Background

The red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) was recorded for the first time in Egypt by Saleh (1992) attacking the date palm Phoenix dactylifera L. at the eastern desert in the newly reclaimed land at Salhya region, Ismailia Governorate. Hallet et al. (1999) mentioned that transportation of infested young palm trees and offshoots introduced the weevil among other areas. For the same factor, the RPW was spread in many areas in Egypt through the last three decades causing destructive damage to date palm plantations. Falerio (2006) reported 17 palm species as hosts for this weevil in about 50% of the date palm growing countries. Generally, chemical insecticides with their known environmental hazards have been used for controlling the adult weevil on the palm tree trucks (Merghem 2011; Abbas 2013). Although sanitation measurements and elimination of infested trees are considered within the control strategies, other effective and environmentally safe control measurements and materials are urgently needed (Abraham et al., 2000). Weevil adult pheromone traps were widely used for monitoring and control of the RPW (Abraham et al., 1998 and 1999 and Falerio et al., 1998). The susceptibility of different stages of the RPW to infection with B. bassiana was studied under laboratory conditions by many authors (Aldossary et al., 2009; El Safty et al., 2009; and Dembilio et al., 2010). Field applications by dusting with B. bassiana conidiospores proved to be successful in controlling adults of the RPW (Sewify and Fouad, 2006; Sewify et al., 2009 and 2014; and El-Akad et al., 2016).

The present work aimed to evaluate the efficacy of a local isolate from the entomopathogenic fungus B. bassiana against larvae and adults of the RPW under laboratory conditions.

Material and methods

Conidiospores production of B. bassiana

An isolate of the fungus B. bassiana isolated from a naturally infected mole cricket (Gryllotalpa gryllotalpa L.) (El Husseini et al. 2008) was cultured on potato dextrose agar (PDA) medium poured into sterilized Petri-dishes (12 cm in diameter). Inoculated Petri-dishes with a spore suspension of B. bassiana were incubated for 15 days at 25 °C. The produced areal conidiospores were harvested from the Petri-dishes by scraping with a spatula and suspended in distilled sterilized water with 0.02% Tween 80 as a wetting agent in the primary stock suspension, following Rombach et al. (1987). The spore count was determined by using a Neubauer Hemocytometer, and the stock was kept in the refrigerator till needed.

Larvae and adults of RPW

Larvae of the RPW were collected from three highly infested date palm trees that suddenly fell on the ground at the Experiment Station at the Faculty of Agriculture, Giza, Egypt, in 2017. A large number of adult weevils were present inside the fallen palm trees associated with many larvae in different instars and cocoons having pupae inside. Adult weevils and larvae were collected by hand and transferred in metal boxes to the laboratory at the Centre of Biological Control, Faculty of Agriculture, Cairo University, Giza, Egypt. Due to the great difference in size and age of the collected larvae, the selected ones were categorized in young (Y) (3rd instar L3) and older (O) as full-grown larvae (7th instar L7). Cut tissue pieces from the inside of the same fallen trees served as a food for both larvae and adults in the laboratory during the experimental period.

Efficacy of B. bassiana versus larvae of RPW

Six concentrations of (6 × 102, 6 × 103, 6 × 104, 6 × 105, 6 × 106, and 2 × 107 spores/ml) were prepared in distilled water from the stock suspension (containing Tween 80) by successive dilutions. A fine perfume atomizer was used to spray the tested spore concentrations directly onto larvae of the two larval instars (L3 and L7). The larvae were treated in five replicates per concentration, each of 10 larvae; i.e., 300 young larvae (L3) and 300 older larvae (L7). After the dryness of the sprayed spore suspension, the treated larvae were transferred, using soft forceps into metal boxes (10 × 15 × 30 cm) with perforated metal cover and provided with cut tissues from date palm trees as food. A control was set of untreated larvae for each instar, i.e., 50 larvae for each and sprayed only with water containing 0.02% Tween 80. The boxes were kept under laboratory conditions of 25 °C and 50–60% RH. Inspection for larval mortality occurred daily for 20 days, and the larvae were provided with food. To prove death by B. bassiana, dead larvae were surface-sterilized for 2–3 s in formaldehyde and rinsed with sterilized distilled water under aseptic conditions. Thereafter, they were placed in sterilized Petri-dishes furnished with wet sterilized filter paper (Merghem, 2011). The Petri-dishes were kept in self-clip closing polyethylene bags to maintain relatively high humidity and kept at room temperature at 25 °C and 50–60% R.H. to allow the development of the fungus.

