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Published in J. Environ. Qual. 33:1376-1386 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Plant and Environment Interactions

Herbicide Selection of Italian Ryegrass under Different Levels of UVB Radiation

M. Alejandra Martínez-Ghersaa,*, Martín M. Vila Aiuba,c, Claudio M. Ghersaa, Pedro Gundela and Emilio H. Satorreb

a IFEVA, Departamento de Recursos Naturales y Ambiente, Universidad de Buenos Aires, Av. San Martin 4453, C1417DSE Buenos Aires, Argentina
b Departamento de Produccion Vegetal, Facultad de Agronomia, Universidad de Buenos Aires, Av. San Martin 4453, C1417DSE Buenos Aires, Argentina
c Present address: University of Western Australia, WA Herbicide Resistance Initiative (WAHRI), Department of Plant Science, 35 Stirling Highway, Crawley, WA 6007, Australia

* Corresponding author (martinez{at}ifeva.edu.ar).

Received for publication June 10, 2003.

   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ultraviolet-B radiation is an environmental stress for plants and this situation could become aggravated in the next decades. In this study we used Italian ryegrass (Lolium multiflorum Lam.) as a model system to test whether an environmental stress derived from global change, such as UVB, can influence the efficacy of control procedures and evolution toward herbicide resistance. We grew three generations of Italian ryegrass plants with and without UVB light and subjected them to a series of diclofop-methyl {(±)-2-[4-(2,4-dichlorophenoxy) phenoxy] propanoic acid, methyl ester} doses. The effect of selection history was tested with herbicide dose response. The effect of herbicide application on plant survival and biomass varied significantly among herbicide doses and with absence or presence of UVB light. In the absence of herbicide, the decrease in individual fecundity with increasing plant density was similar under both no-UVB and UVB light treatments. Only plants growing without UVB light increased production of reproductive structures in response to the decrease in density caused by herbicide application. Our study shows that UVB light was a weak stress factor for the ryegrass plants. However, when herbicide selection pressure was high, UVB light reduced the evolution toward herbicide tolerance. When selection pressure on the parental plants was lower, the two stress factors had a synergistic effect, causing changes in herbicide efficacy that in turn had demographic and evolutionary consequences. In the field, these interactions between stress factors might be of significance for annual weeds in which seed output is a major determinant in fitness.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE IMPACT on biological systems of multiple stresses caused by human activities is far from understood (Mooney and Winner, 1991). Nevertheless, there is some consensus among scientists that broad-scale changes in the environment, including soil water, vegetation, and the atmosphere, will alter the productivity of agroecosystems, influence the selection of crop varieties, and affect the distribution of agricultural pests and their response to control (Vitousek, 1994; Patterson, 1995; Ziska et al., 1999; Sala et al., 2000). One of the most striking environmental global changes is the decrease in column ozone outside of tropical regions (Bojkov et al., 1990; Niu et al., 1992), varying with month, latitude, and longitude. In some regions, scientists have reported a concomitant increase in surface UVB (280–320 nm) due to loss of ozone and its function in protecting against ultraviolet light (Lubin et al., 1989). Ambient UVB radiation is believed to be an environmental stress for plants (Cybulski and Peterjohn, 1999; Hunt and McNeil, 1999). Even with the control of ozone-depleting substances such as chlorofluorocarbons, significant ecological impacts are likely to continue for several decades (Day et al., 1999; Paul and Gwynn-Jones, 2003). Although the effects of UVB light on plants have been extensively reported, it seems critical to diagnose the effects of increasing UVB radiation on how biological systems respond to such management practices as herbicide application.

Weed control, most commonly using herbicides, has become one of the most important activities in crop production because weeds compete with crops and decrease yield (Radosevich et al., 1997). The response of a plant to an applied chemical is influenced by other stresses it is subjected to, before or following treatment (Muzik, 1976; Stanton et al., 2000). Plants are rarely exposed to only a single stress, and adjustments to several concurrent stresses are usually required for attaining high yields (Mooney and Winner, 1991). However, studying the effects of individual stress components has been the most common approach.

