| The modernization of agricultural methods,involving the overuse of chemical fertilizers,pesticides,and high-load operation of soil resources,have led to a decline in soil structure,and soil-borne diseases have become a major obstacle to continuous cropping for agricultural production.Various control strategies have been implemented to treat the diseases,especially the use of chemical fungicides.However,the application of chemical pesticides has harmful effects on human health,resulting in drug resistance of pathogens,disturbance of biodiversity,and seriously polluting the environment.Countries began to restrict the use of chemical pesticides.The application of biocontrol microorganisms and their active metabolites as a means of managing plant diseases,especially soil borne diseases,which has been an active research area for decades.The biological control effect of a single type of microorganism on controlling soil-borne diseases is unstable,easily affected by time and space.Bacillus amyloliquefaciens and Trichoderma longibrachiatum were selected to develop the mixed fermentation technology to apply the control of soil-borne diseases.The specific results are as follows:1.Mixed co-culture fermentation of biocontrol microorganisms to improve the control effects of soil-borne diseaseCompare the antifungal effects of the B.amyloliquefaciens(BCF)and T.longibrachiatum(TCF)monoculture fermentation broth,combined application(BTF),and co-culture fermentation broth(MCF)on the Fusarium oxysporum f.sp.Lycopersici,Botrytis cinerea,Trichothecium roseum,and Collelotrichum gloeosporioides.The result showed that TCF filtrate had no inhibitory effect on the growth of four pathogenic fungi,and the other two treatments could significantly inhibit the growth of them.Among them,BCF had the strongest antibacterial activity(inhibition rates were 89.84%,81.70%,53.22%,and 51.51%respectively),followed by MCF,with inhibition rates of 79.85%,54.04%,36.88%and 33.28%respectively.Furthormore,the effects of four fermentation broths on the spore germination of these pathogenic fungi were found to be similar to the inhibition of mycelium.The BCF group was the strongest,followed by the MCF group;TCF had almost no effect on the spore germination of these pathogenic fungi.Lipopeptide antibiotics are the main antifungal components of Bacillus spp,which were analyzed by HPLC.The results showed that there was no significant change in TCF compared with CK group.Ther chromatographic peaks in both BCF group and MCF group were similar,without increasing or decreasing such substances,but the contents of multiple peaks in MCF group were lower than those in BCF group.However,the metabolome of fermentation broths showed that some small molecular substances with antimicrobial activity,promoting plant growth,and inducing plant resistance increased significantly.MCF fermentation broth,two microorganisms coexisted and antagonistic growth.Pot experiment and field experiment were used to further study the control effect of the above fermentation broth on Tomato Fusarium wilt.The results showed that the fermentation broth was diluted 50 times and irrigated around the rhizosphere of tomato seedlings had inoculated with Fusarium oxysporum f.sp.Lycopersici.On the 9th day of control,almost all plants in the pathogen group were infected,and the disease index was>70%.The biocontrol effect of TCF on Tomato Fusarium wilt was 38.89%;BCF group was 47.22%;MCF group was 62.86%,which was approximately 15.78%higher than that of BTF group(52.78%).Antioxidant enzyme activity analysis:compared with the pathogen group,the activity of superoxide dismutase(SOD),peroxidase(POD),polyphenol oxidase(PPO)and phenylalanine ammonia lyase(PAL)in MCF group could maintain a high level and effectively reduce the content of reactive oxygen species(H2O2 and O2-),to reduce cell membrane permeability(MIL)and malondialdehyde(MDA).The biological control experiment in tomato greenhouse showed that the control effect of MCF applied continuously for two seasons was obvious,and the control effect in the second season was as high as 82.50%.The effect was stable,which could effectively inhibit the outbreak of tomato Fusarium wilt.2.The effects of fermentation broths of biocontrol microorganisms on soil physicochemical properties and soil microbial communitiesA certain concentration of BCF,TCF,BTF,and MCF were applied to the soil,the rhizosphere soil were sampled within 3 weeks to test physicochemical properties,and their rhizosphere bacterial and fungal communities were detected by macrogenomics.Compared with CK group,the application of TCF.BCF,MCF,and BTF could increase the richness and diversity of bacterial community.The changes of bacterial community richness in each group:BCF(2878)>MCF(2829.33)>BTF(2755.33)>TCF(2735)>CK(2609.67).The changes of bacterial community diversity:BCF(9.21)>BTF(9.12)>MCF(9.06)>TCF(9.03)>CK(8.96).Compared with CK group,the application of fermentation broth will reduce the richness and diversity of soil fungal community.The changes of fungal community richness in each group:CK(603.67)>BCF(564.67)>TCF(562.67)>MCF(515.67)>BTF(476.67).Changes of fungal community diversity:CK(5.31)>BCF(5.20)>MCF(4.05)>BTF(3.62)>TCF(2.30).In addition,compared with CK group,the application of BCF will lead to the increase of relative abundance of most pathogenic fungi.The relative abundance of Bacillus sp.in each group was BCF(0.237)>MCF(0.198)>BTF(0.155)>CK(0.011)>TCF(0.009).Compared with CK group,the inoculation of TCF would lead to an extremely significant decrease in soil fungal diversity(2.30),which may be related to the extremely significant increase in the abundance of Trichoderma sp.The