Efficacy of Beauveria bassiana and Metarhizium anisopliae (Ascomycota: Hypocreales) against Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) under controlled and open-field conditions on bitter gourd

Abstract

Background

Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) is the most destructive pest of bitter gourd. Conventionally, it is controlled by chemical insecticides despite their hazardous impacts. Entomopathogenic fungi are considered as eco-friendly and safer alternative of chemical. This study aimed to evaluate the efficacy of Beauveria bassiana and Metarhizium anisopliae against B. cucurbitae on bitter gourd.

Results

The study revealed that B. bassiana and M. anisopliae were efficient against B. cucurbitae in controlled as well as field conditions. Both mode of application (i.e., contact and oral) were found to be effective; however, high efficacies of them were observed through contact application (73.43 and 59.72%), respectively. Pathogenicity of both fungi increased with increase in concentration and time intervals. Under field conditions, significantly low fruit infestations and significantly a high population reduction of B. cucurbitae were observed at 30 DAT when both fungi were applied with 10⁸ CFU/ml concentrations.

Conclusions

Beauveria bassiana and M. anisopliae had the potential to be used against B. cucurbitae; however, the former showed high efficiency comparatively.

Background

Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) is a destructive pest of all cucurbits; however, bitter gourd is preferred one (Qazzaz et al. 2015). Depending upon the crop and environmental conditions, serious economic losses are caused by this pest every year (Dhillon et al. 2005). Despite the hazardous impacts of chemical insecticides, these are still being used to manage B. cucurbitae on a large scale (Abbas et al. 2017). Due to the over-use of insecticides, insect pest resistance, toxic effects on non-targeted organisms and residual effects in soil, water and crops are some of the major issues that have been faced by human beings (Hsu and Feng 2006). Therefore, it is a need of time to explore those pest management strategies which are eco-friendly and easy to adopt.

Entomopathogenic fungi (EPF) are gaining importance as an alternative to chemical insecticides. These are considered natural enemies of a broad range of insects and exist in diverse environments around the globe (Vega et al. 2009). EPF are eco-friendly, and the insect pests' resistance against them has not been reported yet (Hadi et al. 2013).

Fruit flies are susceptible to various EPF; however, Beauveria bassiana and Metarhizium anisopliae are the most efficient ones (Ekesi et al. 2002). These are capable of targeting a wide range of arthropod species and have a good potential to be utilized in IPM programs (Daniel and Wyss 2009). B. bassiana and M. anisopliae have a high pathogenicity against adult fruit flies (Sookar et al. 2008). It is the need of time to check their virulence through different methods against B. cucurbitae (Ortu et al. 2009). In view of the devastating potential of melon fruit flies to infest cucurbits and to reduce the over usage of insecticides, the current study was planned to effectively control B. cucurbitae by the application of EPF under laboratory and field condition. The ultimate goal of the current study is to develop a sound strategy for the management of melon fruit fly through tested the effect of B. bassiana and M. anisopliae against B. cucurbitae.

Methods

Research experiments were conducted in bio-control laboratory and open field during the years of 2017 and 2018.

Rearing of Bactrocera cucurbitae

Rearing of the melon fruit fly was started with infested bitter gourds, collected from local vegetable markets. Transparent plastic cages (45 × 40 × 40 cm) fitted with muslin cloth were used for fruit fly rearing. Autoclaved sand and soil mixture (5-cm-thick layer) was placed in the cages at the bottom, and infested fruits with emerging maggots were placed on it for pupation. Cages were observed on daily basis, and as soon as the pupation completed, sand and soil mixture was sieved out to separate pupae. Collected pupae were then placed in another cage for adult emergence. Emerged adults of B. cucurbitae were identified (on the basis of morphological characteristics) and separated. B. cucurbitae adults were then fed on artificial diet made-up of banana, egg white, yeast, molasses, nutrients, vitamins and honey. After 2 weeks, males and females were released (♂/♀, 1:1) in rearing cages for mating. After 24 h, females were again separated for experimental studies. Laboratory conditions were maintained at 27 ± 2 °C, 65 ± 5% RH and photoperiod of 14:10 (L/D) hours.

