Virulence of Beauveria bassiana Balsamo to red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

The red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), is a cosmopolitan pest of palms. Current management strategies largely depend on insecticides, and use of insect pathogenic fungi is needed in integrated pest management (IPM) programs. The effectiveness of Beauveria bassiana was determined against 3rd instar R. ferrugineus larvae by using the following concentrations, i.e., 3 × 108, 2 × 108, 1 × 108, 1 × 107, and 1 × 106 spores/ml. The lowest LC50 value (1.3×107 spores/ml) was recorded at the Khyber Pakhtunkhwa (KPK) population, followed by those populations from Punjab, Sindh, and Baluchistan, which had LC50 values of 1.5×107, 5.3 ×107, and 1.02 ×108 spores/ml, respectively, on the 7th day post-treatment; the highest mortality rates were 90.0, 85.0, 77.5, and 75.0% for the KPK, Punjab, Sindh, and Baluchistan populations, respectively, at the highest tested concentration of B. bassiana. The findings of this study indicate the potential use of B. bassiana in IPM programs for R. ferrugineus that are more ecologically sound than those programs that are heavily reliant on insecticides.


Background
Phoenix dactylifera L., date palm, is an ancient crop that is cultivated worldwide mostly in tropical regions and can be found as an important part of both large-and small-scale farming (Khushk et al. 2009). The production of dates has been decreasing in Pakistan due to the effect of the red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), which is a major pest of date palm all over the world (Abbas 2013). In the early stage of infestation, it is difficult to detect R. ferrugineus because it is concealed within the tree. In severe infestations, damaged trees can harbor multiple life stages at the same time (Faleiro 2006).
Synthetic insecticides are usually used to control insect pests that may cause resistance development, kill beneficial insects, pollute the atmosphere, and cause human health hazards (Thomas and Read 2007). To mitigate these problems, integrated pest management needs to be adopted such as the use of safer insecticides in combination with biological control agents, for example, entomopathogenic fungi (EPF) (Wraight et al. 2001). The insect pathogenic fungi have significant importance due to their infectious ability to insects (Mustafa and Kaur 2009). Around the globe, Beauveria bassiana Balsamo, Metarhizium anisopliae Sorokin, Isaria fumosorosea Wize, and Beauveria brongniartii Petch are commonly used EPF (de Faria and Wraight 2007;Dembilio et al. 2010) . Fungi are used for the management of various insect orders, e.g., Coleoptera including R. ferrugineus (Hussain et al. 2015).
B. bassiana is eco-friendly, safe in use, and a wellknown biological control agent with a wide host range (Devi and Rao 2006). B. bassiana is a potential tool, and its efficacy differs among adult and larval instars of R. ferrugineus (Lo Verde et al. 2015). The present study was conducted to assess the potential of B. bassiana in managing populations of R. ferrugineus under laboratory conditions from 4 provinces of Pakistan.

Beauveria bassiana
The local soil-extracted isolate of B. bassiana (Bb-01) was obtained from (30°05′11.65″N 71°39′15.65″E) Multan, Punjab, Pakistan, and maintained in the Laboratory of the Insect Microbiology and Biotechnology and was used for the experiments.

Fungal bioassay
Third instar larvae of R. ferrugineus were used in the assays. Each larva was dipped for 10-15s in one of the following concentrations of B. bassiana, i.e., 3 × 10 8 , 2 × 10 8 , 1 × 10 8 , 1 × 10 7 , and 1 × 10 6 spores/ml. All concentrations were prepared in an aqueous solution of 0.1% Tween 80 as defined by Alkhaibari et al. (2017). For each concentration and replicate, 80 larvae were exposed, at 4 replicates. A total of 480 larvae were tested in the bioassay including a control. For the control, only the aqueous solution containing Tween 80 was used. The treated larvae were placed in plastic Petri plates (2.5-cm diameter) with an artificial diet. The data on mortality was recorded on the 3rd, 5th, and 7th day post-treatment.

Statistical analysis
Fungal bioassay data were analyzed by the POLO Plus software (LeOra Software 2005) which yielded LC 50 values, 95% confidence limits (FL), slope ± SE, and chi-square values (χ 2 ). The means for mortality were separated among the treatments by using least significant difference (LSD) test (P ≤ 0.05). All analysis was performed by using Statistix 8.1.

