865 E. and L intron) trn E. crus-galli Echinochloa c RI endonuclease More specifically, from E. crus-galli Tsp 10 Also, chloroplast DNA 8 Vasing.) 2012 Society of Chemical Industry E. oryzicola species. c L-F intergenic spacers and Nucleotide sequencing of three non- trn 7,11 Photini V Mylona, Echinochloa T-L, ∗ Molecular analysis of specific regions of the b in Turkish accessions. b trn 9,10 species. provided information on phylogenetic relationships species discrimination; penoxsulam resistance species discriminated successfully E. oryzoides Laboratory ofThessaloniki, Agronomy, Thessaloniki, Greece School of Agriculture,Agricultural Research Aristotle Centre of Northern Greece, University NAGREF, Greece of Department of Genetics andUniversity Plant of Thessaloniki, Breeding, Thessaloniki, Greece School of Agriculture, Aristotle Correspondence to: Ilias G Eleftherohorinos,of Laboratory Agriculture, of Aristotle Agronomy, University of School Thessaloniki, 541E-mail: 24 [email protected] Thessaloniki, Greece. Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berks, UK ∗ c a d b cross-resistance to ALS-inhibiting herbicides as a result of and fragment length polymorphism (PCR-RFLP)very method has useful proven for discriminating cpDNA provided useful toolsof for species phylogenetic discrimination, relationships analysis Echinochloa and divergence among different sequence comparison between the different species revealed coding regions ( of cpDNA in 30 accessions belongingEchinochloa to nine species of the genus and new tools for species identification. (cpDNA) withowing a to slow thehas evolution maternal been rate inheritance usefulplant and of species. in no the examining organelle recombination relationships genome among different Echinochloa Echinochloa L.), L.), Zea 7 oryzicola has been As regards E. oryzoides Echinochloa var. 3,4 L.), maize ( Glycine max Beta vulgaris and Echinochloa oryzicola Echinochloa oryzicola Sofia L Chatzilazaridou, E. oryzicola a species is still poorly E. crus-galli oryzicola Oryza sativa 50 species that are among Barnyardgrass [ and Ilias G Eleftherohorinos L.), soybean ( Vasing.) and early watergrass ∼ d 1,2 accessions were found to be cross-resistant to penoxsulam, bispyribac-sodium, imazamox, accessions. L.) and sugar beet ( (Stapf) Stapf, Echinochloa E. oryzicola comprises : 865–873 www.soci.org 69 E. oryzicola E. oryzicola 5 Gossypium hirsutum mutation; target-site resistance; restriction digestion; Although some of these taxa can be conventionally 2013; E. phyllopogon 6 and (Ard.) Fritsch.] are also considered to be serious weeds Echinochloa ALS , it has recently become a serious weed of rice fields in accessions. Nicotiana tabacum (L.) Beauv.] is the most common weed species in a wide gene revealed that resistant (R) accessions had a Trp574Leu mutation, which was also confirmed by

L.), cotton (

ALS 2012 Society of Chemical Industry Molecular techniques have offered many new tools for The taxonomy of the E. oryzoides c the most important weed speciestemperate occurring regions in both of tropical and the world. of water-seeded rice in many rice production areas. (Vasinger) T. Koyama, tetraploid var. studying the evolutionaryplants, history as and wellspecies. More species specifically, as a polymerase divergence chain for reaction–restriction of solving taxonomic confusion between understood owinginterspecific to and intraspecific their levels.referred to Thus, morphological as similarity at the (Ard.) Fritsch. Pest Manag Sci 1 INTRODUCTION The genus BACKGROUND: Studies were carried out to elucidate the mechanism of resistance to ALS-inhibiting herbicides in 29 Abstract late watergrass ( Nikolaos S Kaloumenos, Alexios N Polidoros cross-resistance to ALS-inhibiting herbicides in accessions from water-seeded rice fields of northern Greece and to discriminate the (wileyonlinelibrary.com) DOI 10.1002/ps.3450 Target-site mutation associated with Research Article Received: 18 June 2012 Revised: 5 October 2012 Accepted article published: 29 October 2012 Published online in Wiley Online Library: 6 December 2012 range of summer crops such as rice ( crus-galli mays tobacco ( whereas late watergrass ( [ E. oryzicola northern Greece. identified on the basisecological of their habits, spikelet morphological atheir traits significant accurate and identification, amount as the of morphologicalspikelet frequently features skill overlap of within the and is between species/taxa. required for Keywords: foramsulfuron, nicosulfuron andthe rimsulfuron, whereas all accessions were susceptible (S) to profoxydim. Sequencing of RESULTS: Two Trp574Leu mutation. The cpDNA sequence comparisonE. analysis crus-galli is a reliable tool for discrimination of conventionally classified CONCLUSION: This is the first report of digestion. Use of cpDNAoryzicola sequence comparison analysis of E. et al. seeds : 865–873 E. crus-galli 69 C for further ◦ plants that had (Pollacci) Pign.] accessions was accessions were 2013; Echinochloa gene sequencing and E. erecta E.oryzicola ALS Echinochloa Echinochloa Pest Manag Sci of water at 250 kPa pressure. Prior to herbicide 1 − and 11 Pollacci barnyardgrass [ sample (accession A06) was collected from a rice field For each experiment conducted to evaluate resistance to E. oryzicola samples were collected fromcrus-galli the rice fields. Additionally,margin located one in Thessaloniki, as surviving plantsin were the not rice found fields surveyed.to During seed obtain collection, care a waslabelled taken representative as sample separate from accession. each The field, collected which was were initially placedwere in transferred big to plasticthreshed, the bags. placed laboratory, Afterwards, in where the plastic bags they seeds and were stored at air 3–5 dried, treatment, seedlings were thinnedof to each four herbicide per treatment pot. on The the efficacy use in theaccession following susceptible experiments. to ALS- Seedswere purchased and from from ACCase-inhibiting Herbiseed (Twyford, an herbicides Berks,S-control UK) (accession and A05). used During as thematerial 2011 (leaf blades) summer was collected period, from plant survived in two rice fields (notor sampled imazamox before) treatment after respectively. penoxsulam The collectedduring plant the material 2011 summer surveyDNA was extraction, subjected which only was to genomic used for restriction digestion analysis. 