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Introduction: Herbicide application is a highly efficiency method of weed control that boots agricultural output and assures food security. The development of novel herbicides focuses on improved bioactivity and new modes of action. The amino acid biosynthesis was validated as a promising novel mode of action for herbicidal compounds. However, the amino acid biosynthesis enzyme remains largely unexplored for herbicidal targets. Objectives: Serine hydroxymethyl transferase (SHMT) is an essential enzyme in the photorespiratory cycle. The study aims to explore Conyza canadensis SHMT1 (CcSHMT1) as a promising target for herbicide discovery. Methods: Structure determination of CcSHMT1 was resolved by X-ray crystallography. Virtual screening docking experiments were performed with Glide version 5.5. Novel derivatives of dimethyl 2-acetamido terephthalate were further designed, synthesized, and bioassay. The druggability of the inhibitor was evidenced by ultrastructural changes in mitochondria, in vivo and vitro enzyme activity assays, and genetics analysis. Results: CcSHMT1 has a typical PLP-dependent enzyme 3D structure. The dimethyl 2-acetamido terephthalate-containing compounds had herbicidal activity. Dimethyl 2-(2-(4-(2-(4-bromo-2-chlorophenoxy) acetyl)piperazin-1-yl)acetamido) terephthalate (Compound 9ay, EC50 = 193.8 g a.i./ ha) exhibited the highest herbicidal activity on tested weed among the synthesized compounds. Compound 9ay had no obvious adverse effect on the growth of maize and honeybees. Compound 9ay was verified to target CcSHMT1 as an herbicide candidate. Conclusion: A first-in-class CcSHMT1 inhibitor that could be developed as a potent herbicide with a new mode of action and provide an avenue for discovering novel inhibitors of pyridoxal-5-phosphate-dependent enzymes.

S-metolachlor is a selective pre-emergence herbicide used in dryland. However, it is challenging to employ in paddy fields due to its phytotoxic effects on rice. As a common phytohormone, Gibberellin-3 (GA3) is inferred to have the ability to alleviate herbicide phytotoxicity. This study first quantitatively verified the phytotoxicity of s-metolachlor to rice and then demonstrated the mitigative effect of GA3 on these adverse reactions. Furthermore, a transcriptome of rice seedlings subjected to different treatments was constructed to assemble the reference genes, followed by comparative metabolomics and proteomics analyses. Metabolomics revealed an enrichment of flavonoid metabolites in the group of adding GA3, and these flavonoids can eliminate ROS in plants. Proteomics analysis indicated that differential proteins were enriched in the phenylpropanoid biosynthesis pathway responsible for the synthesis of flavonoids and that the functions of most differential proteins are associated with peroxidase. The proteome, combined with the transcriptome, revealed that the expressions of proteins and genes was related to the POD activity in the group of adding GA3. It was speculated that the elimination of ROS is key to alleviating the stress of s-metolachlor on rice growth. It was inferred that the mechanism of GA3 in alleviating the phytotoxicity of the substance s-metolachlor is by increasing the activity of the POD and influencing the growth of rice seedlings through the restoration of flavonoid synthesis. In this study, we screened GA3 as a safener to alleviate the phytotoxicity of s-metolachlor on rice. On this basis, the mechanism of alleviating phytotoxicity was studied. The application range of s-metolachlor might be expanded, providing a new supplementary method for weed control and herbicide resistance management.

The Clearfield® technology is an useful tool for controlling weedy rice due to the effectiveness of imazamox and the cultivation of rice varieties resistant to imidazolines. However, residual imazamox in the soil probably causes phytotoxicity to subsequent non-resistant rice crops. We previously found that pyrroloquinoline quinone (PQQ), a bioactive elicitor, can enhance rice tolerance to imazamox. In this study, we explored the molecular mechanism of PQQ-mediated rice tolerance to imazamox by RNA-seq analysis, real-time quantitative PCR (RT-qPCR) assay, and enzyme activity assay. The results indicated that compared to imazamox at 66.7 mg a.i./L (IMA) alone, rice plants treated with imazamox at 66.7 mg a.i./L and PQQ at 0.66 mg a.i./L (IMA + PQQ) exhibited significantly reduced sensitivity to imazamox. Seven days post-treatment, IMA + PQQ-treated rice plants showed partial chlorosis and leaf curling, but IMA-treated rice plants had severe wilting and died. The fresh weight inhibition rate was 29.3% in the IMA + PQQ group, significantly lower than that of 56.6% in the IMA group alone. RNA-seq analysis showed differentially expressed genes were mainly involved in phenylpropanoid biosynthesis, diterpenoid biosynthesis, and MAPK signaling pathways in response to IMA + PQQ treatment. Both RNA-seq analysis and RT-qPCR assay showed that the expression of OsCATB gene in the catalase (CAT) gene family was upregulated at 12 h, the expression of OsGSTU1 gene was upregulated at 12, 24, and 48 h, while the expressions of OsABCB2, OsABCB11, and OsABCG11 genes were upregulated at 24 and 48 h. Enzyme activity assays revealed that the activity of superoxide dismutase in the IMA + PQQ group was increased by 47.45~120.31% during 12~72 h, compared to that in the IMA group. CAT activity in the IMA + PQQ group was increased by 123.72 and 59.04% at 12 and 48 h, respectively. Moreover, malondialdehyde levels indicative of oxidative damage were consistently lower in IMA + PQQ-treated plants, with a reduction of 46.29, 11.37, and 14.50% at 12, 36, and 72 h, respectively. Overall, these findings discover that PQQ has potential in reducing imazamox phytotoxicity in rice by enhancing antioxidant enzyme activities and regulating oxidative stress responses. They will provide valuable strategies for improving imazamox tolerance in crops.

