RESISTANCE AND DIFFERENTIAL SUSCEPTIBILITY OF Bidens pilosa AND B . subalternans BIOTYPES TO ALS-INHIBITING HERBICIDES

The frequent application of herbicides in agricultural areas may select resistant biotypes in weed populations, whose biological characteristics influence the speed and patterns of resistance. This research aims to charactere, simultaneously, resistance patterns and differential susceptibility of Bidens pilosa and B. subalternans biotypes to ALS-inhibiting herbicides of the imidazolinone and sulfonylurea chemical groups. Six hairy beggarticks biotypes, four suspected resistant and two known susceptible, were treated with eight rates of chlorimuron-ethyl or imazethapyr, in greenhouse conditions. Percent control and percent fresh weight of the plants were evaluated at 28 days after the application. B. subalternans is less susceptible to ALS-inhibiting herbicides than B. pilosa; B. subalternans biotypes were more resistant than B. pilosa biotypes; there are B. pilosa and B. subalternans biotypes with cross resistance to the ALSinhibiting herbicides of the sulfonylurea and imidazolinone groups; there are different patterns of cross resistance to the diverse groups of ALS-inhibiting herbicides.

Weed resistance to herbicides is defined as a natural and inherited capacity of biotypes to survive and reproduce after the application of an herbicide rate that should be lethal to a normal (susceptible) popula- tion of the same species (Christoffoleti & López-Ovejero, 2004).The two most notable weeds that have gained resistance to ALS-inhibiting herbicides in Brazilian soybean fields are Bidens pilosa and B. subalternans (Christoffoleti, 2002;Gelmini et al., 2002).
Morphology and the ecological conditions of habitats occupied by both B. pilosa and B. subalternans are quite similar; these are sympathric species infesting soybean fields (Kissmann & Groth, 1999).Their similarity and overlapping of characters have resulted in imprecise taxonomic characterization, difficulty in establishing clear boundaries, and doubts about the material identification (Grombone-Guaratini et al., 2005a;2005b), even in resistance experiments.These weed species are difficult to control mainly because of their high levels of inbreeding and seed production, and long term survival of seeds on the soil (Grombone-Guaratini et al., 2004).
However, because it is so difficult identify Bidens species accurately, many authors could not say with which species they to dealt with (Monquero et al., 2000;Carvalho et al., 2004).This fact raises questions about the populations used in several published works.The aims of this research were (i) identify and characterize ALS-resistant Bidens biotypes, and (ii) evaluate the degree of resistance and differential susceptibility of B. pilosa and B. subalternans biotypes to chlorimuron-ethyl and imazethapyr.

MATERIAL AND METHODS
Seed origin -Seeds from resistant biotypes of Bidens pilosa and B. subalternans were collected from soybean production areas in São Paulo (SP), Paraná (PR), Mato Grosso (MT), and Mato Grosso do Sul (MS) states, on properties that had been sprayed with ALS-inhibiting herbicides for at least six consecutive years.Susceptible seed samples were collected in the open field in Santa Bárbara do Oeste (SP) and Instituto de Botânica de São Paulo (IBt/SMA), which had never been sprayed with ALS-inhibiting herbicides.For locations and biotype names see Table 1.