2.2 Whole-plant response In three separate experiments,screened 29 tobispyribac-sodium identify and profoxydim, possible and fivewere R selected further accessions accessions evaluatedthe for to sulfonylurea cross-resistance penoxsulam, rimsulfuron. to herbicides All imazamox foramsulfuron, whole-plant and pots nicosulfuron trials (9 were by and each 9 carried pot, by 15 out seeds 10 were cm) inwith placed filled 1 on plastic cm the with of surface and soil:pit theperformed then as same mixture covered needed mixture. 4:1 to Fertilisation achievewere (v/v). and vigorous randomly In irrigation plant placed growth. were in Allfarm a pots (Thessaloniki, net northern Greece) protected and areathe allowed at to 2010 grow the summer during University period.3–5-leaf Herbicides stage were with a applied portableThe POST Netherlands) field using plot a at 2.4 sprayer m the wide (AZO-SPRAYERS, boomfan fitted nozzles with six (Teejet 8002 Spray flat- Systemto Co., deliver Wheaton, 300 L IL) ha and calibrated evaluated 4 weeks after treatment (WAT) by recordingof the number surviving plantsweight and from each determining pot. their Freshto weight above-ground data percentage were fresh then of transformed transformation untreated in analysis of control variance (ANOVA). and used withoutpenoxsulam, further cross-resistance to imazamox orsulfonylurea cross-resistance herbicides, to a completelydesign randomised was experimental used withtreatment. four replications (pots) All fortreatment each experiments herbicide for each includedperformed accession twice, evaluated. an and Each ANOVAwas experiment untreated combined performed was over because control experimentvariances Bartlett’s runs test of indicated(time the that the replicated) data error the obtained were ANOVAs from indicated homogeneous.run no the In significant interaction, treatment two addition,repetitions by data experiments experiment (experimental because were runs).means Differences averaged were among compared overthe treatment LSD at test. the the 5% experiment level of significance using 22 17 in rice www.soci.org NS Kaloumenos E. crus-galli E. oryzicola 2012 Society of Chemical Industry The purpose 26 c – 27 control in the in Turkey has also Echinochloa 19,20,24 17,21 L.) possible with the use Echinochloa accessions to herbicides with For example, an accessions displayed a single E. oryzoides However, in some cases, such accessions. 17 L intron between bases 464 and species had 5–6 A-repeats at the 23 16 spp. were surveyed, and mature trn control after the application of the Oryza sativa in the Echinochloa In addition, rice cultivars makes selective control of , cross-resistance to bispyribac-sodium and 12 (L.) Heyhn. E. phyllopogon 20 Echinochloa  – E. phyllopogon Echinochloa 18 Additionally, the introduction of imidazolinone- and or Echinochloa 15 – and red rice ( spp. after the application of penoxsulam. Therefore, accessions in species. 12 E. oryzicola E. oryzicola E. oryzicola During the 2008 and 2009 growing periods, rice growers from The herbicides bispyribac-sodium, penoxsulam and imazamox The repeated use of herbicides in rice grown globally has The herbicides propanil, pretilachlor, molinate, thiobencarb, water-seeded rice fieldsevolution of of resistance to northern ALS-inhibitingmolecular herbicides. studies Greece Additionally, were carried was out toresistancetoALS-inhibitingherbicidesandtoclassifytheevaluated elucidate due the mechanism to of the Echinochloa northern Greece (countiesunsatisfactory of Thessaloniki and Serres) noticed are acetolactate synthase (ALS;and have EC a 2.2.1.6)-inhibiting propensity herbicides rapidly to select for R weed accessions. resulted in resistant (R) different mechanisms of action. 484, while all other same position. Therefore, the cpDNA sequencebe comparison used could for identification of morphologicallyand classified quinclorac, clomazone,pendimethalin, mefenacet, bispyribac-sodium, fentrazamide,ethyl, cyhalofop-butyl penoxsulam, and oxadiazon, profoxydim control fenoxaprop- effectively. wileyonlinelibrary.com/journal/ps 22.1 MATERIALS AND METHODS Seed sourceA and plant roadside material survey was conductedrice during monoculture fields the located summer in northern of Greece. 2009 in This is confirmedalready developed by R theIn accessions most to fact cases, resistance ALS-inhibiting is thatfollowing herbicides. codons: due Ala122, Pro197, to 126 Ala205, Asp376, point Arg377, Trp574, mutations weedSer653 in and one species of Gly654, the numbered have Arabidopsis on thaliana the basis of the sequence of that nucleotide repeat (A) developed multiple resistance to bensulfuron-methyl, bispyribac- sodium, cyhalofop-butyl and penoxsulam. of the survey was toEchinochloa locate and mark fields with poor control of seeds, before rice harvest, were collectedThessaloniki from and 23 from fields five located fields in located in Serres. In particular, 17 a total of 112poor fields control (92 of in Thessaloniki and 20 in Serres) with resistant Clearfield Echinochloa of imazamox and imazethapyr. recommended rates of penoxsulam.can Although be herbicide attributed failure toor environmental weed conditions, growth when stage, theseresistance herbicide factors could are application explain eliminated, Therefore, the lack whole-plant ofdetermine response satisfactory studies whether weed control. were the conducted reduced to accession from rice grown inhas the Sacramento developed Valley multiple ofthiobencarb, California resistance to clomazone, the fenoxaprop-ethyl,and herbicides penoxsulam. bispyribac-sodium molinate, as that of penoxsulam was due toP450 monooxygenases herbicide (EC 1.14.14.1, metabolism P450s). via cytochrome

