Genetics of Resistance to Acetohydroxyacid Synthase ...

Weed Science 2008 56:000–000

Genetics of Resistance to Acetohydroxyacid Synthase Inhibitors in Populations of Eastern Black Nightshade (Solanum ptychanthum) from Ontario Jamshid Ashigh, Istvan Rajcan, and Franc¸ois J. Tardif*

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Resistance to acetohydroxyacid synthase (AHAS) inhibiting herbicides in populations of eastern black nightshade from Ontario has been linked to an Ala205Val substitution in the AHAS enzyme. The aim of this study was to determine the mechanism of inheritance of AHAS inhibitor resistance and the genetic relationships among resistant (R) and susceptible (S) eastern black nightshade populations from Ontario. Homozygous R and S parental populations were crossed and the inheritance was analyzed in F1 (S 3 R), reciprocal F1 (R 3 S), F2, and backcross (S 3 F1) progenies after application of imazethapyr at 150 g ai ha21. Compared to parental lines, the progenies were rated as R, intermediate (I), and S phenotypes. All the F1 progenies were of the I phenotype. The backcross progenies segregated in a 1:1 (S:I) ratio, and the F2 families segregated in a 1:2:1 (R:I:S) ratio. These results indicate that a single nuclear gene, with incomplete dominance, controls resistance to AHAS-inhibiting herbicides in R population of eastern black nightshade. Random amplified polymorphic DNA (RAPD) markers were screened among 25 R and S populations. The genetic relationship of R and S populations based on RAPD profiles generated from six RAPD primers indicated four groups of populations in which resistance seems to have arisen independently. However, based on similarity coefficients, resistance within three of the groups could have arisen by gene flow. Both similar local selection pressure and gene flow could explain the spread of the Ala205Val substitution in R populations of eastern black nightshade in Ontario. Nomenclature: Imazethapyr; eastern black nightshade, Solanum ptychanthum Dun. SOLPT. Key words: Acetolactate synthase, ALS, RAPD, herbicide resistance, inheritance, genetic diversity.

Acetohydroxyacid synthase (AHAS, also known as acetolactate synthase, ALS) is a plastidic enzyme that is the target site of five different herbicide classes: sulfonylureas (SU), imidazolinones (IMI), triazolopyrimidines, pyrimidinyl-oxybenzoates, and sulfonylamino-carbonyl-triazolinones (Santel 1999; Tranel and Wright 2002). AHAS inhibitors are among the most widely used herbicides worldwide because of their high efficacy and favourable environmental profile (Saari et al. 1994). Unfortunately, AHAS inhibitors are prone to the selection of resistance in weeds and now account for the highest number of resistant biotypes worldwide (Heap 2007). Resistance to AHAS inhibitors in weed species is primarily due to a herbicideinsensitive target site (Saari et al. 1994; Tranel and Wright 2002). Resistant AHAS enzyme is able to maintain its catalytic activity even in the presence of inhibitors at cellular concentrations that are normally lethal. There are many different point mutations in the AHAS gene that code for resistant enzymes. Depending on the particular mutation that is selected, resistance will be confined to one of a few herbicide classes while other mutations will endow weeds with broad-spectrum resistance (Tranel and Wright 2002; Whaley et al. 2007). In weeds, target site based resistance to AHAS inhibitors is controlled by a single nuclear gene. However, the level of dominance varies among plants species or alleles (Tranel and Wright 2002). In prickly lettuce (Lactuca serriola L.), annual sowthistle (Sonchus oleraceus L.), and an eastern black nightshade population from Wisconsin, resistance was incompletely dominant while in kochia (Kochia scoparia L.) and common cocklebur (Xanthium strumarium L.), resistance was fully dominant (Boutsalis and Powles 1995; MallorySmith et al. 1990; Ohmes and Kendig 1999; Saari et al. 1994; Volenberg and Stoltenberg 2002). Herbicide resistance alleles most likely arise in an area through mutations, and after selection, gene flow would DOI: 10.1614/WS-07-140.1 * Department of Plant Agriculture, University of Guelph, Guelph, Ontario, Canada. N1G 2W1. Current address of first author: Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada. N1G 2W1. Corresponding author’s E-mail: [email protected]

