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Seasonal variation of picloram metabolism in broom (Gutierrezia sarothrae) andthreadleaf (Gutierrezia microcephala) snakeweed populations in a common gardenAuthor(s): Larissa A. Gibbs and Tracy M. SterlingSource: Weed Science, 52(2):206-212. 2004.Published By: Weed Science Society of AmericaDOI: http://dx.doi.org/10.1614/WS-03-009R1URL: http://www.bioone.org/doi/full/10.1614/WS-03-009R1

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206 • Weed Science 52, March–April 2004

Weed Science, 52:206–212. 2004

Seasonal variation of picloram metabolism in broom(Gutierrezia sarothrae) and threadleaf (Gutierreziamicrocephala) snakeweed populations in a common garden

Larissa A. GibbsResearch Assistant, Department of Entomology,Plant Pathology and Weed Science, New MexicoState University, Las Cruces, NM 88003

Tracy M. SterlingCorresponding author. Department of Entomology,Plant Pathology and Weed Science, New MexicoState University, Las Cruces, NM 88003;tsterlin@nmsu.edu

Broom and threadleaf snakeweed are major rangeland weeds in the western UnitedStates, and picloram is the major herbicide used for their management. Previouswork has shown that these species are most susceptible to picloram applied in au-tumn or when precipitation is high and that differences in herbicide absorption andtissue sensitivity as measured by picloram-induced ethylene production do not fullyexplain variation in seasonal response. Therefore, the role of picloram metabolismin seasonal susceptibility to picloram was examined. Because snakeweed is charac-terized as highly genetically variable, picloram metabolism was evaluated monthlyfor 3 yr among populations from two species as well. Picloram metabolism wasexamined monthly for 3 yr among two populations of threadleaf and nine popula-tions of broom snakeweed grown in a common garden. Metabolism ranged from 30to 70% of picloram applied, and picloram was converted to two metabolites morepolar than picloram regardless of species or population. Although metabolism wasgreatest in the year with the most precipitation, rate of metabolism was unrelatedto precipitation received in the 7-d period before treatment. Application timing asdefined by a given month or specific phenological stage was not related to the levelof metabolism. We conclude that variation in picloram metabolism is not involvedin differential susceptibility across season or population.

Nomenclature: Picloram; broom snakeweed, Gutierrezia sarothrae (Pursh) Britt. &Rusby GUESA; threadleaf snakeweed, Gutierrezia microcephala (DC.) Gray GUEMI.

Key words: Herbicide metabolism, phenotypic variation, genetic variation.

Broom snakeweed is distributed across the western UnitedStates from northern Mexico to southern Canada (Solbrig1960). Approximately 60% of New Mexico rangeland isinfested by this weed, which interferes with forage produc-tion and is poisonous to livestock (Torell et al. 1988). Sizeand density of broom snakeweed populations have varied inNew Mexico during the last 100 yr in response to manage-ment practices, ecological changes, and environmental con-ditions (McDaniel and Sosebee 1988). Several managementtools can be used to control broom snakeweed, but the pri-mary herbicide used is picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid) (Sterling et al. 1999). In field stud-ies in New Mexico and Texas, broom snakeweed was mostresponsive to picloram applied in its postbloom stage underwarm temperature and moist soil conditions (Sosobee 1985)and from October to December (McDaniel and Duncan1987; McDaniel et al. 2002) or in April and May underhigh moisture and soil temperature conditions (Gesink etal. 1973; Schmutz and Little 1970).

Our previous research has shown that picloram absorp-tion and picloram-induced ethylene production were great-est in July and August, when plants were in the phenologicalstages of shoot regreening or flower bud emergence andwhen temperatures and precipitation were high (Sterling etal. 1996); however, neither of these responses coincided withthe stage when broom snakeweed was most susceptible topicloram in the field (McDaniel and Duncan 1987). Thus,increased picloram absorption and tissue sensitivity, as mea-sured by induction of ethylene, are not the only factors af-

fecting seasonal differences in picloram tolerance in broomsnakeweed. Alternatively, susceptibility differences acrossstages of plant growth may be related more strongly to dif-ferences in picloram translocation during periods of non-structural carbohydrate storage (Lym and Messersmith1991; Sosobee 1985) or to differences in picloram metab-olism to nontoxic metabolites, which is the primary mech-anism of herbicide selectivity (Devine et al. 1993).

