Acetolactate synthase (ALS, EC 2.2.1.6; also referred to as acetohydroxy acid synthase, EC 4.1.3.18) inhibitors have been widely employed in agriculture production since their first commercialization in the 1980s. The advantages of ALS inhibitors include being highly efficient at low rates, safe to mammals, and benign to the environment (Mazur and Falco 1989; Yu and Powles Reference Yu and Powles2014). Resistance was first reported in a prickly lettuce (Lactuca serriola L.) biotype after chlorsulfuron was used for only 5 yr (Mallory-Smith et al. Reference Mallory-Smith, Thill and Dial1990). Worldwide, 159 weed species are ALS resistant (Heap Reference Heap2016). Moreover, the number of ALS-resistant weeds is still increasing. ALS inhibitors inhibit the catalysis of the first process in the biosynthetic pathway of three branched-chain amino acids, valine, leucine, and isoleucine (Duggleby et al. Reference Duggleby, McCourt and Guddat2008; Duggleby and Pang Reference Duggleby and Pang2000; Singh Reference Singh1999), resulting in plant death. ALS is the common target enzyme of five different chemical families of herbicides: sulfonylureas (SUs), imidazolinones (IMIs), sulfonylamino-carbonyltriazolinones (SCTs), triazolopyrimidines (TPs), and the pyrimidinylthio-benzoates (PTBs).
As a group of selective herbicides, ALS inhibitors are continuously prone to the evolution of resistance (Heap Reference Heap2016). The less commonly observed non–target site resistance mechanism involves key enzymes such as glutathione S-transferases and cytochrome P450 monooxygenases (Iwakami et al. Reference Iwakami, Uchino, Kataoka, Shibaike, Watanabe and Inamura2014; Park et al. Reference Park, Fandrich and Mallory-Smith2004; Powles and Yu Reference Powles and Yu2010; Veldhuis et al. Reference Veldhuis, Hall, O’Donovan, Dyer and Hall2000). Many resistance cases have resulted from the incidence of several known point mutations within the ALS gene (Liu et al. Reference Liu, Yuan, Du, Guo, Li, Bi and Wang2015a; Sada et al. Reference Sada, Ikeda and Kizawa2013). To date, 27 different point mutations have been discovered in the ALS gene: Ala-122 (Riar et al. Reference Riar, Norsworthy, Srivastava, Nandula, Bond and Scott2013), Pro-197 (Varanasi et al. Reference Varanasi, Godar, Peterson, Shoup and Jugulam2015), Ala-205 (Brosnan et al. Reference Brosnan, Vargas, Breeden, Grier, Aponte, Tresch and Laforest2016), Asp-376 (Huang et al. Reference Huang, Chen, Zhang, Huang, Wei, Zhou and Wang2015), Arg-377 (Massa et al. Reference Massa, Krenz and Gerhards2011), Trp-574 (Matzrafi et al. Reference Matzrafi, Lazar, Sibony and Rubin2015), Ser-653 (Chen et al. Reference Chen, Huang, Zhang, Huang, Wei, Chen and Wang2015), and Gly-654 (Laplante et al. Reference Laplante, Rajcan and Tardif2009), with the amino acid numbers standardized to the ALS gene sequences of mouse-ear cress [Arabidopsis thaliana (L.) Heynh.]. Superficial similarities were found between SUs and IMIs, suggesting that they have partially overlapping binding sites on the ALS (McCourt et al. Reference McCourt, Pang, King-Scott, Guddat and Duggleby2006). Therefore, certain single point mutations may cause cross-resistance to two or more herbicide classes (Tranel and Wright Reference Tranel and Wright2002).
