| Verticillium wilt of cotton is a very serious soil borne disease,which is caused by Verticillium dahliae.It can cause the decline of cotton yield and fiber quality.Chemical control is easy to cause drug resistance of pathogens and pollute the environment to a certain extent;in the aspect of disease resistance breeding,there is no good resistance resource at present;in recent years,more and more researches have been carried out to control Verticillium wilt by using biocontrol bacteria and fungi,which can improve the soil beneficial microbial population and is friendly to the environment.Previous studies have found that cotton endophytic Chaetomium globosum CEF-082 has an antagonistic effect on V.dahliae,which can effectively reduce the incidence of Cotton Verticillium wilt,and cause cotton plants to produce defensive responses.However,the active substances are not clear,and the more comprehensive defense mechanism is not clear.In view of these two problems,this study has done some exploration.The results are as follows:1.C.globosum CEF-082 can inhibit the growth of V.dahliae and Sclerotium rolfsii.The crude extract of CEF-082 inhibited the growth of the spores and degraded the cell wall of V.dahliae.It improved the sensitivity of Vdahliae to adversity and had no negative effect on the growth of cotton.After high temperature treatment,some substances were inactivated.2.Chaetoviridin A,an active metabolite from CEF-082,can inhibit the growth of V.dahliae.CEF-082 was cultured in PDB,a total of 50 L.The fermentation broth was extracted by ethyl acetate,then separated and purified by silica gel column,Sephadex LH-20,thin layer chromatography and high performance liquid chromatography.Through 13C-NMR and 1HNMR and mass spectrometry identification,an active substance chaetoviridin A was obtained.It can inhibit the germination of micosclerotia,promote the accumulation of H2O2 and NO,and make cell necrosis.The expression of genes related to cell wall synthesis was analyzed by qRT-PCR after the spores of V.dahliae treated with chaetoviridin A.The results showed that chaetoviridin A could inhibit the expression of genes related to cell wall synthesis of V.dahliae,that is,chaetoviridin A could destroy the integrity of the cell wall of V.dahliae.3.CEF-082 can colonize in cotton plants.After the roots of cotton plants inoculated with the spores suspension of CEF-082,CEF-082 could be isolated from the stem of cotton plant two weeks later.It was identified as CEF-082 by morphology and its identification,which indicated that CEF-082 could be colonized in cotton plant.4.CEF-082 could induce the lignification of cotton plants and the production of H2O2 on the leaves.To study the signalling mechanism induced by CEF-082,the transcriptome of cotton seedlings pretreated with CEF-082 was sequenced.The results revealed 5638 DEGs at 24 h post inoculation with CEF-082,and 2921 and 2153 DEGs at 12 and 48 h post inoculation with Verticillium dahliae,respectively.At 24 h post inoculation with CEF-082,KEGG enrichment analysis indicated that the DEGs were enriched in 15 pathways,including plantpathogen interaction,MAPK signalling pathway-plant,flavonoid biosynthesis,phenylpropanoid biosynthesis pathways,and glutathione metabolism,et al.There were 1209 DEGs specifically induced only in cotton plants inoculated with V.dahliae in the presence of the biocontrol fungus CEF-082,and not when cotton plants were only inoculated with V.dahliae at 12 and 48 h.GO analysis revealed that these DEGs were enriched mainly in the following terms:ROS metabolic process,H2O2 metabolic process,defense response,superoxide dismutase activity,and antioxidant activity.There were 96 shared DEGs specifically induced at 12 and 48 h,and they were enriched in glutathione metabolism,flavonoid biosynthesis,plant circadian rhythm and vancomycin resistance.5.By analyzing the DEGs in transcriptome,we screened a gene related to glutathione metabolism Gh_A04G0830,named GhGSTU16,a WRKY family gene Gh_D05G2642,named GhWRKY,and the predicted alpha/beta hydrogen fold gene Gh_A04G0855,named GhPH.These three genes may be related to cotton resistance to Verticillium wilt.6.We analyzed DEGs in transcriptome,screened several genes that were significantly upregulated,related to glutathione synthesis,cloned gene GhGSTU16,and obtained GhGSTU16 gene upstream 1500 bp regulatory sequence from upland cotton genome.It is predicted online that the regulatory region contained MeJA response element,defense and stress response element.7.GhGSTU16 and GhPH genes in upland cotton are related to the resistance against Verticillium wilt.In order to verify the function of gene GhGSTU16,the over expression vector was constructed and transformed into Arabidopsis.Three transgenic lines were obtained.It was found that the resistance to Verticillium wilt was reduced in transgenic Arabidopsis compared with wild type.In order to study the function of these three genes GhGSTU16,GhWRKY and GhPH in cotton,VIGS vectors were constructed for the three genes.The results showed that the expression of these genes in cotton leaves decreased significantly.Compared with the control group,the disease index of gene GhGSTU16 interference group decreased,the disease index of GhWRKY gene interference group did not change significantly,but the disease index of GhPH gene interference group increased.In conclusion,GhGSTU16 and GhPH genes could affect the resistance of cotton plants to Verticillium wilt. |
