Distribution and Mechanism of Japanese Brome (Bromus japonicus) Resistance to ALS-Inhibiting Herbicides in China.

Bromus japonicus is a common monocot weed that occurs in major winter wheat fields in the Huang-Huai-Hai region of China. Pyroxsulam is a highly efficient and safe acetolactate synthase (ALS)-inhibiting herbicide that is widely used to control common weeds in wheat fields. However, B. japonicus populations in China have evolved resistance to pyroxsulam by different mutations in the ALS gene. To understand the resistance distribution, target-site resistance mechanisms, and cross-resistance patterns, 208 B. japonicus populations were collected from eight provinces. In the resistant population screening experiment, 59 populations from six provinces showed different resistance levels to pyroxsulam compared with the susceptible population, of which 17 B. japonicus populations with moderate or high levels of resistance to pyroxsulam were mainly from the Hebei (4), Shandong (4) and Shanxi (9) Provinces. Some resistant populations were selected to investigate the target site-resistance mechanism to the ALS-inhibiting herbicide pyroxsulam. Three pairs of primers were designed to amplify the ALS sequence, which was assembled into the complete ALS sequence with a length of 1932 bp. DNA sequencing of ALS revealed that four different ALS mutations (Pro-197-Ser, Pro-197-Thr, Pro-197-Phe and Asp-376-Glu) were found in 17 moderately or highly resistant populations. Subsequently, five resistant populations, QM21-41 with Pro-197-Ser, QM20-8 with Pro-197-Thr and Pro-197-Phe, and QM21-72, QM21-76 and QM21-79 with Asp-376-Glu mutations in ALS genes, were selected to characterize their cross-resistance patterns to ALS inhibitors. The QM21-41, QM20-8, QM21-72, QM21-76 and QM21-79 populations showed broad-spectrum cross-resistance to pyroxsulam, mesosulfuron-methyl and flucarbazone-sodium. This study is the first to report evolving cross-resistance to ALS-inhibiting herbicides due to Pro-197-Phe mutations in B. japonicus.

Development and inter-laboratory validation of analytical methods for glufosinate and its two metabolites in foods of plant origin.

Glufosinate is widely used to control various weeds. Glufosinate and its main metabolites have become the focus of attention because of their high water solubility and persistence in aquatic systems. Quantification of the agrochemical product and its metabolite residues is essential for the safety of agricultural products. In this study, a highly specific, simple method was developed to directly determine glufosinate and its metabolite residues in 21 plant origin foods by liquid chromatography with tandem mass spectrometry (LC-MS/MS), and it was validated on 11 foods in five laboratories. Finally, the repeatability limit, reproducibility limit, and uncertainty of the method were calculated based on these validated data and used to support the more accurate detection results. Four different chromatographic columns were used to analyze three target compounds, and the anionic polar pesticide column showed the optimum separation and peak shape. Composition of the mobile phase, extraction solvent, and the clean-up procedure were optimized. The developed method was validated on 21 plant origin foods. The average recoveries were 74-115% for all matrices. The validation results of five laboratories showed this method had a good repeatability (RSDr < 9.5%) and reproducibility (RSDR < 18.9%). The method validation parameters met the requirements of guidance established by the European Union (EU) and China for pesticide residue analysis. This methodology can be used for a routine monitoring that performs well for glufosinate and its metabolite residues.

Enhanced metabolic resistance mechanism endows resistance to metamifop in Echinochloa crus-galli (L.) P. Beauv.

Barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.), one of the worst weeds in paddy fields in China, has been frequently reported evolving resistance to acetyl-CoA carboxylase (ACCase) inhibiting herbicides. However, in the previous research, more attention was paid to target-site resistance (TSR) mechanisms, the non-target-site resistance (NTSR) mechanisms have not been well-established. In this study, the potential mechanism of resistance in a metamifop-resistant E. crus-galli collected from Kunshan city, Jiangsu Province, China was investigated. Dose-response assays showed that the phenotypic resistant population (JS-R) has evolved 4.3-fold resistance to metamifop compared with the phenotypic susceptible population (YN-S). The ACCase CT gene sequencing and relative ACCase gene expression levels studies showed that no mutations were detected in the ACCase CT gene in both YN-S and JS-R, and there was no significant difference in the relative ACCase gene expression between YN-S and JS-R. After the pre-processing of glutathione-S-transferase (GSTs) inhibitor NBD-Cl, the resistance level of JS-R to metamifop was reversed 18.73%. Furthermore, the GSTs activity of JS-R plants was significantly enhanced compared to that of YN-S plants. UPLC-MS/MS revealed that JS-R plants had faster metabolic rates to metamifop than YN-S plants. Meanwhile, the JS-R popultion exhibited resistant to cyhalofop-butyl and penoxsulam. In summary, this study presented a novel discovery regarding the global emergence of metabolic resistance to metamifop in E. crus-galli. The low-level resistance observed in the JS-R population was not found to be related to TSR but rather appeared to be primarily associated with the overexpression of genes in the GSTs metabolic enzyme superfamily.

Target gene mutations endowed cross-resistance to acetolactate synthase-inhibiting herbicides in wild Brassica juncea.

Wild Brassica juncea is a troublesome weed that infests wheat fields in China. Two suspected wild B. juncea populations (19-5 and 19-6) resistant to acetolactate synthase (ALS) inhibitors were collected from wheat fields in China. To clarify their resistance profiles and resistance mechanism, the resistance levels of populations 19-5 and 19-6 to ALS-inhibiting herbicides and their underlying target-site resistance mechanism were investigated. The results showed that the 19-5 population exhibited resistance to tribenuron-methyl, pyrithiobac­sodium and florasulam, while the 19-6 population was resistant to tribenuron-methyl, pyrithiobac­sodium, imazethapyr and florasulam. Using the homologous cloning method, two ALS genes were identified in wild B. juncea, with one gene (ALS1) encoding 652 amino acids and the other (ALS2) encoding 655 amino acids. Pro-197-Arg mutation on ALS2 and Trp-574-Leu mutation on ALS1, together with the combination of these two mutations in a single plant, were observed in both 19-5 and 19-6 populations. ALS2 enzymes carrying the Pro-197-Arg mutation were cross-resistant to tribenuron-methyl, pyrithiobac­sodium, imazerthapyr and florasulam, with resistance index (RI) values of 6.23, 32.81, 7.97 and 1162.50, respectively. Similarly, ALS1 enzymes with Trp-574-leu substitutions also displayed high resistance to these four herbicides (RI values ranging from 132.61 to 3375.00). In addition, the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations increased the resistance level of the ALS enzyme to ALS inhibitors, with its RI values 3.83-214.19, 6.88-37.34, 1.91-31.82 and 2.03-5.90-fold higher than a single mutation for tribenuron-methyl, pyrithiobac­sodium, imazerthapyr and florasulam, respectively. Collectively, Pro-197-Arg mutation on ALS2, Trp-574-Leu mutation on ALS1 and the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations in wild B. juncea could endow broad-spectrum resistance to ALS inhibitors, which might provide guides for establishing effective strategies to prevent or delay such resistance evolution in this weed.

Potential Role of EPSPS Mutations in the Resistance of Eleusine indica to Glyphosate.

Gene mutation is a basic evolutionary mechanism in plants under selection pressure of herbicides. Such mutation has pleiotropic effects on plant growth. We systemically investigated the effects of Pro106Leu (P106L), Pro106Ser (P106S), and Thr102Ile + Pro106Ser (TIPS) mutations on EPSPS functionality and fitness traits in Eleusine indica at the biochemical and physiological levels. The affinity of natural EPSPS for glyphosate was 53.8 times higher than that for phosphoenolpyruvate (PEP), as revealed by the dissociation constant; the constant decreased in both the P106L (39.9-fold) and P106S (46.9-fold) mutants but increased in the TIPS (87.5-fold) mutant. The Km (PEP) values of the P106L, P106S, and TIPS mutants were 2.4-, 0.7-, and 4.1-fold higher than that of natural EPSPS, corresponding to resistance levels of 2.5, 1.9, and 11.4, respectively. The catalytic efficiency values (maximum reaction rates) were 0.89-, 0.94-, and 0.26-fold higher than that of natural EPSPS. The levels of metabolites related to amino acids and nucleotides were significantly reduced in the mutated plants. The fitness costs were substantial for the biomass, total leaf area, seed number, and seedling emergence throughout the growth period in the plants with P106L and TIPS mutations. These results provide insights into EPSPS kinetics and their effect on plant growth.

