Ricardo Alcántara-de la Cruz

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico

In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world.

Non-target Site Tolerance Mechanisms Describe Tolerance to Glyphosate in Avena sterilis

Sterile wild oat (Avena sterilis L.) is an autogamous grass established in warm climate regions. This species has been used as a cover crop in Mediterranean perennial crops during the spring period prior to initiating competition with the main crop for water and nutrients. However, such cover crops need to be controlled (by glyphosate or tillage) before the beginning of summer period (due to the possibility of intense drought stress). In 2011, the olive grove farmers of southern Spain expressed dissatisfaction because of the ineffective control with glyphosate on A. sterilis. Experiments were conducted to determine whether the continued use of glyphosate over a 5 year period had selected a new resistant or tolerant species. The GR50 values obtained for A. sterilis were 297.12 and 245.23 g ae ha−1 for exposed (E) and un-exposed (UE) glyphosate accessions, respectively. The spray retention and shikimic acid accumulation exhibited a non-significant difference between the two accessions. The results of 14C- glyphosate absorption was the same in the two accessions (E and UE), while the translocation from the treated leaf to the rest of the shoots and roots was similar in A. sterilis accessions. Glyphosate metabolism to aminomethylphosphonic acid (AMPA) and glyoxylate was similar in both accessions, but increased after treatment with glyphosate, indicating that metabolism plays an important role in tolerance. Both A. sterilis accessions, present similarity in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity enzyme with different glyphosate concentrations and without glyphosate, confirming that both accessions present the same genomic characteristics. The above-mentioned results indicate that innate tolerance to glyphosate in A. sterilis is probably and partly due to reduced herbicide absorption and translocation and metabolism compared to the susceptibility of other grasses weeds like Chloris inflata, Eleusine indica, and Lolium rigidum.

First Resistance Mechanisms Characterization in Glyphosate-Resistant Leptochloa virgata

Leptochloa virgata (L.) P. Beauv. is an annual weed common in citrus groves in the states of Puebla and Veracruz, Mexico limiting their production. Since 2010, several L. virgata populations were identified as being resistant to glyphosate, but studies of their resistance mechanisms developed by this species have been conducted. In this work, three glyphosate-resistant populations (R8, R14, and R15) collected in citrus orchards from Mexico, were used to study their resistance mechanisms comparing them to one susceptible population (S). Dose-response and shikimic acid accumulation assays confirmed the glyphosate resistance of the three resistant populations. Higher doses of up to 720 g ae ha-1 (field dose) were needed to control by 50% plants of resistant populations. The S population absorbed between 7 and 13% more 14C-glyphosate than resistant ones, and translocated up to 32.2% of 14C-glyphosate to the roots at 96 h after treatment (HAT). The R8, R14, and R15 populations translocated only 24.5, 26.5, and 21.9%, respectively. The enzyme activity of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was not different in the S, R8 and R14 populations. The R15 Population exhibited 165.9 times greater EPSPS activity. Additionally, this population showed a higher EPSPS basal activity and a substitution in the codon 106 from Proline to Serine in the EPSPS protein sequence. EPSPS gene expression in the R15 population was similar to that of S population. In conclusion, the three resistant L. virgata populations show reduced absorption and translocation of 14C-glyphosate. Moreover, a mutation and an enhanced EPSPS basal activity at target-site level confers higher resistance to glyphosate. These results describe for the first time the glyphosate resistance mechanisms developed by resistant L. virgata populations of citrus orchards from Mexico.

Pro-106-Ser mutation and EPSPS overexpression acting together simultaneously in glyphosate-resistant goosegrass ( Eleusine indica )

Glyphosate has been used for more than 15 years for weed management in citrus groves in the Gulf of Mexico, at up to 3–4 applications per year. Goosegrass (Eleusine indica (L.) Gaertn.) control has sometimes failed. In this research, the mechanisms governing three goosegrass biotypes (Ein-Or from an orange grove, and Ein-Pl1 and Ein-Pl2 from Persian lime groves) with suspected resistance to glyphosate were characterized and compared to a susceptible biotype (Ein-S). Dose-response and shikimate accumulation assays confirmed resistance of the resistant (R) biotypes. There were no differences in glyphosate absorption, but the R biotypes retained up to 62–78% of the herbicide in the treated leaf at 96 h after treatment (HAT), in comparison to the Ein-S biotype (36%). The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity in the Ein-Or and Ein-S biotypes was over 100-fold lower than the Ein-Pl1 and Ein-Pl2 ones. The latter showed a high EPSPS-basal activity, a mutation at Pro-106-Ser position in the EPSPS gene, and EPSPS overexpression. The EPSPS basal and EPSPS overexpression were positively correlated. The R goosegrass biotypes displayed poor glyphosate translocation. Furthermore, this grassweed showed, for the first time, two mechanisms at the target-site level (Pro-106-Ser mutation + EPSPS overexpression) acting together simultaneously against glyphosate.

