| Chrysanthemum(Chrysanthemum morifolium)originated in China,which has a long cultivation history and a multiple cultivar.It also has high ornamental,edible,tea use and medicinal values.chrysanthemum for tea has a variety of beneficial functional inclusions,which has become an important health drink.In recent years,Alternaria leaf spot disease has occurred frequently in production area of chrysanthemum for tea,especially under the conditions of high temperature and high humidity,which has become a serious disease that severely restricts the large-scale production of chrysanthemum for tea.Irregular spraying of pesticides in chemical control can cause problems such as pesticide residues,environmental pollution and pathogen resistance.Only by identifying high-quality disease-resistant germplasms,clarifying the pathogenic mechanism of Alternaria leaf spot disease pathogens,the disease-resistant mechanism of chrysanthemum for tea and selecting and breeding excellent disease-resistant cultivars with a targeted manner,can we truly solve the problem from the source.Based on this,the identification of resistance to Alternaria leaf spot of 38 tea chrysanthemum cultivars were carried out in this paper,and then the mechanism of resistance to Alternaria leaf spot of chrysanthemum for tea was explored from multiple angles.The main research contents and results are as follows:1.The disease-affected plants were collected from the main production area of’Fubaiju’,and the two pathogens F16 and F20 were separated by the tissue block separation method and cultured until purification.Identification was performed by observing the morphological characteristics of the strain and combining the target gene sequence of 18S rDNA-ITS.At the same time,pathogenicity testing and Koch’s law verification were carried out.It was determined that the two pathogenic strains were Alternaria alternata,and they had no obvious difference pathogenicity on the leaves of‘Chuju’.2.Two pathogenic strains were inoculated in liquid culture medium,the culture filtrate was collected,and sample pretreatment was performed using QuEChERS technology.Qualitative and quantitative detection of toxins in the culture filtrate was performed by UPLC-MS/MS and UPLC to establish a detection system.The test results showed that the content of Tenuazonic Acid(TeA)in the culture filtrate of strain F20 was significantly higher than that of F16,indicating that F20 has a stronger ability to produce toxins and can be used for inoculation of petals and subsequent disease resistance identification.Based on the above detection system,the toxin content in the petals of the chrysanthemum tea was detected,and the results showed that the toxin content in the petals of the tea chrysanthemum showed an increasing trend with the inoculation time.On the 9th day after inoculation,the content of Altenuene(ALT)in petals was significantly higher than that of TeA.3.The 38 chrysanthemum for tea cultivars were evaluated for disease resistance at the seedling stage by the mycelium smear inoculation method.The incidence rate and disease index were calculated according to the disease symptoms and the disease resistance classification was performed based on this.There were 11 cultivars showed‘resistance to disease’,22 cultivars showed‘medium susceptibility’,and 5 cultivars showed’susceptibility’.4.Scanning electron microscope was used to observe the difference of leaf structure characteristics and dynamic changes of leaf stomatal after inoculation with pathogen.The results showed that the resistant cultivars had denser epidermal trichomes density and lower stomata density than the susceptible cultivars.After inoculation with pathogen,the stomata closed first and then opened,and it was found that the pathogen mycelia could invade from the stomata.The above results indicate that the leaf epidermal trichomes density,stomatal density and the degree of stomata opening and closing have a certain correlation with the disease resistance to Alternaria leaf spot disease of chrysanthemum for tea.5.A disease-resistant cultivar’Suju NO.6’ and a disease-susceptible cultivar’Dayangju’ were selected,sampled at 3 time points after inoculation and stained with DAB to observe the distribution of H2O2 in the leaves.The staining results showed that at the three inoculation time points,the leaf staining area of two cultivars showed an upward trend,and the leaf staining area of the susceptible cultivar was significantly larger than that of the resistant cultivar.6.Interaction transcriptome analysis of chrysanthemum for tea and A.alternata samples were performed at 1,12 and 24 hours after inoculation,a total of 27 RNA-seq libraries were constructed.153,532 DEGs were identified in chrysanthemum and 14,932 DEGs were identified in A.alternata.We found that DEGs in chrysanthemum for tea involved in plant-pathogen interactions,cell wall enhancement and decomposition,plant hormone signal transduction and several types of transcription factors.Fungal DEGs were involved in toxin synthesis-related enzymes,cell wall-degrading enzymes,and detoxification(stress tolerance)related genes 24 DEGs from chrysanthemum and A.alternata were verified by qRT-PCR,the results showed that the transcriptome data were reliable.7.Through bioinformatics analysis,45 chrysanthemum chitinase family genes(CmChis)were identified and classified into 5 classes based on conserved domains.The expressions of 9 genes in different tissues in the family and the expression at different time points after inoculation with A.alternata were also analyzed by qRT-PCR.At the same time,the expression level of the CmChi(CHR00076564-RA)in different resistant cultivars of chrysanthemum for tea was also analyzed.The results showed that the 9 selected CmChis were mainly expressed in leaves and 6 CmChis were significantly up-regulated under the condition of A.alternata infection;CmChi(CHR00076564-RA)was found showed up-regulated expression in 6 different cultivars and the expression level of susceptible cultivars was higher than resistant cultivars. |
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