Italian ryegrass is a major grass seed crop and a highly competitive weed in cereal fields (Appleby et al., 1976; Burrill et al., 1988), leading to as much as 60% loss in grain yield (Appleby et al., 1976). Diclofop-methyl has been extensively used to control ryegrass in cereal production in the United States and throughout the world. This had lead to the development of resistant populations (Brewster and Appleby, 1988; Stanger and Appleby, 1989). Genetic, physiological, and ecological research on processes regulating herbicide resistance in ryegrass has been conducted (Ghersa et al., 1994a, 1994b; Powles and Holtum, 1994). Studies on resistance to diclofop-methyl in Italian ryegrass have demonstrated that a single, partially dominant gene encodes resistance to this herbicide, conferring to heterozygous individuals an intermediate tolerance at field-applied rates (Betts et al., 1992). More recently, glyphosate resistance has also been reported in Italian ryegrass (Powles et al., 1998).

Acetyl-coenzyme A carboxylase (ACCase), a key enzyme involved in the biosynthesis of lipids and UVB-absorbing pigments (flavonoids), is the target site of several selective herbicides such as diclofop-methyl (Kobek et al., 1988). There is some evidence to speculate that the combined effects of diclofop-methyl selection and UVB radiation may cause demographic and evolutionary plant responses different from that caused by each stress separately. Diclofop-methyl and UVB light have opposite effects on ACCase. While the enzyme is inhibited in susceptible individuals by diclofop-methyl (Gronwald et al., 1992), previous reports have indicated that total, citosolic, and plastidic activity of ACCase is inducible under UVB exposure (Ebel and Hahlbrock, 1977; Konishi et al., 1996). Moreover, efficacy of ACCase-inhibiting herbicides under field conditions can be improved in the absence of UVB radiation, suggesting that herbicide applications should be performed at late evenings or nights (McMullan, 1996).

To date, there is no published information on the response of annual ryegrass to the combined effects of herbicide application and UVB light environmental stresses. In this study we used Italian ryegrass as a model system to test whether an environmental stress derived from global changes, such as UVB light, can influence the success of herbicides in controlling weeds. We considered the effects of dose on both the efficacy of the control procedures and the plants' evolution toward herbicide resistance. We hypothesized that the efficacy of the herbicide in controlling ryegrass changes with the level of environmental UVB. We further hypothesized that under more stressful conditions (higher levels of UVB radiation), the rate of evolution to herbicide resistance will decrease due to changes in mortality rates and in the fecundity of surviving individuals.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The design of the experiments is summarized in Fig. 1. All experiments were performed during winter and spring of 1996, 1997, and 1998 at the experimental field of Facultad de Agronomía, University of Buenos Aires (34°35' S, 58°29' W).



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  		Fig. 1. Overview of the selection protocol and experimental design. (A) Selection cycles conducted during 1996 and 1997; (B) herbicide screening tests conducted in 1998.

 
Response to Herbicide under UVB Light Environments: Parental Population and Progeny (F1)
A factorial experiment in a randomized block design was performed twice, in 1996 and replicated in 1997, to study the response of a ryegrass population to a series of herbicide doses under different UVB light levels. Thirty 0.25-m2, 15-cm-high wooden boxes filled with organic soil were sown with a commercial accession of endophyte-free Italian ryegrass from Oregon, never exposed to herbicide application, at 1024 seeds m–2, and placed in the field. Seeds from the same parental population were used in both years. We chose an endophyte-free population to avoid plant–fungal interactions, which are known to be affected by UVB (Newsham et al., 1998). Half of the boxes (plots) were randomly assigned to each of the UVB light treatments: ambient light with exclusion of UVB light (UVB–) and ambient solar light (UVB+). All plots were covered immediately after sowing with plastic film that transmitted more than 90% of the photosynthetically active radiation (400–700 nm). The film over the UVB– plots was Mylar-D film (0.1 mm thick; DuPont, Wilmington, DE), which blocks all radiation below 320 nm (see spectrum in Ballaré et al., 1996). The film over the UVB-transparent control plots (UVB+) was Aclar film (0.04 mm thick; Allied Signal Plastics, Morristown, NJ), which has very high transmittance over the whole UV waveband. Sheets of film were draped over 1- x 1-m metal frames that were maintained about 3 cm above the plant canopy during the course of the experiments. Each plot had an individual frame above it, and the sides were left open to allow air circulation. Air temperature under the filters was recorded in the morning and afternoon on three different days using a model 21X Micrologger (Campbell Scientific, Logan, UT), and no differences between plots were detected (not shown). All plots were watered as needed to maintain the soil near field capacity. Experiments started on 13 June 1996 and 10 July 1997.

