Tribenuron Methyl Research Articles

Multiresidue Analysis of Pesticides in Wine and Grape Using Gas Chromatography with Microelectron Capture and Nitrogen–Phosphorus Detection

New multiresidue methods for identification and quantification of over 131 pesticides in wine and grapes have been developed. The analyses were based on gas chromatography with microelectron capture and nitrogen–phosphorus detection (GC-μECD/NPD). Two different extraction protocols (QuEChERS-citrate buffer and unbuffered methods) for wine samples were described. A full validation study of both analytical methods was carried out, including different approaches to cleanup which were evaluated: (a) extracts cleanup via dispersive solid-phase extraction (SPE), (b) the combination of simultaneous extraction with dispersive SPE and (c) extracts without cleanup. Both analytical procedures presented good performance for all analytes, with recoveries in the range of 72–113% and average relative standard deviation (RSD) ≤ 20%. Linearity was studied in the range between 0.009 and 0.023 mg/kg and showed coefficients of determination (R2) ≥ 0.99 for all pesticides. Estimated matrix effects in wine samples which were prepared with the presented methods for most compounds were < 10%. Determination of pesticide residues in grape samples was carried out by the modified QuEChERS approach. Satisfying parameters of recovery and precision at the two spiking levels (LOQ and 100 × LOQ) were achieved almost for all analytes in the range of 70–120% with RSD ≤ 20% (some exceptions were benthiavalicarb isopropyl, carbosulfan, dichlorvos, flazasulfuron, flubendiamide, mevinphos, tetrachlorvinphos and tribenuron methyl). Linearity was observed for all analytes in the concentration range of 0.002–4.158 mg/kg with coefficients of determination ≥ 0.99 except for fenpropidin. The expanded uncertainties were estimated by using a “top-down” approach as being 18% (on average) and 25% (on average) for grape and wine samples, respectively (coverage factor k = 2, confidence level 95%). Finally, the methods were applied with success to determination of pesticide residues in commercial wine and grape fruit samples.

Male Sterility of an AHAS-Mutant Induced by Tribenuron-Methyl Solution Correlated With the Decrease of AHAS Activity in Brassica napus L.

Tribenuron-methyl (TBM), an acetohydroxyacid synthase (AHAS)-inhibiting herbicide, can be used as an efficient chemical hybridization agent to induce male sterility for practical utilization of heterosis in rapeseed (Brassica napus L.). Utilization of rapeseed mutants harboring herbicide-resistant AHAS alleles as the male parent can simplify the hybrid seed production protocol. Here we characterized a novel TBM-resistant mutant K5 derived from an elite rapeseed variety, Zhongshuang No. 9 (ZS9), by ethyl methyl sulfonate mutagenesis. Comparative analysis of three BnAHAS genes (BnAHAS1, BnAHAS2, and BnAHAS3) between the mutant K5 and ZS9 identified a C-to-T transition at 544 from the translation start site in BnAHAS1 in K5 (This resistant allele is referred to as BnAHAS1544T), which resulted in a substitution of proline with serine at 182 in BnAHAS1. Both ZS9 and K5 plants could be induced complete male sterility under TBM treatment (with 0.10 and 20 mg⋅L-1 of TBM, respectively). The relationship between TBM-induced male sterility (Y) and the relative AHAS activity of inflorescences (X) could be described as a modified logistic function, Y = 100-A/(1+Be(-KX)) for the both genotypes, although the obtained constants A, B, and K were different in the functions of ZS9 and K5. Transgenic Arabidopsis plants expressing BnAHAS1544T exhibited a higher TBM resistance of male reproductive organ than wild type, which confirmed that the Pro-182-Ser substitution in BnAHAS1 was responsible for higher TBM-resistance of male reproductive organs. Taken together, our findings provide a novel valuable rapeseed mutant for hybrid breeding by chemical hybridization agents and support the hypothesis that AHAS should be the target of the AHAS-inhibiting herbicide TBM when it is used as chemical hybridization agent in rapeseed.

