Pathogenicity of fungal and bacterial bioinsecticides against adult peach fruit fly,

Background: The peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae), is a serious polyphagous pest of fruits and vegetables. Chemical management of B. zonata in fruits results in toxic residues that have adverse health effects on consumers resulting in increased demand for eco-friendly approaches. Laboratory bioassay was conducted to determine the pathogenicity of fungal and bacterial biopesticides against B. zonata. Results: The pathogenicity of all tested bioinsecticides revealed that the maximum concentration (1 × 10 CFU ml−1) of Metarhizium anisopliae, Beauveria bassiana, Lecanicillium lecanii and Bacillus thuringiensis caused 95.8–100%, 95.9–97.9%, 32.7–39.6%, and 20.0–22.4% mortality in B. zonata, respectively at 7 d post-application interval (PAI) as compared to mortality demonstrated by the same concentration at 5 d PAI. All tested microbial insecticides induced statistically similar mortality in both male and female B. zonata at each concentration for the same PAI. Correlation coefficient (r) values reveal that concentrations of each microbial insecticide had a high positive correlation with mortalities of male and female B. zonata. Regression parameters reveal that concentrations of tested microbial insecticides had significant linear relationship with and explained significant variability in B. zonata mortality (P < 0.05). Results also revealed that M. anisopliae was proved more pathogenic to males and females of B. zonata demonstrating the least LC50 values (5.48 × 10, and 6.17 × 10 CFU ml−1, respectively) 7 d post-application intervals, followed by B. bassiana which explained LC50 value of 1.14 × 10 CFU and 1.15 × 10 CFU ml−1 for B. zonata males and females, respectively, at the same period of application, but less than that of L. lecanii (2.77 × 10 and 1.43 × 10 CFU ml−1) and then B. thuringiensis (3.40 × 10 and 1.39 × 10 CFU ml−1) for the males and females, respectively, at 7 d PAIs. Conclusion: Metarhizium anisopliae incorporated adult diet was proved more effective against B. zonata, followed by B. bassiana, L. lecanii, and B. thuringiensis. Hence, M. anisopliae can be recommended for incorporating in bait-traps to develop attract-and-kill technology for B. zonata.

and wild plants, mainly those with fleshy fruits including guava, mango, peach, apricot, citrus, and figs (El-Minshawy et al. 2018). B. zonata has globally attained the status of economic and quarantine pest. Various published reports reveal that B. zonata is the most dominant, devastating, and abundantly found fruit fly species in different ecological regions of Pakistan infesting variety of fruits and vegetables (Ahmad and Begum 2017). In many countries, management of B. zonata is difficult due to the behavioral, feeding, and biological adaptability of various life stages of fruit fly and the lack of effective broad-spectrum insecticides from markets (Dias et al. 2018).
In developing countries, management of fruit flies mostly depends upon the cover spray of synthetic insecticides because of their quick knockdown impacts (De Bon et al. 2014;Nicholson 2007). Such cover spray not only causes ecological backlashes in fruit flies against insecticides but also induces lethality to non-target beneficial arthropods and phytotoxic effects on plants (Li et al. 2018). Insecticide applications also increase the cost of production and leaves toxic residues in fruits and vegetables causing biomagnification of residues in human (Gogi et al. 2010).
The microbial agents in the form of biopesticides can be a better alternative to synthetic pesticides and an effective part of integrated pest management (IPM) strategies for the control of several agricultural insect pests (Farooq et al. 2020). Spray application and auto-dissemination (through the use of attractive materials/devices to propagate pathogens in target pest populations) (Vega et al. 2007) are used mainly for the introduction of microbial agents into an agro-ecosystem (Talaei-Hassanloui et al. 2007). Entomopathogenic fungi (EPF) typically cause infection when spores come in contact with the arthropod host (Goettel et al. 2008).
EPF have shown very promising results against various species of fruit flies (Soliman et al. 2020). Different strains of Beauveria bassiana, Paecilomyces fumosoroseus, Lecanicillium lecanii, and Metarhizium anisopliae are used for insect pest management (Lacey et al. 2001). Microbial control is a potentially useful method to inhibit fruit flies (Soliman et al. 2020). Recently, entomopathogens as natural enemies have been used to reduce the population of fruit flies, Ceratitis capitata, B. oleae, and Z. cucurbitae (Dias et al. 2018). Studies of some researchers confirmed that M. anisopliae has a very high potential in suppressing fruit flies (Dimbi 2003). Introduction of entomopathogenic bacteria (EPB), Bacillus thuringiensis subspecies darmadiensis, mixed with a protein diet and sugar as a bait was found very effective in killing South American fruit fly, Anastrepha ludens (Martinez et al. 1997).
EPF prove very effective against larvae and pupae of fruit flies when they come in contact with the treated soil (Ekesi et al. 2007). Oral and contact bioassays of B. bassiana and B. brongniartii against B. oleae and C. capitata were found effective for fruit flies (Konstantopoulou and Mazomenos 2005). EPF such as B. bassiana, Isaria fumosorosea, and M. anisopliae demonstrated 90-100% mortality and induced significant impact on the fecundity of European cherry fruit fly, Rhagoletis cerasi, while foliar application of B. bassiana caused 65% of infection in cherry orchards (Daniel and Wyss 2010). Toledo et al. (2017) applied three formulations of B. bassiana by autoinoculation devices and sterilemale-vector technique in coffee orchards for assessing pathogenicity against C. capitata and concluded that application of B. bassiana by later technique proved more effective in the horizontal transmission of conidia to wild-population of C. capitata, but both techniques demonstrated > 90% reduction in C. capitata (Flores et al. 2013). EPF can be applied in form of a bait station against fruit flies (Navarro-Llopis et al. 2015). Application of M. anisopliae-based attractant-contaminant device (ACD) @ 24 ACD ha −1 is an efficient technique for the control of C. capitata up to 3 months when inoculation dishes are replaced mid-season (Navarro-Llopis et al. 2015).
The present research was conducted to evaluate the fungal and bacterial bioinsecticide-based diet (M. anisopliae, B. bassiana, L. lecanii, and B. thuringiensis var kurstaki against both male and female flies of B. zonata under controlled conditions.

