Study On Feasibility Of Controlling Wheat Field Weeds,Mechanism And Environmental Organisms Toxicology Of A New Compound Cypyrafluone

Wheat(Triticum aestivum L.)is one of the most widely grown food crops in China,with an annual planting area of more than 24 million hectares,which plays an important role in China’s food security and food demand.There are various of weed species with strong growth capacity that happened in wheat fields.And they can directly or indirectly affect wheat yield and quality by competing for water,nutrients and occupying ecological space.At present,chemical weed control is still the most important measure for weed control in wheat fields.However,recently years,due to the long-term single application of Acetolactate Synthase(ALS)and Acetyl CoA carboxylase(ACCase)inhibitor herbicides,many weeds have developed resistance to herbicides.Therefore,herbicide with a new site of action is the most cost-effective solution for resistant weeds.In the absence of new herbicide modes of action,HPPD(hydroxyphenylpyruvate dioxidase,HPPD)inhibition herbicides have become the first choice for the development of new herbicide due to their low resistance risks,broad spectrum,high efficiency,safety and environmental friendliness.Cypyrafluone is a patented compound recently developed by Kingagroot Compound Co.,Ltd.And it is speculated that it works by inhibiting the HPPD activity.The aims of this study are to evaluate the novel compound from aspects of safety to different crops,herbicidal activity and the spectrum of weed control,safety to different wheat hybrids and field trials,mechanism of action,residual digestion dynamics in wheat and soil.Moreover,the effects of this compound on soil enzymes and soil microorganisms,and the toxicity,physiological and biochemical effects of this compound on various environmental organisms also studied in this article.It is expected to provide theoretical basis for the feasibility and more scientifically and safely application of cypyrafluone in wheat fields.And the main findings are as follows:1.In order to select suitable crops for cypyrafluone,under the greenhouse conditions,pre-and post-emergence treatments were used to determine the effects of cypyrafluone on fresh weight,dry weight and emergence rate of 11 different crops(Triticum aestivum L.,Oryza sativa L.,Zea may L.,Sorghum bicolor(L.)Moench,Arachis hypogaea L.,Glycine max(L.)Merr.,Allium sativum L.,Cucumis sativus L.,Brassica rapa L.,Spinacia oleracea Linn.,Brassica chinensis L.)at active ingredient dosages of 135,270 and 540g/hm2.Results showed that cypyrafluone was very safe to wheat with the fresh and dry weight inhibition rates of 6.52%～21.48%,2.58～14.31%,1.65%～4.50%,1.85%～4.25%,respectively,and had no effects on the emergence rates.It also showed high safety for garlic,with inhibition rates of 3.89-17.99%;Its safety to Z.may,A.hypogaea,G.max,C.sativus were less than wheat and garlic,with maximum inhibition rates of more than 50%;However,Oryza sativa L.,Sorghum bicolor(L.)Moench,Brassica rapa L.,Spinacia oleracea Linn.and Brassica chinensis L.were the most sensitive to the compound,and the crop plants were significantly bleach and finally died.Results of the above study indicated that cypyrafluone was safe for wheat.Weeds in wheat fields were selected for hercidal activity and weeds spectrum research.The results showed that cypyrafluone had high efficacy against many grass weeds and broadleaf weeds when POST-applied at active ingredient dosages of 90 and 135g/hm2.When treated at two different doses,cypyrafluone was highly effective against 3 kinds of the treated weeds(Alopecurus.aequalis,Myosoton aquaticum,and Descurainia sophia),and all dry weight inhibition rates were more than 90%at 28 days after treatment(DAT).Cypyrafluone also had good efficacy on Alopecurus japonicus,Sclerochloa dura L.,Becmannia syzigachne,Lithospermum arvense L.,Veronica didyma and Capsella bursa-pastor is L.,with dry weight inhibition rates were more than 90%at 135 g a.i./hm2.However,dry weight inhibition rates of Avena fatua L.,Geranium sibiricum L.and Vicia sativa L.were only 50%-60%at 135 g a.i./hm2.Cypyrafluone had the worst efficacy on Bromus japonicus,Aegilops squarrosa L.,Lolium multiflorum,Poa annua L.and Euphorbia helioscopia L.,and the dry weight inhibition rates were less than 30%even under 135 g a.i./hm2.On the basis of the above tests,further evaluation of the safety of the compound for different wheat hybrids was carried out with higher treatment doses of 360 and 540 g a.i./hm2.Results showed that showed that nearly all the tested wheat varieties(Shannong 22,Jinan 17,Shannong 19,Zhengmai 9023,Yannong 19,Ningmai 24,Jimai 22,Liangxing 66,and Tainong 18)were tolerant to cypyrafluone with dry weight inhibition rates of<10%,while Huamai 5,Yangfumai 4,Yangmai 158,Hengguan 35,Haomai 1 and Xinong 979 were more sensitive to cypyrafluone.Based on the results,three wheat hybrids(Jimai 22,Liangxing66,and Tainong 18)and four weed species(Alopecurus aequalis,Alopecurus japonicus,Capsella bursapastoris(L.)and Myosoton aquaticum(L.))were chosen to determine the selective index of POST application.The results showed that cypyrafluone was safe for Jimai 22,Liangxing66,and Tainong 18 against weeds tested in this study with selective index values ranging from 5.7 to 16.6.In order to