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Current status in herbicide resistance in Lolium rigidum in winter cereal fields in Spain: Evolution of resistance 12 years after
Crop Protection 102 (2017) 10e18
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Current status in herbicide resistance in Lolium rigidum in winter cereal fields in Spain: Evolution of resistance 12 years after ~ igo Loureiro a, *, Concepcio n Escorial a, Eva Herna ndez Plaza b, In L. Gonza lez Andújar b, María Cristina Chueca a Jose a b
n Vegetal, Instituto Nacional de Investigacio n y Tecnología Agraria y Alimentaria (INIA), Ctra. La Corun ~ a Km. 7.5, 28040 Madrid, Spain Dpto. Proteccio rdoba, Spain Instituto de Agricultura Sostenible, CSIC, Aptd. 4084, 14080, Co
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 April 2017 Received in revised form 12 July 2017 Accepted 1 August 2017
Lolium rigidum Gaud. is the most prevalent and damaging grass weed of winter cereals in Spain. L. rigidum infestations are frequently treated with herbicides and, consequently, populations have evolved resistance. In 2012e2013 a random survey was conducted across cereal cropping areas of the n and Catalun ~ a regions to establish the distribution and frequency of herbicide resistance in Castilla-Leo L. rigidum populations to chlortoluron (Photosystem II inhibitor), chlorsulfuron (Acetolactate synthase inhibitor) and diclofop-methyl (Acetyl CoA Carboxylase inhibitor), commonly used herbicides for L. rigidum control in Spain. The results of this survey were compared with the results of a previous survey conducted in 2000e02. Resistance to PSII and ALS-inhibiting herbicides was common: 51% and 92% of n and Catalun ~ a respectively were resistant to chlortoluron, the accessions collected from Castilla-Leo while 75% of accessions from both regions were resistant to chlorsulfuron. Resistance to ACCase was ~ a, where 83% of accessions were classified as resistant, than in Castilla-Leo n more widespread in Catalun where 74% of the populations were still classified as susceptible to diclofop-methyl. These results show n since 2000 and to chlortoluron that resistance levels to all three herbicides had increased in Castilla-Leo ~ a. The accessions were also treated with the double dose (2X) of each and chlorsulfuron in Catalun herbicide. The percentage of L. rigidum that now exhibits multiple herbicide resistance has increased ~ a where 75% of the accessions were resistant to multiple herbicides, considerably, especially in Catalun therefore herbicide sustainability and resistance management present a great challenge. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Rigid ryegrass Resistance survey Chlortoluron Chlorsulfuron Diclofop-methyl Resistance mechanisms
1. Introduction The appearance of herbicide resistance is a major concern in agriculture because resistance alters weed control based on herbicides and makes weed control more difficult and more expensive. Weeds adapt to the repeated use of herbicides through the selection of resistance mechanisms that allow the survival of resistant plants. The evolution of herbicide resistance depends on several factors such as the intensity of selection pressure, the biology of weed species and various genetic factors including the frequency of resistance alleles in weed populations, mode of inheritance of resistance and fitness costs associated with resistance. Lolium
* Corresponding author. E-mail addresses: [email protected] (I. Loureiro), [email protected] (C. Escorial), lez [email protected] (E. Hern andez Plaza), [email protected] (J.L. Gonza Andújar), [email protected] (M.C. Chueca). http://dx.doi.org/10.1016/j.cropro.2017.08.001 0261-2194/© 2017 Elsevier Ltd. All rights reserved.
rigidum Gaud. is a native Mediterranean species and is the most important Lolium species in Spain where it is commonly found as a lezmajor weed in winter cereal crops (Recasens et al., 1996; Gonza Andujar and Saavedra, 2003). L. rigidum exhibits several ecological factors such as high genetic variability, plasticity, fecundity and seed survival which have contributed to its success as a major grass weed (Gill, 1996). For years, it has been controlled by various pre- or post-emergence herbicides with different mechanisms of action such as of acetyl CoA carboxylase (ACCase) inhibitors (group A), acetolactate synthase (ALS) inhibitors (group B), and urea/amide photosynthetic inhibitors (group C). HRAC (Herbicide Resistance Action Committee) mode of action (MOA) group classifications are used. The excellent efficacy of these herbicides encouraged their widespread repeated use in several countries. This selection pressure led L. rigidum to become a major problem to evolve resistance to several herbicides (Powles and Yu, 2010). To date, L. rigidum has evolved resistance to 15 herbicide modes of action in 12 countries
I. Loureiro et al. / Crop Protection 102 (2017) 10e18
(Heap, 2017). Surveys conducted in Australia (Owen et al., 2007, 2014; Malone et al., 2014), USA (Rauch et al., 2010) and Spain (Loureiro et al., 2010) have revealed widespread occurrence of herbicide-resistant Lolium species, with biotypes resistant to almost all herbicides available for its control. In Spain, the first cases of failures in L. rigidum control with diclofop-methyl (fop, ACCase inhibitor) and chlortoluron ~a (substituted urea, PS II inhibitor) were cited in fields from Catalun (Recasens et al., 1996), while failures in the control with chlorsuln (de la furon were found in the river Duero region in Castilla-Leo n Carrera et al., 1999). A farmer survey conducted in Castilla-Leo 20 years ago revealed L. rigidum control failures in 4.2% of the fields from the region treated with chlortoluron and in 3.3% of those treated with chlorsulfuron (Fern andez-García, 1998). By that time, chlortoluron, diclofop-methyl and chlorsulfuron herbicides were the most commonly used active ingredients for L. rigidum control in cereals (Taberner, 2001). Previously, a survey of herbicide resistance in L. rigidum conducted in 2000e2002 in these two cereal growing regions (Loureiro et al., 2010) demonstrated that herbicide resistance was not widespread, but there were numerous accessions with certain degree of resistance to chlortoluron (4.7% in n and 10% in Catalun ~ a), chlorsulfuron (10.5% in CastillaCastilla-Leo n and 60% in Catalun ~ a) and also to glyphosate (6.9% in CastillaLeo n). Leo This occurrence of resistance has been increasing as a result of the reliance on the same modes of actions. In 2013, a survey conducted on the use of phytosanitary products in Spain revealed that chlortoluron (260,000 ha), chlorsulfuron (220,000 ha) and diclofop-methyl (550,000 ha) were among the most used active ingredients for grass weed control in cereals (wheat and barley), together with others with the same mode of actions as fenoxapropethyl (group A), metsulfuron-methyl, thifensulfuron-methyl, iodosulfuron-methyl and mesosulfuron-methyl (group B) or isoproturon (group C) (MAPAMA, 2013). Currently there are L. rigidum populations that are resistant to the herbicides containing active ingredients of the groups A, B and C2 (CPRH, 2015). In some cases biotypes with cross- and multiple-resistances have been detected.
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This resistance can be conferred by two mechanisms, mutation(s) in the gene encoding the herbicide target, which decreases the affinity of the target for herbicides (TSR, target-site resistance), or by other mutations that enhance herbicide metabolism and cause a reduction in the amount of herbicide reaching the target (NTSR, lye, 2005, non-target site resistance) (Tranel and Wright, 2002; De 2013). Both mechanisms occur alone or together creating complex genetic linkages for numerous herbicide mode of actions at plant and population level (Petit et al., 2010; Busi et al., 2013). Those situations complicate resistance management and threatens the productivity and sustainability of the cereal farming systems. The problem is even greater considering that no major new site-ofaction herbicide has been introduced into the market for more than 20 years (Duke, 2012). In the present study, a second large-scale random survey of n and L. rigidum across the cereal cropping regions of Castilla-Leo ~ a was conducted to update and quantify the geographical Catalun extent and spectrum of herbicide resistance. 2. Materials and methods 2.1. Plant material Mature seed samples of a total of 89 L. rigidum accessions were collected in field surveys of grass weeds conducted annually between 2012 and 2014. The surveys were conducted randomly across the cereal fields (wheat and barley) of several provinces in n (Avila; n; Palencia; Salamanca; Segovia; Castilla-Leo Burgos; Leo rida (L) in Soria; Valladolid and Zamora) and in the province of Le ~ a, two of the main cereal areas in Spain (Fig. 1). L. rigidum Catalun accessions were collected by driving throughout the regions and stopping at least at 5 km intervals (geo-referenced) to sample the nearest cereal field (Loureiro et al., 2010). A representative random sample of spikes from different plants (25e50) of each accession were harvested at maturity from different field patches. Seeds from all of these plants were manually threshed, bulked and stored for several months before being planted.
n and 12 in Catalun ~ a (in the province of Lleida). The location of each Fig. 1. Geographical origin of the Lolium rigidum populations. 77 populations were collected in Castilla-Leo surveyed population is indicated by a point.
