Evolution of Glyphosate-Resistant Johnsongrass (Sorghum halepense) inGlyphosate-Resistant Soybean

Martin M. Vila-Aiub, Maria C. Balbi, Pedro E. Gundel, Claudio M. Ghersa, and Stephen B. Powles*

In Argentinean crop fields, weed control is mainly achieved by intense use of glyphosate as a nonselective and/or selectiveherbicide. Glyphosate use is very high as more than 95% of the 16 million ha soybean crop consists of glyphosate-resistantcultivars, always treated with this herbicide. From initial success, inconsistent glyphosate control of Johnsongrass, aninvading C4 perennial grass of soybean crops, has become evident to producers from northern Argentina over the last 3 yr.Prior to this, glyphosate provided good control. This study evaluated the nature of these recurrent glyphosate failures inJohnsongrass. Experiments conducted with Johnsongrass plants obtained from seed and rhizome phytomers collected fromfields with intense glyphosate use history showed that these populations showed differential survival and biomassproductivity when glyphosate treated than Johnsongrass plants obtained from similar propagules collected from field siteswith no history of glyphosate use. This empirical evidence establishes that the Johnsongrass survival in glyphosate-treatedtransgenic soybean fields from northern Argentina is due to evolved glyphosate resistance.Nomenclature: Glyphosate; Johnsongrass, Sorghum halepense L. Pers. SORHA; soybean, Glycine max (L.) Merr.Key words: Herbicide selection; resistance; evolution.

Argentina is a major agricultural nation and is the world’ssecond largest producer of genetically modified (GM) crops(James 2006). GM soybean with resistance to the herbicideglyphosate has become universally adopted (99% of thesoybean crop) and has facilitated the expansion of theArgentine soybean area to 16 million ha (40 million Mgproduction) (Argentina Department of Agriculture 2007).Producers have greatly benefited from the massive adoption ofthis technology, which in combination with no till has led tobetter weed control, reduced production costs, and highercrop yields. As a result of this technological adoption, the useof glyphosate has increased from 14 million L in 1996 to175 million L in 2006 (M. Balsa, personal communication).

Continuous use of herbicides with the same mode of actionis a key factor in the evolution of herbicide resistant weedpopulations. The appearance of herbicide resistance is theconsequence of an evolutionary process driven by a strongselection pressure exerted by herbicides on genetically diverseweed populations (Maxwell and Mortimer 1994). Givensufficient genetic diversity in weed populations, resistancealleles will be enriched when herbicides are the main selectiveforce in agricultural systems (Powles et al. 1997).

High efficacy, broad weed spectrum, low mammaliantoxicity, and lack of soil activity are key features that makeglyphosate the world’s most widely used herbicide (Baylis2000). In recent years, populations of several weed specieshave been confirmed as glyphosate resistant in differentcropping systems worldwide (reviewed in Owen and Zelaya2005; Powles and Preston 2006; Powles and Wilcut 2007;Vila-Aiub et al. 2007). Since the first documented case inLolium rigidum Gaudin (Powles et al. 1998), glyphosateresistance evolution rate has been occurring at one weedspecies per year, accounting for a total of 11 species at thepresent time (see Heap 2007). Irrespective of the croppingsystem (fruit orchards, vineyards, cereals, legumes, etc.), thekey element that unequivocally accounts for the evolution of

glyphosate resistant populations of weed species [e.g. L.rigidum, Conyza canadensis (L.) Cronq., Eleusine indica (L.)Gaertn., and Amaranthus rudis Sauer] has been a persistentreliance on glyphosate with low agronomic diversity in weedcontrol systems.

Johnsongrass is a C4 perennial and rhizomatous grass weedwhich reproduces by seeds and rhizomes. It is a tetraallopolyploid (n 5 10, 2n 5 40) (Celarier 1958) andregarded as one of the major weeds of the world (Holmes et al.1977). It was introduced as a forage grass into the USA earlyin the 19th century and into Argentina (from USA) around1910. Johnsongrass became widely naturalized and weedy inboth countries and reports showed the severity and perniciousnature of this weed (McWorther 1971). Considerablevariation in morphology of this weed is known, and it isbelieved that specific traits in Johnsongrass populations mighthave been derived from cultivated sorghum (Celarier 1958),since hybrids between Johnsongrass and cultivated sorghumhave been reported (Hoang-Tang and Liang 1988; Dweikat2005).