relative abundance of Trichoderma in each group:TCF(0.778)>MCF(0.248)>BTF(0.244)>BCF(0.085)>CK(0.026).Compared with CK group,the relative abundance of Flavobacterium,Pseudomonas and Bacillus,which could promote plant growth,were significantly increased after the application of MCF.The reative abundance of some mycorrhizal fungi such as Mortierella and unidenified-glomeromota sp.,and Acaulopage which could prey on protozoa or nematodes were increased significantly in MCF group,and higher than that in BTF group,which may be more conducive to promoting crop growth.Compared with CK group,the application of BTF would cause the abundance of some pathogenic fungi such as Chaetotyrials spp,botryosporium,and Fusarium in soil to decrease significantly,and most of these fungi will also decreased significantly in MCF group.The application of fermentation broths in pot experiment would lead to the decrease of soil pH value.The soil pH of TCF group was significantly lower than that of CK group(P<0.05),approximately 3.99%.TCF group was significantly higher than BCF group,MCF group and BTF group(P<0.05),and there was no significant difference among the three groups(P>0.05).The content of soil organic matter in CK group,TCF group,BCF group,MCF group and BTF group were 9.05%,9.78%,8.23%,9.02%and 8.60%,respectively.Compared with CK group,the application of fermentation broth would increase the content of available nitrate nitrogen.The content of MCF group is the highest,approximately 28.39%and 34.04%higher than TCF group and BCF group,and approximately 13.05%higher than BTF group.There was no significant difference in ammonium nitrogen content among the groups(P>0.05).The content of soil available phosphorus in BTF group was the highest,which was approximately 53.86%,2.73%,34.20%and 18.83%higher than CK group,TCF group,BCF group and MCF group,respectively.The content of soil available potassium was the highest in TCF group,which was approximately 3.55%and 2.91%higher than CK group and MCF group;and it was significantly higher than that in BCF group and BTF group(P<0.05),approximately 14.30%and 9.01%higher,respectively.The application of fermentation broths would lead to the increase of soil invertase,phosphatase and protease.The soil invertases in TCF group,BCF group,MCF group,and BTF group were approximately 31.93%,21.89%,68.10%and 54.76%higher than that in CK group,respectively.The soil phosphatase activity of MCF group was slightly lower than that of TCF(approximately 0.35%);and 2.90%higher than that of BTF group;and significantly higher than CK group and BCF group,approximately 13.64%and 11.72%,respectively.The protease activities in TCF,MCF and BTF groups were significantly higher than those in CK group(P<0.05),approximately 23.48%,29.48%and 22.59%higher,respectively.The protease activities in BCF group and CK group were similar.On the contrary,the urease activity of TCF group,BCF group,MCF group and BTF group decreased by 4.61%,14.49%,4.55%and 16.95%,respectively compared with CK group.The cellulase activities of TCF group and BTF group were similar,decreased by approximately 30.72%and 33.19%,respectively,compared with CK group,followed by the MCF group,with a decrease of approximately 46.39%;BCF group was the lowest.In addition,the catalase activities of the BCF,TCF,MCF,and BTF groups were about 3.74%,5.03%,5.48%and 5.23%lower than that of the CK group,respectively.MCF was applied to kiwifruit orchard,it was found that the richness and diversity of bacterial community after MCF treatment were approximately 8.55%and 0.86%higher than CK group in the first year.In the second year,the richness and diversity of bacterial community in MCF group were approximately 13.45%and 1.88%higher than that in CK group,respectively.For fungal community,the richness and diversity in the first year after MCF treatment were not significantly different from that in CK group.However,in the second year of treatment,the richness of MCF group was slightly lower than that of CK group,but the diversity was approximately 17.71%higher than that of CK group.Comparing the MCF group and BTF group containing two microorganisms,the richness and diversity of bacterial and fungal communities in the two groups were less different.However,the application of MCF could maintain better fungal diversity and fungal richness.In addition,the relative abundance of some beneficial bacteria and mycorrhizal fungi were higher than that of BTF,and the abundance of most pathogens was lower than that of BTF group,which may be more conducive to crop growth and fungal disease control than BTF.3.Structural characterization of extracellular polysaccharide of Collclorichumgloeosporioides and its effect on fruit resistance inductionC.gloeosporioides was isolated from rotten peach fruit.The extracellular polysaccharide was extracted by alcohol precipitation after liquid fermentation.The polysaccharide separated by DEAE Sepharose Fast Flow at the concentration of 0.1 M NaCl was separated and purified.the molecular structure was analyzed by chromatography,spectrum,and nuclear magnetic resonance.A new type of long-chain polysaccharide with three branched chains,named CG-EPS,was obtained,which was mainly composed of mannose and galactose,with a weight average molecular weight of 93208 Da.Spraying 1.5 mg/ml CG-EPS on peach fruit could up-regulate the expression of ethylene synthesis gene in peach and promote the release of ethylene,the release was approximately 23.15%higher than that in CK group.It can accelerate fruit metabolism(CO2 yield),and the CO2 yield was approximately 7.97%higher than that of CK group.CG-EPS can also enhance antioxidant enzymes such as SOD,POD,CAT and APX,as