All experiments were conducted at the laboratories of Plant Protection Department, Desert Research Centre, Egypt (2016–2018).
Collection of soil samples
Soil samples were collected from EL-Khatatba, Egypt, in May 2016. Plants at different fields were examined by the aid of a × 10 magnification lens for spider mite infestation. Samples were collected from the rhizosphere of the spider mite-infested plants at a depth of 10–15 cm under the soil surface. Soil samples (500 g each) were placed in sterilized polyethylene bags and stored at 4 °C until use.
Isolation, purification, and identification of fungi by cultural and morphological methods
Isolation of soil fungi was done by a serial dilution method. Two types of culture media were used for isolation of fungi: potato dextrose agar (PDA) and Czapek’s Dox agar supplemented with yeast extract (5 g/l). All media were supplemented with chloramphenicol antibiotic (25 mg/l). Isolation of fungi was carried out from dilutions of 10−2 and 10−3, where 0.5 ml was taken from each dilution and placed into Petri dishes with a solid media, then spread, using a sterilized glass spreader and was left for 30 min before incubation at 28 °C for 4–7 days. Each morphological distinct, fungal colony was sub-cultured and purified, using standard techniques.
Primary screening of fungal isolates against T. urticae
Sixty-two fungal isolates were screened for their control activity by testing their culture filtrates against adult spider mite females. Four discs (1 cm) were cut from fresh culture of each fungal isolate and inoculated in 100 ml of potato dextrose broth. The broth cultures were incubated in shaking incubator at 28 °C, 150 rpm for 7 days. Fungal cultures were filtered, using sterilized filter papers and centrifuged. Finally, the filtrates were preserved in refrigerator at 4 °C until use within a week to avoid any contamination or alteration of metabolites. Bioassay of fungal filtrates on spider mites was carried out by leaf-dipping method. Three mulberry leaf discs (25 mm) were immersed for 5 s in each fungal filtrate and dried at room temperature. The treated leaf discs were placed on wetted cotton wool in Petri dishes. Each was lined with cotton lining to prevent mites from escaping. Ten females were transferred to each leaf disc, using a fine paintbrush and incubated at 25 °C. Control was immersed in potato dextrose broth and mortality rates were observed after 5 days.
Selection and identification of the most potent isolates
Morphological identification
Four isolates (KF23, KF45, KF40, and KF9) out of 62 were selected for further investigation, as they achieved > 50% mortality in T. urticae. Cultures were grown on Czapek’s yeast extract agar and incubated at 28 °C for 7–10 days. The identification of the most potent isolates was carried out on morphological basis, using culture characteristics, e.g., growth rate, color, and pigmentation, as well as microscopic features, e.g., conidiophores, conidia, production of sclerotia, and dimensions of the different microscopic fungal structures. The results were confirmed by the scientists of Mycological Centre, Assiut University, Egypt.
Effect of crude extract of the selected isolates on adult females of T. urticae
Twelve plugs of freshly prepared culture of each fungal isolate were inoculated in 300 ml potato dextrose broth media and incubated in shaking incubator at 28 °C, 150 rpm for 10 days. After incubation time, each fungal culture was filtered and subjected to extraction with ethyl acetate (1:1) triple. The crude extract was dried, using a rotary evaporator, and stored in a freezer until use. Four concentrations (5, 10, 20, 30 mg/ml) were prepared from the crude extract of each fungal isolate. Bioassay of each extract was undertaken using the leaf dipping method. Leaf discs of 25 mm were immersed in each of the four concentrations, and another disc group was dipped in ethanol and used as a check and left to dry at room temperature. Treated leaf discs were placed on wetted cotton in Petri dishes (9 cm) and lined with a cotton lining to prevent spider mites from escaping, and then 20 females were transferred and incubated at 25 °C. The mortality rates were recorded after 3 days. Each concentration was replicated three times.
Effect of crude extract of the selected isolates on eggs of T. urticae
The ovicidal activity of the previously prepared concentrations of ethyl acetate extract of selected isolates was tested as follows: 10 females were placed on mulberry leaf discs (25 mm), prepared in 9 cm Petri dish to obtain same-aged eggs to be used in the tests. Twenty-four hours later, females were removed from the discs, eggs were counted, and three leaf discs, carrying eggs, were dipped into each concentration. The control discs were dipped in 70% ethanol and were left to dry. Observations continued daily until hatching of eggs in the control group.