Entomopathogenic culture and formulations

Already established culture of EPF (i.e., B. bassiana and M. anisopliae), which was developed from the commercial products Racer® (NCIM 1216) and Pacer® (NCIM 1311), was obtained from Department of Plant Pathology. Obtained fungal culture was streaked on freshly prepared growth media (PDA). The new culture plates were incubated under dark conditions at 28 ± 2 °C for 15 days. The highest concentration (10⁸ CFU/ml) was made by adding freshly harvested conidia in to distilled water. Hemocytometer was used to determine the required conidial concentration. Surfactant (0.05% Tween®80) was also mixed in each conidial suspension to increase the adhesiveness of fungal spores. Remaining 2 concentrations (i.e., 10⁷ and 10⁶ CFU/ml) were made by adding more distilled water in previously described concentration, and again hemocytometer was used to quantify the required conidia. All these formulations were tested against B. cucurbitae.

Laboratory experiments

Two types of bioassays (i.e., contact and oral) were performed under laboratory conditions.

Contact bioassay

Three concentrations (10⁸, 10⁷ and 10⁶ CFU/ml) of each fungi (B. bassiana and M. anisopliae) were prepared in distilled water (Ain et al. 2021). Thirty randomly selected adult flies (15 days old) were anaesthetized by placing in refrigerator at − 4 °C for 40 s (so that the handling would be easy). These 30 flies were placed in a test tube containing 4 ml conidial suspension from each concentration of both fungi. The test tubes containing flies and conidial suspension were shaken for 30 s until each fly attained some treatment. Two types of controls were maintained, i.e., a positive control with Malathion (3 ml/l of water) and a negative control with distilled water (containing 0.05% Tween®80). After the application of treatment, the tubes were placed vertically to allow the run off of excess suspension. Two hours after treatment, the flies were placed in plastic cages for further studies. Data regarding adult mortality were taken every 2 days after treatment (DAT) and consecutively up to 14 days. Factorial design (3-factors) with 5 replications was followed. Percent mortality was corrected using formula provided by Schneider-Orelli (1947).

$${\text{Corrected mortality}}\,\% = \left( {\frac{{{\text{Mt}} - {\text{Mc}}}}{{100 - {\text{Mc}}}} } \right) \times 100$$

where Mt = mortality (%) in treatment and Mc = mortality (%) in control.

Oral bioassay

Three concentrations of each fungus (2 × 10⁸, 2 × 10⁷ and 2 × 10⁶ CFU/ml) were estimated through hemocytometer. These three fungal concentrations were then mixed with artificial diet in (1:1) ratio resulting in 10⁸, 10⁷ and 10⁶ CFU/ml, respectively (Beris et al. 2013). One milliliter of this (suspension and diet mixture) was placed in small plate (3.5 cm diameter) in cage sized (27 × 25 × 25 cm). Fifteen -day-old 30 fruit flies, which were starved for 36 h prior to use in oral bioassay, were released in the cage for feeding on diet and conidial suspension mixture for 12 h and after this mixture was removed and pure diet was given as described by Beris et al. (2013). Two types of controls were maintained, i.e., a positive control with Malathion (1 ml/3 l of water) and a negative control with distilled water (containing 0.05% Tween®80). Data regarding adult mortality were taken every 2 days after treatment (DAT) and consecutively up to 14 days. After recording of each mortality observation, all dead flies were removed and transferred into sterile Petri dishes and wet filter paper was placed at the bottom of Petri plates. Then, these Petri dishes were sealed by using Parafilm, kept at 25 °C in dark and were examined daily for symptoms of mycosis. Factorial design (3-factors) with 5 replications was followed. Mortality was corrected using Schneider-Orelli (1947) formula (as mentioned above).

