Amaranthus palmeri a New Invasive Weed in Spain with Herbicide Resistant Biotypes

: Amaranthus palmeri is the most prominent invasive weed in agricultural land from North America, partly due to its propensity to evolve resistance to multiple herbicide sites of action. In the last two decades, reports of this species have increased throughout the American continent and occasionally in other continents. In 2007, A. palmeri populations were found in three localities in northeastern Spain, and they are still present today. To determine whether these three populations resulted from a common or independent introduction events—and when and from where they could have occurred—research was carried out aiming to characterize the resistance proﬁle and mechanisms to 5-enolpyruvylshikimate-3-phosphate synthase-and acetolactate synthase (ALS)-inhibiting herbicides and to analyze the relationship between these three populations using inter simple sequence repeat DNA ﬁngerprinting. Dose–response trials conﬁrmed that the three populations were susceptible to glyphosate but resistant to nicosulfuron-methyl. Resistance to ALS inhibitors was due to several amino acid substitutions in positions Pro197, Trp574 and Ser653. Moreover, the substitutions Ser653Ile and Pro197Thr are described for the ﬁrst time in this species. At ﬁeld-labeled rates, all populations were fully controlled with alternative herbicides with other sites of action. Amaranthus palmeri individuals were clustered in three groups based on unweighted pair group method with arithmetic mean analysis, which corresponded to the three sampled populations, with a 67% of genetic relationship among them. Considering this high genetic variability and the di ﬀ erent positions and amino acid substations found between populations, it was hypothesized that di ﬀ erent colonization events occurred from the American continent probably prior to the introduction of glyphosate resistant crops. Prevention from new introductions is warranted because new herbicide resistance traits could arrive, complicating the management of this invasive weed species, while managing or eradicating the already established populations.


Introduction
Invasive plant species are a major cause of crop loss and can adversely affect food security [1]. In the United States, crop losses from invasive weeds have been estimated at about US$27,000 million per year [2]. The threat from plant invasive species arriving to countries in which they were previously absent is expected to increase with globalization and connectedness via world trade.
there are reported six codons with mutations in the ALS gene [23]. Finally, non-target site resistance mechanisms to ALS inhibitors have also recently been identified [24].
Several important questions arise as the result of A. palmeri recent introduction and invasion of habitats in NE Spain, which will be addressed in this research: (1) Were the three identified populations originated from a unique initial spread point or did the colonization process take place separately? (2) Do these populations carry herbicide resistance alleles? and (3) When and from where did the first colonization process take place? These and other questions define an interesting scenario for A. palmeri to understand if an initial invasion process took place in summer crops in NE Spain and the potential necessity of developing a pest risk assessment for this species.

Plant Material
In autumn 2016 mature seeds were collected from three A. palmeri populations located in north eastern Spain (Lleida, Menàrguens and Binéfar). Since 2007, these populations were monitored and positively identified every year (until 2019) on roadsides and maize field edges in Menàrguens in Lleida province (41 • 43 N 0 • 45 E), in industrial areas nearby the city of Lleida (41 • 37 N 0 • 39 E), both in Catalonia and road sides of Binéfar (Huesca) in Aragon region (41 • 49 N 0 • 18 E). Seed from an A. palmeri population was collected in autumn 2014 from Bulloch County, Georgia, US (81 • 40 N 32 • 19 E) from a vegetable organic farm with no history of herbicide use in more than twenty years and used as the susceptible (S) standard [25] for the dose-response experiments described below. Seeds were collected separately from at least twenty different mature plants (without herbicide injury) randomly selected and brought to the laboratory; there they were threshed and cleaned and left to dry in paper bags at room temperature until experiments started. Seeds from the S population were collected, dried, and kept under cold storage (4 • C) until used [25].