Many studies have investigated Johnsongrass’s competitiveability in agriculture systems of the Argentine Pampas regionas well as several ecophysiological and demographical featuresof the species (Benech-Arnold et al. 1990; Ghersa et al. 1990,1992). These studies have shown for example, that crop yieldlosses varied between 12 to 95% in corn, 19 to 99% insunflower, and 18 to 94% in soybean for low and highJohnsongrass infestation levels, respectively (Ghersa et al.1985, 1993; Ghersa and Martınez-Ghersa 1991). Highfecundity, seed dormancy, and residual seedbank life allcontribute to survival and weediness of Johnsongrass (Benech-Arnold et al. 1982; Leguizamon 1986). Seed dormancy isimportant in the persistence of Johnsongrass in Argentinecropping systems (Benech-Arnold et al. 1988, 1992), as wellas soil tillage (Van Esso et al. 1986).

The Argentinean Province of Salta is a relatively newcropping area devoted to the production of GM soybean. Inthis subtropical, warm climate region, glyphosate-resistantGM soybean has been grown only since 2000. Glyphosateis being intensively and often exclusively used (an average of4,000 g ae ha21 yr21) as a nonselective and selective herbi-cide in fallow and GM-soybean fields, respectively. Over the

DOI: 10.1614/WS-07-053.1* First, third, and fourth authors: IFEVA–CONICET–Facultad de Agronomıa,

Universidad de Buenos Aires (UBA), Av. San Martın 4453, Buenos Aires (1417),Argentina; second author: Monsanto Argentina, Fontezuela Research Station,Provincia de Buenos Aires, Ruta 8 km 214; fifth author: WAHRI–School ofPlant Biology, The University of Western Australia (UWA), 35 Stirling Hwy,Crawley (6009), WA, Australia. Corresponding author’s E-mail: vila@ifeva.edu.ar

Weed Science 2007 55:566–571

566 N Weed Science 55, November–December 2007

last 3 yr, producers in this region have reported inconsistentcontrol by glyphosate of numerous Johnsongrass populationsin GM-soybean crops and fallow fields. Given the key role forglyphosate in GM-soybean production in Argentina, the goalof this study was to determine whether the reportedJohnsongrass control failures after glyphosate exposure inthe northwest of Argentina are a consequence of resistanceevolution.

Materials and Methods

Plant Material. In January 2006, three different farms nearTartagal (22u399S, 63u499W) (Province of Salta, Argentina)were visited for collection of suspected glyphosate resistantJohnsongrass individuals (rhizomes and seeds) (Figure 1).Anecdotal statements from the operating farmers were thatglyphosate efficacy on this weed had reduced notably in thelast few years. Problem farms had a 6-yr glyphosate use historyof one preseeding fallow application (2,000 g ae ha21) andtwo postemergent applications during GM soybean pro-duction (each of 1,000 g ae ha21). Plant material wascollected from three (designated as A, B, C), two (A, B),and one field corresponding respectively to sites 1 (S1), 2 (S2),and 3 (S3). A Johnsongrass population from the same generalarea but collected in a roadside with no previous history ofglyphosate application served as a reference susceptiblematerial (site 4, S4). Glyphosate resistance evaluation ofcollected material was conducted at the campus of the Facultyof Agronomy, University of Buenos Aires (Buenos Aires,34u359S, 58u359W) and Fontezuela Monsanto ResearchStation (Pergamino, 33u539S, 60u349W). Two Johnsongrasspopulations with no previous exposure to glyphosate selectionwere each collected at Buenos Aires (S5) and Pergamino (S6)

and also served as reference susceptible populations. Allexperiments were carried out between February and Decem-ber 2006.