facilitate the spread of the resistance gene (Jasieniuk et al. 1996; Stankiewiez et al. 2001). Gene flow among and within plant populations occurs through two primary means, pollen and seed movement (Maxwell and Mortimer 1994). Rates of gene flow are generally believed to be higher than rates of mutation and are greatly influenced by the mating system of weed species. When a single dominant gene confers resistance, the resistance trait will spread more rapidly in cross-pollinated species compared to self-pollinated species (Jasieniuk et al. 1996). On average, the rate of gene flow for populations separated by a few hundred meters is thought to be less than 1%, and two orders of magnitude smaller than that for populations separated by 1.5 km or more (Levin 1981; Levin and Kerster 1974). Eastern black nightshade is usually an annual or occasionally a short-lived perennial (Bassett and Munro 1985; Young et al. 2002) and, as most species of Sect. Solanum, is an autogamous plant (Schilling 1978) with chromosome number of 2n 5 24 (Bassett and Munro 1985). In Ontario, eastern black nightshade has been recorded as a ruderal weed since the mid-1850s, however, after the introduction of selective herbicides and possible elimination of more competitive weed species in the early 1960s, it began to invade cultivated fields (Alex 1964). AHAS-inhibiting herbicides are commonly used to control eastern black nightshade in soybean [Glycine max (L.) Merr.] fields (Milliman et al. 2003; Ward and Weaver 1996). However, repetitive use of AHAS-inhibiting herbicides has selected for resistant populations in Ontario (Ashigh and Tardif 2006), Wisconsin (Volenberg et al. 2000), North Dakota (Heap 2007), as well as Illinois and Indiana (Milliman et al. 2003). A single point mutation coding for Ala122Thr in AHAS causes resistance in populations of eastern black nightshade from Illinois and Indiana (Milliman et al. 2003). Surprisingly, an Ala205Val substitution causes resistance in 12 populations from Ontario (Ashigh and Tardif 2007). Also, as we were about to conduct the experiments described in this article, a new R population (SOLPT 24) was brought to us (Table 1). Following AHAS sequencing, it was found to possess the Ashigh et al.: Genetics of AHS resistance

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Table 1. Geographic location of resistant (R) and susceptible (S) eastern black nightshade populations, used for RAPD analysis, in Ontario.

Populations SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT SOLPT

1 3 6 7 8 10 14 22 24 25 26 27 28 2 4 5 9 11 12 13 17 15 16 21 23

Response to imazethapyr at 100 g ai ha21

Location

Latitude/longitude

R R R R R R R R R R R R R S S S S S S S S S S S S

Rodney Strathroy Seaforth Seaforth Seaforth Clinton Eberts Lucknow Fingal Fingal Port Albert Thamesville Clinton Ridgetown Inkerman Seaforth Seaforth Ariss Delaware Burlington Leamington Kingsville Seaforth Williamsburg Apple Hill

42u34900N, 81u409600W 42u57900N, 81u37900W 43u329600N, 81u229600W 43u329600N, 81u229600W 43u329600N, 81u229600W 43u36900N, 81u319600W 42u309060N, 82u109550W 43u57900N, 81u31900W 42u43900N, 81u179600W 42u43900N, 81u179600W 43u529360N, 81u429550W 42u329600N, 81u58900W 43u36900N, 81u319600W 42u259600N, 81u529600W 45u029110N, 75u239410W 43u329600N, 81u229600W 43u329600N, 81u229600W 43u349300N, 80u219580W 42u549330N, 81u259180W 43u19900N, 79u479600W 42u039110N, 82u359590W 42u029110N, 82u449210W 43u329600N, 81u229600W 44u589300N, 75u149370W 45u139160N, 74u469140W

Ala122Thr substitution in the AHAS enzyme (GenBank accession EF656479) (Ashigh 2006). Considering the low number of reported resistant weed species with Ala205Val substitution relative to other substitutions (Tranel and Wright 2002), there are two possible reasons for its high frequency in resistant populations of eastern black nightshade in Ontario. First, it is likely that resistance appeared as separate events on farms where the similar selection pressure (due to similar management strategies) was appropriate for this substitution in different areas. An alternative explanation is that resistance first appeared in one site as a single founder event and further spread to different areas through gene flow. Random amplified polymorphic DNA (RAPD) is a polymerase chain reaction (PCR)-based technique, which does not require DNA sequence information prior to investigation (Williams et al. 1990). Thus, it has been described as one of the most useful techniques for detecting polymorphic DNA in weed species with low genetic variability (Stankiewiez et al. 2001; Williams et al. 1990). There are numerous RAPD primers commercially available, which facilitates the generation of a large amount of data (Dawson et al. 1995). In addition, the determination of the mechanism of inheritance of resistance could be helpful in predicting the impact of management strategies on the fate of the resistant allele. Therefore, the objectives of this study were to determine the inheritance of resistance in populations of eastern black nightshade and to investigate the genetic variability of resistant (R) and susceptible (S) populations of eastern black nightshade from Ontario. Materials and Methods

