| Cucumber wilt is a devastating fungal soil-borne diseases that seriously harms cucumber production.Cucumber wilt is caused by the specific infection of Fusarium oxysporum f.sp.cucumerinum,which can affect the cultivation and yield of cucumber.In production,prevention and control of cucumber fusarium wilt were mainly conducted by means of grafting control,chemical control and other measures to,but these measures also exited their own shortcomings and problems,such as seed-born disease before grafting,causing environmental pollution.In recent years,biological control has received more and more attention because of its safety,efficiency and pollution-free characteristics,and it has gradually become an important prevention and control measure for cucumber wilt.In this study,antagonistic strains of cucumber fusarium wilt were screened and identified by confrontation culture and pot experiment.A good combination of biocontrol bacteria and fungi was selected for the control of cucumber fusarium wilt.By detecting root activity,total active oxygen content,plant defense enzyme activity change and gene expression of enzyme activity of each treated plant,the mechanism of preventing and controlling cucumber fusarium wilt was investigated.The effects of different treatments and combinations of biocontrol bacteria on growth and yield of cucumber under pot and field conditions were determined.The results of experiement show as follows.10 bacterial strains were isolated from the healthy cucumber rhizosphere soil of Fusarium oxysporum in Taian.A bacterial strain J2 with good control effect on cucumber fusarium wilt was screened out through plate confrontation test and prevention and control disease in pot.Mycelia growth inhibition rate of pathogen on plate was 80.95%,and control efficacy was 72.61% in pot.Bacterial strain J2 was identified as Bacillus subtilis by morphological identification,physiological and biochemical identification and molecular biological identification based on 16 S rDNA gene.The mycelia growth inhibited by Bacillus subtilis J2 showed that it had a good inhibition effect on various plant pathogens.Bacillus subtilis J2 had good inhibitory activity against Fusarium oxysporum f.sp.momordicae,pathogen of albizia fusarium wilt,Valsa mali,Valsa sordida,Fusarium gramineam and Colletotrichum gloeosporioides of walnut,with inhibition rates of 81.01%,71.75%,77.48%,83.61%,69.77% and 63.49%,respectively.In this experiment,some basic characteristics of strain J2 were investigated.The content of IAA in the fermentation broth of strain J2 was determined by shaking flask,and the yield of IAA was 19.80 g/mL.The strain J2 could produce protease by detection,.Under the conditions of 28 ?,and 180 r/min fermentation,it was found exponential phase was achieved at 6 h,at the end of the 20 h exponential phase.In order to optimize the biological control technology,improve the effect of biological control and explore induced resistant mechanism of combination strains,we adopted pot experiment to combine the bacillus subtilis J2 with other several fungal antagonist.By comparing with single applying Bacillus subtilis J2,we screened good combination strains which have good effect on cucumber wilt disease.The results of experiment in pot showed that the combination of Bacillus subtilis strain J2 and Chaetomium globosum strain ND35 had a good control effect on cucumber fusarium wilt,and the control effect was better than that of single biocontrol agent.We futher explored the synergy mechanism of Bacillus subtilis strain J2 and strain ND35 combination,the results showed that root activity plays an important role in plant health for the prevention and control of cucumber fusarium wilt.Under the stress of pathogenic fungus,we determined the effect of different treatment on cucumber root activity,the results show that,J2 + ND35 could delay the decline of root activity,keep the root activity at a high level compared with other treatment.Active oxygen reaction is one of the early response of resistance to plant disease,then we analyzed effect of bio-control agent combination of each treatment on cucumber total reactive oxygen content.It was found that J2 + ND35 could enable cucumber response faster to stress and induced cucumber to quickly generate a large number of reactive oxygen species at the early stage of the sampling,but in the middle and later period,its active oxygen content rapidly reduced to FOC level.This study also tested the enzymes activity of several key defense enzymes related to plant disease(CAT,SOD,POD,PAL,PPO),the results show that under the stress of pathogen,the enzymes activity change a general downward trend after rising first.Among them,J2 + ND35 average defense enzyme activity in most of the sampling period to maintain a relatively high level.This study also tested the gene expression of the related enzymes,the results showed that the the gene expression was consistent with the enzyme activity levels,proving that the bio-control agent can affect the enzyme activity in the expression of gene transcription level.Among them,J2 + ND35 average enzyme gene expression levels and defense enzyme activity was significantly higher than other treatments,the result is consistent with the practical prevention effect of the treatment.The experiment in pot showed that the combined treatment had a significant promoting effect on the photosynthetic performance and growth index of cucumber.Compared with CK,chlorophyll A in J2+ND35 increased by 68.22%,net photosynthetic rate increased by 94.38%,and all growth indexes were greatly improved.Field test results showed that the yield of J2+ND35 was 22.65% higher than that of CK.Under the stress of pathogen,combination of bio-control agent J2 + ND35 prevention effect is better than single bio-control agent.Through exploring the mechanisms of combined bio-control microbial on growth promoting,it is speculated that the combined bio-control agents reduce disease and improve the plant disease resistance may be through directing antagonism,promoting plant growth,delaying the root activity dropped,balancing active oxygen metabolism,increasing defense enzyme activity of gene expression,improving the level of enzyme activity and improving the mechanism of defense reaction speed and scope of the plant.This study also laid a theoretical foundation for prevention and control of disease in the field and application of biocontrol agent combinations. |
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