Glyphosate Response of Plants Grown from Rhizomes.Rhizome sections of Johnsongrass individuals collected fromseveral plant stands at sites S1A, S1B, S2A, and S2B(Figure 1) were buried in 15-L pots containing a mixture oforganic soil, sand, and peat moss (1:1:1) and allowed toproduce new rhizomes. The same procedure was conductedfor the known glyphosate susceptible Johnsongrass popula-tions S4 and S5. Rhizomes (3-cm length) containing only onebud (i.e., phytomere) were planted (3-cm depth) in 2-L potscontaining the above substrate. Ten to fifteen rhizomes wereplanted per pot and allowed to sprout. Pots were maintainedoutdoors (normal growing season for Johnsongrass), fertilizedwith commercial urea (N: 46%) and irrigated regularly. Whenshoots (from rhizomes) reached the four- to six-leaf stage and40-cm height they were treated with glyphosate (0, 500,1,000, 2,000, and 4,000 g ae ha21). For field sites S1A andS2A there was only limited plant material so only glyphosateat rates 0, 500, and 1,000 g ae ha21 was used. The potassiumsalt of glyphosate1 (500 g ae L21) was applied using a 20-Lcapacity backpack sprayer with a three-nozzle boom (Lurmark01-F110) delivering a volume of 110 L ha21 at 200 kPa.Glyphosate response curves were obtained by assessing plantsurvival 30 d after glyphosate treatment in a completelyrandomized experimental design with three replicates pertreatment (n 5 3).

Glyphosate Response of Plants Germinated from Seed.The first experiment was conducted using seeds collected fromJohnsongrass plants infesting a field (S1A) in Tartagal.

Figure 1. Field sites (S) in the northwest of Argentina (Province of Salta) where Johnsongrass populations were collected for glyphosate resistance evaluation. Whereassites 1, 2, and 3 denote different farms, A, B, and C correspond to different fields of each farm. S4 represents the location (roadside) where a known glyphosate susceptibleJohnsongrass population was collected.

Vila-Aiub et al.: Glyphosate resistance in Johnsongrass N 567

Collected seeds of glyphosate sensitive population (S5) servedas the control treatment. To overcome seed dormancy, seeds(S1A and S5) were immersed in sodium hypochlorite bleach2

(chlorine 5.5% w/v) for 8 h (Benech-Arnold et al. 1992), thenwashed with tap water and germinated on 0.7% (w/v) agar at12-hourly alternating 30/20 C with a 12-h photoperiod.Seedlings of similar size were transplanted to 2-L pots (10seedlings per pot) under glasshouse conditions (fluctuating30/20 C, day/night) in a completely randomized experimen-tal design with three replicates per treatment (n 5 3). At thefour-leaf stage (40-cm height), seedlings were treated withglyphosate (0, 500, 1,000, 2,000, and 4,000 g ae ha21). Plantsurvival was assessed 30 d after treatment.

A second experiment was conducted employing seedsharvested from rhizomatous plants that had been collectedfrom S2C and S3 field sites (Figure 1) and grown in theglasshouse to seed production. Seeds were collected fromJohnsongrass individuals (S2C and S3) that survived twosequential glyphosate applications of 3,128 g ae ha21. Ma-ture seeds from glyphosate susceptible Johnsongrass popula-tions (S4 and S6) were used for comparison. Suspectedglyphosate resistant seeds collected from both S2C and S3sites were stored dry at 8 C for 4 mo to overcome seeddormancy. Glyphosate response was evaluated under glass-house conditions (26–28 C) using plants at two growthstages: individuals with single 100- to 120-cm high shootswith incipient rhizomes and tillers and 30- to 50-cm tallplants with no rhizome or tiller. Whereas the former stagecorresponded to a thermal time accumulation of 931 degree -days, the latter corresponded to 537 degree days. Bothaccumulated thermal times were estimated using a basetemperature of 15 C (Satorre et al. 1985). A monoamonic saltof glyphosate3 (680 g ae ha21) was applied in this experimentwith a CO2 pressurized backpack sprayer fitted with a TT11001 nozzle delivering 100 L ha21 at 200 kPa.

