Metabolite Variation between Nematode and Bacterial Seed Galls in Comparison to Healthy Seeds of Ryegrass Using Direct Immersion Solid-Phase Microextraction (DI-SPME) Coupled with GC-MS.

Annual ryegrass toxicity (ARGT) is an often-fatal poisoning of livestock that consume annual ryegrass infected by the bacterium Rathayibacter toxicus. This bacterium is carried into the ryegrass by a nematode, Anguina funesta, and produces toxins within seed galls that develop during the flowering to seed maturity stages of the plant. The actual mechanism of biochemical transformation of healthy seeds to nematode and bacterial gall-infected seeds remains unclear and no clear-cut information is available on what type of volatile organic compounds accumulate in the respective galls. Therefore, to fill this research gap, the present study was designed to analyze the chemical differences among nematode galls (A. funesta), bacterial galls (R. toxicus) and healthy seeds of annual ryegrass (Lolium rigidum) by using direct immersion solid-phase microextraction (DI-SPME) coupled with gas chromatography−mass spectrometry (GC-MS). The method was optimized and validated by testing its linearity, sensitivity, and reproducibility. Fifty-seven compounds were identified from all three sources (nematode galls, bacterial galls and healthy seed), and 48 compounds were found to be present at significantly different (p < 0.05) levels in the three groups. Five volatile organic compounds (hexanedioic acid, bis(2-ethylhexyl) ester), (carbonic acid, but-2-yn-1-yl eicosyl ester), (fumaric acid, 2-ethylhexyl tridec-2-yn-1-yl ester), (oct-3-enoylamide, N-methyl-N-undecyl) and hexacosanoic acid are the most frequent indicators of R. toxicus bacterial infection in ryegrass, whereas the presence of 15-methylnonacosane, 13-methylheptacosane, ethyl hexacosyl ether, heptacosyl acetate and heptacosyl trifluoroacetate indicates A. funesta nematode infestation. Metabolites occurring in both bacterial and nematode galls included batilol (stearyl monoglyceride) and 9-octadecenoic acid (Z)-, tetradecyl ester. Among the chemical functional group, esters, fatty acids, and alcohols together contributed more than 70% in healthy seed, whereas this contribution was 61% and 58% in nematode and bacterial galls, respectively. This study demonstrated that DI-SPME is a valid technique to study differentially expressed metabolites in infected and healthy ryegrass seed and may help provide better understanding of the biochemical interactions between plant and pathogen to aid in management of ARGT.

Accumulation and subcellular distribution of cadmium in rygegrass induced by Aspergillus niger TL-F2 and Aspergillus flavus TL-F3.

Although plant growth-promoting fungi can greatly accelerate the ryegrass bioaccumulation of cadmium (Cd), the underlying mechanisms are not yet well documented. Therefore, we performed a 20-days hydroponic experiment to investigate the effects of Aspergillus niger TL-F2 (A. niger TL-F2) and Aspergillus flavus TL-F3 (A. flavus TL-F3) on accumulation/subcellular distribution of Cd by annual ryegrass Dongmu 70 at different Cd concentrations (0, 2.5, and 5 mg L-1). Results indicated that both fungal strains promoted ryegrass biomass/growth by about 60%. Furthermore, we found that ryegrass roots (17.8-37.1 µg pot-1) had a significantly higher capability for Cd uptake than the shoots (1.66-5.45 µg pot-1) (p < 0.05). Of total Cd in ryegrass plants, 44-67% was in soluble form, 24-37% was in cell wall, and 8.5-25.5% was in organelles. Compared with non-fungus ryegrass, cell wall and soluble Cd fractions in fungus-inoculated roots increased and decreased by 13.5-44% and 21.5-26.4%, respectively. Besides, fungus inoculation generally increased the content of cell wall and soluble Cd fractions in ryegrass shoots. Altogether, the study concludes that inoculation of fungus in ryegrass is a promising approach to improve phytoremediation of Cd contaminated environments.Novelty statement Previous study by Han et al. (2018) examined the resistance of ryegrass plant to Cd stress after its inoculation with Aspergillus aculeatus. In this study, using a hydroponic experiment, we examined the effects of co-application of two species of Aspergillus fungi. i.e. A. niger TL-F2 and A. flavus TL-F3 on ryegrass growth/biomass, Cd absorption by ryegrass shoots and roots, and subcellular distribution of Cd in ryegrass roots and shoots.