Continuous Use of Tribenuron-Methyl Selected for Cross-Resistance to Acetolactate Synthase–inhibiting Herbicides in Wild Mustard (Sinapis arvensis)

AbstractWild mustard (Sinapis arvensis L.) is a weed that frequently infests winter wheat (Triticum aestivum L.) fields in Golestan province, Iran. Tribenuron-methyl (TM) has been used recurrently to control this species, thus selecting for resistant S. arvensis populations. The objectives were: (1) to determine the resistance level to TM of 14 putatively resistant (PR) S. arvensis populations, collected from winter wheat fields in Golestan province, Iran, in comparison to one susceptible (S) population; and (2) to characterize the resistance mechanisms and the potential evolution of cross-resistance to other classes of acetolactate synthase (ALS)-inhibiting herbicides in three populations (AL-3, G-5, and Ag-Sr) confirmed as being resistant (R) to TM. The TM doses required to reduce the dry weight of the PR populations by 50% were between 2.2 and 16.8 times higher than those needed for S plants. The ALS enzyme activity assays revealed that the AL-3, G-5, and Ag-Sr populations evolved cross-resistance to the candidate ALS-inhibiting herbicides from the sulfonylureas (SU), triazolopyrimidines (TP), pyrimidinyl-thiobenzoates (PTB), sulfonyl-aminocarbonyl-triazolinone (SCT), and imidazolinones (IMI) classes. No differences in absorption, translocation, or metabolism of [14C]TM between R and S plants were observed, suggesting that these non-target mechanisms were not responsible for the resistance. The ALS gene of the R populations contained the Trp-574-Leu mutation, conferring cross-resistance to the SU, SCT, PTB, TP, and IMI classes. The Trp-574-Leu mutation in the ALS gene conferred cross-resistance to ALS-inhibiting herbicides in S. arvensis from winter wheat fields in Golestan province. This is the first TM resistance case confirmed in this species in Iran.

Preparation of a monolithic magnetic stir bar for the determination of sulfonylurea herbicides coupled with HPLC

A hybrid material of metal organic frameworks (MOFs) and monoliths was developed and used for stir bar sorption extraction (SBSE) of five sulfonylurea herbicides. It was prepared in a glass tube (4 mm I.D.) by thermal polymerization using modified neodymium supermagnetic (Nd2Fe14B) powders as the component substrates, the organic polymerization solutions and ethyl cellulose as the adhesion agent. The prepared stir bar was characterized by scanning electron micrography (SEM) and X-ray diffraction (XRD). It was successfully applied for the determination of sulfonylurea herbicides in soil and lake water samples coupled with high-performance liquid chromatography (HPLC). The extraction and desorption factors affecting the performance of sulfonylurea herbicides were evaluated in detail. Under the optimum conditions, the extraction efficiency of the hybrid stir bar was obviously higher than the blank stir bar without UIO-66(Zr)-NH2. Linear ranges were 10–700 μg/L for thifensulfuron-methyl (TIM), amidosulfuron (AS) and metsulfuron-methyl (MSM) and 10–800 μg/L for sulfosulfuron (SSF) and tribenuron-methyl (TBM), respectively. The obtained detection limits were in the ranges of 0.04–0.84 μg/L with the recoveries of 68.8–98.1% for the real samples. The novel stir bar possessed excellent advantages of both supermagnetic performance and large specific surface areas in the presence of MOFs and monoliths, which was potentially an optimal adsorbent for environmental analysis.

Individual and combined effects of herbicide tribenuron-methyl and fungicide tebuconazole on soil earthworm Eisenia fetida

Earthworms are soil engineers that alter the soil bio-physical properties to favor plant growth whereas pesticides represent a significant threat to their abundance and soil health. Thus, we investigated the toxic effects of tribenuron-methyl (TBM) and tebuconazole (TEB) on the soil earthworm, Eisenia fetida. The TBM demonstrated low toxicity to E. fetida in the contact filter paper and artificial soil tests, with median lethal concentrations (LC50) of 135.6 μg cm−2 at 48 h and 511 mg kg−1 on day 14, respectively. Similarly, TEB also showed low toxicity to E. fetida in the artificial soil test with LC50 of 287 mg kg−1 on day 14. However, TEB was highly toxic to earthworm in the contact filter paper test with LC50 of 5.7 μg cm−2 at 48 h. The mixture of two pesticides had an antagonistic effect on the earthworm. Under 0.1 LC50 of TBM and TEB, either single or combined application of pesticides induced oxidative stress and inhibited cellulase activity in early days of the earthworm exposure. However, both pesticides did not damage the earthworm DNA. Our results suggest that pesticides can negatively affect soil earthworms and provide valuable information regarding the responses of soil biological engineers to the lethal agrochemicals.