Eclipta, distributed widely in the tropical, subtropical, and temperate regions and thriving in moist and dryland fields, is a common annual malignant broadleaf weed belonging to the Asteraceae family. It has gradually become a troublesome weed in several agronomic crops in many countries (Holm et al. Reference Holm, Plucknett, Pancho and Herberger1977), including rice (Oryza sativa L.) (Smith Reference Smith1988), cotton (Gossypium hirsutum L.) (Qiang et al. Reference Qiang, Wei and Hu2000), soybean [Glycine max (L.) Merr.] (Sharma and Amritphale Reference Sharma and Amritphale1988), and peanut (Arachis hypogaea L.) (Altom et al. Reference Altom, Westerman and Murray1995; Wilcut et al. Reference Wilcut, Walls and Horton1991). It not only can directly cause growth and yield reduction but also can endanger crops indirectly by serving as the host of Sclerotinia minor (Melouk et al. Reference Melouk, Damicone and Jackson1992), Amsacta moorei (Galinato et al. Reference Galinato, Moody and Piggin1999), Alternanthera yellow vein virus (He et al. Reference He, Mao, Yu, Wang and Li2008), and Tomato leaf curl New Delhi virus (Haider et al. Reference Haider, Tahir, Latif and Briddon2006). When acting as the host of S. minor, it can reduce production in peanut fields by 25% to 50% (Melouk et al. Reference Melouk, Damicone and Jackson1992). In China, eclipta has widely infested paddy rice fields in Chongqing, Hunan, Hubei, and Jiangsu provinces, and the frequency of occurrence in many areas of the Jiangsu province can reach 100% (Li et al. Reference Li, Zhang and Qiang2009). When ALS inhibitors first appeared as commercial products, they could control this weed effectively. However, under intensive herbicide selection pressure, resistant individuals were repeatedly selected. Farmers have complained recently that the field rate of ALS inhibitors failed to control eclipta in some paddy fields. In our preliminary work in the greenhouse, one ALS inhibitor–resistant population (R) was confirmed.
The aims of this study were (1) to characterize the levels of cross-resistance to four chemical families (SUs, IMIs, TPs, and PTBs) of ALS inhibitors, (2) to determine the difference in ALS sensitivity between susceptible and resistant populations at the biochemical level, and (3) to sequence the ALS gene and detect the ALS mutations conferring resistance.
Materials and Methods
Plant Material
The seeds of two populations were collected in different areas of Jiangsu province in China: one R population identified in our preliminary study and one with known susceptibility (S). The R population seeds were collected in paddy fields with a history of repeated use of pyrazosulfuron-ethyl for more than 10 yr, and the S population seeds were collected in uncultivated areas with no history of herbicide application and used as a control in all biological and molecular assays.
Before planting, seeds were drenched in sterile distilled water at 4 C for 14 d to break dormancy. Then, the germinated seeds were sown into 10.5-cm-diameter pots containing loam soil (organic matter content ≥15%). Pots were placed in the greenhouse (temperature was maintained at 25 to 35 C) and watered and fertilized as required. The seedlings were thinned to 10 evenly sized plants per pot before herbicide application.
Cross-Resistance Whole-Plant Assays
The cross-resistance levels to ALS inhibitors were characterized by whole-plant assays. The herbicides were applied at the 4-leaf stage with a compressed-air, moving-nozzle cabinet sprayer (Compressed air cabinet sprayer 3WPSH-500D, Beijing Research Center for Information Technology in Agriculture, Beijing, China) equipped with one TeeJet® XR8002 flat fan nozzle and calibrated to deliver 367.5 L ha−1 at 0.275 MPa. The applied ALS inhibitors included four SUs (pyrazosulfuron-ethyl, bensulfuron-methyl, metsulfuron-methyl, and tribenuron-methyl), two TPs (pyroxsulam and penoxsulam), two IMIs (imazethapyr and imazapic), and one PTB (bispyribac-sodium); doses are listed in Table 1. A nonionic surfactant solution (a mixture of 78% polyoxyethylene dodecyl ether and 22% water) was added to bispyribac-sodium at 0.1% by volume, and the auxiliary was added to pyroxsulam at 0.1% by volume. The shoot biomass was harvested from ground level 21 d after treatment and dried at 80 C for 48 h, and dry weights were determined. All treatments had three replicates, and each experiment was conducted twice.
Table 1 Basic information concerning ALS inhibitors used and doses applied to eclipta in cross-resistance whole-plant assays.
a WP, wettable powder; WDG, water-dispersible granule; OD, oil dispersion; SC, suspension concentrate; SL, soluble concentrate.
b S, susceptible population; R, resistant population.
c The number in bold represents the field rate (1X).
d PD, pesticide registration number.
In Vitro ALS Activity Assays
Seeds of the eclipta S and R populations were germinated and cultivated under the appropriate conditions described earlier. Fresh plant tissue was harvested at the 4-leaf stage and stored at −80 C. ALS activity was measured based on the method described by Yu et al. (Reference Yu, Friesen, Zhang and Powles2004). Enzyme activity was determined colorimetrically (530 nm) by measuring the amount of acetoin formed. The protein concentration of the crude extract was measured by the Bradford method (Reference Bradford1976). Herbicide doses used for ALS activity assays are listed in Table 2. Three subsamples from each extraction were assayed, and two extractions per population were used.
Table 2 Concentration of ALS inhibitors for eclipta in ALS activity assays in vitro.
a S, susceptible population; R, resistant population.
