Study On The Seed Biology Of The Pseudosclerochloa Kengiana And Its Mechanism Of Resistance To Fenoxaprop-p-ethyl

Keng stiffgrass(Pseudosclerochloa kengiana)is a serious farmland grass weed distributed in winter wheat fields and rice-wheat rotation cropping systems.It has become a troublesome and predominant weed in eastern China.However,the excessive and persistent use of ACCase-inhibiting herbicides has led to the emergence of serious resistance in this species.In this paper,we studied the seed biological characteristics of P.kengiana seeds and its mechanisms of resistance to herbicide fenoxaprop-P-ethyl in wheat field in Huang Huai and Yangtze valley of China.The seed germination and seedling emergence experiments were carried out by petri dish method and final germination was calculated.The dormancy,germination and seed setting characteristics of the seeds were determined.To identify the resistance level and explore the resistance mechanism of P.kengiana to fenoxaprop-P-ethyl,the sensitivities to fenoxaprop-P-ethyl of thirty populations collected from Jiangsu,Anhui,Shandong,Shanghai and Henan province were determined,as well as the cross resistance and multiple resistance of the fenoxaprop-P-ethyl-resistant population.The target enzyme activity,gene sequences and gene expression profiles were detected to explore the target-site resistance(TSR).Simultaneously,we developed a derived cleaved amplified polymorphic sequence(dCAPS)method for detecting the amino acid mutation.Main results were:The experiment of seed germination by petri dish method and final germination was calculated after 14 d.The mature seeds have more one mouth dormancy after collected from fields.The dormancy of seeds could be broken by being buried underground for 30d.The optimum temperature range for seed germination was 15?20 ? and light was not necessary.More than 80%of seeds could be germinated at pH values ranged from 4 to 10.There was no significant difference in seed germination rate in different pH values.P.kengiana was not sensitive to osmotic stress and salt stress.Seedling emergence was higher(55%)when seeds were sown at the depth of 0?1.0 cm.Few seedlings emerged when seeds were planted at a depth of 2.0 cm.This study confirmed that the seed of P.kengiana have more one mouth dormancy and the dormancy of seeds could be broken by being buried underground or being storaged low temperature water.At the same time,the temperature,the water potential,the salt and the depth of buried soil had a certain effect on the seed germination,while the light and PH had no effect on the seed germination.The ED50 value for the presumptive resistant population JYJD-2(159.03 g a.i.ha-1)was significantly higher than the recommended dose(62 g a.i.ha-1),while the perceived susceptible population JYJD-1 showed a relatively lower ED50 value(10.14 g a.i.ha-1).The whole-plant bioassay experiments showed that the JYJD-2 population was highly resistant to fenoxaprop-P-ethyl.Based on the resistance index(RI)values,the JYJD-2 population was confirmed to be 15.68-fold more resistant than the JYJD-1 population.JYJD-1 was found to be sensitive to all herbicides.However,JYJD-2 showed moderate resistance to quizalofop-P-ethyl and pinoxaden,with the RI values of 4.50 and 3.36,respectively.However,it appeared sensitive to fluazifop-P-butyl,haloxyfop-R-methyl,clodinafop-propargyl,clethodim,and sethoxydim,with the RI values of 0.93,0.77,1.30,1.20,and 1.12,respectively.There was no resistance observed to acetolactate synthase(ALS)-inhibiting herbicides,mesosulfuron-methyl,imazapic,and pyroxsulam.For isoproturon,prometryne and glyphosate,JYJD-1 and JYJD-2 showed different sensitivity,with RI values of 2.01,1.42 and 1.01,respectively.Individual plants of each population were assayed for ACCase activity.The average relative ACCase activity for each population was expressed as a percentage of the control assay,as shown in.The IC50 value of wide type population JYJD-1 was 4.81 ?M.However,the IC50 value of the mutant JYJD-2 population(31.16 ?M)was considerably higher than that of the wide type population.The RI of the resistant population was 6.48-fold higher than that of the susceptible population.To analyze the ACCase amino acid sequence,we amplified two different ACCase fragments from both the susceptible and resistant populations.The sequences Acc1;1-1,Accl;2-1 and Accl;1-2,Accl;2-2 were obtained from susceptible and resistant populations,respectively.By comparing the sequence derived from JYJD-1 and JYJD-2,an Trp-1999-Cys substitution was found in JYJD-2,which may be responsible for its resistance to fenoxaprop-P-ethyl.The expression levels of ACCase gene between JYJD-1 and JYJD-2 were measured by qPCR method.At the third day after the fenoxaprop-P-ethyl treatment,the increased expression of ACCase may be involved in fenoxaprop-P-ethyl resistance,of which JYJD-2 was greater than JYJD-12-fold.Using the derived cleaved amplified polymorphic sequence(dCAPS)method,a tryptophan-to-cysteine mutation at codon position 1999(W1999C)was detected in the ACCase gene of the resistant population JYJD-2.Of the 100 JYJD-2 plants tested,we found 47 heterozygous resistant(RS)and 53 homozygous sensitive(SS)individuals.In this paper,the seed dormancy,germination and seed setting characteristics were determined.we report the occurrence of W1999C mutation in the ACCase gene of Keng stiffgrass for the first time,and thereby,elucidated the molecular mechanism underlying fenoxaprop-P-ethyl resistance in Keng stiffgrass.We believed that Keng stiffgrass evolved resistance to fenoxaprop-P-ethyl via TSR caused by a W1999C mutation.We also investigated the cross-and multi-resistance of the fenoxaprop-P-ethyl-resistant Keng stiffgrass carnying a W1999C mutation.This study confirmed that it is not appropriate to use fenoxaprop-P-ethyl,quizalofop-P-ethyl,and pinoxaden to control Keng stiffgrass with W1999C mutation.Simultaneously,we developed a derived cleaved amplified polymorphic sequence(dCAPS)method for detecting the W1999C mutation and the frequency of its occurrence in the ACCase gene of Keng stiffgrass rapidly and accurately.