2.1 Local isolation of entomopathogenic fungi from TSSM and red palm weevil

A total of 80 isolates, comprising 50 adult insect samples of Rhynchophorus ferrugineus (red palm weevil, RPW) and 30 adult mite samples of T. urticae (Koch), were obtained. The R. ferrugineus samples were collected from Riyadh and Qatif regions; and the T. urticae samples were collected from Dammam region. The infected samples were recognized according to the mycelial growth outside the sample’s body (Fig. S1). At each location, cadavers of naturally infected R. ferrugineus were collected and preserved in sterile Petri dishes. Once the samples arrived at the laboratory, fungi were isolated using the (Lo Verde et al., 2015) method with certain modifications. In brief, each sample was socked in 70% alcohol for half a minute.

to sterilize its surface, then washed with sterile distilled water. After one minute, the sample is rinsed in sterile distilled water and treated with 1% sodium hypochlorite. After that, we took pieces of each sample and put them on Sabouraud Dextrose Agar (SDA) with yeast extract (Oxoid). The samples were kept in a 27 ± 2 °C incubator for seven days. After a time of incubation, a small inoculum of the growing mycelium from each sample was transferred into a new SDA plate using a sterilized needle. Cultures were checked daily for the presence of fungal colonies growth (Fig. 1). Once a colony grow it get transferred to SDA or Czapek Dox Agar medium (CZA, Oxoid) for purification (Yousef and Fouly, 2021).

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Fig. 1

Morphological identification of entomopathogenic fungi.

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2.2 Collecting of T. urticae phytophagous mite

At the Agriculture Research and Biological Control Center of King Faisal University located in the Eastern province of Saudi Arabia Tetranychus urticae mite individuals were found in the leaves of bean, eggplant, and cucumber plants grown in greenhouses. At Dammam city, in Imam Abdulrahman bin Faisal University's Basic and Applied Scientific Research Center mite samples were received after they were collected and transferred in plastic bags.

2.3 Preparation of fungal inoculum

The entomopathogenic fungi were cultivated on autoclaved potato dextrose agar, which was supplemented with yeast extract, using the following procedure: A solution was prepared by dissolving potato infusion (200 g/L), dextrose (20 g/L), yeast extract (20 g/L), and agar (20 g/L) in 1L of distilled water. The media were thereafter incubated under controlled conditions of darkness at a temperature of 25 ± 1 °C and relative humidity of 65 ± 5% for a period of 10–14 days to facilitate the development of conidia (Örtücü and Iskender, 2017).

The Neubauer-improved hemocytometer, manufactured in Germany, was used to ascertain the spore concentrations of 1 × 10⁴, 1 × 10⁶, and 1 × 10⁸ (conidia/ml). The conidia were collected by scraping them from the surface cultures and afterwards suspended in 10 ml of sterile distilled water, with a ratio of 0.2 ml of Tween 80 per ml of water. This suspension was prepared in universal flasks containing glass pellets to prevent clumping of the conidia. Subsequently, the produced suspensions underwent filtration using three layers of muslin fabric in order to remove hyphae and conidia that were not well suspended. Subsequently, the spore suspensions were promptly used following their production (Sewify et al., 2015).Fig. 2.

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Fig. 2

Treatment of male and female Tetranychus urticae by Beaveria bassiana.

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2.4 Tetranychus urticae developmental stages

Tomato, eggplant, and pepper as host plants were used to determine how the host plant affected the biology and Life-table parameters of T. urticae. From each leaf sample, four discs were placed on a wet cotton on a petri dish. Then, four T. urticae individuals were placed in the petri dish on each leaf disc (three females and one male). The petri dishes were stored at 28 ± 1 °C and 65 ± 5% RH with a photoperiod of 16L:8D. Cotton was saturated with water every day, and dying leaves were replaced with new ones at regular intervals, so that the leaves would always look and smell fresh. The decaying leaves’ replacement was done until the most significant number of eggs (No. 90) were obtained. Eggs were monitored and TSSM stages were observed until they reached adulthood. The entire lifespan of T. urticae was tracked with twice-daily observations.

