Research On The Protective Effects Of Lentinan Against Fluopyram-induced Toxicity In Caenorhabditis Elegans

The southern root-knot nematode(Meloidogyne incognita)is one of the most important diseases affecting the yield and quality of crops in China.Fluopyram,a novel non-fumigant nematicide,is effective to control M.incognita.However,the application of fluopyram may pose potential risks to the environment and non-target organisms.Caenorhabditis elegans is a typical environmental model organism used for toxicological analysis,and its multiple indicator endpoints can be used for environmental risk assessment of pesticides.It has been reported that sublethal exposure to fluopyram increased the generation of reactive oxygen species(ROS)in C.elegans,induced oxidative stress and caused oxidative damage.When the antioxidant enzymes in C.elegans are insufficient to resist oxidative damage,exogenous antioxidants need to be added to fight against excessive ROS.Lentinan,a β-1,3-D-glucan extracted from Lentinus edodes,has been previously confirmed that it could improve the antioxidant capacity and reduce the oxidative damage in C.elegans.However,the protective effects of lentinan on oxidative stress induced by fluopyram remain largely unknown.Therefore,on the basis of previous studies,this study preliminarily explored the protective effects of lentinan against the fluopyram-induced toxicity in C.elegans.Firstly,the effect of lentinan combined with fluopyram on the control of M.incognita was determined through greenhouse experiments.Then,using C.elegans as a model,the effects of lentinan on the lifespan,reproduction and locomotion of C.elegans under fluopyram stress were observed through the changes of physiological indicators.Then,oxidative stress related indexes were measured,including ROS,malondialdehyde(MDA),catalase(CAT),superoxide dismutase(SOD),glutathione-S-transferase(GST)and succinate dehydrogenase(SDH).Then,the expression of oxidative-stress-related genes daf-16 and skn-1 was determined.Finally,transgenic C.elegans strains were used to study the protective effects of lentinan on fluopyram-induced toxicity in C.elegans.The main results are summarized as follows:(1)In the greenhouse experiments,lentinan could not significantly(p > 0.05)inhibit the number and development of M.incognita in the soil and cucumber roots.However,the number of M.incognita in the soil and cucumber roots decreased significantly(p < 0.05)after treatment with fluopyram,the development of M.incognita was also significantly inhibited.The combination of lentinan and fluopyram did not affect the control effect of fluopyram on M.incognita.These results indicated that the combination of lentinan and fluopyram had no negative effect on the nematocidal activity of fluopyram.(2)The toxicity of C.elegans was determined by 24 well-plate immersion method in vitro.The results showed that fluopyram was significantly(p < 0.001)lethal to the nematodes,and lentinan significantly(p < 0.001)improved the survival rate of the nematodes induced by fluopyram.(3)Results of physiological indexes showed that lentinan was highly efficient on reverting fluopyram-induced inhibition of lifespan.Compared with fluopyram,lifespan was significantly(p < 0.001)increased by 91.46% in the group of fluopyram+lentinan.In addition,lentinan could significantly(p < 0.001)improve the locomotion of nematodes and inhibit the brood size of nematodes induced by fluopyram.These results indicated that lentinan could revert the inhibitory effect on lifespan,locomotive and reproductive capacity of nematodes induced by fluopyram.(4)Biochemical results showed that lentinan could significantly(p < 0.001)reduce the ROS level and MDA content in the nematodes,and increase the activities of antioxidant enzymes SOD,CAT and GST in the nematodes after treatment with fluopyram.In addition,lentinan also significantly(p < 0.001)improved the inhibition of fluopyram target enzyme(succinate dehydrogenase,SDH)in the nematodes.These results indicated that lentinan could significantly(p < 0.001)alleviate the oxidative damage caused by fluopyram,and improve the antioxidant capacity of nematodes.(5)The expression of related genes showed that lentinan upregulated the expression levels of oxidative-stress-related genes daf-16 and skn-1 and their downstream genes(sod-3,hsp-16.2,gst-4 and gcs-1)in the nematodes after exposure to fluopyram.While in the GR1307 [daf-16(mg Df50)] and EU1 [skn-1(Zu67)],fluopyram+lentinan had no significant effects on the daf-16 and skn-1 downstream genes induced by fluopyram.Further studies showed that fluopyram+lentinan not only significantly(p < 0.001)enhanced the fluorescence intensity of SOD-3::GFP and GST-4::GFP,but also significantly(p < 0.001)promoted the nuclear translocation of DAF-16 and SKN-1 in C.elegans.In summary,the results of this study indicated that the combination of lentinan and fluopyram did not affect the nematocidal activity of fluopyram.Then,using C.elegans as model organism,the results demonstrated that lentinan had protective effects on the toxicity of C.elegans induced by fluopyram from the physiological,biochemical and molecular levels,and DAF-16 and SKN-1 pathways were involved.