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Table 1 Herbicides, dosages and application time. PRE ¼ Pre-emergence, POST¼ Post-emergence. Herbicide treatment
Application
Dose
Rate (g a.i. ha1)
HRAC Classification
Chlortoluron (Oracle©, chlortoluron 50%) Chlorsulfuron (Glean©, chlorsulfuron 75%) Diclofop-methyl (Iloxan©, diclofop 36%)
PRE
1 2 1 2 1 2
2000 4000 15 30 540 1080
C2 (Photosynthesis inhibitor) B (ALS inhibitor) A (ACCase inhibitor)
PRE POST
2.2. Whole plant herbicide response
2.3. Data analysis
The experiments were carried out in a greenhouse under controlled conditions (24/16 ± 2 C, day/night temperature) and without additional illumination at the INIA experimental station, Madrid, Spain (40 270 North; 3 440 West). Due to the large number of accessions and space limitation the experiments were carried out between November and March to avoid, as much as possible, excessive seasonal differences. Plants were grown in 1 L pots (12 cm diameter) containing manure, sand and soil (1:1:1 by volume) and watered as required. 100 seeds per pot and 5 replicates were used for each herbicide dose. Germination and seedling emergence in the untreated controls was 73 ± 11% and ensured enough seedlings treated for each accession and herbicide dose. Herbicides were applied using a stationary sprayer with one Teejet 8003 flat fan nozzle delivering 225 L ha1 at 200 kPa. Herbicide rates and times of application are shown in Table 1. Chlortoluron, diclofopmethyl and chlorsulfuron were applied at two doses, one is the dose normally recommended under field conditions for L. rigidum control (1X) and the other is the double dose (2X). The pre-emergence (PRE-) treatments were applied 24 h after sowing while post-emergence (POST-) treatment was applied when plants were at the growth stage Z12 (two leaf stage) according to Zadoks et al. (1974). Six weeks after spraying in the case of PRE-treatments and four weeks in POST-treatment, the number of undamaged plants (plants without symptoms after herbicide application and visually similar in aspect and size to the untreated controls) was counted and all plants of each pot were cut at the soil surface and weighted together. Fresh weight data represent the overall response of all individuals that form each accession, which includes dead, damaged and/or undamaged plants, all contributing to the weight. The fresh weight reduction, calculated for each accession as percentage relative to the untreated controls, was used as a measure of the herbicide resistance. The classification of the L. rigidum accessions in response to the applied herbicides was done according to the “R” resistance rating system described by Moss et al. (1999). If fresh weight reduction was: less than 40%, the accessions were classified as RRR ¼ resistance confirmed, highly likely to reduce herbicide performance; from 40% to 80%, as RR ¼ resistance confirmed, probably reducing herbicide performance; from 80% to 90%, as R? ¼ early indications that resistance may be developing, possibly reducing herbicide performance; more than 90%, as S ¼ susceptible. For chlorsulfuron, the percentage reduction fresh weight value for the susceptible biotype used as control in all experiments was 92%, while for chlortoluron and diclofop-methyl this value was 99%. The percentage of undamaged plants was also calculated for each pot; in the case of PRE-emergence herbicides, the percentage of undamaged plants was calculated relative to the number of seeds emerging in the untreated controls, while in POSTherbicides it was calculated relative to the emerged plants before treatment.
The effect of the accession origin in the response to herbicide applications was tested using ANOVA (Stat Graphics 5.1). Data on the percentage of fresh weight reduction at single dose treatment were square-root transformed (√x/100) prior to the analysis and analysed separately for each herbicide. Correlations between fresh weight and undamaged plants percentages after herbicide applications were analysed using Pearson's correlation coefficient. In order to test the distance relationship between L. rigidum accessions a spatial autocorrelation analyses were undertaken within each region for the response to the three herbicides, both for fresh weight and frequency of undamaged plants. The Moran index was used to estimate the degree of spatial autocorrelation at all sites within the study area. Distance classes of geographical and effective distances between accessions were created following Sturge's rule. The overall significance of the individual correlograms was evaluated using Bonferroni's correction. The Moran's I statistic for spatial autocorrelation is given as:
Pn IðdÞ ¼ n
i¼1
wij ðdÞðyi yÞ yj y P WðdÞ ni¼1 ðyi yÞ2
Pn
j¼1
where n is equal to the total number of considered sampling units, y is the value of the variable in the points i and j (i s j), wij (d) is the spatial weight matrix between i and j within the distance class d and W(d) is the aggregate of all the spatial weights matrix. The Moran's I values range from þ1.0 (perfect positive spatial autocorrelation) and - 1.0 (perfect negative spatial autocorrelation). The value 0 implies perfect spatial randomness. The statistical software package used was PASSAGE v.2 (2.0.8.20) (Rosenberg and Anderson, 2011). 3. Results Figs. 2e4 show the response of each accession to chlortoluron (Fig. 2), chlorsulfuron (Fig. 3) and diclofop-methyl (Fig. 4). The accessions were grouped by region, firstly they were sorted by decreasing fresh weight at 1X dose and secondly by decreasing percentage of undamaged plants. 3.1. Chlortoluron n after preFresh weight of the 77 accessions from Castilla-Leo emergence application of chlortoluron at 2 kg a.i. ha1, a dose normally applied under field conditions, varied from 1% in the most susceptible accession to 79% in the most resistant, with an average fresh weight of 28% and a median fresh weight of 24% (Fig. 2A). Approximately 80% of the accessions contained undamaged plants with percentages ranging 0.2e61% and an average of 9.4%. These frequencies of undamaged plants were positively correlated with the fresh weight values (r ¼ 0.75). In the accessions from ~ a treated with the same dose, fresh weight values varied Catalun
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Fig. 2. Effect of pre-emergence application of chlortoluron on L. rigidum populations. Grey bars represent the undamaged plants while black points represent the fresh weight biomass, both expressed as a percent of control.
from 10% to 80% and averaged 61% (median of 72%). All of them contained undamaged plants averaging 34% (2e70%), approxi n. mately four times more than those accessions from Castilla-Leo This parameter was positively correlated with fresh weight (r ¼ 0.82). When chlortoluron dose was doubled, average fresh weight values varied from 0.2% to 56% and average decreased to 8% n, while they var(median 6%) for the accessions from Castilla-Leo ied from 2 to 71% and averaged 35% (median 33%) for those from ~ a (Fig. 2B). The average frequency of undamaged plants Catalun decreased to 2.9% (0.2e37%) and to 20% (0.4e53%) in each region, respectively. 3.2. Chlorsulfuron n treated with Fresh weight of the accessions from Castilla-Leo chlorsulfuron at 15 g a.i. ha1 ranged from 8% to 100% of the cor~ a, responding controls, and from 24% to 100% for those from Catalun and undamaged plants were present in 75% and 87% of the accessions, respectively in each region (Fig. 3A). The average values for n and Catalun ~ a, 47% and fresh weight were similar for Castilla-Leo 51% respectively (medians of 38 and 41%) and 17% for undamaged plants. The frequencies of undamaged plants were positively
correlated with the fresh weight values obtained at 1X (r ¼ 0.84). At 2X, average fresh weight decreased but they remained at high level: 35% value for both regions, with medians of 21% and 16% for Casn and Catalun ~ a respectively (Fig. 3B). The percentages of tilla-Leo undamaged plants also decreased to average values of 15% n (0.2e98%) and 10% (2e43%) for the accessions from Castilla-Leo ~ a, respectively. and Catalun 3.3. Diclofop-methyl Diclofop-methyl provided a good control of most of the accesn (Fig. 4A). After post-emergence application sions from Castilla-Leo of diclofop-methyl at 540 g a.i.ha1, average values were low, 10% fresh weight (median 5%) and 1.5% for undamaged plants (Fig. 4A). ~ a, 37% for On the other hand, these values were higher in Catalun fresh weight with a median of 42%, and 17% for undamaged plants (Fig. 4A). A similar trend was observed between the responses of the accessions from different origin when dose was doubled (Fig. 4B). The correlation between both values for diclofop-methyl applied at full rate was weaker than for the previous herbicides (r ¼ 0.63 for n and r ¼ 0.69 for those from Catalun ~ a). accessions from Castilla-Leo
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Fig. 3. Effect of post-emergence application of chlorsulfuron on L. rigidum populations. Grey bars represent the undamaged plants while black points represent the fresh weight biomass, both expressed as a percent of control.