Metabolism Difference Is Involved in Mesosulfuron-Methyl Selectivity between Aegilops tauschii and Triticum aestivum.

The acetolactate synthase (ALS) inhibitor mesosulfuron-methyl is currently the only selective herbicide to control Aegilops tauschii in wheat fields; however, the mechanism underlying this selectivity remains unclear. Results showed that the tolerance of Triticum aestivum to mesosulfuron-methyl was much higher than that of A. tauschii. Mesosulfuron-methyl inhibited the in vitro ALS activity of A. tauschii and T. aestivum similarly, but the predicted structural interactions of ALS with mesosulfuron-methyl and induced expression of als were different in the two species. Compared with T. aestivum, A. tauschii was found to absorb more mesosulfuron-methyl and metabolize much less mesosulfuron-methyl. The cytochrome P450 monooxygenase (CYP450) inhibitor, malathion, greatly increased the sensitivity of T. aestivum to mesosulfuron-methyl, while its synergistic effect was smaller in A. tauschii. Finally, 19 P450 genes were selected as candidate genes related with metabolism-based mesosulfuron-methyl selectivity. Collectively, different sensitivities to mesosulfuron-methyl in the two species were likely to be attributed to metabolism variances.

PsbA gene over-expression and enhanced metabolism conferring resistance to atrazine in Commelina communis.

Commelina communis L. is a troublesome weed in agronomic fields and increasingly threatens the yield security of corn in north-eastern China. Previously, we found that a C. communis population (JL-1) has evolved resistance to atrazine. Although the potential genetic and enzymic differences contributing to atrazine resistance in this population have been investigated, the specific molecular mechanisms underlying C. communis resistance are still poorly understood. Here, the expression level of the target gene PsbA and the non-target-site resistance (NTSR) mechanism for this population were studied. The results showed that the decline in chlorophyll content in JL-1 leaves was less than in the susceptible JS-10 population following atrazine treatment. JL-1 exhibited an enhanced expression of the PsbA gene compared with JS-10 of 7.28- and 14.28-fold higher at 0 and 24 h after treatment with atrazine, respectively. The cytochrome P450 monooxygenase (P450) inhibitor piperonyl butoxide (PBO) increased the phytotoxicity of atrazine in both populations of C. communis. Seven candidate genes associated with NTSR of Jl-1 were identified through RNA-seq and validated by quantitative real-time PCR, including 5 upregulated genes involved in herbicide metabolism. In addition, the activities of glutathione S-transferases and P450s in JL-1 were increased compared with JS-10. Collectively, PsbA gene overexpression and enhanced metabolism are likely to be responsible for JL-1 resistance to atrazine.

EST-SSR Markers' Development Based on RNA-Sequencing and Their Application in Population Genetic Structure and Diversity Analysis of Eleusine indica in China.

Goosegrass (Eleusine indica) is one of the worst agricultural weeds in China. Molecular markers were developed for genetic diversity and population structure analyses. In this study, we identified 8391 expressed sequence tag-simple sequence repeat (EST-SSR) markers from the de novo assembled unigenes of E. indica. Mononucleotides were the most abundant type of repeats (3591, 42.79%), followed by trinucleotides (3162, 37.68%). The most dominant mononucleotide and trinucleotide repeat motifs were A/T (3406, 40.59%) and AAT/ATT (103, 1.5%), respectively. Fourteen pairs of EST-SSR primers were verified and used to analyze the genetic diversity and population structure of 59 goosegrass populations. A total of 49 alleles were amplified, with the number of alleles (Na) ranging from two to eleven per locus, and the effective number of alleles (Ne) ranged from 1.07 to 4.53. The average polymorphic information content (PIC) was 0.36. Genetic structure analysis (K = 2) and principal coordinate analysis divided 59 E. indica populations into two groups in a manner similar to the unweighted pair-group method (Dice genetic similarity coefficient = 0.700). This study developed a set of EST-SSR markers in E. indica and successfully analyzed the diversity and population genetic structures of 59 E. indica populations in China.