Comprehensive Transcriptome Analysis Uncovers Distinct Expression Patterns Associated with Early Salinity Stress in Annual Ryegrass (Lolium Multiflorum L.).

Soil salination is likely to reduce crop production worldwide. Annual ryegrass (Lolium multiflorum L.) is one of the most important forages cultivated in temperate and subtropical regions. We performed a time-course comparative transcriptome for salinity-sensitive (SS) and salinity-insensitive (SI) genotypes of the annual ryegrass at six intervals post-stress to describe the transcriptional changes and identify the core genes involved in the early responses to salt stress. Our study generated 215.18 Gb of clean data and identified 7642 DEGs in six pairwise comparisons between the SS and SI genotypes of annual ryegrass. Function enrichment of the DEGs indicated that the differences in lipid, vitamins, and carbohydrate metabolism are responsible for variation in salt tolerance of the SS and SI genotypes. Stage-specific profiles revealed novel regulation mechanisms in salinity stress sensing, phytohormones signaling transduction, and transcriptional regulation of the early salinity responses. High-affinity K+ (HAKs) and high-affinity K1 transporter (HKT1) play different roles in the ionic homeostasis of the two genotypes. Moreover, our results also revealed that transcription factors (TFs), such as WRKYs, ERFs, and MYBs, may have different functions during the early signaling sensing of salt stress, such as WRKYs, ERFs, and MYBs. Generally, our study provides insights into the mechanisms of the early salinity response in the annual ryegrass and accelerates the breeding of salt-tolerant forage.

Comparative Secretome Analyses of Toxigenic and Atoxigenic Rathayibacter Species.

Phytopathogenic Rathayibacter species are unique bacterial plant pathogens because they are obligately vectored by plant parasitic anguinid nematodes to the developing seedheads of forage grasses and cereals. This understudied group of plant-associated Actinomycetes includes the neurotoxigenic plant pathogen R. toxicus, which causes annual ryegrass toxicity in grazing livestock. R. toxicus is currently endemic to Australia and is listed as a plant pathogen select agent by the U.S. Department of Agriculture-Animal and Plant Health Inspection Service. The complex Rathayibacter disease cycle requires intimate interactions with the nematode vector and plant hosts, which warrants an increased understanding of the secretory and surface-associated proteins that mediate these diverse eukaryotic interactions. Here we present the first comparative secretome analysis for this complex, nematode-vectored Rathayibacter genus that compares the three agronomically damaging toxigenic and atoxigenic Rathayibacter species, R. toxicus, R. iranicus, and R. tritici. The exoproteomic comparison identified 1,423 unique proteins between the three species via liquid chromatography-tandem mass spectrometry, leading to the identification of putative pathogenicity-related proteins and proteins that may mediate nematode attachment. Of the uniquely identified proteins, 94 homologous proteins were conserved between the three Rathayibacter exoproteomes and comprised between 43.4 and 58.6% of total protein abundance. Comparative analyses revealed both conserved and uniquely expressed extracellular proteins, which, interestingly, had more similarities to extracellular proteins commonly associated with bacterial animal pathogens than classic plant pathogens. This comparative exoproteome analysis will facilitate the characterization of proteins essential for vector attachment and host colonization and assist in the development of serological diagnostic assays.

Partial Proteome of the Corynetoxin-Producing Gram-Positive Bacterium, Rathayibacter toxicus.