Environmental data were recorded in a meteorological station located 0.5 km from the experimental site. UVB radiation was recorded at noon with clear sky with a biologically weighed Model UV-IL1400A UV-Biometer (International Light Co., Newburyport, MA). The UV meter calibration is corrected for a standard sun defined as the output of the UV radiation model under a 2.7-mm ozone column, 30° solar zenith angle, at sea level and zero albedo. Doses are expressed as MED (minimal erithemal dose) and were defined as equivalent to a biologically effective UVB dose (Madronich, 1992) of 210 J m–2, following manufacturer recommendation (Morys and Berger, 1993; Saul Berger, private communication to the Scientific Advisor of the WMO/SMN Argentina UV Network, Dr. Rubén Piacentini).

Three weeks after sowing, when the plants were at the two-leaf stage, seedling density was measured in a 78.5-cm2 area placed at random in each plot. Plots under UVB– and UVB+ were randomly assigned to a series of herbicide doses: 0, 140, 280, 560 (label dose), and 1120 g a.i. ha–1 of diclofop-methyl (commercial formulation 284 g a.i. L–1, Iloxan; Hoechst-Aventis, Strasbourg, France). Each treatment combination (UVB light x herbicide dose) was replicated three times in both years. Herbicide was applied with a constant-pressure hand sprayer over the entire plot 1 d after density was measured. Seedling survival in the sampling areas was recorded 1 mo after herbicide application, and surviving individuals were harvested. Total dry weight was obtained after oven-drying for 72 h at 70°C.

Once the surviving plants began to flower, we isolated them to prevent pollen transfer between plots. The metal filter frames supported a fiberglass fabric that covered the openings around each plot. This material did not prevent air circulation or light penetration, but it prevented pollen flow among plots.

Another 78.5-cm2 area was sampled in each plot to evaluate seed production. Plots were visited every 10 d from early November until the end of December in both years, and all newly produced spikes were harvested and seed weight recorded. Seeds produced outside of the sampling area were also harvested and stored under laboratory conditions for further use.

Mature seeds harvested from each treatment at the end of 1996 were kept separate. We sampled a subset of the seeds to produce the next generation of seedlings (progeny). Seeds were sown as above in 1997. Each group of seedlings was subjected to the same UVB light x herbicide dose treatments as the corresponding parental plants. Seedling survival, biomass, and seed production of surviving individuals were recorded as described above.

Selection Response to Stress in F1 and F2 Populations
To allow examination of selection responses to the different herbicide and UVB light environmental conditions (selection histories), seeds produced under each treatment by the parental plants in 1996 and 1997 (F1) and those produced by the progeny plants in 1997 (F2) were exposed to a common stress environment.

One hundred seeds of each selection history were sown in organic soil in plastic pots (1.5 L). Initial seedling density was recorded after emergence. Three pots from each selection history were assigned to each of the five diclofop-methyl doses (0, 140, 280, 560, and 1120 g a.i. ha–1). Herbicide was applied at the two- to four-leaf stages, as described before. Seedling survival and biomass of the surviving plants were evaluated.

Data Analysis
All data were analyzed using SAS Version 6.12 statistical package (SAS Institute, 1990). Two- and three-way ANOVA tests were performed on survival and biomass data. We tested the main effects (year, selection history, and herbicide dose) and the interactions using the general linear model procedure. Arcsine transformations were used on all survival data to meet the assumptions of ANOVA. A probability level of P < 0.05 was used to delineate main and interaction treatment differences.

