| Continuous cropping obstacles are common problems in facility agriculture,and the losses caused by soil-borne diseases of facility crops are particularly prominent.It is worth noting that inhibitory soils for root soil-borne diseases will be produced after long-term continuous cropping of some field crops(such as soybeans).According to the co-evolution theory of pathogens and antagonistic microorganisms,long-term continuous cropping can provide time and space conditions for accumulation of antagonistic microorganisms in the soil.However,researches on the formation of soil-borne disease-inhibiting soils and the key role of microbial community in long-term continuous cropping soil in facility agriculture are still rarely reported.Cucumber occupies an important position in the greenhouse vegetable industry,and continuous cropping can cause cucumber wilt with Fusarium oxysporum as the main pathogen.In order to verify that long-term continuous cropping can also accumulate antagonistic microorganisms of soil-borne disease pathogens in the protected soil and form inhibitory soil,a long-term continuous cropping experiment of greenhouse cucumber has been set up since 2013,which was planted twice a year,and the incidence of Fusarium wilt was investigated at a fixed time at seedling stage.In the13th crop of 2019,it was found that the Fusarium wilt disease index of greenhouse cucumber experienced the peak value during short-term continuous cropping,and then fell back to the healthy level of the initial planting period.In order to further analyze the dynamic changes of soil microbial community and the accumulation of antagonistic microorganisms of Fusarium oxysporum in the greenhouse cucumber under long-term continuous cropping,the root cirumference soil samples of greenhouse cucumber from 13 crops for rotation in 7 years of continuous cropping were collected,and real-time fluorescent quantitative PCR and high-throughput sequencing technology were used to study the dynamic changes of soil fungal and bacterial community abundance and diversity.The indigenous antagonistic microorganisms were isolated from the soil samples of the 13th crop of greenhouse cucumber,which could not only inhibit the pathogen Fusarium oxysporum,but also adapt to the salinization environment.Besides,in order to further explore the specific microbial community resources formed by the long-term continuous cropping measures,we aimed for the efficient removal of Fusarium toxin(DON)and found corresponding new species resources in the long-term continuous cropping soil.The main results are as follows:1.After a long-term continuous cropping of greenhouse cucumber,the copies number of fungal ITS gene and the genomic DNA mass of Fusarium in the root cirumference soil decreased from 1.8×10~7(number of copies/gram dry soil)and 54(ng/g soil)in the 3rd crop of continuous cropping to 4.3×10~6(number of copies/gram dry soil)and 15(ng/g soil)in the 13th crop.High-throughput sequencing studies found that the OTU number,Chao 1 index,Simpson index and Shannon index of the fungal ITS sequence in the root cirumference soil of greenhouse cucumber all showed a trend of first increasing and then gradually decreasing after continuous cropping.The results of principal component analysis showed that there were significant differences in fungal community structure similarity between long-term continuous cropping(continuous cropping 11th and 13th crops)and short-term continuous cropping(continuous cropping 3rd,5th,7th and 9th crop).The annotation results based on the level of the fungal order with the highest degree of reliability and completion showed that the relative abundance of Polyporales,Hypocreales and Eurotiales increased significantly after continuous cropping of greenhouse cucumber.In the further screening test of the pathogenic Fusarium oxysporum antagonistic fungal strains,the 10 fungal strains in the preliminary screening all had a certain degree of antagonism,and the strains Trametes hirsuta F1,Flavodon flavus F2 and Phlebia sp.F6 were isolated from the polyporales order mentioned above,The strains Clonostachys sp.F7,F8 were isolated from the polyporales order,and the strains Penicillium sp.F9 and F10 were isolated from the polyporales order.2.After a long-term continuous cropping of greenhouse cucumber,the abundance of bacterial 16S r RNA gene in the root cirumference soil did not change significantly(1.5×10~9-1.8×10~9number of copies/gram dry soil).High-throughput sequencing studies found that the OTU number,Chao 1 index,Simpson index and Shannon index of bacterial 16S r RNA sequence in the root cirumference soil of greenhouse cucumber all showed an increasing trend after continuous cropping.The results of principal component analysis showed that there were significant differences in bacterial community structure similarity between long-term continuous cropping and short-term continuous cropping.The annotation results based on the level of the bacterial order with the highest degree of reliability and completion showed that the relative abundance of Bacillales and Sphingobacteriales increased significantly with the increase of continuous cropping in greenhouse cucumber.In the further screening test of the pathogenic Fusarium oxysporum antagonistic bacterial strains,24 of the 50bacterial strains in the preliminary screening had a certain degree of antagonism,and the strains Bacillus sp.AB1-AB12,Bacillus sp.AB17-22,Cytobacillus sp.AB16,Lysinibacillus sp.AB23 and Lysinibacillus sp.AB24 were isolated from the Bacillus order mentioned above.3.After long-term continuous cropping,there were new microbial resources related to Fusarium toxin metabolism in the root cirumference soil of greenhouse cucumber.Taking the Fusarium toxin(DON)as the object,using a combination of primary screening by enrichment and acclimatization and high-performance liquid chromatography(HPLC)secondary screening,two bacterial strains RV12-1-1 and JB1-3-2 were obtained from the 13th soil sample of the greenhouse cucumber continuous cropping with relatively good detoxification effects.The two strains were identified as model strains of the genus Devosia and Oerskovia by polyphasic taxonomic research system,respectively.Based on the glutathione transferase and glucosyltransferase identified in the genome sequence prediction,it is speculated that the two new bacterial strains using the biodegradation pathway to achieve the removal of DON.In conclusion,long-term continuous cropping can affect the abundance and structure of root cirumference soil microbial community in greenhouse cucumber,enriching a large number of bacterial and fungal strains that have the effect of antagonizing the Fusarium oxysporum,thereby promoting the formation of cucumber wilt inhibitory soil.At the same time,there are many new species of Fusarium toxin degrading bacterial in the root cirumference soil of greenhouse cucumber after long-term continuous cropping.Therefore,the root cirumference soil of greenhouse cucumber after long-term continuous cropping has great potential to provide a variety of agricultural beneficial microbial resources. |
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