Incidence of resistance to ALS and ACCase inhibitors in Echinochloa species and soil microbial composition in Northern Italy.

The increasing amount of weeds surviving herbicide represents a very serious problem for crop management. The interaction between microbial community of soil and herbicide resistance, along with the potential evolutive consequences, are still poorly known and need to be investigated to better understand the impact on agricultural management. In our study, we analyzed the microbial composition of soils in 32 farms, located in the Northern Italy rice-growing area (Lombardy) with the aim to evaluate the relationship between the microbial composition and the incidence of resistance to acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibiting herbicides in Echinochloa species. We observed that the coverage of weeds survived herbicide treatment was higher than 60% in paddy fields with a low microbial biodiversity and less than 5% in those with a high microbial biodiversity. Fungal communities showed a greater reduction in richness than Bacteria. In soils with a reduced microbial diversity, a significant increase of some bacterial and fungal orders (i.e. Lactobacillales, Malasseziales and Diaporthales) was observed. Interestingly, we identified two different microbial profiles linked to the two conditions: high incidence of herbicide resistance (H-HeR) and low incidence of herbicide resistance (L-HeR). Overall, the results we obtained allow us to make hypotheses on the greater or lesser probability of herbicide resistance occurrence based on the composition of the soil microbiome and especially on the degree of biodiversity of the microbial communities.

Enhanced Herbicide Metabolism and Target Site Mutation Enabled the Multiple Resistance to Cyhalofop-butyl, Florpyrauxifen-benzyl, and Penoxsulam in Echinochloa crus-galli.

This study investigated the multiple herbicide resistance (MHR) mechanism of one Echinochloa crus-galli population that was resistant to florpyrauxifen-benzyl (FPB), cyhalofop-butyl (CHB), and penoxsulam (PEX). This population carried an Ala-122-Asn mutation in the acetolactate synthase (ALS) gene but no mutation in acetyl-CoA carboxylase (ACCase) and transport inhibitor response1 (TIR1) genes. The metabolism rate of PEX was 2-fold higher, and the production of florpyrauxifen-acid and cyhalofop-acid was lower in the resistant population. Malathion and 4-chloro-7-nitrobenzoxadiazole (NBD-Cl) could reverse the resistance, suggesting that cytochrome P450 (CYP450) and glutathione S-transferase (GST) contribute to the enhanced metabolism. According to RNA-seq and qRT-PCR validation, two CYP450 genes (CYP71C42 and CYP71D55), one GST gene (GSTT2), two glycosyltransferase genes (rhamnosyltransferase 1 and IAAGLU), and two ABC transporter genes (ABCG1 and ABCG25) were induced by CHB, FPB, and PEX in the resistant population. This study revealed that the target mutant and enhanced metabolism were involved in the MHR mechanism in E. crus-galli.

Discovery of Novel Pyridazine Herbicides Targeting Phytoene Desaturase with Scaffold Hopping.

Phytoene desaturase (PDS) is a critical functional enzyme in blocking ζ-carotene biosynthesis and is one of the bleaching herbicide targets. At present, norflurazon (NRF) is the only commercial pyridazine herbicide targeting PDS. Therefore, developing new and diverse pyridazine herbicides targeting PDS is urgently required. In this study, diflufenican (BF) was used as the lead compound, and a scaffold-hopping strategy was employed to design and synthesize some pyridazine derivatives based on the action mode of BF and PDS. The preemergence herbicidal activity tests revealed that compound 6-chloro-N-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenoxy)pyridazine-4-carboxamide (B1) with 2,4-diF substitution in the benzeneamino ring showed 100% inhibition rates against the roots and stems of Echinochloa crus-galli and Portulaca oleracea at 100 µg/mL, superior to the inhibition rates of BF. Meanwhile, compound B1 demonstrated excellent postemergence herbicidal activity against broadleaf weeds, which was similar to that of BF (inhibition rate of 100%) but superior to that of NRF. This indicated that 6-Cl in the pyridazine ring is the key group for postemergence herbicidal activity. In addition, compound B1 could induce downregulation of PDS gene expression, 15-cis-phytoene accumulation, and Y(II) deficiency and prevent photosynthesis. Therefore, B1 can be considered as a promising candidate for developing high-efficiency PDS inhibitors.

