Mechanism Of Soil Protist-mediated Microbial Interaction Against Fusarium Wilt Under Organic Fertilization

Soil-borne diseases have become one of the main limiting factors of global crop production,seriously threatening the sustainable development of world agriculture.Soil-borne Fusarium wilt,caused by the pathogenic Fusarium oxysporum,has become one of the most serious soil-borne diseases threatening crop safety in the world.There are complex interactions between plants and microbial communities.The composition and function of microbial communities in soil are key factors affecting plant growth and are of great significance for maintaining plant health.The disease-suppressing soil formed in the natural state has a unique microbial community that can effectively inhibit the occurrence of soil-borne diseases;on the contrary,using artificial measures to rationally regulate the composition and function of the soil microbial community can also effectively improve the soil disease-suppressing ability and enhance plant health.Soil microbial community is a complex multi-component collection,including bacteria,fungi and protist,and there are complex interactions between them.At present,the research on soil microbial community and soil-borne disease suppression mostly focuses on bacterial and fungal communities,and lack of protistan communities and the protist-mediated interactions with other microbial communities in soil-borne disease-suppressing.Therefore,it is very important to study the characteristics of soil protistan communities and their mediated mechanisms of microbial community interactions in disease suppression.The laboratory has previously established a field site for long-term application of organic and chemical fertilizers in the continuous cropping banana orchard in Ledong County,Hainan Province.And the study found that,compared with the application of chemical fertilizer,the continuous application of organic fertilizer can effectively change the bacterial and fungal communities and reduce the incidence of banana wilt disease.In this study,the field experiment was taken as the research object,and methods such as traditional plate culture,functional microorganism isolation,real-time quantitative PCR,high-throughput sequencing and metagenomic sequencing were used to reveal the effects of continuous organic fertilization on soil protistan community composition and Fusarium wilt disease of banana,the effects of soil protistan community that regulated by organic fertilization on rhizosphere and endosphere protistan community and its mediated potential mechanism of soil microbial interaction for disease suppression.And the use of protists and Bacillus inoculation experiments to explore the mechanism of protist-mediated disease-suppressive mechanism of microbial interactions.Relevant researches can provide support for artificially regulating soil microbial communities and their interactions,shaping a multi-trophic microbial control network,and inhibiting the occurrence of soil-borne diseases.The main findings of this research are as follows:1.Based on the treatments of continuous application of organic and chemical fertilizers at long-term field experiment site,the contributions of soil bacteria,fungi and protist communities to the control of banana Fusarium wilt after 5 years,7 years and 9 years of application of organic fertilizers were compared.The results showed that among the bacterial,fungal and protistan communities in the soil,the protistan community contributed the most to the control of banana Fusarium wilt.Based on these results,the effects of continuous application of organic fertilizers on soil protistan composition and diversity in banana orchards and the occurrence of banana wilt were studied.Compared with chemical fertilizer treatment,the application of organic fertilizer can significantly reduce the incidence of banana Fusarium wilt and the abundance of Fusarium oxysporum,and change protistan community composition without significant impact on diversity,and alter functional groups of protists and significantly increase the relative abundance of predatory protists.The protistan community composition in soil significantly affects the incidence of banana Fusarium wilt,and there is a significant negative correlation between the relative abundance of predatory protists and the incidence of banana Fusarium wilt.In addition,organic fertilization significantly slowed the decline in the relative abundance of predatory protists in the soil caused by long-term continuous application of chemical fertilizers.The results of random forest model analysis showed that,at the OTU level,the relative abundance of the predatory protist Cercomonas was an important driving factor for predicting the occurrence of banana Fusarium wilt,and the relative abundance of this protistan group was negatively correlated with the abundance of Fusarium oxysporum.In addition,continuous application of organic fertilizer stabilized the relative abundance of the predatory protist(Cercomonas)in soil.2.The effect of organic fertilization on the rhizosphere and endosphere protistan communities was further studied.Based on the fact that rhizosphere protists contributed the most to the inhibition of Fusarium wilt,the potential mechanism of microbial interaction mediated by rhizosphere protists was further studied.The results show that organic fertilization can alter the rhizosphere and endosphere protistan community composition,with rhizosphere protists being the key driver of Fusarium wilt.Among the functional groups of rhizosphere protists,organic fertilizer treatment significantly increased the relative abundance of rhizosphere predatory protists,and there was a significant negative relationship between the relative abundance of rhizosphere predatory protists and the abundance of Fusarium oxysporum.At the OTU level,fertilization treatments had the greatest effect on the relative abundance of Cercomonas taxa in the rhizosphere and the organic treatment significantly increased the relative abundance of Cercomonas taxa in the rhizosphere compared to the chemical fertilizer treatment.In addition,the bipartite model of the food web showed that organic fertilization enhanced the positive interactions between the rhizosphere predatory Cercomonas and Bacillus,and there is a significant negative correlation between the relative abundances of Bacillus and Fusarium oxysporum.Bacillus was the bacterial group most affected by fertilization,and organic fertilization