866 867 of ALS The M C for ◦ 28 L. PCR µ L-R: GGG The DNA C for 30 s, accessions accessions E.oryzicola Briefly, the ◦ trn Echinochloa 29 11 .Becauseno C for 50 s. The  and the down ◦ gene fragment C for 2 min was  species ◦ ALS E. crus-galli E. oryzicola Cfor10 min.Afterwards, ◦ PCR buffer A, 0.2 m Mofeachprimerand1.2 30 µ × ). The primers were the up ; nicosulfuron rates were 40 L of DNA (about 250 ng) was 1 Echinochloa − µ by using CTAB. For each accession, gene sequence of any C per cycle) for 30 s and elongation wileyonlinelibrary.com/journal/ps ◦ 28 ALS accessions) factorial design combined 0.5 L PCR primers. The DNA sequences were − trn C( -CwGGrGTbTCrAGCATCTTC-3 ◦  . Also, the recommended rate of penoxsulam 1 , and the respective rates of rimsulfuron were -CTGGyGCykCTGTGGCyAAC-3 − 1 C for 30 s and elongation at 72  Arabidopsis thaliana ◦ L intron were excised from the gel, and DNA was − L-F: CGA AAT CGG TAG ACG CTA CG, Echinochloa trn trn L total volume contained 1 C for 50 s. The touchdown cycles were followed by 30 µ ◦ L intron ( label), 104, 208 and 416 g AI ha amplification was carried out(Applied Biosystems). in The following a touchdown Veritiwas PCR programme 96-well employed. thermal An cycler initial denaturation at 94 2.7 Amplification and sequencing of the (recommended rate according to Greekand herbicide 320 g label), AI 80, ha 160 followed by ten amplification cycles of denaturation at 94 the amplification products were resolvedand on single a 1.2% bands agarose gel, of the expected size were observed. finalelongationstepwasperformedat72 band slice in a 1.5 mLand centrifuged reaction for 5 tube min was at maximum frozen speed,was in and the liquid recovered liquid nitrogen phase in acrushed new and mixed DNA with collection awhich tube. small was amount The of added agarose sterilefreezing–centrifugation was to distilled water the cycle original wasliquid repeated, tube phase and with was the added a recovered to pipette the tip. DNA The collection tube. 10 (recommended rate according40 to and 80 Greek g AI herbicide ha label), 20, 30 s, annealing at 58 standard amplification cycles of denaturation at 94 primer (D-Down) 5 information regarding the at 72 solution was sent forthe sequencing Department with of no Immunology further andSchool, purification Histocompatibility University to (Medical of Thessaly, Larissa,sequenced Greece). using the DNA was directly aligned and compared using ClustalX 2. along with the untreateddata control obtained (expressed treatment as were percentage of included.from the untreated The the control) experimental(ANOVA) runs using (repetition a in 4rates time) by by were 4 five by analysed 5over experimental (four runs. herbicides by four herbicide 2.6 Molecular identificationLeaves of of individualresistant to penoxsulam (A01, plants A02), two less susceptible fromaccessions (A03, two A04) and two(A05, susceptible A06) wereaccording to Doyle used and Doyle, for DNA extraction. DNA was isolated, the isolation was performed 3 timesdifferent from plants. tissue A taken from quantity three used of as 5 templatetrn in PCR withGAT AGA primers GGG amplifying ACT TGAperform the the AC). PCR cpDNA The reactions KAPA (KAPA Taqof BIOSYSTEMS). PCR The 25 kit PCR was mixture used to annealing at 53 units of KAPAadded Taq to polymerase. The the amount PCR of mixture genomic was DNA 250 ng diluted in 5 each dNTP (New England Biolabs), 0.8 recovered using a modified freeze-squeeze method. bands of the A pair of degenerate primers was designed togene amplify a fragment 336 bp containing thestandardised Trp574 to codon (TGG) (numbering primer (D-Up) 5 ; 1  − SC; OD; 80% 1 1 > − − accession accessions E.oryzicola Echinochloa WG; DuPont 2012 Society of Chemical Industry 40 g L 1 EC; BASF Agro 22.5 g L SL; BASF Agro c − 1  1  − − 80% of the treated . The data obtained , and the respective 1 > 1 E. crus-galli − − E. oryzicola 40 g L 250 g kg 200 g L 80% of the treated plants    < , as well as penoxsulam and 1 − resistance to herbicides www.soci.org SC; Bayer Cropscience Hellas) and 1 accessions along with the S-control − : 865–873 accessions) factorial design combined over 69 N Efthimiadis), cross-resistance to bispyribac- Echinochloa 400 g L +  Echinochloa 2013; accessions (A01, A02, A03, A04, A05). Foramsulfuron OD; K 1 − Echinochloa 80% of the treated plants killed) by 4 times the recommended < Pest Manag Sci 20.4 g L ALS mutation and 2.3 Penoxsulam,resistance bispyribac-sodium and profoxydim Twenty-nine accession (A05) were tested for resistance to penoxsulam (Viper multiple resistance to profoxydim (Aura sodium (Adora Bayer Cropscience Hellas), nicosulfuron (Milagro wasappliedat52(recommendedrateaccordingtoGreekherbicide Agro Hellas) was evaluated againstEchinochloa the five previously described Hellas) applied atherbicide 80 label) (recommended and 160 rate g according AI ha to Greek bispyribac-sodium rates were 30 (recommended rate according to Greek herbicide label), 60 and 