Data from herbicide screening of F1 and F2 populations were evaluated with least square nonlinear regression (PROC NLIN). Regression analysis was further used to fit linear or nonlinear models to fecundity data as appropriate. Nonlinear models were used if ANOVA indicated that higher-order polynomial effects were more significant than linear effects.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Herbicide Efficacy
Survivorship in the First Selection Cycle (Parental Plants)
Average temperature and UVB radiation during the months that experiments were performed are presented in Table 1. Average temperatures in June and July 1996 were lower, and temperatures in December were higher, than temperatures for the corresponding months in the other two years. UVB radiation in the October–December 1996 period was higher than in 1997. In both years of the experiments with parental plants (1996 and 1997), the average efficacy for the herbicide treatments was approximately 90%. However, the effect of UVB and herbicide application on plant survival differed between years, as indicated by a significant three-way interaction (P < 0.05). To explore the nature of these effects, we examined each year separately.


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  		Table 1. Climatic conditions for the years of the experiments.

 
In the first year, average plant survival was 92% for the untreated plots and 10% for the herbicide-treated plots, but survival of treated plants did not differ among herbicide doses or light treatments (Fig. 2A). In the second year, overall survival was higher and more variable than during the first year (Fig. 2B). The response to herbicide dose depended on the UVB light regime to which plants had been exposed (P = 0.04).



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  		Fig. 2. Effect of ultraviolet-B (UVB) radiation on survival of Italian ryegrass seedlings of a parental population when treated with different doses of diclofop-methyl. Bars represent means ± standard errors. Analysis of variance for (A) 1996: UVB light (L), P = 0.18; herbicide dose (D), P = 0.0001; L x D, P = 0.95. Analysis of variance for (B) 1997: L, P = 0.12; D, P = 0.00001; L x D, P = 0.04.

 
Biomass of Individuals in the First Selection Cycle (Parental Plants)
Like survivorship, biomass of the surviving ryegrass plants was significantly affected by the interaction among year, UVB light, and herbicide dose (P < 0.05), which suggests that the consequences of treatment manipulations on the same ryegrass population differed for the two years. Italian ryegrass biomass was not significantly affected by UVB light in either untreated or herbicide-treated plants during 1996 (Fig. 3A). In the second year, the UVB+ treatment produced more variability in final plant sizes among doses than that of the UVB– treatment (Fig. 3B). UVB light increased the effects of herbicide only under low-dose treatments (P = 0.03).



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  		Fig. 3. Effect of ultraviolet-B (UVB) radiation on biomass of seedlings of a parental population of Italian ryegrass surviving treatment with different doses of diclofop-methyl. Bars represent means ± standard errors. Analysis of variance for (A) 1996: UVB light (L), P = 0.13; herbicide dose (D), P = 0.43; L x D, P = 0.76. Analysis of variance for (B) 1997: L, P = 0.39; D, P = 0.10; L x D, P = 0.03.

 
Effect of Herbicide and UVB Light on Fecundity
UVB light and herbicide interacted, modifying the relationship between vegetative and reproductive biomass in parental plants (Fig. 4). The interaction of year and vegetative biomass was not significant for individual reproductive biomass, therefore data from both years were pooled. When plants were not treated with herbicide, UVB light had little impact on fecundity of individual plants (Fig. 4A, difference between slopes, P = 0.19). For herbicide-treated plots without UVB light, there was a correlation between reproductive and vegetative biomass (P = 0.038) (Fig. 4B). When treated plots included UVB+, on the other hand, production of reproductive biomass was relatively low and independent of vegetative biomass (P = 0.17). Consequently, UVB light and diclofop-methyl herbicide interacted to affect the relationship between reproductive biomass per plant and plant density per pot (Fig. 5). In the absence of herbicide, individual fecundity decreased with the increase in density of plants under both UVB– and UVB+ treatments (Fig. 5A). The mortality caused by the herbicide generated a decrease in plant density per pot, but only plants growing under UVB– responded to this decrease by increasing their production of reproductive structures (Fig. 5B).



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  		Fig. 4. The effect of ultraviolet-B (UVB) light on the relationship between reproductive and vegetative biomass per Italian ryegrass plant growing in (A) nontreated (control) plots and (B) herbicide-treated plots. Both parental populations were pooled for analysis. Regression equations: (A) UVB–: y = 0.1354x + 0.0013, R2 = 0.95, P = 0.001, SE of the slope = 0.01; UVB+: y = 0.1102x + 0.0015, R2 = 0.89, P = 0.001, SE of the slope = 0.01; (B) UVB–: y = 0.1011x + 0.0227, R2 = 0.31, P < 0.05, SE of the slope = 0.05; UVB+: y = –0.0461x + 0.0276, R2 = 0.06, P > 0.05.

