The Study On Jacaranda Acutifolia Stem Rot And Its Microecological Regulation By Endophytic Bacterium

Garden plants are an important part of urban ecosystem,which has many functions,such as beautifying the environment,reducing noise,purifying air,forming a good landscape,regulating microclimate and so on.It is not only an important part of urban facilities construction,but also an important part of urban spiritual civilization construction.Jacaranda acutifolia is an important species of garden landscape,mainly distributed in Panxi,southeast Sichuan and Chengdu plain.Jacaranda is applied to urban green space in the form of garden landscape,waterfront landscape,main entrance landscape and road landscape,and there are cultivation and production bases of jacaranda in many areas,which reserve seedling resources for the introduction and application of jacaranda in Sichuan.However,in the growing process of jacaranda,because of the special ecological environment,the leaves,flowers,fruits,branches and roots of jacaranda may suffer from a variety of biological pathogens,which threats the growth of jacaranda and garden scenery.It was found that the new disease of Jacaranda by the continuous fixed-point observation in the sample plot,the pathogens of diseased plants of J.acutifolia were isolated and the pathogenicity was determined,and the phylogeny of the pathogen was emphasized in combination with morphology and r DNA-ITS sequential analysis.On the basis of exploring the effect of pathogen on physiological metabolism of J.acutifolia,the endophytic microorganism with obvious antagonism to the pathogen was screened,in which the correlation with the jacaranda hormone level and the accumulation of root resistance related signaling substances was evaluated.The characteristics of antagonistic substance and the mechanism of action on the fungus of J.acutifolia stem rot were studied,which lays a foundation for the microecological regulation and control of garden plant diseases and the development of dual-purpose biological preparations for medicine and fertilizer.The main research results were following:（1）Fusarium chlamydosporum is the main new pathogen from the stem rot of jacaranda acutifoliaThrough the fixed observation to the fixed sample,a kind of new fungal disease（J.acutifolia stem rot）that infect the stem base of J.acutifolia was first discovered in the cultivation area of Xichang City,Sichuan Province and made a description of symptoms as well.Through diseased plant sampling,separation,single spore culture,and pathogenicity test,two representative strains A1 and B1 were obtained.The morphological characteristics of both strains respectively were consistent in Fusarium oxysporum and Fusarium chlamydosporum.r DNA were amplified by using fungal universal primers ITS1 and ITS4,and then the amplified products were recovered,cloned and sequenced to get the gene fragment of two size 516 and 523 bp respectively.Through landing NCBI to compare by blast,and establishing the phylogenetic tree of the higher homologous Fusarium strains,it was identified that A1 and B1 were F.oxysporum and F.chlamydosporum respectively.Both were diagnosed as the pathogens of J.acutifolia stem rot for the first time,which was first reported at home and abroad.（2）Property of the pathogenic factors are different,Non-protein as mainly substanceThe experiments on metabolites for isolation and purification of Fusarium chlamydosporum and F.solani by thin layer chromatography combined with column chromatography showed that the pathogenic factors of two pathogens were significantly different.Fusarium chlamydosporum can produce protein and non-protein substances,mainly non-protein substances,while Fusarium oxysporum is only partially active in nonprotein.The metabolite activity of Fusarium chlamydosporum was significantly higher than that of Fusarium oxysporum,which was one of the main factors of J.acutifolia stem rot caused by Fusarium chlamydosporum.The analysis of the common non-protein pathogenic factors of the two pathogens showed that there were 3 substances in Fusarium chlamydosporum and only 2 species in Fusarium oxysporum,and the spot area caused by them was significantly greater than that of the latter.Taking the optimum expansion agent of n-butyl alcohol:acetic acid:methanol=4:1:5 as the stationary phase,the nonprotein pathogenic factors of F.chlamydosporum which were purified by chromatography with silica gel H60 could change the membrane permeability（ion leakage enhancement）of jacaranda cortex,and aggravate the degree of membrane lipid peroxidation（increase of malondialdehyde content）,causing serious damage to the membrane,and the hysteretic effect of F.chlamydosporum to pathogenic factors.