Two fungal isolates; BbC1 and M150, available in the laboratory, at the Department of Crop Sciences, Agricultural Entomology, Georg-August University, Germany, were used in the study. Spores were harvested from 2-week old PDA cultures, by adding two drops of Tween 20 to the culture, and then gently scraping the conidia and placing into a 200-ml flask containing 100 ml sterile water. The conidia were harvested by vortexing, followed by filtration of the spore suspension, using a Buchner-type funnel through a 2-layered filter paper (90 mm) to remove mycelial fragments and aggregated conidia. The spore densities were adjusted to 108/ml.
Second larval instar of S. frugiperda were obtained from Bayer Crop Science. The larvae were reared on tomato seedlings for several generations at 25 °C, RH 70%, and at a photoperiod of 8 h. The adults were fed on 30% honey solution and newly hatched larvae were transferred to 200 ml disposable plastic cups and reared on artificial medium (Gupta et al. 2005) until reaching the second larval instar.
Two tomato varieties, Harzfeuer and Matina, originated in the Middle East were sown in pots containing a mixture of peat- moss and sand (3:1). Plants used in experiments were at a similar size and were randomly assigned to each treatment. In three separate repeated experiments, 36 seedlings of each tomato variety were left to grow under greenhouse conditions until the 3rd leaf growth stage. After shaking off excess soil, the roots were rinsed, using running tap water. The roots of 12 seedlings of each tomato variety were dipped in 25 ml of either BbC1 or M150 spore suspensions (108 /ml) for 2 min. Another 12 plants from each variety were dipped in 25 ml of sterile distilled water and used as controls. These plants were then transplanted into 11-cm plastic pots containing the same soil and sand mixture and left to grow under greenhouse conditions for 7 days and used for bioassays.
Determination of endophytic colonization
Re-isolation of fungi on selective media
In order to assess the potential of BbC1 and M150 to endophytically colonize tomato plants, 4 leaf samples/plant were taken both from the inoculated and control plants every 7 days, cut into 4–6 pieces; their surfaces were sterilized, using 70% alcohol for 3 min, 5% sodium hypochlorite for 2 min, and then 70% alcohol for 2 min; and finally, they were rinsed in sterile distilled water 3 times, and then dried out on sterile filter paper. The fragments were then placed on modified selective medium consisting of 39 g PDA, 100 mg NaCl, 50 mg chloramphenicol, and 0.5 mg crystal violet per 1 l. The plates were incubated at 25 °C for 2 weeks. Three replicates of randomly selected leaf fragments of each treatment were assessed for fungal colonization and repeated with each experimental set-up.
Molecular analysis of fungal colonization in tomato plants
DNA extraction and real time PCR were used to assess plants BbC1 and M150 growth inside plant tissues.
The upper 3 newly emerged unfolded leaves leaf samples were picked from inoculated and control plants 10 days after inoculation, and their surfaces were sterilized before freeze-drying and then milled into powder. A 50-mg sample of plant material, in a 2-ml Eppendorf tube, was centrifuged and used for DNA extraction. Total DNA was extracted from the plant materials, using the standard cetyltrimethylammonium bromide (CTAB) extraction protocol (Doyle and Doyle 1987) after which the DNA samples were stored overnight in the fridge. Thereafter, the quality of the extracted DNA was checked, using the electrophoresis by adding 5 μl of extracted DNA and 2 μl loading dye and loading them into 0.8% agarose gel, which was run for 120 min at 60 V then stained with Ethidium bromide and visualized using UV light.
Fungal DNA quantification using the real-time PCR
Specific primers designed for both BbC1 and M150 were used for optimizing the qPCR protocol. Thereafter, standard curves for both fungi were established with a minimum quantity of 200 femtogram/μl. Diluted (1/10 and 1/20) and un-diluted DNA samples were run alongside with the standards, following the optimized qPCR protocol for quantifying the fungal DNA presence in the tested samples.
Introduction of larvae onto inoculated plants
Second-instar larvae (4 day old) of S. frugiperda (one per plant) were introduced onto 12 plants, each of inoculated (that tested positive for fungal DNA) and non-inoculated plants after 14 days of inoculation. One larva was secured per plant, using a clip-cage, and that was moved onto a different leaf on the same plant every 24 h. The plants were arranged in a completely randomized design in a controlled environmental chamber (25 ± 1 °C, RH 80% and L to D 8:16 h). This set-up was repeated 3 times.
Determination of the effect of endophytes on Spodoptera frugiperda larvae
The initial weights of Spodoptera larvae were taken before introduction to the plants. Larvae were weighed every 7 days for 21 days. All larvae were removed from both control plants after 21 days, when the larvae of control plants developed into pupae. Larvae feeding on BbC1- and M150-treated tomato plants never pupated. Dead larvae were removed on a daily basis and placed in dishes with moistened filter paper to evaluate for mycosis.
A factorial ANOVA was used to assess the impact of root-inoculated BbC1 and M150 tomato plants on larval weights. The dependent variables were the weight of larvae, and factors were tomato variety and fungal treatment. Tukey’s test was used for multiple comparisons of means (P < 0.05) (Statistica).