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.

Species distribution modeling combined with environmental DNA analysis to explore distribution of invasive alien mosquitofish (Gambusia affinis) in China.

China has become one of the most serious countries suffering from biological invasions in the world. In the context of global climate change, invasive alien species (IAS) are likely to invade a wider area, posing greater ecological and economic threats in China. Western mosquitofish (Gambusia affinis), which is known as one of the 100 most invasive alien species, has distributed widely in southern China and is gradually spreading to the north, causing serious ecological damage and economic losses. However, its distribution in China is still unclear. Hence, there is an urgent need for a more convenient way to detect and monitor the distribution of G. affinis to put forward specific management. Therefore, we detected the distribution of G. affinis in China under current and future climate change by combing Maxent modeling prediction and eDNA verification, which is a more time-saving and reliable method to estimate the distribution of species. The Maxent modeling showed that G. affinis has a broad habitat suitability in China (especially in southern China) and would continue to spread in the future with ongoing climate change. However, eDNA monitoring showed that occurrences can already be detected in regions that Maxent still categorized as unsuitable. Besides temperature, precipitation and human influence were the most important environmental factors affecting the distribution of G. affinis in China. In addition, by environmental DNA analysis, we verified the presence of G. affinis predicted by Maxent in the Qinling Mountains where the presence of G. affinis had not been previously recorded.

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.

ALS gene overexpression and enhanced metabolism conferring Digitaria sanguinalis resistance to nicosulfuron in China.

Crabgrass (Digitaria sanguinalis) is a common malignant weed in corn fields in China. Recently, the acetolactate synthase (ALS) inhibitor, nicosulfuron, has shown decreasing efficacy against crabgrass. In order to elucidate the molecular basis of resistance to nicosulfuron in crabgrass, we conducted bioassays, combined with gene sequence analysis, relative expression and relative copy number analysis, to characterize resistance in crabgrass populations collected from Beijing, Heilongjiang, Jilin and Anhui provinces. Whole-plant dose-response results indicated that only population collected in Heilongjiang province (HLJ) had developed low level of resistance to nicosulfuron compared with the sensitive population (SD22). No known resistant mutation of ALS gene was found in HLJ population. The real-time fluorescence quantitative PCR results showed that the ALS gene copy number did not differ significantly between the HLJ and SD22 populations. However, the ALS gene expression in the HLJ was 2.07-fold higher than that of the SD22 population at 24 h after treatment with nicosulfuron. Pretreatment with the cytochrome P450 (CYP450) inhibitor malathion, piperonyl butoxide (PBO), and the glutathione S-transferase (GST) inhibitor 4-Chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) all partially reversed HLJ resistance. Among them, the synergistic effect of PBO and nicosulfuron is the most significant. This is the first report of resistance to nicosulfuron in crabgrass through ALS gene overexpression and possible metabolic resistance.

Enhanced absorption in perovskite solar cells by incorporating gold triangle nanostructures.

Perovskite has emerged as an outstanding light-absorbing material, leading to significant advancements in solar cell efficiency. Further improvements can be made by restructuring the internal optical properties of perovskite. In this study, we investigate the impact of gold triangle nanostructures on perovskite absorption rates, and we explore the optimization of surface plasmon resonance to enhance its solar absorption efficiency. Our numerical simulations revealed that stacking gold triangle nanostructures in the perovskite film resulted in a significant increase in its absorption rate. Finally, comparative testing showed that the solar spectral absorption rate of a 200 nm thick perovskite film increased by 41.5%.

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.

Molecular Characterization of Resistance to Nicosulfuron in Setaria viridis.

The green foxtail, Setaria viridis (L.) P. Beauv. (Poales: Poaceae), is a troublesome and widespread grass weed in China. The acetolactate synthase (ALS)-inhibiting herbicide nicosulfuron has been intensively used to manage S. viridis, and this has substantially increased the selection pressure. Here we confirmed a 35.8-fold resistance to nicosulfuron in an S. viridis population (R376 population) from China and characterized the resistance mechanism. Molecular analyses revealed an Asp-376-Glu mutation of the ALS gene in the R376 population. The participation of metabolic resistance in the R376 population was proved by cytochrome P450 monooxygenases (P450) inhibitor pre-treatment and metabolism experiments. To further elucidate the mechanism of metabolic resistance, eighteen genes that could be related to the metabolism of nicosulfuron were obtained bythe RNA sequencing. The results of quantitative real-time PCR validation indicated that three ATP-binding cassette (ABC) transporters (ABE2, ABC15, and ABC15-2), four P450 (C76C2, CYOS, C78A5, and C81Q32), and two UDP-glucosyltransferase (UGT) (UGT13248 and UGT73C3), and one glutathione S-transferases (GST) (GST3) were the major candidates that contributed to metabolic nicosulfuron resistance in S. viridis. However, the specific role of these ten genes in metabolic resistance requires more research. Collectively, ALS gene mutations and enhanced metabolism may be responsible for the resistance of R376 to nicosulfuron.

Field-realistic dose of cefotaxime enhances potential mobility of ß-lactam resistance genes in the gut microbiota of zebrafish (Danio rerio).