120 g AI ha rates of profoxydim wereGreek 200 herbicide (recommended label), 400 rate and according 800 g to AI ha Syngenta Hellas) and rimsulfuron (Rush 2.5 Sulfonylurea cross-resistance Additionally, the efficacy of foramsulfuron (Equip profoxydim applied at the recommended rate. Theand herbicide rates adjuvants used are(expressed described in as Table percentage 1.experimental The of data runs obtained untreated (repetition control)using in from a time) 4 the wereaccessions) by two analysed factorial design 5 (ANOVA) combined over (four experimental runs. herbicide treatments by five from these experiments, asusing described a previously, 3 were analysed byby 3 30 by 30 (three herbicides by three herbicide rates Hellas). The experimentsummer was period. performed Penoxsulam twice wasrate during applied according the at to 2010 40 Greek (recommended herbicide label), 80 and 160 g AI ha (A05) was evaluated in a separate time-replicatedTreatments pot included experiment. imazamox (Pulsar experimental runs. Itfactorial is experiment was worth usedaccessions noting were for screened that in the two separate ANOVA, the experimentsunder conducted although 3 the similar by 30 conditions.experiment 3 More was by specifically, conducted 30 theincluded from 14 first 5 accessions screening May originatingand to the from purchased 20 the S-control June A05 sampledscreening accession, 2010 rice experiment whereas the area and was second 2010 conducted and from included 8 15and accessions the May S-control originating A05 to accession from (for 23of the comparison). The rice June these second run area two separateway experiments as described was previously carried fromaccessions 22 out that June in were to effectively a 6 controlledplants August similar ( 2010. killed) The by the‘S’ recommended rate (A05, in A06), Greece whereas( were the labelled accessions thatrate were of not either controlled herbicideaccessions were that labelled were ‘R’ not controlled (A01,killed) ( by A02). the Finally, recommended rate the but that were controlled ( resistant to penoxsulam (A01, A02), two less susceptible accessions (A03, A04) and one susceptible 2.4 Imazamox cross-resistance The efficacy of imazamox against two of the treated plants killed) by 4 timeslabelled the recommended ‘less rate S’ were (less susceptible) (A03, A04). gof et al. µ 160 : 865–873 E. oryzicola 69 a 2013; L total reaction volume) µ fragment from the wild-type Pest Manag Sci Cin50 software (Bio-Rad Laboratories, ALS ◦  400 800 80 L total reaction volume. The digested at 65 µ 30 accessions by 95–100% and 90–99% 1 accessions by 88–100% by comparison − a accessions by 83–100%. These accessions ). The digestion profile for each accession 1 ) C. The restriction point of this endonuclease, DNA h − 1 E. erecta ◦ − λ allele. Each digestion reaction contained 3 of BSA in 30 (S refers to C or G), overlaps the codon Trp574 1 E. oryzicola − ↓ gmL gof L µ µ µ g E. oryzicola µ RI (restriction endonuclease; New England Biolabs, Ipswich, Herbicide (g AI ha Control (% of untreated) 21 DNA was separated by agarose (2%)at electrophoresis in 95 TAE V buffer forultraviolet 40 (UV) min. light The aftersolution being resulting stained (1 bands with were ethidium bromide visualised under 2.8 Restriction analysis The same PCR productTsp (336 bp) wasUK) subjected for to 4 digestion h by at 65 33.1 RESULTS AND DISCUSSION Penoxsulam,resistance bispyribac-sodium and profoxydim Penoxsulam applied at the recommendedweight rate of reduced the 15 fresh with the untreated control (data not shown).sodiumappliedattherecommendedratereducedthefreshweight Similarly, bispyribac- of nine were labelled ‘S’.recommended However, rate reduced bispyribac-sodium theaccessions applied fresh by weight at of 63–77%.in the six Table These 2)sodium accessions were applied labelled (e.g. at ‘less A03weight S’. the of and the Penoxsulam recommended 11 A04 and rate bispyribac- reduced the fresh and 0.1 once with the forward primerThe sequencing and chromatograms once were visualised with and the editedBioEdit using reverse 7 primer. (Ibis Biosciences, Carlsbad,used CA). to The translate same software thesequences. was DNA The sequences DNA to and thecompared using peptide respective ClustalX 2. sequences peptide were aligned and was compared withcontrol the (A05). A completely respective randomised design digestion wasexperiments,withthreerepetitions.Gelimageswerecapturedwith used for profile these ofa the Kodak S- DS DC40NY), camera (Eastman while Kodak the Company, Rochester, Quantity-One DNA of the PCR product,digesting 1 1 U (the amount of enzyme capable of NNCASTGNN and appears twice in the specific Echinochloa ALS Greece) was used for gel image analysis and band comparisons. accessions as a result of the application of penoxsulam, bispyribac-sodium, 60 120 200 Lof ¨ (Taq) uren, µ a C for 45 s www.soci.org NS Kaloumenos ◦ E. oryzicola Extract II kit H.murinum Echinochloa 2012 Society of Chemical Industry L. (X63553).  