Confirmation of the most potent strain by DNA sequencing
The strain was cultured on PDA overlaid with cellophane at 27 °C for 3–5 days. Total genomic DNA (deoxyribonucleic acid) was directly extracted from fungal mycelia of the KF23 strain, using the Quick-DNA™ Fungal/Bacterial Microprep Kit (Zymo research #D6007). The internal transcribed spacers (ITS1 and ITS4) were amplified using the universal primer pair ITS1 (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al. 1990) in a 50-μl reaction mixture comprising 5 μl of genomic DNA, 1 μl of each primer, 25 μl Maxima Hot Start PCR Master Mix (Thermo K1051), and 18 μl of sterile distilled water. The polymerase chain reaction (PCR) was carried out with the following parameters: an initial denaturation at 95 °C (10 min), 35 cycles of primer denaturation at 95 °C (30s), annealing at 57 °C (1 min), and extension at 72 °C (1 min 30s). A final elongation step was allowed at 72 °C for 10 min. The PCR products were purified using Gene JET™ PCR Purification Kit (Thermo K0701), according to the manufacturer’s instructions. Sequencing was done at GATC Company Germany by using ABI 3730xl DNA sequencer and using forward and reverse primers. The sequence obtained was subjected into Geneious pro 8.9 software and compared to ITS sequences available in Gene Bank by blast analysis. Finally, the phylogenetic tree was constructed to establish the taxonomic rank of the fungus.
Separation, purification, and identification of acaricidal secondary metabolites produced by A. melleus
Preparation of extracellular crude extract from A. melleus
Twenty-five Erlenmeyer flasks (250 ml), each containing 100 ml of PDB media supplemented with sucrose 3% (w/v), ammonium sulfate 0.75% (w/v), at initial pH value 4 and the flasks, were incubated at 30 °C for 6 days under static conditions. After the incubation period, the culture was collected and filtered by muslin cloth, then the clear filtrate was collected and subjected to extraction by Ethyl acetate triple (1:1), then the extraction solvent was dried, using vacuum under reduced pressure at 40 °C, and the crude extract was collected.
Separation of active compounds by thin-layer chromatography
Aluminum sheets (20×20 cm) with pre-coated silica gel 60 F254 (Merck, Germany) was used to separate active metabolites. The crude extract was applied in the form of bands at 1 cm from the bottom of the plate, using a capillary tube. Subsequently, the thin-layer chromatography (TLC) plate was then placed in a glass jar previously saturated with a developing solvent system of toluene to ethyl acetate to formic acid (6:3:1) (v/v). The glass jar was covered tightly. The developing system was left to rise on the TLC plate, until it reached the solvent front. The developed chromatogram was examined visually and observed under short (254 nm) and long (366 nm) wavelength ultraviolet light (UV). The retention factor (Rf) values of the separated spots were determined subsequently. For the detection of bioactive compounds, each band was scraped off the plate, eluted with methanol, and filtered, using a filter paper. The concentration of LC50 was prepared from each band and subjected to assay against T. urticae.
Separation and identification of the most active compound/s
Liquid column chromatography–mass spectrometry (LC-MS/MS) was used to separate active compounds in the highest active band. The band (Rf3) was subjected to identification on Agilent LC-MS/MS (Agilent Technologies, USA) that consists of C18 column model G1316A equipped with a diode array detector (model G1315D) and coupled to a 6420 Triple Quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in positive ionization mode. The mobile phase consisted of 5 mM ammonium formate containing 0.1% formic acid (solvent A) and acetonitrile (solvent B). A total of 5 μl of the sample was injected. The gradient elution program for LC analysis was applied as follows: 0–2 min, 40% B; 2–12 min, 40–85% B; 12–15 min, 85–90% B; 15–35 min, 90–95% B at a flow rate of 0.3 ml min−1 and the column temperature was set to 35 °C. The MS analysis used full-scan mode with the mass range set to 100–1200 m/z in positive and mode. The conditions of the ESI source were as follows: drying gas, high-purity nitrogen (N2), drying gas temperature was set to 350 °C, drying gas flow-rate was 11 L/min, the nebulizing gas (N2) was set at a pressure of 45 psi, a potential of 3500 V for the positive ionization mode was applied to the tip of the capillary, and the fragmentation voltage was 130 V. The acquisition and data analysis were controlled using Agilent LC-MS Software (Agilent, USA). The identification of the separated compounds was carried out by comparing their retention times and mass spectra provided by ESI-MS with the data obtained from previous studies that are carried out on the same A. melleus as well as those of authentic standards when available.
Data analysis
Control is corrected according to Abbott’s formula (Abbott 1925).
$$ \mathrm{Corrected}\ \mathrm{mortality}=\frac{T-C}{100-C}\times 100 $$
where T = dead mites in treatment and C = dead mites in control.
Data obtained from each dose-response bioassay were subjected to probit analysis (Finney 1971) to estimate LC50 values. For mortality and ovicidal activity experiments, the number of dead mite individuals and un-hatched eggs were counted and analyzed by one-way analysis of variance (ANOVA) (P = 0.05). Means were compared by Duncan’s test (Duncan 1955). Also, mortality percentages were calculated and corrected according to Abbott’s formula (Abbott 1925).