 


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  		Fig. 5. The effect of ultraviolet-B (UVB) light on the density-dependent production of reproductive biomass in (A) nontreated (control) plots and (B) herbicide-treated plots. Parental and F1 populations were pooled for analysis. Regression model fitted: y = a exp(–Kx). Parameters for (A) UVB–: a = 0.061, K = 0.017 (SE = 0.01), R2 = 0.88; UVB+: a = 0.045, K = 0.014 (SE = 0.004), R2 = 0.97; parameters for (B) UVB–: a = 0.18, K = 0.12 (SE= 0.055), R2 = 0.47; UVB+: not significant.

 
Survivorship in the Second Selection Cycle (F1 Plants)
After the first selection cycle, herbicide efficacy was reduced significantly; survival values increased more than 50% (Fig. 6). The effect of herbicide dose on plant survival depended on the UVB light environment, as shown by the strong interaction found (P = 0.02). We found no definite pattern in the relationship between herbicide dose and survival rate of the second-generation (F1) plants (Fig. 6).



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  		Fig. 6. Effect of ultraviolet-B (UVB) radiation on survival of Italian ryegrass seedlings of an F1 population when treated with different doses of diclofop-methyl. Bars represent means ± standard errors. Analysis of variance: UVB light (L), P = 0.17; herbicide dose (D), P = 0.39; L x D, P = 0.02.

 
Biomass of Individuals in the Second Selection Cycle (F1 Plants)
The light environment had a significant effect on the average weight of the individuals surviving herbicide application (Fig. 7). Plant biomass was almost 25% greater without UVB light than with it. However, the effect of UVB light on final plant size was not consistent across all herbicide treatments; it was especially evident at the lower herbicide doses (Fig. 7).



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  		Fig. 7. Effect of ultraviolet-B (UVB) radiation on biomass of seedlings of an F1 population of Italian ryegrass surviving treatment with different doses of diclofop-methyl. Parental and F1 populations were exposed to the same UVB light and herbicide environments. Bars represent means ± standard errors. Analysis of variance: UVB light (L), P = 0.0034; herbicide dose (D), P = 0.0007; L x D, P = 0.31.

 
Selection Responses to Stress
F1 Population
There was a significant interaction between parental herbicide doses and herbicide screening dose, showing that one generation of diversifying selection was enough to alter the response to herbicide dose in ryegrass plants. A strong year effect on the survival to the herbicide screening of plants coming from different selection histories (P < 0.001) was found. Separate year analysis revealed a highly significant main effect of UVB light history, suggesting that over all parental and screening herbicide doses, the UVB light environment affected the plants' chances of surviving herbicide application (Fig. 8 and Table 2). Moreover, during the second year of the experiment, the effect of selection history (parental dose) on plant survival varied significantly with light history, as indicated by the interaction term UVB x dose in Table 2 (P = 0.0487). This response was not affected by screening herbicide dose, as suggested by the lack of significance for the three-way interaction for light environment, parental dose, and screening dose (P = 0.5727). We also found evidence of variation in survival to herbicide application associated with the selection history in both years (P = 0.0001) (Table 2).



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  		Fig. 8. Selection of ryegrass populations in different UVB light and herbicide environments as estimated by the survival to herbicide application of F1 seedlings from seeds produced in 1996 and 1997. The figure illustrates the significant interactions between current environment (herbicide screening) and selection history from Table 2. Bars represent means ± standard errors.

 

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  		Table 2. The effect of current environment (herbicide screening) and ultraviolet-B (UVB) light and herbicide exposure (dose) history on survival of F1 ryegrass seedlings from seeds produced in 1996 and 1997 and of F2 ryegrass seedlings from seeds produced in 1997 (screened in 1998).