Statistical Analyses
Nonlinear log-logistic regression analysis was performed to evaluate the data of the aboveground dry biomass and ALS activity using the following model (Equation 1) (Seefeldt et al. Reference Seefeldt, Jensen and Fuerst1995).
$$Y{\,\equals\,}C{\plus}{{D{\,\minus\,}C} \over {1{\plus}\left( {X/\rm ED_{{50}} } \right)^{b} }}$$
In this equation, C is the lower limit, D is the upper limit, b is the slope at ED50 (effective dose producing 50% response). In this study, ED50 represents GR50 (the herbicide dose required for 50% growth reduction) and I50 (the herbicide concentration that inhibited ALS activity by 50%). Y is the response expressed as the percentage of the untreated control at herbicide dose or concentration X. The regression analyses were conducted using Sigma Plot v. 12.0 (Systat Software Inc., San Jose, CA). The resistance index (RI) was determined by dividing the GR50 or I50 of the R population by that of the S population.
ALS Gene Amplification and Sequencing
Genomic DNA Extraction
Fresh leaf tissue from one S population plant and 15 random R population plants was harvested, each sample was placed in plastic bags separately, and then stored at −80 C. Total DNA was isolated with the DNA secure Plant Kit (Tiangen Biotech, Beijing, China). Then, the DNA samples were used immediately for PCR.
Partial ALS Gene Amplification and Sequencing
There are no ALS gene sequences of any Eclipta species available in the National Center for Biotechnology Information (NCBI) GenBank. Based on the sequences of common sunflower (Helianthus annuus L.) (GenBank accession AY541451.1), cocklebur (Xanthium spp.) (GenBank accession U16280.1), and horseweed [Conyza canadensis (L.) Cronq.] (GenBank accession HM067014.1), two pairs of gene-specific primers (1F-1R and 2F-2R) (BGI·Tech, Beijing, China) were designed using Primer Premier v. 5.0 software (Premier Biosoft International, Palo Alto, CA) to amplify the ALS gene (Table 3).
Table 3 Primers designed to amplify the ALS gene.
a F, forward primer; R, reverse primer.
The PCR was conducted in a 15-μl volume containing 0.5 μl genomic DNA (about 75 ng μl−1), 0.3 μl of each primer (10 μM), 1.5 μl 10X PCR Buffer (Mg2+ plus), 1.0 μl dNTPs (2.5 mM), 0.3 μl rTaq DNA polymerase (5 U μl−1) (TaKaRa Biotechnology, Dalian, China), and 11.1 μl ddH2O. The amplification protocol was performed in an Eppendorf AG-22331 Hamburg automated thermal cycler (Eppendorf, Hamburg, Germany) as follows: 10 min denaturation at 94 C; 30 cycles of 0.5 min at 94 C, 0.5 min at annealing temperature, and 1 min at 72 C; then 10 min at 72 C (annealing temperatures for each primer pair are given in Table 3).
PCR products were sequenced commercially, and the sequencing results were aligned using DNAMAN v. 5.2.2 software (Lynnon LLC, San Ramon, CA). The spliced sequences contained domains A, D, F, and B, with high similarity to the ALS genes of other species, as determined by BLAST analysis (NCBI). The sequences were then used for the following assays.
ALS Gene Walking PCR
To amplify the full-length ALS gene, genome walking was used to amplify the 5′ end and 3′ ends of the ALS gene using three nested PCRs. According to the primer design principles of the Genome Walking Kit (TaKaRa Biotechnology, Dalian, China), six primers (5′-SP1, 5′-SP2, 5′-SP3, 3′-SP1, 3′-SP2, 3′-SP3) were designed (Table 3). After three rounds of amplification, the third PCR product was purified and cloned into the pEASY-T1 cloning vector and then transformed into Escherichia coli Trans1-T1 phage-resistant chemically competent cells using the pEASY-T1 Cloning Kit (Beijing TransGen Biotech, Beijing, China). Colonies were chosen, and positive recombinants were sequenced.
Cloning of the ALS Gene Region
After PCR amplification, the sequence of the full-length ALS gene from the S population was confirmed. Based on the obtained sequences, two gene-specific primer pairs: 5′-CCCACCCTGCTTCATCCT-3′ (3F) and 5′-CCGAAACCCATCGCTCCT-3′ (3R); 5′-GGAAGAATAAACAGCCCCAT-3′ (4F) and 5′-AGCCTGCTTACAGAACACAC-3′ (4R) were designed to clone the ALS gene containing eight resistance-endowing amino acid substitutions that have been reported in other resistant species. The PCR reaction volume and reaction condition were the same as that of 1F-1R (or 2F-2R). All PCR products were visualized on a 1% agarose gel with 2,000 bp markers to confirm amplicon size and then sequenced and analyzed.
