Performing Department
(N/A)
Non Technical Summary
Herbicide-resistant Italian ryegrass poses a serious threat to global agriculture, especially in winter crop production. In addition to the genetic factors and unrestricted hybridization, several unique ecological traits make Italian ryegrass a robust invader and a resilient and herbicide-resistance-prone weed at a global scale. It has been reported that a specific microbiome, e.g., a symbiotic-endophyte Epichloë spp. provides Italian ryegrass with increased tolerance to many abiotic stresses, including drought, competition, and sometimes herbicides. Additionally, there is evidence that certain environmental factors can influence the stress response in weed species and, subsequently, the herbicide efficacy, leading to aggravated immunity of the weeds to multiple herbicide chemistries. Although discussions with growers in extension meetings and field visits to wheat-producing counties in Alabama indicate that several Italian ryegrass populations are apparently surviving some of the commonly used herbicides applied at preemergence and postemergence stages in the region, no work has been done to document the current herbicide resistance status in this weed in Alabama or to understand the influence of key driving forces triggering herbicide resistance development and spread in Alabama and beyond. This project aims to evaluate the abundance and association of Epichloë spp. to herbicide resistance development in Italian ryegrass populations in Alabama and understand the interactive effects of soil moisture, texture, temperature, and CO2 levels on herbicide resistance development. Our overall goal is to understand the driving forces of herbicide resistance acquisition and expression in Italian ryegrass to help develop informed management strategies for deaccelerating the resistance evolution and spread.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Goals / Objectives
1. Evaluate the abundance and association of Epichloë spp. to herbicide resistance development in Italian ryegrass populations in AL.Statewide survey and herbicide resistance screening of Italian ryegrass populations.Screening for infection levels and analyzing the genotypic or phenotypic diversity of Epichloë spp. in Italian ryegrass populations.2. Understand the effects of soil moisture, texture, temperature, and CO2 levels on herbicide resistance development.Determine the combined effects of soil texture and moisture stress on endophyte infection and herbicide resistance expression.Determine the interactive effects of temperature and CO2 stress on endophyte infection and herbicide resistance expression.
Project Methods
Approach: Description of ActivitiesObjective 1a: On a statewide effort, the project team has collected, threshed, and stored 15 Italian ryegrass in-field populations that survived the PRE and/or POST herbicide applications, and 50 more populations will be collected to be used in this study. After leaving them at ambient temperature for 3-4 months for seed dormancy release, seeds will be planted in four replications of 20 plants in 30.5 cm x 46 cm trays filled with standard potting mix in greenhouse conditions maintained at 28ºC / 24ºC Day/night temperature and 14-hour photoperiod at the Plant Science Research Center of Auburn University. At a 4-5-leaf stage, Italian ryegrass seedlings will be sprayed with the field recommended rate of five herbicides that have shown the most resistant cases worldwide and suspected cases in AL, namely glyphosate (EPSP Synthase inhibitor), mesosulfuron-methyl (ALS Inhibitor), and diclofop-methyl, sethoxydim, and pinoxaden (Accase Inhibitor). After 28 days of herbicide application, injury and survival/mortality rates will be recorded based on the visual appearance, and biomass from each tray will be collected and dried at 60ºC for three days in an oven.Objective 1b: The presence and viability of Epichloë spp. mycelium (fungal hyphae) will be detected in seeds and plants. Seeds will be soaked in a 5% sodium hydroxide solution followed by washing under tap water, and then stained with aniline blue, boiled, and examined under a stereomicroscope and a compound microscope. The presence of dark blue hyphae around aleurone cells will indicate the presence of the endophyte, but not its viability. To test the viability, seeds infected with mycelium will be germinated and transplanted into pots. Epidermal tissue from leaf sheaths and the nodal region will be examined under a microscope after staining with lactophenol aniline blue for the presence of viable hyphae. Endophyte isolates will be collected to analyze phenotypic and genotypic diversity. To grow a culture from seeds, surface-sterilized seeds will be plated on potato dextrose agar (Difco Laboratories) amended with 0.01% tetracycline (PDAt), followed by incubation for 4 days at 25 °C and examined for endophyte colonies. Endophyte isolates will be identified through multi-locus sequence analysis using the internal transcribed spacer (ITS) region and beta-tubulin primers, following the methods previously used by co-PI Adesemoye to conduct a survey of Fusarium species in Nebraska45. Sequences will be edited through the Seqbuilder and EditSeq modules of the Lasergene Software v14 (DNASTAR, Madison, WI). Subsequently, sequence alignment and assembly will be performed with SeqMan Pro, and if a diverse population is observed, a phylogenetic tree will be created using MegAlign Pro. Once the infection level in seed and plants is quantified across all the Italian ryegrass populations, correlation analyses will be conducted to find any potential association between this endophyte infection and HR expression.Objective 2a: This experiment will be conducted under greenhouse conditions to examine the HR development rate under soil moisture stress across soil types. An Italian ryegrass population collected from Cullman County in AL showing immunity to multiple herbicides will be used for this study. It was tested to be moderately resistant (21-79% injury) to glyphosate applied at field rate. Using this population will enable us to realize the positive or negative influence of the test factors on HR as opposed to the absolutely susceptible or resistant population.The experiment will be conducted in a randomized complete block design (RCBD) in three replications in plastic pots of 25.7 cm top diameter and 19 cm height, filled with standard field soil collected for this study. Factors will be soil type at three levels (clay, silty loam, and loamy sand), soil moisture stress at three levels (100% field capacity, 50%, and 25%), and herbicide at three levels (preferably glyphosate, diclofop-methyl, and pinoxaden). Each plastic pot will be planted with 50 seeds and will be thinned at 3-4-leaf stage to 30 seedlings/pot of the Italian ryegrass population grown in a unique soil type and watered daily to maintain a specific field capacity after thinning. Herbicides will be sprayed at 4-5-leaf stage of the Italian ryegrass seedlings using a calibrated automated spray chamber. Agronomic growth parameters, physiological measurements using LI-6800 Photosynthesis System, and herbicide injury/mortality will be recorded following standard protocols. This study will be repeated for three generations using the progeny seeds from surviving plants of each preceding generation to capture the accumulation of HR over time in response to repeated exposure to three herbicides under different soil moisture stress in diverse soil types.Field capacities will be calculated volumetrically based on the water retained at 24 hours after watering adequately to a specific quantity of soil. The three soil types, which represent three distinct soil series throughout AL, were collected in May 2023 from AU outlying research units: a Fuquay loamy sand (loamy, kaolinitic) from the Wiregrass Research and Extension Center near Headland, AL; a Decatur silt loam (fine, kaolinitic) from the Tennessee Valley Research and Extension Center near Belle Mina, AL; and a Houston clay (very fine, smectitic) from the Black Belt Research and Extension Center near Marion Junction, AL46,47.Objective 2b: This part will be conducted in pots filled with peat-based general-purpose growing medium in the temperature and CO2-regulated plant growth chambers at the USDA-ARS National Soil Dynamics Laboratory, Auburn, AL. It will be laid out in RCBD with four replications. Factors will be CO2 at three levels (ambient CO2, ambient+200 ppm, and ambient+300 ppm), temperatures at three levels (35/30 C Day/night, 30/25 C, and 25/20 C), and herbicide at three levels (select three from the five herbicides mentioned above). Herbicide spraying, agronomic and physiological parameters, and data recording will follow objective 2a. Physiological traits will consist of leaf gas exchange and fluorescence measurements recorded with two LI-6800 units, 2 days before and 5, 10 and 15 days after spraying48,49.