High Antioxidant Ability Confer Resistance to Atrazine in Commelina communis L.

Asiatic dayflower (Commelina communis L.) is a detrimental weed that mainly infests corn and soybean fields in China. Recently, some C. communis populations have exhibited resistance to atrazine, intensifying the difficulties in controlling the weed. However, little is known on the mechanism underlying C. communis resistance to atrazine. Therefore, two populations collected from Jilin (JL-1) and Jiangsu (JS-10) provinces of China were used to evaluate their growth responses to atrazine. The results showed that the JL-1 population displayed a low level of resistance to atrazine compared with JS-10 population, with the resistant index (RI) value of 2.9. To determine if a mutation in the psbA gene was the basis for varied resistance to this herbicide, the full-length gene encoding 353 amino acids with no intron was sequenced by using genome-walking techniques. No mutation known to confer resistance to atrazine was observed in either JL-1 or JS-10 populations. The malondialdehyde (MDA) contents relative to the control group were significantly higher in JS-10 population than in JL-1 population at 7 days after treatment with atrazine, suggesting that atrazine induced severer oxidant damage on JS-10 population. Additionally, significantly enhanced activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX), were detected in the JL-1 population, which was most likely to confer resistance to atrazine. To the best of our knowledge, this is the first investigation into the potential genetic and enzymatic differences contributing to atrazine resistance in this population.

Vegetative and Fecundity Fitness Benefit Found in a Glyphosate-Resistant Eleusine indica Population Caused by 5-Enolpyruvylshikimate-3-Phosphate Synthase Overexpression.

Fitness is an important trait in weed species that have developed herbicide resistance, including resistance to the popular herbicide glyphosate. Fitness cost is commonly found in weeds with glyphosate resistance, which is caused by target-site mutations. In this study, the vegetative and fecundity fitness traits in a glyphosate-resistant (GR) Eleusine indica population caused by 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) overexpression were investigated under glyphosate-free conditions. The results showed that the resistance index of the population resistant (R) to glyphosate compared with that of the population susceptible (WT) to it was approximately 4.0. Furthermore, EPSPS expression level in the R plants was 20.1-82.7 times higher than that in the WT plants. The dry weight of the R population was significantly higher than that of the WT population at the later growth stage after planting; a similar trend was observed for leaf area. In addition, seed production in the R population was 1.4 times higher than that in the WT population. The R and WT populations showed similar maximum germination rates and T50 values. UPLC-MS/MS was performed for the metabolic extracts prepared from the leaves of R and WT populations to address changes in the metabolome. A total of 121 differential metabolites were identified between R and WT individuals. The levels of 6-hydroxy-1H-indole-3-acetamide and indole acetaldehyde, which are associated with auxin synthesis, were significantly higher in plants of the R population than in those of the WT population. However, some secondary metabolite levels were slightly lower in the R population than in the WT population. To conclude, in this study, vegetative and fecundity fitness benefits were found in the GR E. indica population. The results of metabolome analysis indicate that the increase in 6-hydroxy-1H-indole-3-acetamide and indole acetaldehyde levels may be the result of fitness benefit. Further studies should be conducted to confirm the functions of these metabolites.

Selection and Validation of Reference Genes for RT-qPCR Analysis in Aegilops tauschii (Coss.) under Different Abiotic Stresses.