 
The equations fitted to describe the herbicide dose response of the different selected populations (Table 3) are useful to explain the above interactions. The values of the slope and of the model fit (R2) are estimators of the plant response to herbicide dose. A decrease in the value of either of these estimators can be interpreted as a decreased sensitivity in the assayed population to the herbicide-screening test, and thereafter, an increased selection response. In the screening performed with the 1996 F1 population, the steepest slope (–9.60) and the best model fit (0.96) were for the plots without herbicide selection under UVB+. As herbicide selection pressure increased (herbicide dose applied to mother plants), the slope and the model fit decreased, regardless of the light treatment. However, it is important to note that plants selected in the absence of UVB light had lower herbicide response slopes than those selected under UVB+ (Table 3). This difference was especially evident in plants selected under half the label dose (280 g a.i. ha–1).


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  		Table 3. The effect of herbicide and UVB light history (treatments applied to parental generation) on the survivorship of the F1 population of Italian ryegrass seedlings to diclofop-methyl dose screening. Seedlings grew from seeds produced in 1996 and 1997, and bioassays were carried out under the same environmental conditions in 1998.{dagger}

 
The lower selection pressure caused by the herbicide treatments in the 1997 parental generation was reflected in the herbicide dose response of the F1 generation. In this case, the decrease in slope of the response with the increase in selection pressure (parental dose) was much smaller than the decrease found during the first year, regardless of light environment. Again, the UVB environment affected the response of the different ryegrass populations to herbicide, as shown by the difference in model slope. However, it is interesting to note that in this case, slopes for plants under UVB+ were less steep than those for UVB– (Table 3).

F2 Population
A second year of selection under the same herbicide and UVB light conditions further increased the degree of herbicide tolerance in the ryegrass populations (Fig. 9 and Table 2). Again, the effect of the selection history on plant survival changed with the light environment (p = 0.05), independently of the herbicide dose used for the screening test (p = 0.681) (Table 2). When the selection was performed twice in the absence of UVB light (UVB–), both the slopes and the R2 values obtained through the herbicide screening test followed the expected parental dose selection pressures; that is, the highest sensitivity values (slopes) were for the control (no herbicide on parental plants), intermediate values were for the middle herbicide parental doses (140 and 280 g a.i. ha–1), and lowest values were for the highest herbicide parental doses (560 and 1120 g a.i. ha–1) (Table 4). In contrast, when the selection was performed twice in presence of UVB light (UVB+), sensitivity to herbicide was lost at all parental herbicide doses except the highest (1120 g a.i. ha–1), which still responded to the increase in herbicide screening dose with a slope of –3.8 (Table 4).



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  		Fig. 9. Selection of ryegrass populations in different UVB light and herbicide environments as estimated by the survival to herbicide application to F2 seedlings from seeds produced in 1997. The figure illustrates the significant interactions between current environment (herbicide screening) and two cycles of the same selection history from Table 2. Bars represent means ± standard errors.

 

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  		Table 4. The effect of herbicide and UVB light history (treatments applied to parental and F1 populations) on survival to diclofop-methyl dose screening of the F2 population of Italian ryegrass seedlings. Seedlings grew from seeds produced in 1997, and bioassays were carried out during the next growing cycle.{dagger}

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results support the hypothesis that the environmental stress caused by UVB light interacted with diclofop-methyl to affect the efficacy of the herbicide in controlling annual ryegrass, as well as affecting plant growth and reproduction in the plants surviving the treatment. Therefore, UVB light also affected the evolutionary response to herbicide selective pressure.

The effect of UVB light combined with increasing herbicide doses varied between years as well as among doses. Variability of plant response to the combined effects of UVB light and other stress factors is to be expected (Teramura and Sullivan, 1994). In order for UVB radiation to be effective it must first penetrate the leaf, reach sensitive targets, and be absorbed by the chromophores present. A variety of plant responses may alter the penetration of UVB into the mesophyll. Anatomical changes in the epidermal layer, quantitative or qualitative changes in epicuticular waxes, or an increase in leaf thickness or specific leaf weight may reduce penetration of UVB radiation to sensitive targets (Tevini and Steinmuller, 1987). In 1997, UVB light increased herbicide efficacy with intermediate doses (Fig. 2B). In this year, the percent survival of the UVB+ seedlings was statistically equal in the three higher herbicide doses, but lower than for the lowest (140 g a.i. ha–1) dose, whereas for the UVB– seedlings, the percent of survival decreased only with the highest dose.

