Exploring endophyte-mediated resistance response against winter cutworm, Noctua pronuba in cool-season turfgrass systems

Progress report for GW23-248

Project Type: Graduate Student
Funds awarded in 2023: $29,902.00
Projected End Date: 04/01/2025
Grant Recipient: Oregon State University
Region: Western
State: Oregon
Graduate Student:
Principal Investigator:
Dr. Navneet Kaur
Oregon State University
Principal Investigator:
Hannah Rivedal
USDA-ARS National Forage Seed and Cereal Research Unit
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Project Information


Exploring Epichloë endophytes is an exciting frontier for insect pest management in cool-season turfgrass systems in Oregon. In this study, we propose to evaluate endophyte-mediated resistance in commercial cultivars of perennial ryegrass (Lolium perenne), tall fescue (Festuca arundinacea), and fine fescue (Festuca rubra) against an economic insect pest Noctua pronuba (L.) (Lepidoptera: Noctuidae), or winter cutworm, a large yellow underwing moth. The research aims to measure Epichloë endophyte status and insect pest resistance response in commercial cultivars. We will conduct a series of laboratory experiments and then synthesize how these mutualistic associations can be utilized to develop more sustainable pest management practices. The supporting extension objectives will focus on education across the seed production supply chain to improve knowledge on the role of endophytes in sustainable grass seed production, how endophytes are identified and maintained over cropping seasons, and how production practices would need to change to adapt to endophyte-enhanced seed production. The information gained from this project will be used to guide future research efforts on the development of promising cultivar-endophyte relationships. Exploration of endophyte-mediated insect resistance in grass grown for seed crops will allow for more sustainable pest management strategies, with fewer synthetic insecticides, improved profitability due to lower input costs, and greater crop resilience, given the impacts of our changing climate. Together these factors will enhance ecological agricultural practices in seed crops, benefiting growers and potential end-users through enhanced plant protection benefits due to the endophyte-grass symbioses.

Project Objectives:

The research objectives of this study include the following:

  1. To assess winter cutworm survivorship and development time on previously identified cool-season turfgrass cultivars in Oregon State University’s NTEP and A-List trials containing a viable endophyte.
  2. To validate the endophyte-mediated resistance response of promising cultivars to winter cutworms by conducting a series of lab experiments whereby endophytic fungi are removed in known E+ (endophyte -positive) turfgrass cultivars and inoculated in E- (endophyte-negative) turfgrass cultivars.

The educational objectives of this project will focus on the following:

  1. To identify potential marketing strategies for the incorporation of endophyte-positive turfgrass cultivars in grass seed and turfgrass systems.
  2. To conduct an educational workshop to increase the knowledge base within the seed industry regarding the role of beneficial endophytes in plant fitness, how to exploit these endophytes in insect pest management plans, and how to retain endophyte viability throughout the seed supply chain.




Click linked name(s) to expand/collapse or show everyone's info
  • Nicole Anderson (Researcher)
  • Doug Duerst - Producer
  • Julaluck Intasin (Researcher)
  • Alec Kowalewski (Researcher)


Materials and methods:

This study evaluated host plant resistance in terms of winter cutworm feeding response in no-choice assays using commercial perennial ryegrass and tall fescue cultivars with varying endophyte infection levels. 

  1. Assess winter cutworm survivorship and development time on previously identified cool-season turfgrass cultivars in Oregon State University’s NTEP and A-List trials containing a viable endophyte.
  2. Validate the endophyte-mediated resistance response of promising cultivars to winter cutworms by conducting a series of lab experiments whereby endophytic fungi are removed in known E+ (endophyte-positive) turfgrass cultivars and inoculated in E- (endophyte-negative) turfgrass cultivars.

    Cultivars of tall fescue with either a high endophyte infection rate (above 85%) or a low endophyte infection rate (below 20%) were identified as outlined in Obj 1. However, these cultivars were unavailable commercially, so the proposed objectives (listed above) were modified to conduct two no-choice trials in the greenhouse conditions using four cultivars of two grass species (tall fescue, perennial ryegrass), two levels of endophyte infection (high, low), and two fungicide treatments (no fungicide, fungicide), along with simultaneous winter cutworm (N. pronuba) feeding (winter cutworm, control).


Two no-choice trials were conducted between August and November 2023 at the West Greenhouses, Oregon State University, Corvallis, OR.