IntroductionBromus japonicus is one of the most notorious agricultural weeds in China. The long-term use of ALS-inhibiting herbicides has led to rapid evolution of herbicide resistance in B. japonicus. B. japonicus population (BJ-R) surviving mesosulfuron-methyl treatment was collected from wheatland. Here, we aimed to confirm the resistance mechanisms in this putative resistant population.MethodsThe dose-reponse tests were used to test the resistance level of the B. japonicus to ALS-inhibiting herbicides. Pretreatment with P450 and GST inhibitors and GST activity assays were used to determine whether P450 or GST was involved in the resistance of the BJ-R population. Sanger sequencing was used to analyse the ALS mutation of the BJ-R population. RT-qPCR was used to confirm the the expression levels of the ALS gene in mesosulfuron-methyl -resistant (BJ-R) and-susceptible (BJ-S) B. japonicus. An in vitro ALS activity assay was used to determine the ALS activity of the BJ-R and BJ-S populations. Homology modelling and docking were used to determine the binding energy of the BJ-R and BJ-S populations with ALS-inhibiting herbicides.ResultsB. japonicus population (BJ-R) was confirmed to be 454- and 2.7-fold resistant to the SU herbicides mesosulfuron-methyl and nicosulfuron, and 7.3-, 2.3-, 1.1- and 10.8-fold resistant to the IMI herbicide imazamox, the TP herbicide penoxsulam, the PTB herbicide pyribenzoxim and the SCT herbicide flucarbazone-sodium, respectively, compared with its susceptible counterpart (BJ-S). Neither a P450 inhibitor nor a GST inhibitor could reverse the level of resistance to mesosulfuron-methyl in BJ-R. In addition, no significant differences in GST activity were found between the BJ-R and BJ-S. ALS gene sequencing revealed a Pro-197-Thr mutation in BJ-R, and the gene expression had no significant differences between the BJ-R and BJ-S. The ALS activity of BJ-R was 106-fold more tolerant to mesosulfuron-methyl than that of BJ-S. Molecular docking showed that the binding energy of the ALS active site and mesosulfuron-methyl was changed from -6.67 to -4.57 kcal mol-1 due to the mutation at position 197.DiscussionThese results suggested that the Pro-197-Thr mutation was the main reason for the high resistance level of BJ-R to mesosulfuron-methyl. Unlike previous reports of the cross-resistance pattern conferred by this mutation, we firstly documented that the Pro-197-Thr mutation confers broad cross-resistance spectrums to ALS-inhibiting herbicides in B. japonicus.

Rice (Oryza sativa L.) is one of the most important food crops worldwide. However, during direct seeding, rice is extremely vulnerable to flooding stress, which impairs rice’s emergence and seedling growth and results in a significant yield loss. According to our research, chitosan oligosaccharides have the potential to be a chemical seed-soaking agent that greatly increases rice’s resistance to flooding. Chitosan oligosaccharides were able to enhance seed energy supply, osmoregulation, and antioxidant capacity, according to physiological index assessments. Using transcriptome and metabolomic analysis, we discovered that important differential metabolites and genes were involved in the signaling pathway for hormone synthesis and antioxidant capacity. Exogenous chitosan oligosaccharides specifically and significantly inhibit genes linked to auxin, jasmonic acid, and abscisic acid. This suggested that applying chitosan oligosaccharides could stabilize seedling growth and development by controlling associated hormones and reducing flooding stress by enhancing membrane stability and antioxidant capacity. Finally, we verified the effectiveness of exogenous chitosan oligosaccharides imbibed in seeds by field validation, demonstrating that they can enhance rice seedling emergence and growth under flooding stress.

A field trial in Changsha, China, involving the release of a triple Wolbachia-infected strain of the mosquito, Aedes albopictus, demonstrates the feasibility of using an incompatible insect technique to suppress disease vector mosquito populations in urban subtropic environments.

IntroductionLeptochloa chinensis is an annual weed in paddy fields, which can engage in competition with rice, leading to a severe yield reduction. However, theunderlying mechanism governing this interaction remain unknown.MethodsIn this study, we investigated the mutual inhibition between rice and the weed undermono-culture and co-culture conditions. We found that the root exudates of both species played essential roles in mediating the mutual inhibition. Further metabolomic analysis identified a significant number of differential metabolites. These metabolites were predominantly enriched in the phenylpropanoid and flavonoid biosynthesis pathways in weed and rice. Transcriptomic analysis revealed that the differentially expressed genes responding to the interaction were also enriched in these pathways.ResultsPhenylpropanoid and flavonoid biosynthesis pathways are associated with allelopathy, indicating their pivotal role in the response of rice-weed mutual inhibition.DiscussionOur findings shed light on the conserved molecular responses of rice and L. chinensis during theirinteraction, provide evidence to dissect the mechanisms underlying the allelopathic interaction and offer potential strategies for weed management in rice paddies.

The excessive use of chemical herbicides has resulted in evolution of herbicide-resistant weeds. Cytochrome P450 monooxygenases (P450s) are vital detoxification enzymes for herbicide-resistant weeds. Herein, we confirmed a resistant (R) Polypogon fugax population showing resistance to quizalofop-p-ethyl, acetolactate synthase (ALS)-inhibiting herbicide pyroxsulam, and several other ACCase (acetyl-CoA carboxylase)-inhibiting herbicides. Molecular analysis revealed no target-site gene mutations in the R population. Foliar spraying with malathion clearly reversed the quizalofop-p-ethyl phytotoxicity. Higher level of quizalofop-p-ethyl degradation was confirmed in the R population using HPLC analysis. Subsequently, RNA-Seq transcriptome analysis indicated that the overexpression of CYP89A2 gene appeared to be responsible for reducing quizalofop-p-ethyl phytotoxicity. The molecular docking results supported a metabolic effect of CYP89A2 protein on most herbicides tested. Furthermore, we found that low doses of herbicides stimulated the rhizosphere enzyme activities in P. fugax and the increase of rhizosphere dehydrogenase of R population may be related to its resistance mechanism. In summary, our research has shown that metabolic herbicide resistance mediated by CYP89A2, contributes to quizalofop-p-ethyl resistance in P. fugax.