Autophagy contributes to sulfonylurea herbicide tolerance via GCN2-independent regulation of amino acid homeostasis

ABSTRACTSulfonylurea (SU) herbicides inhibit branched-chain amino acid (BCAA) biosynthesis by targeting acetolactate synthase. Plants have evolved target-site resistance and metabolic tolerance to SU herbicides; the GCN2 (general control non-repressible 2) pathway is also involved in SU tolerance. Here, we report a novel SU tolerance mechanism, autophagy, which we call ‘homeostatic tolerance,’ is involved in amino acid signaling in Arabidopsis. The activation and reversion of autophagy and GCN2 by the SU herbicide tribenuron-methyl (TM) and exogenous BCAA, respectively, confirmed that TM-induced BCAA starvation is responsible for the activation of autophagy and GCN2. Genetic and biochemical analyses revealed a lower proportion of free BCAA and more sensitive phenotypes in atg5, atg7, and gcn2 single mutants than in wild-type seedlings after TM treatment; the lowest proportion of free BCAA and the most sensitive phenotypes were found in atg5 gcn2 and atg7 gcn2 double mutants. Immunoblotting and microscopy revealed that TM-induced activation of autophagy and GCN2 signaling do not depend on the presence of each other, and these 2 pathways may serve as mutually compensatory mechanisms against TM. TM inhibited the TOR (target of rapamycin), and activated autophagy in an estradiol-induced TOR RNAi line, suggesting that TM-induced BCAA starvation activates autophagy, probably via TOR inactivation. Autophagy and GCN2 were also activated, and independently contributed to TM tolerance in plants conferring metabolic tolerance. Together, these data suggest that autophagy is a proteolytic process for amino acid recycling and contributes to GCN2-independent SU tolerance, probably by its ability to replenish fresh BCAA.

Improved application of tribenuron-methyl as a chemical hybridizing agent with forchlorfenuron for rapeseed hybrid breeding

Chemicals or agents that can induce plant male sterility are considered chemical hybridizing agents (CHAs) and are quite useful for plant hybrid breeding. Tribenuron-methyl (TBM) can effectively induce male sterility in rapeseed plants. However, applications of TBM in rapeseed breeding practices have not been reported. Here, treatment with the CHA referred to as CHA-TBM, which contained 0.1–0.9 mg/L TBM, induced male sterility in all 22 tested rapeseed inbred lines. However, the highest induced male sterile rates in the tested rapeseed lines were only approximately 54–57% together with poisoned rates of approximately 36–39%, which were resulted from two treatments of CHA-TBM containing 0.5 mg/L TBM at an interval of 9 days beginning at the floral bud stage (FBS) of 2.0–3.5 mm. Such male sterile rates and poisoned rates caused by CHA-TBM indicated that TBM is not an ideal CHA for rapeseed hybrid breeding. The results of many screening experiments showed that forchlorfenuron can alleviate the toxic effects of TBM on rapeseed plants. Two treatments with the CHA referred to as CHA-TBM-FCF, which contained 0.6 mg/L TBM and 2.1 mg/L forchlorfenuron, at an interval of 9 days beginning at the FBS of 2.0–3.5 mm induced male sterility in approximately 96–98% of plants of all the 22 tested rapeseed inbred lines and poisoned only approximately 2–4% of the tested plants. The CHA-TBM-FCF treatment did not affect pistil fertility of the rapeseed plants, and had no carry-over influence on stamen fertility of the rapeseed hybrids produced with this CHA. Some rapeseed hybrids were successfully produced using CHA-TBM-FCF (e.g., Qinrong 1, Qinrong 2, Qinyou 19, Qinyou 33, Qinzayou 4 and Qinzayou 5). The hybridity values of the F1 seeds of the rapeseed hybrids produced using CHA-TBM-FCF were determined to be approximately 93–98% with the simple sequence repeat (SSR) marker techniques. Together, the results indicate that CHA-TBM-FCF containing TBM and forchlorfenuron at suitable concentrations is an ideal CHA for rapeseed hybrid breeding.

