A European biotype of Amaranthus retroflexus cross-resistant to ALS inhibitors and response to alternative herbicides

A European biotype of Amaranthus retroflexus cross-resistant to ALS inhibitors and response to alternative herbicides
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  A European biotype of  Amaranthus retroflexus  cross-resistant to ALS inhibitors and response toalternative herbicides L SCARABEL*, S VAROTTO   & M SATTIN* * Istituto di Biologia Agroambientale e Forestale - CNR, Legnaro, Padova, Italy, and    Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Universita ` di Padova, Legnaro, Padova, Italy Received 24 January 2007Revised version accepted 22 August 2007 Summary An acetolactate synthase (ALS)-resistant  Amaranthusretroflexus  biotype was collected in a soyabean cropafter repeated exposure to imazethapyr and thifensulfu-ron-methyl in north-eastern Italy. Studies were con-ducted to characterise the resistance status anddetermine alternative post-emergence herbicides forcontrolling this biotype. Whole-plant bioassay revealedthat the GR 50  values were 1898- and 293-fold higherthan those observed for the biotype susceptible toimazethapyr and imazamox respectively. The biotypealso displayed high cross-resistance to sulfonylureas.Molecular analysis demonstrated that a single nucleo-tide substitution had occurred in domain B (TGG toTTG at position 574), conferring a change from theamino acid tryptophan to leucine in the resistantbiotype. However, herbicides with other modes of action(PSII, 4-HPPD and PPO inhibitors) provided excellentcontrol. The GR 50  ratios for metribuzin, terbuthylazineand mesotrione were close to 1 and treatments withfomesafen gave 100% control of both susceptible andresistant biotypes at the recommended field dose. Thisstudy documents the first case of an imidazolinone andALS-resistant biotype in European crops and identifiesthe post-emergence herbicide options available formanaging this troublesome weed in soyabean crops.Alternative management strategies are also discussed. Keywords:  common amaranth, redroot pigweed, aceto-lactate synthase resistance, point mutation, soyabean,weed resistance management.S CARABEL  L, V AROTTO  S & S ATTIN  M (2007). A European biotype of   Amaranthus retroflexus  cross-resistant to ALSinhibitors and response to alternative herbicides.  Weed Research  47 , 527–533. Introduction Herbicides are essential tools for weed management inconventional agriculture and among them, acetolactatesynthase (ALS) inhibitors are widely used. This isbecause of their good crop selectivity, excellent efficacyat low rates against a broad spectrum of weeds and lowmammalian toxicity (Saari  et al. , 1994). Since the intro-duction of the first sulfonylurea (SU), the agro-chemicalindustry has continued to discover and develop new SUsandotherALS-herbicidefamilies:imidazolinones(IMIs),triazolopyrimidines (TPs), pyrimidinylthiobenzoatesand sulfonylamino-carbonyl-triazolinones.These herbicides are generally used at low or very lowrate, with most being applied at 5–100 g a.i. ha ) 1 , whichhas significantly contributed towards reducing theamount of herbicide applied to crops (Shaner & Singh,1997). However, because of their high efficacy againstseveral major broadleaf weeds and their very widespreaduse, they impose an intense selection pressure on targetweeds. As a result, there has been rapid growth in thenumber of resistant species and populations withinspecies (Heap, 2007). Integrating herbicides with variouscropping practices and rotating herbicides with differentmodes of action can delay a high frequency of resistantplants being reached (Jasieniuk  et al. , 1996). Correspondence : Laura Scarabel, Istituto di Biologia Agroambientale e Forestale - CNR, viale dell  Universita ` 16, 35020 Legnaro, Padova, Italy.Tel: (+39) 049 8272822; Fax: (+39) 049 8272839; E-mail:   2007 The AuthorsJournal Compilation    2007 European Weed Research Society  Weed Research  47,  527–533  In most cases, field-selected ALS-inhibitor resistanceis due to an altered ALS enzyme that is no longersensitive to the herbicide (i.e. target-site resistance)(Shaner & Singh, 