Plants have evolved circadian clock systems that enable biological processes to occur in tandem with periodic changes in the environment. However, it is largely unknown whether crosstalk occurs between the circadian clock and the response to herbicide in rice. We identified 19 conserved rhythmic metabolites which were response to pesticide application and their metabolic abundance peaked mainly at ZT2 or ZT14-ZT18. We found a series of glyphosate, s-Metolachlor, fenclorim, metcamifen and GA3 response genes were expressed following stable circadian rhythms. In order to determine the patterns of their temporal expression, co-expression network analysis was done on 10,467 genes that were periodically expressed throughout a 24-hour period. Next, we identified 4,031 potential direct target genes of OsCCA1 in using DAP-seq data for OsCCA1. Of these, 339, 22, 53, 53 and 63 genes showed a response to glyphosate, s-Metolachlor, fenclorim, metcamifen and GA3 application, respectively. And they were mainly phased from dusk to midnight. Interestingly, we identified significant OsCCA1 binding peaks in the promoter regions of four herbicide resistance genes, including OsCYP81A12, OsCYP81E22, OsCYP76C2, and OsCYP76C4. Finally, we found that herbicide application could affects the expression of some of the central oscillator genes of the rice circadian clock. Here, we used multi-omics data to reveal the crosstalk between the circadian clock and herbicide response processes at the epigenomics, transcriptome, and metabolome levels in rice. This work will serve as a theoretical guide for identifying rhythmic herbicide targets, leading to the creation of new herbicides or the breeding of crops resistant to herbicides.

IntroductionAlopecurus aequalis is a grass species invading Chinese canola and wheat fields. An A. aequalis KMN-R population surviving mesosulfuron-methyl treatment with recommended rates was acquired from wheatland. Here, we aimed to confirm the resistance profiles of KMN-R to acetolactate synthetase (ALS) inhibiting herbicides and explore the possible resistance mechanisms to mesosulfuron-methyl in this weed population.MethodsThe dose-response tests performed in our study were used to test the toxicity of A. aequalis to ALS-inhibiting herbicides. Sanger sequencing was used to analyze the ALS gene of mesosulfuron-methyl -resistant and -susceptible A. aequalis. RNA sequencing analysis was used to find candidate genes that may confer metabolic resistance to the mesosulfuron-methyl in resistant A. aequalis population. Mesosulfuron-methyl -resistant and -susceptible A. aequalis populations fungal composition was measured via Illumina MiSeq Sequencing.ResultsDose-response results indicated that KMN-R population evolved resistance to mesosulfuron-methyl and other tested ALS-inhibiting herbicides. Known resistance-conferring Trp-574-Leu gene mutation in A. aequalis ALS was detected in the KMN-R population. Pretreatment with 4-chloro-7-nitrobenzoxadiazole reversed mesosulfuron-methyl resistance in KMN-R. Glutathione S-transferases (GST) gene GSTZ2 and GSTT3 were highly expressed in KMN-R population. In addition, we evaluated the alpha diversity in A. aequalis, centering on OTU abundance, equality, and multiplicity, and found that the fungal community composition had more unexplained variance between KMN-R and KMN-S A. aequalis. We also observed higher abundances of specific fungi in KMN-R A. aequalis.DiscussionThe results proved that resistance to mesosulfuron-methyl in A. aequalis KMN-R population is probably caused by target site- and non-target site-based relating GST and provided the basis for further research between fungal interaction and herbicide resistance.

Abstract Background Chinese sprangletop [Leptochloa chinensis (L.) Nees] is an annual malignant weed, which can often be found in paddy fields. Cyhalofop-butyl is a specialized herbicide which is utilized to control L. chinensis. However, in many areas, L. chinensis has become tolerant to this key herbicide due to its continuous long-term use. Results In this study, we utilized a tolerant (LC18002) and a sensitive (LC17041) L. chinensis populations previously identified in our laboratory, which were divided into four different groups. We then employed whole transcriptome analysis to identify candidate genes which may be involved in cyhalofop-butyl tolerance. This analysis resulted in the identification of six possible candidate genes, including three cytochrome P450 genes and three ATP-binding cassette transporter genes. We then carried out a phylogenetic analysis to identify homologs of the differentially expressed cytochrome P450 genes. This phylogenetic analysis indicated that all genes have close homologs in other species, some of which have been implicated in non-target site resistance (NTSR). Conclusions This study is the first to use whole transcriptome analysis to identify herbicide non-target resistance genes in L. chinensis. The differentially expressed genes represent promising targets for better understanding herbicide tolerance in L. chinensis. The six genes belonging to classes already associated in herbicide tolerance may play important roles in the metabolic resistance of L. chinensis to cyhalofop-butyl, although the exact mechanisms require further study.

Abstract Application of a novel bioorganic fertilizer (BIO) has been effectively used to inhibit weeds in rice paddies. To identify changes in soil bacterial community and enzymes in response to BIO treatments, field experiments were carried out in five major rice-growing areas in China. The dominant phylogenetic groups recorded included Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Acidobacteria. Anaeromyxobacter, Bacteroides, Bifidobacterium, Escherichia- Shigella, Geobacter and Haliangium were significantly different between BIO-treatment and untreated control and aided in general function (R), amino acid transport, metabolism (E) and transcription (K) clusters. The soil chemical properties and enzyme activities were less affected by BIO at these study sites. RDA analysis showed that soil bacterial community had a significant positive correlations among northern latitude, eastern longitude, exchangeable K, total K, total P, soil pH, and total N, except for organic matter, hydrolytic N and extractable P. Overall, our work showed that application of BIO does not alter the main community structure and functional diversity of soil bacteria in rice paddies and should be encouraged for use as a sustainable weed management strategy.

Abstract Background Benzobicyclon (BBC) is a β-triketone herbicide (bTH) used in rice paddy fields. It has the advantages of high efficiency, low toxicity, high crop safety, and good environmental compatibility, and shows efficacy against paddy weeds resistant to other types of herbicides. However, as some important indica rice varieties are susceptible to BBC, BBC is currently only registered and applied in japonica rice cultivation areas. Results By analyzing haplotypes of the bTHs broad-spectrum resistance gene HIS1 and phenotypes for BBC in 493 major indica rice accessions in China, we identified a novel non-functional allelic variant of HIS1 in addition to the previously reported 28-bp deletion. Through detection with markers specific to the two non-functional mutations, it was clear that 25.4% of indica conventional varieties, 59.9% of fertility restorers, and 15.9% of sterile lines were susceptible to BBC. In addition, due to natural allelic variations of the HIS1 gene in the sterile and restorer lines, some two-line hybrid sterile lines were sensitive to bTHs, and the corresponding restorers were resistant. We showed the potential effectiveness of using bTHs to address the issue of two-line hybrid rice seed purity stemming from the self-crossing of sterile lines during hybrid rice seed production. Finally, allelic variations of the HIS1 gene may also play an important role in the mechanized seed production of hybrid rice. Conclusions Our findings offer guidance for the application of BBC in indica rice areas and provide a non-transgenic approach to address the seed purity issue of two-line hybrid rice.