From tolerance to resistance: mechanisms governing the differential response to glyphosate in Chloris barbata

BACKGROUND Susceptibility and the mechanism (s) governing tolerance/resistance to glyphosate were characterized in two putative-glyphosate-resistant Chloris barbata populations (R1 and R2), collected in Persian lime orchards from Colima State, Mexico, comparing them with one non-treated population (referred to as S). RESULTS Glyphosate doses required to reduce fresh weight or cause mortality by 50% were 4.2-6.4 times higher in resistant populations than in the S population. The S population accumulated 4.3 and 5.2 times more shikimate than the R2 and R1 populations, respectively. There were no differences in 14 C-glyphosate uptake between R and S populations, but the R plants translocated at least 12% less herbicide to the rest of plant and roots 96 h after treatment. Insignificant amounts of glyphosate were metabolized to aminomethyl phosphonate and glyoxylate in both R and S plants. The 5-enolpyruvylshikimate-3-phosphate synthase gene of the R populations contained the Pro106-Ser mutation, giving them a resistance 12 (R2) and 14.7 (R1) times greater at target-site level compared with the S population. CONCLUSION The Pro106-Ser mutation governs the resistance to glyphosate of the R1 and R2 C barbata populations, but the impaired translocation could contribute to the resistance. These results confirm the first case of glyphosate resistance evolved in this species.

Glyphosate-based herbicides toxicity on life history parameters of zoophytophagous Podisus nigrispinus (Heteroptera: Pentatomidae)

The increase of agricultural areas with glyphosate-resistant (GR) crops, and use of this herbicide in Brazil, makes necessary to assess its impacts on non-target organisms. The objective was to evaluate the development, reproduction and life table parameters of Podisus nigrispinus (Heteroptera: Pentatomidae) reared on GR-soybean plants treated with glyphosate formulations (Zapp-Qi, Roundup-Transorb-R and Roundup-Original) at the recommended field dose (720g acid equivalent ha-1). Glyphosate formulations had no affect on nymph and adult weight of this predator. Fourth instar stage was shortest with Zapp Qi. Egg-adult period was similar between treatments (26 days) with a survival over 90%. Zapp-Qi and Roundup-Transorb-R (potassium-salt: K-salt) reduced the egg, posture and nymph number per female, and the longevity and oviposition periods of this predator. Podisus nigrispinus net reproductive rate was highest in GR-soybean plants treated with Roundup-Original (isopropylamine-salt: IPA-salt). However, the duration of one generation, intrinsic and finite increase rates, and time to duplicate the population, were similar between treatments. Glyphosate toxicity on P. nigrispinus depends of the glyphosate salt type. IPA-salt was least harmless to this predator. Formulations based on K-salt altered its reproductive parameters, however, the development and population dynamic were not affect. Therefore, these glyphosate formulations are compatible with the predator P. nigrispinus with GR-soybean crop.

Side-effects of pesticides on the generalist endoparasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae).

New plant protection strategies focus on minimizing chemical pesticide use and increasing their compatibility with biological control agents. The objective was to evaluate the side-effects of glyphosate, diflubenzuron, malathion, tebuconazole and triflumuron (at 720, 45, 400, 150 and 20 g ai ha−1, respectively), pesticides authorized for soybean crops in Brazil, on the parasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae) reared on Anticarsia gemmatalis (Lepidoptera: Noctuidae). The emergence and female numbers produced per P. elaeisis female were higher in A. gemmatalis pupae from caterpillars fed an artificial diet treated with glyphosate. However, emergence was lower than 50% when the caterpillars were fed on soybean leaves treated with glyphosate offered ad libitum (3–5 times). Palmistichus elaeisis died before parasiting A. gemmatalis pupae treated with malathion. Diflubenzuron reduced the P. elaesis sex ratio in the second generation. Tebuconazole and triflumuron did not cause side-effects on this parasitoid. A continuous exposure to glyphosate by the host may lead to side-effects on P. elaeisis emergence, but its moderate use is acceptable for this parasitoid. Diflubenzuron had severe transgenerational side-effects. Tebuconazole fungicide and triflumuron insecticide are compatible with P. elaeisis in sustainable integrated pest management (IPM) programs, while malathion can not be included in them.