Large variation exists in broom snakeweed vegetative andreproductive traits throughout its range (Sterling et al.2000). Genetics and environment both contribute to thecharacteristics of the phenotype; however, broom snakeweedgenotypes maintain the same phenotype as that observed attheir original collection sites when grown in a common site(Sterling et al. 2000). Isozyme analysis revealed greater var-iability among broom snakeweed populations than withinpopulations (Hou and Sterling 1995). Further, isozymeanalysis determined that broom snakeweed populations weremore genetically diverse than populations of a closely relatedspecies, threadleaf snakeweed (Sterling and Hou 1997). Therole of genetic variation in herbicide susceptibility amongsnakeweed populations is unknown.

Therefore, to better understand the mechanism of snake-weed’s season-dependent susceptibility to picloram, piclorammetabolism was compared throughout three growing sea-sons. In addition, genetic variation among snakeweed pop-ulations with respect to picloram susceptibility was exam-ined among populations of two snakeweed species growingin a common garden.

Gibbs and Sterling: Variation in picloram metabolism • 207

TABLE 1. Time course of picloram uptake and metabolism by excised shoots of five populations from two snakeweed species. Data werepooled over population because there was no population by time interaction, and main effect for time was significant. n 5 60.

Time afterapplication Ratinga Uptake

Distribution

Soluble Insoluble

Metabolism

Metabolite 1 Metabolite 2 Picloram

h no. % applied % recovered % injected

24487296

1.7 db

2.2 c2.8 b3.0 a

22.0 c37.0 b45.9 a47.4 a

95.0 a94.0 ab93.2 bc91.7 c

5.0 c6.0 bc6.8 ab8.3 a

61.3 a54.3 b52.7 b55.4 b

12.0 c14.8 b16.8 a15.8 ab

26.7 c30.9 a30.5 ab28.8 b

a Rating: 1, no epinasty and turgid; 2, slight epinasty and wilt; 3, moderate epinasty and wilt; 4, pronounced epinasty or total desiccation.b Means with the same letter within a column are not significantly different at the 0.05 probability level.

FIGURE 1. Picloram metabolism in broom and threadleaf snakeweed pop-ulations in 1994, 1995, and 1996 after 72 h incubation. Each value rep-resents the mean of six measurements, and error bars are standard error ofthe mean.

Materials and Methods

Common Garden Establishment

A common garden of 10 broom snakeweed populationsfrom New Mexico, Texas, and Wyoming and two threadleafsnakeweed populations from Arizona and New Mexico wasestablished in 1992 at the Leyendecker Plant Science Farmnear Las Cruces, NM. In September 1991 during flowering,50 broom snakeweed plants were collected at random usingthe methods described by Sterling et al. (2000), from eachof the following locations in New Mexico: Bayard (328389N,1088069W), Clovis (348499N, 1038259W), Corona(348169N, 1058239W), Des Moines (368459N, 1038589W),Las Cruces (328359N, 1068509W), Lovington (328569N,1038209W), San Simon Sink (328189N, 1038259W), andTatum (338239N, 1038159W); in Texas: Zavala (29849N,998509W); and in Wyoming: Laramie (418199N, 105849W).Wyoming genotypes did not survive multiple transplantingattempts. Threadleaf snakeweed was collected from theUSDA Jornada Experimental Range near Las Cruces, wherea mixed population of broom and threadleaf snakeweed ex-ists; this was the same site from which Las Cruces broomsnakeweed was collected. Threadleaf snakeweed plants werealso collected from Sasabe, AZ (318329N, 1118309W). De-scription of soil, rainfall, and temperature conditions atthese sites can be found in table 1 in Sterling et al. (2000).