1997). However, a resistance mecha-nism, based on herbicide detoxification, was alsoreported in some monocotyledon weed populations(Kemp  et al. , 1990; Christopher  et al. , 1991; Fischer et al. , 2000; Hidayat & Preston, 2001), but in these casesALS inhibitors were not the selecting agent. Moreoverthe first case of metabolism-based resistance in abroadleaf weed has been reported in  Sinapis arvensis (Veldhuis  et al. , 2000).Target-site resistance is due to one or more pointmutations in the gene  als  coding sequence thatdetermines an amino acid change in the protein(Devine & Eberlein, 1997). Several mutations havebeen documented in the five highly conserved domainsof the  als  gene (for a review, see Tranel & Wright,2002; Heap, 2007). The most frequent are encounteredin domain A at Pro 197 , conferring resistance especiallyto SUs, and in domain B at Trp 574  determining a highlevel of cross-resistance to SUs, IMIs and TPs. InItaly, the constantly increasing reliance on target-sitespecific herbicides such as ALS and ACCase inhibi-tors, as well as on cropping systems with limiteddiversity (i.e. monocultures or short rotations whereherbicides with the same mode of action are used), isleading to a significant increase in the impact of herbicide resistance. The worst situations are relatedto ALS-resistant populations in rice crops ( Alisma plantago-aquatica  L.,  Schoenoplectus mucronatus  (L.)Palla and more recently  Cyperus difformis  L.) (Tabac-chi  et al. , 2004), and to both ALS- and ACCase-resistant populations in durum wheat crops ( Papaverrhoeas  L.,  Lolium  spp.,  Avena sterilis  L. and  Phalaris paradoxa  L.) (Scarabel  et al. , 2004; Sattin, 2005).In Italian maize crops, only three PSII-resistantspecies (i.e.  Solanum nigrum  L.,  Amaranthus hybridus L. and  Chenopodium album  L.) were recorded in theearly 1980s in the most northern part of the Po Valleywhere atrazine was used alone (Porceddu  et al. , 1997).Now, an  Amaranthus retroflexus  L. biotype poorlycontrolled by ALS herbicides has been found in asoyabean crop in north-eastern Italy.  Amaranthus  is acommon annual broad-leaved genus very prone toevolving herbicide resistant populations, with 11 speciesinvolved worldwide (Heap, 2007). Among these, A. retroflexus  and the closely related  A. hybridus  and A. powelii   have evolved cases of resistance to ALSinhibitors in Canada (Ferguson  et al. , 2001; McNaugh-ton  et al. , 2005), USA (Manley  et al. , 1999), Israel(Sibony  et al. , 2001) and Serbia, although no officialreports are available for this latter case. Multipleresistance to ALS and triazine inhibitors have also beenconfirmed in  A. hybridus  in the USA (Maertens  et al. ,2004) and  A. powelii   S. Wats. in Canada (Diebold  et al. ,2003). Amaranthus retroflexus  is a highly competitive C 4 species, difficult to manage in agronomic crops becauseof high seed production, long seed viability with annualchanges in dormancy status (Baskin & Baskin, 1998)and extended germination period. It is a monoeciousspecies, partially or highly self-fertilised with occasionaloutcrossing (Holm  et al. , 1997). However, it has beendemonstrated that different  Amaranthus  species cancross, broadening the genetic variation across species(Wetzel  et al. , 1999). The interspecific transfer of aherbicide-resistance gene from controlled crosses be-tween a dioecious and monoecious  Amaranthus  specieshas been documented (Tranel  et al. , 2002). Morerecently, a large proportion of nonhybrid progeny wasfound in crosses of dioecious  Amaranthus  supporting theoccurrence of unreduced gametes in these species andleading to reconsideration of previously reported data(Trucco  et al. , 2007). The aim of this study was toconfirm the resistance status of the ALS-selected A. retroflexus  biotype, to elucidate the molecular basisof resistance and verify the efficacy of alternative post-emergence herbicides. Materials and methods Plant material  Seeds from  A. retroflexus  plants that had survived atreatment with imazamox plus thifensulfuron-methylwere collected during summer 2003 in north-easternItaly (population 03-07). The soyabean sample field, of about 30 ha, presented