Evaluation Of Hermetia Illucens Fertilizer On Reducing Antibiotic Resistance Genes In Potting Soil

The development of animal husbandry has led to the increase in the use of antibiotics for therapeutic purposes.About 30%-90% of the maternal compounds of antibiotics cannot be absorbed and eventually enter the soil with feces.It can increasing the antibiotics,antibiotic resistance genes(ARGs)and antibiotic resistant bacteria(ARB)in the soil,and seriously threatening soil and food safety.Thus,the development of low ARGs and green fertilizer is an important demand of agricultural fertilizer development.Black solider fly larvae(BSFL)can rapidly transform livestock and poultry feces.Previous studies have shown that BSFL have a good reduction effect on antibiotics and ARGs in livestock and poultry feces.In this study,by establishing tomato pot system,quantitative detection based on gene chip and high-throughput 16 S amplicon detection technology,taking the BSFL fertilizer manure from swine manure and chicken manure as the research object,the reduction of typical ARGs and their transmission were studied.At the same time,the effects on tomato plant growth,soil nutrient composition,key enzyme activity,soil microbial community structure and potential ARB were investigated,which provided theoretical and application basis for further development of fertilizer with low and no ARGs.The main results of this study are as follows:(1)Promoting the growth of tomato plants and improving the soil nutrientsIn this study,a greenhouse tomato potted system was established,BSFL fertilizer and control group of natural fermented fertilizer from swine manure and chicken manure sources was set up.The results showed that compared with the control fertilizer,the application of BSFL fertilizer could effectively promote the growth of tomato plants.The above ground fresh weight,underground fresh weight,plant height and leaf number of tomato in the BSFL fertilizer groups were increased by 440.3%,231.3%,59.4%,218.2%and 365.9%,231.3%,56.3%,138.0%,respectively.The contents of total nitrogen,total phosphorus and total potassium and the activities of urease,dehydrogenase and catalase in the BSFL fertilizer group were increased(> 5%).The BSFL chicken fertilizer was able to provide more potassium(increased by 7.7%),and the BSFL swine fertilizer was able to increase the total nitrogen and total phosphorus(10.7% and 7.8%).Therefore,compared with the control fertilizer,the application of BSFL fertilizer can significantly increase the nitrogen,phosphorus and potassium content and related enzyme activities in soil,so as to better play the fertilizer effect,improve the soil and promote plant growth.(2)Reduce the risk of ARGs import and horizontal transfer in soilHigh-throughput q PCR chip technology was used to detect 8 categories ARGs and 5MGEs in order to study the effects of fertilizer application on ARGs in potting soil.Results showed that ARGs and MGEs input could be reduced by the application of BSFL fertilizer.Compared with the control fertilizer,the total absolute abundance of ARGs and MGEs in BSFL chicken and swine fertilizer soil decreased by 39.2%,55.0%,78.9% and29.4%,58.5%,84.9% and 21.3%,12.0%,12.6% and 35.5%,38.6%,18.5%,after 0 d,60 d and 120 d,respectively.Besides,tnp A-04 and tnp A-05 were the main carriers of ARGs horizontal transfer in soil,and the absolute abundance of ARGs decreased significantly(the maximum reduction rate was 98.5%)in the BSFL fertilize group.Therefore,compared with the control fertilizer,the application of BSFL fertilize have better advantages in reducing ARGs and inhibiting the risk of its transmission.(3)The dynamic succession of soil microbial community and the reduction of potential antibiotic resistant bacteriaIn order to further understand the dynamic rule and mechanism of ARGs reduction in soil environment,we conducted high-throughput 16 S amplicon sequencing and analysis of bacterial community changes in potting soil,and elucidated the reduction of ARB.The results showed that Proteobacteria,Firmicutes and Actinobacteria were the dominant bacteria in soil.Clostridium sensu stricto and Bacillus were the dominant bacteria in soil.Further,the composition of bacteria is revealed through co-occurrence network analysis,Pseudomonas and Thermomonas is the most abundant potential resistant genera in swine and chicken manure systems,and its abundance decreased by 90.8% and 71.6% after 120 days of BSFL fertilization,respectively.In the control group,however,its abundance increased significantly(>1000%).Therefore,the application of BSFL fertilization could effectively reduced the potential ARB abundance of the soil,resulting in the low ARGs abundance.(4)Analysis of the main influencing factors of ARB reductionIn order to reveal the influencing factors and mechanism of ARB reduction caused by the application of BSFL fertilizer,this study monitored the contents of organic matter,total nitrogen,total phosphorus and total potassium in soil,as well as the activities of dehydrogenase,catalase and urease,and carried out the co-occurrence network analysis with ARB.The results showed that two nutrients(organic matter,total potassium)and three enzyme activities(urease,dehydrogenase,catalase)were significantly positively correlated with the abundance of ARB,and the concentration or activity of ARB decreased significantly(11.3%-73.0%)after 120 days of fertilizer application.These nutrients and enzyme may be the main influence factor of ARB reduction.In conclusion,this study has proved that the application of BSFL fertilizer can effectively reduce the abundance of ARB and ARGs in soil and reduce the risk of their transmission,which can be further developed and applied as fertilizer with low resistance.