Collected plants were transplanted into 15-cm-diam, 4.4-L pots containing potting soil1 1 mo after collection andgrown in a greenhouse under natural day length. Plants werefertilized with 20:10:20 (N–P–K)2 at 2.5 g L21 weekly. Sixmonths after transplanting, stem cuttings were establishedfrom collected plants using the methods described by Houand Sterling (1995) and grown to approximately 10-cmheight in a greenhouse for transplanting into the commongarden.

The garden was designed as a randomized complete blockdesign with five blocks of 20 genotypes from each of the 12snakeweed populations. Each block was 74 m2, and plantswere arranged in a grid pattern of 12 rows with 0.9-m rowwidth and 20 plants within each row spaced 0.3 m apart,bordered by an additional row of plants in the same plantingpattern on all four sides. Five clones from each of 20 sur-viving genotypes were transplanted into their respectiveblock in a random pattern in the garden during May andJune 1992. Genotypes and clones that did not survive initialtransplanting were replaced throughout the growing seasonof May through September.

The garden was flood irrigated with approximately 8 cm

208 • Weed Science 52, March–April 2004

TABLE 2. Time course of picloram uptake and metabolism by excised shoots of five populations from two snakeweed species. Data werepooled over time because there was no population by time interaction, and main effect for population was significant. n 5 48.

Species Population Ratinga Uptake

Distribution

Soluble Insoluble

Metabolism

Metabolite 1 Metabolite 2 Picloram

no. % applied % recovered % injected

Threadleaf snakeweed ArizonaLas Cruces

2.3 cdb

2.5 ab33.8 cd31.7 d

93.2 b94.7 a

6.8 b5.3 bc

63.3 a60.7 a

11.7 c12.9 c

25.0 b26.5 b

Broom snakeweed Des MoinesLas CrucesSan Simon Sink

2.7 a2.4 bc2.2 d

44.9 a37.9 bc42.7 ab

89.4 c96.3 a93.3 b

10.6 a3.7 c6.7 b

56.7 b51.8 c46.5 d

12.2 c17.5 b20.5 a

31.1 a30.8 a33.0 a

Contrastc 0.72 , 0.0001 0.09 0.09 , 0.0001 , 0.0001 , 0.0001

a Rating: 1, no epinasty and turgid; 2, slight epinasty and wilt; 3, moderate epinasty and wilt; 4, pronounced epinasty or total desiccation.b Means with the same letter within a column are not significantly different at the 0.05 probability level.c Contrasts between broom and threadleaf snakeweed populations.

TABLE 3. Picloram metabolism by broom and threadleaf snakeweed populations 72 h after treatment. n 5 12.

Species Population Ratinga Uptake

Distribution

Soluble Insoluble

Metabolism

Metabolite 1 Metabolite 2 Picloram

no. % applied % recovered % injected

Broom snakeweed Des MoinesClovisCoronaTatumLovington

3.4 ab

2.8 bc2.9 abc2.5 bcd2.4 cd

50.6 ab32.5 d42.9 abcd34.8 cd41.6 bcd

88.7 c90.6 bc89.5 c89.5 c88.4 c

11.3 ab9.4 abcd

10.5 abc10.5 abc11.6 a

54.4 bc58.3 ab55.4 bc53.7 bc55.5 bc

13.2 cd12.3 cd13.7 cd17.8 b16.0 bc

32.4 abc29.4 bcde30.9 abcd28.5 cde28.6 cde

BayardLas CrucesSan Simon SinkZavala

2.5 bcd2.7 bcd2.4 cd2.3 d

38.6 bcd46.7 abc54.9 a49.6 ab

91.1 bc95.1 a93.9 ab89.5 c

8.9 abcd4.9 ef6.1 def8.1 bcde

54.1 bc42.4 d44.1 d48.3 cd

13.9 cd22.2 a22.5 a18.8 ab

32.0 abc35.4 a33.4 ab32.9 abc

Threadleaf snakeweed Las CrucesSasabe

3.0 ab2.5 bcd

38.7 bcd38.8 bcd

95.5 a92.2 abc

4.5 f7.8 cdef

59.4 ab63.3 a

14.0 cd11.8 d

26.6 de24.9 e

Contrastc 0.16 0.05 0.28 0.23 0.01 0.23 0.002

a Ratings: 1, no epinasty and turgid; 2, slight epinasty and wilt; 3, moderate epinasty and wilt; 4, pronounced epinasty or total desiccation.b Means with the same letter within a column are not significantly different at the 0.05 probability level.c Contrasts between broom and threadleaf snakeweed populations.