Field experiments

During laboratory experiments, the highest mortality causing concentration (i.e., 10⁸ CFU/ml) of each EPF was identified and selected for further trials under field conditions. Each EPF was sprayed directly (Ain et al. 2021) on the crop at rate of 100 l/acre with the help of the knap sack sprayer when bitter gourd fruiting started. Spray was applied on the whole crop, but the soil beneath the plants and bitter gourd fruits were targeted specially. Bitter gourd crop was sown in March at farmers' field, for 2 consecutive years (i.e., 2017 and 2018). ‘Faisalabad Long,’ an indigenous and approved variety, was used. Priming was done by dipping seeds into water for 24 h. Plant-to-plant and row-to-row distances about 2 and 5ft were maintained, respectively. Recommended fertilizers and agronomic practices were applied, following Nawab and Mahmood (2014). When plants started to develop into vine, bamboo poled along with supporting nets were installed. In each unit plot, 10 plants were maintained and each treatment was replicated 5 times with 2 controlled treatments, i.e., positive control and negative control. In positive control, Malathion 57% EC (330 ml/acre), while in negative control only distilled water with 0.05% Tween®80 was used. Yellow sticky traps were hanged in each treatment to note the population of fruit flies before and after treatment. Data regarding fruit infestation (%) and population reduction (%) were taken every 3 days after treatment (DAT) and consecutively up to 30 days. Randomized complete blocked design (RCBD) with 5 replications was followed. Fruit infestation was calculated by the following formula:

$$\text{Fruit infestation }(\%)=\frac{{\text{No of infested fruits}}}{{\text{Total no of fruits}}}\times 100$$

Population reduction was calculated following Henderson and Tilton (1955) equation:

$${\text{Population reduction}}\, \% = \left( {1 - \frac{{{\text{Ta}} \times {\text{Tb}}}}{{{\text{Ca}} \times {\text{Cb}}}} } \right) \times 100$$

where Ta = number of flies stuck on yellow trap after treatment, Tb = number of flies stuck on yellow trap before treatment, Ca = number of flies stuck on yellow trap in control plot after treatment, Cb = number of flies stuck on yellow trap in control plots before treatment.

Statistical analysis

In laboratory experiments, recorded mortality data in each experiment were subjected to analysis of variance (ANOVA), following Tukey–Kramer honest significant difference (HSD) test (α = 0.05). Percent mortality in laboratory experiments was corrected, following Schneider-Orelli (1947) formula, while percent population reduction was calculated in field conditions, following Henderson and Tilton (1955) formula. Analysis of the all the collected data was computed using Statistix 8.1 software (McGraw-Hill 2008), and graphical representations were created on Microsoft Excel (2010).

Results

Laboratory experiments

Contact pathogenicity of B. bassiana and M. anisopliae against B. cucurbitae

Contact pathogenicity of B. bassiana and M. anisopliae differed significantly (DF = 18, F = 3.99, P < 0.05) against B. cucurbitae under 3 different concentrations (10⁸, 10⁷ and 10⁶ CFU/ml) and 7 time intervals (Fig. 1). As shown in the figure, among both the tested fungi B. bassiana induced significantly high pathogenicity (73.42%) 14 days after treatment (DAT) at 10⁸ CFU/ml. However in case of M. anisopliae, maximum mortality (59.72%) was observed under the same time interval and concentration. Furthermore, it can also be seen that mortality induced by both of the fungi was time and concentration dependent. The minimum mortality caused by B. bassiana (5.33%) occurred 4DAT when 10⁶ CFU/ml was applied, while in case of M. anisopliae the lowest mortality induced at the same time and concentration was (2.66%).