Rathayibacter toxicus is a Gram-positive bacterium that is the causative agent of annual ryegrass toxicity (ARGT), a disease that causes devastating losses in the Australian livestock industry. R. toxicus exhibits a complex life cycle, using the nematode Anguina funesta as a physical vector to carry it up to the seed head of the host plant. ARGT is caused by a tunicamycin-like corynetoxin that is produced in R. toxicus-infected seed galls. We analyzed protein expression in R. toxicus under stationary growth phase conditions to obtain a more complete understanding of the biology of this organism and identify potential targets for immunoassay development. A total of 323 unique proteins were identified, including those with putative roles in secondary metabolism and pathogenicity. The proteome analysis for this complex phytopathogenic Gram-positive bacterium will facilitate in the characterization of proteins necessary for host colonization and toxin production, and assist in the development of diagnostic assays. Data are available via ProteomeXchange with identifier PXD004238.

Integrated omics data of two annual ryegrass (Lolium multiflorum L.) genotypes reveals core metabolic processes under drought stress.

BACKGROUND: Annual ryegrass (Lolium multiflorum L.) is a commercially important, widely distributed forage crop that is used in the production of hay and silage worldwide. Drought has been a severe environmental constraint in its production. Nevertheless, only a handful of studies have examined the impact of short-term drought stress on annual ryegrass. The aim of this study was to explore how stress-induced core metabolic processes enhance drought tolerance, or adaptation to drought, in annual ryegrass. RESULTS: We profiled the transcriptomes, proteomes, and metabolomes of two annual ryegrass genotypes: the drought-resistant genotype "Abundant 10" and drought-susceptible genotype "Adrenalin 11." We identified differentially expressed metabolites and their corresponding proteins and transcripts that are involved in 23 core metabolic processes, in response to short-term drought stress. Protein-gene-metabolite correlation networks were built to reveal the relationships between the expression of transcripts, proteins, and metabolites in drought-resistant annual ryegrass. Furthermore, integrated metabolic pathways were used to observe changes in enzymes corresponding with levels of amino acids, lipids, carbohydrate conjugates, nucleosides, alkaloids and their derivatives, and pyridines and their derivatives. The resulting omics data underscored the significance of 23 core metabolic processes on the enhancement of drought tolerance or adaptation to drought in annual ryegrass. CONCLUSIONS: The regulatory networks were inferred using MCoA and correlation analysis to reveal the relationships among the expression of transcripts, proteins, and metabolites that highlight the corresponding elements of these core metabolic pathways. Our results provide valuable insight into the molecular mechanisms of drought resistance, and represent a promising strategy toward the improvement of drought tolerance in annual ryegrass.

Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses.

To select the most stable reference genes in annual ryegrass (Lolium multiflorum), we studied annual ryegrass leaf tissues exposed to various abiotic stresses by qRT-PCR and selected 11 candidate reference genes, i.e., 18S rRNA, E2, GAPDH, eIF4A, HIS3, SAMDC, TBP-1, Unigene71, Unigene77, Unigene755, and Unigene14912. We then used GeNorm, NormFinder, and BestKeeper to analyze the expression stability of these 11 genes, and used RefFinder to comprehensively rank genes according to stability. Under different stress conditions, the most suitable reference genes for studies of leaf tissues of annual ryegrass were different. The expression of the eIF4A gene was the most stable under drought stress. Under saline-alkali stress, Unigene14912 has the highest expression stability. Under acidic aluminum stress, SAMDC expression stability was highest. Under heavy metal stress, Unigene71 expression had the highest stability. According to the software analyses, Unigene14912, HIS3, and eIF4A were the most suitable for analyses of abiotic stress in tissues of annual ryegrass. GAPDH was the least suitable reference gene. In conclusion, selecting appropriate reference genes under abiotic stress not only improves the accuracy of annual ryegrass gene expression analyses, but also provides a theoretical reference for the development of reference genes in plants of the genus Lolium.

Allelopathic interference of alfalfa (Medicago sativa L.) genotypes to annual ryegrass (Lolium rigidum).

