| Chrysanthemum is one of the ten traditional famous flowers in China and the second largest production and sales flower of four cut flowers in the world,it plays an important part in international flower trade and one of the important agricultural products of China's export.However,due to the low temperature and high humidity in spring and autumn under protected cultivation environment,chrysanthemum white rust can easily outbreak and spread heavily.White rust mainly harms young leaves and flower buds and affects the growth of plants,yellow to white teliospore pustules resulted from the infection can seriously affect the quality of products.White rust spread quickly and it is difficult to be controled,which has been recorded as an important quarantine disease in most countries.Most of the domestic chrysanthemum productions enterprises don't have efficient solutions about the prevention and control of this disease;they were mainly relying on several expensive imported fungicides to get some relief,because ordinary fungicides seem almost invalid.However,such chemical methods,not only labor and resource s-consuming,but also increases costs,causes environmental pollution,and pathogens tends to be drug-resistant.Therefore,the prevention and control of white rust has become an obstacle in the chrysanthemum industrialization of production.Creating new germplasm of resistance to white rust has become a great subject in the chrysanthemum resistance breeding.Researches of white rust are still at the beginning.However,closely related species of chrysanthemum have excellent resistant traits,and they haven't been screened for white rust resistance yet.This study was designed to discovery gerrmplasms with white rust resistance from related species of chrysanthemum under artificial inoculation,and to explore the mechanism involved in the resistance to the disease.In this study.19 related species of chrysanthemum were identified for white rust resistance under two independent artificial inoculation experiments.Comparative analysis of leaf structure,antioxidase and defense activities of different resistant germplasmsin response to with white rust inoculation were investigated,and RNA-seq analysis of the C.crassum after artificial inoculation with white rust were conducted.The main findings are as follows:1.Variation in resistance to white rust(Puccinia horiana)among Ajania and Chrysanthemum speciesA panel of 19 accessions of commercial chrysanthemum near-relatives(four Ajania and fifteen Chrysanthemum species)were screened for their reaction to white rust infection in separate greenhouse trials carried out at two independent sites in Eastern China,one in 2010 and the other in 2012.The reaction of the accessions to artificial inoculation ranged from immune to highly susceptible.Accessions of Chrysanthemum indicum,C.yoshinaganthum,C.makinoi var.wakasaense,C.nankingense,C.vestitum,C.lavandulifolium,C.crassum and Ajania tripinnatisecta were immune,and strong resistance was present in C.japonense,C.shimotomaii and A.przewalskii.Most of the accessions behaved similarly in the two trials,but two of the C.indicum accessions produced inconsistent results,each being highly resistant in one trial,but susceptible in the other.2.Leaf morphology and physiological changes in response to Puccnia horiana of two wild chrysanthemum speciesTo better understand the mechanisms of wild chrysanthemum species resistance to this disease,leaf morphological traits,leaf epidermal wax composition and amount of immune(Chrysanthemum makinoi var.wakasaense)and highly susceptible(Ajania shiwogiku var.kinokuniense)to P.horiana species were compared.Scanning electron microscopy revealed trichome density and length and stomata density of two species were markedly different.Leaf epidermal wax composition and amount measured by GC-MS found that C.makinoi var.wakasaense has 2.17 times more total wax load than A.shiwogiku var.kinokuniense and composition were also different,for C.makinoi var.wakasaense was abundant in esters and primary alcohols.Changes induced in leaf superoxide dismutase(EC 1.15.1.1),peroxidase(EC 1.11.1.7),polyphenoloxidase activity(EC 1.14.18.1 or EC 1.10.3.2)and phenylalanine ammonia lyase(EC 4.3.1.5)were also studied within 13 days after inoculation(DAI).In C.makinoi var.wakasaense,the activity of superoxide dismutase and peroxidase enzymes increased rapidly after inoculation,and reached peak at 5,13 days after inoculation in C.makinoi var.wakasaense,respectively.However they remained a low and steady level in the highly susceptible one after inoculation.Two speciess both showed quick increase in polyphenol oxidase activities after P.horiana inoculation,but the activity is higher in C.makinoi var.wakasaense than A.shiwogiku var.kinokuniense.Phenylalanine ammonia lyase reached peak at 3 and 9 DAI in C.makinoi var.wakasaense but remained low activities in A.shiwogiku var.kinokuniense,suggesting involvement of these enzymes in P.horiana resistance.3.Analysis expression profile of immune resistant specie of chrysanthemum induced by white rustThe global expression profiles of Puccnia horiana-infected seedling leaves of an immune resistant germplasm C.crassum were investigated through next generation sequencing technology.A total of 59,969 unigenes were obtained by assembling the inoculated before(0J)reads,inoculated with deionized water(JMixCK)reads and inoculated with Puccnia horiana(JMix)reads respectively and they were compared between two samples though RPKM index to find differentially expressed genes(DEGs).About 5.2 million DEGs were recieved and were annotated by BLAST searching databases of nr,the Clusters of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)with an E-value ?1e-5.With GO enrichment analysis of DEGs,we found that most of the DEGs are related to cellular process,metabolic process and response to stimulus.After futher analyses we found some DEGs are largely induced by inoculationand were annotated to circadian rhythm,phenylpropanoid biosynthesis,plant hormone signal transduction,plant-pathogen interaction pathways,etc. |
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