significantly increased the relative abundance of Bacillus compared to the chemical fertilizer treatment in the rhizosphere.The results of metagenomic sequencing and its random forest model analysis showed that continuous application of organic fertilizer significantly promoted the relative abundance of the rhizosphere microbial Q gene cluster(involved in the biosynthesis of secondary metabolites),which is the key factor to inhibition of Fusarium oxysporum.Among all the genes in the Q gene cluster,the non-ribosomal peptide synthase gene COG1020 contributed the most to the suppression of rhizosphere F.oxysporum,and the continuous application of organic fertilizer also significantly increased its relative abundance.The results of gene microbial traceability showed that the contribution of Bacillus taxa to the relative abundance of Q gene cluster and COG1020 was significantly greater than that of other microbial taxa.Structural equation model analysis and greenhouse experiments showed that rhizosphere predatory protists promoted the expression of secondary metabolite synthesis genes by increasing the proportion of Bacillus in the indigenous bacterial community,and they worked together to inhibit Fusarium oxysporum in the rhizosphere.3.In view of the above-mentioned results of top-down control of rhizosphere bacterial community by predatory protists to inhibit Fusarium oxysporum,further field experiments using bio-organic fertilizer(containing Bacillus)and organic fertilizer were used to study the effect of Bacillus on the composition and function of protistan community,and further analyzed the mechanism of interactions between Bacillus and key protistan groups in suppressing pathogens.The results showed that,compared with organic fertilizers,the application of bio-organic fertilizer(containing Bacillus)significantly reduced the incidence of banana Fusarium wilt,increased the abundance of Bacillus,decreased the abundance of Fusarium oxysporum in the rhizosphere,changed the rhizosphere protistan community composition,and increased the relative abundance of rhizosphere predatory protists.The results of random forest model analysis showed that the relative abundance of rhizosphere predatory protists was a key driver for suppressing banana Fusarium wilt.At the OTU level,the Bacillus of the bio-organic fertilizer drove the increase in the relative abundance of the rhizosphere predatory Cercomonas compared to the organic fertilizer treatment,and the abundance of rhizosphere Bacillus was positively correlated with the relative abundance of Cercomonas.Meanwhile,there was a significant negative correlation between the relative change of rhizosphere predatory Cercomonas and the relative change of rhizosphere Fusarium oxysporum.4.In view of the interactions between the predatory protist and Bacillus found above,the indigenous Bacillus was further isolated and 8 different species of Bacillus were identified by means of morphology and molecular biology.The 8 different species of Bacillus were Bacillus amyloliquefaciens,Bacillus subtilis,Bacillus velezensis,Bacillus methylotrophicus,Bacillus pumilus,Bacillus altitudinis,Bacillus megaterium,Bacillus anthracis.Laboratory and greenhouse pot experiments were further used to explore the interactions between predatory protists and Bacillus and its disease-suppressive mechanism.The results showed that there were different interactions between predatory protists and different Bacillus species,and the strength of the interactions between them was mainly affected by the characteristics of different Bacillus species.The secondary metabolite(antibiotic)production capacity of Bacillus is the internal driving factor affecting the interactions between predatory protists and Bacillus.No matter in the PAS buffer or in the rhizosphere,predatory protists have weak predation intensity on Bacillus with strong secondary metabolite(antibiotic)production capacity.Therefore,we used a wild-type Bacillus strain and its mutant strain(disrupted in the bacillomycin D pathway)to further verify the effect of microbial secondary metabolites(antibiotics)on the interactions between predatory protists and Bacillus.The results showed that the predation intensity of wild-type Bacillus by predatory protists was significantly lower than that of mutant strains,and the antibiotic bacillomycin D produced by Bacillus can inhibit the predation of predatory protists.The greenhouse experiment was further used to analyze the disease-suppressive mechanism of interaction between predatory protists and Bacillus mediated by secondary metabolites(antibiotics).The results showed that the combination of predatory protists and Bacillus with strong secondary metabolite production ability has a strong ability to inhibit rhizosphere Fusarium oxysporum.Further greenhouse experiments using wild-type Bacillus and its mutants(disrupted in the bacillomycin D pathway)and predatory protists have confirmed that the combination of predatory protists and Bacillus with strong secondary metabolite production ability has a strong ability to inhibit rhizosphere Fusarium oxysporum.Therefore,interactions between predatory protists and bacteria mediated by secondary metabolites(such as the antibiotic bacillomycin D)help to increase the number of Bacillus with antagonistic pathogens in the rhizosphere and reduce the number of pathogens in the rhizosphere,promoting plant health.To sum up,the long-term organic fertilizer inputs effectively increased the relative abundance of predatory protists.There are more predatory protists(such as Cercomonas)in the organic fertilizer treatment,which can effectively colonize the rhizosphere.The secondary metabolites of rhizosphere Bacillus,especially the antibiotics with antagonistic effect on pathogens,can effectively resist the predation of predatory protists,and the predatory protists in the rhizosphere further prey on other non-or antibiotic-producing ability weaker Bacillus,vacating the niche for Bacillus with stronger antibiotic-producing ability,promoting its effective colonization in the rhizosphere.Pathogen-antagonizing bacteria(such as Bacillus)promoted by predation by predatory protists(such as Cercomonas)inhibit rhizosphere pathogens(such as Fusarium oxysporum)by secreting more secondary metabolites(antibiotics that have antagonistic effects on pathogens).The interactions between rhizosphere predatory protists and Bacillus can effectively maintain a healthy rhizosphere microbial environment and promote plant health.