Alfalfa (Medicago sativa L.) genotypes at varying densities were investigated for allelopathic impact using annual ryegrass (Lolium rigidum) as the target species in a laboratory bioassay. Three densities (15, 30, and 50 seedlings/beaker) and 40 alfalfa genotypes were evaluated by the equal compartment agar method (ECAM). Alfalfa genotypes displayed a range of allelopathic interference in ryegrass seedlings, reducing root length from 5 to 65%. The growth of ryegrass decreased in response to increasing density of alfalfa seedlings. At the lowest density, Q75 and Titan9 were the least allelopathic genotypes. An overall inhibition index was calculated to rank each alfalfa genotype. Reduction in seed germination of annual ryegrass occurred in the presence of several alfalfa genotypes including Force 10, Haymaster7 and SARDI Five. A comprehensive metabolomic analysis using Quadruple Time of Flight (Q-TOF), was conducted to compare six alfalfa genotypes. Variation in chemical compounds was found between alfalfa root extracts and exudates and also between genotypes. Further individual compound assessments and quantitative study at greater chemical concentrations are needed to clarify the allelopathic activity. Considerable genetic variation exists among alfalfa genotypes for allelopathic activity creating the opportunity for its use in weed suppression through selection.

Bale Location Effects on Nutritive Value and Fermentation Characteristics of Annual Ryegrass Bale Stored in In-line Wrapping Silage.

In southeastern regions of the US, herbage systems are primarily based on grazing or hay feeding with low nutritive value warm-season perennial grasses. Nutritious herbage such as annual ryegrass (Lolium multiflorum Lam.) may be more suitable for preserving as baleage for winter feeding even with more intensive production inputs. Emerging in-line wrapped baleage storage systems featuring rapid wrapping and low polyethylene film requirements need to be tested for consistency of storing nutritive value of a range of annual ryegrass herbage. A ryegrass storage trial was conducted with 24-h wilted 'Marshall' annual ryegrass harvested at booting, heading and anthesis stages using three replicated in-line wrapped tubes containing ten round bales per tube. After a six-month storage period, nutritive value changes and fermentation end products differed significantly by harvest stage but not by bale location. Although wilted annual ryegrass exhibited a restricted fermentation across harvest stages characterized by high pH and low fermentation end product concentrations, butyric acid concentrations were less than 1 g/kg dry matter, and lactic acid was the major organic acid in the bales. Mold coverage and bale aroma did not differ substantially with harvest stage or bale location. Booting and heading stage-harvested ryegrass baleage were superior in nutritive value to anthesis stage-harvested herbage. Based on the investigated nutritive value and fermentation characteristics, individual bale location within in-line tubes did not significantly affect preservation quality of ryegrass round bale silages.

Interactions between prosulfocarb and trifluralin metabolism in annual ryegrass (Lolium rigidum).

BACKGROUND: Cross-resistance between pre-emergence herbicides is developing in Australian populations of annual ryegrass (Lolium rigidum Gaud.). A previous study has reported that selection with prosulfocarb (a pro-herbicide requiring bioactivation to its phytotoxic sulfoxide) can decrease metabolic resistance to trifluralin. Metabolism of prosulfocarb and trifluralin was investigated in L. rigidum populations with different levels of resistance to prosulfocarb, trifluralin and also pyroxasulfone, which is detoxified by glutathione (GSH) conjugation. RESULTS: Coleoptiles and radicles of herbicide-treated seedlings responded differently to the same herbicide. Radicles had a lower capacity for bioactivation of prosulfocarb, and this was correlated with a lower ability to metabolise trifluralin within and among populations. Coleoptile resistance to prosulfocarb sulfoxide was negatively correlated with abundance of a major polar metabolite. There was no evidence of GSH conjugation with the sulfoxide, making any potential links between prosulfocarb and pyroxasulfone resistance less obvious. CONCLUSIONS: Activation and metabolism of prosulfocarb in L. rigidum is complex and differentially regulated in different tissues. Selection with prosulfocarb may ameliorate trifluralin metabolism in the radicles, but the relationship between prosulfocarb and pyroxasulfone resistance is not GSH-mediated. When applying pre-emergence herbicides, care should be taken with the composition of mixtures and rotations to avoid selection of cross-resistance between pyroxasulfone and prosulfocarb. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of Biogas Slurry on the Soil Properties and Microbial Composition in an Annual Ryegrass-Silage Maize Rotation System over a Five-Year Period.

