| Aquilaria sinensis is the the genuine source of agarwood,which is a precious medicinal material and spice.Due to the extremely low yield of natural agarwood,which cannot meet the market demand,conducting research on artificial agarwood formation technology has become a top priority.With the purpose of revealing the mechanism and site of fungus-induced agarwood formation in A.sinensis,while investigating the structural changes of xylem and related biochemical reactions during the agarwood process,the present study was conducted to experiment artificial agarwood formation in A.sinensis using fungus-induced method.The samples were collected regularly and analyzed for structural and histochemical changes in the xylem,while the local chemical composition of the cell walls of different tissue types was analyzed by micro Raman spectroscopy;the characteristic chemical composition of the agarwood was analyzed by gas chromatography-mass spectrometry（GC-MS）;and the changes in the enzymatic activity of the xylem during the process of agarwood.The main findings are as follows.（1）The parenchyma cells near the xylem medulla of A.sinensis seedlings start to produce agarwood after being induced by the fungus.This process included cell death,the conversion of starch granules to soluble sugars,and the production of agarwood resin.The area where agarwood is being produced is the transition zone.With the deepening of the degree of agarwood formation,a large amount of agarwood resin accumulates in the parenchyma cells and vessels,and combines with the xylem.These xylem that have completed agarwood formation is the agarwood zone.After the degree of agarwood formation was further deepened,the fungi in the area near the pith gradually increased,and the enzymes produced by the fungi decomposed the xylem,while the xylem near the pith gradually rotted,eventually forming the decay zone.The area where the outermost layer of xylem does not start to form agarwood is the white zone.The zones differ markedly in structure and histochemistry.After entering the tree,the fungus expanded outward along the xylem vessels and xylem rays,and accumulated in the interxylary phloem and xylem rays and induced the production of agarwood.After the agarwood formation,agarwood resin and other inclusions blocked the pits and squeeze the mycelium to inhibit the fungus from continuing to grow and spread within the tree.The induction of fungi was the key to the formation of agarwood.（2）During the process of agarwood formation in A.sinensis seedlings,the secondary metabolites sesquiterpenoids and chromones were produced during the agarwood formation,among which sesquiterpenoids were produced later,and the number of species and the sum of relative percentages increased with the treatment time,and both reached the peak after 180 days of inoculation.Chromones were produced earlier,while the sum of relative percentages reached the peak after 60 days of inoculation.The characteristic products of agarwood were mainly distributed in the agarwood zone,and a small amount was distributed in the decay zone.The process of agarwood formation was accompanied by the regular changes of the chemical composition of the cell wall.The lignin,which is the main chemical component of the cell wall,had a higher distribution density in the corner of the cell,and the lowest distribution density in the secondary wall of the complex intercellular level.（3）In the process of agarwood formation of A.sinensis seedlings,PAL activity increased sharply in the transition zone,and the content in the transition zone-agarwood zone was stable,whlie decreased significantly in the decay zone.CAT activity increased significantly in the incense zone and decreased in the decay zone,but was still higher than that in the white zone.T-SOD activity increased significantly in the transition zone and agarwood zone,and decreased in the decay zone.MDA content increased significantly in the transition zone and decreased in the agarwood zone and the decay zone.The MDA content increased significantly in the transition zone and decreased in the agarwood zone and decay zone.（4）In the process of adult A.sinensis,the parenchyma cells in the white zone were all active,the average number of nuclei was 14.98 cells/10-2mm2,containing a large number of starch grains,the average number was about 55.04cells/10-2mm2,the average area of soluble sugars was 66.49%,whlie did not contain sesquiterpenes.The cells in the transition zone began to die,the average number of nuclei was 9.06 cells/10-2mm2,the average number of starch grains was about 55.04 cells/10-2mm2./10-2mm2,and the average number of starch grains decreased,the average number of 35.62/10-2mm2,the average area of soluble sugar was 57.96%,while sesquiterpenes began to appear,the average area of sesquiterpenes was 48.50%.All cells in the agarwood zone died,the average number of starch grains was extremely low,the average number of 8.62/10-2mm2,the average area of soluble sugar was while the sesquiterpene content increased significantly with an average color area of 84.61%.The decay zone had no nuclei and soluble sugars,and the starch grain content was extremely low with an average number of 4.17/10-2mm2,whlie the sesquiterpene content was low with an average color area of 6.42%.The process of agarwood of A.sinensis was accompanied by a regular change from starch to soluble sugars,which occurred mainly in parenchyma cells such as the axial parenchyma,xylem rays and axial parenchyma.The interxylary phloem were the most intense transformation zone,providing the structural and material basis for the formation of agarwood resin,and were the core of the production of sesquiterpenes and other characteristic products of agarwood.The aim of this study was to clarify the differences in xylem structure,histochemistry and local chemistry of each stratum during fungal-induced agarwood formation,to clarify the changes in defense-related physiological and biochemical indicators,and to provide the necessary theoretical basis and scientific basis for further investigation into the mechanism of fungal-induced agarwood formation in A.sinensis. |