Aegilops tauschii (Coss.) is an aggressive and serious annual grass weed in China. Its DD genome is a rich source of genetic material and performs better under different abiotic stress conditions (salinity, drought, temperature, etc.). Reverse-transcribed quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for reference gene selection and validation. This work aimed to evaluate the stability of reference gene expression in Ae. tauschii under different abiotic stresses (salinity, drought, hot, and cold) and developmental stages (seedling and development). The results show that the ubiquitin-conjugating enzyme E2 36-like (UBC36) and protein microrchidia 2-like (HSP) are the most stable genes under control and salinity conditions, respectively. Under drought stress conditions, UBC36 is more stable as compared with others. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is the most stable reference gene during heat stress conditions and thioredoxin-like protein (YLS) under cold stress condition. Phosphate2A serine/threonine-protein phosphatase 2A (PP2A) and eukaryotic translation initiation factor 3 (ETIF3) are the most stable genes at seedling and developmental stages. Intracellular transport protein (CAC) is recommended as the most stable gene under different abiotic stresses and at developmental stages. Furthermore, the relative expression levels of NHX1 and DREB under different levels of salinity and drought stress conditions varied with the most (HSP and UBC36) and least (YLS and ACT) stable genes. This study provides reliable reference genes for understanding the tolerance mechanisms in Ae. tauschii under different abiotic stress conditions.

Genetic Diversity and Synergistic Modulation of Salinity Tolerance Genes in Aegilops tauschii Coss.

Aegilops tauschii Coss. (2n = 2x = 14, DD) is a problematic weed and a rich source of genetic material for wheat crop improvement programs. We used physiological traits (plant height, dry weight biomass, Na+ and K+ concentration) and 14 microsatellite markers to evaluate the genetic diversity and salinity tolerance in 40 Ae. tauschii populations. The molecular marker allied with salinity stress showed polymorphisms, and a cluster analysis divided the populations into different groups, which indicated diversity among populations. Results showed that the expression level of AeHKT1;4 and AeNHX1 were significantly induced during salinity stress treatments (50 and 200 mM), while AeHKT1;4 showed relative expression in roots, and AeNHX1 was expressed in leaves under the control conditions. Compared with the control conditions, the expression level of AeHKT1;4 significantly increased 1.7-fold under 50 mM salinity stress and 4.7-fold under 200 mM salinity stress in the roots of Ae. tauschii. AeNHX1 showed a relative expression level of 1.6-fold under 50 mM salinity stress and 4.6-fold under 200 mM salinity stress compared with the control conditions. The results provide strong evidence that, under salinity stress conditions, AeHKT1;4 and AeNHX1 synergistically regulate the Na+ homeostasis through regulating Na+ transport in Ae. tauschii. AeNHX1 sequestrated the Na+ into vacuoles, which control the regulation of Na+ transport from roots to leaves under salinity stress conditions in Ae. tauschii.

Characterization of glyphosate and quizalofop-p-ethyl multiple resistance in Eleusine indica.

Glyphosate and Acetyl-coenzyme A Carboxylase (ACCase) inhibitors are popular herbicides that control goosegrass. However, some populations are difficult to control due to resistance resulting from the increasing selection pressure. The objectives of this research were to detect the multiple resistance levels, resistance mechanisms, and fitness costs of two goosegrass populations collected in China. The resistance indices of two resistant populations (denominated as R1 and R2) to glyphosate were 3.8 and 2.3, respectively; and it was 18.0 and 14.2 to quizalofop-p-ethyl, respectively. Shikimate accumulation in R1 and R2 populations was only 8% of that of the susceptible population after glyphosate treatment. A Pro-106-Ala mutation in EPSPS and an Asp-2078-Gly mutation in ACCase were present in both resistant populations. Both the expression level of EPSPS and ACCase in resistant populations were similar to that of susceptible populations. The leaf area of the individuals in wild-type populations was more than three times of the leaf area in the resistant populations. Similarly, resistant plants were 45-49% shorter, had 70-76% less fresh shoot weight, and 67-69% fewer seeds than wild-type plants. Goosegrass populations have evolved multiple resistance to glyphosate and the ACCase inhibitor quizalofop-p-ethyl in China. The Pro-106-Ala mutation in the EPSPS and the Asp-2078-Gly mutation in the ACCase were responsible for this resistance. In addition, a fitness cost exists in the resistant populations, and more work should conduct to clear which mutation is responsible for the fitness penalty.

Regulation of 2,4-D Isooctyl Ester on Triticum aestivum and Aegilops tauschii Tillering and Endogenous Phytohormonal Responses.

