Optimization Of The Cotton Alternaria Leaf Spot Disease Inoculation Method And Investigation Of The Roles Of MAPK Family Members In Resistance To The Disease

Alternaria Leaf Spot Disease also known as Black leaf spot disease.The spot disease can harm cotton leaves during the whole growth period of cotton,which is the most widely distributed and harmful leaf diseases of cotton with the high popular frequency.However,inoculation of cotton Alternaria leaf spot disease is an extremely hard work,because of the Alternatia spore germinated only in harsh conditions,and the germinated spore only infection of wounded cotton leaves but not infection of healthy cotton leaves.In the earlier studies of cotton Alternaria leaf spot disease in our lab,the disease was successfully inoculated by the application of smearing inoculation method,adjunction with humidity keeping method with loosely sealed plastic bag.But under such conditions,weaknesses are obvious.Such as: the infected leaves were injured uneven and the infected leaves air environment is poor.The leaf spots cannot be ruled out by other mold.Not to mention that this is a labor consuming work.All these shortcomings of inoculation method make the consistency of experimental condition poorer.Therefore,in this paper,optimization of the cotton alternaria leaf spot inoculation method was further developed,the results showed as follows:1.Both in the smearing and spray inoculate methods,the spot disease would only occurred under highly moist conditions.In smearing inoculation method,cotton spot disease only stay in the light green water stains stage in low moist conditions,and the pathogen spores cannot germinate normally,the disease spot does not continue to develop into black wheel pattern;And in spraying inoculation method,cotton leaves can hardly be infected by the pathogen in low moist conditions.2.Under controlling spraying moisturizing conditions,both smearing and spraying inoculation method can make the cotton seedling leaves occurrence of spot disease.The leaf spot was generally larger and the disease was more serious in smearing inoculation method.In contrast,inoculated with the spraying method,the spot of cotton leaf was small,black and rounded,scattered.The disease was gradually developed inward from the edge to the leaf.The disease symptom was also closer to the field.And together with the character of the consistency made the spraying inoculation method,is a much suitable for inoculation of cotton leaf spot disease.3.Smearing method and spraying method under the spray moisturizing conditions,can effectively distinguish the spot disease susceptible varieties of XLZ7、XLZ13 from the spot disease resistant varieties of KV3、XLZ33.In this study,VIGS（virus induced gene silencing）technique was also used to study the function of MAPK gene in cotton resistant to the leaf spot disease,which was carried out by the two different inoculation methods for comparative experiments.The results were as follows:1.Successfully silenced the genes coding MAPK: ADI52623.1G.hirsutum,Gorai.5g011100.1,ADI52622.1G.hirsutum,Gorai.7g050000.1,ABA00652.1G.hirsutum,ADI52627.1G.hirsutum,A₀7602,Gorai.1g158800.1,AET36534.1G.hirsutm.2.For investigation of the roles in resistance to spot disease,nine MAPK genes were silenced respectively and then inoculated using two inoculation methods.The results showed that after silencing the gene ADI52623.1 G.hirsutum,Gorai.5g011100.1,ADI52622.1 G.hirsutum,Gorai.7g050000.1,compared with the control,the disease was no significant difference,indicating that cotton resistance to the spot disease has not been significantly affected;And after silencing the gene ABA00652.1G.hirsutum,ADI52627.1G.hirsutum,A₀7602,Gorai.1g158800.1,AET36534.1G.hirsutm,compared with the control,disease index were increased,indicating that these five MAPK genes may be involved in the regulation of cotton disease resistant and played a important role in the process of signaling pathways.In this study,the cotton leaf spot disease inoculation method was further optimization,and the optimized inoculation method can be used for the further study of cotton leave spot disease.Secondly,the results showed that there some MAPK genes involved in the defense response to cotton leaf spot disease,but the results should be further investigated.With the completion of cotton genome sequencing,all MAPKs genes may be investigated comprehensively for its role in resistance to cotton leave spot disease.