Insecticidal activity of the crude extract from Epichloë bromicola against Rhopalosiphum padi

Rhopalosiphum padi is a important pest in agricultural production. In order to reduce the use of chemical pesticides and improve the control effect, we reported the insecticidal activity of the crude extract from 20 strains Epichloë bromicola isolated from Elymus tangutorum. The results showed that the crude extract of E. bromicola had certain insecticidal activity against R. padi. The experimental results showed that 10 strains with good activity were 2, 3, 5, 7, 8, 11, 16, 18, 12 and 19, and their mortality rate reached more than 50% at 48h. However, the four strains of 13, 14, 15 and 17 had no insecticidal activity, but could promote the growth of R. padi. Among them, strain 7 had the best insecticidal activity at 48h, and the mortality rate reached 88.3%. The weakest insecticidal activity was strain 14, with a mortality rate of 21.7%. Strain 18 had the best insecticidal activity within 24h, with a mortality rate of 33.3%. At the same time, the mortality rate of 48h was significantly higher than that of 24h. In conclusion, these strains with significant insecticidal activity will be of great significance for the biological control of R. padi in the future.


Introduction
Rhopalosiphum padi is a kind of pest that has great influence on agricultural economy and is one of the main causes of crop yield and quality loss in horticulture, grain and trees [1,2] . R. padi sucked the juice of leaf blades, stems and young ears of plants with piercing-sucking mouthparts, resulting in yellowing and curling of the leaf blades (up to 90%), poor plumpness of seeds, transmission of plant viruses, etc [3] . As a result, the wheat reduction reached 10% or above in China every year, especially in the outbreak year (up to 30%). Therefore, R. padi was considered as one of the most important aphids bringing serious damage to wheat in China [4] . It is estimated that aphids cause at least 2 per cent of all insect food loss to the world's crops each year. In addition to removing life fluids from plant screening elements, aphids are very effective vectors for viral diseases: about 60% of plant viruses are transmitted by these insects [5] . Various insecticides can be used to control aphids, but the extensive use of chemical insecticides has led to environmental pollution and the emergence of various aphid resistance populations [6] .
There is an urgent need for a biological pesticide to control R. padi. It has been reported that the secondary metabolites produced by E. bromicola are not toxic to normal animal cells and provide resistance to host plants [7,8] . Using this advantage, we studied the insect resistance of its crude extract.
In this paper, we used the same method to ferment E. bromicola at different altitudes to obtain crude extract, and tested the insecticidal activity of the E. bromicola against R. padi by leaf soaking method.

Materials and methods
These experiments were carried out at the Plant Protection Laboratory, Yuzhong Campus, Lanzhou University, Lanzhou, Gansu province.

Fungal Strain
The fungal strain used in this study was isolated from the Elymus tangutorum. The 20 strains isolated were kept in Institute of Grassland Agriculture Protection, Lanzhou University.

Aphids
The aphid in the experiment was R. padi and provided Institute of Plant Protection, Gansu Academy of Agricultural Sciences.

Preparation of crude extract from E. bromicola
Twenty strains of E. bromicola were cultured on potato dextrose agar (PDA) at 28 ℃ for 15 days as seed, respectively. Agar plugs were used to inoculate 1000-mL Erlenmeyer flasks, each containing 400 mL of M104T (Sorbitol 100 g, Glucose 40 g, Yeast extract 3 g, Glutamic acid 10 g, Tryptophan 0.8 g, MgSO4.7H2O 0.3 g, KH2PO4 1 g). The flasks were placed on an incubatory shaker at 145 rpm and 28 °C for 45 days. The fermentation broth of strain E. bromicola was separated into culture filtrate and mycelia by centrifugation. The culture and the mycelia were extracted with EtOAc (1.2 mL) and MeOH (300 mL) at room temperature (three times), respectively. Then, the EtOAc and MeOH were evaporated to dryness under vacuum on a rotar evaporator, respectively, and were combined to obtain a crude extract.

Insecticidal Assay
The insecticidal activity of crude extract against R. padi was evaluated by leaf-dip method using a previously reported procedure. The assay was repeated in an air-conditioned room at 25 ± 2 °C. Each tested sample was dissolved in acetone at a concentration of 2 g/L and diluted with distilled water containing TW-80 (0.1 mg/L) to obtain a required concentration. Water containing TW-80 (0.1 mg/L) was used as control. Leaf disks (5 cm × 3 cm) were cut from fresh corn leaves and then dipped into the test solution for 6 s. After air-drying the treated leaf disks and 30 R. padi were placed individually into the disks. The mortalities were evaluated 24h and 48h after treatment. All the assays were repeated in triplicate.

Statistical analysis of data
Excel was used to process the original test data and SPSS22.0 was used for significance difference analysis.
The insecticidal activity of these strains was more significant than that of other strains, and the mortality rate of 48h was more than 50%.

Treatment of R. padi with crude extract for 24 hours
The mortality rate of R. padi at 24h was 33.3% and 6.67%, respectively in strain 18 and strain 17. Strain 17 had the same 6.67% as CK, so strain 17 had no inhibition on R. padi at 24h. The mortality rate of R. padi was 33.3%, 31.7%, and 28.3%, respectively in strain 18, 11 and 19.
According to Duncan's multiple comparison method, the difference of killing rate of 30 strains was not significant.

Treatment of R. padi with crude extract for 48 hours
The mortality rate of R. padi at 48 h was 88.3% and 21%， 7%, respectively in strain 7 and strain 14. Strains 14, 15, and 17 had lower mortality rate than CK, so the three strains had the opposite effect and promoted aphid growth.

Comparison of R. padi mortality at 24h and 48h
As shown in Table 1., the mortality rate of 48h was significantly higher than that of 24h. It can be seen from the Fig. 1. that the mortality of strains 3, 5, and 7 at 24h and 48h was significantly different, which was 46.7%, 63.3% and 66.6%, respectively. This indicates that these three strains will play a greater role after 24h, with more obvious insect resistance. The differences of strains 13, 14, and 15 were small, 10%, 8.4% and 11.6%, respectively. Compared with CK by 18.33%, these three strains promoted the growth of R. padi between 24h and 48h.

Conclusion
In this paper, the insecticidal activity of 20 E. bromicola of Elymus tangutorum on R. padi was studied, and the time variables were set for 24h and 48h. We found that E. bromicola were generally resistant to this R. padi and found the crude extract of strain 7 had the best insecticidal activity, up to 88.3%.
Therefore, strain 7 can be used to inhibit R. padi. In the future, this crude extract can be further isolated and purified, and more effective pure compounds can be obtained to further improve the insecticidal activity.