3.4. Classification of the L. rigidum accessions in response to applied herbicides Fig. 5 shows the classification of the L. rigidum accessions in response to applied herbicides according to Moss et al. (1999). At n and present, only 33% of the accessions surveyed in Castilla-Leo screened with the 1X dose of chlortoluron were classified as S. Resistance was confirmed in 51% (39) of the accessions (RRR þ RR) (Fig. 5A). At 2X, 76% of the accessions were susceptible and only 9% ~ a, the behaviour of resiswere classified as RR (Fig. 5B). In Catalun tance was different; resistance was confirmed in 92% of the accessions at 1X (67% RRR and 25% RR, Fig. 5A) while 75% accession remained resistant when the herbicide dose was doubled (Fig. 5B). A significant effect of the province of origin was found on fresh weight reduction data (F9,177 ¼ 5.80, P < 0.001), indicating rida geographic variation in the level of herbicide resistance. Le ~ a) was the province with higher levels of chlortoluron (Catalun resistance, followed by Salamanca, Zamora and Valladolid (Castillan). Leo n were classified as Only 10% of the accessions from Castilla-Leo S when chlorsulfuron was applied at 1X (Fig. 5C), while 75% of the accessions were resistant (RRR þ RR). No S accessions were found
n. When dose was in four surveyed provinces of Castilla-Leo doubled, S accessions increased to 44% (Fig. 5D). There were differences in the response among provinces (F9,88 ¼ 4.41, P < 0.001): Salamanca was the most affected province by chlorsulfuron resis ~ a, no S actance, followed by Avila, Zamora and Soria. In Catalun cessions were detected at 1X. When dose was doubled, while RRR accessions were the same as 1X, S accessions increased to 58% (Fig. 5D). n For diclofop-methyl, 74% of the accessions from Castilla-Leo were classified as S at 1X. There are four provinces with no resistant (RRR or RR) accessions and other provinces as Valladolid, Salan or Zamora which showed up to 37% of manca, Palencia, Leo ~ a, the resistance level resistant accessions (RR) (Fig. 5E). In Catalun to diclofop-methyl was higher, with 83% of the screened accessions being resistant (8% RRR and 75% RR) and only 8% S when the single dose of this herbicide was applied (Fig. 5E). When dose was doubled (Fig. 5F) the percentage of resistant accessions was of 4% n and Catalun ~ a, respectively. and 75% in Castilla-Leo 3.5. Multiple resistance n, 29% exhibited multiple Of the 77 accessions from Castilla-Leo
I. Loureiro et al. / Crop Protection 102 (2017) 10e18
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Fig. 4. Effect of post-emergence application of diclofop-methyl on L. rigidum populations. Grey bars represent the undamaged plants while black points represent the fresh weight biomass, both expressed as a percent of control.
resistance to at least one herbicide within all three groups tested: 25% to any two of the three mode of actions employed and 4% to all of them. The multiple resistance pattern most commonly found was to chlortoluron and chlorsulfuron herbicides, present in almost 19% of the accessions. Multiple resistance levels were higher in ~ a, where 75% of the accessions being multiple resistant. In Catalun this region, 25% of accessions were resistant to the three mode of actions; resistance to chlortoluron and chlorsulfuron was observed in 25% of the accessions while 17% were resistant to chlorsulfuron and diclofop-methyl.
sampling points of 461 km, resulting in a correlogram with 12 distance classes, there was significant spatial autocorrelation (P < 0.001) for the fresh weight variable at normal dose in five ~ a, using distance classes of 3 km distance classes, while in Catalun and a maximum distance between two sampling points of 22 km (7 distance classes), there was significant spatial autocorrelation (P < 0.001) in two distance classes. This indicate different significant hotspots of chlorsulfuron resistance on a local scale, meaning that accessions within site are clustered spatially for their level of resistance. Results were similar when the frequency of undamaged plants was used.
3.6. Autocorrelation analysis Since every accession has been mapped, autocorrelograms within each region have been carried out for the frequency of undamaged plants and for the fresh weight to test the relationship between accessions related to distance. For chlortoluron and diclofop the absence of significant correlograms indicates, in principle, the existence of a random distribution of resistance. Significant spatial autocorrelation was detected only in response to n, using chlorsulfuron in the two evaluated areas. In Castilla-Leo distance classes of 38 km with a maximum distance between two
3.7. Increase in herbicide resistance incidence between 2000e02 and 2012-14 As is shown in Table 2 there is a substantial increase on the resistance levels for the studied herbicides related to 2000-02 n, most accessions were survey (Loureiro et al., 2010). In Castilla-Leo classified as S to herbicides chlortoluron (95.3%) and chlorsulfuron (89.5%) ten years ago. At present, these percentages have been reduced considerably and resistance to both herbicides has increased, only 33% and 10% of the accessions remain S to these
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n (C-L, 77 populations) and Catalun ~ a (province of Le rida, 12 populations) in Fig. 5. Classification of the Lolium rigidum accessions collected in several provinces of Castilla-Leo response to the application of herbicides according to the “R” resistance rating system for designating resistance in screening assays described by Moss et al. (1999).
herbicides, respectively. While any resistance was found for diclofop-methyl in the past, nowadays 26% of the accessions show some level of resistance. In 2000e02, the highest resistance levels were found in Cata~ a: chlortoluron resistance was present in 10% of the accessions lun
and chlorsulfuron resistance was widespread with 60% of the accessions displaying resistance (40% RR and 20% R?). Currently, chlortoluron resistance is almost ten times greater (67% of the accessions RRR and 25% RR) and for chlorsulfuron, all accessions showed resistance: 33% RRR and a further 67% RR and R?, Table 2).
I. Loureiro et al. / Crop Protection 102 (2017) 10e18
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Table 2 n and Catalun ~ a in Change in herbicide resistance status between 2000-2002 and 2012e2014 surveys of Lolium rigidum populations collected from the same areas of Castilla-Leo response to the field dose of the herbicides chlortoluron, chlorsulfuron and diclofop-methyl. Region
Herbicide
Accessions (%) RRR
n Castilla-Leo
~a Catalun
Chlortoluron Chlorsulfuron Diclofop-methyl Chlortoluron Chlorsulfuron Diclofop-methyl
RR
R?