It is necessary to study the combined toxicity of an herbicide and its safener because the two are often used in combination. S-metolachlor and its safener benoxacor have been detected in aquatic environments and can individually damage the oxidative stress system in zebrafish embryos (Danio rerio). However, only their separate toxicity in zebrafish (Danio rerio) embryo development has been reported. This study assessed the combined toxicity of benoxacor and S-metolachlor in zebrafish embryo development, including acute toxicity, developmental toxicity, oxidative damage, and cell apoptosis. The 96-h LC50 values were higher in mixtures of benoxacor and S-metolachlor than in benoxacor alone. The treatments included S-metolachlor, Mix-1 (0.1 mg/L benoxacor + 0.1 mg/L S-metolachlor), Mix-2 (0.1 mg/L benoxacor + 0.3 mg/L S-metolachlor) and benoxacor alone. Embryos exposed to Mix-1 and Mix-2 had lower developmental toxicities, superoxide dismutase (SOD) activity, osx and cat expression levels than those exposed to benoxacor alone. Moreover, glutathione S-transferase (GST), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) activities, and the expressions of tbx16, nrf2, bcl2, and caspase9 were higher in the mixtures than in the benoxacor group. High-throughput RNA sequencing revealed that benoxacor had a greater effect on gene regulation than Mix-1 and Mix-2. The malformation rate, different enrichment gene numbers, and gene expression levels of hatched embryos were higher in Mix-1 than in Mix-2. The results indicate that a mixture of S-metolachlor and benoxacor has antagonistic effects in the early stage of embryo development. The mixtures can break the reactive oxygen species balance, causing abnormal cell apoptosis and developmental malformation in embryos. Besides investigating the combined toxicity of benoxacor and S-metolachlor in zebrafish embryo development, this study provides a risk assessment basis for a herbicide combined with its safener.

Quantification of germination characteristics between herbicide-resistant and -susceptible weeds might provide methods to control resistant weeds and permit better prediction of evolution and persistence of herbicide resistance. This study aimed to compare the germination characteristics of Asian minor bluegrass (Polypogon fugax) populations that are resistant or susceptible to quizalofop-p-ethyl under controlled conditions, which the resistance mechanism is involved in glutathione S-transferases (GSTs) metabolism-based resistance. No major differences in seed germination were found at diverse temperatures, pH ranges, and light conditions. However, a significant difference that seed response to a gradient of osmotic and salt stress between the resistant and susceptible P. fugax populations were found. Two stress response genes (P5CS-1 and CDPK-2) in P. fugax were likely involved in germination rate as well as germination speed in response to these stresses. Subsequently, population verification demonstrated that P5CS-1 and CDPK-2 genes may be linked to the resistance mechanism. Additionally, the two genes play an important role in response to salt stress and osmotic stress as shown by transcript abundance after stress treatments. Our findings suggest that the variation of the germination characteristics in P. fugax associates with the presence of GST-endowed resistance mechanism.

A major challenge in radically alleviating the threats posed by Cd-contaminated paddy fields to human health is to reduce the Cd levels in both soils and rice grains. In this study, the microbial extraction (ME) treatment using a mixotrophic acidophilic consortium was used for the bioremediation of Cd-contaminated soils. The results showed that the ME treatment enhanced the total Cd (40%) and diethylenetriamine pentaacetic acid-soluble Cd (DTPA-Cd, 64%) removal efficiencies in contaminated soils. In addition, ME treatment decreased the levels of Cd acid-soluble and reducible fractions and thereby reduced Cd uptake in rice tissues. Microbial community analysis indicated that the indigenous soil microbial diversity and composition were not changed after the ME treatment, but the relative abundance of functional microbes associated with Cd removal was improved. Notably, soil available nutrient levels were elevated upon inoculation with mixotrophic acidophiles, resulting in an increase in rice growth and grain weight. This study provides a scientific basis for the potential application and evaluation of ME treatment in the field for remediating Cd-contaminated paddy soils.

Abstract Background Fenclorim (Fen) can effectively protect rice from pretilachlor (Pre) injury, but its effects on rice have not been formally evaluated; thus, the Fen mode of action for alleviating the phytotoxicity caused by Pre in rice is not clear. This study aimed to examine the biochemical and physiological effects of Fen on rice and to determine the changes induced by Fen at the transcriptome level. Result The chlorophyll content of rice plants was significantly affected by Pre but not by Fen. The activity of oxidative stress enzymes showed that Fen did not elicit any changes in oxidative stress; however, it reduced lipid peroxidation and oxidative damage induced by Pre. Fen did not affect the uptake of Pre but did affect its persistence in rice. In a transcriptome experiment, Fen upregulated genes in a detoxification pathway. Overall, 25 genes related to detoxification were identified, including P450, GST, and GT. Moreover, qRT-PCR analysis showed that four P450 genes, CYP71Y83, CYP71K14, CYP734A2 and CYP71D55, and two GST genes, GSTU16 and GSTF5, were upregulated by Fen and/or Pre. Conclusion Our work indicates that Fen acts in antioxidative defense in addition to enhancing the metabolism of herbicides in rice.

The response of soil bacterial communities from farmland ecosystems to cadmium (Cd) pollution, in which a steep concentration gradient of more than 100 mg/kg has naturally formed, has not previously been fully reported. In this study, a field investigation was conducted in a typical severe Cd-polluted farmland ecosystem, and the bacterial community response to the steep Cd gradient was analyzed. The results showed that Cd concentration sharply decreased from 159.2 mg/kg to 4.18 mg/kg among four sampling sites alongside an irrigation canal over a distance of 150 m. Bacterial diversity and richness were significantly lower in highly polluted sites, and random forest analysis indicated that Cd gradient played a decisive role in reducing alpha diversity. Redundancy analysis (RDA) and co-occurrence network indicated that the synergistic effects of pH, Cd, and phosphorus were the main drivers shaping community structure. The functional results predicted by BugBase suggested that the bacterial community may adapt to the harsh environment by recruiting Cd-resistant microbes and improving oxidative stress tolerance of the whole community. Cd-resistant microorganisms such as Burkholderia, Bradyrhizobium, and Sulfurifustis, which directly or indirectly participate in diminishing oxidative damage of Cd, may play essential roles in maintaining community stability and might be potential bacterial resources for the bioremediation of Cd pollution.