A UVB effect on average individual aerial biomass was not observed until the second generation (F1) plants (Fig. 3 and 7). Previous studies have shown that UVB may affect the rate of seedling emergence, as well as affecting plant canopy structure (Ballaré et al., 1996; Barnes et al., 1996). The importance of these factors in determining the outcome of intraspecific competition has not yet been determined. However, it is reasonable to think that a delay in the emergence of seedlings and a reduction in growth rate could increase the asymmetry in individual size that occurs as a response to density, which in turn could lead to a greater mortality of the smaller individuals under a UVB+ environment than in the absence of this stress factor.

The effect of the herbicide–UVB light interaction on individual aerial biomass varied between years and among doses. In part, the different responses might be explained by the environmental conditions prevailing during each experiment year, for the periods when seedlings were emerging and herbicide was most lethal for plants. Herbicide was sprayed at the end of June in 1996 but not until the end of July in 1997. The average temperature during June of 1996 was 10°C, with UVB averaging 33.9 J m–2 d–1; during July the temperature remained the same, but UVB radiation dropped to 23.6 (Table 1). During the July–August period of 1997, on the other hand, the average temperature was 12.9°C in the first month, and UVB was 24.4 J m–2 d–1; in August, the average temperature increased by 1.5°C and the UVB radiation level by 10.5 J m–2 d–1 (Table 1). In the 1996 experiment, although no statistical differences were found among treatments, the variation coefficient of the mean for the treatments with the lowest and the highest herbicide doses was lower with UVB light than without it (Fig. 3A). In 1997, the presence of UVB and herbicide interacted to reduce not only the variance of the mean in some dose treatments, but also the biomass at the lower herbicide dose (140 g a.i. ha–1). Moreover, the aerial biomass of individuals receiving the highest herbicide dose (1120 g a.i. ha–1) was greater with UVB light than without it (Fig. 3B). Plant biomass accumulation in response to stress interactions is thus nonlinear. A combination of stress factors or a series of stressful events can reinforce, weaken, mask, or even reverse the response of plants to a single stress factor (Larcher, 1995). Our data suggest that the combined effects of herbicide and UVB stresses will be evident when the herbicide acts as a weak stress factor. In the F1 population subjected to herbicide and UVB treatments in 1997, as the level of one stressor (herbicide concentration, in this case) became so growth-limiting, growth prediction became uncoupled from and thus independent of the other stressor (UVB) (Fig. 7). The effects depended not only on the level of the stressors, but also on the particular ecological scenario (year). Only in 1997, when herbicide selection pressure was low, was there a significant interaction between UVB light and herbicide application that affected both the survival of plants and the biomass of surviving individuals.

The most striking effect of the UVB light–herbicide dose interaction was on the fecundity of individuals in relation to individual biomass and plant density (Fig. 4 and 5). Even though there are examples in the literature indicating increased fecundity per gram biomass in response to other environmental stresses (e.g., water and temperature; Bazzaz, 1996), it is well documented that stresses in general reduce fecundity of individuals (Stanton et al., 2000), as does UVB light alone (Mazza et al., 1999). Similarly, in wild oat (Avena fatua L.), diclofop-methyl not only produces seedling mortality but also reduces plant fecundity (Scursoni et al., 1999). To our knowledge, however, this is the first report on the effects of the UVB light–diclofop-methyl herbicide interaction on individual plant fecundity and plant density responses.

These effects on fecundity may have been related to environmental differences during tiller, flower, and seed production. Temperature and UVB radiation during the October–December period were greater in 1996 than in 1997. These differences may have caused differences in stress levels (Mazza et al., 1999), leading to first year's low average total biomass per plant for surviving plants in the UVB+ treatment (Fig. 3).