（3）The stress of F.chlamydosporum could influence the physiological metabolism and defense enzyme activity of Jacaranda acutifoliaThe determination of pathogen on physiological metabolism of J.acutifolia showed that the threat of pathogenic factors was better than that of F.chlamydosporum.The photosynthetic rate、transpiration rate and chlorophyll content of the jacaranda leaf could be decreased by F.chlamydosporum.The reduction rate was positively correlated with the time of invasion.The longer the invasion time,the greater the reduction rate.The range of the net photosynthetic reduction rate was better than transpiration reduction rate.The soluble sugar content of leaves showed a significant downward trend in the whole period of the test,and the reducing sugar content of leaves increased slowly after the action of pathogenic factors,but the difference was not obvious.The contents of total nucleic acid,DNA and RNA of jacaranda decreased with the time prolonging under the stress of F.chlamydosporum,which was related to the enhanced activity of DNase and RNase.The variation trend of soluble protein content and protease activity of Jacaranda is consistent with metabolism of nucleic acid.The responses of the defense enzyme of Jacaranda to pathogenic factors were as follows:POD activity always showed a upward trend with the inoculation time prolonging and reached up to the level of 262.09 u/g FW after inoculating40d;SOD activity increased after inoculation and reached peak 102.33 u/g FW on the 20th day,then decreased rapidly to the level of activity before inoculation;PPO activity showed a big trend of decreasing after reaching peak 138.52 u/g FW on the 10^thday,but still maintained higher level before inoculation,but CAT activity changed little before and after inoculation,except that a transient rise quickly came back to the state before inoculation.Compared with the effect of pathogenic factors,although there are differences in the effect of F.chlamydosporum on the activity of jacaranda defense enzyme,the effect is not significant at earlier stage,and the effect is similar to that of pathogenic factors during later stage.The physiological responses of Jacaranda that were stressed by F.chlamydosporum had significant correlations with disease index.The contents of soluble sugar,malondialdehyde,chlorophyll,net photosynthetic rate,nucleic acid,protein and the activities of SOD,POD and PPO could be used as indicators for the evaluation of the pathological reaction of jacaranda.（4）Bacillus amyloliquefaciens from the root cortex of Jacaranda acutifolia is an endophytic bacteria with potential biocontrol abilityAfter sterilizating the surface of the root of J.acutifolia,24 strains of endophytic strains,mainly two genus of Bacillus and Pseudomonas,were obtained by dilution plate method.F.chlamydosporum as the tested fungus,the strain zhu66 with inhibition ratio of97.8%was screened from 24 strains by five points confrontation method firstly and plate diffusion method secondly.It was identified as Bacillus amyloliquefaciens by the 16S r DNA sequence and physiological and biochemical tests.The Gen Bank accession number was KJ865860.（5）Bacillus amylonaturiens has the dual function of disease prevention and growth promotion,and has a significant microecological regulation effect in urban green space plants.Greenhouse pot experiment showed that B.amyloliquefaciens could effectively prevent and cure J.acutifolia stem rot.On certain concentration,the concentration was higher,the control effect was better,for 10⁷cfu/m L at the optimal concentration.The control effect of sterilized soil was better than the natural soil.So in the planting,the best way was to sterilize soil or at least root dipping treatment.The control effect of biocontrol strain prior to pathogen was better than that of biocontrol strain and pathogen inoculated at the same time,which had important significance in the productive practice and reflected the control characteristics in which the biological prevention predominates.On the contrary,the control effect of pathogens prior to biocontrol strain was lower.B.amyloliquefaciens not only had biocontrol function,but also could obviously promote the growth of stems,height and crown diameter of J.acutifolia,the treatment of only pathogen,the stem growth of J.acutifolia was severely affected,and the growth rate was the lowest.The stem,height of seeding,crown width was 2.2cm,108cm,45cm respectively.The concentration of antagonistic bacteria was higher,the effect of growth promoting was better.The stem diameter,height of seedling and crownwidth was 4.2-4.3cm,135-136cm and 62-63cm respectively.Field experiment further verifies,the biocontrol effect to J.acutifolia stem rot with different degree had remarkable differences.The preventive effect of ward was very significant,which could have no occurance of stem rot and the rate of control effect reached 100%.There were almost no effect in the severe areas,obvious control effect in the mild areas that reached 74.1%,a certain effect in the moderate areas that was 48.5%,and with the extension of time,the control effect increased,which reflects the ecological sustainable concept of biological control.