Tillering is an important agronomic trait essential for the yield of Triticum aestivum and the propagation of Aegilops tauschii. However, the effect of phytohormones on T. aestivum and Ae. tauschii tillering and the underlying regulatory mechanisms remain poorly understood. In the study, we found that T. aestivum and Ae. tauschii exhibited different tillering sensitivities to the auxin herbicide 2,4-D isooctyl ester. At 3 days post-application, tiller bud growth was inhibited by 77.50% in T. aestivum, corresponding to 2.0-fold greater inhibition than that in Ae. tauschii (38.71%). Transcriptome analysis showed that differentially expressed genes (DEGs) in the T. aestivum response to 2,4-D isooctyl ester were mainly enriched in plant hormone metabolism and signal transduction pathways, but similar changes were not observed in Ae. tauschii. Among that, the auxin biosynthesis and signaling induced by 2,4-D isooctyl ester was quite different between the two species. A total of nine candidate genes involved in varied tillering responses were selected from the DEGs and validated by quantitative real-time PCR. Endogenous hormone levels were assayed to further verify the RNA-seq results. After 2,4-D isooctyl ester treatment, a significant increase in abscisic acid (ABA) levels was observed in T. aestivum, whereas ABA levels were relatively stable in Ae. tauschii. The herbicide induced more cytokinin (CTK) accumulation in Ae. tauschii than in T. aestivum. External ABA clearly restricted tiller bud growth in both T. aestivum and Ae. tauschii, while 6-benzyl aminopurine had no significant effect. These results indicate that ABA and CTK may be related with 2,4-D isooctyl ester-regulated tillering differences between the two species, which will help to further understand the mechanism of the auxin-mediated regulation of tillering.

Herbal Medicine "Shulifenxiao" Formula for Nephrotic Syndrome of Refractory Idiopathic Membranous Nephropathy.

Background: Treatment for adult patients with refractory idiopathic membranous nephropathy (RIMN) by conventional immunosuppressive regimens is not satisfactory. This study aims to evaluate the effectiveness of Chinese herbal medicine, Shulifenxiao formula, as a promising regimen. Methods: A total of 31 RIMN patients resistant to corticosteroid or immunosuppressive agents were retrospectively analyzed. Shulifenxiao treatment lasted a minimum of 12°months in all patients and extended to 24°months in 11 patients. The primary outcomes [the complete remission (CR) and partial remission (PR)] and secondary outcomes (the serum creatinine and estimated glomerular filtration rate (eGFR) levels) were measured at 6, 12, 18, and 24°months. Results: The data provided an average follow-up of 21 ± 9.16°months from baseline. The remission was attained in 25/31 patients (80.7%: CR 29.0% and PR 51.6%) at 12°months and in 10/11 patients (90.9%: CR 54.6% and PR 36.4%) at 24°months, respectively. Proteinuria reduced from 6.02 g/d at baseline to 0.98 g/d at 12°months (p < 0.001) and to 0.27 g/d at 24°months (p = 0.003); serum albumin increased from 28 g/L to 37.2 g/L at 12°months (p < 0.001) and to 41.3 g/L at 24°months (p = 0.003); eGFR improved from 100.25 ml/min/1.73 m2 to 118.39 ml/min/1.73 m2 at 6°months (p < 0.001) and finally to 111.62 ml/min/1.73 m2at 24°months (p = 0.008). Only two patients developed subsequent relapse. Conclusion: Shulifenxiao formula as a clinical cocktail therapy serves as an alternative therapeutic option for steroid and immunosuppressant-resistant RIMN patients, with a favourable safety profile, though further studies are warranted. Clinical Trial registration: http://www.chictr.org.cn, Chinese Clinical Trials Registry [ChiCTR1800019351].

Different leaf-mediated deposition, absorbed and metabolism behaviors of 2,4-D isooctyl ester between Triticum aestivum and Aegilops tauschii Coss.