Plant Material. Seeds of S and R populations of eastern black nightshade were collected from 25 fields in 18 geographic locations in Ontario (Table 1). Berries were collected from 5 0

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to 15 randomly selected plants throughout the field, seeds were extracted by washing the seeds from the berries through a 1.18-mm mesh sieve, air dried, and stored at 5 C. The quantity of seeds was increased by growing plants of all populations in isolation, in a growth room under a 16-h photoperiod at 26 C and an 8-h dark period at 22 C, with a photosynthetic photon flux density (PPFD) of 450 mmol m22 s21 supplied by a mixture of incandescent bulbs and fluorescent tubes. Seeds produced by these populations were collected and stored as described above. Genetic Crosses of the Parental R and S Populations to Generate F1 Families. Seeds of one R (SOLPT 1) and one S (SOLPT 2) population of eastern black nightshade (Table 1) were placed on the surface of commercial potting soil1 in 12.5 by 12.5 by 13.8-cm pots. All the experiments were conducted in a greenhouse under a 16-h photoperiod at 26 C and an 8-h dark period at 18 C, with maximum photosynthetic photon flux density (PPFD) of 1200 mmol m22 s21 supplied by high-pressure sodium lamps and fluorescent tubes. Plants were watered as required (when soil surface was dry) and fertilized weekly using 50 ml of a solution of 1.5 g L21 of 20–20–20 (N–P2O5–K2O) fertilizer. At the cotyledon to oneleaf stage, plants were thinned to one plant per pot. Four R and four S plants were used as parental lines in subsequent experiments. Approximately 1 d before anthesis, 10 flower buds were emasculated using sharp-edged forceps without damaging the stigma. The emasculated flowers of each plant were pollinated by gently rubbing the dehisced anthers of a designated male onto the stigma. Immediately after pollination, flowers were covered with plastic film to avoid cross-pollination. Furthermore, 10 flower buds of each parent plant were covered with plastic film to enforce self-pollination. A total of 40 F1 crosses (S 3 R) and 40 reciprocal F1 crosses (R 3 S) were performed. Mature berries generated from self- and cross-fertilizations were collected separately from each R and S parents approximately 5 wk after pollination, and seeds were extracted and stored as described above. F1 Evaluation and Generation of F2 and Backcross (BC) Families. Three to five seeds of each F1 (S 3 R) and reciprocal F1 (R 3 S) families were placed separately on the surface of each cell of a 72-cell plug flat (53.5 by 28 by 6.5 cm) containing growth media as described above. Seeds of the S population were planted in four 12.5 by 12.5 by 13.8 cm pots. Seedlings were watered and fertilized as described above. At the cotyledon to one-leaf stage, plants were thinned to one plant per cell or pot. After all plants had reached the three- to four-leaf stage, plants from the F1 and reciprocal F1 families, and S plants were treated with imazethapyr at 150 g ai ha21 plus nonionic surfactant2 at 0.25% (v/v) and 28% nitrogen (N) at 1% (v/v), using a laboratory sprayer equipped with an 8002E nozzle3 delivering 210 L ha21 at 276 kPa. Plants were evaluated 20 d after treatment and four surviving F1 plants were transplanted into 12.5 by 12.5 by 13.8 cm pots. Using the same procedures as described above, a total of 40 backcrosses (BC) (S 3 F1) were performed. Mature berries from BCs were collected separately, and seeds extracted and stored as described above. The F2 populations were generated by self-pollinating five F1 and reciprocal F1 plants. Self-pollination was ensured by

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Table 2. Random Amplified Polymorphic DNA (RAPD) primers used in the final molecular marker study. Primer OPB-05 OPE-06 OPE-11 OPE-15 OPG-18 OPH-14

Nucleotide sequences (59 to 39) TGCGCCCTTC AAGACCCCTC GAGTCTCAGG ACGCACAACC GGCTCATGTG ACCAGGTTGG

Table 3. Segregation of AHAS inhibitor resistance phenotype in backcross (BC) families (S 3 F1) of eastern black nightshade 20 days after treatment with imazethapyr at 150 g ai ha21. Phenotypes were visually scored for injury and classified as S (susceptible, total plant necrosis) or I (intermediate, stunted plants with chlorotic apical meristem).