Dose-response curves
Place -The experiment was conducted in a greenhouse at Brazil (22º41'15''S,47º41'15''W and 560 m of altitude) from December/ 2004 to February/2005.Bioassay testing ALS sensitivity to herbicides -Seeds were germinated in plastic boxes (0.11 × 0.11 × 0.03 m) filled with commercial substrate, inside a germination chamber with photoperiod 8h-light, 30°C, and 16h-dark, 20°C.When seedlings were at the cotyledon stage, they were transplanted to 0.5-L plastic pots (four plants per pot), also filled with commercial substrate.
When plants averaged, four leaves, post-emergence herbicide treatments were applied.Spraying was done inside closed spray chamber, using a flat jet spray tip (Teejet 80.02E), calibrated at 0.50 m height above the target surface, and with a relative volume of 200 L ha -1 .After herbicide application, pots were placed in the greenhouse and irrigated on the following day to secure adequate foliar absorption of the molecules.
Trials were set up in randomized block design (n = 4), factorial scheme with six biotypes and eight rates of the herbicide chlorimuron-ethyl (sulfonylurea), and five biotypes and eight rates of the herbicide imazethapyr (imidazolinone).Sufficient seeds did not allow biotype 3 to receive applications of imazethapyr.Rates applied were multiple values of the recommended rate (R) for each herbicide (0.0R; 0.06R; 0.125R; 0.5R; 1R; 2R; 4R and 16R).The recommended rate adopted was 17.5 g ha -1 for chlorimuronethyl, and 100.0 g ha -1 for imazethapyr.Herbicide treatments are listed in Table 2; all treatments received the addition of mineral oil at 0.5% v/v concentration.
Evaluation -Percent control and percent fresh weight were evaluated at 28 days after the application (DAA).Percent control was rated as 0% when the herbicide effect was absent, and 100% when all plants died.Percent fresh weight was obtained based on the pots that received the application rate 0D (check), as 100% of possible weight.Fresh weights were corrected based on the checks within each respective block.Percent correction was used to make up for differences in biotypes growth rates.
Data analysis and elaboration of dose-response curves -Data were initially submitted to ANOVA ("F" test).Treatment showing significant, qualitative effects (biotypes), were compared by the application of Tukey's test (α = 0.05); quantitative effects (rates), were adjusted to a log-logistic model, proposed by Seefeldt et al. (1995), 1 where: y is the variable, x is the herbicide rate (g i.a.ha -1 ); and a, b, c and d are parameters of the curve, that a is the minimum limit of the curve, b is the difference among the maximum and minimum point of the curve, c is the rate that promotes 50% of response from the variable and d is the curve slope around c.
The log-logistic model presents an advantage once one of the parameters of the equation (c) is an estimative of C 50 or GR 50 value (Christoffoleti, 2002).C 50 (control by 50%) or GR 50 (growth reduction by 50%) are the herbicide rate (g i.a.ha -1 ) that promotes 50% of control or weight reduction on plants, respectively (Christoffoleti, 2002;Christoffoleti & López-Ovejero, 2004).From the equations obtained it was also possible to re-calculate mathematically the values of C 50 or GR 50 , which were considered during the discussion (Carvalho et al., 2005).Using the C 50 or GR 50 values, the resistance factor was obtained to all biotypes and all herbicides tested.The resistance factor (C 50 resistant / C 50 susceptible; GR 50 resistant / GR 50 susceptible or simply R/S) is an adimensional number that expresses how many times the necessary rate to control 50% of resistant biotype is superior than the rate that controls 50% of susceptible biotype, of the same species (Christoffoleti, 2002;Hall et al., 1998).