The conditions we created in our experiments had significant effects on fecundity of individual plants. This response strongly supports our prediction that the interactions between environmental stresses and herbicide control practices will have evolutionary consequences. The first evidence of such a consequence was the change in the efficacy of the herbicide on the offspring of the 1996 parental plants (Fig. 6). Herbicide application on the parent populations should have eliminated the individuals susceptible to each dose, therefore 100% of survival to the same herbicide dose was expected in the second generation of plants (F1). Even though the percentage of seedling survival increased for all doses in relation to that of the parents (Fig. 2 and 6), variability among doses and between UVB light environments was high. This is especially true for the plants selected under low herbicide doses. The differential selection was probably due to the fact that in the presence of UVB light, only the more herbicide-tolerant phenotypes produced seeds, whereas in the absence of UVB light, the individuals with a wider tolerance range contributed to the seed pool produced by the plants surviving the herbicide doses. No matter what mechanism is responsible, the reduced fecundity found in some ryegrass plants under UVB (Fig. 5) is likely to have a significant effect on the performance of this species in the natural or agricultural environment. As for other annual species, seed output is a major determinant of ryegrass fitness (Newsham et al., 1998). Moreover, ryegrass species are known to have symbiotic fungal endophytes that increase a plant's tolerance to herbicide (Vila Aiub and Ghersa, 2001) but have a negative effect on fecundity if plants are exposed to UVB light (Newsham et al., 1998), increasing the opportunities for interaction and thus for alternative evolutionary pathways.

Selection caused by high doses of herbicide (1120 g a.i. ha–1) should leave only individuals carrying a resistant homozygous genotype, while lower sublethal doses (mainly 140 g a.i. ha–1) should allow the survival of heterozygous and probably a few susceptible homozygous individuals. Since ryegrass is an obligate outbreeder, when there is a low frequency of herbicide-resistant genes in the population, these genes will be present in individuals with heterozygous genotypes because of the swamping effect of susceptible gametes during reproduction (Mettler et al., 1988). In this case, the highest herbicide dose selection should yield no evolved response. Because the highest dose is lethal for heterozygous individuals, all the resistance genes would be eliminated. In our study, because of the dramatic rate of change in herbicide tolerance for all herbicide doses, it becomes apparent that the ryegrass population had a high initial frequency of individuals with resistant genes. Even so, the herbicide-screening test revealed a clear selection response related to herbicide dose, UVB light environment, and year–environment selection history. Because seedling survival was lower for 1996 parental plants than for those in 1997, regardless of herbicide dose or light environment, the 1996 plants showed a greater selection response in all treatments. In the 1996 experiment, when selection pressure was high, UVB stress reduced the selection response, as shown by the slopes calculated for the regression models fitted to the results of the herbicide-screening test (compare slopes for the UVB– and UVB+ results in the 1996 screening, Table 3). This apparently antagonistic effect of UVB on the evolution of herbicide resistance was still evident in the second generation (F2); that is, one more selection cycle with the highest herbicide dose (1120 g a.i. ha–1) under UVB+ did not increase herbicide tolerance (Table 4). In 1997, with much lower selection pressure on the parental plants, the absolute values of the slopes describing the screening dose response were smaller when there was UVB light stress, suggesting that in this case the UVB stress was synergistic with that imposed by the herbicide, thus augmenting the selection response toward herbicide tolerance (Table 3).

In conclusion, our study shows that ambient UVB light was a weak stress factor for the ryegrass plants. However, when the selection pressure caused by herbicide application was low, the two stress factors had a synergistic effect, causing changes in herbicide efficacy that in turn had demographic and evolutionary consequences. The importance of the interactions between these light environments and herbicide doses for the evolution of herbicide-resistant populations may be greater in populations with lower initial resistant gene frequencies or in cases of tolerance conferred by polygenic systems. Research is needed to assess the practical importance of ongoing weed evolution because it is imperative to preserve the efficacy of acceptable weed control methods by defending these techniques against weed adaptation (Jordan and Jannink, 1997). Our findings suggest that interactions between manmade environmental stressors may be important not only for driving herbicide resistance, but also for the evolution of other phenotypic characteristics in weeds and wild vegetation in agroecosystems. In further studies, we aim to elucidate the mechanisms responsible for the responses to such interactions.


   ACKNOWLEDGMENTS
 
This work was funded by grants UBACYT G068 to C.M. Ghersa, University of Buenos Aires and FONCYT 02090 to E.H. Satorre, Agencia Nacional de Promocion Cientifica y Tecnica. We are grateful to Dr. Ruben Piacentini, Scientific Advisor of the WMO/SMN Argentina UV Network, for his assistance with UV data conversion.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This paper was written while M.A. Martínez-Ghersa and C.M. Ghersa were at the Department of Forest Science, Oregon State University, Corvallis, OR.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 


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