mature plants c accession plants Gaud. (EF411171), Z. mays C for 5 min. Afterwards, ◦ Thermus aquaticus , 5% dimethyl sulfoxide C for 30 s, annealing at L. (AF059600), 2 ◦ Echinochloa ¨ plants from the previously uren, Germany). The purified thermophilic buffer, 5 , elongation at 72 80 160 30 E. oryzicola 1 × − M of each deoxyribonucleotide Lolium rigidum Penoxsulam Bispyribac-sodium Profoxydim Imazamox µ plants (identified on the basis of Cs ◦ a L. (FJ997632) and Echinochloa Hordeumvulgare accession. Extraction was performed from genes from the following Poaceae species: E. oryzicola L. (AF488771), ) 100 100 100 95 100 100 100 100 100 ) 100 100 100 100 100 100 100 100 100 100 100 ) 87 97 99 76 95 99 100 100 100 100 100 ) 85 91 100 76 87 98 100 100 100 100 99 ) 4 19 33 5 17 31 100 100 100 0 0 ) 1 8 16 3 3 12 100 100 100 0 6 ALS Avena fatua PlantIIkit(MACHEREYNAGELGmbH&Co.KG,D accession 40 L of the supplied 10  Fresh weight reduction (% of untreated) of six µ of each primer, 200 Echinochloa L. (AY885674), C with a ramp of 0.2 M C for 3 min, denaturing at 95 ◦ E. crus-galli E. crus-galli E. oryzicola E. oryzicola E. oryzicola E. oryzicola µ ◦ 0.05 The recommended rate on the Greek herbicide label. Leaves of individual LSD A06 ( A05 ( A04 ( A03 ( A02 ( A01 ( a Echinochloa Table 1. profoxydim and imazamox treatments. Mean values are averaged over the two experiments L. Amplifications were carried out in a heated-lid PCR machine triphosphate (dNTP), 1.5 mM of MgCl wileyonlinelibrary.com/journal/ps Bromus tectorum species was available, primer design wassequences based on of the nucleotide and cycling tofinal the elongation step was second performed at 72 denaturing step 34 more times. A (DMSO), 5 DNA sample and 1.2 enzyme units (U) of (MJ Research, model PTC-200) with the following cycle:at denaturing 95 polymerase (DNA polymerase, recombinant; HyTest, Finland) in 50 µ (MACHEREY NAGEL GmbH & Co. KG, D each PCR product was separated on 1.4% agarose gelsaccording and to purified the protocol outlined in the NucleoSpin 59–52 L. (EF540587), O.sativa DNA was sent immediately forImmunology sequencing and Histocompatibility to (Medical School, the University Department of of Thessaly, Larissa, Greece). Each PCR product was sequenced twice, before rice harvest)fields that treated had with the survived penoxsulamimazamox in (A07 (A08 putative each R putative of accession) Rwas or the extracted accession) from leaf two samples recommended taken rice from rate. threeof individual plants DNA each 100 mg ground leafbromide tissue (CTAB) method using according to the the protocolNucleoSpin cetyl outlined in trimethylammonium the described six accessionsused (A01, for A02, A03, DNAcollected A04, extraction. from A05, In the A06)(A01 particular, two and were A02) leaf resistant thatafter blade survived treatment tissue with with no was theplant visible material penoxsulam was adverse collected recommended from symptoms the two rate.accessions less (A03, sensitive Also, A04) and from the two barnyardgrass S(A05, accessions A06) prior toextracted herbicide from treatments. leaf Infield tissue addition, survey that DNA from was was collected during the 2011 the morphological traits on remaining Germany). The polymerase chain reaction (PCR) mixtureof consisted 0.8

868 869 E. E. 20 in the – (syn. 18 Echinochloa It is worth A01 and A02 23 E. oryzicola species E. oryzicola E. oryzicola A01 and A02 accessions by 21 Echinochloa – 17 cross-resistance to ALS-inhibiting ) accession from the Sacramento wileyonlinelibrary.com/journal/ps in Turkey and for A01 and A02 accession plants reduced 31,32 accessions indicates that the reported E. oryzicola A01 and A02 accessions strongly supports A01 and A02 accessions suggests evolution E. oryzicola E. oryzoides accession was found to be cross-resistant to R accessions to profoxydim in Greece could be E. phyllopogon E. oryzicola Echinochloa ’[porgn:__txid45618]’’ retrieved 63 sequences that ) accession was reported R to the ACCase-inhibiting E. oryzicola E. oryzicola (syn. E. oryzicola Echinochloa Thepoorefficacyofforamsulfuron,nicosulfuronandrimsulfuron The high level of profoxydim efficacy against the 30 The fact that imazamox applied at 2 times the recommended The study on penoxsulam and bispyribac-sodium efficacy Echinochloa accession plants indicates clearly that these accessions, into addition their previouslyand bispyribac-sodium, confirmed have cross-resistance alsoimazamox. developed to cross-resistance penoxsulam to against the the evidence that these accessions, inconfirmed addition to their cross-resistance previously toand imazamox, penoxsulam, have bispyribac-sodium sulfonylurea also herbicides registered developed for cross-resistance usecross-resistance in to corn. has This these been pattern of associatedTrp574, with which mutation usually confers ininhibiting herbicides, cross-resistance position whereas the to respective mutation different inPro197 position usually ALS- causes resistance mainly to sulfonylurea herbicides, as shown for other dicot and grass species. their fresh weight by 3–29%, whereasreduction the due respective to fresh weight 4 timesFinally, their penoxsulam recommended applied rate at wasthe the 41–46%. fresh recommended weight rate of reduced the rate did not have any effect on the 3.3 Molecular identificationA of searchDecember at 2011) the‘ with NCBI the nucleotide database query (performed ‘‘trnL in AND intron AND applied on the 12 and 14% respectively, whereas 4reduced times their the fresh recommended weight rate by 39 and 37% respectively. against the unsatisfactory control bycould the be attributed farmers toapplication in or weed unsuitable environmental most growth conditions. However, stage, sampled the lack improper fields of herbicide acceptableagainst penoxsulam the and bispyribac-sodium efficacy of cross-resistance to these herbicides. Thisthe could herbicide be selection attributed pressure to imposed by theapplied rice monoculture as main practicerepeated use in of these the two herbicides samplingan for a area, 6 year along period. Similarly, withbispyribac-sodium and the penoxsulam by other researchers. herbicides penoxsulam (triazolopyrimidines), bispyribac-sodium (pyrimidinylthiobenzoates), imazamoxformsulfuron, nicosulfuron (imidazolinones)reported and for and the rimsulfuron firstcross-resistance (sulfonylureas) time only worldwide. to is Similar