Dose-Response Trials
Dose-response experiments were conducted in 2018 using the three Spanish A. palmeri populations and the S standard population from US. Seeds were sown in aluminum trays with peat. Trays were placed in a growth chamber at 15/25 • C day/night and a 12/12 h photoperiod under 350-µmol m −2 s −1 photosynthetic photon-flux density. After 14 d, seedlings were transplanted to 8 by 8 by 8 cm plastic pots filled with a mixture of silty loam soil, sand and peat (1.3:1:1 by volume). Three seedlings were transplanted per pot and later thinned to two. Pots were brought to a greenhouse at the University of Lleida, Spain (41.629 • N, 0.598 • E). At the 5-to 6-leaf stage (5 to 6 cm), the ALS inhibitor nicosulfuron-methyl and the EPSPS inhibitor glyphosate were applied separately to different pots at the rates 0, 7.5, 15, 30, 60, 120, 240 g a.i. ha −1 and 0, 75, 150, 300, 600 and 1200 g a.i. ha −1 , respectively. A total of four replicates (pots) were included for each dose in a completely randomized design, and the experiment was repeated. Herbicides were applied using a precision bench sprayer delivering 200 L ha −1 at a pressure of 215 kPa. Pots were placed in the greenhouse and were regularly watered. At 4 weeks after treatment, weed control efficacy in terms of mortality (%) from each dose was evaluated for each pot and aboveground dry weight evaluated after 24 h at 72 • C in an oven. The data were expressed as a percentage of dry weight reduction compared to the untreated control. Plants without green tissues were classified as dead.

Alternative Herbicides
As described above, several alternative herbicides were applied at field recommended rates to the three Spanish A. palmeri populations to assess their sensitivity to different chemical options available in the market for weed control in maize crop. Additionally, thifensulfuron-methyl (group B, according to HRAC, Herbicide Resistance Action Committee) was also applied at field rate to study potential cross-resistance to other sulfonylurea ALS inhibitors. The herbicides S-metolachlor (group K3, S-metolachlor 96%, Dual Gold, Syngenta) and isoxaflutole (group F2, isoxaflutole 24%, Spade Flexx, Bayer CropScience) were applied preemergence (BBCH 00). Dicamba (group O, dicamba 48%, Banvel-D, Syngenta), sulcotrione (group F2, sulcotrione 30%, Decano, Ascenza Agro), mesotrione (group F2, mesotrione 10%, Callisto 100, Syngenta) and thifensulfuron (thifensulfuron 50%, Harmony 50, FMC Agricultural Solutions) were applied postemergence as described for the dose-response experiments. Five seeds were sown for the preemergence treatments, which all germinated after in the corresponding untreated controls. A total of four replicates (pots) were included for each herbicide in a completely randomized design. The experiment was not repeated. Weed control efficacy in terms of mortality (%) was assessed 4 weeks after treatments. Plants were scored as dead when were all necrotic or alive if they were undamaged or clearly still growing with green parts.

ALS Gene Sequencing
DNA from the leaf fragment of new grown plants was extracted using the Speedtools Plant DNA Extraction Kit (Biotools B&M Labs S.A., Valle de Tobalina, Madrid, Spain) and the DNA sample concentration was measured in a NanoDrop Thermo Scientific spectrophotometer (Thermo Fisher, Nano Drop Products, Wilmington, DE, USA). Each DNA sample was diluted to a final concentration of 10 ng µL −1 , which was immediately used for the polymerase chain reaction (PCR) test or stored at −20 • C until use. All mutations conferring ALS resistance in A. palmeri in positions Ser653, Trp574, Pro197, Pro197, Ala122 and Ala122 of CAD and BE domains [26] were analyzed for all the samples. Fragments of the ALS gene that included the regions of those codons were amplified using two pairs of primers (Table 1)

ISSR DNA Fingerprinting
Seeds from individual plants were germinated in the greenhouse to generate 20 plants of each Spanish A. palmeri population. The DNA material from each plant was extracted from young leaves using the commercial kit Speedtools Plant DNA Extraction Kit (Biotools B&M Labs S.A., Valle de Tobalina, Madrid, Spain). For the analyses with the molecular markers, seven primers of the (inter simple sequence repeat) ISSR type were used (Table 2)

