# Endophytic Colonization of Pepper (_Capsicum annum_) Controls Aphids (_Myzus persicae_ Sulzer)
## 2 Materials and Methods  
### Fungi, Insect and Plant Material  
Local fungal strains of _B. bassiana_ (strain name: _GBBSTTS_), _I. fumosorsea_ (strain name: _RHZ4 RAS_) and _M. anisopliae_ (strain name: _Z3 RAS_) from the Achaia region, Greece, were used. Soil samples were collected from random regions in the prefecture of Achaia. We chose to retrieve fungi from their natural habitat, as opposed to using laboratory strains, as the former are expected to have higher endophytic ability. To collect the soil samples, the surface litter was removed, and the soil was dug to a depth of 10 cm with a soil core borer. The samples, which weighed 300 g each, were placed in plastic bags and stored at 4 \({}^{\circ}\)C until they were transferred to the laboratory for further processing. As the fungi contained in the soil samples would later be retrieved using 3 rd-instar _Galleria mellonella_ L. larvae (Lepidoptera: Pyralidae) as baits, the collected soil was placed on rough cardboard on the laboratory stalls for 24 h to reduce its humidity. This was necessary as in conditions of excess humidity, the entomopathogenic nematodes present in the soil would attack and kill the larvae of the insect bait before the entomopathogenic fungi [15]. Once the soil was dry, it was sieved and placed in petri dishes (10 g of soil from each soil sample) where 10 _G. mellonella_ larvae were also inserted. This was repeated three times (every 10 days), meaning that by the end of the experiment, 30 _G. mellonella_ individuals were tested in total per soil sample. The _G. mellonella_ individuals were kindly provided by the Benaki Phytopathological Institute, Athens. Once in the petri dishes, the _G. mellonella_ individuals were left in special dark chambers at a temperature of \(25\pm 1\)\({}^{\circ}\)C for 14 days. Those insects which exhibited fungal infection were then isolated in different petri dishes on a layer of the nutrient Sabouraud dextrose agar or SDA (Sabouraud Dextrose Agar, OXOID LTD). Alternatively, fungal conidia which had been removed from the infected insects, were cultivated on the same material. Conidia were required to prepare fungal solutions which would later be sprayed on the pepper plants. The petri dishes were kept at a temperature of \(25\pm 1\)\({}^{\circ}\)C in the dark, to achieve the incubation and development of the fungi. When a fungus developed, it was isolated once more in order to avoid infestation and to obtain a pure fungal colony. This was achieved by subculturing of the fungal hyphae. Isolates were maintained in petri dishes on a nutrient SDA material at \(25\pm 1\)\({}^{\circ}\)C and were renewed every month for the duration of the experiment. Fifteen days after the pure colony was last isolated, fresh conidia were collected from the SDA cultures and transferred to a 500 mL glass beaker with 100 mL sterile distilled water containing 0.05% Tergitol NP9. The conidial suspension was filtered across 10-12 layers of sterile cloth to remove hyphal debris and prepared for the spraying of pepper plants by mixing the solution with a magnetic stirrer for 5 min [16]. Suspensions were adjusted at \(1\times 10^{8}\) conidia per milliliter according to [PERSON] et al. [17], using a Neubauer hemocytometer (TIEFE 0,100 mm 1/400 9 mm). The viability of conidia, which was over 95% in all cases, was determined after 24 h by repeating a germination test for each stock suspension as described by [PERSON] and [PERSON] [15], in order to ensure the constancy of the viability assessments. The colony of the aphids (collected from greenhouse pepper plants) was kept on pepper plants in a growth chamber (PHC Europe/Sanyo/Panasonic Biomedical MLR-352-PE), in controlled environmental conditions (\(27\pm 1\)\({}^{\circ}\)C, \(65\pm 5\%\) r.h., 16:8 light-night) throughout their biological cycle. We used the pepper plant variety Stauros Peloponnesus (_Capsicum annum_ L. Solanales: Solanaceae).