Identification of some native entomopathogenic fungal species and their pathogenicity against Apple Blossom Beetle, Tropinota (Epicometis) hirta (Poda, 1761) (Coleoptera: Cetoniidae) adults

Entomopathogenic fungi (EPFs) have more important role in biological control of the important insect pests. In the present study 15 EPF isolates, selected from the EPF culture collection, were identified using molecular methods and their effectiveness against adults of Apple Blossom Beetle (ABB) [Tropinota (= Epicometis) hirta (Poda, 1761) (Coleoptera: Cetoniidae)] was investigated. To determine virulence of isolates against ABB adults, primarily a single-concentration trial was conducted at 1 × 107 conidia ml−1. Furthermore, concentration-mortality trials were conducted with some isolates that were determined to have a high effect, employing concentrations of 1 × 106, 1 × 108, and 1 × 109 conidia ml−1. For isolates tested in concentration-mortality trials, LT30, LT50 and LT90 values at a concentration of 1 × 108 conidia ml−1 were calculated using probit analysis. Molecular diagnostic tests revealed that the 13 isolates were Beauveria bassiana, 1 isolate was Clonostachys rosea, and 1 isolate was Purpureocillium lilacinum. According to the single- concentration trial data, some isolates with high efficacy were selected (GN16, GN22-1, GN29-1, HP3-1 and HP33-1) and concentration-mortality trials were established with these isolates using 1 × 106, 1 × 108 and 1 × 109 conidia ml−1 concentrations. According to the results of the concentration-mortality trials, all isolates used in 1 × 108 and 1 × 109 conidia ml−1 concentrations started to show a significant effect from the 11th day, and at the end of the 17th day, the effect in all concentrations was over 90% (except 1 × 106 conidia ml−1 concentration of GN22-1). According to the data obtained, all isolates used in concentration-mortality trials were effective against ABB adults. With these isolates, more detailed studies should be conducted under orchard conditions.


Background
There are many factors that limit fruit production. Among them, diseases, pests and weeds have an important place. When these factors are not managed properly, significant economic losses occur. Apple Blossom Beetle (ABB) Tropinota (= Epicometis) hirta (Poda, 1761) (Coleoptera: Cetoniidae), is one of the fruit pests (Yaşar and Dahham 2019). It is an important species that spreads to across the Palaearctic region and to almost all regions of Turkey (Aydın 2011). In the spring, ABB adults cause significant damage by feeding on different parts of the flowers of the plants, especially fruit trees, Page 2 of 8 Uçar et al. Egyptian Journal of Biological Pest Control (2022) 32:95 strawberries and roses (Aslan and Aslan 2015). They live on leaves during non-flowering periods (Atanasova et al. 2017). Kutinkova and Andreev (2004) reported that the ABB is one of the most important pests of cherry trees in Bulgaria that causes up to 70% damage by feeding on the flowers of young cherry trees. Chemical control against ABB adults is not recommended due to the presence of pollinators and beneficial insects in the orchards during the flowering period of fruit trees. As a result, eco-friendly control strategies are needed to limit the damage caused by ABB while also protecting the non-target species (Yaşar and Dahham 2019). Biological control with entomopathogenic microorganisms is one of the most effective non-chemical pest control approaches (Mantzoukas and Eliopoulos 2020). EPFs are common in terrestrial environments and serve a crucial role in insect population control (Khan et al. 2012). Approximately 750 EPF species have been found to infect a wide variety of insects and mites (Inglis et al. 2001). Strains of EPFs are concentrated in Hypocreales (various genera), Onygenales (Ascosphaera genus), Entomophthorales, and Neozygitales (Entomophthoromycota) (Mora et al. 2017). These biocontrol agents are the most important of all microbial biocontrol agents because they can be mass-produced easily, quickly, and cheaply, and they can be utilized with the same technological methods as traditional contact insecticides. Many commercial mycoinsecticides are being developed from EPFs however most of these products are based in only four genera of entomopathogenic fungi: Beauveria, Metarhizium, Lecanicillium and Isaria (Vega et al. 2009).
In this study, EPF isolates selected from the culture collection in Mycology Laboratory (Tokat Gaziosmanpaşa University, Faculty of Agriculture, Plant Protection Department) were identified using morphological and molecular methods and their virulence against adults of ABB was investigated under laboratory conditions.

Fungal isolates
Fifteen EPF isolates were chosen for the current investigation from the fungal culture collection of the Tokat Gaziosmanpasa University, Faculty of Agriculture, Department of Plant Protection in Tokat, Turkey. These isolates were initially isolated from Hypera postica (Gyllenhal 1813) (Coleoptera, Curculionidae) and Gonioctena fornicata (Brüggemann 1873) (Coleoptera, Chrysomelidae) adults gathered from alfalfa fields in Tokat Province, Turkey (Baysal 2017).

Morphological characterization
Fifteen isolates were preliminarily identified using morphological characteristics such as appearance of the fungal infection, colony shape, spore size and spores shape (Humber 1997).