Susceptibility of American burnweed (Erechtities hieraciifolius) to herbicides and clipping in wild blueberry (Vaccinium angustifolium Ait.)

American burnweed is an increasingly common annual weed in wild blueberry fields in Atlantic Canada. Knowledge of susceptibility to several common herbicides used in wild blueberry, however, is lacking, and it is unclear how burnweed responds to clipping. Using greenhouse-grown plants, the objectives of this research were to evaluate burnweed susceptibility to various herbicides registered in wild blueberry and to determine the effect of clipping on burnweed lateral branch and flower production. Postemergence broadcast applications of mesotrione, flumioxazin, glufosinate, clopyralid, foramsulfuron, glyphosate, tribenuron methyl, nicosulfuron/rimsulfuron, terbacil, and hexazinone provided >90% control of burnweed. Postemergence spot applications of clopyralid, glyphosate, mesotrione, and nicosulfuron/rimsulfuron to 24 ± 0.3 cm tall burnweed plants caused 60%–97% injury, but <50% of treated plants died. In contrast, spot applications of glufosinate, tribenuron methyl, and foramsulfuron provided complete control of treated plants. Efficacy of postemergence broadcast mesotrione and foramsulfuron applications declined with increasing burnweed plant height, although foramsulfuron provided greater control of plants >10 cm in height than mesotrione. A tank mixture of mesotrione and foramsulfuron was effective on burnweed and may be useful for controlling plants of varying size with broadcast applications in the field. Removal of 25%, 50%, and 75% of aboveground burnweed stems with clipping increased lateral branch numbers relative to untreated control plants, and 100% removal of the aboveground stem was required to prevent lateral branching and flowering. Stem removal through mowing under field conditions will likely have limited effects on burnweed, and herbicides are recommended for burnweed management in wild blueberry.

Degradation and adsorption of tebuconazole and tribenuron-methyl in wheat soil, alone and in combination

In practice, pesticides are usually simultaneously applied or one after another for crop protection. This often leads to a coexistence of pesticide mixtures in soil. One of the most important processes influencing the behavior of a pesticide in the environment is its degradation and adsorption in soil. Thus, the degradation and adsorption behaviors of tebuconazole (TEB) and tribenuron-methyl (TBM) alone and combined application in wheat soil (pH 7.28) were studied. The concentrations of TEB and TBM in soil were determined using a quick, easy, cheap, efficient, rugged and safe method (QuEChERS) with ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Results showed that TEB has longer half-lives compared with TBM in the tested soils when they were applied individually. The degradation of TEB was markedly affected by concentration and soil microorganisms. In the non-sterilized soil, half-lives of 0.8 and 80 mg kg-1 TEB were 16.4 and 69.3 d, respectively, and half-lives of 0.8 and 80 mg kg-1 TEB were 20.4 and 73.5 d in the sterilized soil, respectively. The degradation of TBM was not affected by TEB in both soils; however, TBM could reduce the degradation of TEB. Compared to TEB alone, the rates of degradation of 80 mg kg-1 TEB in presence of 32 mg kg-1 TBM were reduced 15.9% and 18.2% in non-sterilized and sterilized soils, respectively. Soil adsorption affinity of TEB was significantly higher than TBM. When used combined, adsorption capacity of TEB was not affected by TBM. However, the adsorption of TBM was obviously inhibited in presence of TEB.

Effect of sulfonylurea tribenuron methyl herbicide on soil Actinobacteria growth and characterization of resistant strains.