Soil health is seriously threatened by the overuse of chemical fertilizers in agricultural management. Biogas slurry is often seen as an organic fertilizer resource that is rich in nutrients, and its use has the goal of lowering the amount of chemical fertilizers used while preserving crop yields and soil health. However, the application of continuous biogas slurry has not yet been studied for its long-term impact on soil nutrients and microbial communities in a rotation system of annual ryegrass-silage maize (Zea mays). This study aimed to investigate the impacts on the chemical properties and microbial community of farmland soils to which chemical fertilizer (NPK) (225 kg ha-1), biogas slurry (150 t ha-1), and a combination (49.5 t ha-1 biogas slurry + 150 kg ha-1 chemical fertilizer) were applied for five years. The results indicated that compared to the control group, the long-term application of biogas slurry significantly increased the SOC, TN, AP, and AK values by 45.93%, 39.52%, 174.73%, and 161.54%, respectively; it neutralized acidic soil and increased the soil pH. TN, SOC, pH, and AP are all important environmental factors that influence the structural composition of the soil's bacterial and fungal communities. Chemical fertilizer application significantly increased the diversity of the bacterial community. Variation was observed in the composition of soil bacterial and fungal communities among the different treatments. The structure and diversity of soil microbes are affected by different methods of fertilization; the application of biogas slurry not only increases the contents of soil nutrients but also regulates the soil's bacterial and fungal community structures. Therefore, biogas slurry can serve as a sustainable management measure and offers an alternative to the application of chemical fertilizers for sustainable intensification.

Root Influences Rhizosphere Hydraulic Properties through Soil Organic Carbon and Microbial Activity.

Processes of water retention and movement and the hydraulic conductivity are altered in the rhizosphere. The aim of this study was to investigate the physical-hydric properties of soil aggregates in the rhizosphere of annual ryegrass (Lolium multiflorum) cropped in a Kandiudalfic Eutrudox, taking into account aspects related to soil aggregate stability. Soil aggregates from rhizosphere soil (RZS) and soil between plant rows (SBP) were used to determine soil water retention curves (SWRCs) and saturated hydraulic conductivity (Ksat). In addition, properties related to soil aggregate stability, such as water-dispersible clay, soil organic carbon (SOC), and microbial activity, were also assessed. The higher microbial activity observed in the RZS was facilitated by increased SOC and microbial activity, resulting in improved soil aggregation (less water-dispersible clay). For nearly all measured matric potentials, RZS had a higher water content than SBP. This was attributed to the stability of aggregates, increase in SOC content, and the root exudates, which improved soil water retention. The increase in total porosity in RZS was associated with improved soil aggregation, which prevents deterioration of the soil pore space and results in higher Ksat and hydraulic conductivity as a function of the effective relative saturation in RZS compared to SBP.

Effects of Coumarin on Rhizosphere Microbiome and Metabolome of Lolium multiflorum.