well as defense related enzyme activities such as CHI and GNS,to up-regulate the expression level of its related genes,better inhibit the invasion of pathogenic fungi,improve DPPH free radical scavenging activity,and reduce the reactive oxygen species(H2O2 and O2-).The antioxidant enzymes such as SOD,POD,CAT and APX,and defense related enzyme activities such as CHI and GNS and other defense related enzyme activities,and up regulate the expression level of its related genes,better inhibit the invasion of pathogens,improve DPPH free radical scavenging activity,and reduce reactive oxygen species(H2O2 and O2-)4.Effects of mixed culture fermentation of biocontrol microorganism on postharvest fruit quality and shelf life.The effects of mixed fermentation broth on fruit quality and shelf life were studied on tomato and kiwifruit.The spraying(SMF)with 200 times dilution of MCF,rhizosphere irrigation(IMF)with 100 times dilution of MCF,and combined spraying and irrigation(SIF)would effect fruit quality and shelf life.The maturation process of SMF group and IMF group were approximately 12.75%and 20.81%slower than that of CK group on the 10th day of storage.The maturation process of SIF group was approximately 29.93%lower than that of CK group.The degree of lesion and shrinkage in SIF group was significantly lighter than that in CK group on the 25th day of storage.The changes of fruit firmness and weight loss corresponded with the maturation process.Although there was no significant difference in soluble solids(SSC)and total organic acids during storage,the contents of total phenols and total flavonoids of SMF,IMF,and SIF were approximately 15.08%,6.67%and 24.27%higher than that of CK group on the 10th day of storage,,respectively.The total flavonoids were approximately 21.99%,17.07%and 56.89%higher than CK group,which may be related to better prolonging fruit storage and slowing down fruit senescence.The SIF treatment was applied to kiwifruit trees with approximately 2L irrigation(TiL)and approximately 4L irrigation(T4L)per square meter.The results showed that the fruit size and fresh fruit weight of treatment group,especially T4L treatment group,were increased compared with CK group.On the 10th day of storage,the fruit firmness of treatment group(TiL and T4L)was higher than that of CK group,approximately 47.70%and 63.85%higher,respectively.The fruit weight loss was lower than that of CK group,especially T4L group.On the 10th and 15th day of storage,the ethylene release of the treatment group was significantly lower than that of CK group.On the 10th day,the ethylene yield of T4L group was approximately 16.36%lower than that of CK group.The respiratory metabolism(CO2 production)of TiL and T4L treatment groups were also lower than that of the control group,approximately 5.20%and 16.50%lower,respectively,indicating that the treatment group can effectively slow down the respiratory metabolism of fruits.During frut storage,the activities of enzymatic and non-enzymatic antioxidant systems such as total phenol content,total flavonoids content and SOD and POD activities in the treatment group were higher that in CK group,which seemed to be more conducive to deal with their physiological aging and adverse external environment.Proteins play different biological functions during fruit ripening,and are synthesized and degraded specifically at different stages of fruit ripening.The color of some protein bands increased with the concentration of fermentation broth and storage time,which affected the shelf life and quality of fruits.LC-MS/MS metabonomics analysis showed that the number of significantly up-regulated metabolites in T4L group was greater than that of significantly down regulated metabolites.These up-regulated metabolites were mainly related to amino acid synthesis and metabolism pathway,flavonoid and flavonoid synthesis pathway,nucleotide metabolism pathway,zeatin biosynthesis pathway,glucosinolate synthesis pathway,glucosinolate biosynthesis,aminoacyl-tRNA biosynthesis,and other metabolic pathways may lead to higher content of some known or unknown nutrients,which may be related to the improvement of fruit quality or the prolongation of fruit shelf life.In conclusion,the combined treatment of whole plant spraying and rhizosphere irrigation with MCF,could enhance the fruit quality and prolong the shelf life,which may be related to reducing the invasion of pathogenic microbiology,inducing resistance,and promoting growth.This agronomic measure could promote the more reasonable and effective utilisation of biocontrol microorganisms on fruit quality and preservation.In conclusion,MCF containing two biocontrol microorganisms with different mechanisms,had the potential to improve the survival and biocontrol of dominant strains,and could effectively improve the control effect of tomato Fusarium wilt.Improve soil physicochemical properties,increase the richness and diversity of soil bacterial community,and.reduce the richness and diversity of fungal community.MCF can also increase the relative abundance of beneficial bacteria and mycorrhizal fungi,reduce the abundance of most pathogenic fungi,and improve the control of soil-borne diseases and promote crop growth.The application of MCF to tomato and kiwifruit plants by spraying and root irrigation before harvest could improve the postharvest fruit quality and prolong the fruit shelf life.This study provides a new strategy and theoretical basis for the control of plant diseases by microorganisms in agricultural production,improving crop quality and prolonging storage shelf life which is of great significance to the development of sustainable agriculture. |
PDF Full Text Request