Resistance Mechanism Of Two Species And Occurrence Pattern Of The Quinclorac-resistant Echinochloa In China

Echinochloa species,noxious weeds,are widely distributed in the paddy field across China.The application of herbicides mainly targets to barnyard grass.In particular,synthetic auxin-type herbicide quinclorac was early used extensively to control the weeds.Since herbicides were over-used in the long term,Echinochloa species have been suspected of evolving into herbicide resistance.Hence,an investigation of herbicide-resistance was conducted.Seeds of different Echinochloa species were collected from seventy different sites in China during fall 2016.A total of nine Echinochloa species or varieties were found from 13 Chinese provinces.All the collected species were subjected to herbicide resistance bioassay against quinclorac,cyhalofop-butyl,bensulfuron-methyl,bispyribac sodium,pyrazosulfuron,penoxsulam,and metamifop.Bioassay results at EC50 values in Petri plates and pots showed that herbicide resistance proportion of Echinochloa species among all collected sites was 31%-with E.phyllopogon having highest share of 19%in herbicide resistance followed by E.crus-galli var.mitis(6%),E.crus-galli var.zelayensis(3%),E.crus-galli(3%),and E.oryzoides(1.4%).The distribution pattern of herbicide-resistant Echinochloa species varied among different provinces.Herbicide-resistant species were mostly confined to North-eastern provinces(Heilongjiang.Jilin,and Liaoning)and eastern provinces(Zhejiang,Jiangsu,Anhui and Jiangxi).The five Echinochloa species(E.phyllopogon,E.crus-galli,E.crusgalli var.mitis,E.crus-galli var.zelayensis,and E.oryzoides)evolved into resistance to quinclorac,bensulfuron-methyl,bispyribac sodium,pyrazosulfuron,but all the species were susceptible to cyhalofop-butyl,penoxsulam,and metamifop.The occurrence proportion of herbicide-resistant species based on total collected samples of the same species were as follow;E.phyllopogon 65%,E.crus-galli var.zelayensis 40%,E.crusgalli var.mitis 21%.E.crus-galli 18%,and E.oryzoides 12%.Among the species mentioned above,E.crus-galli var.mitis and E.oryzoides have rarely been reported for herbicide resistance,and no case of quinclorac resistance has been reported so far.It was first exhibited four biotypes of E.crus-galli var.mitis that evolved into resistance to quinclorac ranking as JS01-R>AH01-R>JS02-R>JX01-R(27,18,17,12-times resistant than susceptible ones)from three provinces of China,along with one biotype of E.oryzoides(21-times resistant than susceptible ones).Further study was conducted to explore the potential mechanism causing quinclorac resistance in E.crus-galli var.mitis and E.oryzoides.Results revealed that ethylene production in these biotypes was negatively correlated with resistance level and positively correlated with growth inhibition.Determination of the related ethylene response pathway exhibited resistance in biotypes that recorded a decline in 1-aminocyclopropane-1carboxylic acid(ACC)content,ACC synthase oxidase activities,and less inducible ACS and ACO genes expressions than the susceptible biotypes,suggesting that there was a positive correlation between quinclorac resistance and ethylene biosynthesis inhibition.Cyanides produced during the ethylene biosynthesis pathway were potentially degraded by the activity of β-cyanoalanine synthase(β-CAS).Resistant biotypes exhibited higher β-CAS activity(～2-fold)than the susceptible ones.Nucleotide changes were found in the EcCAS gene of resistant biotypes as compared to sensitive ones that caused three amino acid substitutions(Asn-105-Lys,Gln-195-Glu,and Gly-298-Val)in E.crus-galli var.mitis,and two amino acid substitutions(Met-287-Lys and Thr-352-Ala)in E.oryzoides.That resulted in alteration of enzyme structure,increased binding residues in the active site with its cofactor,and decreased binding free energy(depicted by simulation modeling);hence,its activity was higher in resistant biotypes.Moreover,these mutations increased the structural stability of the enzyme.In view of the positive correlation between ethylene biosynthesis inhibition and cyanide degradation with resistance level,it is concluded that the alteration in ethylene response pathway or at least variation in ACC synthase and ACC oxidase enzyme activities-due to less relative expression of ACS and ACO genes and enhanced β-CAS activity,as well as mutation and increased relative expression of EcCAS gene-can be considered as a probable mechanism of quinclorac resistance in E.crus-galli var.mitis and E.oryzoides.