2. Li Honglian Wang Min Yuan Hongxia Dept. of plant protection ， Henan Agricultural University ， Zhengzhou 450002 ， China Study on the effect and Mechanism of Chemical Elicitors Induced Resistance on Cotton to Verticillium Wilt

3. This Program funded by ： Henan Outstanding Youth Fund ， and The Fund for CAS Trial Station of Agricultural Ecology

4. Part1. Background

5. Verticillium wilt is a most important cotton disease in China and caused enormous losses. — over 50% cotton fields are diseased ； — hundreds million ￥ loss in average every year

6. Because it is a soil-borne and vascular disease, the control of Verticillium wilt is very difficult.

7. Resistant cultivars is not available in most cases in China No effective fungicides to control Verticillium wilt up to now Agricultural practices such as crop rotation also is available in some cotton planting areas Biocontrol still has a long-way to use practically in field So We need fund new method to control this disease ！

8. Collection of Chemical Elicitors Security test 2. Outline ： Test induced effect in pot Test induced effect in flied mechanism Inhibition effect on pathogen Changes of enmyces ， Protein ， pathotoxin

9. Part2. Materials and methods

10. 2.1 Materials Chemical Elicitors ： ★ Chemical Elicitors ： Chitosan, SA （ 2-(acetyloxyl)-Salicylic acid ） ethephon CCC （ chlorocholine chloride ） Oligoglucose, BTH (Benzothiadiazole-7-carbothioic), Procaine hydrochloride. Oxalic acid DPC (N,N-dimethyl peperidinium chloride) ★ Cotton cultivar ： Zhongmian 17 ， susceptible ★ Pathogen strain ： Verticillium dahliae HN98-1

11. 2.2 Using of Chemical Elicitors Chitosan 100μg/ml 2 times SA 100μg/ml 2 times Oligoglucose 1000μg/ml 2 times BTH 100μg/ml 2 times Procaine hydrochloride 80μg/ml 2 times Oxalic acid 100μg/ml 2 times DPC 50μg/ml 1 times ethephon 50μg/ml 1 times CCC 100μg/ml 1 times 2 times ： seedling stage + square stage ； 1 times ： seedling stage

12. The inhibition of chemical elicitors to the growth of mycelium and spores germination of Verticillium dahilae were tested in vitro. growth of mycelium ： —— mycelium dish spores germination ： ——suspend drop 2.3 The inhibition of chemical elicitors to the growth of mycelium and spores germination

13. 2.4 The effect of chemical elicitors to biochemistry of cotton plants — Peroxidase(POD) ， Phenylalanine aminolyase (PAL) activity and the Contents of hemi-gossypol were measured by spectrophotometer ； — Water-solubility proteins was analyzed by SDS-PAGE

14. Part 3. Results

15. 3.1 The effect of chemical elicitors on control cotton Verticillium wilt 3.1.1 in pot 3.1.2 in field

16. Table 1. The effect of chemical elicitors on control cotton Verticillium wilt （ in pot ， bell stage ） TreatmentsIncidence%Index of disease effect % Chitosan2.71.4*79.7 SA8.93.352.5 Oligoglucose7.83.648.1 BTH15.84.830.6 Procaine hydrochloride8.33.154.8 Oxalic acid6.73.351.7 DPC3.31.7*75.8 ethephon3.32.5*63.8 CCC11.74.735.2 Non-treatment （ C K ） 11.76.9 ——

17. Table 2. The effect of chemical elicitors on control cotton Verticillium wilt （ in pot ， harvest stage ） TreatmentsIncidence%Index of disease effect % Chitosan23.412.8**63.1 SA43.527.520.7 Oligoglucose37.014.3*58.7 BTH29.216.0*53.8 Procaine hydrochloride47.618.3*47.3 Oxalic acid36.718.3*47.3 DPC29.917.8*51.6 ethephon48.323.532.4 CCC13.410.7**69.3 Non-treatment （ C K ） 61.834.7 ——

18. Fig.1 The effect of chemical elicitors on control cotton Verticillium wilt （ in pot)

19. Table 3. The effect of chemical elicitors on control cotton Verticillium wilt （ in field ， bell stage ） TreatmentsIncidence%Index of disease effect % Chitosan20.57.4**65.4 SA42.513.835.7 Oligoglucose52.916.224.3 BTH21.68.1*62.1 Procaine hydrochloride10.84.7*78.0 Oxalic acid8.82.986.4 DPC47.415.8*62.6 ethephon20.07.565.0 CCC40.015.7**26.6 Non-treatment （ C K ） 61.834.7 ——