Insect collection and rearing method

Winter cutworm, N. pronuba, eggs were collected from commercial grass seed fields in Oregon, USA (45°11'14.4"N 123°14'59.7"W and 44°33'12.3"N 123°18'02.8"W) from August to September 2023. Eggs were checked for parasitism before the introduction into a laboratory for rearing. Healthy eggs and collections without any prior exposure to insecticides were used in the laboratory colonies. The insect colonies were maintained on a general-purpose lepidopteran diet (Frontier Scientific Inc., Newark, DE) using a method slightly modified (Hervet et al. 2016) under a laboratory condition (20 ± 2 °C; 9:15 (L:D) h photoperiod; 70% humidity). One liter of diet was prepared as per the protocol modified from the recipe provided by Frontier Scientific. 875 ml of DI water was boiled in a beaker on a hotplate with a stirring bar, after which 105 g of dry mix were slowly added to the beaker. A rubber spatula was used to vigorously mix the slurry to prevent burning on the bottom of the beaker. Once the diet was fully mixed, 19 g of agar was added to the mixture, followed by vigorous mixing for five minutes. The diet was then transferred into 1 oz-cups, filling them up to ¼ of their volume. 

Plants and seed sources

Cultivars of tall fescue and perennial ryegrass with either a high endophyte infection rate (above 85%) or a low endophyte infection rate (below 20%) were identified. Two cultivars of each grass species-endophyte combination (e.g. tall fescue-high endophyte, tall fescue-low endophyte, perennial ryegrass-high endophyte, perennial ryegrass-low endophyte) were used in the experiment.

Seeds of the eight cultivars of both grass species were obtained from five different seed storage and distribution facilities in Oregon during Spring 2023, as detailed in Table 1 . The seeds were stored at 4°C when not in use. These seeds were sown in a 40-cell tray insert (150 cm3 per cell) containing RESiLIENCE® potting mix (comprising 35-45% Canadian sphagnum peat moss, processed softwood bark, perlite, coir, and dolomite) (Sun Gro Horticulture, Agawam, MA). Five seeds per cultivar were sown in each cell at a depth of 1 cm. One week after germination, seedlings were manually thinned to one per cell. The grasses were maintained under a 16:8h L:D photoperiod with supplemental lighting from 1000 W sodium vapor bulbs and watered as needed. Day and night temperatures were set at 25 and 20°C, respectively. Another set of the same seeds was treated with a systemic fungicide (Banner Maxx II) containing propiconazole as the active ingredient (Syngenta, Wilmington, DE) to create fungicide-treated counterparts. The seeds were soaked in the fungicide solution (0.5 g a.i .) for 25 min and air-dried on paper towels overnight. The appropriate fungicide rate for controlling endophytes in seedlings was previously determined. Fungicide-treated seeds were planted and maintained in the greenhouse as described above.   

Insect survivorship and development

Eight to nine weeks after the seeds germinated, five plant plugs of each cultivar-fungicide treatment combination were transferred into a 5-gallon plastic storage tote filled with potting mix. The container bases and lids were drilled with six (size) and two holes (size), respectively, to enhance ventilation. Meshes were glued over the holes on the lid and placed between the base and potting mix to prevent larval escape. The potting mix consisted of a 1:1 ratio of G&B Simples Seed Starter Mix (comprising peat moss, perlite, pumice, washed sand) (Kellogg Garden Products, Carson, CA) and autoclaved kiln-dried sand (Marion Ag Services, Inc., St. Paul, OR).

Five second to third instar winter cutworms (with a total weight ranging from 0.14 – 0.55g for trial 1 and 0.33 – 1.13g for trial 2) were added to each plastic container containing five transferred test plants. Every three days after the insect release, the numbers of observed live and dead larvae were recorded, and the aboveground grass area was photographed. The winter cutworm larvae were allowed to feed on the grasses for 14 days before removing them from the arena. Missing larvae were considered dead, and all live larvae were weighed. Each plant was cut at ground level and weighted before three representative tillers were taken and stored in an Agdia’s mesh sample bag at -20°C for endophyte detection using molecular methods (Schardl et al., 2012; Vivuk et al., 2019) (Table 1). This experiment was repeated twice.