Data Analysis
Data from dose-response experiments were analyzed using a nonlinear regression model (1) with SigmaPlot 11.0 (Systat Software, Inc., San Jose, CA, USA). The herbicide rate required for 50% growth reduction of plants (GR 50 ) or 50% of plant mortality (LD 50 ) was calculated with the use of a four parameter logistic curve: where c = the lower limit adjusted to 0, d = the upper limit adjusted to 100 and b = the slope at the LD 50 or GR 50 . In this regression equation, the herbicide rate (g a.i. ha −1 ) was the independent variable (x) and the plants' dry weight expressed as percentage of the untreated control or mortality were the dependent variables (y). The resistance index (RI) was computed as GR 50 (R)/GR 50 (S) or LD 50 (R)/LD 50 (S). Weed control efficacies (% of mortality) of alternative herbicides experiment were analyzed with one-way ANOVA considering population as factor for each herbicide tested.
The results of sequencing were visualized using CHROMAS 2.6.6 software (Technelysium Pty Ltd., South Brisbane, Australia). Subsequently, these sequences were aligned using the CLUSTAL OMEGA software (EMBL-EBI, Hinxton, UK). Heterozygous base pairs were identified in the sequence trace files by manual inspection.
Genetic relationships among the studied populations were assessed using the unbiased genetic identity coefficient (I) of Nei [27], which ranges from I = 0.0 for populations with no genetic similarity to I = 1.0 for populations that are genetically identical. The phylogenetic analysis was based on the unweighted pair group method with arithmetic mean (UPGMA) for the DNA fingerprint data using the software Numeric Taxonomy System (NTSYS)-pc 2.2 (Exeter Software, Exeter, UK) [28].

Dose-Response Trials
The populations Binéfar, Menàrguens and Lleida were 6.8-, 4.0-and 11.0-fold more R to nicosulfuron-methyl than S plants in terms of mortality (Table 3, Figure 1). Only the S standard population was fully controlled at the field dose. The LD 50 values ranged from 19 for the S population to 209 g a.i. ha −1 for the most R one. Considering the dry weight reduction (%) compared to untreated controls, RI were 4.8, 3.7 and 12.1, respectively, for the same populations, with GR 50 values between 22 (S population) and 271 g a.i. ha −1 . Table 3. Estimated slopes (b), plant mortality (LD 50 ) and growth reduction of plants (GR 50 ), p-values (level of significance of curves fitting) and resistance index (RI) values for mortality (%) and dry weight reduction (%) to nicosulfuron-methyl and glyphosate for several Amaranthus palmeri populations in 2018: three from Spain and one susceptible (S) standard from US.

Mortality (%)
Dry Weight Reduction (%) For glyphosate, RI were 1.4, 1.7 and 1.0 for Binéfar, Menàrguens and Lleida populations, respectively, in terms of mortality, while considering the percentage of dry weight reduction RI were 1.0, 1.5 and 1.4 (Table 3). LD50 values ranged from 106 to 179 and from 57 to 89 g a.i. ha −1 for GR50. Finally, no population survived the field recommended dose (Figure 1). Figure 1. Dose-response regression curves (2018) for mortality (above graphs) and dry weight (below graphs) of S standard (Georgia, US) and Spanish (Binéfar, Menàrguens and Lleida) Amaranthus palmeri populations to nicosulfuron-methyl (left) and glyphosate (right) (log scale). Data for dry weight were expressed as percentage of the mean dry weight of untreated control plants. Grey arrows represent field dose for each herbicide. Figure 1. Dose-response regression curves (2018) for mortality (above graphs) and dry weight (below graphs) of S standard (Georgia, US) and Spanish (Binéfar, Menàrguens and Lleida) Amaranthus palmeri populations to nicosulfuron-methyl (left) and glyphosate (right) (log scale). Data for dry weight were expressed as percentage of the mean dry weight of untreated control plants. Grey arrows represent field dose for each herbicide.