2.5 Reproduction, lifespan, and life tables of T. urticae

In this study, we used the same cultivars to assess T. urticae longevity and reproductive success. When the mites reached adulthood, two pairs (male and female) for mating occurrence were transferred to a new disc and kept together for the experiment using a 16L:8D photoperiod, 28 ± 1 °C, and 65 ± 5% relative humidity. Eggs were counted daily until all pairs in the experiment had died with maintaining the leaves' disc freshness condition. Using the developmental period of immature stages, the survival rate of immature females to adulthood, and the sex ratio of the progeny, the Birch (Birch, 1948) approach determined the life-table characteristics for each treatment group.

2.6 Molecular identification of fungi

Total genomic DNA was extracted from the isolated fungus using the Yeast/Bact Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The fungal isolate's 18S rRNA gene was then amplified using an absolute master mix (MoleQule-On, Auckland, New Zealand) with 10 µM of both 18S rRNA Forward (5′-GCTTAATTTGACTCAACACGGGA-3′) and Reverse primers (5′-AGCTATCAATCTGTCAATCCTGTC-3′) (Applied Biosystems, Foster City, California, USA). The Polymerase Chain Reaction (PCR) was performed on the master mixes as follows: (a) a 10-minute initial denaturation at 95 °C; (b) 35 cycles of denaturation at 95 °C for 1 min, annealing at 56 °C for 1.15 min, and extension at 72 °C for 2 min; and (c) a 5-minute final extension at 72 °C. The PCR amplicons were then electrophoretically separated on a 2% agarose gel (Alshammary et al., 2023). Following visual inspection of the PCR results, the amplicons were purified with the QI quick PCR Purification Kit (Qiagen, Hilden, Germany). Prior to 18S rRNA sequencing of isolated fungi, the purified PCR amplicons’ quantity and purity were measured using the Nanodrop 8000 V2.3.2 spectrophotometer. Using 3500 genetic analyzer, Sanger sequencing analysis was performed using the recent study (Alshammary et al., 2023) between 18S rRNA gene sequences of the fungal isolates. All of the fungus isolates' unprocessed 18S rRNA gene sequences were aligned in Bio Edit. The BLASTN program from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/blast) was used to compare the obtained sequences to those in the GenBank database. When identity and query cover were between 98% and 100%, sequences were deemed to be significantly aligned.

2.7 Screening fungi for extracellular enzymes production

Lipase assay. The fungi were examined for their lipolytic activity using two agar mediums: phenol red (Triyaswati and Ilmi, 2020) and Tributyrin (TBA, Himedia) (Wadia and Jain, 2017). The phenol red agar medium was prepared by dissolving 0.4 g/L phenol red, 20 ml/L olive oil, 10 ml/L Tween 80, 1 g/L CaCl₂ and 20 g/L Agar in 970 ml distilled water; pH was adjusted to 7. The TBA medium was prepared by dissolving 5.0 g/L Peptone, 3.0 g/L yeast extract, 15.0 g/L Agar, and 10.0 ml/L tributyrin glycerol tributyrate in 990 ml distilled water; pH was adjusted to 7.5. Both agar mediums were inoculated with fungi isolates and incubated for 7 days at 27 °C. After incubation, the diameter of formed halo zone around the fungal colony was measured (in mm) as an indicator of the lipolytic activity of the fungi.

Protease assay. Using Czapek Dox Agar medium, the fungi were screened for their proteolytic activity on skimmed milk according to (Ben Mefteh et al., 2019) procedure with some modifications. procedure with some modifications. In brief, the agar medium was prepared by stirring 2.5 g/L NaNO₃, 1 g/L KH₂PO₄, 0.5 g/L MgSO₄·7H₂O, 0.5 g/L KCl, 0.01 g/L FeSO₄·7H₂O, 30 g/L Sucrose, and 20 g/L Agar in 800 ml of distilled water. The pH was adjusted to 7.0 then sterilized at 121 °C in an autoclave. After sterilization, 200 ml/L skimmed milk was added to the slightly cooled medium before solidification in a fume hood cabinet. The Czapek Dox Agar medium was poured into sterile petri dishes. After cooling, fungi were inoculated and incubated for 7 days at 27 °C. After incubation, the halo zone diameter formed around the fungal colony was measured (in mm) as an indicator of the proteolytic activity.