Mass rearing of Bactrocera zonata
Guava fruits infested with fruit flies were collected from different orchards in Faisalabad. Bactrocera zonata was identified on the basis of four morphological characteristics as described by White and Elson-Harris (1996). The infested fruits were taken into the laboratory and kept in card boxes half-filled with sieved and sterilized sand. Pupae were collected from sand by using a fine-mesh sieve after a week. The pupae were kept in the domeshaped rearing cages till the adult emergence. The cages were provided with the spongy strips soaked with the adult diet containing honey, protein and sugar solution (1 part sugar and 9 parts water) mixed in a 1:1:9 ratio. These strips were suspended after soaking in an adult diet solution. The fresh, properly cleaned and washed guava fruits were brought to the laboratory and hanged inside the rearing cage for eggs collection. Then, after 3 d, fruits were shifted from rearing cage to card boxes having Page 3 of 20 Nisar et al. Egyptian Journal of Biological Pest Control (2021) 31:146 sterilized sand for attaining the next progeny. This procedure was used to mass culture B. zonata.

Preparation and pouring of ¼ SDAY media for culturing fungal strains
A quantity of 16.25 g Saburaud dextrose agar, 11.25 g agar, and 1.25 g yeast was added in 1 liter of distilled water and autoclaved at 20 psi and 121 °C for 20 min. After autoclaving, the media was poured into Petri plates and was allowed to cool at room temperature.

Culturing of fungal strains
One gram of powder of each of the commercially available strains i.e., B. bassiana, M. anisopilae and L. lecanii was added in 1 ml of distilled water separately in 15-ml vortex tubes to prepare conidial suspension and covered with aluminum foil. Each vortex tube was vortexed for 1 min and then 1 ml of conidial suspension was taken and sprinkled onto a separate ¼ SDAY media plate for inoculation. After inoculation on ¼ SDAY media plates, the conidial suspension was spread on the media plate with the help of a sterile inoculating loop and then plates were incubated at 28 °C for 20-30 d.

