| Fusarium head blight(FHB)in wheat is a worldwide fungal disease,which can lead to the decline and complete loss of yield,and occurs in all the wheat growing regions around the world.In recent years,with the increase of fungicide use time and dose,FHB pathogens have developed resistance to carbendazim.Moreover,carbendazim-resistant frequency and range have increased year by year,and the disease control efficacy has significantly decreased.However,people have also tried to control FHB by increasing the dosage of fungicides,which further aggravated the pollution of pesticide residues and ecological environment.In recent years,new respiratory inhibitors,such as succinate dehydrogenase inhibitors(SDHIs)and quinone outside inhibitors(QoIs),have appeared in the market of fungicides.Previous studies from Fungicide Biology Laboratory of Nanjing Agricultural University found that azoxystrobin had a good control effect on FHB,but it could stimulate DON biosynthesis,and increase the amount of DON produced per mycelial weight significantly.Based on the above research results,whether different varieties of QoIs exhibit food safety problems caused by mytotoxin contamination in the control of FHB and the molecular regulation mechanism of QoIs on DON biosynthesis has becomed an important scientific issue throghtout the world.In this study,the mycelial growth inhibition and spore germination inhibition methods were used to determine the fungicidal activity of six QoIs(pyraclostrobin,coumoxystrobin,picoxystrobin,fluoxastrobin,azoxystrobin and fenaminstrobin)against the 32 F.graminearum strains,respectively.The results showed that,for the mycelial growth inhibition,the EC50 values of pyraclostrobin,fenaminstrobin,picoxystrobin,coumoxystrobin,fluoxastrobin and azoxystrobin against the 32 strains ranged from 0.0492 to 0.1557 μg/mL,0.0199 to 0.4158 μg/mL,0.0532 to 0.5093 μg/mL,0.0433 to 0.9855μg/mL,0.2680 to 1.7754 μg/mL,and 0.2739 to 1.2403 μg/mL,respectively,the average EC50 values were 0.0859 μg/mL,0.1115 μg/mL,0.1220 μg/mL,0.2902 μg/mL,0.8413μg/mL and 0.9663 μg/mL,respectively.For the spore germination inhibition,the EC50 values of picoxystrobin,pyraclostrobin,fenaminstrobin,coumoxystrobin,azoxystrobin and fluoxastrobin against the 32 strains ranged from 0.0642 to 0.1352 μg/mL,from 0.0379 to 0.1953 μg/mL,from 0.0784 to 0.2509 μg/mL,from 0.0810 to 0.3518 μg/mL,from 0.2065 to 1.8422 μg/mL,from 0.2453 to 1.9818 μg/mL,respectively.The average EC50 values were 0.0940 μg/mL,0.0948 μg/mL,0.1248 μg/mL,0.2149 μg/mL,0.6101 μg/mL,0.9260μg/mL,respectively.The above results showed that,Qois exhibit excellent fungicidal activities on either mycelial growth or spore germination of F graminearum and have the broad application prospect in the control of FHB.In order to reveal the regulatory mechanism of QoIs on DON biosynthesis in F.graminearum,in this study,six QoIs,pyraclostrobin,coumoxystrobin,picoxystrobin,fluoxastrobin,azoxystrobin and fenaminstrobin,were used to determine the regulatory effect on DON biosynthesis against 3 F.graminearum strains,including FgS-1,FaS-5 and FaR-11.The results showed the corresponding EC50,10×EC50,100×EC50 values of six QoIs could accelerate DON biosynthesis in F.graminearum,0.51-8.7 times higher than the control groups,and significantly up-regulated TRI5 expression,which is the key gene in DON biosynthesis pathway.Moreover,QoIs could significantly increase acetyl coenzyme A(acetyl-CoA),a precusor substance of DON biosynthesis pathway which further confirmed that QoIs with the corresponding EC50,10×EC50,100×EC50 values had a stimulating effect on DON biosynthesis in F.graminearum.In order to further reveal the effect of QoIs on the pharmacological meshinasm of F.graminearum,we measured the biological characteristics of the selected strains,including cell membrane permeability,respiratory rate,ATP content,acetyl-CoA content,hydrogen peroxide content,and pyruvate content.The results showed that QoIs with the corresponding EC50,10×EC50,100×EC50 values could increase cell membrane permeability,reduce respiratory rate by 20.0%-46.0%,decrease the ATP content by 8.9%-73.7%,and decrease hydrogen peroxide content by 7.8%-77.2%in F.graminearum.QoIs could inhibit mitochondrial respiratory by targeting the oxidation site(Qo site)of the reduced coenzyme Q of cytochrome b(cytb)in mitochondrial respiratory chain.However,the effects of QoIs on mitochondrial morphology is not clear.It has been reported that,toxisomes is a special globular structure around the nucleus formed by recombinant endoplasmic reticulum under DON-producing conditions,which is the suborganelle of DON biosynthesis.In this study,the confocal microscope was used to study the formation of toxisomes and the effects of mitochondrial morphology in F.graminearum as affected by QoIs,and the transmission electron microscopy was used to observe the structure of mitochondria as affected by QoIs.The results showed that,under confocal microscope both number and fluorescence intensity of toxisomes increased after treated with the corresponding EC50,10×EC50,100×EC50 values of six Qois,and the translational level of toxisomes formation associated protein TRI was obviously increased by Western Blot.Moreover,mitochondria were markedly fragmented by QoIs.According to the structure of mitochondria photographed by transmission electron microscope,there are many cavities in the mitochondria inside,that is,and the mitochondria performed with dissolving,deforming or other forms after treated by QoIs.The above results further showed that QoIs could affect the formation of toxisomes,cause the fragmented mitochondria,and disrupt the dynamic homeostasis of mitochondria.In order to further test the effort of QoIs on DON biosynthesis in wheat grain,we conducted the simulated infection test of wheat grain.The results showed that,the amount of mycelia in wheat grain was decreased by 60.0%-90.1%after QoIs treatment compared with non-fungicide controls.There are two methods to evaluate DON levels:the ratio of DON content to wheat grain weight and the ratio of DON content to dry mycelial weight.These results showed that other five QoIs with the corresponding EC50,10×EC50,100×EC50 values significantly increase DON levels for both the ratios of DON content to wheat grain weight and DON content to dry mycelial weight except for picoxystrobin.Picoxystrobin did not increase DON levels for the ratio of DON content to wheat grain weight significantly,while significantly increased DON levels for the ratio of DON content to dry mycelial weight.Therefore,QoIs can significantly reduce the amount of infected mycelia in wheat grain,but can significantly enhance DON biosynthesis and aggravate mycotoxin contamination in wheat grains.In conclusion,QoIs targets cytochrome b and c1 complex of respiratory chain,inhibiting the respiration and ATP production in F.graminearum and reducing the acetyl-CoA content,which is precusor substance of DON biosynthesis.Meanwhile,the structure of endoplasmic reticulum was affected,leaded to remodeling and formed the toxisomes,therby increasing DON biosynthesis.Although QoIs have good fungicidal activity against F.graminearum,they have the risk of enhancing DON biosynthesis.Thus,this study clarified the effects of several QoIs with high activity against F.graminearum on DON biosynthesis,which will provide a theoretical basis for screening the chemical or biological factors inhibiting DON biosynthesis.The results of this study not only have important theoretical significance for the development and safety evaluation of QoIs,but also have important theoretical and application values for evaluating the safety of other respiratory inhibitors and guiding pesticide registration and safe use of these fungicides. |
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