S
2000
2012
2000
2012
2000
2012
2000
2012
0.0 1.2 0.0 0.0 0.0 e
7.6 27.1 0.0 66.7 33.3 8.3
1.2 3.5 0.0 10.0 40.0 e
43.4 47.5 12.6 25.0 41.7 75.0
3.5 5.8 0.0 0.0 20.0 e
15.9 15.6 13.4 0.0 25.0 8.3
95.3 89.5 100.0 90.0 40.0 e
33.1 9.7 74.0 8.3 0.0 8.3
4. Discussion n, in the 12e14 The current survey show that, in Castilla-Leo years since the last random survey was conducted, the resistance status of L. rigidum has changed from very low level of resistance (0e5%) to high levels of resistance (RRR and RR) (13e74%). The highest percentages of resistant L. rigidum accessions were found in cereal fields of Salamanca, Zamora or Palencia provinces. Under semiarid climate, cereal yields are medium-low which do not allow farmers for a major input on chemicals, so preference is common for the least expensive effective herbicides. In this situation, with few alternatives in the crop rotation and in the availability of herbicides, pre-emergence herbicides are commonly applied. These facts explains the higher incidence of resistance to chlortoluron and chlorsulfuron herbicides than to diclofop-methyl, applied at post emergence. All the screened accessions from Avila, Soria and Segovia provinces were susceptible to diclofop-methyl, so in these areas diclofop-methyl may be included as one of the management options for control of the resistant biotypes to the other herbicides. ~ a, diclofop-methyl response was not analyzed in our In Catalun previous survey although problems of resistance were known (Taberner, 2005). At present, 83% of the screened accessions show diclofop-methyl resistance, while resistant accessions have increased from 10% to 92% for chlortoluron and from 40% to 75% for chlorsulfuron. In this region, cereal yields are higher and thus profits, with a greater margin for the use of herbicides imposing a higher selection pressure. For both regions the cost effectiveness of the studied herbicides and their broad-spectrum weed control that include, L. rigidum and other grasses such as Bromus spp. and Avena spp. (Escorial et al., 2011) or even some common annual dicotyledonous species (Papaver rhoeas, Polygonunm aviculare, Salsola kali or Matricaria spp.), promoted their widespread use alone or in mixtures. Therefore, they contributed to the selection of resistant biotypes and to the spread of resistance, which is faster in genetically diverse and cross-pollinated weeds, such as species of the Lolium genera. This increase in the frequency of resistance has occurred in Australia, where L. rigidum resistance was firstly reported to ACCase and ALS inhibiting herbicides (Heap and Knight, 1982, 1986) and has been intensively monitored. In the 1998 survey, 50e60% of the L. rigidum accessions from the randomly surveyed zones were classified as resistant or developing resistance to ACCase and ALS inhibiting herbicides (Llewellyn and Powles, 2001), while over the subsequent 5-year period it increased to 70e90% (Owen et al., 2007), and after 12 years to > 95% (Owen et al., 2014). L. multiflorum Lam. (Italian ryegrass), a close relative L. rigidum, is also a weed management problem in different cropping areas of North America, with populations resistant to many mode of action and some of them exhibiting cross- and/or multiple herbicide resistance (Chandi et al., 2011; Hulting et al., 2012; Liu et al., 2014). Studies conducted in the Pacific Northwest of the USA indicated
that 73% of L. multiflorum accessions were resistant to the aryloxyphenoxypropionate herbicides as diclofop-methyl and 39% to the sulfonylurea herbicides as chlorsulfuron (Rauch et al., 2010). This species is also a problematic resistant grass weed in cereals in many European countries including Germany, Denmark or Italy (Sattin, 2005) and in the United Kingdom, where 70% of the Lolium spp. populations were resistant to at least one herbicide (Hull et al., 2014). Herbicide applications are designed by growers at the level of each individual field. As a consequence, herbicide selective pressure varies among agricultural fields and regions so local evolution of resistance is expected. Our results show special spatial correlation for chlorsulfuron resistance suggesting that these accessions were likely exposed to similar herbicide regimes and/or are close enough for gene flow. Either scenario is equally plausible: the frequent use of a similar herbicide regime to control L. rigidum in different fields and/or resistance alleles being dispersed through either pollen or seed, and this is of utmost importance mainly in accessions harbouring plants with TSR, as could be the case for this herbicide. In a previously conducted study on pollen mediated gene flow in L. rigidum under semi-arid conditions, 0.1% cross-pollination rate would be predicted at 100 m (Loureiro et al., 2016), which delivers to an initial frequency of 1 herbicide-resistant individual in 1000 S seeds, a rate higher than the spontaneous mutation rate in weed populations which is generally assumed to be about 106 (Maxwell and Mortimer, 1994). Busi et al. (2008) reported that pollenmediated gene flow in L. rigidum can occur up to 3 km from the nearest pollen source. Although resistance development in every accession is a unique case, these spatial scales are of great relevance for resistance spread and management, which should consider more than just the single resistant accession. From the data presented and taking into account knowledge from literature, we show that herbicide resistance in L. rigidum evaluated by random surveys has increased significantly in 12 years in cereal crops in Spain. Further studies are being conducted to ascertain the resistance mechanisms present in the resistant accessions. The resistance mechanisms for the three studied herbicides in this species could be determined as being TSR and NTSR (Han et al., 2016). Both types of resistance, could be present in one or many populations, and sometimes accompanied by multiple and cross-resistance (Beckie and Tardif, 2012). In addition, spatial correlation, irrespective of its origin that could be similar herbicide selection pressures or resistance dispersal by gene flow, was evidenced in this study for chlorsulfuron treated accessions. This worrying scenario shows that the resistance management is becoming increasingly complex. Nevertheless other active ingredients and non-chemical control measures are available for resistant L. rigidum control that could be used in an integrated weed management. This situation needs to be addressed quickly and efficiently to avoid this scenario to turn dramatic, if no adequate measures are adopted.
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Acknowledgements This work was funded by the Ministerio de Economía y Competitividad (MINECO) of Spain, project AGL2011-23542. We thank to Zeus Hornos del Monte and Ana Mateo for technical assistance. References Beckie, H.J., Tardif, F.J., 2012. Herbicide cross resistance in weeds. Crop Prot. 35, 15e28. Busi, R., Yu, Q., Barret-Lennard, R., Powles, S., 2008. Long distance pollen-mediated flow of herbicide resistance genes in Lolium rigidum. Theor. Appl. Genet. 117, 1281e1290. Busi, R., Vila-Aiub, M., Beckie, H.J., Gaines, T.A., Goggin, D., Kaundun, S.S., et al., 2013. Herbicide-resistant weeds: from research and knowledge to future needs. Evol. Appl. 6, 1218e1221. Chandi, A., York, A.C., Jordan, D.L., Beam, J.B., 2011. Resistance to acetolactate synthase and acetyl Co-A carboxylase inhibitors in North Carolina Italian ryegrass (Lolium perenne). Weed Technol. 25, 659e666. para la Prevencio n de la Resistencia a Herbicidas. http://semh. CPRH, 2015. Comite net/wp-content/uploads/2016/01/Ficha-Lolium-CPRH-definitiva.pdf. (Accessed 29 March 2017). de la Carrera, N., Villarroya, M., Chueca, M.C., García-Baudín, J.M., 1999. Evaluation of Lolium rigidum biotypes resistance to chlorsulfuron: useful parameters. Proc. Brighton Crop Prot. Conf. Weeds 153e158. lye, C., 2013. Unravelling the genetic bases of non-target-site-based resistance De (NTSR) to herbicides: a major challenge for weed science in the forthcoming decade. Pest. Manag. Sci. 69, 176e187. lye, C., 2005. Weed resistance to acetyl coenzyme A carboxylase inhibitors: an De update. Weed Sci. 53, 728e746. Duke, S.O., 2012. Why have no new herbicide modes of action appeared in recent years? Pest. Manag. Sci. 68, 505e512. Escorial, C., Loureiro, I., Rodríguez-García, E., Chueca, C., 2011. Population variability in the response of ripgut brome (Bromus diandrus) to sulfosulfuron and glyphosate herbicides. Weed Sci. 59, 107e112. ndez-García, J.C., 1998. Problem Ferna atica de las malas hierbas y el empleo de n. MS Thesis. Universidad de Leo n, herbicidas en los cereales paja de Castilla-Leo Leon. Spain. Gill, G.S., 1996. Why annual ryegrass is a problem in Australian agriculture. Plant Prot. Quart. 11, 193e194. lez-Andujar, J.L., Saavedra, M., 2003. Spatial distribution of annual grass weed Gonza populations in winter cereals. Crop Prot. 22, 629e633. Han, H., Yu, Q., Owen, M.J., Cawthray, G.R., Powles, S.B., 2016. Widespread occurrence of both metabolic and target-site herbicide resistance mechanisms in Lolium rigidum populations. Pest Manag. Sci. 72, 255e263. Heap, I., 2017. International survey of herbicide resistant weeds. www.weedscience. com. (Accessed 29 March 2017). Heap, I., Knight, R., 1982. A population of ryegrass tolerant to the herbicide diclofopmethyl. J. Aust. Inst. Agric. Sci. 48, 156e157. Heap, I., Knight, R., 1986. The occurrence of herbicide cross-resistance in a population of annual ryegrass, Lolium rigidum, resistant to diclofop-methyl. Aust. J. Agric. Res. 37, 149e156. Hull, R., Tatnell, L.V., Cook, S.K., Beffa, R., Moss, S.R., 2014. Current status of herbicide-resistant weeds in the UK. Asp. Appl. Biol. 127, 261e272. Hulting, A.G., Dauer, J.T., Hinds-Cook, B., Curtis, D., Koepke-Hill, R.M., MallorySmith, C., 2012. Management of Italian ryegrass (Lolium perenne ssp. multiflorum) in western Oregon with preemergence applications of pyroxasulfone in winter wheat. Weed Technol. 26, 230e235.