Fig. 1

Contact pathogenicity of Beauveria bassiana and Metarhizium anisopliae against Bactrocera cucurbitae at three concentrations (10⁶, 10⁷ and 10⁸ CFU/ml)

Oral pathogenicity of B. bassiana and M. anisopliae against B. cucurbitae

The pathogenicity of entomopathogenic fungi, i.e., B. bassiana, M. anisopliae, was also evaluated by adopting oral bioassay procedure. The virulence posed by both fungi was again concentration and time dependent. It can be seen from Fig. 2 that a significant difference existed among the virulent effect imposed by each fungi in melon fruit fly (DF = 18, F = 2.44, P < 0.05). The highest mortality (51.41%) was recorded when B. bassiana was applied at the rate of 10⁸ CFU/ml 14DAT and in case of M. anisopliae at the same time and concentration the peak lethality induced was (45.14%). Furthermore, the lowest mortality induced by B. bassiana and M. anisopliae was 4.66 and 4.06% 4 DAT, respectively, when 10⁶ CFU/ml were offered to B. cucurbitae orally.

Fig. 2

Oral pathogenicity of Beauveria bassiana and Metarhizium anisopliae against Bactrocera cucurbitae at three concentrations (10⁶, 10⁷ and 10⁸ CFU/ml)

In case of B. bassiana, comparative study of contact and oral bioassay against melon fruit fly showed a significant difference (DF = 18, F = 19.32, P < 0.05). In contact application, when 10⁸ CFU/ml were applied, B. bassiana was more pathogenic (73.42% mortality) 14 DAT, while in case of the oral bioassay, mortality was (51.41%) as shown in Fig. 3. On other hand, when M. anisopliae was used as an oral bioassay and contact bioassay differed significantly (DF = 18, F = 18.89, P < 0.05) against the melon fruit fly (Fig. 4). Contact bioassay of M. anisopliae gave significantly a high mortality (59.72%) 14 DAT under the application 10⁸ CFU/ml suspension whereas it was significant low (45.14%) in case of oral bioassay.

Fig. 3

Contact and oral pathogenicity of Beauveria bassiana against Bactrocera. cucurbitae at three concentrations (10⁶, 10⁷ and 10⁸ CFU/ml)

Fig. 4

Contact and oral pathogenicity of Metarhizium anisopliae against Bactrocera cucurbitae at three concentrations (10⁶, 10⁷ and 10⁸ CFU/ml)

Field experiments

Impacts of B. bassiana and M. anisopliae on fruit infestation by B. cucurbitae

Effect of the EPF on percent fruit infestation caused by B. cucurbitae is presented in Table 1. Fruit infestation against the application of B. bassiana and M. anisopliae differed significantly (DF = 3, F = 25.27, P < 0.05) 3DAT. Results revealed that percent fruit infestation due to melon fruit fly 3DAT was significantly high (43.25, 49.71%) when plants were exposed to B. bassiana and M. anisopliae, respectively, while infestation was relatively low in the control. However, as shown in Table 1 that at this time interval Malathion effectively reduced the fruit infestation (31.67%). Furthermore, the time interval increased the effectiveness of both EPF started to increase, while Malathion lost its efficacy as time lapsed. 12DAT results showed that fruit infestation in Malathion treated plant started to increase and reached 24.04%; however, in case of B. bassiana M. anisopliae the fruits’ infestation was (26.67, 36.31%) significantly lower than 3DAT. After 30 days of treatment, application EPF treated fruits showed significantly difference (DF = 3, F = 495.11, P < 0.05), high reduction in percent fruit infestation as, 7.11% fruit infestation recorded under B. bassiana, while M. anisopliae was able to reduce infestation about 15.14% and for Malathion it was 51.09%.

Table 1 Impact of entomopathogenic fungi (Beauveria bassiana and Metarhizium anisopliae) on fruit infestation (%) by Bactrocera cucurbitae under field conditions (pooled data 2017 and 2018)

Impacts of B. bassiana and M. anisopliae on population reduction of B. cucurbitae

Effect of both EPF at 10⁸ CFU/ml concentration was also examined on percent corrected population reduction at different time intervals under field conditions (Table 2). Three days after treatments application, a population reduction was significantly different (DF = 2, F = 48.40, P < 0.05) as 39.1% was observed in Malathion treated plot, while in case of EPF population reduction was significantly high (26.47%), when B. bassiana was sprayed, whereas it was significantly low (21.08%) in case of M. anisopliae. Population reduction against the application of Malathion remained higher than EPF up to 12DAT. Anyhow, 18 DAT population reduction (%) in EPF treated plots increased by Malathion treated plot (DF = 2, F = 96.82, P < 0.05). It was significantly high (56.68%) in case of B. bassiana, and significantly low (41.34 and 30.62%) under treatment of M. anisopliae and Malathion, respectively. Maximum population reduction (68.59%) was recorded 30 DAT (DF = 2, P < 0.05), when B. bassiana was applied, while at the same time interval against the application of M. anisopliae and Malathion, it was (48.60 and 13.13%), respectively.

