Host plant, mite and predator
P. vulgaris seeds of Derakhshan variety were sterilized using a one percent sodium hypochlorite solution. Then they were planted in 15 cm diameter pots with 20 cm height, and filled with equal proportions of autoclaved field soil, Perlite, and Peat moss. These pots were kept in climate control rooms (25 ± 2°C, 65 ± 5% relative humidity and a photoperiod of 16L:8D h). A population of urticae was inoculated on these bean plants after two weeks. They were kept in the laboratory mentioned above conditions for two months.
A colony of P. persimilis was obtained from Koppert Biological System (Spidex®). This predator was grown on T. urticae-filled kidney bean leaves placed on plastic sheets on water-soaked foams. These foams were placed in half-filled plastic boxes with water. New kidney bean leaves containing preys were added to this arena daily. The edges of plastic sheets were covered with moist tissue papers and stored in laboratory settings for two months to prevent predators from escaping and kept for two months in laboratory conditions (25 ± 2ºC, 65 ± 5% relative humidity and a photoperiod of 16L: 8D h) (Walzer and Schausberger 1999). This population was used as a stock colony.
Plant probiotic bacteria
B. pumilus INR7 were kindly provided by J.W. Kloepper (Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama). Bacillus velezensis FOL were obtained from the culture collection of the Department of Plant Protection, College of Agriculture, Razi University.
Both of these Bacillus strains were cultivated in nutrients for 48 hours at room temperature (25 ± 2ºC), with constant shaking at 150 rpm. Then, the bacterial suspension was centrifuged at 5000 rpm for 20 minutes and the pellet was suspended in physiological saline. The bacterial concentration was adjusted to \({10}^{9}\) CFU\({\text{m}\text{l}}^{-1}\) was later used as bacterial inoculum. P. vulgaris cv Derakhshan germinated seeds were sterilized and soaked in 5ml of this bacterial suspension for 30 minutes before being planted in 14cm tall Plastic pots with an 18 cm diameter. These pots were filled with an equal mixture of sterilized field soil, sand, and peat moss and kept in a growth chamber (25 ± 1ºC, 65 ± 5% relative humidity and a photoperiod of 16L: 8D h).
Herbivore induced plant volatiles treatments
A concentration of 100 µM Methyl salicylate, methyl jasmonate, indole and 3-pentanol were prepared in 0.02% Tween 20. Sterilized germinated seeds of P. vulgaris cv Sterilized germinated seeds of P. vulgaris cv. Derakhshan were separately soaked in these emulsions for an hour and planted in pots of 14 cm in height and 18 cm in diameter afterward. The plants soaked in 0.02% Tween 20 served as the control group. The pots of each treatment were kept in separate growth chambers (25 ± 1ºC, 65 ± 5% relative humidity and a photoperiod of 16L: 8D h). After ten days, the young plants were sprayed with five ml of each HIPV emulsion. The plants were ready for functional response experiments the next day.
Functional response
A population of 100 P. persimilis females were randomly selected and transferred to a new arena to obtain a same-aged predator colony. The predatory mites were allowed to lay eggs for 24 hours, then removed from the arena. These arenas were kept in a growth chamber (25 ± 1ºC, 65 ± 5% relative humidity and a photoperiod of 16L: 8D h) and monitored daily. Newly emerged female adults were transferred to the new arena and starved for one day. These 1-day-old adult female individuals were used for functional response experiments.
The experimental unit was a 3.5 cm diameter petri dish placed on the lower side of an attached kidney bean leaf. During 24 hours, the 1-day-old adult female individuals of P. persimilis were given eight densities (2, 5, 10,20,30,40,50 and 60) of T. urticae eggs in ten replications. Gravid females of T. urticae were transferred to the leaves, allowed to oviposit for 24 hours, and then removed to simulate natural conditions (such as the existence of web and egg placement). The placed eggs were tallied on each leaf, and if the number was too high or low, some eggs were added or removed. One 1-day-old adult female individual of P. persimilis was added to each replication from the same aged colony, and the predators were removed after 24 hours. The intact remaining eggs were counted afterward.
Data analysis
The functional response data were analyzed in two steps (Juliano 2001). For the first step, we used the logistic regression of the proportion of prey consumed (Ne/N0) as a function of initial density (N0) to determine the type of the functional response.
$$\frac{{N}_{e}}{{N}_{0}}=\frac{exp({P}_{0}+{P}_{1}{N}_{0}+{P}_{2}{N}_{0}^{2}+{P}_{3}{N}_{0}^{3})}{1+exp({P}_{0}+{P}_{1}{N}_{0}+{P}_{2}{N}_{0}^{2}+{P}_{3}{N}_{0}^{3})}$$
1
Where (Ne/N0) is the probability, a prey will be consumed, and P0, P1, P2 and P3 are the intercepts, linear, quadratic and cubic coefficients, respectively, estimated using the maximum likelihood method (Xiao and Fadamiro 2010). The type of functional response was determined by fitting data to the model (1). If the function is negative (P1 < 0), the functional response is type II, which means the amount of consumed prey, decreases monotonically as the number of prey increases. predator displays a type III functional response if the function is positive(P1 > 0 and P2 < 0) (Juliano 2001). The predator displays a type III functional response, if the function is positive. The handling time (Th) and the attack rate (a) coefficients of a type II response were estimated using an explicit deterministic model in the second stage (Royama 1971; Rogers 1972).
$${N}_{e}={N}_{1}\left[1-exp\left(a\left({T}_{h}{N}_{a}-T\right)\right)\right]$$
2
Where Ne is the number of preys consumed; N0 is the initial number of preys; Th is the handling time, and T is the predator's total time. Statistical paired comparisons of parameters of the functional responses for different treatments and the control were performed using the indicator variable method (Juliano 2001).
$${N}_{a}={N}_{0}\{1-\text{exp}\left[-\left(a+\left({D}_{a}\left(j\right)\right)\left(T-\left({T}_{h}+{D}_{Th}\left(j\right)\right){N}_{a}\right)\right]\right\}$$
3
Where j is an indicator variable with a value of 0 for the first data series and 1 for the second data series. The parameters Da and DTh estimate the differences between the values of the parameters a and Th, respectively, of the data sets being compared. The null hypothesis that DTh comprises 0 is tested to see if there is a substantial difference between the two treatments (Juliano 2001). The data were analyzed using SAS software (SAS 9.4). Using the predicted Th, the maximum predation rate (T/ Th) was computed, the maximum number of prey that a predator can consume in 24 hours (Hassell 2000).