Liu, M., Hulting, A.G., Mallory-Smith, C.A., 2014. Characterization of multipleherbicide-resistant Italian ryegrass (Lolium perenne spp. multiflorum). Pest Manag. Sci. 70, 1145e1150. Llewellyn, R., Powles, S.B., 2001. High levels of herbicide resistance in rigid ryegrass (Lolium rigidum) across the western Australian wheat belt. Weed Technol. 15, 242e248. Loureiro, I., Escorial, M.C., Chueca, M.C., 2016. Pollen-mediated movement of herbicide resistance genes in Lolium rigidum. PLoS One 11 (6), e0157892. http:// dx.doi.org/10.1371/journal.pone.0157892. Loureiro, I., Rodriguez-García, E., Escorial, M.C., García-Baudin, J.M., Gonz alezAndújar, J.L., Chueca, M.C., 2010. Distribution and frequency of resistance to four herbicide modes of action in Lolium rigidum Gaud. accessions randomly collected in winter cereal fields in Spain. Crop Prot. 29, 1248e1256. Malone, J.M., Boutsalis, P., Baker, J., Preston, C., 2014. Distribution of herbicideresistant acetyl-coenzyme A carboxylase alleles in Lolium rigidum across rain cropping areas of South Australia. Weed Res. 54, 78e86. n y Medio Ambiente), MAPAMA (Ministerio de Agricultura y Pesca, Alimentacio n de Productos Fitosanitarios. http://www.mapama. 2013. Encuesta de Utilizacio gob.es/es/estadistica/temas/estadisticas-agrarias/agricultura/estadisticasmedios-produccion/fitosanitarios.aspx#para4. (Accessed 29 March 2017). Maxwell, B.D., Mortimer, A.M., 1994. Selection for herbicide resistance. In: Powles, S.B., Holtum, J.A.M. (Eds.), Herbicide Resistance in Plants: Biology and Biochemistry. CRC Press Inc., Baton Rouge, USA, pp. 1e25. Moss, S.R., Clarke, J.H., Blair, A.M., Culley, T.N., Read, M.A., Ryan, R.J., Turner, M., 1999. The occurrence of herbicide-resistant grass-weeds in the United Kingdom and a new system for designating resistance in screening assays. In: BCPC (Ed.), The Brighton Conference - Weeds, England, pp. 179e184. Owen, M., Walsh, M., Llewellyn, R., Powles, S.B., 2007. Widespread occurrence of multiple herbicide resistance in Western Australian annual ryegrass (Lolium rigidum) populations. Aust. J. Agric. Res. 58, 711e718. Owen, M.J., Martinez, N.J., Powles, S.B., 2014. Multiple herbicide-resistant Lolium rigidum (annual ryegrass) now dominates across the Western Australia grain belt. Weed Res. 54, 314e324. lye, C., 2010. Complex genetic control of nonPetit, C., Duhieu, B., Boucansaud, K., De target-site-based resistance to herbicides inhibiting acetyl-coenzyme A carboxylase and acetolactate-synthase in Alopecurus myosuroides Huds. Plant Sci. 178, 501e509. Powles, S.B., Yu, Q., 2010. Evolution in action: plants resistant to herbicides. Ann. Rev. Plant Biol. 61, 317e347. Rosenberg, M.S., Anderson, C.D., 2011. PASSaGE: pattern analysis, spatial statistics and geographic exegesis. Version 2 Methods Ecol. Evol. 2, 229e232. Rauch, T.A., Thill, D.C., Gersdorf, S.A., Price, W.J., 2010. Widespread occurrence of herbicide-resistant Italian ryegrass (Lolium multiflorum) in Northern Idaho and Eastern Washington. Weed Technol. 24, 281e288. Recasens, J., Riba, F., Izquierdo, J., Forn, R., Taberner, A., 1996. Gramíneas infestantes ~ a. I.T.E.A 2, 116e130. de los cereales de invierno de Catalun Sattin, M., 2005. Herbicide Resistance in Europe: an Overview. Proc. British Crop Production Council International Conference Crop Science & Technology, Glasgow, UK, pp. 131e138, 1236 pp. Taberner, A., 2001. Biología de Lolium rigidum Gaud. como planta infestante del n al establecimiento de me todos de control. MS cultivo de cebada. Aplicacio rida, Le rida. Spain. Thesis. Universidad de Le Taberner, A., 2005. Estado de las resistencias de las malas hierbas a los herbicidas en ~ a. La actividad del Comite de Prevencio n de las Resistencias a Herbicidas, Espan ~ a 173, 92e96. CPRH. Phytoma Espan Tranel, P.J., Wright, T.R., 2002. Resistance of weeds to ALS-inhibiting herbicides: what have we learned? Weed Sci. 50, 700e712. Zadoks, J.C., Chang, T.T., Konzak, C.F., 1974. A decimal code for the growth stages of cereals. Weed Res. 14, 415e421.
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Current status in herbicide resistance in Lolium rigidum in winter cereal fields in Spain: Evolution of resistance 12 years after ~ igo Loureiro a, *, Concepcio n Escorial a, Eva Herna ndez Plaza b, In L. Gonza lez Andújar b, María Cristina Chueca a Jose a b
n Vegetal, Instituto Nacional de Investigacio n y Tecnología Agraria y Alimentaria (INIA), Ctra. La Corun ~ a Km. 7.5, 28040 Madrid, Spain Dpto. Proteccio rdoba, Spain Instituto de Agricultura Sostenible, CSIC, Aptd. 4084, 14080, Co
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 April 2017 Received in revised form 12 July 2017 Accepted 1 August 2017
Lolium rigidum Gaud. is the most prevalent and damaging grass weed of winter cereals in Spain. L. rigidum infestations are frequently treated with herbicides and, consequently, populations have evolved resistance. In 2012e2013 a random survey was conducted across cereal cropping areas of the n and Catalun ~ a regions to establish the distribution and frequency of herbicide resistance in Castilla-Leo L. rigidum populations to chlortoluron (Photosystem II inhibitor), chlorsulfuron (Acetolactate synthase inhibitor) and diclofop-methyl (Acetyl CoA Carboxylase inhibitor), commonly used herbicides for L. rigidum control in Spain. The results of this survey were compared with the results of a previous survey conducted in 2000e02. Resistance to PSII and ALS-inhibiting herbicides was common: 51% and 92% of n and Catalun ~ a respectively were resistant to chlortoluron, the accessions collected from Castilla-Leo while 75% of accessions from both regions were resistant to chlorsulfuron. Resistance to ACCase was ~ a, where 83% of accessions were classified as resistant, than in Castilla-Leo n more widespread in Catalun where 74% of the populations were still classified as susceptible to diclofop-methyl. These results show n since 2000 and to chlortoluron that resistance levels to all three herbicides had increased in Castilla-Leo ~ a. The accessions were also treated with the double dose (2X) of each and chlorsulfuron in Catalun herbicide. The percentage of L. rigidum that now exhibits multiple herbicide resistance has increased ~ a where 75% of the accessions were resistant to multiple herbicides, considerably, especially in Catalun therefore herbicide sustainability and resistance management present a great challenge. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Rigid ryegrass Resistance survey Chlortoluron Chlorsulfuron Diclofop-methyl Resistance mechanisms
1. Introduction The appearance of herbicide resistance is a major concern in agriculture because resistance alters weed control based on herbicides and makes weed control more difficult and more expensive. Weeds adapt to the repeated use of herbicides through the selection of resistance mechanisms that allow the survival of resistant plants. The evolution of herbicide resistance depends on several factors such as the intensity of selection pressure, the biology of weed species and various genetic factors including the frequency of resistance alleles in weed populations, mode of inheritance of resistance and fitness costs associated with resistance. Lolium
* Corresponding author. E-mail addresses: [email protected] (I. Loureiro), [email protected] (C. Escorial), lez [email protected] (E. Hern andez Plaza), [email protected] (J.L. Gonza Andújar), [email protected] (M.C. Chueca). http://dx.doi.org/10.1016/j.cropro.2017.08.001 0261-2194/© 2017 Elsevier Ltd. All rights reserved.
