The Research On Characteristics And Mechanisms Of Phenanthrene Degradation By Rice-endophytic Fungus Phomopsis Liquidambari Combination

Polycyclic aromatic hydrocarbons(PAHs)are persistent environmental pollutants because of their high toxicity(carcinogenic,teratogenic,mutagenic),difficult degradation and long degradation cycles.Phenanthrene(PHE)is a typical low molecular weight PAH containing three fused benzene rings.It has a high content in the polluted environment and can easily be distributed from water into organisms,sediments,and organic matter.PHE cannot be effectively degraded in plants and it is greatly hazardous for food safety and human health.At present,plant-microorganism combined remediation,which combined the advantages of microbial remediation and phytoremediation,has become an advantageous way to relieve the pollution of PHE in vivo and in vitro.In our previous works,we isolated a broad-spectrum endophytic fungus called Phomopsis liquidambari(B3)from the inner bark of Bischofia polycarpam’s stem.Our study has found that P.liquidambari can degrade the pollution of PHE in vitro.At the same time,it can successfully establish a symbiotic relationship with rice(Oryza sativa L.),an important food crop,and this combination has the potential to degrade PAHs in the environment.Therefore,the above results indicated that the combined system established by B3 and rice has beneficial effects on effectively degrading PHE pollution in vitro and in vivo of rice.However,the mechanisms for remediation of PHE by endophytic fungi B3 and the combined system remained unclear.Therefore,our study attempted to explore the effects and potential mechanisms of B3 on bioremediation of PHE from the aspects of degradation pathways by P.liquidambari and the role of rice and B3 by the combined system.We conducted pure culture experiment in vitro to confirm the ability of remediation of PHE contamination by B3 in vitro and explore the mechanism of degradation.The results showed that B3 can use PHE as carbon and energy source to grow,and the degradation rate of PHE reached 77%.Two degradation pathways were proposed,and the changes of four key degradative enzyme activities in them(P450 monooxygenase,epoxide hydrolase,dehydrogenase and catechol 2,3-dioxygenase)agreed with trends of corresponding enzyme genes expression,which increased firstly and then decreased.Above results laid a foundation for the research of bioremediation by combined system.We established two systems of PHE pollution in vivo and in vitro of rice to study the degrading-ability and mechanism that the combined system how to cope with PHE contamination stress.The measurement of physiological and biochemical indicators related to rice growth,energy production and resistance indicators reflected the role of rice in the remediation by combined system.The role of B3 was explored by measuring degradation enzymatic activities and corresponding gene expression associated with B3.The results showed that the combined system could degrade the PHE contamination in vitro and in vivo of rice,but the mechanisms existed similarities and differences between in vivo and in vitro.The combined system can enhance bioremediation by promoting plant growth,increasing root activity and chlorophyll content.However,during the PHE remediation in vitro of rice,chlorophyll content was significantly different with non-inoculated treatment in the whole experiment,while the significant difference occured after 21 days when remediation in vivo.Combined system did not significantly increase the activities of PPO and decrease the content of MDA,but it increased the SOD and POD activity during the remediation in vitro of rice;however,the content of MDA decreased,SOD activity increased significantly,and the PPO activity increased significantly in shoot when remediation in vivo.There was no significant difference in POD activity.The combined system can increase the ATP content in the shoot and root of rice at the seedling stage,but this system did not significantly increase the NADPH content in shoot during the remediation in vitro of rice.The expression of rice biomarker gene clearly showed the toxic effects on rice under PHE stress,the expression in the combined system was significantly lower than that of the non-inoculated treatment.Changes of phenanthrene-degrading genes of B3 in the combined system were consistent with the trends of their corresponding enzymatic activities,and the phenanthrenedegrading enzyme activities and gene expression levels in roots of rice were higher than those in shoot.During the remediation in vitro of rice,combined system improved the bacterial community in rice rhizosphere but inhibited the change of the fungal community.However,it improved both bacterial and fungal community structure of rice rhizosphere during the remediation in vivo of rice.A pot experiment was conducted to explore the application effect of biodegradation of PHE by combined system during the whole rice breeding period via four treatments(R,R+P,R+B3+P and R+inactivated B3+P)and different concentration of PHE contamination(50 mg kg-1,100 mg kg-1,200 mg kg-1).The results showed that the combined system had a significant effect on the remediation for low concentration of PHE in the soil,and the accumulation of PHE in the tissues of rice was significantly reduced in the combined remediation treatment at mature period.Inoculation with B3 could promote rice growth and increase yield,and the grain biomass of ripening stage increased by 20%compared with the non-inoculated treatment.The combined system may enhance the bioremediation ability by promoting the activities of soil defense enzymes(PPO and POD),as well as improving bacterial community structure and inhibiting the changes of fungal community structure in the soil.