Design, Synthesis, and Herbicidal Evaluation of Pyrrolidinone-Containing 2-Phenylpyridine Derivatives as Novel Protoporphyrinogen Oxidase Inhibitors.

In this work, a series of pyrrolidinone-containing 2-phenylpyridine derivatives were synthesized and evaluated as novel protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors for herbicide development. At 150 g ai/ha, compounds 4d, 4f, and 4l can inhibit the grassy weeds of Echinochloa crus-galli (EC), Digitaria sanguinalis (DS), and Lolium perenne (LP) with a range of 60 to 90%. Remarkably, at 9.375 g ai/ha, these compounds showed 100% inhibition effects against broadleaf weeds of Amaranthus retroflexus (AR) and Abutilon theophrasti (AT), which were comparable to the performance of the commercial herbicides flumioxazin (FLU) and saflufenacil (SAF) and better than that of acifluorfen (ACI). Molecular docking analyses revealed significant hydrogen bonding and π-π stacking interactions between compounds 4d and 4l with Arg98, Asn67, and Phe392, respectively. Additionally, representative compounds were chosen for in vivo assessment of PPO inhibitory activity, with compounds 4d, 4f, and 4l demonstrating excellent inhibitory effects. Notably, compounds 4d and 4l induced the accumulation of reactive oxygen species (ROS) and a reduction in the chlorophyll (Chl) content. Consequently, compounds 4d, 4f, and 4l are promising lead candidates for the development of novel PPO herbicides.

The effect of caraway oil-loaded bio-nanoemulsions on the growth and performance of barnyard grass and maize.

A proper formulation is crucial to improve the herbicidal effects of essential oils and their selectivity. In this study, we investigated the physicochemical properties of bio-based nanoemulsions (CNs) containing several concentrations of caraway (Carum carvi) essential oil stabilized with Eco Tween 80, as a surfactant, maintaining 1:1 proportions. Detailed physicochemical characteristics of the CNs revealed that their properties were most desired at 2% of the oil and surfactant, i.e., the smallest droplet size, polydispersity index, and viscosity. The CNs caused biochemical changes in maize and barnyard grass (Echinochloa crus-galli) seedlings, however, to a different extent. Barnyard grass has overall metabolism (measured as a thermal power) decreased by 39-82% when exposed to the CNs. The CNs triggered changes in the content and composition of carbohydrates in the endosperm of both species' seedlings in a dose-response manner. The foliar application of CNs caused significant damage to tissues of young maize and barnyard grass plants. The effective dose of the CN (ED50, causing a 50% damage) was 5% and 17.5% oil in CN for barnyard grass and maize tissues, respectively. Spraying CNs also decreased relative water content in leaves and affected the efficiency of photosynthesis by disturbing the electron transport chain. We found that barnyard grass was significantly more susceptible to the foliar application of CNs than maize, which could be used to selectively control this species in maize crops. However, further studies are needed to verify this hypothesis under field conditions.

Glutathione S-transferase activity facilitates rice tolerance to the barnyard grass root exudate DIMBOA.

BACKGROUND: In paddy fields, the noxious weed barnyard grass secretes 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) to interfere with rice growth. Rice is unable to synthesize DIMBOA. Rice cultivars with high or low levels of allelopathy may respond differently to DIMBOA. RESULTS: In this study, we found that low concentrations of DIMBOA (≤ 0.06 mM) promoted seedling growth in allelopathic rice PI312777, while DIMBOA (≤ 0.08 mM) had no significant influence on the nonallelopathic rice Lemont. DIMBOA treatment caused changes in the expression of a large number of glutathione S-transferase (GST) proteins, which resulting in enrichment of the glutathione metabolic pathway. This pathway facilitates plant detoxification of heterologous substances. The basal levels of GST activity in Lemont were significantly higher than those in PI312777, while GST activity in PI312777 was slightly induced by increasing DIMBOA concentrations. Overexpression of GST genes (Os09g0367700 and Os01g0949800) in these two cultivars enhanced rice resistance to DIMBOA. CONCLUSIONS: Taken together, our results indicated that different rice accessions with different levels of allelopathy have variable tolerance to DIMBOA. Lemont had higher GST activity, which helped it tolerate DIMBOA, while PI312777 had lower GST activity that was more inducible. The enhancement of GST expression facilitates rice tolerance to DIMBOA toxins from barnyard grass root exudates.