rigidum Gaud. is a native Mediterranean species and is the most important Lolium species in Spain where it is commonly found as a lezmajor weed in winter cereal crops (Recasens et al., 1996; Gonza Andujar and Saavedra, 2003). L. rigidum exhibits several ecological factors such as high genetic variability, plasticity, fecundity and seed survival which have contributed to its success as a major grass weed (Gill, 1996). For years, it has been controlled by various pre- or post-emergence herbicides with different mechanisms of action such as of acetyl CoA carboxylase (ACCase) inhibitors (group A), acetolactate synthase (ALS) inhibitors (group B), and urea/amide photosynthetic inhibitors (group C). HRAC (Herbicide Resistance Action Committee) mode of action (MOA) group classifications are used. The excellent efficacy of these herbicides encouraged their widespread repeated use in several countries. This selection pressure led L. rigidum to become a major problem to evolve resistance to several herbicides (Powles and Yu, 2010). To date, L. rigidum has evolved resistance to 15 herbicide modes of action in 12 countries
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(Heap, 2017). Surveys conducted in Australia (Owen et al., 2007, 2014; Malone et al., 2014), USA (Rauch et al., 2010) and Spain (Loureiro et al., 2010) have revealed widespread occurrence of herbicide-resistant Lolium species, with biotypes resistant to almost all herbicides available for its control. In Spain, the first cases of failures in L. rigidum control with diclofop-methyl (fop, ACCase inhibitor) and chlortoluron ~a (substituted urea, PS II inhibitor) were cited in fields from Catalun (Recasens et al., 1996), while failures in the control with chlorsuln (de la furon were found in the river Duero region in Castilla-Leo n Carrera et al., 1999). A farmer survey conducted in Castilla-Leo 20 years ago revealed L. rigidum control failures in 4.2% of the fields from the region treated with chlortoluron and in 3.3% of those treated with chlorsulfuron (Fern andez-García, 1998). By that time, chlortoluron, diclofop-methyl and chlorsulfuron herbicides were the most commonly used active ingredients for L. rigidum control in cereals (Taberner, 2001). Previously, a survey of herbicide resistance in L. rigidum conducted in 2000e2002 in these two cereal growing regions (Loureiro et al., 2010) demonstrated that herbicide resistance was not widespread, but there were numerous accessions with certain degree of resistance to chlortoluron (4.7% in n and 10% in Catalun ~ a), chlorsulfuron (10.5% in CastillaCastilla-Leo n and 60% in Catalun ~ a) and also to glyphosate (6.9% in CastillaLeo n). Leo This occurrence of resistance has been increasing as a result of the reliance on the same modes of actions. In 2013, a survey conducted on the use of phytosanitary products in Spain revealed that chlortoluron (260,000 ha), chlorsulfuron (220,000 ha) and diclofop-methyl (550,000 ha) were among the most used active ingredients for grass weed control in cereals (wheat and barley), together with others with the same mode of actions as fenoxapropethyl (group A), metsulfuron-methyl, thifensulfuron-methyl, iodosulfuron-methyl and mesosulfuron-methyl (group B) or isoproturon (group C) (MAPAMA, 2013). Currently there are L. rigidum populations that are resistant to the herbicides containing active ingredients of the groups A, B and C2 (CPRH, 2015). In some cases biotypes with cross- and multiple-resistances have been detected.
11
This resistance can be conferred by two mechanisms, mutation(s) in the gene encoding the herbicide target, which decreases the affinity of the target for herbicides (TSR, target-site resistance), or by other mutations that enhance herbicide metabolism and cause a reduction in the amount of herbicide reaching the target (NTSR, lye, 2005, non-target site resistance) (Tranel and Wright, 2002; De 2013). Both mechanisms occur alone or together creating complex genetic linkages for numerous herbicide mode of actions at plant and population level (Petit et al., 2010; Busi et al., 2013). Those situations complicate resistance management and threatens the productivity and sustainability of the cereal farming systems. The problem is even greater considering that no major new site-ofaction herbicide has been introduced into the market for more than 20 years (Duke, 2012). In the present study, a second large-scale random survey of n and L. rigidum across the cereal cropping regions of Castilla-Leo ~ a was conducted to update and quantify the geographical Catalun extent and spectrum of herbicide resistance. 2. Materials and methods 2.1. Plant material Mature seed samples of a total of 89 L. rigidum accessions were collected in field surveys of grass weeds conducted annually between 2012 and 2014. The surveys were conducted randomly across the cereal fields (wheat and barley) of several provinces in n (Avila; n; Palencia; Salamanca; Segovia; Castilla-Leo Burgos; Leo rida (L) in Soria; Valladolid and Zamora) and in the province of Le ~ a, two of the main cereal areas in Spain (Fig. 1). L. rigidum Catalun accessions were collected by driving throughout the regions and stopping at least at 5 km intervals (geo-referenced) to sample the nearest cereal field (Loureiro et al., 2010). A representative random sample of spikes from different plants (25e50) of each accession were harvested at maturity from different field patches. Seeds from all of these plants were manually threshed, bulked and stored for several months before being planted.
n and 12 in Catalun ~ a (in the province of Lleida). The location of each Fig. 1. Geographical origin of the Lolium rigidum populations. 77 populations were collected in Castilla-Leo surveyed population is indicated by a point.
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Table 1 Herbicides, dosages and application time. PRE ¼ Pre-emergence, POST¼ Post-emergence. Herbicide treatment
Application
Dose
Rate (g a.i. ha1)
HRAC Classification
Chlortoluron (Oracle©, chlortoluron 50%) Chlorsulfuron (Glean©, chlorsulfuron 75%) Diclofop-methyl (Iloxan©, diclofop 36%)
PRE
1 2 1 2 1 2
2000 4000 15 30 540 1080
C2 (Photosynthesis inhibitor) B (ALS inhibitor) A (ACCase inhibitor)
PRE POST
2.2. Whole plant herbicide response
2.3. Data analysis
The experiments were carried out in a greenhouse under controlled conditions (24/16 ± 2 C, day/night temperature) and without additional illumination at the INIA experimental station, Madrid, Spain (40 270 North; 3 440 West). Due to the large number of accessions and space limitation the experiments were carried out between November and March to avoid, as much as possible, excessive seasonal differences. Plants were grown in 1 L pots (12 cm diameter) containing manure, sand and soil (1:1:1 by volume) and watered as required. 100 seeds per pot and 5 replicates were used for each herbicide dose. Germination and seedling emergence in the untreated controls was 73 ± 11% and ensured enough seedlings treated for each accession and herbicide dose. Herbicides were applied using a stationary sprayer with one Teejet 8003 flat fan nozzle delivering 225 L ha1 at 200 kPa. Herbicide rates and times of application are shown in Table 1. Chlortoluron, diclofopmethyl and chlorsulfuron were applied at two doses, one is the dose normally recommended under field conditions for L. rigidum control (1X) and the other is the double dose (2X). The pre-emergence (PRE-) treatments were applied 24 h after sowing while post-emergence (POST-) treatment was applied when plants were at the growth stage Z12 (two leaf stage) according to Zadoks et al. (1974). Six weeks after spraying in the case of PRE-treatments and four weeks in POST-treatment, the number of undamaged plants (plants without symptoms after herbicide application and visually similar in aspect and size to the untreated controls) was counted and all plants of each pot were cut at the soil surface and weighted together. Fresh weight data represent the overall response of all individuals that form each accession, which includes dead, damaged and/or undamaged plants, all contributing to the weight. The fresh weight reduction, calculated for each accession as percentage relative to the untreated controls, was used as a measure of the herbicide resistance. The classification of the L. rigidum accessions in response to the applied herbicides was done according to the “R” resistance rating system described by Moss et al. (1999). If fresh weight reduction was: less than 40%, the accessions were classified as RRR ¼ resistance confirmed, highly likely to reduce herbicide performance; from 40% to 80%, as RR ¼ resistance confirmed, probably reducing herbicide performance; from 80% to 90%, as R? ¼ early indications that resistance may be developing, possibly reducing herbicide performance; more than 90%, as S ¼ susceptible. For chlorsulfuron, the percentage reduction fresh weight value for the susceptible biotype used as control in all experiments was 92%, while for chlortoluron and diclofop-methyl this value was 99%. The percentage of undamaged plants was also calculated for each pot; in the case of PRE-emergence herbicides, the percentage of undamaged plants was calculated relative to the number of seeds emerging in the untreated controls, while in POSTherbicides it was calculated relative to the emerged plants before treatment.
