Efficacy of single and combined applications of entomopathogenic fungi and nematodes against the pupae of colorado potato beetle ( Leptinotarsa decemlineata [Say]), (Coleoptera: Chrysomelidae)

Background: The lethal effects of different entomopathogenic fungi (EPF) and nematodes (EPN) on the pupae of the Colorado Potato Beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) were investigated in a two-step study when used separately or together. In the first step, Nostalgist ( Beauveria bassiana strain Bb-1, Agrobest Co.), Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae), Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae) (Bioglobal Co.), 2 Turkish isolates of Beauveria bassiana BIM-001 and BY2 were applied separately to the pupal stage of pest in the soil. In the experiments, 3 different concentrations (150, 200, and 250 million infective juveniles (IJs)/100 l water) of EPN and a single concentration (1x10 8 spores/ml for and maximum recommended field dose 1 × 10 8 CFU/ml) of EPF were used. In the second step, BIM-001 and BY2 isolates of B. bassiana were combined with both EPN species; S. feltiae + B . bassiana BIM-001, S. feltiae + B . bassiana BY2, H. bacteriophora + B . bassiana BIM-001, H. bacteriophora + B . bassiana BY2 were applied to pupae. At this stage, 150 million IJs/100 l water which was the maximum recommended field concentration for EPN and 1 × 10 8 spores/ml for local EPF isolates were used. Results: According to the study results, pupae deaths were significantly higher in Nostalgist (74%) than those of B . bassiana BIM-001, B . bassiana BY2, S . feltiae , and H . bacteriophora ( P <0.05). While both EPN caused higher mortality than B . bassiana BY2, B . bassiana BIM-001 had the highest mortality rates. The combination of S. feltiae + B. bassiana BIM-001 at the highest mortality rate (80%) was found statistically different and significant from other EPF + EPN treatments on pupae. Moreover, there were non-statistical differences between the other EPF and EPN combinations. Conclusion: It was found that the combined use of EPF and EPN was more effective on the pupae of L . decemlineata than a single application. According to the results of the study, it may be promising to use EPN and EPF together for the control of the Colorado Potato Beetle pupae.


Background
The Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) is an important pest that causes significant economic losses in plants belonging to Solanaceae (Balaško et al. 2020). Colorado potato beetle spends its pre-pupal stage on the soil surface and Page 2 of 6 Uzun Yiğit et al. Egyptian Journal of Biological Pest Control (2022) 32:81 then enters 4-8 cm below the soil and starts its the pupal stage after approximately the 2nd day (Kekillioğlu and Yılmaz, 2018). It has been known that the entomopathogenic nematodes (EPNs) exist as natural microorganisms in a complex environment such as the soil. The capability of these EPNs is to actively seek and infect their hosts and also survive in the soil for a long time under optimal conditions, and also their absence of negative effects on non-target organisms provides significant advantages in pest control. In recent years, the importance of EPNs has been standing out due to the aforementioned features in the biological control of harmful species found under the soil surface, especially in certain life periods (Özdemir and Bayram 2017). The 2 genera, Steinernema and Heterorhabditis spp., are actively used commercially in pest control (Özdemir and Bayram 2017). EPFs have a large share in the world biopesticide market today (Bugti et al. 2020). There are an estimated 750 species of EPF in about 90 genera (Abd El-Ghany, 2015). Numerous EPF species belonging to Beauveria, Metarhizium, Lecanicillium, and Isaria are widely used commercially due to their easier mass production (Rai et al. 2014). In 2 decades, insecticides containing neonicotinoids have been widely used for controlling of the Colorado potato beetle around the world. The resistance to neonicotinoids and the negative effects of these compounds on other organisms in the agro-ecosystem reduced the chance of sustainable management of the pest (Oberemok et al. 2018). The use of entomopathogenic organisms in pest control has become inevitable for the many reasons presented above.
As known, the lethal effects of EPF and EPN on L. decemlineata are higher, especially in pre-adult stages (Ropek and Kołodziejczyk 2018). Use of the entomopathogenic organisms on the pupal stage of this pest has not been studied. Therefore, the lethal effects of EPF and EPN on pupae of the Colorado potato beetle, separately or combined, were investigated in the present study.

Experiment 1
The EPF and EPN were applied separately to the pupae of L. decemlineata, and their lethal effects were determined. For this purpose, individuals in the 4th larval stage of the Colorado potato beetle were transferred to plastic boxes (500 ml, 8 × 12 × 8.5 cm), which had a standard soil substrate (Zemek et al. 2017) moistened with sterile distilled water (12.5 ml). These boxes were divided into 5 separate groups with sterile cardboard. Only one 4th instar larva was left in each compartment (total 5 larvae for one box), and it was observed whether these individuals penetrated into the soil after 24 hrs. It is known that the 4th instar larvae of the Colorado potato beetle usually enter the pupal stage approximately 48 hrs after they throw themselves into the soil (Kekillioğlu and Yılmaz 2018). Therefore, fungi and nematode applications were carried out 3 days after these larvae were transferred to the plastic boxes at 5 ml (pre-pupa) per compartment (Table 1). Each compartment was moistened with 5 ml of sterile distilled water before 24 h from the applications.