Remote Sensing Evaluation Drone Herbicide Application Effectiveness for Controlling Echinochloa spp. in Rice Crop in Valencia (Spain).

Rice (Oryza sativa L.) is a staple cereal in the diet of more than half of the world's population. Within the European Union, Spain is a leader in rice production due to its climate and tradition, accounting for 26% of total EU production in 2020. The Valencian rice area covers around 15,000 hectares and is strongly influenced by biotic and abiotic factors. An important biotic factor affecting rice production is weeds, which compete with rice for sunlight, water and nutrients. The dominant weed in Spain is Echinochloa spp., although wild rice is becoming increasingly important. Rice cultivation in Valencia takes place in the area of L'Albufera de Valencia, which is a natural park, i.e., a special protection area. In this natural area, the use of phytosanitary products is limited, so it is necessary to use the minimum amount possible. Therefore, the objective of this work is to evaluate the possibility of using remote sensing effectively to determine the effectiveness of the application of the herbicide cyhalofop-butyl by drone for the control of Echinochloa spp. in rice crops in Valencia. The results will be compared with those obtained by using sterilisation machines (electric backpack sprayers) to apply the herbicide. To evaluate the effectiveness of the application, the reflectance obtained by the satellite sensors in the red and near infrared (NIR) wavelengths, as well as the normalised difference vegetation index (NDVI), were used. The remote sensing results were analysed and complemented by the number of rice plants and weeds per area, plant dry weight, leaf area, BBCH phenological state, SPAD index values, chlorophyll content and relative growth rate. Remote sensing is validated as an effective tool for determining the efficacy of an herbicide in controlling weeds applied by both the drone and the electric backpack sprayer. The weeds slowed down their development after the treatment. Depending on the phenological state of the crop and the active ingredient of the herbicide, these results are applicable to other areas with different climatic and environmental conditions.

Bioprospection of Phytotoxic Plant-Derived Eudesmanolides and Guaianolides for the Control of Amaranthus viridis, Echinochloa crus-galli, and Lolium perenne Weeds.

The phytotoxicities of a selection of eudesmanolides and guaianolides, including natural products and new derivatives obtained by semisynthesis from plant-isolated sesquiterpene lactones, were evaluated in bioassays against three weeds of concern in agriculture (Amaranthus viridis L., Echinochloa crus-galli L., and Lolium perenne L.). Both eudesmanolides and guaianolides were active against the root and shoot growth of all the species, with the eudesmanolides generally showing improved activities. The IC50 values obtained for the herbicide employed as positive control (on root and shoot growth, respectively, A. viridis: 27.8 and 85.7 µM; E. crus-galli: 167.5 and 288.2 µM; L. perenne: 99.1 and 571.4 µM) were improved in most of the cases. Structure-activity relationships were discussed, finding that hydroxylation of the A-ring and C-13 as well as the position, number, and orientation of the hydroxyl groups and the presence of an unsaturated carbonyl group can significantly influence the level of phytotoxicity. γ-Cyclocostunolide was the most active compound in the series, followed by others such as dehydrozaluzanin C and α-cyclocostunolide (outstanding their IC50 values on A. viridis)─natural products that can therefore be suggested as models for herbicide development if further research indicates effectiveness on a larger scale and environmental safety in ecotoxicological assessments.

Elicitation of nutritional, antioxidant, and antidiabetic potential of barnyard millet (Echinochloa esculenta (A. Braun) H. Scholz) sprouts and microgreens through in vitro bio-accessibility assessment.