Herbicide resistance to chemical herbicide is a global issue that presents an ongoing threat to grain production. Though it has been frequently implicated that the production of detoxification enzymes increased in resistance development, the mechanisms for overexpression of these genes employed by herbicide-resistant weeds remain complicated. In this study, a mesosulfuron-methyl resistant Beckmannia syzigachne population (R) was found to be cross-resistant to another herbicide pyriminobac-methyl. No known target-site mutations were detected in the R population. In contrast, the decreased uptake and enhanced metabolic rates of mesosulfuron-methyl were detected in the R than the susceptible (S) population. Two candidate ATP-binding cassette (ABC) transporter genes (ABCB25 and ABCC14) that were constitutively up-regulated in the R population were identified by RNA-sequencing and validated by RT-qPCR. Alteration of antioxidant enzyme activities and gene expressions implied that mesosulfuron-methyl-induced antioxidant defenses provoked reactive oxygen species (ROS) burst. ROS scavenger assay showed that ROS induces ABCB25 and ABCC14 expression. This study reported for the first time that ABC transporters mediated non-target-site resistance contributes to mesosulfuron-methyl resistance in a B. syzigachne population, and implicated that ROS burst might be involved in the overexpression of ABC transporter genes in weeds.

Cadmium (Cd) speciation ratio in arable land determines the Cd exposure risk and Cd uptake in crops. However, the driving mechanisms of Cd speciation change on the vertical scale of paddy fields remain poorly understood. In this study, the effects of plow layer and plow pan on Cd speciation distribution were investigated in a long-term Cd-contaminated rice ecosystem. The Cd accumulative effect within rice grain was enhanced with high levels of activated Cd speciation ratios in soils. Activated Cd speciation ratios were higher in plow layer soils, while stabilized Cd speciation ratios were elevated in plow pan soils. Soil physicochemical properties and soil microbes synergistically affected the Cd speciation changes in different ways between the two soil layers. Soil pH and organic elements in plow layer environment directly hindered the transformation of stabilized Cd speciation, while in plow pan environment, soil pH and organic elements indirectly decreased activated Cd speciation ratios and resulted in the accumulation of stabilized Cd speciation via regulating the predominant bacterial taxa. This study will improve our understanding of how soil environments regulate Cd speciation distributions in rice ecosystems and help to seek effective remediation methods of Cd-contaminated paddy fields to reduce the Cd accumulation in rice.

Alopecurus japonicus has been a serious weed across China and developed resistance to the acetyl-CoA carboxylase (ACCase)-inhibiting herbicide. The A. japonicus ACCase gene mutations accounting for target-site resistance (TSR) have been clarified, while non-target-site resistance (NTSR) is not distinct. Here, dose–response testing indicated that an A. japonicus population (R) was resistant to four ACCase-inhibiting herbicides fenoxaprop-P-ethyl, sethoxydim, clethodim, and pinoxaden. Compared with herbicide-susceptible A. japonicus (S), no known resistant mutations for TSR in ACCase were identified in the R population using sequencing. To investigate the NTSR mechanism, increased metabolism of fenoxaprop-P-ethyl was detected in the R population using high-performance liquid chromatography (HPLC) analysis. Notably, resistance cannot be reversed by P450 and GST inhibitors. RNA-seq was performed to further explore the resistance mechanisms, and eight candidate contigs (four glycosyl transferases (GT) and four ATP-binding cassette (ABC) transporters) were chosen and their expression patterns were validated using RT-qPCR. Three GT and three ABC transporter contigs were constitutively upregulated in the R population. In short, six contigs expressed highly in the R population causing enhanced fenoxaprop-P-ethyl metabolism appear to be involved in fenoxaprop-P-ethyl resistance.

To enhance quinclorac potency, twenty-five derivatives were synthesized containing 3-methyl-1H-pyrazol-5-yl by intermediate derivatization methods (IDMs). These compounds were confirmed by melting point (mp), 1HNMR, 13CNMR, and HRMS. The compound 1,3-dimethyl-1H-pyrazol-5-yl 3,7-dichloroquinoline-8-carboxylate (10a) was determined by X-ray diffraction. The activity of these compounds substituent on the phenyl was: electron-drawing group > neutral group > donor-drawing group, the results was like that of substituted benzyl group on pyrazole. The herbicidal activity assays showed that compounds 1-(2-fluorophenyl)-3-methyl-1H-pyrazol-5-yl 3,7-dichloroquinoline-8-carboxylate (8l, EC50 = 10.53 g/ha) and 10a (EC50 = 10.37 g/ha) had an excellent inhibition effect on barnyard grass in greenhouse experiment. Greenhouse safety experiment of rice exhibited almost no difference in plant height and fresh weight treated 10a at stage 1∼2-leaf of rice after 14 days but 8l had a detrimental effect. Two season field assays showed 10a herbicidal activity on barnyard grass at 150 g/ha as equal as 300 g/ha quinclorac in fields in 2019 and 2020. The study demonstrated that 10a could be further researched as a potential herbicide to control barnyard grass in fields.