Differential Resistance Mechanisms to Glyphosate Result in Fitness Cost for Lolium perenne and L. multiflorum

Multiple mechanisms of resistance to glyphosate are exhibited by populations of Lolium spp. worldwide. Association of resistance with growth and reproductive fitness is an important predictor for long-term success of glyphosate-resistant (R) versus glyphosate-susceptible (S) biotypes. Numerous studies were conducted on R- and S-biotypes of Italian ryegrass (Lolium multiflorum) and perennial ryegrass (L. perenne) to characterize the underlying mechanism(s) of glyphosate resistance and associate this with growth and reproductive fitness. L. perenne expressed both altered uptake and translocation as well as a genetic change at 106-Pro to –Ser, This pattern for two resistance mechanisms is unique. L. multiflorum also exhibited altered uptake and translocation as well as duplication of EPSPS gene copies. Reduced plant biomass and height for R-versus S-biotypes of both species was evident over two growing seasons. This resulted in S- versus R- L. multiflorum producing up to 47 and 38% more seeds in 2014 and 2015, respectively. S- L. perenne produced up to 20 and 30% more seeds in 2014 and 2015, respectively. Both non-target site and target-site mechanisms of glyphosate resistance can render Lolium spp. at a competitive disadvantage. This has long-term implications for the success of glyphosate-resistant plants in the absence of selection pressure.

Genetic Relationships between Tropical Sprangletop (Leptochloa virgata) Populations from Mexico: Understanding Glyphosate Resistance Spread

The susceptibility to glyphosate and genetic diversity based on intersimple sequence repeat markers were characterized for 17 tropical sprangletop populations collected from two separate regions mainly in Persian lime groves in Veracruz, Mexico. The whole-plant dose response together with shikimic acid assays indicated different levels of glyphosate resistance in those populations. Genetic diversity values (h) estimated using POPGENE ranged from 0.119 to 0.198 and 0.117 to 0.214 within susceptible and resistant populations, respectively. The average genetic diversity (HS) within the susceptible populations was 0.157, and the total genetic diversity (HT) was 0.218. The HS of the resistant populations was 0.144, and the HT was 0.186. The analysis of molecular variance based on the response to glyphosate indicated that most of the genetic variation was found within groups of susceptible and resistant populations (90% of the genetic variation), whereas 10% or less was among groups. The high level of genetic diversity between glyphosate-resistant tropical sprangletop populations from distant and adjacent locations is likely due to both short- and long-distance seed dispersal and independent evolutionary events in tropical sprangletop populations among Persian lime groves in Veracruz. Nomenclature: Glyphosate; tropical sprangletop, Leptochloa virgata (L.) P. Beauv.; Persian lime, Citrus latifolia Tan.

Underlying Resistance Mechanisms in the Cynosurus echinatus Biotype to Acetyl CoA Carboxylase-Inhibiting Herbicides

Hedgehog dogtail (Cynosurus echinatus) is an annual grass, native to Europe, but also widely distributed in North and South America, South Africa, and Australia. Two hedgehog dogtail biotypes, one diclofop-methyl (DM)-resistant and one DM-susceptible were studied in detail for experimental dose-response resistance mechanisms. Herbicide rates that inhibited shoot growth by 50% (GR50) were determined for DM, being the resistance factor (GR50R/GR50S) of 43.81. When amitrole (Cyt. P450 inhibitor) was applied before treatment with DM, the R biotype growth was significantly inhibited (GR50 of 1019.9 g ai ha-1) compared with the GR50 (1484.6 g ai ha-1) found for the R biotype without pretreatment with amitrole. However, GR50 values for S biotype do not vary with or without amitrole pretreatment. Dose-response experiments carried out to evaluate cross-resistance, showed resistance to aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ) inhibiting herbicides. Both R and S biotypes had a similar 14C-DM uptake and translocation. The herbicide was poorly distributed among leaves, the rest of the shoot and roots with unappreciable acropetal and/or basipetal DM translocation at 96 h after treatment (HAT). The metabolism of 14C-DM, D-acid and D-conjugate metabolites were identified by thin-layer chromatography. The results showed that DM resistance in C. echinatus is likely due to enhanced herbicide metabolism, involving Cyt. P450 as was demonstrated by indirect assays (amitrole pretreatment). The ACCase in vitro assays showed that the target site was very sensitive to APP, CHD and PPZ herbicides in the C. echinatus S biotype, while the R biotype was insensitive to the previously mentioned herbicides. DNA sequencing studies confirmed that C. echinatus cross-resistance to ACCase inhibitors has been conferred by specific ACCase double point mutations Ile-2041-Asn and Cys-2088-Arg.