Repeated application of pesticides disturbs microbial communities and cause dysfunctions on soil biological processes. Granstar® 75 DF is one of the most used sulfonylurea herbicides on cereal crops; it contains 75% of tribenuron-methyl. Assessing the changes on soil microbiota, particularly on the most abundant bacterial groups, will be a useful approach to determine the impact of Granstar® herbicide. For this purpose, we analyzed Actinobacteria, which are known for their diversity, abundance, and aptitude to resist to xenobiotic substances. Using a selective medium for Actinobacteria, 42 strains were isolated from both untreated and Granstar® treated soils. The number of isolates recovered from the treated agricultural soil was fewer than that isolated from the corresponding untreated soil, suggesting a negative effect of Granstar® herbicide on Actinobacteria community. Even so, the number of strains isolated from untreated and treated forest soil was quite similar. Among the isolates, resistant strains, tolerating high doses of Granstar® ranging from 0.3 to 0.6% (v/v), were obtained. The two most resistant strains (SRK12 and SRK17) were isolated from treated soils showing the importance of prior exposure to herbicides for bacterial adaptation. SRK12 and SRK17 strains showed different morphological features. The phylogenetic analysis, based on 16S rRNA gene sequencing, clustered the SRK12 strain with four Streptomyces type strains (S. vinaceusdrappus, S. mutabilis, S. ghanaensis and S. enissocaesilis), while SRK17 strain was closely related to Streptomyces africanus. Both strains were unable to grow on tribenuron methyl as unique source of carbon, despite its advanced dissipation. On the other hand, when glucose was added to tribenuron methyl, the bacterial development was evident with even an improvement of the tribenuron methyl degradation. In all cases, as tribenuron methyl disappeared, two compounds were detected with increased concentrations. These by-products appeared to be persistent and were not degraded either chemically or by the studied strains. Based on these observations, we suggested that bacterial activity on carbon substrates could be directly involved in the partial breakdown of tribenuron methyl, by generating the required acidity for the first step of the hydrolysis. Such a process would be interesting to consider in bioremediation of neutral and alkaline tribenuron methyl-polluted soils.

Tribenuron methyl exposure inhibits oogenesis in zebrafish Danio rerio (Hamilton, 1822)

Tribenuron methyl is a herbicide that is used to control annual and perennial dicots in cereals and fallow land. Pesticides can access aquatic ecosystems via groundwater and affect water quality and aquatic life. Our study evaluated histopathological effects of tribenuron methyl on ovary of the zebrafish Danio rerio (Hamilton, 1822). Ovary of the fishes in control group not exposed to the herbicide showed normal histological architecture. In the fishes exposed to tribenuron methyl, vacuolisation, edema and dysmorphism were recorded. Significant decrease in the number of primary and mature oocytes as well as dose dependent increase in the number of atretic oocytes were recorded in fishes exposed to tribenuron methyl.

The influence of humic acids extracted from Chaouia soil on the behavior of transition metal ions and pesticides

Humic acid (HA) and fulvic acid (FA) exist ubiquitously in environments and have a variety of functional groups, which allow them to complex with metal ions and pesticides. Furthermore, these interactions can not only alter the environmental behavior, but also influence the removal and transportation of those pollutants. The study of the interaction between Cu (II), VO (II) and Mn (II) with HAs provides environmental information on the oxidation states of paramagnetic metals and their mechanisms of binding to humic acids. Electron spin resonance (ESR) study demonstrates that VO (II) and Cu (II) ions are bound with oxygen ligands to HAs, while the Mn (II) complex occurs as [Mn(H 2 O) 6 ] 2+ . Additionally, Cu (II) ions are more strongly bound than Mn (II) ions to soil HAs. The effect of the presence of HA on photolysis of Tribenuron-methyl (TRB) and Imazapyr (IMAZ) herbicides in water was studied by irradiation of different mixtures of HA/herbicide (0.5:1 and 1:1 by volume). The obtained results clearly demonstrate that HA substances exhibit a screening effect on the photochemical degradation of the two herbicides. The protective effect of HAs on the TRB and IMAZ degradation could be explained with an inclusion and/or adsorption of the herbicide molecules in the humic matrix.

Determination of Two Sulfonylurea Herbicides Residues in Soil Environment Using HPLC and Phytotoxicity of These Herbicides by Lentil Bioassay.

A HPLC-UV detection system was used for determination of sulfosulfuron and tribenuron methyl residues from soils. The soils were fortified with sulfosulfuron and tribenuron methyl at rates of 26 and 15g a.i. ha-1 respectively and samples were taken randomly on 0 (2h), 1, 2, 4, 10, 20, 40, 60, 90 and 120 days after treatment. The final extracts were prepared for analysis by HPLC. The results showed that degradation of both herbicides in the silty loam soil was faster than sandy loam soil. Half-life of sulfosulfuron was ranged from 5.37 to 10.82 days however this value for tribenuron methyl was ranged from 3.23 to 5.72 days on different soils. The residue of both herbicides at 120 days after application in wheat field had no toxicitic effect on lentil. It was concluded that HPLC analysis procedure was an appropriate method for determination of these herbicides from soils.