water once per month during the 1992 growing season, re-sulting in approximately 48 cm additional moisture. Thegarden was irrigated twice in 1994 (March 18, June 4) andonce per year in 1995 (March 30) and 1996 (February 24)to a depth of 7 to 10 cm. To manage grass and some broad-leaf weeds, preemergent applications of metolachlor (2-chlo-ro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methyle-thyl)acetamide) at 2.2 kg ai ha21 and trifluralin (2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine) at 0.8 kg aiha21 were raked to a depth of approximately 2.5 cm justbefore the early irrigation event each year.

Picloram Screening

Stem cuttings were established from plants growing in thecommon garden using the methods described by Hou andSterling (1995) and maintained in the greenhouse undernatural day length with no supplemental lighting. Whenrooted cuttings had reached approximately 10-cm height,formulated picloram was applied to eight individuals fromeach species or population at 0 and 0.28 kg ae ha21 in acarrier volume of 187 kg ha21 using a CO2 backpack spray-er. Plants were rated over 30 d for epinasty. Thirty days aftertreatment, shoot tissue was excised and dried at 60 C. Dry

weights were recorded and data expressed as a percentage ofthe control.

Picloram Absorption by LeavesAbsorption of picloram by broom and threadleaf snake-

weed leaf tissue was compared using tissue from plantsgrowing at a common site near Las Cruces, NM (328329N,1068549W). From each of 15 plants within a species, onestem, approximately 10 cm long, was excised, the cut stemend was immediately submerged in 15-ml vials containing2 ml distilled water, and all samples were brought to thelaboratory before treatment of individual leaves with 14C-picloram using the methods of Sterling and Lownds (1992).Twenty-four hours after foliar treatment, external picloramwas rinsed from the leaf surfaces and tissue radiolabel con-tent determined using the methods of Sterling and Lownds(1992).

Picloram Metabolism in Excised TissuesIncubation and Tissue Processing

For each experiment below, individual garden plants fromeach replicate and target population or species were selected

Gibbs and Sterling: Variation in picloram metabolism • 209

TABLE 4. Monthly sums of precipitation and irrigation events in1994, 1995, and 1996.

Month 1994 1995 199640-yr

averagea

mm

JanuaryFebruaryMarchAprilMay

1.08.17.1b

6.118.0

21.812.7

0.8b

0.50.0

4.10.5b

1.84.60.0

12.48.45.84.87.9

JuneJulyAugustSeptember

1.0b

5.11.00.0

25.47.6

17.532.8

17.574.918.054.1

19.337.655.432.8

OctoberNovemberDecemberTotal

16.015.236.1

114.8

0.03.3

12.2134.6

4.13.02.5

185.2

22.611.918.5

237.4

a Data from Western Regional Climate Center, Station 298535, New Mex-ico State University, http://www.wrcc.dri.edu/summary/climsmnm.html.

b Irrigation events were estimated at a depth of 80 to 100 mm.

to evaluate picloram metabolism. Samples were collectedfrom plants in a single block, and only plants with less than50% necrotic tissue were selected for treatment. One 10- to15-cm-long cutting was excised from each target plant andplaced immediately in a vial containing 3 ml of water treat-ed with approximately 8,300 Bq of 14C-picloram, ring-la-beled in positions 2 and 6 with a specific activity of 1.013 109 Bq mmol21. Control vials contained 14C-piclorambut no plant tissue. Excised tissue was incubated in the lab-oratory at room temperature and lighting for the lengths oftime described below. Visible injury was recorded twice aday using a rating scale of 1 to 4: 1, no epinasty and turgid;2, slight epinasty and wilt; 3, moderate epinasty and wilt;4, pronounced epinasty or total desiccation. Water was add-ed to the cuttings as needed until harvest.