Population

Total plants Family classified

Phenotype S

I

df

x2 (1:1)a Probabilityb

-----No. of plants ---BC (S 3 F1)

enclosing plants before anthesis within polyethylene pollinating bags4 (91 by 183 cm). Mature berries from plants were collected and seeds were extracted and stored as described above. Evaluation of Herbicide Resistance in BC and F2 Families. For the segregation experiments, three to four seeds of each BC and F2 family were placed at the soil surface of each cell of a 72-cell plug flat containing growth media as described above. As control for each BC and F2 family, nine separate cells were planted each with the seeds from the corresponding self-pollinated R and S parental lines. Seedlings were thinned and grown as stated above. At the three- to four-leaf stage, plants were treated with imazethapyr at 150 g ai ha21 as described above. Plants from BC and F2 families were compared to the parental R and S populations and visually scored for injury 20 d after treatment and classified as R (no imazethapyr injury), S (total plant necrosis), or intermediate (I, stunted plants with chlorotic apical meristem). The observed segregation pattern was compared to expected ratio of 1:2:1 (R:I:S) for F2 and of 1:1 (I:S) for BC families and subjected to Pearson chi-square (x2) goodness of fit test. A heterogeneity test was performed to pool the data among BC and F2 families from different crosses (a 5 0.05) (Bowley 1999). The experiments were arranged as randomized complete block design with families as repetitions (blocks). All computations were performed using Proc FREQ of SAS5 (ver 8.2). RAPD Analysis. The RAPD analysis was based on the methods described by Stankiewicz et al. (2001) with modifications as described below. For each of 25 eastern black nightshade populations, leaves from two individual eight- to ten-leaf stage plants were harvested and stored separately at 280 C. Frozen leaf tissue was freeze-dried for 36 h and ground with a mortar and pestle. DNA was extracted from the ground leaf tissue according to protocol outlined in the DNA extraction kit.6 Isolated DNA was quantified by measuring the absorbance at 260 nm, and diluted with distilled water to an approximate concentration of 10 ng ml21 and stored at 220 C. Initial RAPD profiles were generated using 160 RAPD primers (OPA, OPB, OPC, OPD, OPE, OPF, OPG, and OPH),7 and DNA from one randomly selected individual from four populations (SOLPT 2, 4, 6, and 14). Amplification reactions contained 30 ng of genomic DNA, 260 nM of primer, 3 mM MgCl2, 200 mM of each deoxynucleoside triphosphate, 1.25 unit of Platinum Taq DNA polymerase,8 and 2.5 ml of manufacturer supplied buffer in a final volume of 25 ml. Polymerase chain reactions were performed in a thermal cycler.9 Reaction mixtures were denatured at 94 C for 3 min, then subjected to 40 cycles of amplifications with the

Total Pooled Heterogeneity

1 2 3 4

72 72 72 72

39 28 34 40

33 44 38 32

1 1 1 1

0.500 3.556 0.222 0.889

0.4795 0.0593 0.6374 0.3458

288

141

147

4 1 3

5.167 0.125 5.042

0.2706 0.7237 0.1687

a Chi-square values are the results of tests for goodness of fit to a 1:1 (I:S) segregation model. The null hypothesis, i.e., the observed frequencies are in accordance with expected frequencies, is accepted in all cases. b The type I error rate if the null hypothesis is rejected based on the statistic (Bowley 1999).