Recent studies indicated that ‘inert ingredients’ exert negative effects on the environment. Herbicide safeners are classed as ‘inert ingredients’, which increase the selectivity and detoxification of herbicides. However, little attention has been focused on the environmental behavior of herbicide safeners. AD-67 (AD), fenclorim (FM), and flurazole (FZ) are three commercially available herbicide safeners. In this study, zebrafish embryos were used as a model to investigate the potential developmental toxicity of these three safeners. The results showed that AD, FM, and FZ 96 h-LC50 values were 2.52, 1.26, and 2.01 mg/L, respectively. Significant decreased body lengths were observed in embryos after 96 h of exposure to 0.2 mg/L FM and FZ. Hatching rates significantly increased at 24 h and decreased at 96 h in all treatment groups (0.02 mg/L AD, 0.2 mg/L AD, 0.02 mg/L AD, 0.2 mg/L FZ, as well as 0.01 and 0.1 mg/L FM). No significant (p > 0.05) changes in heartbeat numbers (60 s), but clearly increased malformation rates were observed in response to safeners exposure. Furthermore, embryos showed signs of oxidative stress, such as decreased activities of superoxide dismutase, altered activities of glutathione reductase and catalase and cell apoptosis. The gene transcription related to body malformation (egf, krt 17, and tbx 16) and cell apoptosis (bcl 2 and bax) changed in treated groups. These genes have been connected to potential toxicological mechanisms. These results indicate that the herbicide safeners AD, FM, and FZ induced developmental toxicities in zebrafish embryos. This study is the first report of the toxicity of safeners in the development of zebrafish embryos. The results may be useful for assessing the risk of herbicides safeners in the aquatic ecosystem.

This study focused on cadmium (Cd) uptake by two rice varieties, Yuzhenxiang (YZX) and Xiangwanxian 12 (XWX), which differ in their capacity to accumulate Cd, i.e., XWX > YZX. Treatments with three different gradients of soil Cd concentrations showed that with the increase in soil Cd concentration gradient, the Cd content in each rice plant organ also increased, i.e., Cd-3 > Cd-2 > Cd-1. The trend in the Cd content of each organ was such that the farther the organ from the root, the lower its Cd content, i.e., root > stem and sheath > leaf > grain. We observed that for all four growth stages, the booting stage is the key stage in terms of Cd absorption, where the highest levels of accumulation are observed, that is, booting stage > full heading stage > tillering stage > maturity stage. Of the two cultivars, XWX had higher SOD, POD, and CAT activities but lower MDA content. In contaminated soils, SOD, POD, and CAT activities increased gradually with the increase in Cd concentration, while MDA content decreased, which indicated that the low Cd variety XWX had an advantage over the high Cd variety YZX. Through the comparative analysis of photosynthetic physiology, it was found that the low-Cd-accumulating rice variety XWX appeared more tolerant to Cd, while the high-Cd-accumulating rice YZX was more sensitive. Therefore, the low Cd rice variety XWX was more suitable for planting safe rice in Cd-polluted paddy fields.

Cadmium (Cd) pollution in paddy soil is an increasingly serious issue in rice production. It has been reported that there is a higher or lower grain Cd accumulation in the rice cultivars Yuzhenxiang (YZX) or Xiangwanxian 12 (XWX), respectively. To better manage the Cd pollution problem, the genes that might play vital roles in governing the difference in root Cd responses between these two rice cultivars were examined. In this study, the results of RNA sequencing (RNA-seq) showed that there were 341 and 161 differentially expressed genes in the roots of YZX and XWX after Cd exposure, respectively. Among these genes, 7 genes, such as Os06g0196300 (OsJ_019618), Os07g0570700 (OsJ_24808), ADI1, GDCSH, HSFB2C, PEX11-4, and CLPB1, possessed higher degree nodes with each other, through interaction analysis by the STRING (search tool for the retrieval of interacting genes/proteins) software, suggesting that they might play vital roles in Cd response. Based on GO enrichment analysis, 41 differently expressed genes after Cd treatment in YZX or XWX were identified to be related to Cd response. Through comparative transcriptomic analysis, 257 genes might be associated with the root Cd response difference between YZX and XWX. Furthermore, we supposed that ADI1, CFBP1, PEX11-4, OsJ_019618, OsJ_24808, GDCSH, CLPB1, LAC6, and WNK3 might be implicated in Cd response based on the combined analysis of RT-qPCR, interaction, and GO annotation analysis. In conclusion, the numerous genes that might be related to Cd stress response and root Cd response difference between YZX and XWX at the booting stage may be of benefit for the development of rice varieties with low Cd consumption.

The strain B-4, isolated from a field in Changsha (China), presents strong antifungal activities, as identified by the Kirby–Bauer test, especially for pathogens that harm crops. Here, we obtained the complete genome sequence of the strain B-4 by Pacific Biosciences single-molecule real-time sequencing, making it well analyzed for understanding mechanisms and creating biological agents. Its 3,919-kb circular chromosome genome has 3,725 protein-coding genes [coding sequences (CDSs)] and 46.7% guanine–cytosine content. A comparative genome analysis of B-4 with other published strains (including Bacillus velezensis, Bacillus amyloliquefaciens, and Bacillus subtilis) revealed that the strain B-4 is a B. velezensis strain. These different strains have 2,889 CDSs in common, whereas 179 CDSs were found to be unique in the strain B-4, which is a far greater number than that in other strains. Regarding the antifungal activities of B-4, we were specifically concerned with the genes involved in the biosynthesis of secondary metabolites. In total, more than 19.56% of the genome was annotated to 12 gene clusters relating to synthesis of antimicrobial metabolites, which contained various enzyme-encoding operons for non-ribosomal peptide synthetases, polyketide synthases, and lantipeptide synthesis proteins. They were all considered to be related to the production of bacteriostatic substances or stimulation of induced systemic resistance by bacterial metabolites. These situations also present an advantage over those of other strains for biocontrol potential. We provide evidence that the biological control effect of the strain B-4, as demonstrated in antibacterial activity experiments and predicted from the complete genome sequence analysis, provides the basis for research promoting agricultural research on sustainable development, especially the contribution of biotechnology to agriculture.

Barnyardgrass is a problematic agricultural weed. Here, via genomic analysis, Guo et al. identify genes potentially underlying its success, including a gene cluster activated in response to co-cultivation with rice that leads to synthesis of the allelochemical DIMBOA.