Rhizosphere microorganisms can help plants absorb nutrients, coordinate their growth, and improve their environmental adaptability. Coumarin can act as a signaling molecule that regulates the interaction between commensals, pathogens, and plants. In this study, we elucidate the effect of coumarin on plant root microorganisms. To provide a theoretical basis for the development of coumarin-derived compounds as biological pesticides, we determined the effect of coumarin on the root secondary metabolism and rhizosphere microbial community of annual ryegrass (Lolium multiflorum Lam.). We observed that a 200 mg/kg coumarin treatment had a negligible effect on the rhizosphere soil bacterial species of the annual ryegrass rhizosphere, though it exhibited a significant effect on the abundance of bacteria in the rhizospheric microbial community. Under coumarin-induced allelopathic stress, annual ryegrass can stimulate the colonization of beneficial flora in the root rhizosphere; however, certain pathogenic bacteria, such as Aquicella species, also multiply in large numbers in such conditions, which may be one of the main reasons for a sharp decline in the annual ryegrass biomass production. Further, metabolomics analysis revealed that the 200 mg/kg coumarin treatment triggered the accumulation of a total of 351 metabolites, of which 284 were found to be significantly upregulated, while 67 metabolites were significantly downregulated in the T200 group (treated with 200 mg/kg coumarin) compared to the CK group (control group) (p < 0.05). Further, the differentially expressed metabolites were primarily associated with 20 metabolic pathways, including phenylpropanoid biosynthesis, flavonoid biosynthesis, glutathione metabolism, etc. We found significant alterations in the phenylpropanoid biosynthesis and purine metabolism pathways (p < 0.05). In addition, there were significant differences between the rhizosphere soil bacterial community and root metabolites. Furthermore, changes in the bacterial abundance disrupted the balance of the rhizosphere micro-ecosystem and indirectly regulated the level of root metabolites. The current study paves the way towards comprehensively understanding the specific relationship between the root metabolite levels and the abundance of the rhizosphere microbial community.

Evaluation of a Saccharomyces cerevisiae Fermentation Product on the Feedlot Performance and Carcass Merit of Hair Lambs Offered an Annual Ryegrass-Hay-Based Finishing Diet: A Pilot Study.

Hair sheep production has increased in recent years, which has resulted in an increased presence in feedlots. Additionally, grass-based finishing systems for ruminant animal production have increased. Data are limited for finishing hair lambs on diets based on cool-season hay. The objective was to evaluate a Saccharomyces cerevisiae fermentation product (SCFP) on the feedlot performance and carcass characteristics of Katahdin lambs offered an annual ryegrass (Lolium multiflorum)-hay-based diet. Twenty-four Katahdin lambs (21.5 ± 2.5 kg BW) were assigned to either the control (CON) or the yeast-supplemented group (SCFP) in a completely randomized design. Lambs were offered a 14% crude protein total mixed ration diet based on annual ryegrass hay. The SCFP group also received the yeast supplement in their diet. Lambs in the SCFP group had a higher final body weight (p < 0.01) and ADG (p = 0.01). Less maximum and total energy were required to cut SCFP lamb meat compared to CON lamb meat (p < 0.03). Results from this study indicated that SCFP supplementation may prove to be beneficial in hair lamb finishing diets. Future research will need to specifically evaluate the use of these products in hair lambs with a larger sample size.

Enhanced production of water-soluble cinmethylin metabolites by Lolium rigidum populations with reduced cinmethylin sensitivity.

BACKGROUND: Cinmethylin, a pre-emergence herbicide inhibiting fatty acid thioesterase activity, has recently been introduced to Australian cereal cropping for the control of Lolium rigidum Gaud. (annual ryegrass). To date, there have been no confirmed cases of cinmethylin resistance identified in this species, but some populations exhibit reduced sensitivity to this herbicide. To explore the mechanism which contributes to reduced sensitivity of annual ryegrass to cinmethylin, the extent and nature of cinmethylin metabolism, using carbon-14 (14 C)-labelled herbicide, were analysed in three reduced-sensitivity annual ryegrass populations, alongside a susceptible population and cinmethylin-tolerant wheat as controls. RESULTS: All samples showed the same metabolite profile, with the extent of production of a specific water-soluble metabolite being correlated to the level of herbicide sensitivity. Application of the cytochrome P450 inhibitor phorate caused a decrease in water-soluble metabolite production as well as seedling growth in the presence of cinmethylin, indicating that reduced cinmethylin sensitivity in annual ryegrass could be wholly or partially due to oxidative modification of cinmethylin. CONCLUSION: Because annual ryegrass has the potential to metabolize cinmethylin in the same way as wheat, careful stewardship is required to ensure the longevity of this herbicide. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of different nitrogen doses and cultivars on fermentation quality and nutritive value of Italian ryegrass (Lolium multiflorum Lam.) silages.