Virulence of B. bassiana against R. ferrugineus
The virulence of B. bassiana on larvae of R. ferrugineus and the LC 50 values were estimated for populations of the weevil collected from 4 provinces in Pakistan. The lowest LC 50 value (1.3×10 7 spores/ml) was recorded at the KPK population, followed by those populations from Punjab, Sindh, and Baluchistan, which had LC 50 values of 1.5×10 7 , 5.3 ×10 7 , and 1.02 ×10 8 spores/ml, respectively (Table 1).

Virulence of B. bassiana against R. ferrugineus on different days post-treatment
The highest percent mortality of the weevil larvae were 57.5, 53.75, 52.5, and 50.0% on the 3rd day posttreatment, for the Punjab, KPK, Sindh, and Baluchistan populations, respectively (Table 2). On the 5th day posttreatment, correspondent percentages were 70.0, 67.5, 65.0, and 53.75% for the KPK, Punjab, Sindh, and Baluchistan populations, respectively (Table 3). On the 7th day post-treatment, respective mortality rates were 90.0, 85.0, 77.5, and 75.0% for the KPK, Punjab, Sindh, and Baluchistan populations, respectively (Table 4). These percent mortality rates occurred at the highest tested concentration of B. bassiana.

Discussion
The date growers mostly rely on the insecticides due to its rapid and spectacular results against R. ferrugineus. Development of an alternative strategy such as introduction of insect pathogenic fungi is necessary as a major component of IPM. EPF have proved to be an effective tool for the control of insect pests due to larvicidal potential and less toxicity to non-target species (Freed et al. 2012). B. bassiana is the most important EPF of Coleopterans (Cottrell and Shapiro-Ilan 2003) and showed promising results against the larvae of R. ferrugineus (Hajjar et al. 2015). In the present study, the pathogenicity of B. bassiana was evaluated against larvae of R. ferrugineus collected from 4 provinces of Pakistan. The highest percent mortality was found in the KPK population (90%), followed by the Punjab (85%), Sindh (77.5%), and Baluchistan (75%) populations at the highest concentration, 7th day post-treatment. These rates of mortality on day 7 suggest that the fungal spores successfully penetrated the cuticle of the larvae and were able to grow vegetatively inside the larvae, causing death due to a lack of nutrition (Inglis et al. 2001).
The outcomes of this study were consistent with Gindin et al. (2006) who examined the susceptibility of R. ferrugineus to B. bassiana at the concentration 2×10 7 spores/ml that caused 100% larval mortality within 6-7 days. Obtained results are also supported by the studies of Hou et al. (2018) who found that percent mortality of adult R. ferrugineus increased with increased conidial concentration applied. The highest concentration tested, 7.85 × 10 8 spores/ml, resulted in the highest mortality of the adult weevils. The findings were also supported by Yasin et al. (2019), who reported >75% adult and >88% larval mortality against R. ferrugineus by using B. bassiana. Collectively, the results of these studies suggest that mortality of various life stages of R. ferrugineus increased with increasing time after exposure and concentration of B. bassiana. Francardi et al. (2012) found that treatment of both larvae and adults of R. ferrugineus with B. bassiana resulted in 100% larval and 90% adult mortality under laboratory conditions. Moreover, El Husseini (2019) reported B. bassiana to be pathogenic against the 3rd instar larvae of R. ferrugineus causing 100% mortality. However, Lo Verde et al. (2015) found higher efficacy of B. bassiana against weevil larvae than the adults. The high efficacy of B. bassiana against R. ferrugineus was demonstrated in field trials (Abbas 2013). In the present study, B. bassiana treatment resulted in 90% larval mortality, whereas in earlier studies, this treatment provided 45% mortality (Francardi et al. 2013). Findings of current research provided novel options to develop very effective biocontrol agents based on fungi. This research will facilitate productive date industry with the use of an efficient tactic for the management of R. ferrugineus.

Conclusion
In conclusion, the present study described B. bassiana pathogenicity in R. ferrugineus. B. bassiana with its novel mode of action properties is a good alternative to chemical control as it tremendously reduced the survival of R. ferrugineus. This research also opens novel options to develop mycoinsecticides that might provide date industry with an efficient technique for management of R. ferrugineus.