Influence of Nitrogen Rate, Seeding Rate, and Weed Removal Timing on Weed Interference in Barley and Effect of Nitrogen on Weed Response to Herbicides

Field experiments were conducted at the Montana State University Southern Agricultural Research Center, Huntley, MT, in 2011 through 2013 to determine the effect of nitrogen (N) rate, seeding rate, and weed removal timing on weed interference in barley. A delay in weed removal timing from the 3- to 4-leaf (LF) stage to the 8- to 10-LF stage of barley resulted in up to 3.5-fold increase in total weed biomass and 10% reduction in barley biomass, and this was unaffected by a N rate that ranged from 56 (low) to 168 (high) kg ha−1. The effect of N rate on barley biomass was more pronounced when weed removal was delayed from the 3- to 4-LF stage to the 8- to 10-LF stage of barley and in nontreated plots. Increasing the barley seeding rate from 38 to 152 kg ha−1increased the barley plant density by 50%, biomass by 13%, and grain yield by 29%, averaged over N rates and weed removal timing. On the basis of 5 and 10% levels of acceptable yield loss, the addition of ≥112 kg N ha−1delayed the critical timing of weed removal by at least 1.3 wk in barley, compared with the 56 kg N ha−1rate. A medium or high N rate prevented reduction in barley grain quality (plumpness and test weight) observed when the seeding rate was increased from 38 to 76 or 152 kg ha−1at the low N rate. In a separate greenhouse study, the effect of N rate on the effectiveness of various herbicides for controlling wild oat, green foxtail, kochia, or Russian thistle was investigated. Results highlighted that wild oat or green foxtail grown under 56 kg N ha−1(low N) soil required 1.4 to 2.6 times higher doses of clodinafop, fenoxaprop, flucarbazone, glyphosate, glufosinate, pinoxaden, or tralkoxydim for 50% reduction in shoot dry weights (GR50) compared with plants grown under 168 kg N ha−1(high N). Similarly, a reduced efficacy of thifensulfuron methyl + tribenuron methyl, metsulfuron methyl, or bromoxynil+pyrasulfotole was observed (evident from the GR50values) for kochia or Russian thistle grown under low- vs. high-N soil. Information gained from this research will aid in developing cost-effective, integrated weed management (IWM) strategies in cereals and in educating growers on the importance of fertilizer N management as a component of IWM programs.

Evaluation of four different crops’ sensitivity to sulfosulfuron and tribenuron methyl soil residues

ABSTRACTOn the basis of greenhouse bioassays, the sensitivities of root and shoot biomass of barley (Hordeum vulgare L.), corn (Zea mays L.), oilseed rape (Brassica napus L.) and sugar beet (Beta vulgaris L.) to soil-incorporated sulfosulfuron and tribenuron methyl were assessed. Shoot and root biomass production was measured 30 days after emergence. Plant responses, including roots and shoots dry weight per pot, were described by a three parameter log-logistic regression model as a function of sulfosulfuron or tribenuron methyl doses and the relative sensitivities were calculated at the various ED-levels with their associated 95% confidence intervals. The most precise ED-levels were that at ED50 and sensitivity rank was oilseed rape, sugar beet, corn and barley, whatever the chosen response level and herbicide. We calculated relative sensitivities at ED10, ED50 and ED90 for the species of barley, the most tolerant crop, as reference. Comparison of relative sensitivity of crops to both herbicides showed that the sensitivity of these crops was much larger for tribenuron methyl than for sulfosulfuron. Oilseed rape was the most sensitive species (ED50 = 0.202 and 0.179 µg kg−1 soil for root dry weight (RDW) response to sulfosulfuron and tribenuron methyl, respectively) while barley was the most tolerant one (ED50 = 1.008 and 3.68 µg kg−1soil for RDW response to sulfosulfuron and tribenuron methyl, respectively). Sugar beet and corn had intermediate sensitivity. Also, we demonstrated how important it is to show the confidence intervals of relative sensitivities. In several instances the relative sensitivities, even numerically large, were not significantly different from 1.00. We demonstrate that classifying biotypes as resistant to a herbicide requires the threshold of resistance/susceptible (R/S) of 4.00 only be accepted if confidence intervals do not cover 1.00.

