| As an important vegetable crop in the world,Raphanus sativus is vulnerable to biological stress such as Alternaria.Black spot caused by Alternaria brassicae infection seriously affects the yield and characters of Raphanus sativus,Traditional control methods have high cost and high pollution.Inducing endogenous immunity is one of the important measures to explore the green development of industry.so induction of endogenous immunity is an important measure to explore the green development of industry.Melatonin,as a ubiquitous amine growth regulator,is ubiquitous in the biological world,and has the function of improving crop resistance to biological stress.Exogenous spraying can affect both host and pathogen physiology on susceptible plants,and the interaction mode between host and pathogen mediated by exogenous spraying remains to be unclear.It is of great significance to study the role of melatonin in regulating microbial life activities for revealing its mechanism of improving host disease resistance.In this study,"Jiangnanyuanbai"Raphanus sativus seedlings and A.brassicae（Berk.）Sacc,the pathogen of Raphanus sativus black spot in Weining County,Guizhou Province,were used as materials,Three groups of experiments were set up:1.the same amount of melatonin 0,50,100,500,1000 and1500μM were sprayed on plant leaves,to study the mechanism of its effect on Radish Growth and immunity;2.Exogenous melatonin with the same gradient concentration was applied to A.brassicae（Berk.）SACC monoclonal antibody to study the effects of melatonin on colony growth,spore development and pathogenicity;3.Spraying exogenous melatonin with the same gradient concentration on the diseased radish seedlings to study the action mode of melatonin on radish disease resistance.At the same time,the phenotype was verified at the transcriptional level.The main results are as follows:1.Spray the same amount of melatonin with gradient concentration of 0-1500μM to the leaves of radish seedlings,measure the biomass,total chlorophyll content,APX enzyme activity,H2O2Content,endogenous melatonin content and subcellular structure of samples in each group,and make statistics on the incidence of samples inoculated with Alternaria Brassica.It is proved that the concentration of melatonin is within the range of 50-500μM,Seedling growth and immunity increased with the increase of spraying concentration,among which 500μM treatment group had the best indexes,while 1000-1500μM melatonin treatment reduced radish indexes,indicating that melatonin can regulate Radish Growth and immunity through a dose-dependent mode.In this study,eight DEGs related to photosynthetic regulation（Ubr）and stress resistance（Aos1,Prp1,Zat-like,Jub1-like,Edr1,Usp and Eda）were screened by RNA SEQ transcriptome analysis of 0,500 and 1500μM treatment groups.The expression pattern of DEGs also showed a significant dose-dependent effect..2.When melatonin of 0,50,100,500,1000 and 1500μM was applied to Alternaria Brassica,the results showed that the effect of melatonin of gradient concentration on colony growth,spore differentiation and pathogenicity showed significant dose effect,50-500μM can significantly promote the above indicators,and500μM has the best effect,while high concentration（1000-1500μM）It has obvious inhibitory effect.The effects of gradient concentration melatonin on colony growth,spore differentiation and virulence of Alternaria Brassica showed a significant dose-dependent effect,50-500μM can significantly promote the above indicators,and500μM has the best effect,while high concentration（1000-1500μM）It has obvious inhibitory effect.By comparing 0,500 and 1500μM The samples of treatment group were sequenced and analyzed by transcriptome sequencing,and nine differentially expressed genes that may be involved in the regulation of exogenous melatonin dose effect were screened,including aflatoxin biosynthesis（Sld1 and Sld2）,MAPK signaling pathway（glp）,linoleic acid metabolism（hp1）,non reducing polyketide synthase synthesis（Nps）,heme peroxidase synthesis（hp2）Selenium metabolism（thr）,endoplasmic reticulum protein processing（khp）and glycolysis/gluconeogenesis（ppl）related genes affect Alternaria proliferation（thr and glp）,pathogenicity（Sld1、Sld2、Nps、ppl and glp）,stress（khp）and antioxidation（hp1 and hp2）.The expression of DEGs also showed significant dose effect in different concentrations,which was consistent with the trend of physiological measurement results.The above results show that exogenous melatonin can“promote the growth and development of Alternaria Brassica at low concentration and inhibit the growth and development of Alternaria Brassica at high concentration”.3.The results of interaction experiments showed that low concentration of melatonin could significantly improve the resistance of Raphanus sativus to bacteria,and 500μM showed the best performance,while 1000 and 1500μM had no obvious effect,and the effect of melatonin was obviously dose-dependent.Dual RNA-seq showed that the ratio of Raphanus sativus genomic reads was more than 88%,the ratio of Alternaria gene reads was less than 0.06%,and the number of Raphanus sativus DEGs was significantly higher than that of Alternaria alternata.The expression levels of Raphanus redox reaction related enzymes,ethylene-like reaction transcription factors,defense-related proteins,glutathione metabolism related enzymes of Alternaria,sugar transporters,isocitrate lyase,phosphoryl mutase and2-methylcitrate dehydratase synthesis related genes were verified,of which expression pattern were basically consistent with dose-dependent pattern,and most genes were related to positive regulation of their development and resistance.The above results indicated that melatonin could promote the growth of Raphanus and Alternaria at the concentration of 500μM,but because the biomass of Raphanus was larger than that of Alternaria,melatonin affected the host more than that of Alternaria,and finally the resistance of Raphanus to Alternaria increased.This study expands and supplements the functional analysis of melatonin in horticultural crops,and provids a theoretical basis for the application and popularization of melatonin in commercial production of crops. |
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