Harvesting the conidia and preparation of different concentrations
The fungal culture was harvested by flooding 5 ml of 0.04% (vol:vol) sterile polysorbate-20 (Tween 20, Sigma-Aldrich) solution in water (0.4 ml Tween-20 in 999.6 ml H 2 O, autoclaved for 20 min at 121 °C) on culture-plate and fungal conidia were harvested from media/cultureplate with the help of loop for detaching the conidia from hyphae. The resulting suspension was poured into a 15 ml sterile conical tube which was vortexed to disrupt clumping. This conidial suspension was used as a stock solution. A volume of 100 µl was taken from the stock solution and added into 900 µl of 0.04% Tween-20 in a vortex tube. Again 100 µl of this diluted stock solution was taken added to 900 µl of 0.04% Tween-20 in a vortex tube. A volume of 10 µl of second time the diluted stock solution was micropipette, spelled out on the counting chamber of hemocytometer and covered with glass cover. Then the number of conidia was counted on the counting chamber of the hemocytometer under a microscope (hemocytometer count). The conidial concentration of the stock solution was calculated by the following formula (Iqbal et al. 2020): The final volume of stock solution required to prepare each concentration was determined by the following formula (Iqbal et al. 2020): where V Final = Final volume of stock solution needed to prepare required concentration; V Stock = Volume of stock solution; C Stock = Concentration of stock solution; C Final = Final concentration to be prepared.

Conidial viability test
Conidial viability was assessed by plating 100 µl of the second dilution of stock solution (100-fold dilution) on ¼ SDAY media. The media plates were then incubated for 24 h at 28 °C. Then, three random groups of 100 conidia were inspected. Germination of conidia was considered only when germ-tube grew longer than half of the diameter of the conidium projects from it (Parsa et al. 2013). After counting the germinating conidia, percent germination was estimated by the following formula (Iqbal et al. 2020): The whole of the above-mentioned procedural protocols was used for the commercial formulation of each tested EPF to prepare their respective eleven concentrations (1 × 10 8 , 1 × 10 7 , 1 × 10 6 , 1 × 10 5 , 1 × 10 4 and 1 × 10 3 CFU ml −1 ). In the case of each fungi > 90% conidial germination was estimated. So, the bioassay study of each EPF was conducted against B. zonata adults.

Concentration of stock solution
= Haemocytometer count × 10 4 × Dilution factor Percent germination = Germinating spores Total spores in group × 100 Page 4 of 20 Nisar et al. Egyptian Journal of Biological Pest Control (2021) 31:146 Bacterial culturing, harvesting, concentrations preparation, and viability test Same procedures and protocols, as used for fungi, were used for culturing, harvesting, concentrations preparation and viability testing of bacteria. The growth media used for bacterial culturing was broth media.

Bioassay study
A solution of 1 ml of each treatment (concentration) was pipetted onto an adult diet (honey, egg yolk, protein hydrolysate, and sugar water solution) in disposable cups having lids. The solution was then admixed with fruit fly adult diet with the help of a sterilized loop. The treatment-baited adult diet was lapped partially on the walls of the treatment unit (plastic jar) as well as placed inside the treatment unit in a disposable cup. A mixed population of newly emerged 50 males and 50 females adults of B. zonata were aspirated from culture and released into treatment unit which was maintained at 28 °C and 70% ± 5 RH for 24 h. The flies were let to feed on a treatment-baited adult diet for 24 h. After an exposure period of 24 h, the flies were transferred to the fruit fly adult rearing unit (plastic jars) having above-mentioned normal fruit fly adults that were maintained at 28 ± 2° C for 14 d. The mortality of adult flies of B. zonata was recorded after 5 d and 7 d. The dead flies were placed on respective growth media to promote the growth of fungal mycelia (mycosis) from treated flies and confirm that the death of flies is caused by a fungal infection.

Data analysis
Mortality data were transformed into percent corrected mortality by Abbot Formula (Abbott 1925): This transformed corrected mortality data were analyzed by ANOVA at 5% probability level with STA-TISTICA-10 software to compute various ANOVA parameters and means for various independent variables (treatments). Tukey's honestly significant difference (HSD) test was performed to compare the mean values of significant treatments (Danho et al. 2002).
LC 50 , LC 75 , LC 95 , LT 50 and LT 90 values and their associated significant descriptive parameters (values of degree of freedom, P value, fiducial limits, Chi-square, and slope) were computed for each bioinsecticides by applying probit analysis on mortality data using the Minitab Statistical Program (Finney 1971 Linear regression and Pearson correlation analyses were also performed at α value of 5% to establish regression between B. zonata mortality and concentrations. The coefficient of determination (R 2 ), coefficient of correlation, and linear regression equation were computed to assess the nature and strength of association between concentrations of each bioinsecticide and B. zonata adult mortality. Scatter diagrams were also plotted for each bioinsecticide to determine the trend of the fitted simple regression line of Ŷ (mortality) on X (concentration) of each bioinsecticide.