Chitinase assay. Following (Homthong et al., 2016) with some modifications, we synthesized a wet colloidal chitin from the chitin of shrimp shells (Hi-Media). Moist colloidal chitin was prepared from the chitin of shrimp shells (Hi-Media) Following (Homthong et al., 2016) method with some modifications. Briefly in a 1000 ml beaker, 10 g of chitin powder was added softly to 200 ml concentrated hydrochloric acid (HCl) with continuous vigorous magnetic stirring for 3 h, where the solution became very viscous. With constant stirring, the solution was transferred into 1 L of cooled distilled water. The suspension was kept overnight under static conditions at 4 °C to facilitate better precipitation of colloidal chitin. The following day, chitin suspension was washed 10 times through cycles of centrifugation and precipitation at 4 °C. In the first washing cycle, the suspension was centrifuged at 10,000 rpm for 20 min. Precipitate was then collected and washed initially with 1 N NaOH. The second cycle of washing was at 8000 rpm for 15 min and the precipitate was washed with tap water. The rest of the washing cycles were also with tap water but at 5000 rpm for 10 min instead; until the chitin suspension reached a pH of 5.5 to 7. The obtained moist colloidal chitin suspension was placed in a 100 ml glass beaker covered with two layers of aluminum foil and sterilized by chitin agar medium was prepared and used to screen the fungi chitinolytic activity. The agar was prepared according to (Chi, 2015) procedure, where 2.0 % of the prepared moist colloidal chitin was dissolved with 0.7 g/L K₂HPO₄, 0.3 g/L KH₂PO₄, 0.5 g/L MgSO₄·5H₂O, 0.01 g/L FeSO₄.7H₂0, 0.001 g/L ZnSO₄, 0.001 g/L MnCl₂, and 20 g/L Agar in 1000 ml of distilled water. The medium pH was adjusted to 7.0 ± 0.2. Fungi were inoculated and incubated for 7 days at 30 °C. After incubation, the clear zone diameter formed around the fungal colony was measured (in mm) to indicate the fungi chitinolytic activity.

2.8 Treatment of Tetranychus urticae with Beaveria bassiana

Fifteen adult females and males of T. urticae, were individually treated on a pepper leaf disc (3 cm in diameter) and maintained on a moist cotton-wool pad in a 15 cm Petri dish. Each plate has 6 duplicate tablets. Direct spray was delivered to the body surface of mites from each dosage concentration using a hand sprayer at 25–30 cm, and control duplicates were sprayed with water. After application, using a microscope counted live and dead mites every day for 7 days. Cadavers were designated “mycotic” if fungal growths occurred in damp Petri dishes after three days in the same conditions (Sewify et al., 2015) Fig. 2.

2.9 Statistical analysis

The ANOVA test analyzed the obtained data, and Tukey HSD compared the means (IBM Crop. Released, 2014 SPSS 22.0). The values are expressed as means ± standard error (SE). Moreover, T. urticae Life-table parameters were calculated using TWO SEX-MSChart (Murthy and Bleakley, 2012).

3.1 Isolation of host-associated fungal isolates

The collected 80 samples of insects and mites were showing disease symptoms (e.g., slow movement, the appearance of mycelium on the surface body, change in color and swelling of the individual). As a result, 8 fungal isolates were recovered from these samples collected from Dammam, Qatif, and Riyadh. Interestingly, a single isolate of Beauveria sp., Aspergillus sp., and Penicillium sp. were found on the RPW from Qatif. Whereas two Scopulariopsis species were found from the Riyadh RPW samples. On the other hand, three isolates of Fusarium spp. were obtained: two found on the RPW in Qatif and one on the TSSMs in Dammam (Table 1). Similarly, Qayyum et al (Qayyum et al., 2021) isolated 94 entomopathogenic and opportunistic from 225 cadavers of insects in Multan, Pakistan. Almost half of these were harbored by Coleoptera, an order of insects includes RPW.

