Preliminary Study On The Interaction Mechanism Between Dendrobium Officinale And Anthracnose

ObjectiveDendrobium officinal Kimura et Migo is a perennial herb belonging to the genus Dendrobium,which has the effect of invigorating stomach,promoting saliva,nourishing yin and clearing away heat.Dendrobium officinale used fresh or dried stems as medicine.It is a valuable Chinese medicinal herb for both medicine and food.The wild resources of Dendrobium officinal distributed in Guangdong,Fujian,Anhui,Zhejiang,Guangxi,Yunnan and other regions of the humid and warm environment.Wild resources are becoming less and less,with demand increasing,Dendrobium ofcinal cultivated area continues to expand,intensive cultivation,coupled with poor ventilation,the growth environment is generally high temperature and high humidity,become a hotbed of various harmful pathogens.In recent years,artificial cultivation of Dendrobium officinal disease is increasing year by year,and is not easy to control,larger losses.In addition to the current of spraying pesticides,there is no effective prevention measures,disease prevention and control has become a key link in the planting industry,if you want to happen fast and efficient disease control must first understand the pathogens causing diseases,in order to put forward the corresponding prevention measures are efficient and easy.Therefore,Comprehensive morphological characteristics,cultural characteristics,this study Illumina HiSeq high-throughput sequencing and bioinformatics analysis of Dendrobium species and determine the pathogen,biological characteristics research of pathogenic species,provide the basis for disease prevention and control;and studies on metabolism of Dendrobium group anthrax infection after learning and proteomics,and to explore related diseases the metabolite and protein and its possible mechanism,lay the foundation for the cultivation of Dendrobium officinal resistant varieties.MethodsIn this study,through the establishment of Dendrobium officinal pathogen identification system,biological characteristics and system of Dendrobium anthracnose pathogen infection,metabolic anthrax physiology and proteomics,investigate and disease related metabolites,protein and its possible mechanism.Specific work includes:1.The establishment of identification system of bacteria isolated from Dendrobium officinal:isolation and purification of pathogenic strains from Dendrobium officinal leaf anthracnose,the pathogenicity of Dendrobium officinal for determination showed that the pathogenic bacteria,by observing the disease symptom,culture traits,Illumina HiSeq morphology,high-throughput sequencing and bioinformatics analysis methods to determine the species of pathogenic bacteria.2.According to the results of isolation and identification of pathogenic bacteria,the representative strains of anthrax pathogen were selected and transferred to the PDA medium plate.The test design of different medium,temperature,pH value,light,carbon source,nitrogen source and culture conditions of single factor 3-8 levels,effects of various factors on the growth of pathogenic bacteria,obtained the optimum conditions of their respective.3.Susceptible leaves of Dendrobium officinale collected by Colletotrichum gloeosporioides treatment,the sample pretreatment,GC/MS analysis,bioinformatics analysis to find out the differences of metabolites,preliminary study of different metabolites and its mechanism involved in disease.4.Susceptible leaves of Dendrobium officinale collected by Colletotrichum gloeosporioides treatment,the sample preparation,protein,SDS-PAGE protein electrophoresis sample detection,reductive alkylation and enzymolysis,reversed-phase chromatographic separation,protein identification,bioinformatics analysis to find out the differences of protein,explore the differences in proteins involved in disease and its mechanism.Results1.The establishment of identification system of pathogenic isolates of Dendrobium officinal:comprehensive Dendrobium pathogenic fungi culture characteristics,morphological characteristics,pathogenicity and Illumina HiSeq high-throughput sequencing and genome-wide phylogenetic tree of each representative strains were identified as white or brown root rot pathogen a hard shell bacteria(Rosellinia necatrix),Rosellinia necatrix,Colletotrichum gloeosporioides,Diaporthe ampelina,Podospora anserina,Glomerella cingulata,shell beans(Macrophomina phaseolina),Diaporthe ampelina.The DFW07-B strain failed to identify the species,but whether the new species was still uncertain,further studies are needed.The root rot pathogen of brown or a hard shell bacteria(Rosellinia necatrix),Diaporthe