RESULTS AND DISCUSSION
Bidens pilosa and B. subalternans show morphological differences mainly in the cypselas: in Bidens pilosa the majority of cypselas have three awns, while in B. subalternans they have four.The angle between the awns and the cypsela is about 135º in B. pilosa, while in B. subalternans the angle is about 180º.In B. pilosa, the fruit surface is scabrous and minutely tuberculate in almost all its length, with trichomes arising from the tubercles.In B. subalternans, the surface of the cypselas is scabrous, lacks tubercles and has trichomes concentrated in the apex.Both species are widely distributed in agricultural areas and along roadsides.
According to Kissmann & Groth (1999), B. pilosa and B. subalternans are sympathric, soybean crop pests.However, in this research, we identified by morphological seed and seedling characters, all resistant seeds from Paraná as B. pilosa, and all resistant seeds from Mato Grosso, Mato Grosso do Sul and São Paulo as B. subalternans.
For both herbicides, in all evaluations, there were significant interactions between the rates applied and Bidens biotypes, justifying factorial decomposition.Table 3 presents the parameters of log-logistic model of two variables studied for the herbicides chlorimuron-ethyl and imazethapyr.These parameters allowed fitting plant control or fresh weight (%) as function of herbicides rates, as presented on Figures 1  to 4. Least significant differences (LSD) of Tukey's test (α = 0.05) comparing biotypes, also are presented in the figures.
Control results indicated that the B. subalternans susceptible biotype is more tolerant than the B. pilosa susceptible biotype, and that B. subalternans resistant biotypes display higher level of resistance than B. pilosa resistant biotypes.Neither chlorimuron-ethyl nor imazethapyr was effective in controlling resistant biotypes of B. pilosa and B. subalternans (Figures 1 and 2).However, both herbicides were effective in controlling the susceptible biotypes of B. pilosa and B. subalternans at recommended rates.C 50 and GR 50 values showed differences between   the resistant (R) and susceptible (S) biotypes of both species (Table 4).According to Kissmann & Groth (1999), B. subalternans has higher levels of tolerance to herbicides than B. pilosa.B. subalternans from Mato Grosso and Mato Grosso do Sul had highest levels of resistance to chlorimuron-ethyl.Rates up to 70 g ha -1 of this herbicide were used to obtain controls of 50% in these populations.To reach control of 50% in B. pilosa population, rates of the same herbicide used were lower (Table 4).Control tests with imazethapyr showed that B. subalternans from São Paulo displayed highest lev-    els of resistance.Rates up to 140 g ha -1 of the herbicide were used to obtain control of 50% in these populations (Table 4).These results are similar to those reported by Gelmini et al. (2002), but are in disagreement with Christoffoleti (2002) and Monquero et al. (2000), whose data showed that B. pilosa biotype presented higher levels of resistance to imazethapyr.Cross-resistance between the two ALS-inhibiting herbicides (the imidazolinones -IMIs and the sulfonylurea -SUL) was registered for all B. pilosa and B. subalternans suspect biotypes studied.Both species had different degrees of cross-resistance to ALS-inhib- iting herbicides.These data agree with results of Christoffoleti (2002) and Monquero et al. (2003) for B. pilosa, andGelmini et al. (2002) for B. subalternans.
There was percent fresh weight reduction with increasing herbicides rates (Figures 3 and 4).Once again, on the supposed resistant biotypes, the rates needed to reduce fresh weight were greater in comparison to the susceptible biotypes.For this variable, the B. subalternans biotype from Mato Grosso do Sul was the least sensitive to the herbicide chlorimuron-ethyl (Figure 3), while B. subalternans biotype from São Paulo was the least sensitive to the herbicide imazethapyr (Figure 4).
Resistance in many weeds has been attributed to single point mutations, which can occur at multiple sites in the ALS gene (Tan & Medd, 2002).Base changes in at least four protein domains have been associated with in vivo resistance in field plants (Wright et al., 1998).The most common mutation in biotypes selected by sulfonylureas is in the highly conserved Domain A, that codes for 13 amino acids, where any alteration of the codon for Pro confers resistance, primarily, to sulfonylureas (SUL) and triazolopyrimidines (TP) (Guttieri et al., 1992).A Trp→Leu mutation in Domain B has been associated with broad cross-resistance to representatives of all four families of ALSinhibiting chemicals (Bernasconi et al., 1995;Woodworth et al., 1996a).In Domain C, an Ala→Thr mutation appears to confer resistance only to imidazolinones (IMI) (Bernasconi et al., 1995), whilst an Ala→Val substitution in Domain D is reported to confer broad cross-resistance (Woodworth et al., 1996b), as in the case of the mutation in Domain B.
Many mutations have been documented for ALS, confering different resistance levels to various classes of inhibitors; resistant biotypes to ALS inhibiting herbicides show cross-resistance to members of chemical families with the same action mechanism (Tranel & Wright, 2002).Many works have shown that ALS-resistant weed biotypes have cross-resistance to herbicides that belong to chemical group that selected them, and diverse levels of cross-resistance to other herbicide groups with the same action mechanism (Rizzardi et al., 2002).It is probably a consequence of the selection pressure imposed by the main herbicide used, which selected those mutations especially related with its chemical group.This can, at least in part, explain results of this work and the great diversity of results reported in the literature.There are many ALS amino acid substitutions that confer herbicide resistance (Tranel & Wright, 2002); however we did not find any papers that reported data for the B. pilosa-B.subalternans complex.
The majority of the resistance cases of hairy beggarticks to ALS-inhibiting herbicides occur in Bidens subalternans, not in B. pilosa, as previously thought.The confirmation that susceptible biotypes of B. subaternans are more tolerant to these herbicides than B. pilosa reveals how important species identification is to effective control.The identification of point mutations in the ALS gene domains can clarify the resistance patterns and give insights on the necessity of alternative systems that prevent or delay the emergence of resistance.

Table 2 -
Treatments with multiples of the recommended rates of the herbicides applied on Bidens pilosa and B. subalternans biotypes.

Table 3 -
Log logistic model 1 parameters to Bidens biotypes for all variables evaluated, when treated with chlorimuronethyl or imazethapyr.

Table 4 -
C 50 , GR 50 and resistance factor (R/S) for resistant and susceptible biotypes of Bidens pilosa and B. subalternans.