Virulence effect of Metarhizium anisopliae (Met.) and Beauveria bassiana (Bals.) fungi against the peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae)

The peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae), is a key pest of fruits in Egypt. Insect-pathogenic fungi are one of the biocontrol agents that increasingly substitute the traditional pesticides to overcome pesticide risks. Therefore, the present study aims to assess the fungal virulence of Beauveria bassiana (Balsamo) and Metarhizium anisopliae (Metchnikoff) against B. zonata pupae. Also, extended pathogenicity effect of these fungi on adult flies was studied. The results showed that M. anisopliae fungus had more pathogenicity to B. zonata pupae on the 2nd, 3rd, and 5th days post-treatment than B. bassiana. Pathogenicity fungal effects of treated larvae extended to the surviving adults. Fungal concentration and post-exposure interval reversely impacted the pupae by 63.88 and 63.59% mortality in the case of M. anisopliae and B. bassiana, respectively. The lethal concentration of treated fly by M. anisopliae (LC50 = 9.5 × 106 conidia/ml and LC90 = 9.9 × 107 conidia/ml) was lower than that of B. bassiana (LC50 = 5.1 × 107 conidia/ml and LC90 = 1.9 × 109 conidia/ml). Median lethal time (LT50) value was fungal species-dependent, and concentration. Metarhizium anisopliae was more virulent than B. bassiana; the lowest LT50 value was 9.48 days by M. anisopliae and 13.33 days by B. bassiana, depending on the fungal tested concentration of 2.3 × 106 conidia/ml. The tested entomopathogenic fungi could be considered promising biocontrol agents against B. zonata and could be used for fly suppression through soil application in IPM programs.


Background
Tephritid fruit flies are a group of economic insect pests that attack fruits and certain vegetables worldwide, causing direct and indirect economic injury (Lysandrou 2009). The genus Bactrocera has a wide range of hosts and invasive capabilities of certain species that are considered a severe threat to horticultural crops (Clarke et al. 2005). The peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae), is one of the significant economic pests present in West Asia, North Africa, and Southern Europe (Eppo 2010). It attacks over 40 host plants (Delrio and Cocco 2012) between fruits and vegetables. Furthermore, according to the European and Mediterranean Plant Protection Organization, it is classified as a quarantine A1 pest (Eppo 2010).
Extensive applications of conventional pesticides against fruit flies generate major environmental problems (Magaña et al. 2007), such as resistance development of field strain of the fly. Several efforts have been carried out to limit the usage of toxic pesticides to manage this pest, such as employing abiotic factors, soil water content, soil compaction, and biopesticides alternative to traditional insecticides (El-Gendy and AbdAllah 2020; El-Gendy et al. 2021).
Over the last decades, entomopathogenic fungi (EPF) have played a significant role in the natural biological control of many insects (Burges 1981). Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Clavicipitaceae) and Metarhizium anisopliae (Metchnikoff ) Sorokin (Hypocreales: Clavicipitaceae) have an essential role in insect pests' control. B. bassiana is the only insect pathogen that can directly penetrate into its hosts through their cuticles and provides good prospects for managing B. zonata puparia in the soil (soil inocula) (Garrido-Jurado et al. 2011). B. bassiana is a safe biocontrol organism on non-target insects and mammals, including people (Zimmermann 2007). B. zonata adults and pupae were found susceptible to the EPF, such as M. anisopliae and B. bassiana (Rashad et al. 2015). Extended pathogenicity of B. bassiana and M. anisopliae to surviving adult flies treated in the larval stage is limited. Therefore, the present trial aimed to assess the pathogenicity of the EPF, B. bassiana and M. anisopliae, against B. zonata pupae and their extended potential effect on the adult flies under laboratory conditions.

Target pest
The peach fruit fly, B. zonata, was obtained from the Eradiation of Peach Fruit Fly Laboratory at Damanhour, El-Beheira Governorate, Egypt, which was reared according to El-Gendy (2002).