Plant survival and biomass was determined 28 d aftertreatment. Aboveground biomass was excised at the soilsurface and dried at 45 C for 4 wk, weighed and thencalculated as a percentage of the mean dry biomass of theuntreated control plants. A completely randomized experi-mental design with three replicates (n 5 3) was conducted.Each experimental unit consisted of eight plants each in 0.5-Lpots containing organic soil, peat moss, vermiculite, andperlite (50:10:20:20). Pots were watered to field capacity.

Statistical Analysis. Analysis of variance (ANOVA) wasperformed in all experiments to determine principal effects ofphenotype (population origin) and herbicide dose on assessed

variables (plant survival and biomass). When necessary, plantsurvival (%) and biomass (as % of untreated plants) data wereangular-transformed (y 5 arcsine !x) to increase normalityand variance homogeneity (Sokal and Rohlf 1969). The LD50

or GR50 which respectively account for the herbicide raterequired to cause 50% survival and biomass reduction wasestimated for the Johnsongrass populations under glyphosateselection by nonlinear regression analysis using the logisticmodel (SigmaPlot4 V9.01):

S or B %ð Þ~ a.

1 z x�

LD or GRð Þ50

� �b ½1�

where S and B denote respectively the survival and biomass ofplants as percentage at herbicide rate x, a is the maximumplant survival or biomass attained (%), and b is the slope atLD50 or GR50.

Results and Discussion

Glyphosate Response of Plants Grown from Rhizomes.Johnsongrass individuals collected from field sites witha history of glyphosate use showed a different response toglyphosate treatment than individuals collected from sitesnever exposed to glyphosate (P , 0.001). Plants from soybeanfields with a glyphosate selection history of 6 yr, hada significantly higher plant survival to glyphosate rates 500,1,000, and 2,000 g ae ha21 than those from fields with noglyphosate selection history (Table 1, Figure 2). The logisticmodel provided a significant fit (P , 0.0001) to plant survivalin relation to glyphosate rates for Johnsongrass individualscollected from field sites S1B (R2 5 83%) and S2B (R2 590%). Estimated LD50 values were 2,182 6 244 g ae ha21

and 1,263 6 139 g ae ha21, respectively (Figure 2). Gly-phosate provided excellent control of S4 and S5 individuals(i.e., no survival was observed at all applied glyphosate rates)(Figure 2, Table 1), and then LD50 was less than the lowestglyphosate concentration used (500 g ae ha21).

Glyphosate Response of Plants Germinated from Seeds. Inboth experiments plant survival and growth of Johnsongrassindividuals collected from field sites with previous glyphosateselection history (S1A, S2C, and S3) after glyphosate exposurewas significantly different (P , 0.001) to that of plants fromthe sites with no glyphosate use history (S4, S5, and S6).Glyphosate failed to adequately control plants germinatedfrom seed collected from sites where glyphosate failures hadbeen reported compared to susceptible seedlings (Figures 3and 4).

As expected, the growth stage of Johnsongrass individualshad a significant overall effect on glyphosate response (P ,0.001) regardless of the herbicide history of the field. Older,taller plants with a few tillers and rhizome required moreglyphosate to be controlled than small plants with no tillers orrhizomes (Table 2, Figure 4). However, it is noteworthy thatsurvival of adult plants originating from sites S2C and S3 washigher than that of plants from field sites S4 and S6, which arehighly herbicide susceptible (Figure 4A vs. 4B).

Except for the case in which the populations had no historyof glyphosate selection and therefore plants were highlysusceptible (S5) (Figure 3) and when plant survival was veryhigh (S2C) (Figure 4B), data fitted the regression models wellfor both survival and plant growth (P , 0.0001, R2 5 0.74–

Table 1. Glyphosate response of rhizomatous Johnsongrass plants collected fromfield sites S1A and S2A of the northwest of Argentina (Province of Salta) withreported glyphosate control failures. S4 and S5 represent sites from where knownglyphosate susceptible populations were collected.