（6）Endophytic Bacillus amyloliquefaciens have important influences on the levels of endogenous hormone and the accumulation of resistance-related signal substances in jacarandaThe effect of B.amylonaturfaciens on the endogenous hormones of jacaranda was determined by indirect enzyme-linked immunoassay.The responses of the four hormones to the stress of B.amylonaturfaciens and F.chlamydosporum showed different manifestations,and different inoculation methods of B.amylonaturfaciens significantly affected the hormone level of jacaranda root system.Inoculating B.amylonaturfaciens only,the two hormone levels（IAA and GAs）existing in the root system of jacaranda were significantly higher than the control and other treatment;the ABA level had no significant difference among treatment;B.amylonaturfaciens did not stimulate or decrease the ZR in the root;and the reaction to antagonistic bacteria,pathogenic fungus and the Collaborative inoculation of them was not large.B.amylonaturfaciens inoculation can increase the level of ZR hormone,but have no relativity with the challenge inoculation method of F.chlamydosporum.B.amylonaturfaciens on the accumulation of SA and H₂O₂existing in the root of jacaranda was determined by HPLC and spectrophotometer respectively,and early bioinoculation treatment could stimulate the generation of H₂O₂in the root of jacaranda.The content of H₂O₂in the root of jacaranda inoculated at 6h by the three methods of pathogenic fungus,biocontrol bacteria,biocontrol bacteria and pathogenic fungus at the same time was significantly higher than that of the water control,while at12-24h,the content of H₂O₂treated by pathogenic fungus was similar to that of the control.The content treated by biocontrol bacteria,biocontrol bacteria and pathogenic fungus at the same time was still significantly higher than that of the control.After 48h,the content by the three measures was not significantly different from that of the water control,which was returned to the pre-inoculation level.The three inoculation methods could stimulate the total SA content in the root,and the early activation of pathogenic fungus is significant.After reaching the peak at 12h,it gradually decreased with time prolonging,and it was lower than the control at 72h.The SA content of challenge inoculation was at the maximum excitation,which fluctuated with time after reaching the highest level at 6h,and closed to the control at the later stage.Although the bioinoculation at the initial stage could not significantly stimulate SA,which reachend the peak at 24h and decreased,all of them were significantly higher than the control.The content of SA varied with different types.The treatment of biocontrol strain mainly stimulate the stable production of high-level free SA of jacaranda for a long time,the treatment of biocontrol strain and pathogen inoculated mainly stimulate the free SA of jacaranda at the early stage,and the treatment of pathogen inoculated could stimulate equal free and combined SA of jacaranda at the early generation.（7）B.amylonaturfaciens produced a kind of novel antibacterial protein with strong inhibitory activity on Fusarium chlamydosporumThe crude protein,which had antibacterial activity,were obtained by the fractionation method of ammonium sulfate from the sterile fermentation of B.amylonaturfaciens.On the basis of obtaining highly active mixed-proteins by weak anion exchange chromatography,pure antibacterial proteins with strong inhibitory activity against Fusarium chlamydosporum were obtained through Sephadex g-50 molecular sieve gel.After dialysis concentration,SDS-PAGE electrophoresis and comus brilliant blue staining,only one band was shown,which was completely coincident with the 29k Da band of Marker.According to the Standard protein molecular weight and Standard curve of relative mobility,the apparent molecular weight of the antibacterial protein was calculated to be 29k Da.The N-terminal eight-amino-acid sequence,NH₂-Gly-Arg-Pro-Leu-Pro-Leu-Ala-Ala,was determined using the automatic Edman degradation method.A sequence alignment search was performed in the NCBI BLAST protein database.A 100%homology was found with the amino acid residue sequence at position 154–161 of the hypothetical protein from Chromobacterium sp.LK1.However,no known antimicrobial protein with high sequence homology was found,which indicated that the antimicrobial protein obtained in this study constitutes a novel protein moiety.