Results and Discussion
Cross-Resistance Whole-Plant Assays
As expected, the results of whole-plant assay showed that the S population was very sensitive to all herbicides, whereas the R population was broadly resistant to all herbicides (the GR50 values of S and R populations to pyrazosulfuron-ethyl, bensulfuron-methyl, metsulfuron-methyl, tribenuron-methyl, pyroxsulam, penoxsulam, bispyribac-sodium, imazethapyr, imazapic were 0.21 and 28.05, 0.13 and 22.37, 0.07 and 2.12, 0.20 and 39.01, 0.40 and 39.02, 0.22 and 6.68, 0.20 and 33.15, 4.35and 44.43, 0.40 and 7.63 g a.i. ha−1, respectively) (Table 4). Obviously, in whole-plant assays, the GR50 values for the R population were higher than those for the S population for all herbicides. Moreover, the R population was highly resistant to four SUs (pyrazosulfuron-ethyl, 134-fold; bensulfuron-methyl, 172-fold; metsulfuron-methyl, 30-fold; and tribenuron-methyl, 195-fold), two TPs (pyroxsulam, 98-fold; and penoxsulam, 30-fold), and one PTB (bispyribac-sodium, 166-fold), and moderately resistant to two IMIs (imazethapyr, 10-fold; imazapic, 19-fold) (Figure 1; Table 4).
Figure 1 Dose–response curves of shoot dry weight of susceptible (S) and resistant (R) populations to different doses of ALS inhibitors. Error bars represented the SD.
Table 4 Dose–response parameters of eclipta populations in cross-resistance whole-plant assays.
a S population, susceptible population; R population, resistant population.
b GR50, herbicide dose required to decrease shoot dry weight by 50% compared with the untreated control; SE, standard error.
c Resistance index (RI) was determined by calculating the ratio of the GR50 values of resistant population to that of the susceptible population.
Continuous application of ALS inhibitors for several years has resulted in the rapid evolution of herbicide resistance (Heap Reference Heap2016). Several weed species in paddy fields have been reported to be resistant to ALS inhibitors, including monochoria [Monochoria vaginalis (Burm. f.) Kunth] (Wang et al. Reference Wang, Lin, Li, Ito and Itoh2004), Monochoria korsakowii Regel & Maack (Wu et al. Reference Wu, Cao and Liu2007), barnyardgrass [Echinochloa crus-galli (L.) Beauv.] (Yu et al. Reference Yu, Ge, Liu and Li2010), Ammannia arenaria Kunth (Wang et al. Reference Wang, Xu, Zhu, Liu and Wang2013), and threeleaf arrowhead (Sagittaria trifolia L.) (Liu et al. Reference Liu, Liu and Gao2015b). So studying the resistance mechanism of an important malignant broadleaf weed such as the eclipta R population is essential to develop reasonable herbicide-use programs to delay or slow the evolution of resistance.
In Vitro ALS Activity Assays
The results of in vitro ALS activity assays indicated that ALS isolated from the R population was much less susceptible to all herbicides than that isolated from the S population. As shown in Table 5, the I50 values of S and R populations to respective pyrazosulfuron-ethyl, bensulfuron-methyl, metsulfuron-methyl, tribenuron-methyl, pyroxsulam, penoxsulam, bispyribac-sodium, imazethapyr, and imazapic were 0.13 and 15.18, 0.08 and 14.21, 0.04 and 2.54, 0.12 and 9.28, 0.08 and 23.46, 0.16 and 4.45, 0.17 and 33.40, 0.19 and 2.25, 0.19 and 3.47 μM, respectively, and the RI values ranged from 12 to 293 across all the above herbicides (Figure 2; Table 5). The results from the inhibition of ALS activity by ALS inhibitors were similar to the results of whole-plant assays in the greenhouse. More interestingly, the RI values of the R population for SUs, TPs, and PTBs were generally an order of magnitude more resistant than those for IMIs in whole-plant assays and in vitro ALS activity assays.
Figure 2 Dose–response curves of ALS activity of susceptible (S) and resistant (R) populations to different doses of ALS inhibitors. Error bars represented the SD.
Table 5 Dose-response parameters of eclipta population in ALS activity assays in vitro.
a S population, susceptible population; R population, resistant population.
b I50, herbicide dose required to inhibit the ALS activity by 50% compared with the untreated control; SE, standard error.
c Resistance index (RI) was determined by calculating the ratio of the I50 values of resistant population to that of the susceptible population.