Glyphosate

Collection field sites of Johnsongrass populations

S1A S2A S4 S5

g ae ha21 ---------------------------------------------% survivala ---------------------------------------------

0 100 a 100 a 100 a 100 a500 85 a 94 b 0 c 0 c

1,000 77 a 74 a 0 b 0 b

a Different superscript letters within rows denote significant differences inplant survival among populations according to Bonferroni’s test (P , 0.01).

568 N Weed Science 55, November–December 2007

0.99). Estimated LD50 and GR50 parameters were signifi-cantly greater for all populations originating from fields whereglyphosate failures had been reported than for susceptibleJohnsongrass populations (Figure 3, Table 2). The highersurvival and growth observed in all the indicated Johnsongrass

populations was accounted for the combination of shootdamage and tiller sprouting. Resistant plants at glyphosaterates lower than 1,000 g ae ha21 showed no damage to themain shoot. At higher rates, the main shoot was damaged, butnew tillers sprouted from the crown. Susceptible plantsshowed damage at all glyphosate rates and no sprouting fromthe crown (data not shown).

The results of this study establish that Johnsongrass controlfailures at commercial field rates of glyphosate (1,000 g aeha21) in the Province of Salta (Argentina) are a consequenceof evolved heritable resistance to glyphosate. Differences inthe glyphosate response profiles between all Johnsongrasspopulations collected from field sites with glyphosate selectionhistory and those populations from sites with no selectionhistory are evident (Figures 2, 3, and 4) and further supportedby the 3.5- to 10.5-fold glyphosate resistance index contrast(Table 2). Glyphosate resistant Johnsongrass phenotypes haveevolved following continuous use of glyphosate.

Sustainable GM-soybean production in Argentinean agri-culture and elsewhere in the world requires conservingglyphosate as a precious and valuable tool for weed control.It is now clear that evolved glyphosate resistance in weedspecies is a biological reality and therefore agronomicmanagement practices to minimize and manage glyphosateresistance must be taken in the short term if this preciousherbicide resource is to be sustained.

Sources of Materials

1 Glyphosate herbicide, TouchdownH, Syngenta Agro Argentina,Av. Del Libertador 1855 (B1638BGE), Vicente Lopez, Provincia deBuenos Aires, Argentina.

Figure 3. Glyphosate response curves of Johnsongrass plants (N) at four-leaf stageand 40-cm height derived from seeds collected in a genetically modified (GM)-soybean field (site S1A) of the northwest of Argentina (Province of Salta). Seedscollected from known glyphosate susceptible plants (population collected in siteS5) (#) were also evaluated for glyphosate response. Symbols are mean observedplant survival (%) (n 5 3) 6 standard error of the mean. Dashed line representspredicted survival values derived from non-linear regression analysis for thepopulation of site S1A using the logistic model (N) y 5 99.8/1 + (x/710)3.7.Dotted lines join actual survival values of S4 and S5 glyphosate susceptiblepopulations.

Figure 2. Dose–survival response curves for Johnsongrass populations following increasing glyphosate rates. Glyphosate responses correspond to rhizomatous plants atfour- to six-leaf stage and 40-cm height collected from plant stands in sites S1B (N) and S2B (m) of GM-soybean fields of the Province of Salta (Argentina) that hadsurvived several glyphosate applications. Two known glyphosate susceptible Johnsongrass populations collected from sites S4 (#) and S5 (#) were also evaluated.Symbols denote mean observed plant survival (%) (n 5 3) 6 standard error of the mean. Dashed lines represent predicted survival values derived from nonlinearregression analysis using the logistic model: S1B (N) y 5 73.3/1 + (x/2,182)5.7 and S2B (m) y 5 84.7/1 + (x/1,263)3.8. Dotted lines join actual survival values of S4 andS5 glyphosate susceptible populations.