Alternative Herbicides
For glyphosate, RI were 1.4, 1.7 and 1.0 for Binéfar, Menàrguens and Lleida populations, respectively, in terms of mortality, while considering the percentage of dry weight reduction RI were 1.0, 1.5 and 1.4 (Table 3). LD 50 values ranged from 106 to 179 and from 57 to 89 g a.i. ha −1 for GR 50 . Finally, no population survived the field recommended dose (Figure 1).

Alternative Herbicides
The effectiveness of all alternative herbicides tested was 100% of mortality (Table 4). At the field recommended rates applied, both treatments in pre-emergence (S-metolachlor and isoxaflutole) or in postemergence (mesotrione, sulcotrione or dicamba), reached full control of the three studied Spanish A. palmeri populations. On the other hand, when ALS-inhibiting herbicides were applied in postemergence, both nicosulfuron and thifensulfuron (another sulfonylurea) achieved very poor control, with mortality ranging from 8% to 56% and from 25% to 58%, for each herbicide respectively, depending on the population. Significant differences between populations were only found for ALS-inhibiting herbicides: Lleida population showed the lowest and highest mortality, for thifensulfuron and nicosulfuron, respectively. Table 4. Mortality (%) of three Spanish Amaranthus palmeri populations for several herbicides applied either in pre-or postemergence at field recommended rates (1x) in 2014. Herbicide Resistance Action Committee (HRAC) Groups and Sites of Action (SoA) for each herbicide are indicated in the first two columns.

ALS Gene Sequencing
Substitutions at codons Pro197 of the CAD domain and Trp574 and Ser653 of the BE domain were found in plants of the three analyzed Spanish A. palmeri populations (Table 5). Of the 20 analyzed plants for each population, 9, 11 and 13 individuals for Lleida, Binéfar and Menàrguens populations, respectively, did not have any substitution in codons conferring resistance to ALS-inhibiting herbicides. One amino-acid replacement was identified in position 574 (Tryptophan by Leucine), two different replacements in position 653 (Serine by Asparagine or Isoleucine) and also two different replacements in position 197 (Proline by Threonine or Serine). In Lleida, both heterozygous and homozygous plants for substitution Trp574Leu were found, while only homozygous plants for Ser653Asn were found. No substitution in position 197 was found.

ISSR DNA Fingerprint
The total number of polymorphic markers obtained with the seven ISSR primers used was 256. As observed in Figure 2, all the plants studied shared a genetic relationship of 0.67, showing a genetic variation of 33% between all the individuals analyzed. Two big distinct clusters were observed at a similarity level of 0.62. The first cluster contained most of the plants belonging to localities of Lleida and Menàrguens, while in the second cluster all individuals sampled in Binéfar were found (Group 3, Figure 2), excepting two found in Group 2 ( Figure 2). In the first cluster, at a coefficient level of 0.72, two groups were identified, one with plants sampled in Lleida, Group 1 (

ISSR DNA Fingerprint
The total number of polymorphic markers obtained with the seven ISSR primers used was 256. As observed in Figure 2, all the plants studied shared a genetic relationship of 0.67, showing a genetic variation of 33% between all the individuals analyzed. Two big distinct clusters were observed at a similarity level of 0.62. The first cluster contained most of the plants belonging to localities of Lleida and Menàrguens, while in the second cluster all individuals sampled in Binéfar were found (Group 3, Figure 2), excepting two found in Group 2 ( Figure 2). In the first cluster, at a coefficient level of 0.72, two groups were identified, one with plants sampled in Lleida, Group 1 (  Unweighted pair group method with arithmetic mean (UPGMA) dendrogram using distance of Nei [27] showing the molecular relationships using seven inter simple sequence repeat (ISSR) between 60 plants from three Spanish Amaranthus palmeri populations. Lle1-Lle20: plants from Lleida; Me1-Me20: plants from Menàrguens; Bi1-Bi20: plants from Binéfar.
Finally, there were three plants that were not found among the previously described groups (Figure 2, bottom part). All of them were sampled in the locality of Menàrguens. Unweighted pair group method with arithmetic mean (UPGMA) dendrogram using distance of Nei [27] showing the molecular relationships using seven inter simple sequence repeat (ISSR) between 60 plants from three Spanish Amaranthus palmeri populations. Lle1-Lle20: plants from Lleida; Me1-Me20: plants from Menàrguens; Bi1-Bi20: plants from Binéfar.
Finally, there were three plants that were not found among the previously described groups (Figure 2, bottom part). All of them were sampled in the locality of Menàrguens.