This study has redirected focus towards the untapped potential of millets, exploring their utilization as small-scale vegetables like sprouts and microgreens. This study assessed the metabolite profiles and therapeutic efficacy of barnyard millets as sprouts and microgreens for antioxidant, anti-diabetic, and bioaccessibility properties. Based on the study, sprouts contained 456.52 mg GE/g of starch and microgreens contained 470.04 mg GE/g of carbohydrates, whereas the gastric phase of microgreens showed 426.85 mg BSAE/g, 397.6 mg LE/g, 348.19 g RE/g, and 307.40 g AAE/g of proteins, amino acids, vitamin A and vitamin C respectively. Secondary metabolites were significantly concentrated in the microgreen stage which is responsible for their increased antioxidant and antidiabetic potential than sprouts. This study validated the therapeutic and nutritional value of millet sprouts and microgreens by demonstrating their significant nutritional composition.

Multi-omics analysis identifies EcCS4 is negatively regulated in response to phytotoxin isovaleric acid stress in Echinochloa crus-galli.

BACKGROUND: Knowledge of herbicidal targets is critical for weed management and food safety. The phytotoxin isovaleric acid (ISA) is effective against weeds with a broad spectrum, carries low environmental risks, and is thus an excellent herbicide lead. However, the biochemical and molecular mechanisms underlying the action of ISA remain unclear. RESULTS: Multi-omics data showed that acetyl coenzyme A (acetyl-CoA) was the key affected metabolite, and that citrate synthase (CS) 4 was substantially down-regulated under ISA treatment in Echinochloa crus-galli leaves. In particular, the transcript level of EcCS4 was the most significantly regulated among the six genes involved in the top 10 different pathways. The EcCS4 encodes a protein of 472 amino acids and is localized to the cell membrane and mitochondria, similar to the CS4s of other plants. The protein content of EcCS4 was down-regulated after ISA treatment at 0.5 h. ISA markedly inhibited the CS4 activity in vitro in a concentration-dependent manner (IC50 = 41.35 µM). In addition, the transgenic rice plants overexpressing EcCS4 (IC50 = 111.8 mM for OECS4-8 line) were more sensitive, whereas loss-of-function rice mutant lines (IC50 = 746.5 mM for oscs4-19) were more resistant to ISA, compared to wild type (WT) plants (IC50 = 355.6 mM). CONCLUSION: CS4 was first reported as a negative regulator of plant responses to ISA. These results highlight that CS4 is a candidate target gene for the development of novel herbicides and for breeding herbicide-resistant crops. © 2023 Society of Chemical Industry.

Boosting underwater germination in Echinochloa colona seeds: the impact of high amplitude alternating temperatures and potassium nitrate osmopriming.

Underwater germination could risk seedling survival, suggesting the need for control through seed perception of environmental cues. These cues include diurnally alternating temperatures tied to drained soils or shallow water tables. We examined high-amplitude alternating temperatures impact on underwater germination. Besides, the conditions experimented by seeds in the soil (e.g. hydration/dehydration phases) change their germinability so we tested if osmopriming could affect underwater germination. We worked with Echinochloa colona seedlots from extensive crop fields, exposing seeds to sequential submergence and drained treatments in combination with cues that promote germination. While a 10°C difference between maximum and minimum daily temperatures maximised germination in drained conditions, higher amplitudes (>15°C) alternating temperatures promoted E. colona underwater germination under hypoxic water (pO2 <4.1kPa). KNO3 osmopriming in drained conditions promoted later underwater germination even under hypoxic water; however, PEG 6000 osmopriming induced seeds to enter secondary dormancy inhibiting underwater germination. KNO3 improved E. colona underwater germination under air-equilibrated floodwater (pO2 : 16.5-17.4kPa) yet not under hypoxic conditions. This suggests that germination can proceed in flooded nitrate-fertile soils as long as it remains aerobic. Hypoxic submergence did not inhibit the induction of hypersensitivity to light in E. colona seeds. This research expands our understanding of wetland seed germination ecophysiology, shedding light on the inducible nature of underwater germination in hydrophyte weeds.

Syntenic analysis of ACCase loci and target-site-resistance mutations in cyhalofop-butyl resistant Echinochloa crus-galli var. crus-galli in Japan.

