Using predatory species and entomopathogenic fungi as alternatives to chemical pesticides in green bean field

Background Green bean, Phaseolus vulgaris L. (Fam.: Leguminosae) is a widely consumed grain legume prized for its edible seeds and pods. It is susceptible to infestations with various pests as insects and mites throughout the growing season. In this study, the efficacy of the predatory species, Chrysoperla carnea (Stephens), Phytoseiulus persimilis Athias-Henriot and the entomopathogenic fungus (EPF) Metarhizium anisopliae (Hypocreales: Clavicipitaceae), as well as conventional pesticides, Mospilan and Vertimec, were evaluated against the most important pests, mainly the whitefly, Bemisia tabaci (Genn.) and the two-spotted spider mite, Tetranychus urticae Koch, infesting the green beans cultivated at two locations Giza and El-Menoufia Governorates in Egypt. Results The findings demonstrate that treatments using C. carnea and M. anisopliae effectively reduced the whitefly population, while pesticide treatments were comparatively less effective. In the Giza plots, at the end of the experiments, the use of both M. anisopliae and C. carnea showed high reductions in whitefly population (85.57 and 84.87%), respectively, while in El-Menoufia, C. carnea (97.74%) was the most effective treatment followed by M. anisopliae (90.32%). Pesticide treatment in this case yielded a reduction rate of (22.76 and 59.67%) in Giza and El-Menoufia plots, respectively. However, for spider mite control, P. persimilis proved to be the most effective treatment in Giza and El-Menoufia plots, reducing the spider mite population to 98.44 and 96.14%, respectively. Metarhizium anisopliae treatment also displayed moderate effectiveness, with reduction rates of 75.62 and 75.37% in Giza and El-Menoufia plots, respectively. In comparison, pesticide treatment showed low effectiveness, with reduction rates of only 23.92 and 53.16% in the two locations, respectively. Conclusion Applications of the predator, C. carnea and the EPF, M. anisopliae were highly effective in reducing the population of whitefly, while the predator mite P. persimilis proved to be the most effective for controlling the spider mites. Overall, the study suggests that biocontrol agents, such as the predators and the EPF, can be considered as alternatives to synthetic chemical pesticides for controlling pests infesting green beans.

fried, or ground into flour.Additionally, crop residues such as dried pods and stems (straw) and processing byproducts like discarded pods and pod extremities can be used as fodder for livestock (Wortmann 2006).Overall, green beans are a versatile and nutritious crop with a range of uses for both human consumption and animal feed.However, the use of pesticides in bean cultivation can pose risks to human health, animal health and the environment.Therefore, it is essential to ensure that bean crops are grown and processed, using sustainable and environmentally friendly practices, such as integrated pest management and organic farming methods.
Green bean is usually infested with a variety of pests throughout its growing season, including the two-spotted mite, whitefly, aphids, leaf miners, leafhoppers and thrips.These insect pests and mites can cause an extensive damage to the bean pods, affecting both their quantity and quality.As a result, the infestation often leads to reduced yields, which can have a significant impact on the overall productivity of the crop (El-lakwah et al. 2010).
Use of synthetic pesticides has been the primary method for managing pests, but they had led to challenges in controlling the whitefly, Bemisia tabaci Genn.(Hemiptera: Aleyrodidae).This main pest poses a significant threat to the vegetable industry due to its resistance to many pesticides and its role as a vector for numerous plant viruses (Gerling 1990).Unfortunately, the extensive use of synthetic insecticides has resulted in B. tabaci developing resistance to a wide range of insecticides, making it difficult to manage (Patra and Hath 2022).Similarly, the two-spotted spider mite, Tetranychus urticae Koch, has also developed resistance to various insecticides and acaricides, including organophosphates (OPs), carbamates, synthetic pyrethroids and even some recently developed compounds (Xu et al. 2018).
Mixed infestations of insects and mites are commonly occurring, and the traditional method of controlling them involves the use of large amounts of insecticides and acaricides.It is essential to adopt effective nonchemical control measures of both pests.One promising alternative is the use of biocontrol agents, such as predators, parasitoids and microbial entomopathogenics.These agents can be effective in reducing the pest population and minimizing the harmful side effects of pesticides (Batta 2003).
Tetranychus urticae and B. tabaci are significant pests that can cause substantial damage to green bean crops if not managed effectively.Due to avoid potential harm to human health and the environment from conventional pesticide treatments, this study aimed to assess the efficacy of EPF, M. anisopliae and two predatory species, P. persimilis and C. carnea, for controlling major pests in green bean fields, and to compare their performance with conventional pesticide treatments.Therefore, this study's findings will be of great value to farmers and agricultural practitioners seeking to implement safe sustainable and effective pest management strategies.

