Fenoxaprop-P-Ethyl-Resistant Beckmania Syzigache In Wheat Fields And The Resistance Mechanisms

Beckmannia Syzigachne compete with wheat(Triticum aestivum L.)and oilseed rape(Brassica napus L.)in China,where it exists as an annual winter grass weed with a wide distribution,most prominently in the Yangtze Delta and Southwest Region.Fenoxaprop-P-ethyl,a ACCase(acetyl-CoA carboxylase)-inhibiting herbicide,was first introduced to world agriculture in the 1980s,and has been widely used to control a variety of grass weeds for nearly 30 years.Because of their frequent use,resistance has evolved in a large number of weeds,with altered ACCase activity functioning as a widespread resistance mechanism.Therefore,it is important to illuminate the sensitivity of B.syzigachne poppulations in wheat fields in East China and reveal the resistance mechanisms of fenoxaprop-P-ethyl-resistant B.syzigachne.Main results of the present study are:1.Whole-plant pot dose-response testsWhole-plant pot bioassay were conducted to evaluate the dose-response to fenoxaprop-P-ethyl of 70 B.syzigachne populations collected from Jiangsu and Anhui.Dose-response tests suggested that 27 populations were resistant to fenoxaprop-P-ethyl,while the other 43 populations were sensitive.According to ED50(effective dose of 50%inhibition in plant growth),the RI(resistance index)between the most resistant population(JYJS,ED50＝2560 g a.i.ha-1)and the most sensitive population(AFCJ,ED50=44g a.i.ha-1)was 58.18-fold.By contrast,the recommended field dose of fenoxaprop-P-ethyl used was 62.0 ga.i.ha-1.Whole-plant pot dose-response tests suggested that populations JCWL,JCJT,JYJD were resistant to 5 other APP herbicides(clodinafop-propargyl,fluazifop-P-butyl,quizalofop-P-ethyl,haloxyfop-R-methyl and metamifop),a DEN herbicide pinoxaden,and a CHD herbicide sethoxydim,but not to a CHD herbicides clethodim.These populations were not resistant to sulfosulfuron,mesosullfuron-methyl,flucarbazone-sodium,pyroxsulam,acetochlor,prometryne,chlorotoluron and glyphosate.2.Target-site resistance mechanisms in fenoxaprop-P-ethyl-resistant B.syzigachneFive mutations,I1781L,W2027C,I2041A,D2078G and G2096A,were found in more than 300 fenoxaprop-P-ethyl-resistant B.syzigachne plants from 27 populations.The mutations consist of an A to T in the codon for Ile-1781,replacing Ile with Leu;G to C in the codon for Trp-2027,replacing Trp with Cys;T to A in the codon for Ile-2041,replacing Ile with Asn;A to G in the codon for Asp-2078,replacing Asp with Gly;and G to C in the codon for Gly-2096,replacing Gly with Ala.The study of target enzyme shows that,at different herbicide concentration,the activity of ACCase between the resistant and sensitive populations were different.When the concentration was lower than 0.1μmol·L-1,the activity of sensitive populations was significantly higher than that in resistant populations;while if the concentration was higher than 0.1 μmol·L-1,the result was completely different.With the change of inhibitor concentration,the ACCase activity in two groups shows different trends,we found that the sensitive population’s ACCase activity significantly decreased with the increasing of inhibitor concentration,but the decline of resistant populations were not very significant.Overall,the IC50 inhibiting ACCase activity of sensitive populations is 0.11 μmol·L-1,and the resistant populations are 0.76-1.63 μmol·L-1,which are 6.9-14.8 fold higher than the sensitive populations.The expression level of ACCase gene in resistant populations were significantly higher than that in the sensitive population before and after fenoxaprop-P-ethyl treatment,indicating that the over-expression of ACCase gene in the resistant populations also play an important role in fenoxaprop-P-ethyl resistance.3.Non-target-site resistance mechanisms in fenoxaprop-P-ethyl-resistant B.syzigachnePBO and ABT alone did not have a major impact on plants.Treatment with either PBO combined with fenoxaprop-P-ethyl or ABT combined with fenoxaprop-P-ethyl increased toxicity JCWL,JCJT,JYJD populations,with a reduction of ED50 value,while the resulting ED50 value were still higher than either that of the susceptible population,AFCJ,or the recommended dose of fenoxaprop-P-ethyl.The use of cytochrome P450 inhibitors was shown to slightly reduce the ED50 value of fenoxaprop-P-ethyl-resistant populations,which inferred non-target-site resistance(NTSR)was involved in fenoxaprop-P-ethyl-resistance in B.syzigachne.The study of activity of metabolizing enzymes shows that,after herbicide treatment,the activity of cytochrome P450 in resistant and sensitive populations