bispyribac-sodium and results penoxsulamalso concerning were reported for noting that the accessions studied suggests thatdeveloped resistance none to this of ACCase-inhibiting herbicide. these Theof lack accessions has attributed to theof lower its selection very pressure limitedoryzicola imposed use in as the a rice result area. On the other hand, an Sacramento Valley of California. Valley inherbicide California fenoxaprop-p-ethyl, whereas another wasphyllopogon found Rherbicide cyhalofop-butyl. to the ACCase-inhibiting ) accessions E.crus-galli 1 − 2012 Society of Chemical Industry Echinochloa c accessions (A05 accession plants, 4.4 E. oryzicola 20 40 80 80 160 320 80 160 320 104 208 416 Control (% of untreated) E. crus-galli a a a a E. oryzicola 10 40 52 40 ) 100 100 100 100 ) 100 100 100 100 ) 100 100 100 100 ) 100 100 100 100 124 60 47 )132741 )143143 )) 97 86 98 44 100 100 100 38 )123539 )141837 100 ) 100 ) 91 88 97 90 93 99 100 100 )) 3) 7 17) 98 30 44 90 100 46 100 100 100 90 100 83 100 234 51 39 )293742 )172234 )) 99 100 100 100 100 100 100 100 resistance to herbicides www.soci.org A03 and A04 accessions by 97–99% accessions (A01, A02) only by 16–33% and A03 and A04 E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. oryzicola E. crus-galli E. crus-galli E. crus-galli E. crus-galli : 865–873 69 Echinochloa A01 ( A02 ( A03 ( A04 ( A01 ( A02 ( A03 ( A04 ( A01 ( A02 ( A03 ( A04 ( A01 ( A02 ( A03 ( A04 ( A05 ( A05 ( A05 ( A05 ( E. oryzicola E. oryzicola 2013; E. crus-galli Fresh weight reduction (% of untreated) of five 0.05 The recommended rate on the Greek herbicide label. Foramsulfuron, nicosulfuron and rimsulfuron applied at the Rimsulfuron Penoxsulam LSD Nicosulfuron a accessions as a result of the application ofrimsulfuron foramsulfuron, nicosulfuron, and penoxsulam treatments.over Mean the values two experiments. are averaged HerbicideForamsulfuron Accession Rate (g AI ha Table 2. Pest Manag Sci 3.2 Imazamox andThe sulfonylurea cross-resistance recommended rate ofof imazamox the reduced A05 the fresh weight and A06) (Table 1). and 12–31% respectively (Table 2). These accessions‘R’. were Finally, labelled profoxydim applied at thethe recommended fresh rate weight reduced of all evaluated accessionsA02, by A03 95–100% and (e.g. A04) A01, (Table 1),in and all a herbicide high treatments resulted level of control of the two respectively compared withshown). Nevertheless, the both untreated penoxsulamapplied and control at bispyribac-sodium 4 (dataweight times of not two the recommended rate reduced the fresh ALS mutation and recommended rate reduced the fresh weight of the A05 whereas 2 times the recommended rate reduced their freshby weight 0 and 6% respectively. accession by 100% compared with the untreatedSimilarly, control (Table the 2). same rate ofweight the three of herbicides the reduced theand fresh 86–100% respectively. However, their recommended rate by 99–100% by comparison withHowever, the untreated the control same (Table 2). the herbicide fresh weight rate of did the A01 not and A02 have any effect on ALS et al. (A03, GTGGA GTGGA GTGGA GTGGA GTGGA GTGGA GTGGA GTGGA GTGGA GTGGA GTGGA GTGGA GTGGA : 865–873 AGTGGA E. crus-galli E. crus-galli -GTGGA -GTGGA 69 accessions. Echinochloa gene fragment E. oryzicola 2013; ALS E. oryzicola (A05, A06) accessions is in along with the six TACTTTAAAAAA-----GTGGA TACTTTTAAAAA----- TACTTTTAAAAA----- TACTTTAAAAAA----- TACTTTAAAAAA----- TACTTTTAAAAA----- TACTTTAAAAAA----- TACTTTTAAAAA----- TACTTTAAAAAA----- TACTTTTAAAAA----- TACTTTAAAAAA----- TACTTTAAAAAA----- TACTTAAAAAAAAAAAAGTGGA TACTTAAAAAAAAAAAAGTGGA TACTTTAAAAAAAAA TACTTAAAAAAAAAA-GTGGA TACTTAAAAAAAAAA TACTTTTAAAAA----- TACTTTTAAAAA----- TACTTAAAAAAAAAAAAGTGGA TACTTAAAAAAAAAAAAGTGGA TACTTAAAAAAAAAAAAGTGGA TACTTAAAAAAAAAAA Pest Manag Sci (A01 and A02) and putative R accessions studied. In particular, L intron sequences retrieved from accessions studied. The sequences gene from the less S Echinochloa ALS E. crus-galli (A03 and A04) accessions did not show E. oryzicola Echinochloa Echinochloa trn Echinochloa gene fragment obtained from three individuals of gene sequence X51514. As nucleotide and amino ALS (A05 and A06) accessions. Nevertheless, nucleotide (A07 and A08) accessions revealed a heterozygous E. oryzicola gene from the S The lack of point mutations at codon 574 of the sequenced 3.4 Amplification and sequencing of the ALS any mutation atE. the crus-galli 574polymorphisms codon when were compared observedfragment, which resulted with in in amino acid the substitutions not related otherherbicide to S resistance (Fig. 1). regions It is worth noting of that the S the The use of degenerate primersthe targeted led to successful amplification of (A05 and A06) accession revealed more nucleotide polymorphisms irrelevant to herbicide resistance than the agreement with presentexperiments. findings Also, of the the absencecodon of of whole-plant the point sequenced response mutationsA04) at accessions the is same inplant agreement response with experiment the and suggests findings thatrecorded of their as less the regrowth sensitivity whole- might beand due bispyribac-sodium to resistance an mechanism unknown and penoxsulam not due to an E. oryzicola mutation in R and putative Rof accessions codon at 574. the second In nucleotide particular,substituted by the thymine codon’s (T) second and guanineamino thus (G) resulted acid in was the Trp574 substitutionthe (TGG) by less S Leu (TTG) (Trp574Leu). However, each of the eight of the PCR product correspond toequivalent a to 320 bp nucleotide coding positions region 1879Arabidopsis (Fig. ALS to 1), 2198 of theacid standard sequence variation was notof detected among each the