Mortality of Bactrocera zonata exposed to fungal and bacterial bioinsecticides at different post-application intervals
The mortality results depict that all tested fungal and bacterial bioinsecticides demonstrated significantly different mortality against B. zonata at two PAIs (P < 0.05) (Figs. 1, 2, 3 and 4). An exposure interval and concentration-dependent mortality in both sexes of B. zonata was explained by all tested bioinsecticides.

Regression and correlation between mortality of Bactrocera zonata and concentrations of fungal and bacterial bioinsecticides
The probability values for correlation (P < 0.05) confirm that an association existed between concentrations and mortalities of male and female flies of B. zonata for M. anisopliae, B. bassiana, L. lecanii, and B. thuringiensis (Figs. 5,6,7 and 8).
The values of 95% confidence interval (CI) for correlation coefficient (r) explain that correlation between concentrations and mortalities of male and female B. zonata varied significantly for two PAI (5 and 10 d) for M. anisopliae, B. bassiana, L. lecanii, and B. thuringiensis as none of their 95% CI value overlap with each other (Figs. 5,6,7 and 8).
Regression parameters and scatter diagrams reveal that concentrations of all test bioinsecticides had a significant linear relationship and explained significant variability in mortality of male and female B. zonata (P < 0.05) (Figs. 5, 6, 7 and 8).
All the tested entomopathogenic formulations exhibited less LC 50 values against B. zonata males and hence    proved more toxic for males than to females of B. zonata. The results also exhibited that pathogenicity of all the tested EPs increased with increasing exposure interval, being significantly higher at 7 d PAIs and lower at 5 d PAIs (Table 1-4).

LT values of fungal and bacterial bioinsecticides against Bactrocera zonata exposed to different post-application intervals
The results of lethal times ( Table 5).