shows different trends.With the extension of herbicide treatment,the maximum value of the activity in resistant populations were significantly higher than that of the sensitive population.The study of GST(glutathione S-transferases)activity shows that after herbicide treatment,the activity of sensitive population showed declining trend,while in the entire treatment time,the activity of resistant populations was always higher than sensitive populations.To reveal the mechanisms of metabolic resistance,the genes likely involved in herbicide metabolism(e.g.cytochrome P450s,esterases,hydrolases,oxidases,peroxidases,glutathione-S-transferases,glycosyltransferases and transporter proteins)were isolated using transcriptome sequencing,in combination with RT-PCR(reverse transcription PCR)and RACE(rapid-amplification of cDNA ends).Consequently,we established that herbicide metabolizing enzyme library containing at least 333 genes,and each of these genes was cloned and the sequence and the expression level compared between the fenoxaprop-P-ethyl-resistant and susceptible populations.Fifteen metabolic enzyme genes were found to be possibly involved in fenoxaprop-P-ethyl-resistance.In addition,we found 5 metabolizing enzyme genes that have different gene sequence in plants of susceptible versus resistant B.syzigachne populations.These genes may be major candidates for herbicide metabolic resistance.This established metabolic enzyme library represents an important step forward towards a better understanding of herbicide metabolism and metabolic resistance in this and possibly in other closely related weed species.We then constructed small RNA libraries and subjected them to deep sequencing and bioinformatics analyses.Forty known and 36 potentially novel,predicted miRNAs were successfully identified.Of these,we identified 4 conserved,predicted candidate NTSR-determinant miRNAs and their potential corresponding target genes,as well as 4 novel potential miRNAs with high count.Target gene prediction and annotation indicated that these 8 differentially expressed miRNAs potentially play a role in regulating specific stress-responsive genes,very likely related to herbicide resistance.Expression profiles were determined with quantitative real-time PCR.The present study is a novel,large-scale characterization of weed miRNAs.The results should further our understanding of miRNA expression profiles associated with herbicide resistance,allowing for the development of more effective weed management strategies.The nziR397 gene from B.syzigachne(referred to as bsy-miR397)was functionally characterized to determine its role in regulating fenoxaprop-P-ethyl resistance.We showed that(1)bsy-miR397 transcript level is constitutively higher in resistant than in sensitive B.syzigachne plants,whereas bsy-Laccase expression and activity show the opposite trend,and(2)bsy-miR397 suppresses the expression of bsy-Laccase in tobacco,indicating that it negatively regulates bsy-Laccase at the transcriptional level.We found evidences that miR397/laccase regulation might be involved in fenoxaprop-P-ethyl NTSR.First,the rice transgenic line overexpressing OsmiR397 showed improved fenoxaprop-P-ethyl tolerance.Second,following activation of bsy-Laccase gene expression by CuSO4 treatment,fenoxaprop resistance in B.syzigachne tended to decrease.Therefore,we suggest that bsy-miR397 might play a role in fenoxaprop-P-ethyl NTSR in B.syzigachne by down-regulating laccase expression,potentially leading to the enhanced expression of 3 oxidases/peroxidases genes to introduce an active moiety into herbicide molecules in Phase-2 metabolism.Bsy-miR397,bsy-Laccase,and other regulatory components might form a regulatory network to detoxify fenoxaprop-P-ethyl in B.syzigachne,supported by the differential expression of a further transcription factors and oxidases/peroxidases in the rice transgenic line overexpressing OsmiR397.To our knowledge,this is the first case of how down-regulation of a gene(laccase)can enhance NTSR.Our findings might form a basis for future studies for understanding and managing complex NTSR in weedy plant species.In the three fenoxaprop-P-ethyl-resistant B.syzigachne populations(JCWL,JCJT,JYJD)identified in this study,the resistance mechanisms to fenoxaprop-P-ethyl are diverse involving both TSR and multiple NTSR mechanisms.TSR to fenoxaprop-P-ethyl in this species is due to point mutations in the ACCase CT domain.However,NTSR also exists in populations R1,R2,and R3 and is more complex,as we have detected structural changes and altered expression levels of 15 genes encoding metabolizing enzymes.In