individuals accession,each the accession. Nevertheless, consensusobserved among nucleotide sequence the polymorphisms is were the presented sequence alignment for comparison(A05 between and the A06) S and R ) E. E. E. (2), and GenBank Acc. No. Acc. GenBank AB353425 AB223107 AB353427 AB353419 AB223115 AB223111 AB353421 AB353428 AB353429 AB223117 AB353420 Alignment Sequence AB353430 AB223091 AB353422 AB223118 AB223120 AB223098 E. crus- (1) and www.soci.org NS Kaloumenos had ten A- and one E. crus-galli Echinochloa Echinochloa 2012 Society of Chemical Industry L intron of cpDNA of 13 species of the genus E. picta c E. phyllopogon E. crus-galli trn species, whereas (33 accessions), E. crus-pavonis (1), var. formosensis (2), (2), species. Also, the less E. obtusiflora E. stagnina and one var. crus-galli (3), species. Therefore, these E. oryzicola E. crus-galli E. turneriana ) and A06 accessions (collected E. crus-galli E. stagnina E. oryzicola The same alignment performed (1), (3), (2), E. crus-galli E. oryzicola 33 , whereas the samples taken from A05 E. crus-galli (4), E. crus-galli E. colona ) ) ) formosensis praticola motif (and also the remaining species with 5–6 E. phyllopogon species var var E. frumentacea E. esculenta E. phyllopogon (1), species and the sequenced six samples of this study, species and accessions: A01 and A02 accessions with cross-resistance to ALS- Alignment of the A-repeat region of the (2), accessions. Similar results were reported by Yamaguchi (1). The retrieved sequences, aligned by using MEGA5 (10), accession had 11 A-repeats, one and E. crus-galli E. crus-galli E. oryzicola E. oryzicola) E. oryzicola) E. oryzicola) who found that the nucleotide sequencing of cpDNA in E. crus-galli 11 var. praticola (3), . E. crus-galli E. crus-galli E. crus-galli E. crus-galli E. crus-pavonis E. colona E. esculenta E. frumentacea E. oryzoides E. picta E. stagnina E. turneriana E. walteri E. oryzicola E. phyllopogon Echinochloa Echinochloa E. stagnina E-obtusiflora E. oryzicola A05 ( A06 ( A01 ( A02 ( A03 ( A04 ( These findings of molecular differentiation of the six samples examined. The alignment is representative of the one performed with 63 accessions of Table 3. the NCBI nucleotide database and shows all the observed variation in the A-repeat region of this sequence E. walteri software, revealed that nine accession contained 12 A-repeats, one oryzicola repeats, while the remaining sequences containedat 5–6 the A-repeats respective positions. wileyonlinelibrary.com/journal/ps were classified according toEchinochloa the source annotationoryzicola as different galli E. oryzoides E. obtusiflora 30 accessions belonging to nine speciesprovided of information the on phylogenetic genus relationships and new tools for species identification. in the A-repeatEchinochloa region, including representative accessions for and A06 accession plants have five A-repeats matching theof pattern the indicated that the samplesaccession plants taken contain from 12 A-repeats A01, matching A02, the A03 pattern of and A04 oryzicola A-repeats) (Table 3). inhibiting herbicides belong to accessions studiedEchinochloa strongly support the evidence that the E. oryzicola et al were found toresults belong indicate to that theused cpDNA for identification sequence of comparison conventionally classified could be S A03 and A04 accessionsthe belong S to A05 (purchased as from the field margin and classified conventionally as

870 871 35 Alopecurus (A01, A02), two also reported 34 . et al ´ elye and Boucansaud E. oryzicola accessions, which caused the wileyonlinelibrary.com/journal/ps using primers designed from sequence E. oryzicola (A05, A06) accessions. Observed polymorphisms L. rigidum E. crus-galli mutation in four gene of ALS information of related grasstime worldwide. weed This taxa method enabled is thean successful reported detection of for the first information of related taxa. Additionally, D reported thepolymorphic development sequence (dCAPS) method of thatdetect made a Trp574Leu it possible or to derived Pro197Thr in cleaved most black-grass amplified ( amino acid substitution Trp574Leu conferringmost cross-resistance to of the ALS-inhibiting herbicides. Tan successful determination ofALS the Trp574Leu mutation in the gene fragments, originating from two resistant (R) ALS ALS (A03, A04) and two S gene ALS sequences 2012 Society of Chemical Industry E. oryzicola c E. oryzicola ALS resistance to herbicides www.soci.org (A01, A02, A07, A08) accessions supports : 865–873 (A07, A08), two less susceptible (S) 69 Echinochloa species using primers designed from sequence 2013; E. oryzicola E. oryzicola Nucleotide and deduced amino acid sequence alignment of Echinochloa The successful amplification and sequencing of the alleles encoding an amino-aciddetection of replacement the Trp574 at mutation in one codon of thethat 574. two the alleles The suggests 12 R sequenced individualfor plants the were resistant heterozygous Trp574Leu (RS) allele. the evidence thatresistance their to (A01, ALS-inhibiting A02) herbicides was previously due detected to cross- mutant are marked in bold. The codon positions refer to the wild-type mouse-ear cress ALS gene (GenBank: X51514). of Pest Manag Sci altered target-site mechanism of action. However, theTrp574Leu presence mutation of in the 12 analysed from the R Figure 1. putative R ALS mutation and E. in (L.) ALS et al. acces- An Jard Weed Sci Proc Conf Molecular Weed Biol : 865–873 Echinochloa accessions species from 69 Echinochloa :11–17 (2002). E. crus-galli 2 The World’s Worst 2013; E. oryzicola ) and early watergrass and Echinochloa accessions had an E. oryzicola accessions in rice. Vasing. and ; A07 and A08: putative R accessions inferred from internal Weed Biol Manag en el suroeste de Europa. Pest Manag Sci , University Hawaii Press, Honolulu, accessions, measures should be E. crus-galli :416–423 (2008). :1086–1093 (2006). E. oryzicola RI endonuclease distinguished the ˜ nos, Laguna, Philippines, pp. 291–306 E. oryzicola 56 54 Echinochloa Tsp accessions examined in whole-plant ) species in Turkish rice production. , ed. by Soltis DE and Doyle JJ. Chapman Echinochloa oryzicola Echinochloa accessions and confirmed the sequencing ,LosBa Weed Sci Weed Sci Echinochloa oryzicola E. oryzicola ´ enero Echinochloaoryzicola :91–108 (1981). Echinochloa 38 and :233–242 (2008). 