Generation of tribenuron‐methyl herbicide‐resistant OsCYP81A6‐expressing rapeseed (Brassica napus L.) plants for hybrid seed production using chemical‐induced male sterility

AbstractChemical‐induced male sterility (CIMS) is a method for hybrid rapeseed (Brassica napus L.) production. Some sulphonylurea herbicides such as tribenuron‐methyl (TBM) are used as chemical hybridization agents (CHAs) in CIMS systems. However, the male parents must be protected from herbicide injury with a shield during spraying of the female parents with CHAs to induce male sterility. Thus, using herbicide‐resistant rapeseed lines as the male parents can significantly simplify the seed production procedure and reduce the cost in hybrid seed production. A rice cytochrome P450 hydroxylase, OsCYP81A6, has been previously characterized to confer resistance to bentazon and sulphonylurea herbicides. We demonstrate here that the introduction of OsCYP81A6 renders rapeseed plants resistant to TBM. Compared with wild‐type plants, the transgenic plants displayed normal stamen development and male fertility when treated with 0.05 mg/l of TBM, the dose used for inducing male sterility in hybrid seed production. These results indicate that the OsCYP81A6‐expressing rapeseed plants can be used as the male parents for hybrid rapeseed production using CIMS.

Multiple Mechanisms Increase Levels of Resistance in Rapistrum rugosum to ALS Herbicides.

Rapistrum rugosum (turnip weed) is a common weed of wheat fields in Iran, which is most often controlled by tribenuron-methyl (TM), a sulfonylurea (SU) belonging to the acetolactate synthase (ALS) inhibiting herbicides group. Several cases of unexplained control failure of R. rugosum by TM have been seen, especially in Golestan province-Iran. Hence, there is lack of research in evaluation of the level of resistance of the R. rugosum populations to TM, using whole plant dose-response and enzyme assays, then investigating some potential resistance mechanisms Results revealed that the resistance factor (RF) for resistant (R) populations was 2.5–6.6 fold higher than susceptible (S) plant. Neither foliar retention, nor 14C-TM absorption and translocation were the mechanisms responsible for resistance in turnip weed. Metabolism of TM was the second resistant mechanism in two populations (Ag-R5 and G-1), in which three metabolites were found. The concentration of TM for 50% inhibition of ALS enzyme activity in vitro showed a high level of resistance to the herbicide (RFs were from 28 to 38) and cross-resistance to sulfonyl-aminocarbonyl-triazolinone (SCT), pyrimidinyl-thiobenzoate (PTB) and triazolopyrimidine (TP), with no cross-resistance to imidazolinone (IMI). Substitution Pro 197 to Ser 197 provided resistance to four of five ALS-inhibiting herbicides including SU, TP, PTB, and SCT with no resistance to IMI. These results documented the first case of R. rugosum resistant population worldwide and demonstrated that both RST and NRST mechanisms are involved to the resistance level to TM.

Molecular Basis of Evolved Resistance to Glyphosate and Acetolactate Synthase-Inhibitor Herbicides in Kochia (Kochia scoparia) Accessions from Montana

