Response Of Daylily Autumn Red To Low Temperature Stress And Isolation Of Pathogen Causing Leaf Blight And Screening Of Control Agents

Daylily is one of the three traditional roots internationally recognized flowers,has three thousand years of cultivation history in our country,is called the mother flower,and has beautiful symbolic meaning.Daylily,as one of the most important plants in garden and horticulture,has not only ornamental value but also a high value in food and medicine.However,in the application of daylily,abiotic stresses are often important factors restricting the development of the daylily industry.Therefore,it is of great practical significance to study the stress related to daylily.’Autumn Red’is a kind of diploid perennial daylily variety,colorful flowers,large flowers,and a large number of,with high ornamental value;’Autumn Red’with heat resistance,resistance to moisture and drought,the outstanding art features;’Autumn Red’can grow in most cold tolerant areas with low care requirements.Vacuolar invertase is a class of sucrose invertase,which plays an important role in plant sucrose hydrolysis.In this study,the vacuole invertase genes that may be involved in the response of daylily’autumn red’to low temperature stress were identified by low temperature stress treatment and transcriptome sequencing,and bioinformatics analysis was conducted.Transgenic experiments were conducted to verify the response of Hf VIN2 to low temperature stress.A kind of hemerocallis leaf blight caused by fuscus culum was found and the optimal fungicide was selected.The main experimental results are as follows::（1）Six vacuolar invertase genes were screened from the transcriptome database of daylily’Autumn Red’.After PCR amplification and sequencing verification,they were named Hf VIN2～Hf VIN7.The ORF of the six genes ranged from 1767bp to 1944bp,and the number of amino acids encoded ranged from 588 to 655 The similarity between the amino acid sequences encoded by the six genes was more than 70%,among which the amino acid sequences encoded by Hf VIN2 and Hf VIN4 had the highest similarity（99.10%）.The amino acids encoded by Hf VIN2 and Hf VIN7 have the lowest similarity,which is 72.90%.（2）The obtained vacuole invertase gene sequences were analyzed,and the results showed that the six vacuole invertases were stable hydrophilic proteins,with the Glyco-32superfamily and DUF3357 family.Amino acid sequences encoded by vacuole invertase genes of different plants were selected from NCBI to construct a phylogenetic tree.It was found that the amino acid sequences encoded by vacuole invertase genes of’Autumn Red’daylily were clustered in a clade,among which Hf VIN2 and Hf VIN3 were closely related,and Hf VIN4and Hf VIN5 were closely related.Hf VIN6 and Hf VIN7 are closely related,and this clade contains only one distinct species of agave.Conserved Motif analysis was performed on the six Hf VIN gene-encoded proteins by MEME.All sequences were identified with 9 conserved motifs.All the Hf VINs were located in the vacuole.Transmembrane structure analysis showed that Hf VIN6 and Hf VIN7 had no transmembrane structure,while the other members had transmembrane structures.The prediction results of high protein structure showed that high protein structure of Hf VINs had high similarity.（3）The phenotypic changes of’Autumn Red’daylily were observed and recorded under low-temperature stress.The soluble protein content and soluble sugar content of collected leaf tissues were determined and the fluorescence quantitative analysis of vacuole invertase gene expression was performed.The results showed that the phenotypic characteristics of’Autumn Red’leaves didn’t change significantly at 10℃compared with 15℃（control temperature）.After treatment at 5℃for 24h,the leaf tip turned yellow and the leaf color deepened.Treated at 0℃for 24 h,the leaves appeared water-stained with low-temperature damage,and the tip of the leaves began to wilt.After the treatment of low-temperature stress,the above-ground part of the hemerocallis plants can grow new leaves quickly,and the recovery condition is good.In the process of temperature decrease,soluble protein content,and soluble sugar content decreased first and then increased.Fluorescence quantitative analysis showed that the relative expression levels of Hf VIN2,Hf VIN6,and Hf VIN7genes all increased with the decrease in temperature,while the relative expression levels of Hf VIN3,Hf VIN4,and Hf VIN5genes increased first and then decreased.（4）The optimal genetic transformation system of daylily callus was optimized by the Agrobacterium-mediated method.The optimal culture medium for induction of daylily callus was MS11.74 g/L,sucrose30g/L,agar7 g/L,6-BA3 mg/L,NAA2 mg/L（p H5.8）,the number of callus subgenerations was determined to be less than three times.Hf VIN2 was introduced into wild-type tobacco plants by agrobacterium-mediated method,the transgenic tobacco DNA was extracted after differentiation and screening,and the target fragment was amplified by PCR and compared with the positive control.The identification results showed that Hf VIN2 had been successfully expressed in wild-type tobacco plants.Transgenic tobacco showed better cold resistance than wild-type tobacco under low-temperature stress,suggesting that the Hf VIN2 gene may be a positive regulator of cold tolerance of daylily.（5）The leaves of daylily’Autumn Red’suspected to be infected with leaf blight were collected and purified pathogenic bacteria were isolated.The fungal colony was snowy white radiate,the conidial period was like wheat ear,there were clusters of ovate or nearly oval conidial period,the spore wall was black,the contents were transparent,the spore size was about（3.63-7.27）μm×（3.63-7.27）μm,the spore wall was about 1.82μm.The phylogenetic tree was constructed according to ITS sequence of pathogenic bacteria,and combined with morphological characteristics,the strain was identified as Limonomyces culmigenus.Three fungicides,chlorothalonil,and copper calcium sulfate,were used to study the control effect.The semi-lethal concentration(EC₅₀)of 75%Chlorothalonil against fusarium culum was0.1147 mg/L,which showed a stronger inhibitory effect compared with the other two fungicides.50%humus EC₅₀and 77%calcium cupric sulfate EC₅₀are more than 30 times75%chlorothalonil EC₅₀,showing moderate inhibitory effects.Chlorothalonil was selected as the preferred fungicide.