20. Table4. The effect of chemical elicitors on control cotton Verticillium wilt （ in field ， harvest stage ） TreatmentsIncidence%Index of disease effect % Chitosan57.533.830.3 SA42.526.345.5 Oligoglucose52.931.634.8 BTH62.538.321.0 Procaine hydrochloride38.219.160.5 Oxalic acid47.133.830.3 DPC57.633.331.3 ethephon56.838.520.3 CCC57.133.630.7 Non-treatment （ C K ） 69.048.3 ——

21. Fig.2 The effect of chemical elicitors on control cotton Verticillium wilt （ in field ）

22. 3.2 The inhibition of chemical elicitors to pathogen 3.2.1 The effect of chemical elicitors on growth of mycelium of Verticillium dahliae 3.2.2 The effect of chemical elicitors on spores germination of Verticillium dahliae

23. Table 5 The effect of chemical elicitors on growth of mycelium of Verticillium dahliae Treatmentsconsistency （ μg/ml ） Dia. Of Mycelium （ cm ） effect % Chitosan 1003.85-13.23 SA 1003.253.53 Oligoglucose 1003.82-12.35 BTH 1002.8915.00 DPC 1003.68-8.23 ethephon 1003.62-6.47 Non-treatment （ C K ） 3.40 ——

24. BTH Oligoglucose Fig.3-1 The effect of chemical elicitors on growth of mycelium of Verticillium dahliae BTH Oligoglucose

25. SA ethephon Fig.3-2 The effect of chemical elicitors on growth of mycelium of Verticillium dahliae SA ethephon

26. Chitosan DPC Fig.3-3 The effect of chemical elicitors on growth of mycelium of Verticillium dahliae Chitosan DPC

27. Table 6 The effect of chemical elicitors on spores germination of Verticillium dahliae Treatmentsconsistency （ μg/ml ） rate of spores germination% effect % Chitosan10020.035.1 SA10033.5-8.8 Oligoglucose10030.80.0 BTH10025.317.9 DPC10049.4-60.3 ethephon10018.041.6 Non-treatment （ C K ） 30.8 ——

28. 3.3 The effect of chemical elicitors to biochemistry of cotton plants 1 Phenylalanine aminolyase (PAL) activity 2 Peroxidase(POD) activity 3 The Contents of hemigossypol

29. Fig.4 The activity of Phenylalanine aminolyase (PAL) after inducing treatment h

30. Fig.5 The activity of Peroxidase(POD) after inducing treatment

31. Fig.6 The Contents of hemigossypol (  g/g) after inducing treatment d

32. 3.4 The change of Water-solubility proteins in cotton leave （ SDS-PAGE ）

33. Fig.7 The compose of water-solubility proteins in cotton leaves （ SDS-PAGE ）

35. Fig.8 The compose of water-solubility proteins in cotton leaves （ after 24h ） C K

36. SA

37. ethephon

38. BTH

39. Chitosan

40. marker

41. Fig.9 The compose of water-solubility proteins in cotton leaves （ after 5d ）

42. The compose bands of water-solubility proteins in cotton leaves （ after 5d ）

43. CK Fig.10 The scanning of water-solubility protein bands in cotton leaves （ SDS-PAGE ） （ after 5d ）

44. BTH

45. SA

46. Chitosan

47. Part 4 Summary and Discussion

48. The effect of 9 chemical elicitors on control cotton Verticillium wilt were investigated in boll and harvest stages respectively in pots and plots. The experimental results show that every kind of elicitor can control this disease in some degree. In pots ， chitosan was the best ， and in field, procaine hydrochloride was the best.

49. Experimental results indicated chemical elicitor is potential and valuable measure to control cotton Verticillium wilt. But ， the effect is unstable in different cases and different cotton living-stages. So the effect and using method of chemical elicitor need to do more systemic tests.

50. The inhibition of different chemical elicitor to the growth of mycelium and spores germination of Verticillium dahilae were measured in vitro. The results indicated that different chemical elicitor had low inhibition to Verticillium dahilae in the using consistency. Furthermore, some elicitors facilitated the mycelium growth and spores germination of pathogen. Therefore ， the effect of chemical elicitor on disease isn’t caused by their inhibition to pathogen.

51. The biochemical reaction after induced treatment by chemical elicitors was tested. The results showed that ： —Activities of phenylalanine aminolyase(PAL) 、 peroxidase(POD) both increased markedly than non-treatment （ CK ） —The hemi-gossypol phytoalexin also accumulated after treatment; —The compose of the water-solubility proteins in cotton leaves was changed and produced some new specific protein bands.

52. The biochemical reaction in cotton after induced treatment by chemical elicitors was similar to the reaction inoculated by Verticillium dahilae. Base on former research ， we consider that the control effect of chemical elicitors on cotton Verticillium wilt was caused by activation resistant metabolizability of cotton plant 。