Experiment 2
In experiment 2, lethal effects were detected in the pupal stage, as a result of the combined use of BIM-001 and BY2 local isolates of B. bassiana and EPN. Experiment 2 was also designed the same as in experiment 1, and the EPF and EPN applications were made (Table 2).
Both experiments were carried out with 10 replications according to the randomized plot design. All experiments were carried out in the climatized chambers at 25 ± 1 °C, 75 ± 5% humidity, and 16: 8 photoperiod conditions. The pupal development process can vary between 5 and 8 days under the stated conditions (Kekillioğlu and Yılmaz 2018), and accordingly, the dead-alive counts were done on the 7th day after the applications, and the mortality rates were calculated (Choo et al. 2002). To figure out whether the pupae were dead poked with a fine brush. Those wiggled ones were taken into different boxes and the adult emergence was awaited. Pupae that did not wiggle were kept in Petri dishes with a moist filter paper for re-isolation of EPF. The re-isolation was performed on all pupae that died in EPF applications, according to Meng et al. (2017). Those containing EPF spores were recorded as dead due to EPF. In addition, each cadaver in EPN treatments was taken into the White Trap to reisolate of EPNs. Nematode emergence was observed after 15 days in the White Traps (Kaya and Stock 1997). The mean number of re-isolated juveniles per dead pupa was recorded for each application dose of EPN. Since Nostalgist is a commercial product based on EPF (Beauveria bassiana strain Bb-1), it contains additional ingredients. Therefore, only local isolates were used in combined studies that included EPF and EPN to eliminate interference that may be caused by the ingredients of the commercial product. Besides, adult emergence was recorded after applications and the number of adult individuals emerged from the pupa was recorded in both experiments. Adult emergences are given as percentage (%) for each application.

Statistical analysis
The assumption of normality of obtained data was checked with the test of Kolmogorov-Smirnov. In addition, square root transformation was applied to the numbers of the re-isolated nematodes in the 3 rd juvenile stage that was nonparametric. Then, these data were subjected to one-way ANOVA, followed by Tukey's HSD (honestly significant difference) test (P< 0.05). All data were analyzed using SPSS 20.0 software.

Experiment 1
Mortality rates in all EPF and EPN applications were found higher and significant than the control (P< 0.05), and the mortality percentage on Nostalgist (74%) reached a higher mortality rate than all applications. Additionally, non-significant difference was observed between the recommended highest concentrations (200 and 250 million IJs/100 l water) of H. bacteriophora (40 and 44%, respectively) and B. bassiana BIM-001 (50%). It was found that there was non-statistical difference between the recommended concentration of H. bacteriophora (150 million IJs/100 I water) and the all application concentrations of S. feltiae (Table 3). While both EPN caused higher mortality than B. bassiana BY2, B. bassiana BIM-001 had the highest mortality rates.
On the other hand, it was found that the mean number of 3 rd juveniles that were re-isolated from dead pupae in experiment 1 was increased linearly with the application concentrations of both nematode species. While the reisolated juvenile numbers in 250 million concentration were higher and more significant than other doses of H. bacteriophora, this was not the same for the S. feltiae. In H. bacteriophora, the number of juveniles obtained at 200 million concentration (256.70 ± 10.73) was significantly higher than 150 million concentration (192.87 ± 6.09) (P< 0.05). In addition, the difference between the number of 3rd juveniles obtained at 200 million (234.55 ± 4.80) and 250 million concentrations (247.34 ± 8.80) of S. feltiae was significant (P< 0.05) than 150 million concentration, but was not differed from each other (P˃ 0.05) (Table 4).
When EPF and EPN were applied separately, adult emergence was determined for Nostalgist, B. bassiana

Experiment 2
Lethal effects on pupae of the Colorado potato beetle were determined when EPF and EPN were applied simultaneously in the experiment 2. In the present experiment, the mortality rate was significantly higher (80%) than other EPF-EPN combinations (combination of S. feltiae and B. bassiana BIM-001). Moreover, there were nonstatistical differences between the other EPF and EPN combinations. According to the present results, simultaneous applications of EPF and EPN caused a higher mortality on the pest than the applications of these organisms alone. In the combination of S. feltiae and B. bassiana BIM-001, the mortality was 80% on the pupae, and these rates were 50 and 22%, respectively, when EPF and EPN were applied separately. Beauveria bassiana BY2 was found to cause low mortality rates when applied simultaneously with EPN relative to other isolate (P< 0.05) (

