| Aspergillus fungi are widely distributed filamentous fungi that exist in many different habitats in nature.Aspergillus fungi can produce various types of physiologically active metabolites.For thousands of years,Aspergillus have been an important fungus in food processing,fermentation industry and agricultural production.The chemical structure of Aspergillus metabolites is complex,diverse,and novel.These metabolites have various functions,such as antibacterial,anticancer,antioxidation,antiviral activities,antitumor,antidiabetes,lower blood lipids and so on.In addition,Aspergillus fungi are also widely used in agriculture,including in the field of biological fertilizers and in the field of biological control.Rice bacterial leaf blight?BB?caused by Xanthomonas oryzae pv.oryzae?Xoo?is one of the bacterial diseases with high destruction potential in rice-growing regions worldwide,and it is a threat to rice.The bacteria spread via water and invade rice through small holes or wounds in the leaves,resulting in a 10%to 50%reduction of yield or even crop failure.Thus,minimizing the economic losses resulting from rice BB is a pressing issue.In recent years,with the adjustment of rice planting structure and the changes of cultivars,especially frequent occurrence of catastrophic weather such as typhoon and rain,and the gradual accumulation of bacterial sources,the occurrence of bacterial blight in rice has increased again.Therefore,it is significant for agriculture reproduction to seek the methods of preventing rice BB.In this study,68 Aspergillus strains isolated from different habitats and 158Aspergillus strains preserved in the laboratory were used as materials to screen and obtain Aspergillus sclerotiorum As-75 which against Xoo.First,to screen for Aspergillus activity against Xoo.Isolated and purified from decaying fruits,moldy food,soil,air and other habitats,then according to their natural growth state and microstructural characteristics to identify 68 Aspergillus spp..The 68strains of Aspergillus strain collected and 158 Aspergillus strains preserved in the laboratory were used as test strains,and the physiological strain of Xoo P6 were used as indicator bacteria,one strain of Aspergillus that exhibited the strongest inhibitory effect against Xoo were identified by primary screening?Co-culture method?and secondary screening?Oxford cup method?,and its inhibition zone diameter was 31.3mm.According to morphologic features,microstructural characteristics,the results of physiological and biochemical experiments and ITS rDNA gene sequencing,compared their homology and constructed phylogenetic tree.Finally,the target fungus As-75 was identified as Aspergillus sclerotiorum,named Aspergillus sclerotiorum As-75.Second,analyzed the antimicrobial spectrum of A.sclerotiorum As-75fermentation filtrate.Selected seven plant pathogenic bacteria and nine plant pathogenic fungi as test strains.Uesd the physiological strain of Xoo P6 as indicator bacteria and determine the antimicrobial spectrum of A.sclerotiorum As-75fermentation filtrate.The antimicrobial analyses revealed that the fermentation filtrate well exerted antibacterial activity against Xanthomonas oryzae pv.oryzicola,Xanthomonas axonopodis pv.glycines,Pseudomonas syringae pv.tabaci,Pseudomonas syringa pv.tomato,Xanthomonas campestris pv.campestris and Ralstonia solanacearum.However,the fermentation filtrate exhibited no antibacterial activity against Pseudomonas syringae pv.glycinea.In addition,it exerted good antifungal activity against Fusarium solani,Gibberella fujikuroi,Gaeumannomyces graminis,Botryosphaeria berengerianade Notaris,Magnaporthe grisea;it exerted weak antifungal activity against Fusarium oxysporumf.sp.momordicae,Colletotrichum orbiculare,Alternaria solani,while it is of no effect to Fusarium pseudograminearum.Third,the antimicrobial different temperatures stability,different illumination hours and types stability and different pH stability of A.sclerotiorum As-75fermentation filtrate was tested by using Xoo P6 as the indicator bacteria.The test results showed that the fermentation filtrate of strain As-75 displayed maximum stability when under the 4000 lx fluorescent illumination,temperature is below 60?and pH is 6.5;the diameter of inhibition zone reduced when the tempreture was up to60?;under UV lamps?40 w?,the stability decreased with the irradiation time,but this decrease was small.When the pH is lower than or higher than pH 6.5,the stability of A.sclerotiorum As-75 fermentation filtrate was decreased.Fourth,in order to improve the antibacterial ability of the fermentation filtrate of A.sclerotiorum As-75 strain to Xoo P6,its original fermentation process?including medium formulation and culture conditions?was optimized by single factor optimization and response surface optimization.Finally,the optimum fermentation conditions for obtaining the secretion ability of antibacterial active substance of strain As-75 were?based on liquid fermentation medium?:glucose is carbon source,the soybean meal is nitrogen source,concentration of carbon source is 9.46%,concentration of nitrogen source is 0.57%,nitrogen ratio is 10:0.6,initial pH is 6.8,fermentation temperature is 28?,shaking speed is 171 r/min,fungal age is 6.05,inoculum amount is 5%.It was verified that under the above fermentation process,the antibacterial ability of strain As-75 fermentation filtrate was significantly improved,and the inhibition zone diameter was 47.9 mm.Fifth,a large amount of 30 L strain As-75 fermentation filtrate was fermented by a shaker,and 27 L of the fermentation filtrate was filtered by a vacuum pump,and dried by a rotary evaporator to obtain 15.4 g of orange-yellow ethyl acetate residue.A variety of active substances were obtained by silica gel column chromatography and gel column chromatography.The Xoo P6 race was used as an indicator,and the monomeric compound with strong antibacterial ability was obtained by the Oxford cup method.The diameter of the inhibition zone was 40.1 mm.Through nuclear magnetic resonance and electrospray ionization mass spectrometry?ESI-MS?analyses,thetargetcompoundwasidentifiedas?2Z?-2-butenedioic acid-2-?1-methylethenyl?-4-methyl ester;its molecular weight of 170.1622 daltons and formula of C8H10O4 identify.Sixth,preventative and curative effect trials:determined the preventative and curative effects of fermentation filtrate of A.sclerotiorum As-75.The leaves of the ZF802,Nipponbare,IR24,Wuyunjing21 and Zhonghuan 11 were artificially infected by Xoo.The experiment involved two control groups:a distilled water control group and an Xoo P6 control group.The experiment involved three treatment groups:method?1?,method?2?and method?3?.Method?1?:first,treated with fermentation broth,then infected with Xoo P6 physiological race;method?2?:first,infected with Xoo P6 physiological race,and sprayed with fermentation broth;method?3?:the leaves were first treated with bismerthiazol?chemicals?and the leaves were infested with Xoo P6 races.Trials of the preventative and curative effects demonstrated that the inhibition rate of leaf lesions in the five rice lines was up to 77.55%.Compared with the Xoo P6 control group,the disease index was reduced by up to 50%.And the control effect was up to 59.52%.In addition,through the comparison of biological liquid and chemical preparations,it is found that with the same concentration,the effects of the two are close to each other,and the biological liquid has better control effect.Combined with the results of the control experiment,it can be found that although the effects of the method?1?and?2?are better than those of the P6 control,the method?1?procedure?prevention?had a more significant effect than the method?2?procedure?curative?in preventing disease.Therefore,it is more effective to take preventive measures in the prevention and control of rice bacterial blight in agriculture.Seventh,preliminary assessment of ecological security.By investigating the effect of strain As-75 on seed germination rate and the toxicological effect on earthworm,it was shown that the fermentation filtrate had no toxic effect on rice seed germination of fthe five rice varieties;it had no lethal effect on earthworm and had no effect on earthworm activity.Therefore,preliminary assessment of the fermentation filtrate of strain As-75 is ecologically safe. |
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