| It is difficult to avoid the continuous cropping of orchards during the renewal and reconstruction of orchards due to limited by land resources,which leads to the widespread occurrence of apple replant disease(ARD).The ARD has become a key limiting factor restricting the renewal and transformation of apple orchards and seriously hinder the sustained and healthy development of apple industry in China.In this study,earthworms were used as the optimal fermentation precursor protein,the fermentation conditions were optimized,the fermentation products were developed,the effect of preventing and controlling ARD was studied,and the antibacterial mechanism of the fermentation products were initially explored.The main results were as follows:1.Earthworm,fish and soybean meal could inhibit the growth of four pathogenic fungi caused ARD.Among them,the effect of earthworm fermentation products was the most obvious,and the inhibition rates of Fusarium oxysporum,Fusarium solani,Fusarium fusobacterium and Fusarium moniliforme were 71.48%,65.56%,65.93%and 70.74%,respectively.The effect of soybean fermentation products was the second,and the inhibition rate of four harmful Fusarium was between 48%to 54%,while that of fish fermentation products was the worst,with an inhibition rate of 31%to 46%.The pot experiment results showed that the fermentation products of three different protein materials improved the replanted soil environment and promoted the growth of Malus hupehensis Rehd.seedlings,among which earthworm fermentation products had the best effect.Earthworm fermentation products promoted the number of bacteria in replanted soil,which increased by 84%compared with the control,and had a significant effect on reducing the number of harmful fungi,F.oxysporum,F.solani,F.fusobacterium and F.moniliforme were decreased by 59.2%,52.0%,64.9%and 67.1%,respectively,compared with CK.The earthworm fermentation products also increased the soil enzyme activities and reduced the contents of phenolic acids in replanted soil.The biomass of Malus hupehensis Rehd.seedlings in the treatments of earthworm and soybean meal were significantly higher than that of CK,and the dry weight of Malus hupehensis Rehd.seedlings were 90.22%and 43.05%higher than CK,respectively.The fermentation products also promoted the growth of plant roots,as well as increasing the root antioxidant enzymes and root respiration rate.The total root length,surface area,root volume and root tip numbers of earthworm fermentation products were 147.21%,225.87%,299.85%and 291.03%higher than those of CK,respectively.Compared with CK,the activities of SOD,POD and CAT,and root respiration rate increased by 46.90%,133.94%,197.69%and 113.23%,respectively.2.The optimal fermentation conditions for earthworm were as follows:fresh earthworms and water were mixed at a ratio of 4:1,then crushed,pasteurized and connected with the fermentation bacteria,the ratio of fermentation bacteria and tissue homogenate was 1:1000.The effective viable number of Bacillus subtilis was 1×10~9 CFU/m L,the spore concentration of Aspergillus niger was 1.2×10~9 CFU/m L,the initial p H value was 7,and aerobic fermentation at 37?for 10 days.3.Earthworm fermentation products can inhibit the growth and reproduction of pathogenic fungi by inhibiting the mycelium growth of pathogenic fungi,destroying the integrity of mycelium,and inhibiting the spore germination.Earthworm fermentation products,sterilized earthworm fermentation products and earthworm homogenates have inhibitory effects on the growth of pathogenic fungi and the germination of spores.Earthworm fermentation products had the best effects,and the inhibition rates to four species of Fusarium were 83.81%,83.79%,84.03%and 86.31%,respectively;and the inhibition rates to germination of spores were 83.83%,83.15%,87.32%and 84.81%,respectively.The mycelium morphology of F.solani treated with earthworm fermentation products and the control were observed by scanning electron microscope.It was found that the mycelium integrity of F.solani was lost,grew messy and shriveled.Some mycelium was broken and damaged,and the growth points of the mycelium were also appeared abnormal and damage,then the content was overflow after treating with earthworm fermentation products.After separation and purification of earthworm fermentation products,the small molecule active peptide dry powder with a molecular weight of less than 1500 Da was obtained,which still had high antibacterial activity,and the inhibition rates of F.oxysporum,F.solani,F.fusobacterium and F.moniliforme were 78.33%,72.32%,65.50%and 69.99%respectively.4.The effects of earthworm fermentation products on the growth of Malus hupehensis Rehd.seedlings and replanted soil environment were studied under potted conditions.The results showed that earthworm fermentation products significantly promoted the growth of Malus hupehensis Rehd.seedlings under replanted conditions and