OBJECTIVE: The fermentation profile and silage quality of 3 Italian ryegrass (Lolium multiflorum Lam.) cultivars (cvs. Devis, Hellen, and Trinova) treated with 5 nitrogen doses (0, 50, 100, 150, and 200 kg/ha) were evaluated. METHODS: The experiment was laid out in split plot in randomized complete block design with three replications. Annual ryegrass cultivars used in this study have been commonly grown in Turkey. Nitrogen doses were set in main plot and cultivars in split plot in the field. Plants were harvested at full-flowering stage with dry matter content about 220 g/kg for first cutting and 260 g/kg for second cutting. Harvested plants were chopped theoretically into 2 to 3 cm lengths for ensiling. Chopped fresh materials were ensilaged by compressing in 2 L plastic jars about 3±0.1 kg. RESULTS: Effects of N doses on dry matter, neutral detergent fiber, acid detergent fiber, dry matter digestibility, relative feed value, crude protein, pH, ammonia nitrogen, lactic acid, acetic acid, and lactic acid/acetic acid were statistically significant while water soluble carbohydrate, ash and organic matter were not statistically different. Ammonia nitrogen, crude protein, ash, organic matter, lactic acid, and lactic acid/acetic acid were affected by cultivars, but the other parameters were not. Increasing nitrogen applications positively affected the chemical composition of annual ryegrass silage. The significant increase in protein content was remarkable, however, silage fermentation properties were adversely affected by the increasing nitrogen dose. CONCLUSION: It can be recommended 150 kg/ha nitrogen dose for annual ryegrass harvested at full blooming stage. Even though the silage fermentation properties of the used cultivars were similar, cv. Devis gave better results than the others in terms of silage pH and relative feed value.

Target-site resistance to trifluralin is more prevalent in annual ryegrass populations from Western Australia.

BACKGROUND: Trifluralin is widely used in Australia as one of the important pre-emergence herbicides to control annual ryegrass (Lolium rigidum Gaud.) populations. Trifluralin resistance evolution and mechanisms have been identified in some ryegrass populations. RESULTS: In this study, 21 putative resistant field survey populations from Western Australian were screened with trifluralin, and 90% (19 of 21) contained individuals surviving 480 g ha-1 trifluralin treatment. Twelve populations contained individuals possessing the known α-tubulin resistance mutations at Val-202, Thr-239 and Arg-243 in TUA4 (alpha-tubulin 4 n), plus multiple potential resistance mutations in TUA4 pending genetic confirmation. Three populations had only individuals carrying newly identified (but uncharacterized) mutations in TUA3/TUA4. Radioactive work found that six populations evolved metabolic resistance to trifluralin, and at least four of them also possessed the known and/or putative target-site mutations. CONCLUSION: These results confirm that a high incidence of resistance to the dinitroaniline herbicide (trifluralin) is present, and target-site tubulin mutations make a major contribution to resistance in these annual ryegrass populations. Co-evolution of both target-site and non-target-site resistance to per-emergence herbicides warrants diverse management tactics.

Design, synthesis and herbicidal activities of p-menth-3-en-1-amine thiourea derivatives.

An efficient method for the synthesis of 1-isopropyl-4-isothiocyanato-4-methylcyclohex-1-ene has been developed with p-menth-3-en-1-amine as raw material. Next, a number of p-menth-3-en-1-amine thiourea derivatives (4a-f) have been obtained from the reactions between 1-isopropyl-4-isothiocyanato-4-methylcyclohex-1-ene and cyclohexylamine or the reactions between p-menth-3-en-1-amine and different isothiocyanates. Then, the herbicidal activities of these compounds were evaluated. The result indicated that some of these newly synthesised compounds displayed remarkable herbicidal activities against annual ryegrass. In addition, the derivative containing cyclohexane ring (4a) displayed much higher herbicidal activity against annual ryegrass than the derivatives containing benzene ring.