Vila-Aiub et al.: Glyphosate resistance in Johnsongrass N 569

Figure 4. Glyphosate dose–survival (A and B) and dose–growth (C and D) response curves of Johnsongrass populations. Treated plants derive from seeds collected fromindividuals that survived two glyphosate applications under glasshouse conditions and originally harvested from GM-soybean fields (sites S2C [¤] and S3 [&]; Provinceof Salta, Argentina). Known glyphosate susceptible Johnsongrass populations collected from site S4 (#) and S6 (n) were evaluated. Glyphosate responses correspond toeither 30- to 50-cm tall plants with no rhizome or tiller (A and C) or individuals with single 100- to 120-cm high shoots (incipient rhizomes and tillers) (B and D).Symbols denote mean observed plant survival (%) (n 5 3) 6 standard error of the mean. Broken lines represent predicted survival values derived from nonlinearregression analysis: (A) S2C (¤) y 5 89.8/1 + (x/937)1.8; S3 (&) y 5 91.4/1 + (x/876.0)1.7; S4 (#) y 5 89.5/1 + (x/135.4)1.8; S6 (n) y 5 89.7/1 + (x/238.3)2.5; (B) S2C(¤) nonsignificant parameters estimation; S3 (&) y 5 90.3/1 + (x/5,095.0)2.6; S4 (#) y 5 91.7/1 + (x/964.1)2.0; S6 (n) y 5 92.2/1 + (x/935.9)1.8; (C) S2C (¤)y 5 99.5/1 + (x/652.9)2.4; S3 (&) y 5 100/1 + (x/542.9)3.3; S4 (#) y 5 99.9/1 + (x/75.9)2.3; S6 (n) y 5 99.9/1 + (x/62.2)1.2; (D) S2C (¤) y 5 100/1 + (x/2,804.6)0.9; S3(&) y 5 100/1 + (x/2,242.9)0.95; S4 (#) y 5 100/1 + (x/635.5)1.7; S6 (n) y 5 97.1/1 + (x/603.7)2.4.

Table 2. Estimates and standard errors (SE) of LD50 and GR50 parameters after nonlinear regression analysis (see Equation 1) for Johnsongrass populations collectedfrom genetically modified (GM)-soybean fields (S2C and S3, see Figure 1) at two growth stages (i.e. young and adult). S4 and S6 denote known glyphosate susceptibleJohnsongrass populations.a

Parameter

Collection field sites of Johnsongrass populations

S4 S6 S3 S2C S4 S6 S3 S2C

g ae ha21 ----------------------------------------------------Young stage -------------------------------------------------- -------------------------------------------------------Adult stage -------------------------------------------------------

LD50 135 (44) 238 (59) 876 (151) 937 (141) 964 (115) 936 (87) 5,095 (548) .4,000R/S (LD50) 3.7–6.5 3.9–6.9 5.2–5.4GR50 76 (28) 62 (14) 543 (14) 653 (44) 636 (66) 604 (72) 2,243 (492) 2,805 (555)R/S (GR50) 7.1–8.7 8.6–10.5 3.5–3.7 4.4–4.6

a The growth stages are characterized by 30- to 50-cm tall plants with no rhizome or tiller (young stage) and individuals with single 100- to 120-cm high shoots(incipient rhizomes and tillers) (adult stage). Values in parenthesis denote SE of the mean. R/S is the ratio range for LD50 or GR50 of the populations originated in fieldsS2C and S3 to that of each of the known glyphosate susceptible populations.

570 N Weed Science 55, November–December 2007

2 Sodium hypochlorite bleach, AyudınH.3 Glyphosate herbicide, Roundup Ultra MaxH Monsanto Argen-

tina, Maipu 1210 (1066), Buenos Aires, Argentina.4 SigmaPlot 9.01, Systat Software Inc. SigmaPlot 2004 for

Windows. SPSS Inc., 444 North Michigan Avenue, Chicago, IL60611.

Acknowledgments

Syngenta Agro Argentina financially supported the experimentsconducted at the Faculty of Agronomy, University of Buenos Aires,through a joint research collaboration between both institutions. Wethank Osvaldo Figueroa from Syngenta Agro Argentina for hisexpertise and help to collect the plant material in Tartagal (Provinceof Salta, Argentina). German Baldi kindly contributed to the designand editing of the georeferences of the collection field sites in theprovided map. We thank Lorena Garcia, Camila Malaspina, andFederico Garat from Monsanto Argentina for their help in theconduction of the trials and material collection.

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Received March 15, 2007, and approved August 6, 2007.

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