Tausch's goatgrass (Aegilops tauschii Coss.), is a major weed species, infesting wheat (Triticum aestivum) fields in China. 2,4-D isooctyl ester is widely used for broadleaf weed control and selected as a tool to study the differences between, A. tauschii and T. aestivum. In this study, we measured the growth responses of these species to 2,4-D isooctyl ester and found that T. aestivum was more sensitive to the herbicide than A. tauschii. To clarify the reasons for this difference, we measured the leaf-mediated deposition, absorption and metabolism of 2,4-D isooctyl ester and the expression of auxin receptor transport inhibitor response (TIR1) gene in T. aestivum and A. tauschii. The results indicated that the deposition of 2,4-D isooctyl ester droplets may be lower on A. tauschii than on T. aestivum, because of the increased contact angle and greater density of trichomes on the leaves of the former. A distinct increase in 2,4-D isooctyl ester uptake was detected in T. aestivum during the entire experimental period, and the rate was 2.2-fold greater than that in A. tauschii at 6 h after treatment. Compared with A. tauschii, T. aestivum exhibited a greater accumulation of primary metabolite 2,4-D in plants, which may be responsible for the different responses of the two species. Additionally, the absolute expression level of TIR1 was clearly greater in T. aestivum than that in A. tauschii. These data will be helpful to further understand the differences between T. aestivum and A. tauschii, which may provide a unique perspective for the development and identification of new target compounds that are effective against this weed species.

RNA-Seq transcriptome analysis to identify candidate genes involved in non-target site-based mesosulfuron-methyl resistance in Beckmannia syzigachne.

American sloughgrass (Beckmannia syzigachne Steud.) has become a dominant weed in fields with rice-wheat rotation. Moreover, herbicide resistance has rendered weed control difficult. We identified a biotype showing resistance to ALS inhibitor mesosulfuron-methyl with a resistant index 3.3, but without any ALS mutation. This study aims to identify and confirm the factors associated with non-target site resistance of this biotype to mesosulfuron-methyl using RNA-Seq. 118,111 unigenes were assembled, and 50.9% of these were annotated across seven databases. Eleven contigs related to metabolic resistance were identified based on differential expression via RNA-Seq which include a novel resistance-related transcription factor (MYC3) and two disease resistance proteins were also identified (At1g58602 and At1g15890). Fold changes in expression of these genes in comparison M-R vs. M-S ranged from 3.9 to 11.6, as confirmed by qPCR. The expression of a contig annotated as cytochrome P450 (CYP86B1) in resistant individuals was over 3 times higher than that in sensitive individuals at 0-72 h after mesosulfuron-methyl treatment. A similar trend was noted for three other genes annotated as glutathione S-transferase (GST), namely GST-T3, GST-U6, and GST-U14; the expression of GST-U6 in resistant individuals was up to 142.3 times higher than that in sensitive individuals at 24 h after mesosulfuron-methyl treatment. In addition, GST activity in resistant individuals was 2.1 to 5.3 times higher than that in sensitive individuals. The GR50 of resistant biotype decreased from 24.4 to 11.3 g a.i. ha-1 after P450 inhibitor malathion treatment. This study identified a cytochrome P450 gene CYP86B1 and three GST genes GST-T3, GST-U6, and GST-U14 that have higher expression in mesosulfuron-methyl resistant B. syzigachne, suggesting that both P450- and GST-based activities could be involved in resistance.

Pro-197-Ser Mutation in ALS and High-Level GST Activities: Multiple Resistance to ALS and ACCase Inhibitors in Beckmannia syzigachne.