BACKGROUND: Recently, suspected cyhalofop-butyl-resistant populations of allohexaploid weed Echinochloa crus-galli var. crus-galli were discovered in rice fields in Aichi Prefecture, Japan. Analyzing the target-site ACCase genes of cyhalofop-butyl helps understand the resistance mechanism. However, in E. crus-galli, the presence of multiple ACCase genes and the lack of detailed gene investigations have complicated the analysis of target-site genes. Therefore, in this study, we characterized the herbicide response of E. crus-galli lines and thoroughly characterized the ACCase genes, including the evaluation of gene mutations in the ACCase genes of each line. RESULT: Four suspected resistant lines collected from Aichi Prefecture showed varying degrees of resistance to cyhalofop-butyl and other FOP-class ACCase inhibitors but were sensitive to herbicides with other modes of action. Through genomic analysis, six ACCase loci were identified in the E. crus-galli genome. We renamed each gene based on its syntenic relationship with other ACCase genes in the Poaceae species. RNA-sequencing analysis revealed that all ACCase genes, except the pseudogenized copy ACCase2A, were transcribed at a similar level in the shoots of E. crus-galli. Mutations known to confer resistance to FOP-class herbicides, that is W1999C, W2027C/S and I2041N, were found in all resistant lines in either ACCase1A, ACCase1B or ACCase2C. CONCLUSION: In this study, we found that the E. crus-galli lines were resistant exclusively to ACCase-inhibiting herbicides, with a target-site resistance mutation in the ACCase gene. Characterization of ACCase loci in E. crus-galli provides a basis for further research on ACCase herbicide resistance in Echinochloa spp. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Synthesis, Herbicidal Activity against Barnyard Grass, and Photolytic Behavior of Aryl 2,6-Dipyrimidinoxybenzoates.

In this study, we investigated the characteristics and herbicidal potential of bispyribac phenolic esters, which belong to the 2-(pyrimidin-2-yloxy)benzoic acid (PYB) class of acetohydroxyacid synthase (AHAS-)-inhibiting herbicides. These herbicides are primarily used for controlling Poaceae and broadleaf weeds. Among them, bispyribac-sodium stands out as a representative in this class. Surprisingly, other bispyribac esters, including alkanol and phenol esters exhibit considerably reduced herbicidal activity compared to bispyribac-sodium. In contrast, oxime esters (e.g., pyribenzoxim) demonstrate high activity. To further understand and develop novel PYB herbicides, we synthesized and screened a series of bispyribac phenolic esters while investigating their photochemical behaviors. Several compounds displayed excellent herbicidal activity, with compounds Ia-19 and Ic showing impressive 90% effective dosages for fresh weight inhibition of barnyard grass, measuring 0.55 and 0.60 g a.i./hm2, respectively. These values were approximately half of bispyribac-sodium or pyribenzoxim. The results indicate that the herbicidal activity of phenolic esters is influenced by both their binding ability to the AHAS enzyme and their decomposition into bispyribac acid. For instance, bispyribac phenol ester exhibited considerably reduced receptor affinity compared to bispyribac-sodium, and faced challenges in transforming into bispyribac acid, explaining its diminished herbicidal activity. However, introducing a photosensitive nitro group led to a complete transformation. This modification improved its affinity with AHAS and accelerated its decomposition into bispyribac acid, further accelerated by photocatalysis. Consequently, nitro-containing compounds displayed heightened herbicidal activity. The findings from this study open possibilities for structural optimization of phenolic esters through quantitative structure-activity relationship analysis, potentially regulating their activity-releasing period. Furthermore, the high activity of aromatic heterocyclic esters offers new insights into developing novel PYB herbicides.

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.

Telomere-to-telomere genome assembly of an allotetraploid pernicious weed, Echinochloa phyllopogon.

Echinochloa phyllopogon is an allotetraploid pernicious weed species found in rice fields worldwide that often exhibit resistance to multiple herbicides. An accurate genome sequence is essential to comprehensively understand the genetic basis underlying the traits of this species. Here, the telomere-to-telomere genome sequence of E. phyllopogon was presented. Eighteen chromosome sequences spanning 1.0 Gb were constructed using the PacBio highly fidelity long technology. Of the 18 chromosomes, 12 sequences were entirely assembled into telomere-to-telomere and gap-free contigs, whereas the remaining six sequences were constructed at the chromosomal level with only eight gaps. The sequences were assigned to the A and B genome with total lengths of 453 and 520 Mb, respectively. Repetitive sequences occupied 42.93% of the A genome and 48.47% of the B genome, although 32,337, and 30,889 high-confidence genes were predicted in the A and B genomes, respectively. This suggested that genome extensions and gene disruptions caused by repeated sequence accumulation often occur in the B genome before polyploidization to establish a tetraploid genome. The highly accurate and comprehensive genome sequence could be a milestone in understanding the molecular mechanisms of the pernicious traits and in developing effective weed control strategies to avoid yield loss in rice production.