a patchy  A. retroflexus  infesta-tion and had been treated with ALS inhibitors for atleast 5 years (Table 1). Seeds from a susceptible popu-lation (99-01) of the same species were collected in thesame region as population 03-07 from a location thathad never been treated with ALS inhibitors. Both seedsamples were cleaned and stored at room temperature.Records of 5 year herbicides use in the sampled fieldwere obtained from farmers. Table 1  Crops and herbicide use history of the site where seeds of the suspected ALS-resistant  A. retroflexus  population werecollected Year Crop Herbicides applied1999 Soyabean Imazethapyr + thifensulfuron-methyl2000 Soyabean Imazethapyr + thifensulfuron-methyl2001 Soyabean Imazethapyr + thifensulfuron-methyl2002 Maize Sulfonylurea2003 Soyabean Imazamox + thifensulfuron-methyl 528  L Scarabel  et al.   2007 The AuthorsJournal Compilation    2007 European Weed Research Society  Weed Research  47,  527–533  Greenhouse whole-plant bioassays Screening experiment  A preliminary screening was performed to confirm theresistant or susceptible status of both populations and toobtain indications on the herbicide dose ranges to usefor the subsequent dose–response experiments.Seeds were sown in Petri dishes containing agar(0.6%) and placed in a germination cabinet at 15  ⁄   25  Cnight  ⁄   day and 12 h photoperiod with neon tubesproviding a photosynthetic photon flux density of 15–30  l mol m ) 2 s ) 1 . Pre-germinated seeds were trans-planted into plastic boxes (24 cm  ·  30 cm  ·  9 cm) filledwith a substrate (60% silty loam soil, 15% sand, 15%perlite and 10% peat by volume) and placed in agreenhouse at Legnaro, north-eastern Italy (45  21 ¢ N,11  58 ¢ E).A week after transplanting, plants were thinned to 20per box. The experimental layout was a complete rando-mised design with three replicates. Three- to four-leaf stage plants, corresponding to growth stage 13–14 of theextended BBCH Scale (Hess  et al. , 1997), were treatedwith post-emergence herbicides at recommended fielddose (1 · ) and three times that (3 · ), along with recom-mended surfactants. ALS-inhibitor herbicides: imazeth-apyr (Overtop 35 LC, 35 g a.i. L ) 1 , LC; Dupont),imazamox (Altorex, 40 g a.i. L ) 1 , LC; BASF), oxasulfu-ron (Dynam, 750 g a.i. L ) 1 , WDG; Syngenta), nicosul-furon (Ghibli, 40 g a.i. L ) 1 , SC; Syngenta) andthifensulfuron-methyl (Harmony, 750 g a.i. L ) 1 , DF;Dupont); PSII inhibitors: metribuzin (Niber WG, 350 ga.i. L ) 1 , WG; SIAPA) and terbuthylazine (Click 50 FL,560 g a.i. L ) 1 , FL; SIPCAM); 4-hydroxyphenyl-pyru-vate-dioxygenase inhibitors (bleaching herbicides): mes-otrione (Callisto, 100 g a.i. L ) 1 , SC; Syngenta) and aprotoporphyrinogen oxidase (PPO) inhibitor: fomesafen(Flex, 250 g a.i. L ) 1 , SC; Syngenta).Herbicides were applied using a precision benchsprayer delivering 300 L ha ) 1 at a pressure of 215 kPaand speed of about 0.75 m s ) 1 , with a boom equippedwith three flat-fan (extended range) hydraulic nozzles(Teejet, 11002). Plants were watered daily as required.Just prior to treatment, the number of plants wascounted in each box. Three weeks after herbicideapplication, the number of surviving plants and abiomass visual score (0 for dead plants, 10 for plantssimilar to the untreated check) were recorded. Plantsthat showed no active growth, regardless of colour,were considered to be dead. The screening was carriedout during spring  ⁄   summer. The temperature in thegreenhouse varied between 15 and 20  C and from25  C to 34  C, during the night and day respectively.Standard error (SE) was calculated for each meandata. Dose–response experiment  A greenhouse dose–response experiment was performedon the resistant and susceptible populations with all theherbicides used in the preliminary screening, fomesafenexcepted. Six to seven herbicide doses (plus an untreatedcheck) were used, ranging from 1 ·  (i.e. recommendedfield dose) to 8 ·  (thifensulfuron-methyl), 16 ·  (oxasulfu-ron and nicosulfuron) or 32 ·  (imazethapyr and imaza-mox) for the resistant population and from 1  ⁄   32 ·  to 1 · for the susceptible population. The number of survivingplantsandshootfreshweightwererecorded3 