In most studies of resistance mechanisms, reduced susceptibility in vitro translates to herbicide resistance in the whole plant. In this study, the reduced susceptibility of ALS from the R population was in accordance with previous research of SU-R water starwort [Myosoton aquaticum (L.) Moench] (Liu et al. Reference Liu, Bi, Li, Yuan, Du and Wang2013) and ALS-R corn poppy (Papaver rhoeas L.) (Kaloumenos et al. Reference Kaloumenos, Adamouli, Dordas and Eleftherohorinos2011).
ALS Gene Amplification and Sequencing
Full-length ALS gene sequences of eclipta without any introns were obtained, and their sequences were highly similar to the ALS genes of sunflower (87%), cocklebur (86%), horseweed (83%), and mayweed chamomile (Anthemis cotula L.) (80%). Using 3F-3R and 4F-4R primers and 1,416- and 847-base pair fragments that included the eight target sites, all fragments were amplified, respectively. Compared with the sequences of the S population, one missense mutation at position 197 was detected in the ALS gene of all plants tested in the R population. This single nucleotide replacement in the ALS gene of the R population resulted in an amino acid substitution of proline (CCC) to serine (TCC) at position 197 (Figure 3). The corresponding sequence chromatograms revealed no double peaks at that site in any resistant plant tested, indicating the mutation was homozygous and inherited steadily as a single dominant monogenic trait (Figure 3).
Figure 3 The sequence chromatograms of eclipta ALS gene sequence at position 197 in susceptible (S) and resistant (R) populations.
This is not the first instance of detection of the Pro-197-Ser substitution, and this substitution endowing herbicide cross-resistance has been reported in other weed species: prostrate pigweed (Amaranthus blitoides S. Wats.) (Sibony and Rubin Reference Sibony and Rubin2003), wild radish (Raphanus raphanistrum L.) (Yu et al. Reference Yu, Han, Li, Purba, Walsh and Powles2012), corn poppy (Kaloumenos et al. Reference Kaloumenos, Adamouli, Dordas and Eleftherohorinos2011), downy brome (Bromus tectorum L.) (Park and Mallory-Smith Reference Park and Mallory-Smith2004), wild mustard (Sinapis arvensis L.) (Warwick et al. Reference Warwick, Sauder and Beckie2005), mayweed chamomile (Intanon et al. Reference Intanon, Perez-Jones, Hulting and Mallory-Smith2011), and water starwort (Liu et al. Reference Liu, Bi, Li, Yuan, Du and Wang2013). In these weed species, this amino acid substitution was correlated with broad cross-resistance to SUs, TPs, PTBs, or SCTs but low or negligible resistance to IMI herbicides. In this study, the eclipta R population had broad cross-resistance to SUs, TPs, and PTBs and moderate resistance to IMIs.
The Pro-197 position in the ALS gene exhibits the highest mutation frequency in amino acid mutations causing ALS resistance in weeds. Substitution types at position 197 occur frequently in cases of resistant weeds, and the Pro has been mutated to Thr, His, Arg, Leu, Gln, Ser, Ala, Ile, Asn, Glu, and Tyr (Heap Reference Heap2016). Different cross-resistance patterns were determined by weed species, mutation types, ALS inhibitor classes, and certain herbicides of a given class (Yu and Powles Reference Yu and Powles2014). The Pro-197-His substitution in smallflower umbrella sedge (Cyperus difformis L.) resulted in broad cross-resistance to SUs (halosulfuron, RI=21), PTBs (bispyribac-sodium, RI=71), IMIs (imazamox, RI=12), and TPs (penoxsulam, RI=22) (Tehranchian et al. Reference Tehranchian, Riar, Norsworthy, Nandula, McElroy, Chen and Scott2015). The Pro-197-Arg substitution in henbit (Lamium amplexicaule L.) resulted in cross-resistance to SUs (chlorsulfuron, RI>1000) and SCTs (propoxycarbazone-sodium, RI=331) but also resulted in sensitivity to IMIs (imazamox) (Varanasi et al. Reference Varanasi, Godar, Peterson, Shoup and Jugulam2015).
This study is the first to confirm the cross-resistance of eclipta to ALS inhibitors due to a Pro-197-Ser point mutation in Jiangsu province in China. Target site–based resistance was conferred by the Pro-197-Ser substitution in the ALS gene, resulting in the insensitivity of isolated ALS to ALS inhibitors. The eclipta populations resistant to ALS inhibitors are becoming a serious constraint in major rice production areas in China, so effective measures to manage the weed resistance are desperately needed.
Acknowledgments
This work was financed by the Special Fund for Agro-scientific Research in the Public Interest (201303031).