Incubation ended when surface herbicide residue was re-moved with two 1-ml rinses of 50% (v/v) aqueous metha-nol. Radioactive content of rinsate was quantitated usingliquid scintillation spectrometry3. Tissue was weighed andfrozen at 220 C until extraction. Radiolabel was extractedfrom the entire tissue sample in methanol–water (80:20, v/v) using a Ten-Broeck tissue homogenizer. The ground tis-sue was centrifuged at 1,450 3 g for 15 min. The sedi-mented plant debris (insoluble fraction) was resuspendedtwice in 2 ml methanol–water (80:20, v/v) and centrifugedto remove soluble radiolabel from the insoluble fraction.Previous work by Sterling and Jochem (1995) revealed thatsubsequent extraction of insoluble fraction from plant tissuewith hexane yielded no additional radiolabel. Radioactivityin the insoluble fraction was measured by tissue combustionusing a biological oxidizer.4 The clear supernatant was evap-orated to dryness under an air stream at 25 C. Concentrateswere resuspended in 4 ml methanol–water (80:20, v/v) andfiltered through 0.2 mM nylon membranes5 to remove par-ticulate matter before high-performance liquid chromatog-raphy (HPLC) analysis.

Metabolites of picloram were separated using techniquesmodified from Frear et al. (1989). An HPLC6 system witha 254-nm UV detector and an in-line liquid radioactivitymonitor7 was used for 14C-metabolite separation and quan-

titation. Samples and 12C- and 14C-picloram standards wereinjected individually onto a C18 reversed-phase HPLC col-umn8 and separated from metabolites of picloram using oneof two HPLC systems at a flow rate of 1.5 ml min21. SystemA was an isocratic mobile phase of acetonitrile–water (80:20, v/v) (aqueous phase contained 4% by volume of glacialacetic acid). System B was a 20-min linear gradient wheremobile Phase A (4% glacial acetic acid, 0.1% ammoniumacetate)–Phase B (acetonitrile) changed from 100:0 to 40:60. Both systems detected three 14C peaks with retentiontimes for metabolite 1, metabolite 2, and unmetabolizedpicloram and standards of 2:11, 3:46, and 5:20 min in Sys-tem A and 3:52, 5:54, and 10:16 min in System B, respec-tively. Radioactivity levels for any metabolites detected incontrol vials were subtracted from those in treated tissue.Metabolites were not identified.

Time Course of Picloram Metabolism

Two populations of threadleaf snakeweed collected fromSasabe, AZ, and Las Cruces, NM, and three populations ofbroom snakeweed collected from Des Moines, Las Cruces,and San Simon Sink, NM, were selected for comparing pi-cloram metabolism over incubation time. These accessionswere chosen to compare picloram metabolism between spe-cies and among populations from a range of locations andgrowth habits; Des Moines was the northernmost broomsnakeweed population, possessing the smallest canopy sizeof all the populations, and San Simon Sink was the south-ernmost broom snakeweed population, possessing one of thelargest canopy sizes among the populations (Sterling et al.2000). The unique architecture of each population did notvary even after 9 yr in a common environment (Sterling etal. 2000). Plants were sampled in July and September 1996and were incubated with picloram for 24, 48, 72, or 96 hand processed as described above.

Seasonal Variation in Picloram Metabolism

Two populations of threadleaf snakeweed, collected fromSasabe, AZ, and Las Cruces, NM, and three populations ofbroom snakeweed collected from Des Moines, Las Cruces,and San Simon Sink, NM, were selected to track variationin picloram metabolism throughout the growing season forthe reasons given above. Plants were sampled monthlythroughout each growing season for 3 yr consecutively (Mayto November 1994, April to October 1995, and April toOctober 1996), incubated with picloram for 72 h, and pro-cessed as described above.