ampelina,Podospora anserina,Glomerella cingulata,shell beans(Macrophomina phaseolina)sphaerica is a new record of pathogenic fungi of Dendrobium officinal.2.Study on biological characteristics of pathogenic bacteria of Dendrobium officinal:Dendrobium officinal anthracnose was caused by Colletotrichum gloeosporioides.The most suitable medium for mycelia growth and spore production was PDA and PSA culture medium.The mycelia growth temperature ranges from 10℃ to 35℃,the optimum temperature range is 25℃ to 30℃,The Optimum spore production temperature is 20℃.The suitable pH values for mycelium growth and spore production were 4～9 and 7.Light is conducive to the growth of the mycelium,the darkness is conducive to the production of spores.The Optimum carbon source are glucose,sucrose,maltose for mycelia growth,the most suitable sporulation of carbon source is soluble starch.The most favorable nitrogen source for mycelial growth and sporulation is peptone.The lethal temperature and time of hyphae and spores are 55℃.The results show that PDA was suitable for mycelial growth.When the temperature is 28℃,the dense mycelia grew fastest and produced highest perithecia.The light can promote the perithecia.Mycelium growth rate was the fastest When the pH value was 8.The yield of perithecia was highest When the pH value was 9.Sorbitol was the best for mycelia growth.The mycelia is dense in the maltose carbon source media and perithecia production reached the highest.Mycelia had a highest efficiency using to peptone.The water bath lethal temperature of mycelium was 50℃.3.Dendrobium officinal infection metabolic anthrax studies:The multivariate statistical analysis models were established successfully,and 84 different metabolites were screened out from the identified metabolites by means of single dimensional statistical analysis.The results showed that the results of the statistical analysis were as follows:1.In addition to susceptible materials containing imino two acetic acid(iminodiacetic acid)83 metabolites abroad,and 36 of them were susceptible group specific metabolites,different up trend was most susceptible material content in the rest of the 47 metabolites,decreased only 4.May be correlated with the course of the metabolites have hydantoin,spermidine,Xiong Guogan,trehalose-6-phosphoric acid,catechol,hippuric acid,oxalic acid,and tartaric acid,malonic acid,catechol has aggravated the condition of Dendrobium officinal trend,further analysis and research.4.Dendrobium officinal infection proteomics research on anthrax:Successfully established analysis of Dendrobium susceptible group-normal group proteomics system iTRAQ technology,identified 3956 protein,protein trusted number 2246,screened 133 susceptible group normal group differentially expressed proteins,67 up-regulated protein,down regulated protein 66.GO function classification and enrichment show that these proteins mainly involved in the transport activity,catalytic activity and enzyme activity regulation,KEGG pathway significantly enriched in metabolic pathways,secondary metabolite biosynthesis,carbon metabolism,endoplasmic reticulum protein processing,glyoxylate metabolism,photosynthesis,photosynthetic organisms in carbon fixation.To find out the heat shock protein 70,sucrose synthase,antifungal protein,chlorophyll a/b binding protein related to the course of disease,and provide the basis for the study of the disease and the breeding of resistant varieties.In Dendrobium officinal susceptible leaves as experimental materials,Illumina HiSeq high-throughput sequencing technology to establish Dendrobium officinal pathogen identification system based on the identification of 7 pathogenic fungi,of which 6 species are new record species of Dendrobium officinal pathogenic bacteria.At the same time provide comprehensive genetic information of the pathogenic fungi,laying the foundation for the later cultivation of Dendrobium officinal resistant varieties.Study on the biological characteristics of Dendrobium Colletotrichum gloeosporioides and Glomerella cingulata has carried on a more comprehensive system,determine the most suitable bacterial growth medium,pH value,temperature,light,carbon source,nitrogen source culture conditions,provide reference for the prevention and treatment of Dendrobium officinal anthrax.Based on GC/MS,iTRAQ in different treatment groups respectively(disease group and normal group)of Dendrobium offcinal leaf samples of metabolomics and proteomics analysis,and identify metabolites and proteins and related diseases,provide the basis for the molecular level study of Dendrobium officinal diseases and breeding resistant varieties.