Table 2 Impact of entomopathogenic fungi (Beauveria bassiana and Metarhizium anisopliae) on corrected population reduction (%) of Bactrocera cucurbitae under field conditions (pooled data 2017 and 2018)

Discussion

EPF have a wide range of hosts, and their mode of action may be different (Sujeetha and Sahayaraj 2014). Unlike the chemical insecticides, EPF are safe for human being (Zimmermann 2007). Due to their unique and pests controlling features, they can be used as one of the most potential bio-control agent and can also be used efficiently in organic farming (Litwin et al. 2020).

This study was conducted to evaluate the efficacy of B. bassiana and M. anisopliae against B. cucurbitae under controlled and field conditions. Results showed that both fungi had the potential to be used against B. cucurbitae. The study conducted by Gul et al. (2015) and Qazzaz et al. (2015) also strengthens the above statement as they also found that these fungi could be used against fruit flies efficiently. However, in the present study, it was examined that B. bassiana had more virulent effect than M. anisopliae against B. cucurbitae. Finding regarding the more pathogenicity of B. cucurbitae in this study is also supported by the research work of (Mehdi and Al-Fadili 2020) who reported that under laboratory conditions isolates of B. bassiana were more virulent than the isolated of M. anisopliae.

During the present studies, contact and oral mode of applications were also tested and it was found that the contact mode was more suitable comparatively. Similar findings were also reported by Konstantopoulou and Mazomenos (2005) that the isolates of B. bassiana were more virulent against adult fruit flies than the isolates of other fungi. Obtained findings were also supported by Gul et al. (2015), who evaluated the pathogenicity of EPF against fruit flies (B. zonata) through different application methods and they found that these fungi were more virulence in contact method than the oral method. Mainly these fungi caused pathogenicity by adhering to the hosts and then penetrate in their bodies (Litwin et al. 2020). Conventionally, it was considered that EPF are pathogenic only in contact application methods, but during recent studies it has been found that other application methods can be used (Mannino et al. 2019). Anyhow the reason of low pathogenicity of fungi in oral bioassay may be that these fungi mostly kill their host arthropods by penetrating their cuticle as described by Mora et al. (2018) that in most cases EPF spores adhere with the cuticle of an insect, then germination start and ultimately by penetrating in the cuticle, spores disseminate in the body of insects.

Mode of application, dose rate, environmental conditions and isolates of fungi are important factors for causing pathogenicity in host pests (Rizvi et al. 2009). In this study, a maximum pathogenicity was attained at the highest concentration 1 × 10⁸. Similar findings were revealed by Mar and Lumyong (2012). Three concentrations (10⁶, 10⁷ and 10⁸ CFU/ml) were used in this study, and their efficacy was checked after 2 days up to 2 weeks in laboratory experiment and the most promising results were given by the highest concentration. Trinh et al. (2020) also used the same concentration against aphid, and his results were supportive to the obtained findings that the efficacy of EPF has increased by increasing the concentration. Time interval is also an important for EPF, as the maximum mortality was observed at 14 DAT. This result also corroborates with the results of Nazir et al. (2019) who reported that after applying the EPF, with the passage of time mortality of insects was increased.

Conclusions

Both EPF (i.e., B. bassiana and M. anisopliae) had the potential to be used against B. cucurbitae. However, B. bassiana showed a high efficacy than M. anisopliae. Moreover, contact application method was found to be more effective than the oral application.