The rapid evolution and spread of glyphosate-resistant (GR) kochia in the Northern Great Plains is an increasing threat to GR cropping systems and conservation tillage practices common in this region. GR kochia accessions with 4.6- to 11-fold levels of resistance to glyphosate have recently been reported in Montana. Those GR kochia accessions were also suspected to be resistant to acetolactate synthase (ALS) inhibitors, i.e., multiple herbicide-resistant (MHR) kochia. In this research, the level of resistance to the ALS-inhibitor herbicides (sulfonylureas) and the molecular mechanisms conferring resistance to glyphosate and ALS-inhibitor herbicides in MHR kochia was investigated. On the basis of whole-plant dose–response assays, MHR kochia accessions (GIL01, JOP01, and CHES01) were 9.3- to 30-fold more resistant to premixed thifensulfuron methyl + tribenuron methyl + metsulfuron methyl than the susceptible (SUS) accession. In an in vivo leaf-disk shikimate assay, MHR plants accumulated less shikimate than the SUS plants at a discriminate dose of 100 μM glyphosate. Sequencing of the conserved region ofEPSPSrevealed no target-site mutation at Thr102or Pro106residue. MHR kochia accessions had increased relativeEPSPSgene copies (~ 4 to 10) compared with the SUS accession (single copy). Furthermore, MHR kochia accumulated higher EPSPS protein compared with the SUS plants. Resistance to the ALS-inhibitor herbicides was conferred by Pro197amino acid substitution (proline to glutamine).EPSPSgene amplification and a single target-site mutation at Pro197inALSgene confer resistance to glyphosate and ALS-inhibitor herbicides, respectively, in MHR kochia accessions from Montana. This is the first confirmation of occurrence of MHR kochia in Montana.

Grain Yield and Quality of Foxtail Millet (Setaria italica L.) in Response to Tribenuron-Methyl.

Foxtail millet (Setaria italica L.) is cultivated around the world for human and animal consumption. There is no suitable herbicide available for weed control in foxtail millet fields during the post-emergence stage. In this study, we investigated the effect and safety of the post-emergence herbicide tribenuron-methyl (TBM) on foxtail millet in terms of grain yield and quality using a split-plot field design. Field experiments were conducted using two varieties in 2013 and 2014, i.e., high-yielding hybrid Zhangzagu 10 and high-quality conventional Jingu 21. TBM treatments at 11.25 to 90 g ai ha−1 reduced root and shoot biomass and grain yield to varying degrees. In each of the two years, grain yield declined by 50.2% in Zhangzagu 10 with a herbicide dosage of 45 g ai ha−1 and by 45.2% in Jingu 21 with a herbicide dosage of 22.5 g ai ha−1 (recommended dosage). Yield reduction was due to lower grains per panicle, 1000-grain weight, panicle length, and panicle diameter. Grain yield was positively correlated with grains per panicle and 1000-grain weight, but not with panicles ha−1. With respect to grain protein content at 22.5 g ai ha−1, Zhangzagu 10 was similar to the control, whereas Jingu 21 was markedly lower. An increase in TBM dosage led to a decrease in grain Mn, Cu, Fe, and Zn concentrations. In conclusion, the recommended dosage of TBM was relatively safe for Zhangzagu 10, but not for Jingu 21. Additionally, the hybrid variety Zhangzagu 10 had a greater tolerance to TBM than the conventional variety Jingu 21.

Tribenuron-Methyl Induces Male Sterility through Anther-Specific Inhibition of Acetolactate Synthase Leading to Autophagic Cell Death

Tribenuron-methyl (TM) is a powerful sulfonylurea herbicide that inhibits branched-chain amino acid (BCAA) biosynthesis by targeting the catalytic subunit (CSR1) of acetolactate synthase (ALS). Selective induction of male sterility by foliar spraying of TM at low doses has been widely used for hybrid seed production in rapeseed (Brassica napus); however, the underlying mechanism remains unknown. Here, we report greater TM accumulation and subsequent stronger ALS inhibition and BCAA starvation in anthers than in leaves and stems after TM application. Constitutive or anther-specific expression of csr1-1D (a CSR1 mutant) eliminated anther-selective ALS inhibition and reversed the TM-induced male sterile phenotype in both rapeseed and Arabidopsis. The results of TM daub-stem experiments, combined with the observations of little TM accumulation in anthers and reversion of TM-induced male sterility by targeted expression of the TM metabolism gene Bel in either the mesophyll or phloem, suggested that foliar-sprayed TM was polar-transported to anthers mainly through the mesophyll and phloem. Microscopy and immunoblotting revealed that autophagy, a bulk degradation process induced during cell death, was elevated in TM-induced male sterile anthers and by anther-specific knockdown of ALS. Moreover, TM-induced pollen abortion was significantly inhibited by the autophagy inhibitor 3-MA. These data suggested that TM was polar-transported to anthers, resulting in BCAA starvation via anther-specific ALS inhibition and, ultimately, autophagic cell death in anthers.