Discussion
Many studies have been conducted to determine the effects of different strains of B. bassiana on the adult, larval, pupal stages, and hatching rates of the Colorado potato beetle (Atanasova and Vasilev 2020). Among these researches, the results of the applications made against the pupa stage under the soil are remarkable. It has been reported that the pupal stage of the pest is sensitive to soil applications and these applications can reduce the next adults' emergence up to 74% (Klinger 2003). Cantwell et al. (1986) recorded the adult emergence 38.8-28-62.6-43.6%, respectively, when the rates of 7.5 and 75 g/m 2 of B. bassiana were applied to the pupal    (Wright et al. 1987). In the present study, adult emergence was determined as 70-78% and 56-74% for S. feltiae and H. bacteriophora, respectively. Additionally, EPNs were also re-isolated from dead pupae and it was proved that used EPNs caused death in the pupal stage. Although EPNs cause low mortality rates, the presence of EPNs in pupal cadavers suggests the probability of being the source of inoculum. It is also known that EPF and EPN generally showed synergistic interactions in pest management and can be used together successfully (Shahid et al. 2012). When the combination of EPF and EPN was applied to the pre-adult stages, this caused lethal effects on different harmful species. In the same way, Ibrahim et al. (2019) recorded 100% mortality in the larvae of Galleria mellonella L. (Lepidoptera: Pyralidae) 2 days after the application of the combination of H. zealandica (7 IJ/larva) and B. bassiana (3.1 × 10 2 conidia/ml). The highest mortality rate in pre-adult periods was observed when H. bacteriophora and B. bassiana (WG-11) were combined. In the present study, H. bacteriophora + B. bassiana BIM-001 that were applied simultaneously caused 76% mortality on pupae of Colorado potato beetle, when applied separately and led to 50 and 26%.
In studies on the Colorado potato beetle, Hussein et al. (2016) found that the mortality rate on the last larval stage of L. decemlineata was 98% on the 7th day in Petri dishes when the combination of S. feltiae with Isaria fumosorosea CCM 8367 were applied. Zemek et al. (2017) also determined that the mortality rate was 44 and 45%, respectively, when I. fumosorosea CCM 8367 and S. feltiae were applied separately to the last stage larvae of L. decemlineata in the potting soil. When I. fumosorosea CCM 8367 and S. feltiae were applied together, the mortality rate was 84%. In the present study, when S. feltiae and B. bassiana BIM-001 were applied simultaneously to the pupae of the Colorado potato beetle, the mortality rate was found as 80%. Additionally, the mortality rates were 50 and 22% in B. bassiana BIM-001 and S. feltiae applications, respectively. Heterorhabditis bacteriophora and B. bassiana BIM-001 that applied simultaneously caused 76% mortality in the pupa of the Colorado potato beetle. When these EPF and EPN were applied separately, the rates were 50 and 26%. Obtained results of this study showed that the BY2 isolate of B. bassiana caused lower mortality rates than BIM-001 isolated from Colorado potato beetle, both singly and when combined with nematodes. Kamionek et al. (1974) found that when EPFs and EPNs were applied simultaneously against insects of the order Coleoptera or Lepidoptera in Petri dishes, the lethal infection time was shorter than when the fungus was applied alone. This may be since EPNs release bacteria shortly after entering the insect body. Ansari et al. (2008) reported 100% larval mortality when EPNs were applied 7 to 14 days after M. anisopliae application. Dlamini et al. (2020) determined a synergistic effect and the highest mortality in the application of Steinernema yirgalemense 1 or 2 weeks after the application with different isolates of B. bassiana and M. anisopliae on Phlyctinus callosus (Schönherr) (Coleoptera: Curculionidae) larvae. These studies showed that more successful results were obtained when EPN was applied after EPF application. However, in the present study, it was determined that the mortality rate increased significantly even in simultaneous applications. The mortality rate was lower than H. bacteriophora when S. feltiae was separately applied to the pupae of the Colorado Potato Beetle. When B. bassiana isolates (BIM-001, BY2) were combined with S. feltiae, death of pupae reached higher rates than the combination of the EPF isolates with H. bacteriophora. Additionally, it was determined that mortality rates of pupae increased in treatments where B. bassiana isolates were applied simultaneously with S. feltiae. Moreover, BY2 isolate of B. bassiana also had a low mortality rate when used alone, while a high mortality rate was observed when combined with S. feltiae. Consequently, it can be concluded that there was a synergistic relationship between S. feltiae and B. bassiana isolates (BIM-001, BY2).

Conclusions
The combination of the EPF and EPN was more effective than single applications against the pre-adult stages of different agricultural pests. Similarly, in this study, they were tested together, and up to 80% of death obtained under laboratory conditions. It was determined that the use of EPF and EPN combination led to higher mortality rates in the pupal stage of the Colorado potato beetle. The use of entomopathogens is a valuable and an alternative tool in pest control. The results suggest that the combined