## 2 Materials and Methods
### Entomopathogenic Fungi
#### 2.1.1 Isolation and Identification of Entomopathogenic Fungi
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Fungal Species** & **Isolate** & **Insect Bait** & **Collection Site** &
\begin{tabular}{c} **Percent** \\ **Identity** \\ \end{tabular} & **Blast ID Number** \\ \hline _Beauveria bassiana_ & BB K & _Tribolium confusum_ & Moires—Crete, Greece & 98.5 & H9 WJ96N9013 \\ _Beauveria bassiana_ & 2 & _Sitophilus granarius_ & Elo sAyias—Patras, W. Greece & 98.0 & H9 PGGF6301R \\ _Beauveria bassiana_ & 147 & _Sitophilus granarius_ & Dasylylio—Patras, W. Greece & 98.0 & H9 PPYWZ0013 \\ _Beauveria bassiana_ & 65 & _Sitophilus granarius_ & Dasylio—Patras, W. Greece & 98.2 & H9 PX08B6016 \\ _Beauveria bassiana_ & 149 & _Sitophilus granarius_ & Dasylio—Patras, W. Greece & 99.8 & H9 PZCCA601R \\ _Beauveria bassiana_ & BB A & _Galeria mellonella_ & Athens—Central & 20140422 CS6P8\_H02\_2016-655 & 98.9 & 20140422 CS6P8\_H02\_2016-04-27 \\ _Beauveria bassiana_ & BB AC & _Tribolium confusum_ & Glafkos—Patras, W. Greece & 98.9 & 20170105 CS6P2\_F02\_2017-01-11 \\ _Beauveria bassiana_ & BB Kar & _Galeria mellonella_ & Hleia—Greece & 98.6 & 20180422 CS7P4\_C08\_2021-03-12 \\ _Beauveria varroae_ & 23 & _Sitophilus granarius_ & Elo sAyias—Patras, W. Greece & 99.6 & 1 EZT99P1 GenBank \\ _Cordyceps blackwelliae_ & 64 & _Sitophilus granarius_ & Dasylio—Patras, W. Greece & 96.8 & 8E2M17 WB01R \\ _Cordyceps fumosorosea_ & IS & _Galeria melonella_ & Stefanos—Central & 96.9 & 20170105 CS10P1\_G01\_2017-01-11 \\ _Metathizium anisopliae_ & MET A & _Rhyzopertha domitica_ & Zavlani—Patras, W. Greece & 99.1 & 20140422 CS3P2\_C02\_2016-04-27 \\ _Metathizium anisopliae_ & MET B & _Pledia interpunctella_ & Glafkos—Patras, W. Greece & 99.4 & 20140422 CS3P3\_C02\_2016-04-27 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Isolates of various entomopathogenic fungal species (EPF) that were tested in the present study. All collected fungal isolates were lab cultured and stored at 25 \({}^{\circ}\)C on SDA plates. The isolates, after being subcultured several times (at least three) on plates with SDA to ensure purity and monosporic cultures, were morphologically identified using a ZEISS Primo Star microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) at \(\times\)400 magnification. At the end of the growth period, transparent tape was used to stick the sporulation structure down from the edge, followed by staining with phenol cotton blue reagent (Sigma-Aldrich 61335, St. Louis, MI, USA). The selected isolates were stored in the microorganism's repository in the Department of Agriculture, University of Ioannina. The conidia were scraped from the surface of the dead insects using a sterile loop and were transferred to SDA plates [33]. Molecular identification of tested EPF was performed following the method by [PERSON] and [PERSON] [34]. A fragment of the ITS spacer region was expanded by applying universal primer sets ITS4 (5\({}^{\prime}\)-TCCTCGCCTTATTGATATGC-3\({}^{\prime}\)) and ITS5 (5\({}^{\prime}\)-GGAAGTAAAAAGTCGTAAC AAGG-3\({}^{\prime}\)). PCR reactions (25 \(\upmu\)L) were performed using Taq 2\(\times\) Master Mix (M0270) and included working concentrations of 1\(\times\) Master Mix, 0.5 \(\upmu\)M of each primer, and 50 ng of template gDNA. The PCR protocol for amplification of the ITS regions included 33 cycles, at 95 \({}^{\circ}\)C for 30 \(s\), 55 \({}^{\circ}\)C for 40 s, and 68 \({}^{\circ}\)C for 1 min, followed by a final elongation at 68 \({}^{\circ}\)C for 5 min. PCR products were kept at 4 \({}^{\circ}\)C. The quantity and quality of PCR products were determined by gel electrophoresis using 1% agarose gel, which was stained with SYBR Safe DNA Gel Stain (Invitrogen, Waltham, MA, USA) and visualized under UV light (BIO-RAD, Molecular Imager Gel Doc XR System, Hercules, CA, USA). The purification and the sequencing of the amplified products took place at Eurofins Genomics (Ebersberg, Germany).