With large amounts of cephalosporin end up in natural ecosystems, water has been acknowledged as the large reservoir of ß-lactam resistance over the past decades. However, there is still insufficient knowledge available on the function of the living organisms to the transmission of antibiotic resistance. For this reason, in this study, using adult zebrafish (Danio rerio) as animal model, exposing them to environmentally relevant dose of cefotaxime for 150 days, we asked whether cefotaxime contamination accelerated ß-lactam resistance in gut microbiota as well as its potential transmission. Results showed that some of ß-lactam resistance genes (ßRGs) were intrinsic embedded in intestinal microbiome of zebrafish even without antibiotic stressor. Across cefotaxime treatment, the abundance of most ßRGs in fish gut microbiome decreased apparently in the short term firstly, and then increased with the prolonged exposure, forming distinctly divergent ßRG profiles with antibiotic-untreated zebrafish. Meanwhile, with the rising concentration of cefotaxime, the range of ßRGs' host-taxa expanded and the co-occurrence relationships of mobile genetics elements (MGEs) with ßRGs intensified, indicating the enhancement of ßRGs' mobility in gut microbiome when the fish suffered from cefotaxime contamination. Furthermore, the path of partial least squares path modeling (PLS-PM) gave an integral assessment on the specific causality of cefotaxime treatment to ßRG profiles, showing that cefotaxime-mediated ßRGs variation was most ascribed to the alteration of MGEs under cefotaxime stress, followed by bacterial community, functioning both direct influence as ßRG-hosts and indirect effects via affecting MGEs. Finally, pathogenic bacteria Aeromonas was identified as the critical host for multiple ßRGs in fish guts, and its ß-lactam resistance increased over the duration time of cefotaxime exposure, suggesting the potential spreading risks for the antibiotic-resistant pathogens from environmental ecosystems to clinic. Overall, our finding emphasized cefotaxime contamination in aquatic surroundings could enhance the ß-lactam resistance and its transmission mobility in fish bodies.

Nonsteroidal anti-inflammatory drug diclofenac accelerates the emergence of antibiotic resistance via mutagenesis.

Overuse of antimicrobial agents are generally considered to be a key factor in the occurrence of antibiotic resistance bacteria (ARB). Nevertheless, it is unclear whether ARB can be induced by non-antibiotic chemicals such as nonsteroidal anti-inflammatory drug (NSAID). Thus, the objective of this study is to investigate whether NSAID diclofenac (DCF) promote the emergence of antibiotic resistance in Escherichia coli K12 MG1655. Our results suggested that DCF induced the occurrence of ARB which showed hereditary stability of resistance. Meanwhile, gene variation was identified on chromosome of the ARB, and DCF can cause bacterial oxidative stress and SOS response. Subsequently, transcriptional levels of antioxidant (soxS, sodA, sodC, gor, katG, ahpF) and SOS (recA, lexA, uvrA, uvrB, ruvA, ruvB, dinB, umuC, polB) system-related genes were enhanced. However, the expression of related genes cannot be increased in high-dosage treatment compared with low-dosage samples because of cytotoxicity and cellular damage. Simultaneously, high-dosage DCF decreased the mutation frequency but enhanced the resistance of mutants. Our findings expand our knowledge of the promoting effect on the emergence of ARB caused by DCF. More attention and regulations should be given to these potential ecological and health risks for widespread DCF.

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.

Investigating the Metabolic Mesosulfuron-Methyl Resistance in Aegilops tauschii Coss. By Transcriptome Sequencing Combined with the Reference Genome.

Aegilops tauschii Coss. is a malignant weed in wheat fields in China, its herbicide resistance has been threatening crop production. This study identified one mesosulfuron-methyl-resistant(R) population, JJMHN2018-05 (R), without target resistance mutations. To fully understand the resistance mechanism, non-target site resistance was investigated by using transcriptome sequencing combined with a reference genome. Results showed that the cytochrome P450 monooxygenase (P450) inhibitor malathion significantly increased the mesosulfuron-methyl sensitivity in R plants, and greater herbicide-induced glutathione S-transferase (GST) activity was also confirmed. Liquid chromatography with tandem mass spectrometry analysis further supported the enhanced mesosulfuron-methyl metabolism in R plants. Gene expression data analysis and qRT-PCR validation indicated that eight P450s, six GSTs, two glycosyltransferases (GTs), four peroxidases, and one aldo-keto reductase (AKRs) stably upregulated in R plants. This research demonstrates that the P450s and GSTs involved in enhanced mesosulfuron-methyl metabolism contribute to mesosulfuron-methyl resistance in A. tauschii and identifies potential contributors from metabolic enzyme families.

Metagenomic insights into comparative study of nitrogen metabolic potential and microbial community between primitive and urban river sediments.

As a result of anthropogenic pollution, the nitrogen nutrients load in urban rivers has increased, potentially raising the risk of river eutrophication. Here, we studied how anthropogenic impacts alter nitrogen metabolism in river sediments by comparing the metagenomic function of microbial communities between relatively primitive and human-disturbed sediments. The contents of organic matter (OM), total nitrogen (TN), NO3--N and NO2--N were higher in primitive site than in polluted sites, which might be due to vegetation density, sediment type, hydrology, etc. Whereas, NH4+-N content was higher in midstream and downstream, indicating that nitrogen loading increased in the anthropogenic regions and subsequently leading higher NH4+-N. Hierarchical cluster analyses revealed significant changes in the community structure and functional potential between the primitive and human-affected sites. Metagenomic analysis demonstrated that Demequina, Streptomyces, Rubrobacter and Dechloromonas were the predominant denitrifiers. Ardenticatena and Dechloromonas species were the most important contributors to dissimilatory nitrate reduction. Furthermore, anthropogenic pollution significantly increased their abundance, and resulting in a decrease in NO3-, NO2--N and an increase in NH4+-N contents. Additionally, the SOX metabolism of Dechloromonas and Sulfuritalea may involve in the sulfur-dependent autotrophic denitrification process by coupling the conversion of thiosulfate to sulfate with the reduction of NO3--N to N2. From pristine to anthropogenic pollution sediments, the major nitrifying bacteria harboring Hao transitioned from Nitrospira to Nitrosomonas. This study sheds light on the consequences of anthropogenic activities on nitrogen metabolism in river sediments, allowing for better management of nitrogen pollution and eutrophication in river.

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.