Insect culture
Colonies of T. granarium, T. castaneum and C. maculatus were collected initially from the Entomology Laboratory, Faculty of Agriculture, Iraq. Insects of each species (50 male and female pairs) were reared on their main stored product in 300-ml plastic jars, secured with a muslin cloth and rubber bands and maintained at 30±2°C and 65±3% RH at continuous darkness for 2 generations. Grains were sterilized by placing them in a freezer (Samsung Ltd, Thailand) at −20°C for at least 2 days before each experiment. T. granarium was reared on whole sterilized wheat grains, C. maculatus was reared on sterilized cowpeas (Vigna unguiculata L. var. Parastoo) and T. castaneum was reared on sterilized wheat flour. Adults were removed after 5 days, and wheat grains, cowpea grains and wheat flour with the insect eggs were maintained under the laboratory conditions described above. Same-age cohorts of adult T. granarium, T. castaneum, or C. maculatus were obtained after 40–45 days and used in the treatments.
Source and preparation of C. rosea isolates
Two isolates of C. rosea [AA80 (MT366561) and AA82 (MT366214)] were obtained from dead M. persicae adults, collected from a greenhouse at the Faculty of Agriculture. Isolates were identified as C. rosea, following general and specific identification keys of Schroers et al. (1999). DNA amplification, sequencing and phylogenetic analysis technique were used to confirm morphological identification. Both isolates were cultivated on Sabouraud dextrose peptone yeast extract agar (SDAY) at 25°C. Aerial conidia were harvested from 14-day-old cultures by adding 15 ml of 0.01% Tween 80 to culture agar Petri dishes and gently scraping the surface of the cultures with a sterile inoculating loop to dislodge the conidia from the surface of the agar plates. The conidial suspension was pipetted from the Petri dish and filtered through 3 layers of cheesecloth. The number of conidia in the suspension was estimated, using a haemocytometer (Neubauer improved, Superior Marienfeld, Germany). The resulted suspension was diluted to the desired concentrations with 0.01% Tween 80 as required. The viability of the conidia was determined by spraying (0.1 ml of 1 × 106 conidia ml−1) on a sterile Petri dish with 1.5% SDAY. The dishes were sealed by a parafilm and incubated at 20°C, 90±2% RH, and a photoperiod of 16:8 (L:D) h. After 24 h, the number of germinated spores per 100 spores of each Petri dish was assessed under the microscope (×400 magnification). Germination was considered positive when the length of germ tube was at least half of the spore length. The viability exceeded 96% for both isolates.
Virulence of C. rosea isolates
Ten Petri dish replicates, each with 5 pairs (male and female adults), were used for each EPF isolate and control (0.02% sterile aqueous Tween 80 only) for either T. granarium, T. castaneum, or C. maculatus. Whatman No. 1 filter paper was placed in a Petri dish. Afterwards, pairs of each of T. granarium, T. castaneum, or C. maculatus were released into the dishes and sprayed with 2 ml of conidial concentrations of each C. rosea isolate (1 × 106 or 1 × 108 conidia ml−1), using 1l handheld sprayer and air-dried for 30 min at room temperature. Then, 5 g of sterilized wheat grain, cowpea grain or wheat flour were added to each Petri dish. All Petri dishes were sealed by a parafilm and incubated at 25±2°C, 70±2% RH and a photoperiod of 12:12 (L:D) h. Mortality rate was recorded daily for 6 days only, because the number of dead T. granarium and C. maculatus in the control significantly increased after 6 days of treatment, which was likely to influence the death rate of the fungus-treated insects. Dead insects were surface sterilized by rinsing twice with 70% ethanol for 30 sec, then rinsed with sterilized distilled water, before being placed on water agar (3 g of agar/l of water) in 9-cm Petri dishes for 5 days to confirm infection by EPF (Mohammed and Hatcher 2016). Cadavers were regarded as died from infection with these fungi, if conidia were recovered from the cadavers. The median lethal time (LT50) values for each fungal isolate were calculated. The entire experiment was repeated twice.
Effect of C. rosea treatment on the fecundity of coleopteran stored pests
The effect of infection with C. rosea isolates on the fecundity was determined for T. granarium, T. castaneum and C. maculatus adult females, male-female (1–2 days old) pairs of each insect species (each pair was a replicate). The experiment followed the method described in the ‘Virulence of C. rosea isolates’ section, where each pair was sprayed by 2 ml of each fungal isolate at a concentration of 1 × 106 conidia ml−1. Insects in the control treatment were treated by 0.02% sterile aqueous Tween 80 only. Each treatment was replicated 20 times. The number of eggs produced by each female was recorded daily until the female’s death using a magnifying lens.
Statistical analysis
Cumulative mortality was corrected for natural death in the control using Abbott’s formula (Abbott 1925). Normal distribution of data was assessed, using the Shapiro-Wilk test. Corrected mortalities were login transformed when necessary to meet the assumption of normality. Data used to determine the LT50 values of C. rosea isolates were calculated, using the probit analysis method. Two-factor repeated measurement was used to determine the effect of fungal isolate and insect species on the efficacy of C. rosea isolates. The effect of fungal infection on the fecundity of either T. granarium, T. castaneum or C. maculatus adult females was analysed separately using one-way repeated measurement ANOVA. Mean comparisons were performed using LSD test at 5% level of significance (P < .05).