American sloughgrass (Beckmannia syzigachne Steud.) is one of the most troublesome weeds infesting wheat and canola fields in China. Some biotypes cannot be controlled, either by acetolactate synthase (ALS) or acetyl coenzyme A carboxylase (ACCase) inhibitors, which are the main herbicides for controlling this weed. However, very few studies have investigated multiple resistance mechanism in B. syzigachne. In this study, a B. syzigachne biotype with a high resistance to ALS inhibitors we have reported was also showed relatively lower resistance to ACCase inhibitors, with a resistance index around 7. RNA-seq analysis was used to investigate the factors responsible for multiple resistance, and 60,108 unigenes were assembled by de novo transcriptome assembly and then annotated across eight databases. A Pro-197-Ser mutation was identified in the ALS gene by SNPs analysis and validated by PCR, while no mutation was identified in the ACCase gene. Nineteen candidate metabolic genes were screened and their overexpression was confirmed by qPCR. The expression of GST-T3 and GST-U6 in resistant plants ranged from 7.5- to 109.4-folds than that in susceptible ones at different times after two kinds of herbicide treatment. In addition, GST activities in resistant plants were 3.0-5.0 times higher than that in susceptible plants. Other novel resistance factors also showed high correlation with multiple resistance which included four genes encoding disease resistance proteins, a transcription factor (MYC3), and one gene conferring blight resistance. In this research, a B. syzigachne biotype was confirmed to have evolved multiple resistance to ACCase and ALS inhibitors. The Pro-197-Ser mutation in ALS gene and high-level GST activities were confirmed responsible for the multiple resistance. Characterized disease-resistance proteins, transcription factor, and blight-resistance proteins may play an essential role in these multiple herbicide resistance.

Effects of plant density on tillering in the weed grass Aegilops tauschii Coss. and its phytohormonal regulation.

Aegilops tauschii Coss, a notorious wheat field weed, poses a serious threat to wheat in China. Tillers are an important agronomic tool for yield. In this study, a total of 12 Ae. tauschii populations were collected from China to investigate the effect of plant density on tiller occurrence and its phytohormonal regulation. We assayed the growth parameters of Ae. tauschii and the levels of endogenous hormones at different plant densities. The results showed that plant density had a significant effect on the quantity and quality of Ae. tauschii seeds produced per plant. In particular, the tiller and spike numbers per plant were negatively affected by plant density (P < 0.0001). The contents of 13 endogenous hormones in the tiller nodes changed in response to plant density. Among them, indole-3-acetic acid (IAA) and gibberellin (GA) positively responded to plant density. However, the reverse result was found for cytokinin (CTK). Interestingly, phylogenetic tree analysis of auxin (AeYUCCA), CK (AeIPT) and GA (AeCPS) biosynthesis related genes found that phylogenies in the Gramineae for the three different genes were various, hinting at horizontal gene transfer. Moreover, the dynamics of the expression of AeYUCCA, AeIPT and AeCPS were roughly consistent with their phytohormone contents during tillering stage. When externally sprayed on plants of Ae. tauschii, 2,4-D isooctyl ester and GA3 markedly reduced its tillering while 6-BA had no significant effect.

Regulation of Aegilops tauschii Coss Tiller Bud Growth by Plant Density: Transcriptomic, Physiological and Phytohormonal Responses.

Aegilops tauschii Coss is one of the most hazardous weeds that severely infests wheat fields in China. The tillering ability of Ae. tauschii strongly affects the occurrence and spread by influencing its seed output. In this study, Ae. tauschii was sown at low plant density (LPD) and high plant density (HPD) to investigate the effect of plant density on tiller bud outgrowth and its potential regulators using RNA-Seq. Additionally, the chlorophyll content and photosynthesis, soluble sugar and phytohormone levels were also determined at different plant densities. The results showed that an increased plant density significantly inhibited the elongation of tiller buds in the axil of the first leaf at 15 days after planting, with 7.69 mm at LPD and 1.69 mm at HPD. A total of seven putative tiller-related genes were selected and validated using quantitative real-time PCR. Furthermore, chlorophyll levels, photosynthetic efficiency, and soluble sugar contents were distinctly inhibited by HPD in Ae. tauschii, which may be responsible for the restriction of tiller bud growth. In addition, differentially expressed genes (DEGs) were markedly enriched in indole-3-acetic acid (IAA), abscisic acid (ABA), and gibberellin metabolism and signaling. Accordingly, the levels of ABA and gibberellin A3 in Ae. tauschii were strikingly higher at HPD compared with those at LPD, yet the reverse tendency was observed for IAA. Undoubtedly, such results will be highly beneficial for illuminating the underlying regulators of the Ae. tauschii tillering response to plant density and may provide new ideas for the control of this weed in the future.