Discussion
Many investigations demonstrate the significant role of entomopathogenic microbes as bioagents against tephritid fruit pests. The entomopathogenic microbes prove virulent against different stages (maggots, pupae, and adults) when exposed via different routes of exposure (Soliman et al. 2020). However, the pathogenicity of the entomopathogenic microbes on target insects and other arthropods varies significantly (Soliman et al. 2020).
In the present work, the pathogenicity of various EPF and bacteria was evaluated against B. zonata for biological control of this species. Results showed that M. anisopliae, B. bassiana, L. lecanii, and B. thuringiensis exhibited varied pathogenicity against B. zonata at different exposure periods. These results are in the light of findings of Iqbal et al. (2020) who studied that B. cucurbitae exhibited significantly varied mortality toward various EPF and EPB at various exposure intervals. Varied pathogenicity of M. anisopliae, B. bassiana, L. lecanii, and B. thuringiensis against B. zonata is also in the lights of various other studies in which toxicity of EPF (Soliman et al. 2020) and EPB (Cossentine et al. 2016) was assessed against various fruit fly species.
These results are in agreement with Soliman et al. (2020), who reported that local strains of M. anisopliae were found effective in its virulence to kill different life stages of C. capitata. Ekesi et al. (2003) also confirmed that isolates of M. anisopliae exposed to late 3 rd instar larvae of C. capitata and C. fasciventris in sand and caused a significant reduction in adult emergence and a corresponding large mortality on puparia of both species. All isolates also induced large deferred mortality in emerging adults following treatment as late third instar larvae. Wang et al. (2021) found that M. anisopliae Ma04 presented the highest virulence against B. dorsalis. Results of virulence bioassay indicated that the LC 50 values of M. anisopliae Ma04 against B. dorsalis declined from 5.2 × 10 28 to 5.2 × 10 7 conidia ml −1 over a 1-10 d period post adult emergence, and the LT 50 values decreased from 5.25 to 2.78 d with the concentrations of conidial suspension increasing from 1.0 × 10 8 to 1.0 × 10 10 conidia ml −1 . Therefore, M. anisopliae Ma04 had a greater potential for B. dorsalis control.
The results of the present study revealed that at LC 50 concentration, all the tested entomopathogenic formulations exhibited less LC 50 values against male B. zonata and hence proved more toxic for males than to females of this species. However, the studies conducted by Chergui et al. (2020) showed that B. bassiana was Page 13 of 20 Nisar et al. Egyptian Journal of Biological Pest Control (2021) 31:146      virulent to adults of C. capitata, where females were less susceptible than male flies of this species in both oral and contact bioassays, which is contrary to our results. Reason for this variation might be due to difference in strains of EPF and species of fruit fly. Varied mortality at different exposure periods in both male and female sexes of B. zonata caused by EPs in the present studies might be due to variation in virulence factors i.e., spore germination, hyphal growth, bacterialbudding, toxins etc. during the different growth period of tested entomopathogens. In this study B. zonata was least susceptible to B. thuringiensis as compared to EPFs i.e., B. bassiana, M. anisopliae, and L. lecanii. Similar results were also observed by Iqbal et al. (2020) who tested these entomopathogens against B. cucurbitae.
The results also exhibited that pathogenicity of all the tested EPs increased with increasing exposure interval, being significantly higher at 7 d post-application intervals and lower at 5 d post-application intervals. These results were also supported by Ekesi et al. (2001) who figured out that maximum mortality of B. cucurbitae was recorded at the highest concentration (10 8 spore ml −1 ) while mortality rate decreased gradually as concentration decreased. Similar results were also reported by Amala et al. (2013) who demonstrated that after 5 and 7 d of treatment, maximum mortality of B. cucurbitae was observed when treated with Paecilomyces lilacinus at the highest concentration (2.4 × 10 9 spores ml −1 ). This variation in the highest concentration reported by Amala et al. (2013) and present results has attributed the difference in EPF used by Amala et al. (2013) and in these studies.
LC 50 values of all tested microbial insecticides were found time-dependent and decreased with increased post-exposure interval. The LC 50 and LC 90 results of present experiment for male and female B. zonata are partially following those of Imoulan and Elmeziane (2014) who documented LC 50 values of 2.85 × 10 3 and 3.16 × 10 3 spores ml −1 for male and female fruit flies, respectively. These results are not consistent with those of Alberola et al. (1999) who reported that mortality rate increased with time. Aboussaid et al. (2010) reported that adults and larvae of C. capitata were susceptible to different strains of B. thuringiensis and maximum mortality was observed after 5 to 6 d after treatment. The results regarding LT 50 of present experiment are supported by Davidson and Chandler (2005) who reported a time-dependent infection and mortality of fungal and bacterial-based products against insects in the laboratory. The results of present experiments are partially consistent with those of various scientists (Mahmoud 2009) who documented strong potential of EPF against tephritid flies within 4-8 d after application at LC 50 concentrations.

Conclusions
Based on the tested pathogens when incorporated in adult diets, it can be concluded that M. anisopliae, proved highly virulence against B. zonata, followed by B. bassiana, L. lecanii, and B. thuringiensis. Hence, M. anisopliae can be recommended for incorporation in B. zonata baits or pheromone traps to develop attractand-kill technology.
Abbreviations LC: Lethal concentration; LC 50 : Lethal concentration required to kill 50% of insects; LC 75 : Lethal concentration required to kill 75% of insects; LC 95 : Lethal concentration required to kill 95% of insects; CFU ml −1 : Colony-forming unit per millimeter; EPF: Entomopathogenic fungi; EPB: Entomopathogenic bacteria; EPs: Entomopathogens; ACD: Attractant-contaminant device; IPM: Integrated pest management; CFU g −1 : Colony-forming unit per gram; psi: Pound per square inch; SDAY: Sabouraud dextrose agar yeast; V Final : Final volume of stock solution needed to prepare required concentration; V Stock : Volume of stock solution; C Stock : Concentration of stock solution; C Final : Final concentration to be prepared; LT: Lethal time; LT 50 : Lethal concentration required to kill 50% of insects; LT 75 : Lethal concentration required to kill 75% of insects; LT 95 : Lethal concentration required to kill 95% of insects; PAIs: Post-application intervals; EPs: Entomopathogens; r: Correlation coefficient; CI: Confidence interval; R 2 : Coefficient of determination; χ 2 : Chi-square value; df: Degree of freedom; P value: Probability value; FL: Fiducial limit; SE: Standard error.