Target-Site and Metabolic Resistance Mechanisms to Penoxsulam in Late Watergrass (Echinochloa phyllopogon) in China.

Echinochloa phyllopogon, a malignant weed in Northeast China's paddy fields, is currently presenting escalating resistance concerns. Our study centered on the HJHL-715 E. phyllopogon population, which showed heightened resistance to penoxsulam, through a whole-plant bioassay. Pretreatment with a P450 inhibitor malathion significantly increased penoxsulam sensitivity in resistant plants. In order to determine the resistance mechanism of the resistant population, we purified the resistant population from individual plants and isolated target-site resistance (TSR) and nontarget-site resistance (NTSR) materials. Pro-197-Thr and Trp-574-Leu mutations in acetolactate synthase (ALS) 1 and ALS2 of the resistant population drove reduced sensitivity of penoxsulam to the target-site ALS, the primary resistance mechanisms. To fully understand the NTSR mechanism, NTSR materials were investigated by using RNA-sequencing (RNA-seq) combined with a reference genome. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis further supported the enhanced penoxsulam metabolism in NTSR materials. Gene expression data and quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation confirmed 29 overexpressed genes under penoxsulam treatment, with 16 genes concurrently upregulated with quinclorac and metamifop treatment. Overall, our study confirmed coexisting TSR and NTSR mechanisms in E. phyllopogon's resistance to ALS inhibitors.

Selection and validation of reliable reference genes for quantitative real-time PCR in Barnyard millet (Echinochloa spp.) under varied abiotic stress conditions.

Quantitative real-time polymerase chain reaction (RT-qPCR) using a stable reference gene is widely used for gene expression research. Barnyard millet (Echinochloa spp.) is an ancient crop in Asia and Africa that is widely cultivated for food and fodder. It thrives well under drought, salinity, cold, and heat environmental conditions, besides adapting to any soil type. To date, there are no gene expression studies performed to identify the potential candidate gene responsible for stress response in barnyard millet, due to lack of reference gene. Here, 10 candidate reference genes, Actin (ACT), α-tubulin (α-TUB), ß-tubulin (ß-TUB), RNA pol II (RP II), elongation factor-1 alpha (EF-1α), adenine phosphoribosyltransferase (APRT), TATA-binding protein-like factor (TLF), ubiquitin-conjugating enzyme 2 (UBC2), ubiquitin-conjugating enzyme E2L5 (UBC5) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were selected from mRNA sequences of E. crus-galli and E. colona var frumentacea. Five statistical algorithms (geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder) were applied to determine the expression stabilities of these genes in barnyard millet grown under four different abiotic stress (drought, salinity, cold and heat) exposed at different time points. The UBC5 and ɑ-TUB in drought, GAPDH in salinity, GAPDH and APRT in cold, and EF-1α and RP II in heat were the most stable reference genes, whereas ß-TUB was the least stable irrespective of stress conditions applied. Further Vn/Vn + 1 analysis revealed two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with Cu-ZnSOD (SOD1) in the plants exposed to different abiotic stress conditions. The results revealed that the relative quantification of the SOD1 gene varied according to reference genes and the number of reference genes used, thus highlighting the importance of the choice of a reference gene in such experiments. This study provides a foundational framework for standardizing RT-qPCR analyses, enabling accurate gene expression profiling in barnyard millet.

Inter-household transfers of material goods among Sama "sea nomads" of the Philippines: Reciprocity, helping, signaling, or something else?