Discussion
Dose-response trials in the greenhouse confirmed that all three A. palmeri populations established in northeastern Spain were S to glyphosate but R to ALS-inhibiting herbicides. For the EPSPS inhibitor glyphosate, GR 50 values estimated in this research were within the range (i.e., 66 to 194 g a.i. ha −1 ) of other A. palmeri S populations reported in the literature [29,30]. In addition, LD 50 values around 100 g a.i. ha −1 estimated in previous studies for glyphosate S populations [31] were in accordance with those found here.
Concerning the ALS inhibitor nicosulfuron, in terms of mortality, LD 50 for the S population (19 g a.i. ha −1 ) was in the range of other studies (<18 g a.i. ha −1 ) [32]. Accordingly, GR 50 values to nicosulfuron (percent of fresh weight reduction) of 18 g a.i. ha −1 were reported in S populations from Argentine [32], similar to those found in this study (22 g a.i. ha −1 ). For the R populations, the GR 50 values obtained here (83 to 271 g a.i. ha −1 ) were also in the range of other A. palmeri populations R to nicosulfuron, though usually they can be higher (>350 g a.i. ha −1 ) [32]. In addition, RI above 20 are usually estimated in other A. palmeri populations R to different ALS-inhibiting herbicides, both based on LD 50 or GR 50 [22]. Relative low resistance levels to nicosulfuron found in these three A. palmeri populations established in Spain could be explained by the relative high frequency of S plants found (around 50%) based on the sequencing of the ALS gene ( Table 5).
The molecular analyses of ALS gene sequence revealed target site mutations in the three A. palmeri populations that confer resistance to ALS inhibitors, corroborating the results from the dose-response experiments. Amino acid changes were found in positions Pro197, Trp574 and Ser653, already described in this species [20]. On the other hand, the specific amino acid substitutions Pro197Thr and Ser653Ile, found in plants from Binéfar and Menàrguens and in one homozygous plant from Menàrguens, respectively, are-to our knowledge-the first report worldwide for A. palmeri. To date, the Ser653Ile substitution has been found only in Setaria viridis [20]. Changes in positions Pro197 and Trp574 are expected to persist in these populations, since mutations at these points do not represent a major fitness cost [33]. The latter, in fact, was the only position changed in all the three populations, potentially conferring cross-resistance to all ALS chemistries [33]. Finally, it might be important to highlight that the percent mortalities observed matched the percent of plants genotyped as R. Of the plants analyzed, 55%, 45% and 35% had amino acid substitutions in the ALS gene for Lleida, Binéfar and Menárguens populations, respectively (Table 5). These percentages roughly correlated with estimated mortalities at field rate ( Figure 1, Table 4). This would rule out the presence of non-target site resistance to ALS inhibitors, but it should be further confirmed.
All three A. palmeri populations established in northeastern Spain and R to ALS-inhibiting herbicides, were fully controlled with other herbicides at field rates from three different modes of action (Table 4). Therefore, there is still the chance for managing the population in Menàrguens that has already infested a maize field. On the other hand, it is important to notice that this species is able to develop resistance to all tested sites of action herein [20], so an adequate integrated weed management should be required. Among chemical strategies, rotating sites of action (SoA), herbicide mixtures with different (SoA) and sequential applications should be considered [34]. Amon non-chemical and curative strategies, scouting for further resistance, cultural techniques such as seeding dates/crop rotations, tillage/hand weeding, edge management control and plantings to reduce invasion potential should be implemented [34]. Finally, cross-resistance to the sulfonylurea thifensulfuron was observed at field rate, as expected by the point mutations detected in these populations [33].
