Target-site and non-target-site resistance mechanisms confer multiple resistance to glyphosate and 2,4-D in Carduus acanthoides.

Carduus acanthoides L. is mainly a range-land weed, but in the 2010s has begun to invade GM crop production systems in Córdoba (Argentina), where glyphosate and 2,4-D have been commonly applied. In 2020, C. acanthoides was found with multiple resistance to these two herbicides. In this study, the mechanisms that confer multiple resistance to glyphosate and 2,4-D, were characterized in one resistant (R) population of C. acanthoides in comparison to a susceptible (S) population. No differences in 14C-herbicide absorption and translocation were observed between R and S populations. In addition, 14C-glyphosate was well translocated to the shoots (∼30%) and roots (∼16%) in both R and S plants, while most of 14C-2,4-D remained restricted in the treated leaf. Glyphosate metabolism did not contribute to resistance of the R population; however, as corroborated by malathion pretreatment, the mechanism of resistance to 2,4-D was enhanced metabolism (63% of the herbicide) mediated by cytochrome P450 (Cyt-P450). No differences were found in baseline EPSPS activity, copy number, and/or gene expression between the R and S populations, but a Pro-106-Ser mutation in EPSPS was present in the R population. Multiple resistances in the R population of C. acanthoides from Argentina were governed by target site resistance (a Pro-106 mutation for glyphosate) and non-target site resistance (Cyt-P450-based metabolic resistance for 2,4-D) mechanisms. This is the first case of resistance to glyphosate and 2,4-D confirmed for this weed in the world.

Apigenin Isolated from Carduus crispus Protects against H2O2-Induced Oxidative Damage and Spermatogenic Expression Changes in GC-2spd Sperm Cells.

Testicular oxidative stress is one of the most common factors underlying male infertility. Welted thistle, Carduus crispus Linn., and its bioactive principles are attracting scientific interest in treating male reproductive dysfunctions. Here, the protective effects of apigenin isolated from C. crispus against oxidative damage induced by hydrogen peroxide (H2O2) and dysregulation in spermatogenesis associated parameters in testicular sperm cells was investigated. Cell viabilities, ROS scavenging effects, and spermatogenic associated molecular expressions were measured by MTT, DCF-DA, Western blotting and real-time RT-PCR, respectively. A single peak with 100% purity of apigenin was obtained in HPLC conditions. Apigenin treated alone (2.5, 5, 10 and 20 µM) did not exhibit cytotoxicity, but inhibited the H2O2-induced cellular damage and elevated ROS levels significantly (p < 0.05 at 5, 10 and 20 µM) and dose-dependently. Further, H2O2-induced down-regulation of antioxidant (glutathione S-transferases m5, glutathione peroxidase 4, and peroxiredoxin 3) and spermatogenesis-associated (nectin-2 and phosphorylated-cAMP response element-binding protein) molecular expression in GC-2spd cells were attenuated by apigenin at both protein and mRNA levels (p < 0.05). In conclusion, our study showed that apigenin isolated from C. crispus might be an effective agent that can protect ROS-induced testicular dysfunctions.

Roots of the invasive species Carduus nutans L. and C. acanthoides L. produce large amounts of aplotaxene, a possible allelochemical.

The invasive thistle Carduus nutans has been reported to be allelopathic, yet no allelochemicals have been identified from the species. In a search for allelochemicals from C. nutans and the closely related invasive species C. acanthoides, bioassay-guided fractionation of roots and leaves of each species were conducted. Only dichloromethane extracts of the roots of both species contained a phytotoxin (aplotaxene, (Z,Z,Z)-heptadeca-1,8,11,14-tetraene) with sufficient total activity to potentially act as an allelochemical. Aplotaxene made up 0.44 % of the weight of greenhouse-grown C. acanthoides roots (ca. 20 mM in the plant) and was not found in leaves of either species. It inhibited growth of lettuce 50 % (I 50) in soil at a concentration of ca. 0.5 mg g(-1) of dry soil (ca. 6.5 mM in soil moisture). These values gave a total activity in soil value (molar concentration in the plant divided by the molarity required for 50 % growth inhibition in soil = 3.08) similar to those of some established allelochemicals. The aplotaxene I 50 for duckweed (Lemna paucicostata) in nutrient solution was less than 0.333 mM, and the compound caused cellular leakage of cucumber cotyledon discs in darkness and light at similar concentrations. Soil in which C. acanthoides had grown contained aplotaxene at a lower concentration than necessary for biological activity in our short-term soil bioassays, but these levels might have activity over longer periods of time and might be an underestimate of concentrations in undisturbed and/or rhizosphere soil.

Direct and indirect effects of the synthetic-auxin herbicide dicamba on two lepidopteran species.

Herbicides are the most commonly applied pesticides in agroecosystems, and therefore pose potentially significant ecotoxicological risks to plants and insects. Glyphosate is the most common herbicide worldwide, and glyphosate-resistant weeds are quickly becoming serious challenges in some agroecosystems. Because of this resistance epidemic and the recent development of crops with resistance to dicamba or 2,4-D, herbicide-use patterns are likely to change. Presently, dicamba and 2,4-D cause most herbicide-drift damage to nontarget plants despite limited agricultural usage, but the effects of these synthetic auxin herbicides on insects have been poorly explored. To understand the influence of dicamba on insects, we applied several sublethal, drift-level rates of dicamba to soybean, Glycine max L., and Carduus thistle, and measured growth and survival of Helicoverpa zea (Boddie) and Vanessa cardui (L.) larvae, respectively. For thistle, we measured percent nitrogen content before and after dicamba application. We also performed direct toxicity bioassays on the two caterpillar species with several rates of dicamba. Dicamba was not directly toxic to larvae of either species, and H. zea showed no negative effects when feeding on soybeans dosed with dicamba. We did, however, detect significant negative, indirect effects of higher rates of dicamba on V. cardui larval and pupal mass, total nitrogen of thistles post application, and thistle biomass in the presence of V. cardui larvae. Notably, thistle biomass was not related to dicamba dose in absence of larvae. Our results indicate that dicamba can indirectly influence the performance of some caterpillar species, possibly by altering plant nutritional content.