3.2 Identification of fungi

3.3 T. urticae Koch and the biology of TSSM

Tetranychus urticae was shown to have a five-stage life cycle (egg, larva, protonymph, deutonymph, and adult) on all the solanaceous plants examined under a 16L:8D photoperiod at temperatures of 28 ± 1 °C and relative humidity of 65 ± 5% RH (Fig. 2 and S5). These plants significantly altered the Life-table aspects of the species, including its growth, reproduction, and overall health. Previous findings by support these results on different host plants, tomato, eggplant, pepper, bean, and squash at 25 ± 1 °C, in addition to Awad (Awad et al., 2018) on tomato, eggplant, and pepper at 27 ± 4 °C, Mohamed (Mohamed et al., 2020) also, on tomato, eggplant, and pepper at 27 °C; moreover, a study (Praslička and Huszár, 2004) on Phaseolus vulgaris, Cucumis sativus and Capsicum annuum at different temperature 15, 30 and 35 °C.

3.4 Female T. urticae longevity and life duration

The effects of temperature (28 ± 1 °C), relative humidity (65 ± 5%), and photoperiod (16L:8D) on the reproductive phases, longevity, and life span of T. urticae females are shown in Table 5 and Fig.S8. The pre-oviposition period on pepper was 1.72 days, on tomato it was 2.22 days, and on eggplant it was 2.55 days (F2,57 = 9.922, P = 0.000). Comparable results were found when using tomato (1.07–1.15 days), eggplant (1.18–1.9 days), pepper (1.08–2.50 days), cucumber (0.79–1.13 days), and beans (1.26–1.44) as the host plants. The oviposition period averaged 7.90 days, 9.50 days, and 9.55 days on pepper, tomato, and eggplant, respectively (F2,57 = 4.008, P = 0.024).

The obtained results were less or more than the values obtained in previous studies on different solanaceous plants. Mohamed (Mohamed et al., 2020) were (7.43 days) on eggplant, (5.63 days) on tomato and (3.00 days) on pepper (Awad et al., 2018) (11.13 days) on eggplant, (9.93 days) on tomato and (8.61 days) on pepper (Nasr et al., 2019), was (10.33 days) on tomato due to the quality of the host plant can affect the fecundity rate of their herbivores, maybe due to its chemical composition such as nitrogen, phosphorus, and potassium (Helle and Sabelis, 1985).

3.5 Life-table parameters of T. urticae

Laboratory testing of T. urticae on solanaceous plants was conducted at 28 ± 1 °C, 65 ± 5% RH, and 16L:8D photoperiod (Table 7). Infestations of TSSM varied in their GRR between solanaceous plants, where the highest values were recorded with spider mites reared on tomato as (29.75 ± 3.79 eggs), followed by eggplant (24.37 ± 3.29 eggs), while the lowest values were obtained on pepper (13.75 ± 0.0114 eggs).

3.6 Screening of fungal isolates for enzymes

3.7 The efficacy of infection with the entomopathogenic fungus Beauveria bassiana

on males and females of the two-spotted spider mite Tetranychus urticae was evaluated under laboratory conditions at a temperature of 25 ± 1 °C with a relative humidity of 65 ± 5% and a photoperiod of 16:8 h (L:D). Three concentrations (1 × 104, 1 × 106, and 1 × 108 conidia/ml) of B. bassiana were used (Table 9). The ability of the fungus to kill the mites was very high. At all concentrations, the fungus completely killed all individuals. Many researchers have obtained results similar findings have been reported by Al Alawi (Al-Alawi, 2019) and Irigarai et al., (Irigaray et al., 2003) in T. urticae, as well as in other arthropods, such as the spider mite Tetranychus evansi (Wekesa et al., 2006), and tick species (Samish et al., 2001; Gindin et al., 2002).