Barnyardgrass (Echinochloa crus-galli) is one of the most troublesome herbicide-resistant weeds worldwide that interferes with rice growth and rice yield. Here we provide the data from a comparative proteomic analysis of leaves in resistant (R) and susceptible (S) biotypes of Echinochloa crus-galli both with and without multi-herbicide treatment in two independent biological experiments using iTRAQ. The distribution of length and number of peptides, mass and sequence coverage of proteins were presented, and the repeatability of the replicates was analyzed. 1342 differential accumulated proteins were identified from 2248 unique peptides by searching uniprot database and data analysis. These results are associated with the research article "Quantitative proteomics reveals ecological fitness cost of multi-herbicide resistant barnyardgrass (Echinochloa crus-galli L.)" (X. Yang, Z. Zhang, T. Gu, M. Dong, Q. Peng, L. Bai, Y Li, 2017) [1].

The use of herbicide safeners can significantly alleviate herbicide injury to protect crop plants and expand the application scope of the existing herbicides in the field. Sanshools, which are well known as spices, are N-alkyl substituted compounds extracted from the Zanthoxylum species and have several essential physiological and pharmacological functions. Sanshools display excellent safener activity for the herbicide metolachlor in rice seedlings. However, the high cost of sanshools extraction and difficulties in the synthesis of their complicated chemical structures limit their utilization in agricultural fields. Thus, the present study designed and synthesized various N-alkyl amide derivatives via the scaffold-hopping strategy to solve the challenge of complicated structures and find novel potential safeners for the herbicide metolachlor. In total, 33 N-alkyl amide derivatives (2a–k, 3a–k, and 4a–k) were synthesized using amines and saturated and unsaturated fatty acids as starting materials through acylation and condensation. The identity of all the target compounds was well confirmed by 1H-NMR, 13C-NMR, and high-resolution mass spectrometry (HRMS). The primary evaluation of safener activities for the compounds by the agar method indicated that most of the target compounds could protect rice seedlings from injury caused by metolachlor. Notably, compounds 2k and 4k displayed excellent herbicide safener activities on plant height and demonstrated relatively similar activities to the commercialized compound dichlormid. Moreover, we showed that compounds 2k and 4k had higher glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO) activities in rice seedlings, compared to the metolachlor treatment. In particular, 2k and 4k are safer for aquatic organisms than dichlormid. Results from the current work exhibit that compounds 2k and 4k have excellent crop safener activities toward rice and can, thus, be promising candidates for further structural optimization in rice protection.

Water and N are the most important factors affecting ramie (Boehmeria nivea (L.) Gaudich) growth. In this study, de novo transcriptome assembly and Tandem Mass Tags (TMT) based quantitative proteome analysis of ramie under nitrogen and water co-limitation conditions were performed, and exposed to treatments, including drought and N-deficit (WdNd), proper water but N-deficit (WNd), proper N but drought (WdN), and proper N and water (CK), respectively. A total of 64,848 unigenes (41.92% of total unigenes) were annotated in at least one database, including NCBI non-redundant protein sequences (Nr), Swiss-Prot, Protein family (Pfam), Gene Ontology (GO) and KEGG Orthology (KO), and 4268 protein groups were identified. Most significant changes in transcript levels happened under water-limited conditions, but most significant changes in protein level happened under water-limited conditions only with proper N. Poor correlation between differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) was observed in ramie responding to the treatments. DEG/DEP regulation patterns related to major metabolic processes responding to water and N deficiency were analyzed, including photosynthesis, ethylene responding, glycolysis, and nitrogen metabolism. Moreover, 41 DEGs and 61 DEPs involved in regulating adaptation of ramie under water and N stresses were provided in the study, including DEGs/DEPs related to UDP—glucuronosyhransferase (UGT), ATP synthase, and carbonate dehydratase. The strong dependency of N-response of ramie on water conditions at the gene and protein levels was highlighted. Advices for simultaneously improving water and N efficiency in ramie were also provided, especially in breeding N efficient varieties with drought resistance. This study provided extensive new information on the transcriptome, proteome, their correlation, and diversification in ramie responding to water and N co-limitation.

Salicylic acid (SA) is an endogenous plant hormone that has a wide range of pharmacological effects. Studies have indicated that SA has herbicide safening activity. In this study, the herbicide safening activity of SA and 20 substituted molecules were tested on agar-cultured rice. Biological assay results indicated that SA and substituted SA had a low inhibitory effect on the growth of rice seedlings (Oryza sativa), and partially alleviated the effects of metolachlor toxicity. Moreover, at 0.25 mg L−1, the safening effect of compounds l and u lessened the effects of metolachlor phytotoxicity on plant height and fresh weight when compared to the effects of the control, fenclorim. The effects of metolachlor toxicity were reduced on root length due to the safening effects of compounds l, n, and u; these effects were greater than those of fenclorim. These compounds could facilitate the development of novel herbicide safeners.

Dimethoate, a systemic insecticide, has been used extensively in vegetable production. Insecticide residues in treated vegetables, however, pose a potential risk to consumers. Photocatalytic degradation is a new alternative to managing pesticide residues. In this study, the degradation of dimethoate in Bok choy was investigated under the field conditions using cerium-doped nano titanium dioxide (TiO2/Ce) hydrosol as a photocatalyst. The results show that TiO2/Ce hydrosol can accelerate the degradation of dimethoate in Bok choy. Specifically, the application of TiO2/Ce hydrosol significantly increased the reactive oxygen species (ROS) contents in the treated Bok choy, which speeds up the degradation of dimethoate. Ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) analysis detected three major degradation products, including omethoate, O,O,S-trimethyl thiophosphorothioate, and 1,2-Bis (acetyl-N-methyl-) methane disulfide. Two potential photodegradation pathways have been proposed based on the intermediate products. To understand the relationship between photodegradation and the molecular structure of target insecticides, we investigated the bond length, Mulliken atomic charge and frontier electron density of dimethoate using ab initio quantum analysis. These results suggest the P = S, P-S and S-C of dimethoate are the initiation sites for the photocatalytic reaction in Bok choy, which is consistent with our empirical data.

