| Pine wilt disease caused by pine wood nematode(PWN)gives rise to devastating ecological disasters and major economic losses to the forestry of the incoming area,causing a lot of attention in many countries in the world.It is listed as a key quarantine object.Pine wood nematode is China’s largest forestry invasion pest,and its population growth and spread is extremely rapid.At present,the occurrence area in China has expanded from southern pine forest to the whole country.At present,the prevention and control strategy is mainly to clean up dead wood and control the vector beetle,and there is no effective and directly applicable method for prevention and control of PWN.Due to the adverse effects of chemical pesticides on human health,environment and biology,biological control of PWN is of great strategic significance to the healthy development of national sustainable forestry.Esteya vermicola is currently the most extensively studied PWN biocontrol fungus,and is the world ’s first reported parasitic fungus of PWN.E.vermicola attracts PWN by releasing volatile substances,and adheres and infects PWN with crescent spores.The indoor control effect shows that E.vermicola is a PWN biocontrol fungus with great potential.However,the transcriptional expression of E.vermicola in response to PWN during the attraction phase is unclear,and the transcriptional and metabolic differences of E.vermicola under different nutritional conditions are also unclear.In this thesis,E.vermicola is studied at the stage of attracting PWN,revealing the changes in the transcriptional expression of E.vermicola in the presence of PWN,and digging out the key genetic factors of E.vermicola’s response to PWN;also studying the expression of infection PWN-related genes and metabolism of E.vermicola under different nutritional conditions(carbon and nitrogen).The research in this paper provides valuable genetic resources for the research of genetically improved highvirulence E.vermicola,an important biocontrol fungus of PWN.The main findings are as follows:(1)Through PWN induced E.vermicola experiment design and test,E.vermicola phenotype with obvious difference happened after induction.After PWN induced E.vermicola,the back of the E.vermicola colonies began to turn black on the 1st day,and the colony growth accelerated and aerial mycelium was thickened,the front of the colonies gradually turned green,and the biomass increaseed.And E.vermicola continued to change with the extension of induction time.The growth rate of the treatment group which was induced by PWN was greater than that of the control group in the first four days.The difference in colony diameter between the control group and the treatment group was only about 0.2 cm.From the 4th day,it started to grow rapidly until the 10 th day.The treated group had a diameter of 2.6 cm larger than that of the control group,and is 50% larger than the control.The colonies induced by PWN turned green as a whole,while the control was light green at the edges and white at the center.(2)E.vermicola rapidly propagated and extended through quorum sensing to enhance the area and intensity of capturing PWN after capturing the signal of PWN;E.vermicola quickly adjusted metabolism and quickly conducted the PWN signal to E.vermicola through MAPK signaling pathway,and up regulated the expression of the receptor genes that accepted PWN pheromone;E.vermicola up regulated the expression of GLEYA protein domain,CFEM protein domain,WSC protein domain and lectin and other cell surface adhesion proteins,which is better preparation for adhesion to PWN;E.vermicola up regulated expression of subtilisin,serine protease S28,cutinase transcription factor,and PWN body wall degrading enzyme to prepare for penetration of PWN cuticle;melanin synthetic genes that contribute to the pathogenicity of fungi were also significantly up regulated;E.vermicola may release volatile substances through insect pheromone synthesis pathways to attract PWN.(3)Transcriptome data of PWN biocontrol fungus E.vermicola under nitrogen-rich medium carbon-rich medium were obtained,and differentially expressed genes and specifically expressed genes were analyzed.Not only did the E.vermicola fungus grow rapidly on nitrogen-rich medium,but the total number of genes and the specifically expressed genes expressed by E.vermicola fungus on nitrogen-rich medium were higher than those on carbon-rich medium.Related genes such as subtilisin and toxin synthesis with nematicidal effect were significantly up regulated on nitrogen-rich medium.Nitrogen-rich culture conditions were conducive to the extended growth of E.vermicola mycelium and the expression of highly virulent genes.(4)E.vermicola metabolites were significantly different under different culture conditions of carbon-rich and nitrogen-rich.Phosphoguanidinoacetate and p-cresol sulfate were unique metabolites produced in large quantities under nitrogen-rich culture conditions,5628 times and5438 times of those under carbon-rich culture respectively.Nitrogen-rich culture conditions produced more abundant metabolites than carbon-rich culture conditions.Most metabolites were up-regulated under nitrogen-rich conditions,and the biosynthetic metabolic pathways of ubiquinone and other terpenoid quinones were significantly enriched.The metabolites photopigment,indole,allantoin and trehalose were significantly up regulated under nitrogen-rich conditions.Photopigment and indole may be signal molecules between bacteria and fungi,and allantoin and trehalose may be supplied as nitrogen-rich and carbon-rich sources for utilization of E.vermicola fungus intracellular symbiotic bacteria.In summary,this article analyzed the gene expression of E.vermicola in the presence of PWN but not in contact,and explored the receptor gene and signal transduction pathway of E.vermicola corresponding PWN signal,and genes related to adhesion and infection.The research provides a batch of valuable genetic resources for the strong attraction and high virulence genetic improvement of E.vermicola.Through a comparative study on the growth,transcription and metabolism of E.vermicola under carbon-rich and nitrogen-rich nutrition,a nitrogen source medium suitable for the rapid growth of E.vermicola and the expression of highly virulent factors was found.It provides a theoretical basis for the large-scale cultivation and application of E.vermicola in the wild,and discusses the possible signaling molecules between the biocontrol fungus E.vermicola and its intracellular symbiotic bacteria. |
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