The effect of the accession origin in the response to herbicide applications was tested using ANOVA (Stat Graphics 5.1). Data on the percentage of fresh weight reduction at single dose treatment were square-root transformed (√x/100) prior to the analysis and analysed separately for each herbicide. Correlations between fresh weight and undamaged plants percentages after herbicide applications were analysed using Pearson's correlation coefficient. In order to test the distance relationship between L. rigidum accessions a spatial autocorrelation analyses were undertaken within each region for the response to the three herbicides, both for fresh weight and frequency of undamaged plants. The Moran index was used to estimate the degree of spatial autocorrelation at all sites within the study area. Distance classes of geographical and effective distances between accessions were created following Sturge's rule. The overall significance of the individual correlograms was evaluated using Bonferroni's correction. The Moran's I statistic for spatial autocorrelation is given as:
Pn IðdÞ ¼ n
i¼1
wij ðdÞðyi yÞ yj y P WðdÞ ni¼1 ðyi yÞ2
Pn
j¼1
where n is equal to the total number of considered sampling units, y is the value of the variable in the points i and j (i s j), wij (d) is the spatial weight matrix between i and j within the distance class d and W(d) is the aggregate of all the spatial weights matrix. The Moran's I values range from þ1.0 (perfect positive spatial autocorrelation) and - 1.0 (perfect negative spatial autocorrelation). The value 0 implies perfect spatial randomness. The statistical software package used was PASSAGE v.2 (2.0.8.20) (Rosenberg and Anderson, 2011). 3. Results Figs. 2e4 show the response of each accession to chlortoluron (Fig. 2), chlorsulfuron (Fig. 3) and diclofop-methyl (Fig. 4). The accessions were grouped by region, firstly they were sorted by decreasing fresh weight at 1X dose and secondly by decreasing percentage of undamaged plants. 3.1. Chlortoluron n after preFresh weight of the 77 accessions from Castilla-Leo emergence application of chlortoluron at 2 kg a.i. ha1, a dose normally applied under field conditions, varied from 1% in the most susceptible accession to 79% in the most resistant, with an average fresh weight of 28% and a median fresh weight of 24% (Fig. 2A). Approximately 80% of the accessions contained undamaged plants with percentages ranging 0.2e61% and an average of 9.4%. These frequencies of undamaged plants were positively correlated with the fresh weight values (r ¼ 0.75). In the accessions from ~ a treated with the same dose, fresh weight values varied Catalun
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13
Fig. 2. Effect of pre-emergence application of chlortoluron on L. rigidum populations. Grey bars represent the undamaged plants while black points represent the fresh weight biomass, both expressed as a percent of control.
from 10% to 80% and averaged 61% (median of 72%). All of them contained undamaged plants averaging 34% (2e70%), approxi n. mately four times more than those accessions from Castilla-Leo This parameter was positively correlated with fresh weight (r ¼ 0.82). When chlortoluron dose was doubled, average fresh weight values varied from 0.2% to 56% and average decreased to 8% n, while they var(median 6%) for the accessions from Castilla-Leo ied from 2 to 71% and averaged 35% (median 33%) for those from ~ a (Fig. 2B). The average frequency of undamaged plants Catalun decreased to 2.9% (0.2e37%) and to 20% (0.4e53%) in each region, respectively. 3.2. Chlorsulfuron n treated with Fresh weight of the accessions from Castilla-Leo chlorsulfuron at 15 g a.i. ha1 ranged from 8% to 100% of the cor~ a, responding controls, and from 24% to 100% for those from Catalun and undamaged plants were present in 75% and 87% of the accessions, respectively in each region (Fig. 3A). The average values for n and Catalun ~ a, 47% and fresh weight were similar for Castilla-Leo 51% respectively (medians of 38 and 41%) and 17% for undamaged plants. The frequencies of undamaged plants were positively
correlated with the fresh weight values obtained at 1X (r ¼ 0.84). At 2X, average fresh weight decreased but they remained at high level: 35% value for both regions, with medians of 21% and 16% for Casn and Catalun ~ a respectively (Fig. 3B). The percentages of tilla-Leo undamaged plants also decreased to average values of 15% n (0.2e98%) and 10% (2e43%) for the accessions from Castilla-Leo ~ a, respectively. and Catalun 3.3. Diclofop-methyl Diclofop-methyl provided a good control of most of the accesn (Fig. 4A). After post-emergence application sions from Castilla-Leo of diclofop-methyl at 540 g a.i.ha1, average values were low, 10% fresh weight (median 5%) and 1.5% for undamaged plants (Fig. 4A). ~ a, 37% for On the other hand, these values were higher in Catalun fresh weight with a median of 42%, and 17% for undamaged plants (Fig. 4A). A similar trend was observed between the responses of the accessions from different origin when dose was doubled (Fig. 4B). The correlation between both values for diclofop-methyl applied at full rate was weaker than for the previous herbicides (r ¼ 0.63 for n and r ¼ 0.69 for those from Catalun ~ a). accessions from Castilla-Leo
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Fig. 3. Effect of post-emergence application of chlorsulfuron on L. rigidum populations. Grey bars represent the undamaged plants while black points represent the fresh weight biomass, both expressed as a percent of control.
3.4. Classification of the L. rigidum accessions in response to applied herbicides Fig. 5 shows the classification of the L. rigidum accessions in response to applied herbicides according to Moss et al. (1999). At n and present, only 33% of the accessions surveyed in Castilla-Leo screened with the 1X dose of chlortoluron were classified as S. Resistance was confirmed in 51% (39) of the accessions (RRR þ RR) (Fig. 5A). At 2X, 76% of the accessions were susceptible and only 9% ~ a, the behaviour of resiswere classified as RR (Fig. 5B). In Catalun tance was different; resistance was confirmed in 92% of the accessions at 1X (67% RRR and 25% RR, Fig. 5A) while 75% accession remained resistant when the herbicide dose was doubled (Fig. 5B). A significant effect of the province of origin was found on fresh weight reduction data (F9,177 ¼ 5.80, P < 0.001), indicating rida geographic variation in the level of herbicide resistance. Le ~ a) was the province with higher levels of chlortoluron (Catalun resistance, followed by Salamanca, Zamora and Valladolid (Castillan). Leo n were classified as Only 10% of the accessions from Castilla-Leo S when chlorsulfuron was applied at 1X (Fig. 5C), while 75% of the accessions were resistant (RRR þ RR). No S accessions were found
n. When dose was in four surveyed provinces of Castilla-Leo doubled, S accessions increased to 44% (Fig. 5D). There were differences in the response among provinces (F9,88 ¼ 4.41, P < 0.001): Salamanca was the most affected province by chlorsulfuron resis ~ a, no S actance, followed by Avila, Zamora and Soria. In Catalun cessions were detected at 1X. When dose was doubled, while RRR accessions were the same as 1X, S accessions increased to 58% (Fig. 5D). n For diclofop-methyl, 74% of the accessions from Castilla-Leo were classified as S at 1X. There are four provinces with no resistant (RRR or RR) accessions and other provinces as Valladolid, Salan or Zamora which showed up to 37% of manca, Palencia, Leo ~ a, the resistance level resistant accessions (RR) (Fig. 5E). In Catalun to diclofop-methyl was higher, with 83% of the screened accessions being resistant (8% RRR and 75% RR) and only 8% S when the single dose of this herbicide was applied (Fig. 5E). When dose was doubled (Fig. 5F) the percentage of resistant accessions was of 4% n and Catalun ~ a, respectively. and 75% in Castilla-Leo 3.5. Multiple resistance n, 29% exhibited multiple Of the 77 accessions from Castilla-Leo
I. Loureiro et al. / Crop Protection 102 (2017) 10e18
15
Fig. 4. Effect of post-emergence application of diclofop-methyl on L. rigidum populations. Grey bars represent the undamaged plants while black points represent the fresh weight biomass, both expressed as a percent of control.
resistance to at least one herbicide within all three groups tested: 25% to any two of the three mode of actions employed and 4% to all of them. The multiple resistance pattern most commonly found was to chlortoluron and chlorsulfuron herbicides, present in almost 19% of the accessions. Multiple resistance levels were higher in ~ a, where 75% of the accessions being multiple resistant. In Catalun this region, 25% of accessions were resistant to the three mode of actions; resistance to chlortoluron and chlorsulfuron was observed in 25% of the accessions while 17% were resistant to chlorsulfuron and diclofop-methyl.
sampling points of 461 km, resulting in a correlogram with 12 distance classes, there was significant spatial autocorrelation (P < 0.001) for the fresh weight variable at normal dose in five ~ a, using distance classes of 3 km distance classes, while in Catalun and a maximum distance between two sampling points of 22 km (7 distance classes), there was significant spatial autocorrelation (P < 0.001) in two distance classes. This indicate different significant hotspots of chlorsulfuron resistance on a local scale, meaning that accessions within site are clustered spatially for their level of resistance. Results were similar when the frequency of undamaged plants was used.