Sources of bioagents
Larvae of the predatory C. carnea were obtained from the Center of Bio-Organic Agricultural Services (CBAS) in Aswan, Egypt.In this study, eggs of the angoumois grain moth, Sitotroga cerealella (Oliver), were used, as prey, for C. carnea larvae.The cocoons of C. carnea were collected daily and transferred to a plastic jar.The adults were placed in a 2-l transparent plastic jar covered with black muslin cloth fixed with rubber bands, for egg laying.The predatory adults were fed on artificial diet containing yeast, honey, pollen and water (1:1:1:1) and pasted on horizontal plastic strips placed in an adult rearing cage.Wet-soaked cotton was placed inside the jar to provide moisture.The rearing was carried out under the controlled conditions of 25 ± 2 °C, 60% R.H. and 16:8 photoperiods (L/D).The rearing was continued throughout the experimental period to ensure that second instar larvae of C. carnea were readily available for release in the field.
The formulation of M. anisopliae (Bio-Magic ® ) which was used against T. urticae and B. tabaci was manufactured by Gaara Establishment for Import and Export, Egypt.It was available as a Powder package containing spores and mycelial fragments (1 × 10 9 CFU's/gm).

Pesticides
The pesticides used were the synthetic neonicotinoid Mospilan 25% SP and the acaricide-insecticide Vertimec 1.8% EC.

Experimental design
The study was conducted at two farms located in the Giza and El-Menoufia governorates of Egypt.The farms were planted with green bean seedlings of the Almonte and Paulista varieties on November 24th and December 25th, 2021, in the Giza and El-Menoufia governorates, respectively.The daily weather conditions, including minimum and maximum temperatures and relative humidity, were provided by the Central Laboratory for Agricultural Climate (CLAC) located in Dokki, Egypt.A randomized complete block design with five replications was used for each treatment in the experiment.Each experimental unit (plot) was 10 m 2 to accommodate 30 plants spaced at 0.5 m × 0.5 m.A two-meter-wide walkway was used to separate the plots to prevent any treatments from drifting.
To control B. tabaci, five-second larval instars of C. carnea per plant were released in the field, while for T. urticae, ten adults of P. persimilis were released per plant.The M. anisopliae formulation was applied as a foliar spray (6 gm/liter) to control both pests.The pesticides used were synthetic neonicotinoid Mospilan 25% SP at 25gm/100l and the acaricide-insecticide Vertimec 1.8% EC at 75cm/150l.
Metarhizium anisopliae and pesticides were applied using a separate Pomsan sprayer 10L for each treatment (model: K-93), with a nozzle size of 1 mm.Once the pests' infestations were detected, the treatments were applied 3 times at 14-day intervals to control the infestations.

Assessment of the effectiveness of treatments
To evaluate the effectiveness of all treatments, data were collected by inspecting 25 plants chosen at random from each treatment (five plants from each replicate) weekly started from seedling stage until harvest.Population density of pests was determined by counting the number of pests on three leaves that were randomly selected from the top, middle and lower levels of each plant.Square inch lens with 10X magnification was used for inspection.The total number of B. tabaci nymphs and pupae and the mobile stages of T. urticae was recorded.

Statistical analysis
Collected data were coded and entered into the statistical package SPSS version 22. Quantitative variables were described in terms of mean and standard deviation.To test significant differences among treatments, Analysis of variance (ANOVA) was conducted, followed by posthoc Tukey's (HSD) with a significance level of p < 0.05.This was done to reject the null hypothesis and confirm the presence of significant variance among treatments' groups.
The percentage of reduction in pests' populations was calculated per plant leaf as a mean from each plant compared to the control.These percentages were estimated according to Henderson and Tilton (1955): no. in co.before treatment X no. in T after treatment no. in Co. after treatment X no. in T before treatment * 100 where no.= pest population, Co. = control, and T = treated.This formula allowed for a standardized method of calculating the reduction in pest populations and enabled comparison among different treatments.The data were statistically analyzed by correlation analysis between weather parameters and pest populations.