Molecular endophyte determination methods

Total nucleic acids were extracted from frozen plant material using the protocol modified from Dellaporta et al. (1983). The modification involved the use of drill press attached with an Agdia tissue homogenizer (Agdia, Inc., Elkhart, IN) to grind samples in the Agdia’s mesh sample bags. The endophyte infection status, infection rate, and chemotype were determined using a high throughput multiplex PCR method described by Takach et al. (2012). The translation elongation factor 1-alpha (tefA) and the following alkaloid biosynthesis-related genes: peramine synthetase (perA), lolC, dmaW, and idtG were amplified from total DNA. All genes have been widely used for endophyte detection and chemotypic analysis of endophytes worldwide (Schardl et al., 2013). The primers used for the multiplex PCR reaction are listed in Table 2. The PCR reactions with a total volume of 25 µl each contained 5× Green GoTaq reaction buffer, 10 μM of each deoxynucleoside triphosphate (dNTP), 1.0-U GoTaq DNA polymerase (Promega Corp., Madison, WI), and each target-specific primer (10 µM). Amplification conditions were two min of initial denaturation at 94°C, 30 cycles of 15s at 94°C, 30s at 56°C, and one min at 72°C, followed by seven min at 72°C (Takach et al., 2012). PCR products were visualized via gel electrophoresis with a 1.5% agarose gel in 1X Tris-borate-EDTA (TBE) buffer following ethidium bromide staining and UV transillumination. The expected fragment sizes for each amplicon was presented in Table 2. Each chemotype was characterized using banding patterns.

Table 1 seed source and cultivar profiles

Grass Species


Endophyte Level provided by the Breeder

Seed Supplier

Tall Fescue



AMPAC Seed Co.

Tall Fescue



Columbia Seeds, LLC

Tall Fescue



AMPAC Seed Co.

Tall Fescue



AMPAC Seed Co.

Perennial Ryegrass

Furlong (LTP-FCB)


Lebanon Seaboard Corp.

Perennial Ryegrass

Tee-Me-Up (BSP-25)


Bailey Seed & Grain, LLC

Perennial Ryegrass



Ledeboer Seed, LLC

Perennial Ryegrass



Ledeboer Seed, LLC



Table 1. Primers used in the multiplex PCR.

Locus Primer  Primer name Primer sequence gDNA size
TefA tef1-­exon1d-­1 GGG TAA GGA CGA AAA GAC TCA 860
tef1-­exon6u-­1 CGG CAG CGA TAA TCA GGA TAG  

Representative PCR products were purified with the PCR purification kits and sent for sequencing to generate sequence data of Epichloë strains (wild type or novel) among promising cultivars identified during the insect-feeding bioassays.


Participation Summary
3 Producers participating in research

Research Outcomes

Recommendations for sustainable agricultural production and future research:

Intasin_ESA meeting_final

  • Preliminary results indicated no significant difference in mean insect mortality and weight gain between grass cultivars with high endophyte infection and those with low endophyte infection for tall fescue and perennial ryegrass included in this study. No treatment effect, i.e., fungicide treatment to eradicate endophytes, grass species, and endophyte infection level, on insect mortality and feeding damage was observed (P>0.05).
  • The next step is to confirm the differences between endophyte infection levels of plant samples used in this study using PCR methods and determine their relationships to N. pronuba feeding. The results will help inform if the grass-endophyte association can be practically utilized for N. pronuba control in turf systems
1 Grant received that built upon this project
1 New working collaborations

Education and Outreach

3 Consultations
1 Curricula, factsheets or educational tools
1 Workshop field days

Participation Summary:

3 Farmers participated
1 Ag professionals participated
Education and outreach methods and analyses:

Graduate student Pear Intasin hosted a table at the 2023 Hyslop field day on May 24, 2023, and interviewed a few growers and grass seed industry representatives about their perceptions on utilizing endophyte-mediated insect resistance in cool-season grass species. Pear also prepared a poster to present at the Annual Ryegrass Meeting in Albany in January 2024, but the event was canceled due to inclement weather and icy roads. 

Other presentations to peers included:

Intasin, P., N. Kaur, N.P. Anderson, A. Kowalewski, A. Willette, and H.M. Riveldal. 2024. Evaluating endophyte-mediated resistance against winter cutworm Noctua pronuba in cool-season turfgrass. APS Pacific Division and Conference on Soilborne Plant Pathogens, March 2023, Corvallis, OR. 

Intasin, P., N. Kaur, N.P. Anderson, A. Kowalewski, A. Willette, and H.M. Riveldal. 2024. Evaluating endophyte-mediated resistance against winter cutworm Noctua pronuba in cool-season turfgrass. Pacific Branch ESA Meeting, April 2024, Big Island, Hawaii.


Education and outreach results:

Pear Intasin finished the greenhouse trials during winter and is now wrapping up the molecular analysis. Our team will disseminate the research findings during the 2024 Hyslop field day and the grower meetings in the fall of 2024.  

1 Farmers intend/plan to change their practice(s)
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.