Mite collection and maintenance
Infested date-palm fruits with O. afrasiaticus were collected from the National Center for Palms and Dates, Al-Ahsa, KSA and King Faisal University Research areas, KSA in 2017. The mites were incubated under laboratory conditions (25 ± 1 °C, 62.5 ± 12.5 % R.H., and a 16:8 h (L:D) photoperiod).
Biological control agent
Entomopathogenic fungus, Metarhizium anisopliae, isolates (2049, 8762, and 7234) were used to evaluate their potential against O. afrasiaticus. EPF isolates were grown on potato dextrose agar (Oxoid, Hampshire, UK) in Petri plates (115 × 20 mm). After inoculation, cultures were kept at 25 ± 0.5 °C, 70% ± 5% RH in complete darkness.
Evaluation of virulence factors
A 24-day-old culture of each tested isolate was harvested, using a sterile scalpel to prepare conidial suspension, using 0.05% Tween 80 (Sigma-Aldrich Cat # P4780). Conidial suspension with a concentration of 1 × 107 conidia/ml was prepared under a compound microscope (× 400), using a Neubauer hemocytometer (Wertheim, Germany). The conidial viability of each M. anisopliae isolate was determined by inoculating 5 PDA-Petri dishes (5.5 cm) with 50 μl conidial suspension (1 × 107 spores/ml). Viability data were calculated after 18 h post-inoculation as described by Hussain et al. (2010). Each experimental unit’s percent viability was calculated by viewing 100 conidia at × 400 magnification under compound microscope.
Cuticle degrading protease activity
The protease (Pr1) activity of the conidial suspensions of all tested isolates was assayed by recording the release of nitroanilide (NA) at 405 nm, using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate as described by Hussain et al. (2015).
Laboratory pathogenicity bioassays
The pathogenicity of each isolate of M. anisopliae was calculated on the basis of LT50 (lethal time to 50% mortality) and LC50 (lethal concentration to 50% mortality). The LT50 values for all tested isolates were calculated by infecting the date-palm dust mite with a single concentration. In brief, conidial suspension with a single concentration (1 × 107 conidia/ml), separately prepared for each isolate, was used to dip pesticide-free date-palm leaf disks (length 7.5 × width 4 cm). After air-drying, 25 mite individuals (deutonymphs), with the aid of a camel-hair brush, were transferred on the leaves, surrounded with a wet cotton. Control treatment of the date-palm leaf disks was likewise prepared using 0.05% Tween 80. Five replicates, using 5 different populations, were prepared and incubated at 25 ± 1 °C, 62.5 ± 12.5% RH, and a 16:8 h (L:D) photoperiod. In addition, the study was repeated in the next season. Conidial response in terms of host mortality was recorded daily to calculate LT50 values, using Probit analysis.
In the case of LC50 determination, 5 concentrations (1 × 105, 1 × 106, 1 × 107, 1 × 108, and 1 × 109 conidia/ml) for each isolate were prepared in 0.05% Tween 80. Pesticide-free date-palm leaf disks (length 7.5 × width 4cm) were separately dipped into the 5 different concentrations. Twenty-five date-palm dust mites (deutonymphs) were carefully transferred on dampened cotton, using a camel-hair brush. In the case of the control, leaf disks were treated by a 0.05% Tween 80 solution. Five replicates were prepared using five different populations. Each experimental unit was incubated under controlled conditions, disclosed in the above section. Concentration-mortality response was recorded every 24 h to calculate the LC50, using Probit analysis. In both tests, dead mites were surface-sterilized and shifted into Petri dishes lined with dampened sterile filter paper. Mycosis of the inoculating fungal isolate was confirmed by microscopic examination.
Antioxidant defense mechanism of date palm dust mites
A single concentration (1 × 107 conidia/ml) of each tested isolate was used to infect the mites. Approximately, 1000 deutonymphs were allowed to feed for 9 days on conidial suspension treated with date-palm leaf disks. Overall, 3 mite samples (3, 6, and 9 days) with a 3-day difference for enzyme analysis were taken for homogenization under ice-cold potassium phosphate buffer in a glass homogenizer. After centrifugation, each supernatant was used to quantify protein, using Bradford (1976) methodology.
Catalase (CAT) activity was calculated, following the Aebi (1984) methodology. In brief, 25 μl of the sample was mixed with 665 μl of 50 mM PBS buffer and 10 μl of H2O2 to determine their absorbance in the spectrophotometer at 240 nm. Five replicates were likewise prepared.
Superoxide dismutase (SOD) activity of each sample was calculated with the Beauchamp and Fridovich (1971) methodology, mainly based on the generation of superoxide anions in the presence of light in response to reduce flavins. Five replicates were separately likewise prepared.
Glutathione S-transferase (GST) activities were calculated following the Habig et al. (1974) protocol, using chloro-dinitro-benzene (CDNB) and dichloro-nitrobenzene (DCNB). The homogenated sample was added into the microplate reader for 5 min for equilibration in the presence of CDNB and reduced glutathione. Absorbance was recorded in the spectrophotometer at 340 nm. Five replicates were prepared.
The data on conidial viability (%) and Pr1 activities (μm NA released/ml/min) were analyzed by one-way ANOVA. The mortality data of date-palm dust mites were angularly transformed. Abbott’s formula was used to correct the mortality rates of the mites (Abbott 1925). The data of the mortality rates and of the enzymatic activities of CAT, SOD, and GST relative to control treatment were analyzed by repeated measures ANOVA. Differences among the means were compared with Fisher’s LSD test (Statistix 2003).