Genetic Variation in Picloram Metabolism

Samples collected in July and September 1996 from twopopulations of threadleaf snakeweed, Sasabe, AZ, and LasCruces, NM, and nine populations of broom snakeweed,Bayard, Clovis, Corona, Des Moines, Las Cruces, Loving-ton, San Simon Sink, and Tatum, NM, and Zavala, TX,were incubated with picloram for 72 h and processed asdescribed above.

StatisticsEach experiment was performed using a randomized com-

plete block design and a factorial arrangement of treatments

210 • Weed Science 52, March–April 2004

TABLE 5. Dry weight response of snakeweed plants establishedfrom cuttings to picloram at 0.28 kg ha21 30 d after treatment. n5 16.

Species Population Dry weight

% control

Broom snakeweed Des MoinesClovisCoronaTatumLovingtonBayardLas CrucesSan Simon Sink

30 da

54 b50 bc54 bc75 a58 b40 cd58 b

Threadleaf snakeweed Las Cruces 50 bc

a Means with the same letter are not significantly different at the 0.05probability level.

with at least four replicates and was repeated. Data weresubjected to analysis of variance, and means were separatedwith Fisher’s Protected LSD (a 5 0.05). Contrasts betweensnakeweed species were conducted. Data from repeated ex-periments were combined, unless noted otherwise, becausethere were no interactions. All statistical computations werecarried out using SAS (1991).

Results and Discussion

Time Course of Picloram Metabolism

Herbicide injury to excised tissues increased over time re-gardless of population or snakeweed species (Table 1). Theamount of 14C in the insoluble fraction increased over time,with the greatest amount of 8.3% accumulated after 96 h(Table 1). Picloram metabolism was greatest between 48 and96 h, with 69 and 71% converted to metabolite, respectively;the distribution of metabolites was unchanged after 48 h.Because absorption was greatest at 72 h after treatment, the72-h incubation time was chosen for subsequent experiments.Also, because the absorbed picloram caused injury in all ex-periments (Tables 1–3), yet was metabolized regardless of spe-cies or time, this delivery method provided an effective doseand valid means of comparing metabolism in populations andspecies.

Picloram was converted to two metabolites by bothsnakeweed species (Table 1). Both metabolites had a shorterretention time on the HPLC column than picloram, whichsuggests that they have greater polarity than picloram. Upto 61% of the recovered radiolabel was converted to themore polar metabolite 1, whereas only 12 to 17% of piclo-ram was converted to metabolite 2.

The identity of picloram metabolites in these snakeweedspecies was not determined, but many polar metabolites ofpicloram have been characterized as glucose conjugates.Frear et al. (1989) found several N-glucoside conjugates,glucose esters, and gentiobiose esters of picloram in leafyspurge (Euphorbia esula L.). Hallmen (1974) found that alarge fraction of picloram was bound in water-soluble com-plexes in both sunflower (Helianthus annuus L. var. uniflo-rus) and rape (Brassica napus L. cv. ‘Nilla’). Up to 40% ofpicloram activity in barley (Hordeum vulgare L.) and 25%in Canada thistle (Cirsium arvense L.) was conjugated withsugars (Sharma and Vanden Born 1973). Other studies haveidentified acid-labile N-glucosides of picloram in sunflower(Chkanikov et al. 1983), alkali-labile glucose esters in otherplants (Kudaikina et al. 1981), conjugates of picloram withmustard oils in radish (Raphinus sativus L.) and mustard(Brassica kaber L.) plants (Chkanikov et al. 1984), or water-soluble conjugates that yielded the carboxylic acid amide ofpicloram upon ammonolysis (Hall and VandenBorn 1988).

Seasonal Variation in Picloram Metabolism

Our earlier results suggested that seasonal susceptibility topicloram did not involve differential foliar absorption or tis-sue sensitivity as measured by ethylene induction (Sterlinget al. 1996). Results from this 3-yr study suggest that me-tabolism is not involved as well. For all variables exceptabsorption, there was a year by population by date inter-action; therefore, yearly results are presented separately (Fig-ure 1). Because all other variables responded similarly com-

pared with other experiments (Table 1), only picloram re-maining will be discussed.