Evaluation of nitrogen-delivery methods for stocker cattle grazing annual ryegrass.

A 2-yr grazing experiment was conducted to evaluate efficacy of nitrogen (N) fertilization, interseeded legumes, and protein supplementation for N delivery to stocker cattle grazing annual ryegrass (Lolium multiflorum). Each year, 90 steers (initial BW, 241 ± 13 kg) were assigned to the following N-delivery methods, with or without monensin fed in a free-choice mineral supplement as a 5 × 2 factorial arrangement of treatments: ryegrass fertilized with 112 kg N/ha (NFERT); ryegrass interseeded with crimson clover (CC, Trifolium incarnatum); ryegrass interseeded with arrowleaf clover (AC, Trifolium vesiculosum); ryegrass plus distillers dried grains with solubles (DDGS) supplemented at 0.65% BW daily; and ryegrass plus whole cottonseed (WCS) supplemented at 0.65% BW daily. Pastures within the interseeded-clover and protein-supplementation treatments were fertilized with 56 kg N/ha at time of establishment. Steers were weighed every 28 d, and forage mass (FM, kg DM/ha) was measured concurrently using the destructive harvest/disk meter double-sampling method. Each of 30 0.81-ha paddocks was stocked initially with 3 "tester" steers, and stocking density (steers/ha) was adjusted using "put-and-take steers" based on changes in FM and steer BW in order to maintain a uniform forage allowance (FA) of 1 kg DM/kg steer BW. Grazing was discontinued on May 11, 2016 in Yr 1 and May 10, 2017 in Yr 2 following 140 and 84 d of grazing, respectively. Data were analyzed as a completely randomized design with repeated measures for which pasture (n = 3) was the experimental unit. Ionophore inclusion did not affect (P > 0.10) any variable measured. Mean FM differed (P < 0.0001) between years and among N-delivery methods (P < 0.10), and mean FA differed (P = 0.005) among N-delivery methods. Steer ADG differed among N-delivery methods (P = 0.02) and between years (P < 0.001), whereas total gain/ha differed (P < 0.0008) among N-delivery methods, but not between years (P = 0.78). Stocking density differed among N-delivery methods (P = 0.02) and between years (P < 0.0001), and grazing-days/ha differed between years (P < 0.0001) and among N-delivery methods (P = 0.001). Results indicate that supplementation with a high-protein by-product feed for cattle grazing annual ryegrass maintained ADG, total gain/ha and grazing-days/ha compared with N-fertilized annual ryegrass, and increased ADG, total gain/ha, and grazing-days over interseeded legumes.

Forage Characteristics and Grazing Preference of Cover Crops in Equine Pasture Systems.

Cover crops are commonly used to provide environmental benefits and can extend the grazing season, but have not been explored in horse pastures. The objectives of this research were to evaluate forage mass, forage nutrient composition, and preference of annual ryegrass, winter rye, berseem clover, purple top turnip, and daikon radish under horse grazing. Cover crops were seeded in monoculture and mixtures in August 2018 and 2019 as a randomized complete block with four replicates and grazed by four adult horses. Prior to grazing, forages were sampled to determine herbage and root mass and nutrient composition. After grazing, forages were visually assessed for the percentage of removal on a scale of 0 to 100% to estimate preference. Data was analyzed using an analysis of variance and linear regression; significance was set at P ≤ .05. Berseem clover was the lowest producing forage (590 to 1,869 kg ha-1 dry matter; P ≤.001), while minimal differences in herbage mass were observed among the other cover crops. All forages met digestible energy (>2.17 Mcal kg-1) and crude protein (>19%) requirements for idle, adult horses. Berseem clover was most preferred (>73% removal) while turnip and radish were the least preferred (<19% removal; P ≤.001). Winter rye and annual ryegrass in monoculture and when seeded with berseem clover were moderately preferred (20%-68% removal). Placing a priority on preference, berseem clover, annual ryegrass, and winter rye appear to be suitable cover crops to extend the grazing season in horse pastures.