Herbicide resistance can be either target-site or non-target-site based. The molecular mechanisms underlying non-target-site resistance (NTSR) are poorly understood, especially at the level of gene expression regulation. MicroRNAs (miRNAs) represent key post-transcriptional regulators of eukaryotic gene expression and play important roles in stress responses. In this study, the miR397 gene from Beckmannia syzigachne (referred to as bsy-miR397) was functionally characterized to determine its role in regulating fenoxaprop-P-ethyl resistance. We showed that (1) bsy-miR397 transcript level is constitutively higher in resistant than in sensitive B. syzigachne plants, whereas bsy-Laccase expression and activity show the opposite trend, and (2) bsy-miR397 suppresses the expression of bsy-Laccase in tobacco, indicating that it negatively regulates bsy-Laccase at the transcriptional level. We found evidences that miR397/laccase regulation might be involved in fenoxaprop-P-ethyl NTSR. First, the rice transgenic line overexpressing OXmiR397 showed improved fenoxaprop-P-ethyl tolerance. Second, following activation of bsy-Laccase gene expression by CuSO4 treatment, fenoxaprop resistance in B. syzigachne tended to decrease. Therefore, we suggest that bsy-miR397 might play a role in fenoxaprop-P-ethyl NTSR in B. syzigachne by down-regulating laccase expression, potentially leading to the enhanced expression of three oxidases/peroxidases genes to introduce an active moiety into herbicide molecules in Phase-2 metabolism. Bsy-miR397, bsy-Laccase, and other regulatory components might form a regulatory network to detoxify fenoxaprop-P-ethyl in B. syzigachne, supported by the differential expression of transcription factors and oxidases/peroxidases in the rice transgenic line overexpressing OXmiR397. This implies how down-regulation of a gene (laccase) can enhance NTSR. Our findings shed light on the daunting task of understanding and managing complex NTSR in weedy plant species.

Quinclorac is a widely used herbicide in rice filed. Unfortunately, quinclorac residues are phytotoxic to many crops/vegetables. The degradation of quinclorac in nature is very slow. On the other hand, degradation of quinclorac using bacteria can be an effective and efficient method to reduce its contamination. In this study, we isolated a quinclorac bioremediation bacterium strain F4 from quinclorac contaminated soils. Based on morphological characteristics and 16S rRNA gene sequence analysis, we identified strain F4 as Mycobacterium sp. We investigated the effects of temperature, pH, inoculation size and initial quinclorac concentration on growth and degrading efficiency of F4 and determined the optimal quinclorac degrading condition of F4. Under optimal degrading conditions, F4 degraded 97.38% of quinclorac from an initial concentration of 50 mg/L in seven days. Our indoor pot experiment demonstrated that the degradation products were non-phytotoxic to tobacco. After analyzing the quinclorac degradation products of F4, we proposed that F4 could employ two pathways to degrade quinclorac: one is through methylation, the other is through dechlorination. Furthermore, we reconstructed the whole genome of F4 through single molecular sequencing and de novo assembly. We identified 77 methyltransferases and eight dehalogenases in the F4 genome to support our hypothesized degradation path.

A novel method to quickly prepare molecularly imprinted polymer (MIP) monolithic fibres is developed using microwave irradiation. The well-known water-compatibility problem of MIP is overcome by liquid-liquid-solid microextraction (LLSME). The atrazine (ATR) MIP fibre was obtained after silica was etched away with a controlled length of 1 cm, and subsequently characterized by scanning electron microscopy. Main factors affecting the selective extraction, including extraction time, desorption time, extraction and desorption solvents, were investigated for LLSME procedures in detail. The selectivity was also evaluated using the ATR template molecule and its structurally related compounds, including 2-amino-4-methoxy-6-methyl-1,3,5-triazine, terbuthylazine, ametryn and metribuzin pesticides. The extraction ratio of target pesticides in ATR-imprinted polymeric monolithic fibre was increased to more than 10 times than that in non-imprinted polymeric monolithic fibre. The resultant fibres coupled by HPLC were successfully applied to detect ATR and its analogue pesticides with recoveries in the range of 68.3–92.6%. In addition, the LLSME technique was proved to be effective in extracting spiked TRI, ATR, AME and TER in lake water. This method provided a satisfactory solution for the simple, rapid, selective and sensitive pre-treatment of trace herbicides in various aqueous samples. The results demonstrate that the proposed technique could solve the water compatibility problem when the MIP monolithic fibre was exposed directly to a non-polar solvent above the aqueous solution.

In this study, we isolated an endophytic quinclorac-degrading bacterium strain Q3 from the root of tobacco grown in quinclorac contaminated soil. Based on morphological characteristics, Biolog identification, and 16S rDNA sequence analysis, we identified strain Q3 as Bacillus megaterium. We investigated the effects of temperature, pH, inoculation size, and initial quinclorac concentration on growth and degrading efficiency of Q3. Under the optimal degrading condition, Q3 could degrade 93% of quinclorac from the initial concentration of 20 mg/L in seven days. We analyzed the degradation products of quinclorac using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The major degradation products by Q3 were different from those of previously identified quinclorac degrading strains, which suggests that Q3 may employ new pathways for quinclorac degradation. Our indoor pot experiments demonstrated that Q3 can effectively alleviate the quinclorac phytotoxicity in tobacco. As the first endophytic microbial that is capable of degrading quinclorac, Q3 can be a good bioremediation bacterium for quinclorac phytotoxicity.

BackgroundAccurate evaluation of gene expression requires normalization relative to the expression of reliable reference genes. Expression levels of "classical" reference genes can differ, however, across experimental conditions. Although quantitative real-time PCR (qRT-PCR) has been used extensively to decipher gene function in the sweetpotato whitefly Bemisia tabaci, a world-wide pest in many agricultural systems, the stability of its reference genes has rarely been validated.ResultsIn this study, 15 candidate reference genes from B. tabaci were evaluated using two Excel-based algorithms geNorm and Normfinder under a diverse set of biotic and abiotic conditions. At least two reference genes were selected to normalize gene expressions in B. tabaci under experimental conditions. Specifically, for biotic conditions including host plant, acquisition of a plant virus, developmental stage, tissue (body region of the adult), and whitefly biotype, ribosomal protein L29 was the most stable reference gene. In contrast, the expression of elongation factor 1 alpha, peptidylprolyl isomerase A, NADH dehydrogenase, succinate dehydrogenase complex subunit A and heat shock protein 40 were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility.ConclusionOur finding is the first step toward establishing a standardized quantitative real-time PCR procedure following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guideline in an agriculturally important insect pest, and provides a solid foundation for future RNA interference based functional study in B. tabaci.