortality is influenced by the quadratic response surface model of concentration and temperature, so that increasing the concentration caused increase in the mortality. Also increasing the temperature caused a decline in mortality rate.