8 Echinochloa :525–530 (2012). Echinochloa oryzoides (1983). Bot Madr and molecular markers for classification of northern Greece. crus-galli transcribed spacer andManag chloroplast DNA sequences. Italian rice fields. physiological variation among species of the genus Weeds: Distribution and Biology Hawaii, 609 pp. (1977). a special reference toWeed their Control occurrence in as Rice weeds of rice. identification of Beauv. using alength polymorphism polymerase technique. chain reaction–restriction fragment of late watergrass( ( Systematics of Plants and Hall, New York, NY, pp. 1–13 (1992). 60 phylogeny: present status and future prospects, in accessions. 3 Carretero JL, El g 4 Tabacchi M, Mantegazza R, Spada A and Ferrero A, Morphological traits 6 Aoki D and Yamaguchi H, Genetic relationship between 5 Damalas CA, Dhima KV and Eleftherohorinos IG, Morphological and 2 Michael PW, Taxonomy and distribution of Echinochloa species with 1 Holm LG, Plucknett DL, Pancho JV and Herberger JP, 7 Yasuda K, Yano A, Nakayama Y and Yamaguchi H, Molecular 8 Mennan H and Kaya-Altop E, Molecular techniques for discrimination 9 Clegg MT and Zurawski G, Chloroplast DNA and the study of plant REFERENCES 4 CONCLUSIONS This work clearly demonstrated that two taken for rotationalaction use to reduce of selection pressure on herbicides athus single with mode delay of the action different evolution and of modes R of RfromtheS analysis. The use of cpDNAfied sequence the comparison conventionally classified analysis identi- sions. Therefore, taking into consideration theresistance high to ALS-inhibiting level herbicides of as a cross- result ofmutation the Trp574Leu present in out of the 30 response experiments have developedinhibiting herbicides cross-resistance penoxsulam, to bispyribac-sodium, imazamox, ALS- foramsulfuron, nicosulfuron and rimsulfuron. The DNA sequencing revealed that all plants of thepoint mutation R at codon 574 that resulted in thetution amino Trp574Leu. acid The use substi- of accessions. A01 and A02: resistant (R) E. E. crus-galli ALS also 36 . fragment However, accession www.soci.org NS Kaloumenos E. oryzicola et al Echinochloa Echinochloa 2012 Society of Chemical Industry ALS 17,23 c (A05 and A06) RI endonuclease ; A05 and A06: S fragment (336 bp) Tsp E. oryzicola fragment from the S ALS ALS E. crus-galli E. oryzicola (A01 and A02) and putative R alleles. Also, the similar restriction (A03 and A04) accessions to that of plants the Pro-197-Ala, Pro-197-Arg, RI) of the eight sequenced ALS (A03andA04)accessionsweresimilarto Tsp E. oryzicola L. rigidum E. oryzicola 19,20,24 fragment from the S target-site resistance is in contrast to results E.oryzicola Huds.) plants analysed from a field where the ALS (A07 and A08) accessions suggests the coexistence (A05 and A06) accessions resulted in the same restriction Digestion profile ( ; A03 and A04: less susceptible (S) fragment from the R (A01 and A02) as well as from the puta- RI endonuclease (restriction enzyme) was chosen because E. oryzicola Tsp ALS The same restriction profile (two products of 115 and 221 bp) of The high level of cross-resistance to ALS-inhibiting herbicides profilewithtwoproducts(115and221 bp).Also,therestrictionpro-filesofthelessS the digested it fully digests the SR sequence sequence with at a the mutation Trp574digestion performed within codon, resulted in the but different profiles restriction not for R site. the andsions. Thus, S In acces- particular, the the digestion of the crus-galli thoseoftheSaccessions(A05andA06).Incontrast,thedigestionofthe tive R (A07 and A08)(336 bp) accessions and revealed two an digestion products intact with 115 and 226 bp (Fig. 2). of both R and SRS). alleles These in findings the indicated R that individuals the (heterozygous use plants, of E. oryzicola distinguished successfully the R and putative R accessions fromS the and less S accessions, whichandthusstronglysupportedthehypothesisthatthismethodcould confirmed the sequencing analysis be used to identify mutations atspecies the without Trp574 the codon need in for DNA sequencing. wileyonlinelibrary.com/journal/ps 3.5 Restriction analysis The accessions suggests the integrityabsence of of mutation in the both restriction site and the and two digestion products (115 andfragment 226 from bp) in the the R digested profile of the less S the S accessions supports the574. absence In of contrast, any the mutation presence at of codon an intact myosuroides Figure 2. ALS-inhibiting herbicides failed after 3 years of use. Yu reported by other researchers who found an with cross-resistance to bispyribac-sodium andresult penoxsulam of as enhanced a herbicidemonooxygenases. metabolism via cytochrome P450 this oryzicola developed cleaved amplified polymorphic sequence (CAPS) mark- ers for detecting in as a result of the Trp574Leu mutation present in the Pro-197-Gln, Pro-197-Leu, Pro-197-Ser and Trp-574-Leu mutations. accessions is consistent with other results reported.

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