evaluate the herbicidal effect of cypyrafluone on resistance weeds,Alopecurus aequalis and Alopecurus japonicus with different resistant populations(ACCaseR,ALS-R and ACCase+ALS-R)were selected for testing.The results showed that at 135 and 180 g a.i./hm2,cypyrafluone had good herbicidal activity agains different resistance populations of Alopecurus aequalis and Alopecurus japonicus,with dry weight inhibition of 91-94%and 84-95%,respectively.However,fenoxapro-P-ethyl application at 50 g a.i./hm2 only caused 1418%and 13-16%dry weight inhibition to Alopecurus aequalis(ACCase-R and ACCase+ALSR)and Alopecurus japonicus(ALS-R and ACCase+ALS-R),respectively;mesosulfuronmethyl application at 9 g a.i./hm2 only caused 11-14%and 17-18%dry weight inhibition to Alopecurus aequalis(ALS-R and ACC+ALS-R)and Alopecurus japonicus(ALS-R and ACCase+ALS-R),respectively.2.Field experiments(2 years(2017～2018 and 2018～2019),2 places(Suzhou,Anhui and Tai’an,Shandong),3 growth stages of wheat(tillering,jointing stage,and booting stage))were carried out to evaluate the herbicidal efficacy of cypyrafluone on weeds and the safety to wheat.The results showed that cypyrafluone application at 90-270 g a.i./hm2 provided an over 90%(reduction in plant biomass)control efficacy against Alopecurus aequalis,Descurainia Sophia and Myosoton aquaticum when applied at tillering and jointing stage.Cypyrafluone had worse herbicidal efficacy when applied at booting stage and reduction in plant biomass were only about 30%at 90 g a.i./hm2.Cypyrafluone(135,270,and 540 g a.i./hm2)also had a certain yield increase effect on wheat,with yield increase of more than 10%.But when the concentration reached 540 g a.i./hm2,this compound caused slight injury to wheat,manifested as slight whitening at the leaf source and the heart of wheat leaves.But as time goes on,the bleach symptoms gradually disappear,and the injury would not affect wheat yield.From the results of this experiment,it can be recommended that the best application period of cypyrafluone was wheat tillering stage or jointing stage,and the application dose is 90～180 g a.i./hm2.These results obtained from greenhouse and filed experiments indicated that cypyrafluone had great potential for weed management in wheat production systems in China,such as Alopecurus aequalis,Descurainia Sophia,Myosoton aquaticum,Capsella bursa-pastoris and Alopecurus japonicus.3.Two experimental methods were selected to study the mechanism of cypyrafluone.Arabidopsis thaliana(6-7 leaf stages)was treated with cypyrafluone at concentrations of 180,270,and 540 g a.i/hm2.The expression of HPPD in Arabidopsis thaliana was quantitatively determined by fluorescence at 6,12,24,48 and 72 hours after treatment.The results showed that:the expression of HPPD decreased with increasing the dose of cypyrafluone at the same treatment time.At the same dosage,the expression of HPPD also decreased with prolonging the treatment time.The results showed that cypyrafluone could inhibit HPPD expression in Arabidopsis thaliana.Mesotrione(traditional HPPD inhibitor)was selected to determine the inhibitory constant(Ki)value against Arabidopsis thaliana HPPD(AtHPPD)of cypyrafluone.Results showed that:cypyrafluone displayed strong inhibition against AtHPPD with Ki value of 0.0114 ± 0.0006μM,which was about half of mesotrione(Ki=0.0055 ± 0.0006μM).The results further proved that cypyrafluone is a novel HPPD inhibitor.The results of above test results showed that cypyrafluone had broad range of applications in the future as an HPPD inhibitor herbicide that was safe to wheat and effective for wheat weeds.Therefore,to applied more scientifically and safely,the analysis of cypyrafluone residue and the research on the impact on the environment and biology were carried out.4.By optimizing the chromatography and mass spectrometry conditions,an analytical method for determining cypyrafluone in soil,wheat plant and grain was developed,and a field experiment was carried out to research the residues of cypyrafluone in wheat and soil.The results showed that the degradation of cypyrafluone in wheat plants and soil both met the first-degree dynamics matrix and constant.The half-life of cypyrafluone in soil and whaet plants were 3.45-3.55 and 5.50-5.59 days,respectively.At harvest time,the residues of cypyrafluone in wheat plants,soil and grains were below 0.01mg/kg.This analytical method was fast,simple,accurate and reliable.These results provided theoretical data to help establish principles related to the safe use of cypyrafluone.5.In order to better evaluate the environmental impact of this compound after application,this paper has preliminary studied the influence of cypyrafluone on soil enzyme activity and microorganism quantity in two different soils(brown-loam soil and lime concretion black soil)at concentrations of 0.1,1,and 5mg/kg soil.Results indicated that cypyrafluone initially inhibited soil protease,urease,and sucrase activity and this effect increased with concentration.During the later stages of incubation,inhibitory effects gradually weakened and by the end of the experiment(45 days),enzyme activities were restored to control levels.Catalase activity was stimulated by cypyrafluone,this stimulation effect decreased during the later stages of the experiment;however,catalase activity was still significantly higher as compared to the control at the end of the experiment.Bacteria and actinomycetes quantities were decreased by cypyrafluone(10 days).As incubation progressed,microorganism quantities in lower concentration groups(0.1 and lmg/kg soil)were restored to control levels,while those of the high concentration group(5mg/kg soil)did not fully recover.Cypyrafluone did not significantly impact fungi quantity.6.Tests were conducted to evaluate the acute toxicity of cypyrafluone(technical and formulation)to 7 environmental organisms.The results showed that:in addition to the lowly toxic of cypyrafluone(technical)to Coturnix japonica,the acute toxicity tests of cypyrafluone on Apis mellifera L.