following thermal cycles: denaturation for 1 min at 94 C, annealing for 1 min at 35 C, and extension for 2 min at 72 C, finally 7 min extension at 72 C. Amplification products were resolved by electrophoresis on 1.5% (w/v) agarose gels cast with 7.5 ml ethidium bromide (10 mM) for 3 h at 130 V. Fifteen primers, which produced polymorphic banding pattern in tested individuals, were selected for analysis of entire sample set of the populations. However, prior to this analysis, to account for the intrapopulation variation (Moodie et al. 1997), DNA from individuals within each population was pooled. Based on reproducible banding patterns between reactions, six primers were chosen for final analysis (Table 2). Furthermore, DNA from a hairy nightshade (Solanum sarrachoides Sendt.) population from Kettleby, Ontario (44u009200N, 79u349250W) was used as an out-group to verify the reliability of RAPD results (Stankiewicz et al. 2001). According to the presence or absence of each unequivocally visible RAPD marker (compared to all populations), a binary matrix of 1 and 0 was constructed for each population sample. The data matrix was used to construct a similarity matrix, using simple matching coefficient. The genetic similarity dendrogram was constructed by further analysis of similarity data, using the UPGMA (unweighted pair group method for arithmetic averages) cluster analysis in the NTSYS-PC 2.02 (Rohlf 1997) computer program. Results and Discussion

Evaluation of F1 Progenies. All F1 and reciprocal F1 progeny plants survived 150 g ai ha21 imazethapyr, however, the surviving plants were stunted and had chlorotic apical meristem (data not shown). The absence of reciprocal differences in F1 plants indicated nuclear control of resistance in AHAS inhibitor resistant eastern black nightshade. Segregation Pattern of BC and F2 Families. Imazethapyr application at 150 g ha21 resulted in two distinct phenotypic responses in BC families (I and S) (Table 3) and three phenotypic responses in F2 families (R, I, and S) (Table 4). The BC progeny segregated in a 1:1 (I:S) ratio, while the F2 progeny segregated in a 1:2:1 (R:I:S) ratio. Moreover, the test of heterogeneity indicated that the BC families were Ashigh et al.: Genetics of AHS resistance


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Table 4. Segregation of AHAS inhibitor resistance phenotype in F2 families of eastern black nightshade 20 days after treatment with imazethapyr at 150 g ai ha21. Phenotypes were visually scored for injury and classified as R (resistant, no imazethapyr injury), S (susceptible, total plant necrosis), or I (intermediate, stunted plants with chlorotic apical meristem).

Population

Total plants Family classified

Phenotype S

I

R

x2 df (1:2:1)a Probabilityb

----- No. of plants ---F2 (S 3 R)

F2 (R 3 S)

Total Pooled Heterogeneity

1 2 3 4 1 2 3 4

72 72 72 72 72 72 72 72

10 16 17 16 19 13 17 10

40 34 40 40 38 42 34 39

22 22 15 16 15 17 21 23

576

124

307

145

2 2 2 2 2 2 2 2

4.889 1.222 1 0.889 0.667 2.444 0.667 5.194

0.0868 0.543 0.6065 0.6412 0.7165 0.2946 0.7165 0.0745

16 16.972 2 4.038 14 12.934

0.3874 0.1328 0.5317

a Chi-square values are the results of tests for goodness of fit to a 1:2:1 (S:I:R) segregation model. The null hypothesis, i.e., the observed frequencies are in accordance with expected frequencies, is accepted in all cases. b The type I error rate if the null hypothesis is rejected based on the statistic (Bowley 1999).

homogeneous for the 1:1 segregation pattern, and all eight F2 families were homogeneous for the 1:2:1 (R:I:S) segregation pattern, indicating that a single gene with incomplete dominance controls resistance to AHAS-inhibiting herbicides in R populations of eastern black nightshade from Ontario. These results are in agreement with the results from the study on the inheritance of resistance in AHAS inhibitor resistance eastern black nightshade population from Wisconsin (Volenberg and Stoltenberg 2002), however, the type of substitution causing AHAS inhibitor resistance in that population has not been determined. Furthermore, resistance due to the Ala205Val substitution has been reported as partially dominant in sunflower (Helianthus annuus L.) populations (Kolkman et al. 2004). RAPD Analysis. The six selected RAPD primers generated a total of 45 bands (loci), from which 35 bands were polymorphic across all nightshade populations (including the hairy nightshade population) whereas 10 polymorphic bands were specific only to populations of eastern black nightshade (data not shown). An example of RAPD profiles generated in nightshade individuals using the primer OPH-14 is shown in Figure 1. The genetic similarity dendrogram constructed from the comparative data indicated four groups of eastern black nightshade populations with similarity coefficients of less than 0.95 (Figure 2). The genetic similarity coefficients that determined subdivision within each of the groups were higher than 0.95. The first group consisted of seven R populations and one S population. Ten S and two R populations were included in the second group. The third group contained three R and one S population and, interestingly, the fourth group contained the only R population (SOLPT 24) with the Ala122Thr substitution (Figure 2). The RAPD profile of nightshade populations showed that eastern black nightshade populations were more related to each other than to hairy nightshade, confirming the reliability of RAPD-generated profiles. The different groups of resistant populations, as defined by RAPD analysis, tended to show geographical clustering 0