effectively improve the replanted soil microbial environment.The number of bacteria in apple replanted soil increased by 102.5%,and the number of actinomycetes increased by 92.5%compared with the control after applying the fermentation products of earthworms.The four species of harmful Fusarium decreased by 67.3%,13.9%,84.2%and 24.1%,respectively;earthworm fermentation products effectively reduced the content of phenolic acids in replanted soil,in which the contents of phlorizin,phloretin and p-hydroxybenzoic acid decreased by 74.2%,48.9%and 67.0%,respectively;the root growth,root respiration rate and antioxidant enzyme activities were also promoted significantly,and the root respiration rate,SOD,POD and CAT activities increased by 69.4%,81.2%,81.1%and 161.5%,respectively;The addition of earthworm fermentation products greatly promoted the growth of Malus hupehensis Rehd.seedlings,and the dry weight of the plant was 2.58 times than that of CK;at the same time,the photosynthetic efficiency and chlorophyll content of the leaves were increased by 46.5%,and the contents of chlorophyll a and chlorophyll b of the leaves were 1.38 and 1.65 times higher than those of the control group,respectively.5.Field test results showed that earthworm fermentation products promoted the growth of replanted young trees and improved the soil environment and microbial community structure in replanted orchards.Earthworm fermentation products significantly promoted the biomass of replanted young trees in Dashaling,Fengmaozhai and Wantou village.The year when the apple orchards were establishment,the plant heights of the replanted young apple trees were increased by 16.25%,15.38%,and 15.65%compared with the control in the three places.The total number of bacteria and actinomycetes in the replanted soils was increased,but the number of soil fungi was reduced,changing the community structure after applying the earthworm fermentation products,and the abundance of Mortierella was 172.4%higher than that of continuous cropping control.The earthworm fermentation products improved the fruit qualities to a certain extent,in which the single fruit weight increased by 58.9%,the soluble solids increased by 26.8%,and the fruit firmness decreased by 24.6%.6.Under potted conditions,the effects of vermicompost on the growth of Malus hupehensis Rehd.seedlings and replanted soil environment were explored.It was found that vermicompost significantly promoted the growth of Malus hupehensis Rehd.seedlings and improved the soil environment of continuous cropping.Compared with CK,the dry weight of Malus hupehensis Rehd.seedlings treated with vermicompost(YF)and sterilized vermicompost(MYF)significantly increased by 351.1%and 348.2%.Both vermicompost(YF)and sterilized vermicompost(MYF)treatments significantly promoted the root growth of Malus hupehensis Rehd.seedlings,increased the activity of antioxidant enzymes and root respiration rate,and there was no significant difference between the them.The number of bacteria and actinomycetes in soil treated with YF increased by 107.8%and 97.7%respectively,and the number of fungi decreased by 17.1%.The results of real-time fluorescence quantitative PCR showed that the number of F.oxysporum in replanted soil treated with vermicompost YF and MYF were decreased by 51.0%and 57.6%respectively,and there was no significant difference between the two treatments.The activities of soil phosphatase,urease,sucrase and catalase in YF were 97.9%,540.9%,213.1%and 109.4%higher than those in CK,respectively.7.Field test results showed that the mixed application of earthworm fermentation products and vermicompost could significantly improve the soil environment and microbial community structure in replanted apple orchards,as well as promoting the growth of replanted young apple trees.The number of bacteria and actinomycetes treated with the mixed application increased by 73.98%and 106.45%,and the number of fungi decreased by 30.82%compared with CK;the promoting effect of the mixed application of earthworm fermentation products and vermicompost on the activities of urease,phosphatase and sucrase were obvious,which increased by 210.34%,59.47%and 84.24%respectively compared with CK,but it was not obvious on catalase activity;the mixed application also effectively reduced the content of phenolic acids in soil,the contents of phlorizin,phloretin,cinnamic acid,benzoic acid and ferulic acid decreased by 23.18%,42.91%,52.30%,36.74%and 36.30%respectively in 2018;the growth of young apple trees were promoted significantly treated with the mixed application of earthworm fermentation products and vermicompost,in the same year,the plant height,stem diameter,amounts of shoots and branch length of the fruit trees increased by16.31%,14.38%,17.92%and 15.41%respectively compared with CK,which increased by15.65%,9.4%,21.65%and 16.18%in 2019,respectively. |
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