(acute oral and contact toxicity),Brachydanio rerio,Daphnia magna Straus,Bombyx mori,Eisenia foetida,and Trichogramma ostriniae were all limit tests.The acute toxicity test of cypyrafluone(formulation)on Eisenia foetida was a limit test,and the acute toxicity on Apis mellifera L.(acute oral and contact toxicity),Coturnix japonica and Bombyx mori were lowly toxic,and the risk was relatively low.It was moderately toxic to Brachydanio rerio and Daphnia magna Straus.All above test results showed that cypyrafluone was safer for variety of environmental organisms,which build a good foundation for the popularization and application of the herbicide.7.Although the acute toxicity test of cypyrafluone(technical and formulation)on Eisenia foetida were limit tests,considering extreme conditions such as accidental release of the compound,terrestrial organisms such as earthworms may be exposed to the agent environment for a long time.Therefore,further experiments were conducted to determine the effects of this herbicide on the ecological toxicity,DNA damage and growth of earthworm.The results showed that after exposure to cypyrafluone,it would cause oxidative stress on Eisenia foetida,which would cause a large accumulation of reactive oxygen species(ROS)and malondialdehyde(MDA)content,and then gradually decrease,but still higher than the control.However,with the extension of the test time,the content of ROS and MDA at lower concentrations could be restored to control levels.While the contents at high concentration was still slightly higher than the control.Moreover,cypyrafluone would inhibit the activities of related antioxidant enzymes(Superoxide Dismutase(SOD),Catalase(CAT),Peroxidase(POD))and eventually returned to normal levels.While the activity of glutathione S-transferase(GST)was first induced and then gradually decreased.Cypyrafluone caused the Eisenia foetida body cavity of cell DNA damage,and the damage would gradually increase with prolonging the exposure time and increasing the treated concentrations,showing a significant dose-effect and time-effect relationship.Additionally,cypyrafluone also could affect the normal growth of Eisenia foetida,and the increase in the weight of Eisenia foetida gradually decreases with the increase of the herbicide concentration.8.In addition to evaluate the toxic effects of cypyrafluone on terrestrial organism earthworm,tests were also carried out to study the effects of cypyrafluone on Chlorella vulgaris(C.vulgaris)from aspects of the algal cell growth,photosynthetic activity,oxidative stress and cell morphology under different doses and exposure times.Exposed to 5-50mg/L cypyrafluone showed a progressive inhibition of cell growth with 96 h EC50 value was 14.92mg/L.Further determinations were performed using 5 and 15mg/L concentration.Result showed that cypyrafluone affected the algal photo synthetic capacity,and the decreased pigment content and down-regulation of photosynthesis-related genes(psaB,psbC,rbcL)expression confirmed this result.Cypyrafluone induced oxidative stress,the levels of ROS and MDA in algae increased,and the antioxidant enzyme activities were also altered.Furthermore,the herbicide damaged the algal cellular structure,which is characterized by plasmolysis,blurred organelles and disruption of the chloroplast structure.Flow cytometric analysis also showed that cypyrafluone caused unevenly growth and interior irregularities of algal cells.The apparent propidium iodide(PI)influx also confirmed that the herbicide damaged the membrane integrity.The results of this study provide a more comprehensive theoretical basis for the safer and scientific application of cypyrafluone in the future and its impact on the environment after application,and also provide a reference for evaluating the physiological and biochemical effects of this compound on weeds in the future.Based on the results of the all above tests,it can be concluded that cypyrafluone is a novel HPPD-inhibition herbicide,was very safe to wheat,and was highly effective against both grass(Alopecurus aequalis Sobol.,Alopecurus japonicus Steud)and broadleaf(Descurainia sophia(L.),Myosoton aquaticum(L.)and Capsella bursa-pastoris Medic.)weeds.It was clear that cypyrafluone was a readily degradable herbicide and half-life of it was about 3-6 days.Moreover,cypyrafluone was safer to variety of environmental organisms,but also had adverse effects on soil enzyme activities,microorganism quantity,earthworm and C.vulgaris at limit dosages.From this article,we can know that cypyrafluone has broad application prospects as a high-efficiency,broad-spectrum and safe herbicide in wheat fields.