Molecular characterization DNA isolation and PCR studies
DNA isolation of the EPF isolates was performed according to the "Genomic DNA Purification Kit Thermo Fisher Scientific" method. ITS5 (5′-GGA AGT AAA AGT CGT AAC AAGG-3′) as the forward primer and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) as reverse primers were used for ITS PCR-amplification (White et al. 1990). For PCR, 1 µl template DNA, 2.5 µl 10X Taq Buffer, 0.2 µl dNTP (25 mM), 0.5 µl ITS5 primer (100 pmol), 0.5 µl ITS4 primer (100 pmol), 1.5 µl MgCl2 (25 mM), 0.25 µl Taq polymerase enzyme (Thermo) and 1 µl of Dimethylsulfoxside (DMSO) were mixed in PCR tubes and made up to 25 µl with distilled water and placed in the termocycler (Techne Prime). Thirty-five cycles were performed in thermocycler following the denaturation at 95 °C for 5 min, 95 °C for 1 min, 55 °C for 55 s, 72 °C for 2 min, with a final extension at 72 °C for 10 min (Sevim et al. 2014). The PCR products obtained as a result of PCR were subjected to electrophoresis at 100 V for 1 h in an agarose gel containing 10 mg/ml ethidium bromide, prepared at a rate of 1.2% and visualized under the imaging device (Sevim et al. 2014).

Phylogenetic analysis
For phylogenetic studies, the products obtained at the end of PCR were sent to Atlas Biotechnology (Ankara-Turkey) for the sequencing. The data obtained at the end of the sequencing were analysed with the MEGAX (Kumar et al. 2018) computer program. The data were then compared with reference isolates registered in the National Center for Biotechnology Information (NCBI) gene bank, and molecular identification of the isolates was made.

Inoculum preparation from entomopathogenic fungal isolates
Fungal isolates were cultured on Potato Dextrose Agar (PDA) in Petri dishes. A small part of mycelia was taken from each fungal isolates and inoculated in of PDA and incubated at 25 ± 2 °C with a 16/8 (L/D) photoperiod. At the end of the 21-day incubation period, spores were harvested with 10 ml of sterilized water containing 0.02% Tween 80. The conidial suspensions were filtered through 3 layers of sterile muslin to remove particles and then conidial concentration adjusted to 1 × 10 6 , 1 × 10 7 , 1 × 10 8 and 1 × 10 9 conidia ml −1 (Saruhan et al. 2017).

Bioassays
To determine virulence of isolates against ABB adults, primarily a single-concentration trial was conducted at 1 × 10 7 conidia ml −1 . To test the effect of each of the isolates, ABB adults were immersed into 1 × 10 7 conidia ml −1 suspension of each isolate for 10 s. and transferred into a Petri dish (5 adults per plate) containing fresh apple leaf and flower. The control group was treated with sterile water including 0.02% Tween 80. Mortality of the adults was assessed on the 3rd, 5th, 7th, 9th, 13th, 15th and 17th days of incubation periods. Furthermore, concentration-mortality trials were conducted similar to single-concentration trials. These tests were performed with some isolates that were determined to have a high effect, employing concentrations of 1 × 10 6 , 1 × 10 8 , and 1 × 10 9 conidia ml −1 . A completely randomized block design with 5 replications was used for the experiments and replicated 2 times.

Statistical analysis
Data was analysed with analysis of variance (ANOVA) and the means were compared to Tukey's multiple comparison test by using the MINITAB Release 16 packet program. To determine the statistical interactions between treatments, MINITAB Release 16 was used with a general linear model. For isolates tested in concentration-mortality trials, LT 30 , LT 50 and LT 90 values at a concentration of 1 × 10 8 conidia ml −1 were calculated using probit analysis.

Molecular identification of the fungi
The 15 isolates used in the study were identified by molecular methods, and 13 isolates were Beauveria bassiana, 1 isolate was Clonostachys rosae and 1 isolate was Purpureocillium lilacinum (Table 1; Fig. 1).
A complete ANOVA analysis was performed to determine the effects of isolate, concentration, and time for ABB. Isolate*concentration, isolate*time, Fig. 1 Phylogenetic tree constructed based on ITS region of the obtained isolates and references using the Neighbor-Joining method. The isolates obtained in this study are shown in blue. Obtained Beauveria bassiana, Purpureocillium lilacinum and Clonostachys rosea isolates are indicated using a black-filled circle, black-filled square, black-filled triangle respectively     et al. Egyptian Journal of Biological Pest Control (2022) 32:95 concentration*time, isolate* concentration*time interactions were statistically significant (Table 8).
When the values were examined, it was seen that GN29-1 isolate caused the fastest effect in all LT rates among the isolates applied against ABB (LT 30 : 5.897, LT 50 : 6.817, LT 90 : 9.715). HP33-1 isolate is the second isolate that causes the fastest death in respect of LT 30 (6.722) and LT 50 (8.060) rates. HP3-1 isolate was found to be the second isolate causing the fastest mortality in terms of LT 90 (12.333) rate. The slowest effect in all LT values was determined in GN22-1 isolate (LT 30 : 7.651, LT 50 : 9.416, LT 90 : 15.638) (Table 9).

Discussion
The results showed that all B. bassiana EPF isolates used in concentration-mortality trials were significantly effective at 1 × 10 8 and 1 × 10 9 conidia ml −1 concentrations from the 11th day. This effect increased above 80% in all isolates by the end of the 13th day. 17 days after postinoculation, it was observed that the effect exceeded 90%, including the lowest concentration (except 1 × 10 6 conidia ml −1 concentration of GN22-1). Only one investigation had reported the susceptibility of ABB to EPFs. In that study, similar to the present, it was reported that the effectiveness of B. bassiana and Fusarium sp. against