The extent to which humans share with both kin and non-kin is a defining characteristic of our species. Evolutionary research suggests that pervasive reliance on inter-individual transfers of goods and services may have evolved to support a cooperative breeding adaptation in humans. However, while intensive food sharing between individuals and families has frequently been investigated in small-scale human societies, a comprehensive analysis of the daily transfers of all material goods has not been attempted. Likewise, while much previous research on cooperative transfers focused on terrestrial foraging populations, less attention is paid to other small-scale economic modalities traditionally inhabited by humans. Drawing on over three years' worth of interviews and observational data from a community of primarily ethnic Sama people residing along the coast of Southern Mindanao Island in the Philippines, this paper examines the overall transfer patterns of material goods in a marine foraging economy. A quantitative description of resource acquisition is followed by an in-depth exploration of the characteristics of individual households and household dyads who gave and/or received more during the study period. Results indicate that a household's age and income are consistently correlated with increased inflow and outflow of material goods. Results also suggest differential motivations underlie inter-household sharing of food, money, and other goods in the study community. Most importantly, we find that both daily and long-term reciprocity overwhelmingly drive sharing within household dyads in the study community, despite secondary effects of kinship, relative need, and relative household age between household dyads.

Multiple resistance of Echinochloa phyllopogon to synthetic auxin, ALS-, and ACCase-inhibiting herbicides in Northeast China.

Echinochloa phyllopogon is a self-pollinating allotetraploid weed and a serious threat to global rice production. One sensitive and three multiple-resistant populations collected from two provinces of Northeast China were used to analyze the mechanism of multiple resistance of E. phyllopogon to penoxsulam, metamifop, and quinclorac. Compared with the sensitive population LN12, LN1 showed higher resistance to these three herbicides; LN24 showed medium resistance to penoxsulam and metamifop and higher resistance to quinclorac (274-fold); HLJ4 showed low resistance to penoxsulam and high resistance to metamifop and quinclorac. Target sequence analysis showed no mutations in acetolactate synthase or acetyl-CoA carboxylase genes. In-vitro enzyme activity analysis showed that the activity of the target enzyme of multiple herbicide-resistant populations was similar to that of the sensitive population. The P450 inhibitor, malathion, noticeably increased the sensitivity of LN1, LN24, and HLJ4 to penoxsulam, LN1 to metamifop, and HLJ4 to quinclorac. Under all four treatments, the GSTs activities of resistant and sensitive populations showed an increasing trend from day 1 to day 5, but the sensitivity and activity of GSTs were higher in the multiple-resistant population than that in the sensitive population LN12. This study identified the development of multiple-resistant E. phyllopogon populations that pose a serious threat to rice production in rice fields in Northeast China, preliminarily confirming that multiple-resistance was likely due to non-target-site resistance mechanisms. These populations of E. phyllopogon are likely to be more difficult to control.

Metabolic Exploration for Cyhalofop-Butyl Antagonism in Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] Following Pretreatment of Malathion.

The present study investigated the effects of broad-spectrum metabolic inhibitors malathion (cytochrome P450 inhibitor) and/or 4-chloro-7-nitrobenzofurazan (NBD-Cl; glutathione S-transferase inhibitor) on the metabolism of cyhalofop-butyl (CyB) in barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] biotypes confirmed previously with multiple resistance to two herbicides CyB and florpyrauxifen-benzyl. The metabolic inhibitors were not effective at recovering the sensitivity of resistant barnyardgrass biotypes to CyB treated at the labeled rate (313 g ai ha-1). Rather, treatment with malathion followed by CyB caused antagonism, reducing the efficacy of CyB and promoting the growth of resistant biotypes. Pretreatment with malathion did not influence absorption/translocation of the applied form CyB and its conversion to the active herbicide form cyhalofop-acid (CyA), in both susceptible and resistant biotypes. In contrast, metabolism of the applied form (CyB) decreased 1.5 to 10.5 times by the malathion pretreatment. Taken together, the maintained CyA production against the reduced CyB metabolism could be the mechanism to account for the cause of CyB antagonism observed in barnyardgrass following malathion pretreatment. Additionally, the evolution of CyB resistance in barnyardgrass might be associated with reduced production of CyA in resistant biotypes, independent of activities of cytochrome P450 or GST enzymes.