Picloram metabolism was greatest in 1996 (P , 0.0001)(Figure 1), the year with the most total precipitation (Table4); however, there was no relationship between the amountof precipitation in the 7-d period before initiation of eachmonthly treatment and the amount of picloram metabolized(r 2 5 0.017). Additionally, among years, no single monthpossessed a greater level of metabolism (Figure 1). The mostmetabolism of picloram occurred in August through Octo-ber 1994, August through October 1995, and October1996. Picloram applied in August rarely affords control,whereas picloram applied in October or November providesthe greatest control (McDaniel and Duncan 1987; Mc-Daniel et al. 2002). Because picloram metabolism is maxi-mal during periods when application of picloram is mostsuccessful, picloram metabolism plays little role in seasonaltolerance to picloram.

There was a population by year interaction (P 5 0.0016)for absorption of picloram by excised tissues (data notshown). For all years, absorption was greatest (up to 80%of applied) in April and May for all populations except DesMoines (data not shown). Cut stems of Des Moines plantsabsorbed 25 to 50% of applied picloram regardless ofmonth; other populations absorbed similar levels from Junethrough November. Similarly, when this same technique wasused on other picloram-sensitive species, excised tissue oflocoweeds (Oxytropis sericea Nutt. and Astragalus mollissimusTorr.) absorbed 57% of applied picloram (Sterling and Jo-chem 1995).

Genetic Variation in Picloram MetabolismPicloram metabolism was greater in threadleaf snakeweed

populations, with only 25 to 26% of picloram remainingcompared with broom snakeweed populations with 29 to35% remaining (Table 2, P , 0.0001; Table 3, P 50.0021). Despite threadleaf snakeweed metabolizing andconverting more picloram to metabolite 1 than broomsnakeweed, excised tissues from the two species did not varyin their susceptibility to picloram. These results are consis-tent with field (McDaniel et al. 2002) and greenhouse (Ta-ble 5) trials, where both species grew together and differ-ences in susceptibility to picloram did not differ.

Excised stems of broom snakeweed absorbed more piclo-

Gibbs and Sterling: Variation in picloram metabolism • 211

ram than those of threadleaf snakeweed (Tables 2 and 3).Likewise, foliar absorption of picloram by broom snakeweed(15.9% applied) was greater than that by threadleaf snake-weed (10.9% applied) (LSD0.05 5 2.8, n 5 30). These smalldifferences in picloram absorption apparently were notenough to alter herbicide action. Consistent with these sim-ilarities between species, broom and threadleaf snakeweedsdid not differ in the amount of 14C in the insoluble fraction.

Although susceptibility of excised tissues to picloram didnot differ between snakeweed species (Tables 2 and 3), therewas variation in susceptibility to picloram among excisedtissues from broom snakeweed populations (Table 3). Tissuefrom the Des Moines population was most susceptible, andtissues from those populations collected south of DesMoines were less susceptible. Des Moines plants establishedfrom cuttings were also the most susceptible of broomsnakeweed populations to picloram (Table 5). Variation inpicloram susceptibility among broom snakeweed popula-tions is consistent with the high level of genetic variationidentified using isozyme analyses (Hou and Sterling 1995);however, metabolism of picloram by Des Moines was similarto that by other broom snakeweed populations (Tables 2and 3), suggesting that differential metabolism is not in-volved in susceptibility differences among broom snakeweedpopulations.

Absorption by excised tissues of broom snakeweed pop-ulations ranged from 33 to 55% of applied (Tables 2 and3); these levels are similar to those reached in other speciesusing this method (Sterling and Jochem 1995). Of the her-bicide absorbed by broom snakeweed stem tissues, up to11% was detected in the insoluble fraction in Des Moinesand as little as 4% in Las Cruces broom snakeweed (Table3). These findings confirm that differential absorption andincorporation into insoluble residues, another mechanism ofherbicide tolerance (Devine et al. 1993), do not contributeto differential response of broom snakeweed populations topicloram.