weeksafterthe herbicide treatments. Given that no mortality wasrecordedfortheuntreatedplants,survivalwascalculatedas a percentage of the number of plants recorded in eachplastic box just before the treatment. The experimentallayout was a completely randomised design with threereplicates. Identical experimental procedures to those forthescreeningwerefollowed,theonlydifferencebeingthatplants were thinned to fifteen per box.A log-logistic equation was fitted to the data (Seefeldt et al. , 1995). Regression analysis was performed with BIOASSAY 97, an EXCEL   VBA macro (Onofri, 2001).The data proved to be non homogeneous and therefore,after an analysis of the residuals, the data weretransformed by using a  k  of 0.25 in the Box-Coxtransformation. The equation was re-parameterised toobtain GR 50  (herbicide dose that causes 50% shootfresh weight reduction) and ED 50  (herbicide dose thatcauses 50% survival) as equation parameters. Standarderrors of the parameters were calculated. For both thepercentage of survival and percentage of fresh weightdata, the upper asymptote was set at 100. Resistantratios were calculated from the GR 50  and ED 50  values(GR 50 R  ⁄   GR 50 S, ED 50 R  ⁄   ED 50 S). Molecular basis of resistance  Genomic DNA was extracted from five individual plantsthat survived the treatment with imazethapyr at fielddose (1 · ) and from three plants sampled from theuntreated susceptible check using the Nucleon Phyto-pure extraction Kit (Amersham International, Buck-inghamshire, UK). All extractions using 0.4 g of freshplant tissue were made following the manufacturer  sinstructions, apart from the addition of twice thesuggested volume of suspension resin. The final elutionstep was performed in 500  l L of sterile water.Specific primers were designed on regions of highhomology among the als gene sequences of different Amaranthus  species available in GenBank:  Amaranthus sp. U55852,  A. retroflexus  AF363369,  A. quitensis  KunthAJ514935,  A. powellii   S.Wats. AF363370 and  A. blito-ides  S. Watson AY124583. The primer combinationAMA-3F (5 ¢ -GCGATGTTCTCGTTGAAGCTC-3 ¢ )  ⁄   ALS resistance in  Amaranthus retroflexus  529   2007 The AuthorsJournal Compilation    2007 European Weed Research Society  Weed Research  47,  527–533  AMA-3 (5 ¢ -AACAGGTCCAGGTCTACCAGA-3 ¢ )produced a genomic fragment at the 5 ¢  end of the alsgene, whereas the combination AMA-2F (5 ¢ -TC-CCGGTTAAAATCATGCTC-3 ¢ )  ⁄   AMA-2R (5 ¢ -CTT-CTTCCATCACCCTCTGT-3 ¢ ) amplified at the 3 ¢  endof the gene. Amplifications of each gene region for eachplant were performed as separate reactions. These PCRreactions were carried out in 50  l L with 100 ng of genomic DNA template, 0.6  l M  of each primer, 0.2 m M of each dNTP and 1  l L of proofreading Advantage 2Polymerase mix (BD Biosciences Clontech, Palo Alto,CA, USA). Touchdown PCR reactions were carried outin a Thermal Cycler 2400 (Applied Biosystems, FosterCity, CA, USA) for 15 s at 94  C, then six cycles for 10 sat 94  C, 1 min at 62  C, followed by 30 cycles for 10 s at94  C and 1 min at 58  C. Final extension time was10 min at 70  C. Primers AMA-3F and AMA-3R wereused to amplify a genomic fragment of 451 bp contain-ing domains A, C and D of the gene, while primersAMA-2F and AMA-2R produced a fragment of 336 bpcontaining the B and E domains. The amplified productswere fractioned through 1% agarose gels and the desiredfragments were excised and purified using a DNA gelextraction kit (Millipore, Billerica, MA, USA). Thesequencing reaction was prepared using the Big DyeTerminator Ready Reaction Kit (Applied Biosystems)and nucleotide sequencing of the DNA fragments wasperformed by an ABI PRISM 310 Genetic Analyser(Applied Biosystems). Forward and reverse primerswere used to sequence both DNA strands for eachALS region at least twice in each orientation. Nucleotidesequences were aligned and compared using Lasergenesoftware  DNASTAR . Results and discussion Greenhouse bioassays  The preliminary screening confirmed that the twopopulations of   A. retroflexus  displayed very differentsusceptibility to the ALS inhibitors tested (data notshown). The susceptible population was totally