Li Honglian Wang Min Yuan Hongxia Dept. of plant protection ， Henan Agricultural University ，

1. 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

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

3. Part1.Background

4. 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

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

6. Resistant cultivars is not available in most cases in China • No effective fungicides to control Verticillium wiltup tonow • 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 ！

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

8. Part2. Materials and methods

9. ★ 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 2.1 Materials

10. 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 2.2 Using of Chemical Elicitors

11. 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

12. 2.4The 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

13. Part3. Results

14. 3.1The effect of chemical elicitors on control cotton Verticillium wilt 3.1.1 in pot 3.1.2 in field

15. Table 1. The effect of chemical elicitors on control cotton Verticillium wilt（in pot ，bell stage）

16. Table 2. The effect of chemical elicitors on control cotton Verticillium wilt（in pot ，harvest stage）

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

18. Table 3. The effect of chemical elicitors on control cotton Verticillium wilt（in field ，bell stage）

19. Table4. The effect of chemical elicitors on control cotton Verticillium wilt（in field ，harvest stage）

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

21. 3.2The inhibition of chemical elicitors to pathogen 3.2.1The effect of chemical elicitors on growth of mycelium of Verticilliumdahliae 3.2.2The effect of chemical elicitors on spores germination of Verticillium dahliae

22. Table 5 The effect of chemical elicitors on growth of mycelium of Verticilliumdahliae

23. BTHOligoglucose Oligoglucose BTH Fig.3-1 The effect of chemical elicitors on growth of mycelium of Verticilliumdahliae

24. SA ethephon ethephon SA Fig.3-2 The effect of chemical elicitors on growth of mycelium of Verticilliumdahliae

25. Chitosan DPC DPC Chitosan Fig.3-3 The effect of chemical elicitors on growth of mycelium of Verticilliumdahliae

26. Table 6 The effect of chemical elicitors on spores germination of Verticillium dahliae

27. 3.3The effect of chemical elicitors to biochemistry of cotton plants1Phenylalanine aminolyase (PAL) activity 2 Peroxidase(POD)activity3 The Contents of hemigossypol

28. Fig.4 Theactivity of Phenylalanine aminolyase (PAL) after inducing treatment h

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

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

31. 3.4 The change of Water-solubility proteins in cotton leave（ SDS-PAGE）

32. Fig.7 The compose of water-solubility proteins in cotton leaves （ SDS-PAGE） 1 2 3 3 4 4 5 5 6 5

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

35. SA

36. ethephon

37. BTH BTH

38. Chitosan

39. marker

40. Fig.9The compose of water-solubility proteins in cotton leaves （after 5d） 1 1 2 2 3 3 4 4

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

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

43. BTH

44. SA SA

45. Chitosan

46. Part4 Summary and Discussion

47. 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.

48. 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.

49. 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 elicitoron disease isn’t caused by their inhibition to pathogen.

50. 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.