Picloram remaining varied only from 29 to 35% of re-covered among broom snakeweed populations. Threadleafsnakeweed consistently metabolized approximately 5 to 10%more picloram than broom snakeweed tissue yet was as sus-ceptible as broom snakeweed when excised tissues weretreated (Tables 2 and 3), in greenhouse screening trials (Ta-ble 5) and in field applications at a site where both speciesgrow (McDaniel et al. 2002). These results suggest that me-tabolism does not play a large role in differential tolerancebetween species or among populations. In fact, the smallamounts of parent picloram remaining in all species andpopulations under all environments imply that one or bothof the metabolites are actually toxic or the herbicide is soactive that only a small amount of parent herbicide is need-ed to cause a herbicidal response.

All three metabolism experiments confirmed that snake-weed species and populations metabolize picloram. Regard-less of species, population, or season, snakeweed cuttingsmetabolized approximately 30 to 70% of absorbed picloramto metabolites more polar than the parent. Therefore, it isdoubtful that picloram metabolism explains differential sus-ceptibility across species or time. Picloram metabolism didnot differ between picloram-resistant and -susceptible to-bacco (Nicotiana tabacum L.) cell lines (Chaleff 1980). Con-versely, significantly more 14C-picloram was converted to a

water-soluble metabolite in rapeseed than in sunflower, eventhough both species are susceptible to picloram (Hall andVandenBorn 1988). Mitchell and Stephenson (1973) dis-covered that picloram-susceptible red maple (Acer rubrumL.), and picloram-tolerant white ash (Fraxinum americanaL.) formed metabolites at the same rate, which could notaccount for the selective toxicity of picloram between thetwo species.

In general, herbicide metabolism is a primary mechanismof selectivity for plants (Devine et al. 1993); however, me-tabolism of picloram did not explain differential toleranceof broom or threadleaf snakeweed to picloram throughoutthe growing season. Metabolism differences between speciesand among populations were minor, supporting the rela-tively small differences in susceptibility observed betweenspecies and among populations. Because we previouslyfound that foliar absorption or differences in tissue sensitiv-ity as measured by ethylene induction do not correlate withseasonal differences in tolerance (Sterling et al. 1996), otherparameters including translocation patterns at differing phe-nological stages may be involved. This possibility needs fur-ther investigation.

Sources of Materials1 Terra-Lite Metro Mix 350, W. R. Grace & Co., 6401 Poplar

Avenue, Memphis, TN 38119.2 Peters Fertilizer Products, J. R. Peters, Inc., 6656 Grant Way,

Allentown, PA 18106.3 Model 1900 TR, PerkinElmer Life and Analytical Sciences,

2200 Warrenville Road, Downers Grove, IL 60515.4 Model OX400, R. J. Harvey Instrument Corp., 123 Patterson

Street, Hillsdale, NJ 07642.5 Acrodisc 13, Gelman Laboratory, 600 South Wagner Road,

Ann Arbor, MI 48103-9019.6 HPLC system, Millipore Corp., 80 Ashby Road, Bedford, MA

01730-2271.7 b-RAM flow-through detector, IN/US Systems, 5809 North

50th Street, Tampa, FL 33610-4809.8 Radial-Pak cartridge, Nova-Pak C18, 4 m, Waters Corp., 34

Maple Street, Milford, MA 01757.

Acknowledgments

We express our appreciation to Dr. Jill Schroeder for plot spaceat the Weed Science field laboratory; to Chris Canavan, CathyWelker, Evelyn Eichler, Ruth Parreira, Alice Janaveris, and RolandMaynard for technical assistance; and to the many undergraduateswho helped care for the snakeweed garden. We also thank Dr. JackDeLoach, USDA, Temple, TX, for providing the Texas plants, Dr.Tom Whitson, University of Wyoming at Laramie, for the Wyo-ming plants, and Dr. David Thompson, New Mexico State Uni-versity, for collecting the Arizona plants. This work was supportedby the New Mexico Agricultural Experimental Station and theUSDA-CSRS special grant 89-34196-4872. We also appreciategreatly the radiolabeled picloram and standards from DowAgro-Sciences.

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Received January 14, 2003, and approved August 16, 2003.