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Figure 1. The random amplified polymorphic DNA (RAPD) profile generated with primer OPH-14. The first lane represents the molecular size markers (100bp ladder). The successive lanes each represent the amplification products of pooled DNA from individuals within resistant (R) and susceptible (S) populations of eastern black nightshade (SOLPT) (lanes 2 to 26) and hairy nightshade (SOLSA) (lane 27). The polymorphic product of large molecular weight is present in SOLPT populations 14, 2, 1 and 27.

(Figure 3). For example, group 1 comprised seven R populations (SOLPT 6, 7, 8, 10, 22, 26, and 28), which were collected from an area approximately 50 by 60 km, on the east shore of Lake Huron (Figure 3). The only S population (SOLPT 11) from that group was found about 100 km east. Group 3 also was geographically clustered. It comprised three R (SOLPT 1, 14 and 27) and one S (SOLPT 2) populations in the southwest of Ontario on the north shore of Lake Erie (Figure 3). Group 2 showed much more widespread distribution with populations found in the extreme southwest and east parts of the province. This group was comprised of two R populations (SOLPT 3 and 25) from proximal areas and 10 S populations (SOLPT 4, 5, 9, 12, 13, 15, 16, 17, 21, and 23). Population SOLPT 24 (group 4) was also located in the southwest of the province. The close proximity of the R populations within group 1, group 2, and group 3 strongly suggest that local gene flow contributed to the resistance spreading from at least three single resistance selection events. Therefore, both similar local selection pressure in different fields and gene flow could explain the spread of the Ala205Val substitution in R populations of eastern black nightshade in Ontario.

Figure 2. Genetic similarity dendrogram showing the relationships between resistant (R) and susceptible (S) eastern black nightshade populations (SOLPT) (Table 1) and a hairy nightshade (SOLSA) population.

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the important factors in the spread of triazine resistant populations of black nightshade (Solanum nigrum L.) from France to Poland (Stankiewicz et al. 2001). A single nuclear gene with incomplete dominance controls resistance to AHAS inhibitors in population SOLPT 1. This is in agreement with resistance being conferred by a point mutation in the AHAS gene coding for an Ala205Val substitution. Up to 12 populations in Ontario are resistant due to this mutation (Ashigh and Tardif 2007) and it appears that a combination of separate local selection and gene flow account for their geographic distribution. Our results highlight the fact that both phenomena can explain resistance occurring in a field and need to be taken into account in the prevention and management of resistance.

Figure 3. Geographic locations of four groups of eastern black nightshade (SOLPT) populations in Ontario. Eastern black nightshade groups were determined using RAPD analysis. Group one with seven AHAS inhibitor resistant (R) ( ) and one AHAS inhibitor susceptible (S) (#) populations, group two with two R (m) and 10 S (n) populations, group three with three R (¤) and one S (e) populations and group four with only one R ( ) population.