Results
The study recorded weather data during field applications, including maximum temperature ranged from 37.53 to 11.76 °C, minimum temperatures of 19.9-4.1 °C and a relative humidity ranged from 82.81 to 53.83% in the Giza Governorate from November 2021 until March 2022, while in the El-Menoufia Governorate the maximum temperature ranged from 42.72 to 15.14 °C), minimum temperatures of 19.78-8 °C and relative humidity of 81.62-57.92%from December 2021 until April 2022.These data indicated that the conditions seem to be similar in both locations.
The data underwent statistical analysis involving correlation analysis between weather parameters and pest populations.In the Giza plots, a correlation was observed between maximum temperature (r = 0.0175 and 0.215), minimum temperature (r = 0.0023 and 0.219) and relative humidity (r = 0.654 and 0.392) with whitefly and mite populations, respectively.
In El-Menoufia plots, a correlation was identified between maximum temperature (r = 0.34 and 0.29) and minimum temperature (r = 0.284 and 0.229) with whitefly and mite populations, respectively.Furthermore, a negative correlation was observed between relative humidity (r = − 0.058 and − 0.115) and whitefly and mite populations, respectively.
In Giza plots, the whitefly was occurred first in small numbers during the 4th week after planting, and its population gradually increased over time.Prior to the beginning of treatments, the whitefly population varied between 6.48 ± 2.1, 5.96 ± 1.4, 5.96 ± 2.4 and 6.1 ± 1.9 nymphs and pupae/leaf in control, pesticides, M. anisopliae and C. carnea treated plots, respectively.Three applications of pesticides (Mospilan 25% SP), M. anisopliae and C. carnea were applied on the 5th, 7th and 9th weeks after planting.On the 12th week after planting, the whitefly population reached (50.8 ± 1.6, 36.99 ± 2.3, 14.1 ± 1.7 and 9.44 ± 1.2 nymphs and pupae/leaf ) in the control, pesticides, M. anisopliae and C. carnea plots, respectively.After the third application, the whitefly population in M. anisopliae and C. carnea plots had reduced to 9 ± 4.1 and 8.95 ± 3.1 nymphs and pupae/leaf, respectively.However, in pesticides and control plots, they increased to 48.2 ± 15.74 and 62.4 ± 20.7 nymphs and pupae/leaf, respectively (Fig. 1).
Significant differences were recorded among different treatments (F = 8.31, df = (3), p < 0.05).Non-significant differences were found between control and pesticides plots and M. anisopliae and C. carnea plots at p < 0.05.However, there was a significant difference between pesticides and M. anisopliae plots and pesticides and C. carnea plots at p < 0.05.
The pesticides treatment had the lowest reduction in the whitefly population after the third application, reaching (22.76%).The whitefly population reduction was the highest in M. anisopliae and C. carnea plots, with their proportions being close to each other, reaching (85.57and 84.87%), respectively (Fig. 2).
In El-Menoufia plots, also, the whitefly was first observed in small numbers during the 4th week after planting and varied between 0.5 ± 0.2, 0.56 ± 0.4, 0.7 ± 0.3 and 0.62 ± 0.3 nymphs and pupae/leaf in control, pesticides, M. anisopliae and C. carnea plots, respectively (Fig. 3).A total of three applications of Mospilan 25% SP, M. anisopliae and C. carnea in the 5th, 7th and 9th weeks after planting were applied.
Data analysis revealed significant differences between control and M. anisopliae and control and P. persimilis at p < 0.05.Non-significant difference was found between control and pesticides and pesticides and M. anisopliae at p < 0.05.
The treatment with P. persimilis resulted in the highest reduction in spider mite population (96.14%) followed by M. anisopliae treatment (75.37%) and then the pesticides treatment (53.16%) (Fig. 8).The results indicated that the biocontrol agent P. persimilis was the most promising treatment for spider mite control, as it resulted in the highest reduction in mite populations.