addition,bsy-miR397 down-regulating laccase expression,as described in the present study,represents the first example of how down-regulation of a gene(laccase)can enhance NTSR in B.syzigachne.Knowledge of these resistance-regulating mechanisms is important for designing effective weed control strategies to manage and delay the onset of resistance to fenoxaprop-P-ethyl.4.dCAPS and LAMP methods obtained with the five assays targeting acetyl-coA carboxylase(ACCase)from B.syzigachne plants carrying mutant ACCase alleles.Nineteen of B.syzigachne that were resistant to fenoxaprop-P-ethyl collected in 2013 and contained the ACCase substitution were identified.Conventional PCR and derived cleaved amplified polymorphic sequence(dCAPS)methods were used to detect the mutation.Additionally,a rapid nucleic acid detection method,loop-mediated isothermal amplification(LAMP),was successfully developed and used to detect the genetic mutation underlying the ACCase substitution in the B.syzigachne ACCase enzyme.This report is the first to describe the application of a LAMP assay for mutation detection in herbicide-resistant weeds.The assay does not require specialized equipment:only a standard laboratory bath is needed.This technique could be employed for detecting the I1781L substitution in B.syzigachne plants and seeds.With a method composed of LAMP and dCAPS,19 fenoxaprop-P-ethyl-resistant B.syzigachne populations collected in 2013 were studied.An effective method,composed of LAMP and dCAPS,to detect five mutations,I1781L,W2027C,I2041A,D2078G and G2096A,in B.syzigachne populations was developed.With this method,a B.syzigachne population resistant to fenoxaprop-P-ethyl can be studied to confirm its constitution.And we determined that the resistance level might be relevant to the mutation type and mutation frequency.The type of mutation and its frequency in fenoxaprop-P-ethyl-resistant B.syzigachne populations can be confirmed to provide appropriate herbicide management.5.Effect of environment conditions on the germination characteristics of B.syzigachneThis study also aims to compare the germination characteristics of three pairs of resistant/susceptible B.syzigachne biotypes,each having a similar genetic background.A series of experiments was performed under controlled conditions to investigate seed germination characteristics of the three B.syzigachne biotypes showing target-site herbicide resistance,which was conferred by three homozygous mutant ACCase alleles(Ile-1781-Leu,Ile-2041-Asn and Gly-2096-Ala).Lines consisting of each herbicide-resistant biotype with different ACCase mutations and their relative susceptible biotypes were used in the comparative experiments.No major differences in seed germination were found at diverse temperatures,pH range,and salinity levels between the resistant and susceptible biotypes.Seed germination in both the seven B.syzigachne biotypes(the control and the three resistant and susceptible varieties each)decreased sharply when the osmotic potential was reduced from 0 to-0.5 MPa.The germination of the biotypes was>90%when the osmotic stress was-0.1 MPa.When the osmotic potential was reduced to-0.2 MPa and-0.3 MPa,the final germination rate of the JYDX-1781SS biotyp was higher than that of the JYDX-1781RR biotype.At-0.2 MPa and-0.3 MPa,the final germination rates of the remaining biotypes were similar those of the JYDX-1781SS biotype and higher than that of the JYDX-1781RR biotype).At-0.4 MPa,the final germination rate of the JYDX-1781SS biotype was considerably higher than that of the JYDX-1781RR biotype,whereas that of the SHQP-2041SS biotype was considerably lower than that of the SHQP-2041RR biotype.At-0.5 MPa,the final germination rate of the JYSC-2096SS biotype was considerably higher than that of the JYSC-2096RR biotype,whereas the JYDX-1781RR biotype did not germinate.In addition,no germination occurred at osmotic stress of less than-0.5 MPa.The expression of EXPB7 did not change consistent among the S,JYDX-1781SS,SHQP-2041SS and JYSC-2096SS biotypes.In addition,its expression was about 14.37 times lower in the JYDX-1781RR biotype than in the JYDX-1781SS biotype,about 7.03 times higher in the SHQP-2041RR biotype than in the SHQP-2041SS biotype,and about 4.48 times lower in the JYSC-2096RR biotype than in the JYSC-2096SS biotype.The expression pattern of the EXPB7 gene in the seven B.syzigachne biotypes was consistent with the germination speed and final germination rate in response to osmotic stress,indicating that EXPB7 might be involved in the germination speed and response to osmotic stress during seed germination in B.syzigachne.