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In Ontario, imazethapyr is a registered herbicide with high efficacy for control of eastern black nightshade in soybean. Based on our previous studies at the whole plant and enzyme levels (Ashigh and Tardif 2006, 2007), SOLPT 1 with the Ala205Val substitution shows high level resistance to the IMI imazethapyr and low level resistance to the SU primisulfuron. Therefore, since all R populations have been collected from soybean fields, imazethapyr may have played a major role in the selection of eastern black nightshade populations with Ala205Val substitution. In contrast, the Ala122Thr substitution has been shown to endow higher resistance level to IMI herbicides with none to very low levels of cross-resistance to SU herbicides (Milliman et al. 2003; Volenberg et al. 2000). Therefore, it could be logical to expect the selection of Ala122Thr substitution when the primary selector is an IMI herbicide. However, the selection of eastern black nightshade populations with Ala205Val substitution might have been influenced by both IMI and SU herbicides. While the SU herbicides registered for use in soybeans are less efficacious on nightshades, they might still induce low, albeit sufficient pressure for selection of plants with Ala205Val substitution (with low levels of resistance to these herbicides). Plants with Ala122Thr substitution (with none to very low levels of resistance to these herbicides) would however be selected against. Eastern black nightshade is an autogamous plant (Schilling 1978). However, under favorable conditions, the rate of outcrossing in this species could be as high as 17% (Hermanutz 1991). Thus, pollen movement could be one reason for local spread of resistance within the population groups. However, the spread of resistant plants was most likely through seed dispersal via crop seed contamination, manure spreading, or transport in harvest equipment. However, migratory birds might have also contributed to long distance spread. It has been suggested that anthocyanin pigments in nightshade berries could act as attractants for birds (Stankiewicz et al. 2001). Furthermore, the distribution of 13 populations belonging to Group 2 on the north shores of Lake Erie and Lake Ontario corresponds to one of the principal routes of bird migration in the Atlantic Flyway (Anonymous 2007). In addition, it has been suggested that bird-assisted seed dispersal has been one of

Sources of Materials 1

Sunshine LA4 MIX AGGREGATE PLUS (55–65% Canadian sphagnum peat moss, perlite, dolomite, calcite, vermiculite, and wetting agent), Sun Gro Horticulture Inc., Vancouver, BC, Canada V7X 1T2. 2 Nonionic surfactant, Agral 90, 90% nonylphenoxy-polyethoxyethanol, Norac Concepts Inc., Orleans, ON, Canada K1C 7H8. 3 Teejet 8002EH nozzle tip, Spraying Systems Co., Wheaton, IL 60189. 4 Delnet pollinating bags. Delstar Technologies Inc. 601, Industrial Drive, Middletown, DE 19709. 5 SAS Institute Inc., Cary, NC, USA. 6 FastDNA Kit, Qbiogene, 15 Morgan, Irvine, CA 92618-2005. 7 Operon Technologies Inc. 1000, Atlantic Ave. Suite 108. Alameda, CA 94501. 8 Platinum Taq DNA Polymerase, GIBCO, Invitrogen Canada Inc., 2270 Industrial Street, Burlington, ON L7P 1A1, Canada. 9 RoboCycler 96 Temperature Cycler, Stratagene, 11011 North Torrey Pines Road, La Jolla, CA 92037.

Acknowledgments The authors thank Mr. Chris Grainger and Mr. Peter Smith for their technical assistance. This work was supported by grants from the Ontario Ministry of Agriculture, Food, and Rural Affairs and the Ontario Soybean Growers. The postgraduate scholarship to J. Ashigh from Syngenta and the National Science and Engineering Research Council of Canada is gratefully acknowledged.

Literature Cited Alex, J. F. 1964. Weeds of tomato and corn fields in two regions of Ontario. Weed Res. 4:308–318. Anonymous. 2007. North American Migration Flyways. http://www.birdnature. com/flyways.html. Accessed: August 2, 2007. Ashigh, J. 2006. Resistance to acetohydroxyacid synthase-inhibiting herbicides in populations of eastern black nightshade (Solanum ptycanthum Dun.) from Ontario. Ph.D. dissertation. Guelph, ON, Canada: University of Guelph. 198 p. Ashigh, J. and F. J. Tardif. 2006. ALS-inhibitor resistance in populations of eastern black nightshade (Solanum ptycanthum) from Ontario. Weed Technol. 20:308–314. Ashigh, J. and F. J. Tardif. 2007. An Ala205Val substitution in scetohydroxyacid synthase of eastern black nightshade (Solanum ptychanthum) reduces sensitivity to herbicides and feedback inhibition. Weed Sci. In press. Bassett, I. J. and D. B. Munro. 1985. The biology of Canadian weeds. 67. Solanum ptycanthum Dun., S. nigrum L. and S. sarrachoides Sendt. Can. J. Plant Sci. 65:401–414. Boutsalis, P. and S. B. Powles. 1995. Inheritance and mechanism of resistance to herbicides inhibiting acetolactate synthase in Sonchus oleraceus L. Theor. Appl. Genet. 91:242–247.

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