Discussion
The findings of this study suggest that using the predator C. carnea and the EPF M. anisopliae can be effective tools for reducing whitefly populations in green bean fields, providing a promising alternative to synthetic chemical pesticides.
Several studies have reported the effectiveness of M. anisopliae in controlling whitefly populations in the field.Abdel-Raheem and Al-Keridis (2017) observed that Beauveria bassiana, M. anisopliae and Lecanicillium lecani isolates were promising fungal biocontrol agent for the whitefly control in the field.Similarly, Flores et al. (2012) reported that M. anisopliae was significantly more effective against eggs, first, second and third nymphal instars and pupae of the whitefly B. tabaci.Mixed applications of M. anisopliae and B. bassiana were found to maximize the likelihood of control of all stages of B. tabaci.Additionally, Alghamdi et al. (2018) found successfully suppressed of the aphid, Aphis gossypii Glov and the whitefly B. tabaci populations on sweet pepper and squash plants in open fields, through the releases of C. carnea.Also, Zaki et al. (1999) observed that different releasing rates of C. carnea induced highly significant reduction of A. gossypii and B. tabaci on various vegetable crops.These studies provided further support for the use of C. carnea and M. anisopliae as effective biocontrol agents for managing whitefly populations in agricultural fields.
The results of the present study also demonstrated the potential effectiveness of P. persimilis and M. anisopliae as treatments for managing spider mite populations in green bean fields.Notably, P. persimilis was found to be more effective in reducing the population of T. urticae than M. anisopliae These findings are consistent with previous researches that have shown the efficacy of these natural enemies in controlling mite infestations in various crops.Abdel-Aziz (2016) found that releasing six individuals of P. persimilis per plant can be an effective approach for controlling populations of T. urticae.Similarly, Tiftikçi et al. (2020) (Vapcomic) in reducing the population of T. urticae on kidney beans and sugar snap peas.It was showed that P. persimilis was the most effective treatment followed by Vapcomic, B. bassiana and Typhlodromips swirskii.Bugeme et al. (2015) reported that M. anisopliae isolate ICIPE78 could be an alternative to acaricides for managing T. urticae on common bean in the screen house and field experiments.Shaef Ullah and Lim (2017) found that a single application of B. bassiana was effective in controlling T. urticae and reduced the egg and adult populations of initially, but mite populations rebounded again after few days.Phytoseiulus persimilis at the highest release rate eliminated the mite population completely, while the lowest release rate failed to control the spider mites.The combined application of B. bassiana and low release rate of P. persimilis also successfully controlled T. urticae population, with the lowest corrected leaf damage (1.5%).Batta (2003) reported that M. anisopliae had a great potential for controlling whitefly B. tabaci and the spider mite T. cinnabarinus, particularly when applied in an invert emulsion formulation.However, further studies are necessary to clarify the effect of the fungus on the mite predator to ensure safe application of these treatments together.
Based on the results of this study, both the combination of the mite predator P. persimilis and the EPF M. anisopliae can be used for controlling the whitefly and mites, but the necessary studies have to be done to clarify the effect of the fungus on the mite predator so that they can be applied together safely.

Conclusion
The findings of this study demonstrated the potential of using the predator C. carnea, the predatory mite P. persimilis and the EPF M. anisopliae for controlling some of the main pests in green bean fields.These natural enemies offered a promising alternative to synthetic chemical pesticides for managing pests' infestations in the crop.Further research is needed to determine the optimal application rates and conditions for these treatments, as well as their compatibility when used together.

Fig. 1
Fig.1Impact of the treatments on weekly average number of whitefly per leaf during the green bean season in Giza plots

Fig. 2 Fig. 3
Fig. 2 Percentage of reduction in whitefly population after applied pesticides, Metarhizium anisopliae and Chrysoperla carnea during the green bean season in Giza plots

Fig. 4 Fig. 5
Fig. 4 Percentage of reduction in whitefly population after applied pesticides, Metarhizium anisopliae and Chrysoperla carnea during the green bean season in El-Menoufia plots

Fig. 6 Fig. 7
Fig. 6 Percentage of reduction in spider mite population after applied pesticides, Metarhizium anisopliae and Phytoseiulus persimilis during the green bean season in Giza plots

Fig. 8
Fig. 8 Percentage of reduction in spider mite population after applied pesticides, Metarhizium anisopliae and Phytoseiulus persimilis during the green bean season in El-Menoufia plots