con-trolled by all herbicides sprayed at the recommendedfield dose. Population 03-07 appeared to be highlyresistant to both IMI herbicides (more than 90%survival at dose 1 ·  and between 70% and 95% at dose3 · ). The response was more variable for the SUs,showing a control percentage of between 20% and 65%at recommended field dose. Population 03-07 was fullycontrolled by all non-ALS herbicides used at therecommended field dose.The results of the screening were fully confirmed bythe dose–response experiment (Fig. 1; Table 2). Thesusceptible population was entirely controlled with half the recommended field dose of imazethapyr, whereasonly 27% of the plants of the resistant biotype werecontrolled at dose 32 ·  (1120 g a.i. ha ) 1 , Fig. 1). Survivalpercentage and shoot fresh weight gave similar results,so only the latter are reported in Table 2. The SE of GR 50  and ED 50  are generally rather low and indicatethat the log-logistic equation fitted the data accurately.However, it was sometimes impossible to fit the curvebecause of a very high resistance level. Population 03-07was >1898- and 293-fold more resistant to imazethapyrand imazamox, respectively, than the susceptible popu- 01020304050607080901000.01 0.1 1 10 100 1000 10 000 Imazethapyr (g a.i. ha –1 )    S  u  r  v   i  v  a   l   (   %   o   f  u  n   t  r  e  a   t  e   d   ) 0 Fig. 1  Effect of imazethapyr (predominant selecting agent) onsurvival of   Amaranthus retroflexus -resistant ( d ) and susceptible( s ) biotypes 22 days after treatment. Given the very low controllevel of the resistant biotype at even the highest herbicide dose(32 · ), the fitting of the log-logistic equation was impossible.Vertical bars represent the standard errors. ED 50  of Spopulation = 1.88 ± 0.902. Table 2  GR 50  values (herbicide dose causing 50% reduction of shoot fresh weight relative to untreated control), relative standarderror (SE) and resistant ratio of the ALS-resistant (03-07) andsusceptible (99-01) biotypes of   A. retroflexus  treated with ALSinhibitors Herbicide PopulationGR 50 (g a.i. ha ) 1 ) SEResistantratioImazethapyr 03-07 >1120* – >189899-01 0.59 0.046 –Imazamox 03-07 167.2 50.6 29399-01 0.57 0.105 –Nicosulfuron 03-07 139.2 8.49 33.999-01 4.11 0.657 –Oxasulfuron 03-07 >1200* – >21599-01 5.58 1.198 –Thifensulfuron-methyl03-07 >60* – >42999-01 0.14 0.006 – *The highest herbicide dose is reported where the reduction inshoot fresh weight never reached 50%, so it was impossible to fitthe log-logistic equation. 530  L Scarabel  et al.   2007 The AuthorsJournal Compilation    2007 European Weed Research Society  Weed Research  47,  527–533  lation. The resistant ratios for nicosulfuron, oxasulfuronand thifensulfuron-methyl were 34, >215 and >429(Table 2) respectively. Except for imazamox, the valuesof the resistant ratios based on plant survival (ED 50 ,data not shown) were generally lower than thosecalculated for shoot fresh weight. Different resistancelevels within the same chemical family have already beendocumented in other studies (Devine  et al. , 1991; Sibony& Rubin, 2003). This suggests that herbicide–enzymeinteractions are complex and one particular pointmutation in the  als  gene can diversely affect the bindingof each herbicide molecule; given this situation it isdifficult to precisely predict the cross-resistance pattern.The highest resistance ratios observed for imazethapyrand thifensulfuron-methyl are probably related to thefact that these herbicides were the selecting agents.While the lower resistant level induced by nicosulfuronis likely due to the intrinsic herbicide characteristics, herbicide not specific for dicotyledons. The occur-rence of the resistant  A. retroflexus  population in thesoyabean crop after as few as four or five herbicideapplications fits well the classical model of the develop-ment of resistance from selection pressure imposed forfour to seven applications of the selection agent(s) (Saari et al. , 1994). Similarly, the sulfometuron-selected  A.retroflexus  biotype reported by Sibony  et al.  (2001)occurred after three consecutive years of treatments withsulfometuron-methyl combined with simazine. Molecular basis of resistance  The very high levels of resistance, plus cross-resistancebetween SUs and IMIs, strongly suggested that theresistance mechanism was likely to be the site of actionmediated. The responsible mutation was identified bysequencing two regions of the als gene. These regionsinclude the five conserved domains where mutationsconferring resistance are usually located. The combina-tion of specific primers AMA-3R and AMA-3F ampli-fied a fragment of 451 bp containing domains A, C andD, while combination of primers AMA-2R and AMA-2F amplified a genomic fragment of 336 bp containingthe conserved domains B and E of the als gene.Combining the two fragments, a contig of 787 basepairs was sequenced and aligned. Alignment of thenucleotide sequences of the ALS-amplified regions of theresistant and susceptible populations showed only onenucleotide substitution coding for an amino acid changeat position 574 in domain B (referred to as  Arabidopsisthaliana  (L.) Heynh sequence). The susceptible biotypehad a TGG encoding tryptophan residues, while theresistant biotype had a TTG encoding a leucine. All fiveresistant plants analysed presented the same pointmutation and were homozygous at this locus. No otherpolymorphisms were identified within the 787 bp alssequence. The leucine to tryptophan substitution foundat position 574 has been shown to confer a broadspectrum of resistance to ALS herbicides in  Amaranthus sp. and other weed biotypes (Bernasconi  et al. , 1995;Foes  et al. , 1998; Boutsalis  et al. , 1999; Maertens  et al. ,2004; McNaughton  et al. , 2005). Consistently, theItalian  A. retroflexus  biotype displayed cross-resistanceto both SU and IMI herbicides and it is likely that the  driving   selecting agents of population 03-07 were IMIherbicides. Instead, most resistant biotypes selected bySUs have a mutation in position Pro 197  (Tranel  et al. ,2007), which usually confers no or little resistance toIMIs. A partial exception to this pattern was reported bySibony  et al.  (2001) for a population of   A. retroflexus found in Israel. The population had been selected bysulfometuron-methyl and, as expected, the mutationinvolved was at position Pro 197 , although it is unclear onhow many plants the molecular test had been carriedout. However, the Israeli biotype showed a broad cross-resistance between ALS inhibitors, with highly variableresistance levels among herbicides. These and otherresults published so far point to the existence of exceptions to the general   model   associating a particularmutation to a given resistance pattern.It can be concluded that the leucine to tryptophansubstitution at position 574 is the molecular basis for theALS resistance in  A. retroflexus . The type of reproduc-tive system in  A. retroflexus  (partially or highly self-fertilised with occasional outcrossing) facilitates theincrease in the frequency of the dominant allele andmight explain why all five tested plants were homozy-gous at the ALS locus. Management of ALS-resistant   A. retroflexus Several non-ALS-inhibiting herbicides effective against A. retroflexus  and used in soyabean and maize cropswere tested. The GR 50  of susceptible and resistantpopulations calculated for metribuzin, terbuthylazineand mesotrione were similar, determining resistantratios close to 1 (Table 3). The screening with fomesafenalso showed 100% control of both susceptible andresistant biotypes (data not shown) at doses 1 ·  and 3 · .The results proved that herbicides with differentmodes of action belonging to the PSII, 4-HPPD andPPO inhibitors adequately controlled the ALS-resistantpopulation 03-07. Therefore bentazone (a PSII inhibi-tor) and fomesafen could be applied in combinationwith ALS inhibitors to control this troublesome weed insoyabean. However, to manage resistant weed popula-tions it is important that the herbicide tank mixturescontain herbicides with different modes of action, havesimilar persistence and control the same target weeds ALS resistance in  Amaranthus retroflexus  531   2007 The AuthorsJournal Compilation    2007 European Weed Research Society  Weed Research  47,  527–533
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