| The Eurasian woodwasp, Sirex noctilio F. (Hymenoptera; Symphyta; Siricidae) is a wood boring pest that has wide host ranges. Recorded hosts of S. noctilio have included species of Pinus, Abies, Larix, Picea, Pseudotsuga and Araucaria. Of these, Pinus species are most preferred.S. noctilio is native to Eurasia and North Africa, where it always attacks dead and dying Pinus species and is generally considered to be a secondary pest of low economic importance. However, with the development of global economic integral, the economic trade contacts of the international community, including cross-border trade of logs and other wooded products, are increasingly frequent. During the twentieth century S. noctilio has been accidentally introduced to several countries in the Southern Hemisphere and gradually spread to many countries and regions in Oceania, South America and Southern Africa. In these regions, S. noctilio attacks vigorous pine trees and thus causes considerable damage and cost to the local economies. For its highly destructive ability and risk to the forest ecosystem health, S. noctilio has been listed as one kind of the most important forest plant quarantine pests in the world by Food and Agriculture Organization.The Eurasian woodwasp Sirex noctilio Fabricius was first detected in Daqing, Heilongjiang Province, in the northeast region of China in August,2013. S. noctilio’s terrible attacks on host tree are in fact the act of its distinctive oviposition:Female insects drill 1 to 5 small holes in the host tree through the bark to the cambium, injecting phytotoxic venom together with the arthrospores of A. areolatum, and sometimes an egg, into the holes. Together, the three pathogenic factors, Sirex larvae, symbiotic fungus and phytotoxic venom act to weaken or even kill the tree. In this research we focused on the collaborative damage effect of the invasive Sirex noctilio and its fungal symbiont Amylostereum areolatum on host tree. The main research works were as followings:Based on morphological characteristics of the culture and ITS sequence, the fungal isolates obtained from the mycangia of adult S. noctilio females were identified. The biological and ecological characteristics of the successfully obtained isolates of A. areolatum on defined solid and liquid medium were investigated. Specifically, we examined the interrelationship between the insect venom and fungus by analyzing laccase activity from A. areolatum YQL03 in combination with several different concentrations of Sirex venom, as the inducer. Besides, the effect of Sirex venom on the laccase gene expression in A. areolatum YQL03 was also determined. Main conclusions are as follow:1. The location, patterns and structure of the main reproductive organs, mycangia, venom gland and venom reservoir of the adult S. noctilio female were determined by anatomical methods. The mode of connection structure, through which the ovaries, mycangia and venom reservoir of the female can be linked together, resembled that of T-branch pipe. It was just the well adapted "T-branch pipe" structure formed in the reproductive system of the female that assured the act of Sirex’s distinctive oviposition, in which all the materials could be deposited simultaneously into the host tree. Totally,7 isolates of A. areolatum was successfully obtained from the mycangia of adult S. noctilio females. All the seven isolates shared high sequence identity (≥99%) and belonged to one clone of A. areolatum.2. The response of the fungal isolate A. areolatum YQL03 to the factors in culture conditions, such as temperature, C/N ratio and pH value, was tested. The optimum growth temperature, the suitable C/N ratio and pH value in medium for A. areolatum YQL03 were determined. The biological and ecological characteristics of isolates of A. areolatum on defined solid and liquid medium were preliminarily made clear.3. It was found that the Sirex venom had some kind of prominent stimulating effect on the extracellular laccase production by the fungal isolate A. areolatum YQL03. With the method of spectrophotometry in which 2,2’-azino-bis (3-ethylbenzthiazoIine-6-sulfonate) (ABTS) was used as a reducing agent, the bioassay on the laccase activity of Sirex venom showed that the native venom contained laccase. The extracellular laccase production from the fungal isolate A. areolatum YQL03 was noticeably stimulated by both types of venom solution, regardless of dosage, with laccase production being significantly higher than the control throughout the whole incubation period after adding venom solution.4. The laccase gene lacYQL03B from the fungal isolate A. areolatum YQL03 was successfully cloned. The qRT-PCR analysis determined that the transcription level of the laccase gene lacYQL03B could be prominently enhanced by Sirex venom, which might partly reveal the molecular mechanism underlying the phenomenon that the laccase activity in the fungus was prominently stimulated by Sirex venom. Using RACE-PCR technology, the full-length cDNA of the laccase gene lacYQL03B was obtained. lacYQL03B encoded a putative protein of 509 aa with 20 aa as the signal peptide sequence. The structural analysis of the nucleotide sequence of lacYQL03B and the corresponding amino acid sequence encoded was conducted. Alignment of deduced amino acid sequence of the laccase gene lacYQL03B with those of other related fungal species was also conducted.5. The transcriptome profiling of the fungal isolate A. areolatum YQL03 was conducted. The difference between the two samples (sample 1 as the blank, sample 2 with Sirex venom as inducer) on transcriptional level of the whole fungal genome was determined. The differently expressed genes between the two samples were succeeded in annotation in nucleic and protein database. The pathogenicity-related genes and gene families, especially those involved in carbohydrate decomposition and lignin-degrading, were given further functional prediction and annotation. The results provide the molecular foundation for the further understanding of the pathogenic mechanism of A. areolatum and the collaborative damage effect of the Sirex venom and A. areolatum on host tree.The collaborative damage effect of the invasive Sirex noctilio and its fungal symbiont Amylostereum areolatum on host tree has been studies in this thesis. S. noctilio could effectively regulate the laccase gene transcription and the secretion of laccase of its mutualistic fungus A. areolatum through Sirex venom. During the regulation Sirex venom could prominently stimulate the transcription level of laccase gene from A. areolatum, leading to the corresponding enhanced laccase production by the fungus. It was just the Sirex venom that assisted the fungus in degrading the lignocelluloses of the host tree, by which the fungus established a productive infection, resulting in the decline of the tree vigor in a short time. In turn, Sirex larvae relied on the fungus for wood decomposition and food. Meanwhile, the wood-decaying symbiont dried the wood substrate providing a more suitable micro-environment for egg and larvae development. In conclusion, focusing on the stimulating effect of Sirex venom on the laccase gene transcription and laccase activity in A. areolatum, our present study has given a relatively reasonable interpretation on the collaborative damage effect of S. noctilio and its fungal symbiont A. areolatum on host tree. |