Gene flow, diversity analyses, tracking herbicide R biotypes, population structure and genetic mapping is feasible for invasive weeds using ISSRs markers [35]. Three groups of A. palmeri plants were distinguishable according the UPGMA analyses, and most sampled plants in each locality were properly classified in each group (Figure 2). The three A. palmeri populations (groups) had a genetic divergence of 33%, while between Lleida and Menàrguens divergence it was slightly lower (28%). This genetic differences among populations suggest that plants from these three localities are not closely related [36,37] and might come from different colonization events. Accordingly, the occurrence of different and multiple amino acid substitutions among R plants of the three A. palmeri populations suggests a complex and different evolutionary history of these resistance traits. While not definitive, the absence of mutations in position Pro197 in the Lleida population (present in the other two) or also the absence in position Ser653 in the Binéfar population, indicates potential different arrivals to the country and from different sources. For example, the absence of mutations in Ser653 in A. palmeri from Argentina and the presence of both Trp574 and Ser653 in plants from Brazil, suggested that independent introductions of the species occurred in the two countries [38]. Moreover, changes in different positions in the ALS gene also suggest different herbicide selection pressures and cropping systems. The selection of ALS alleles conferring more imidazolinone-specific herbicide resistance, such as Ser653, point out to soybean as the predominant crop and imazethapyr treatments, while changes in position Pro197 to the use of sulfonylureas in maize, as suggested for A. tuberculatus [39]. Therefore, the hypothesis that not only of different introduction events but also different sources is reinforced for these three A. palmeri populations established in NE Spain. Finally, it has to be stressed that there were nine years between the likely introduction of these populations and sample collection. Therefore, it cannot be ruled out that populations from the same source may have diverged through new mutation events.
As proven in this research, all populations were glyphosate S, so the introduction events might have occurred before glyphosate resistance started to evolve in this species in North America. The main selecting agent for the resistance to this herbicide in A. palmeri were glyphosate R crops and the first reported case was in 2006 [40]. Hence, the arrival of A. palmeri to Spain might be already quite old and might have occurred before 2006.
This research reports for the first time the presence of herbicide resistance biotypes of A. palmeri, not only in Spain, but also in Europe. The occurrence of herbicide R A. palmeri is very threatening to the sustainability of both irrigated arable crops and natural ecosystems. The species is starting to enter some maize fields and, in this sense, the Servei de Sanitat Vegetal from the Department of Agricultura, Ramaderia, Pesca i Alimentació of the Generalitat de Catalunya and also the Servicio de Sanidad Vegetal of the Gobierno de Aragon, are already undertaking the corresponding legislative measures to contain and eradicate these populations. Recently, an Order at the Regional Catalan level (DOGC number 7959, 13th September 2019) was issued [41] declaring the presence of A. palmeri and stating that it is of public interest to establish the required measures to control it [42]. Unfortunately, based on our own monitoring efforts, we have found new A. palmeri populations around the studied area (no more available data). Thus, this aggressive species is spreading, maybe through harvesting machinery and irrigation channels. Hence, it is urgent to undertake a pest risk assessment to develop appropriate management and eradication strategies in accordance with particular characteristics of Spanish and European ecological and agricultural systems. Moreover, in the Region of Extremadura (800 km away), the species has already been detected (Dr. Osuna, personal communication). Preliminary studies have confirmed the presence of the amino acid substitution Trp574Leu in this region (personal communication). Finally, ALS R A. palmeri plants have already arrived to Italy too (Dr. Donato Loddo, personal communication). Therefore, it is clear that several introduction and dispersal processes across Europe are occurring, probably through contaminated grain from America and will continue if preventive measures are not quickly implemented. The prevention of further introductions is warranted, since this species is able to evolve resistance to a wide range of sites of action and glyphosate resistance is already spread across America [20]. Hence, new arrivals with new herbicide R traits would make its management even more difficult and probably require more aggressive and expensive control measures.