Fungi used
Two commercial fungus formulations of B. bassiana and M. anisopliae (WP 2.5%, 2.3 × 10 8 conidia/gm) were tested against B. zonata in the present study. B. bassiana strain was originally isolated from the red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), in Ismailia governorate, while M. anisopliae was isolated from the cotton whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), in Sharkiah governorate, and both were identified in the fungal center of Faculty of Science, Assiut University, Egypt (Ibrahim 2006). Fungal formulations were gained from Bioinsecticides Production Unit, Plant Protection Research Institute, Agricultural Research Center, Giza, Egypt.

Virulence assay of EPF, M. anisopliae and B. bassiana, against B. zonata
Laboratory assay of the EPF, M. anisopliae and B. bassiana, was carried out against the 3rd larval instar of B. zonata (full-grown larvae) under laboratory conditions of 25 ± 2º Ϲ, and 70-80% RH. The experiments were applied in plastic cups (250 cm 3 ) closed at the top with a cloth net (1 mm mesh). The cups contained 75 gm of sterilized sand, autoclaved at 105ºC for 24 h. The fungi were dissolved in tap water. Each fungus was assayed in 5 concentrations, one with a recommended concentration (6 × 10 5 conidia/ml), and the others were above and below the recommended concentration (2.0 × 10 5 , 4.0 × 10 5 , 1.2 × 10 6 , and 2.3 × 10 6 conidia/ml), in addition to the control treatment (water only).
One hundred larvae were used per each soil treatment (concentration) in 5 replicates, 20 larvae each. Ten milliliters of fungal suspension per replication was dropped on the soil by the plastic pipette 3 ml. Larvae, full-grown, were transferred to the cups and freely allowed to pupate in the soil. After 2, 3, and 5 days of treatment, the treatments were inspected, and the dead pupae were recorded. On the 7th day, the remained pupae were transferred to Petri dishes until flies' emergence. The newly emerged flies were transferred into plastic cups (250 cm 3 ) covered with a cloth net, supplied with a source of food and water, as previously described. Treatments were checked daily, and the dead flies were removed to Petri dishes containing wet filter papers to confirm that death was caused by the fungal infection.

Determination of the lethal time for 50% (LT 50 ) and 90% (LT 90 ) of the individuals
Time-mortality (median time values associated with 50 and 90% mortality of flies, LT 50 and LT 90 ) of B. zonata exposed to the above-mentioned fungal concentrations of M. anisopliae and B. bassiana was determined.

Statistical analysis
Mortality percentages of B. zonata were subjected to two-way analysis of variance (ANOVA), using CoStat Software (2008) Version 6.4. Means were compared by the Tukey-Kramer test at the 5% probability level. LC 50 and LC 90 and LT 50 and LT 90 values were determined by probit analysis (Finney 1952), using the Ldp-Line program (Bakr 2007).

Virulence of B. bassiana and M. anisopliae against B. zonata pupae and adult stages The cumulative mortality of B. zonata pupae of various time intervals
As mentioned above, the EPF, B. bassiana and M. anisopliae, were assayed on the full-grown larvae of B. zonata in 5 fungal concentrations: 2.0 × 10 5 , 4.0 × 10 5 , 6.0 × 10 5 , 1.2 × 10 6 and 2.3 × 10 6 conidia/ml, using soil treatment application. It was indicated that no fly's mortality was recorded during the experimental period, either during the pupal or adult stages in the control treatment. As shown in Fig. 1a, b, pupal mortality was dependent on fungal species and concentration, as well as post-treatment time. No fungal effect was detected on B. zonata pupae after two days of treatment with low concentrations of M. anisopliae and B. bassiana (2.0 × 10 5 and 4.0 × 10 5 conidia/ml, respectively). Pupal mortality began at the concentration of 6 × 10 5 conidia/ml, with 5 and 2.5% mortality, respectively, by M. anisopliae and B. bassiana. Mortality of pupae increased gradually, with increasing the fungal concentration of 1.2 × 10 6 and 2.3 × 10 6 conidia/ml, to achieve 7.5 and 12.5% mortality by M. anisopliae and 5 and 7.5% by B. bassiana, respectively. These mortalities varied significantly among fungal concentrations based on fungus species (M. anisopliae: F = 7.68, P = 0.005 and B. bassiana: F = 2.88, P = 0.04).
Pupal mortality was affected by the fungal concentration and post-exposure time by 63.88% with M. anisopliae and 63.59% with B. bassiana of the total factors affected pupal mortality, according to determination coefficient (r 2 ).