3.6. Autocorrelation analysis Since every accession has been mapped, autocorrelograms within each region have been carried out for the frequency of undamaged plants and for the fresh weight to test the relationship between accessions related to distance. For chlortoluron and diclofop the absence of significant correlograms indicates, in principle, the existence of a random distribution of resistance. Significant spatial autocorrelation was detected only in response to n, using chlorsulfuron in the two evaluated areas. In Castilla-Leo distance classes of 38 km with a maximum distance between two
3.7. Increase in herbicide resistance incidence between 2000e02 and 2012-14 As is shown in Table 2 there is a substantial increase on the resistance levels for the studied herbicides related to 2000-02 n, most accessions were survey (Loureiro et al., 2010). In Castilla-Leo classified as S to herbicides chlortoluron (95.3%) and chlorsulfuron (89.5%) ten years ago. At present, these percentages have been reduced considerably and resistance to both herbicides has increased, only 33% and 10% of the accessions remain S to these
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n (C-L, 77 populations) and Catalun ~ a (province of Le rida, 12 populations) in Fig. 5. Classification of the Lolium rigidum accessions collected in several provinces of Castilla-Leo response to the application of herbicides according to the “R” resistance rating system for designating resistance in screening assays described by Moss et al. (1999).
herbicides, respectively. While any resistance was found for diclofop-methyl in the past, nowadays 26% of the accessions show some level of resistance. In 2000e02, the highest resistance levels were found in Cata~ a: chlortoluron resistance was present in 10% of the accessions lun
and chlorsulfuron resistance was widespread with 60% of the accessions displaying resistance (40% RR and 20% R?). Currently, chlortoluron resistance is almost ten times greater (67% of the accessions RRR and 25% RR) and for chlorsulfuron, all accessions showed resistance: 33% RRR and a further 67% RR and R?, Table 2).
I. Loureiro et al. / Crop Protection 102 (2017) 10e18
17
Table 2 n and Catalun ~ a in Change in herbicide resistance status between 2000-2002 and 2012e2014 surveys of Lolium rigidum populations collected from the same areas of Castilla-Leo response to the field dose of the herbicides chlortoluron, chlorsulfuron and diclofop-methyl. Region
Herbicide
Accessions (%) RRR
n Castilla-Leo
~a Catalun
Chlortoluron Chlorsulfuron Diclofop-methyl Chlortoluron Chlorsulfuron Diclofop-methyl
RR
R?
S
2000
2012
2000
2012
2000
2012
2000
2012
0.0 1.2 0.0 0.0 0.0 e
7.6 27.1 0.0 66.7 33.3 8.3
1.2 3.5 0.0 10.0 40.0 e
43.4 47.5 12.6 25.0 41.7 75.0
3.5 5.8 0.0 0.0 20.0 e
15.9 15.6 13.4 0.0 25.0 8.3
95.3 89.5 100.0 90.0 40.0 e
33.1 9.7 74.0 8.3 0.0 8.3
4. Discussion n, in the 12e14 The current survey show that, in Castilla-Leo years since the last random survey was conducted, the resistance status of L. rigidum has changed from very low level of resistance (0e5%) to high levels of resistance (RRR and RR) (13e74%). The highest percentages of resistant L. rigidum accessions were found in cereal fields of Salamanca, Zamora or Palencia provinces. Under semiarid climate, cereal yields are medium-low which do not allow farmers for a major input on chemicals, so preference is common for the least expensive effective herbicides. In this situation, with few alternatives in the crop rotation and in the availability of herbicides, pre-emergence herbicides are commonly applied. These facts explains the higher incidence of resistance to chlortoluron and chlorsulfuron herbicides than to diclofop-methyl, applied at post emergence. All the screened accessions from Avila, Soria and Segovia provinces were susceptible to diclofop-methyl, so in these areas diclofop-methyl may be included as one of the management options for control of the resistant biotypes to the other herbicides. ~ a, diclofop-methyl response was not analyzed in our In Catalun previous survey although problems of resistance were known (Taberner, 2005). At present, 83% of the screened accessions show diclofop-methyl resistance, while resistant accessions have increased from 10% to 92% for chlortoluron and from 40% to 75% for chlorsulfuron. In this region, cereal yields are higher and thus profits, with a greater margin for the use of herbicides imposing a higher selection pressure. For both regions the cost effectiveness of the studied herbicides and their broad-spectrum weed control that include, L. rigidum and other grasses such as Bromus spp. and Avena spp. (Escorial et al., 2011) or even some common annual dicotyledonous species (Papaver rhoeas, Polygonunm aviculare, Salsola kali or Matricaria spp.), promoted their widespread use alone or in mixtures. Therefore, they contributed to the selection of resistant biotypes and to the spread of resistance, which is faster in genetically diverse and cross-pollinated weeds, such as species of the Lolium genera. This increase in the frequency of resistance has occurred in Australia, where L. rigidum resistance was firstly reported to ACCase and ALS inhibiting herbicides (Heap and Knight, 1982, 1986) and has been intensively monitored. In the 1998 survey, 50e60% of the L. rigidum accessions from the randomly surveyed zones were classified as resistant or developing resistance to ACCase and ALS inhibiting herbicides (Llewellyn and Powles, 2001), while over the subsequent 5-year period it increased to 70e90% (Owen et al., 2007), and after 12 years to > 95% (Owen et al., 2014). L. multiflorum Lam. (Italian ryegrass), a close relative L. rigidum, is also a weed management problem in different cropping areas of North America, with populations resistant to many mode of action and some of them exhibiting cross- and/or multiple herbicide resistance (Chandi et al., 2011; Hulting et al., 2012; Liu et al., 2014). Studies conducted in the Pacific Northwest of the USA indicated
that 73% of L. multiflorum accessions were resistant to the aryloxyphenoxypropionate herbicides as diclofop-methyl and 39% to the sulfonylurea herbicides as chlorsulfuron (Rauch et al., 2010). This species is also a problematic resistant grass weed in cereals in many European countries including Germany, Denmark or Italy (Sattin, 2005) and in the United Kingdom, where 70% of the Lolium spp. populations were resistant to at least one herbicide (Hull et al., 2014). Herbicide applications are designed by growers at the level of each individual field. As a consequence, herbicide selective pressure varies among agricultural fields and regions so local evolution of resistance is expected. Our results show special spatial correlation for chlorsulfuron resistance suggesting that these accessions were likely exposed to similar herbicide regimes and/or are close enough for gene flow. Either scenario is equally plausible: the frequent use of a similar herbicide regime to control L. rigidum in different fields and/or resistance alleles being dispersed through either pollen or seed, and this is of utmost importance mainly in accessions harbouring plants with TSR, as could be the case for this herbicide. In a previously conducted study on pollen mediated gene flow in L. rigidum under semi-arid conditions, 0.1% cross-pollination rate would be predicted at 100 m (Loureiro et al., 2016), which delivers to an initial frequency of 1 herbicide-resistant individual in 1000 S seeds, a rate higher than the spontaneous mutation rate in weed populations which is generally assumed to be about 106 (Maxwell and Mortimer, 1994). Busi et al. (2008) reported that pollenmediated gene flow in L. rigidum can occur up to 3 km from the nearest pollen source. Although resistance development in every accession is a unique case, these spatial scales are of great relevance for resistance spread and management, which should consider more than just the single resistant accession. From the data presented and taking into account knowledge from literature, we show that herbicide resistance in L. rigidum evaluated by random surveys has increased significantly in 12 years in cereal crops in Spain. Further studies are being conducted to ascertain the resistance mechanisms present in the resistant accessions. The resistance mechanisms for the three studied herbicides in this species could be determined as being TSR and NTSR (Han et al., 2016). Both types of resistance, could be present in one or many populations, and sometimes accompanied by multiple and cross-resistance (Beckie and Tardif, 2012). In addition, spatial correlation, irrespective of its origin that could be similar herbicide selection pressures or resistance dispersal by gene flow, was evidenced in this study for chlorsulfuron treated accessions. This worrying scenario shows that the resistance management is becoming increasingly complex. Nevertheless other active ingredients and non-chemical control measures are available for resistant L. rigidum control that could be used in an integrated weed management. This situation needs to be addressed quickly and efficiently to avoid this scenario to turn dramatic, if no adequate measures are adopted.
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