Efficacy of B. bassiana versus adults of RPW

Five concentrations of (6 × 103, 6 × 104, 6 × 105, 6 × 106, and 2 × 107 spores/ml) were tested against the adults. The conidiospore suspensions were applied directly on the adult weevils in Petri-dishes as mentioned earlier. After the dryness of the sprayed suspension, the weevils were transferred into metal boxes (the same as in case of the larvae) and inspected daily among a period of 10 days to supply with food if needed and to record the mortality. The dead adult weevils were treated as that of the dead larvae to prove death by B. bassiana.

Statistical analysis

LC50 and LC90 were calculated, using the software “Ldp Line” software (Bakr, 2005).

Results and discussion

Susceptibility of RPW larvae to B. bassiana

Results showed that the 3rd instar larvae of RPW were highly susceptible to B. bassiana as (100%) mortality was recorded on the 11th day post treatment for the concentrations (6 × 102 and 6 × 103 spores/ml), on the 10th day for the concentrations (6 × 104 and 6 × 105 spores/ml), and on the 7th day post treatment for the concentrations (6 × 106 and 6 × 107 spores/ml) (Table 1). Calculated LC50 and LC90 values were 3.9 × 102 (slope 0.436) and 3.7 × 102 spores/ml for larvae of L3, respectively, based on mortality values at the 6th day post treatment. The older instars (L7) showed mortality rates ranged between 45–46% for the concentrations 6 × 102 and 6 × 103 spores/ml and 70–75% for the concentrations 6 × 106 and 6 × 107 spores/ml, respectively. The calculated LC50 was 3.6 × 102 spores/ml (slope 0.160), and the LC90 was 3.7 × 103 spores/ml based on mortality values on the 12th day post treatment. Meanwhile, the survived treated larvae of (L7) formed their cocoons and developed to pupae; but all pupae and newly formed adults died inside their cocoons with developed symptoms of the White Muscardine caused by infection with B. bassiana (Fig. 1). Accordingly, all treated RPW larvae (L3 and L7) were killed by all tested concentrations of B. bassiana conidiospores. The results revealed a shorter time to kill the young larvae by increasing the fungus concentration from 11 days (6 × 102 spores/ml) to 7 days (6 × 107 spores/ml). The present findings are in line with those of Gindin et al. (2006) and Merghem (2011).

Table 1 Mortality % among larvae of the RPW R. ferrugineus treated with different concentrations of B. bassiana conidiospores and resulted pupae
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Fig. 1
figure 1

Dead pupa of RPW R. ferrugineus inside cocoon with developed mycosis after treatment with B. bassiana conidiospores in the larval stage

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Susceptibility of RPW adults to B. bassiana

Adults of RPW were found to be highly susceptible to infection with B. bassiana (Table 2). All treated adult weevils were killed (100% mortality) on the 10th, 9th, 8th, and 7th days post treatment at the concentrations (6 × 103, 6 × 104, 6 × 105, 6 × 106, and 6 × 107 spores/ml), respectively. The calculated LC50 was 5.8 × 101 (slope 0.328) and the LC90 was 4.7 × 103 spores/ml. All dead adult weevils were kept for proving death with B. bassiana developed the typical symptom of the White Muscardine. The present results are generally common for EPF against different insect species recorded by many authors, e.g., De la Rosa et al. (2000), Cruz et al. (2005), Ferron (2015), and Jayaprakash and Saranraj (2017). Such high susceptibility of both RPW larvae and adults to the infection with B. bassiana may explain the successful field control trials of RPW with B. bassiana and also with Metarhizium anisopliae in Egypt and other countries (Merghem 2011 and Abbas 2013).

Table 2 Mortality percentage among adults of Rhynchophorus ferrugineus treated with different concentrations of Beauveria bassiana conidiospores
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Conclusion

Treatment of adults and larvae of the red palm weevil (RPW) R. ferrugineus with the conidiospores of the entomopathogenic fungus Beauveria bassiana resulted in a reasonable mortality among larvae and 100% mortality in contaminated adults. The obtained results confirmed the possibility of using this fungus at least in controlling adults of the RPW by contamination with the conidia of the fungus. In nature, contamination of insects with fungi occurs by contacting the conidiospores of the fungus. This could be reached by dusting the palm trees with the conidia of the fungus to spread the disease among the weevil adults and later to their progeny inside the host palm.