Cumulative mortality of adult fly of B. zonata at various time intervals
The effect of EPF continued to the adult fly stage. M. anisopliae was more effective than B. bassiana based on mortality rates that appeared in the adult stage (Fig. 2a, b). On the adult's 5th day, fly mortality rate was 50% (F = 19.11, P = 0.0000) after treating M. anisopliae and 27.5% (F = 14.73, P = 0.0000) after treating with B. bassiana. As both fly age and fungal concentration increased, the fly mortality rate increased. Fungal treatments of M. anisopliae (2.0 × 10 5 -2.3 × 10 6 conidia/ ml) achieved significant mortality effects of 20 to 57.5% (F = 21.15, P = 0.0000), 35-65% (F = 26.93, P = 0.000),

B. bassiana
5.1 × 10 7 1.9 × 10 9 0.69 6.0 0.71 0.82 ± 0.50 Page 5 of 7 El-Gendy et al. Egyptian Journal of Biological Pest Control (2022) 32:43 Discussion Entomopathogenic fungi (EPF) have received growing interest as microbial agents to control insect pests during the last decades. The pathogenicity of EPF, B. bassiana and M. anisopliae, was assayed on the full-grown larvae of B. zonata in 5 concentrations: 2.0 × 10 5 , 4.0 × 10 5 , 6.0 × 10 5 , 1.2 × 10 6 , and 2.3 × 10 6 conidia/ml, using soil treatment application. The findings revealed that mortality rates of B. zonata pupae were significantly related to the fungal concentration and fungal species. These findings were confirmed by B. zonata and B. cucurbitae (Coquillett) adult flies, i.e., as the mortality rates of the pests varied based on fungus species and its isolated race (Sookar et al. 2008). The variation in mortality rates of the tested fly stages between both fungal species revealed that B. zonata pupae were much tolerant to the fungal insecticide than in the adult fly stage. They were also more tolerant to B. bassiana than M. anisopliae. In a similar vein, Mahmoud (2009)  On the other hand, fungal mortality rates for B. zonata pupae increased as the fungal concentration and exposure-time interval increased, with higher mortality rates at M. anisopliae than B. bassiana. In parallel with the results of Rashad et al. (2015), the pupal mortality rates of B. zonata increased significantly by increasing the concentration of M. anisopliae and B. bassiana fungi at the same time of exposure. Similar results were recorded on the Medfly, Ceratitis capitata (Wiedemann), as the M. anisopliae fungus was superior pathogenically to the pupae than B. bassiana at the same fungal concentration along with different tested times (Soliman et al. 2020).
Fungal mortality effects of M. anisopliae and B. bassiana on B. zonata pupae appeared on the 2nd day of treatment with little effects to be increased with time post-treatment increased to 27.5% by treatment of M. anisopliae and 17.5% by B. bassiana on the 5th day of pupal age. Obtained results showed differences in pupal mortality rates during the different test periods and between fungal species. However, mortality rates may be different according to the fruit fly species and race. In parallel, Attia (2018) reported 37.75-55.02% mortality in the pupal stage of B. zonata treated with 1-4gm/l of B. bassiana. On C. capitata pupae, M. anisopliae and B. bassiana resulted in about 94% mortality (Ekesi et al. 2002). However, no fungal effect of B. bassiana was detected on Anastrepha ludens (Loew) pupae (Aluja 1993).
Pathogenicity effect of M. anisopliae and B. bassiana extended to the adult fly stage; M. anisopliae fungus was more fatality than B. bassiana. In parallel with the present results, in earlier studies on B. zonata fly, M. anisopliae fungus was superior in the fatality rates on the fly than B. bassiana fungus (Ibrahim et al. 2014). However, Hussein et al. (2018) reported that B. bassiana was more effective than M. anisopliae on the immature stages and adult flies of B. zonata. Furthermore, Rashad et al. (2015) found a significant influence of the soil application of B. Bassiana fungus on mortality rates in adult flies B. zonata, compared to M. anisopliae fungus.
Mortality rates of the adult fly of B. zonata were significantly related to the concentrations of fungi and according to adult stage and fungus species. Similar to the present results, the correlation coefficient values of microbial-insecticide concentrations of B. bassiana and