Source: PENNSYLVANIA STATE UNIVERSITY submitted to
FLAVONOIDS FOR RESISTANCE AGAINST PLANT PESTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1020993
Grant No.
2019-70006-30442
Cumulative Award Amt.
$325,000.00
Proposal No.
2019-02925
Multistate No.
(N/A)
Project Start Date
Sep 1, 2019
Project End Date
Aug 31, 2024
Grant Year
2019
Program Code
[ARDP]- Applied Research and Development Program
Project Director
Chopra, S.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Plant Science
Non Technical Summary
Corn and sorghum, two annual cereals can be grown in temperate and tropical climatic zones and are thus a very dependable source of food, feed and fuel related products. An ongoing challenge is imposed by myriad of pests and pathogens that attack these cereal crops. In a modern agricultural system, insect pests are tackled by the use of synthetic chemicals, many of which are unsafe to human and environmental health. Safe crop protection strategies are essential for sustainable production of economically important crops. In this project we propose to develop: 1. insect resistant corn and sorghum breeding lines carrying plant natural compounds (flavonoids) and 2. Isolate and purify flavonoids from corn and sorghum to develop biopesticides.Our preliminary and published results have established that the use of these flavonoids is effective against pathogens and pests of corn and sorghum and other specialty crops. Thus, this project is consistent with the national research and technology development goal of the IPM Roadmap aiming to "develop low-risk suppression tactics, including use of biopesticides, biological control and products of both traditional breeding and molecular genetic technology." This project, involves a team of geneticists, entomologists, and growers. Project will provide opportunities of training students in an interdisciplinary team. They will develop expertise in the area of plant natural compound chemistry, genetic mechanisms of pest resistance as well as implementation of novel and safe IPM tools and tactics.
Animal Health Component
50%
Research Effort Categories
Basic
45%
Applied
50%
Developmental
5%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2161480108120%
2161520108020%
2163110310020%
2165220310020%
2021510108120%
Knowledge Area
216 - Integrated Pest Management Systems; 202 - Plant Genetic Resources;

Subject Of Investigation
1480 - Sweetcorn; 1510 - Corn; 1520 - Grain sorghum; 3110 - Insects; 5220 - Pesticides;

Field Of Science
1081 - Breeding; 1080 - Genetics; 3100 - Management;
Goals / Objectives
This is a CPPM ARDP Applied Research project addressing program area priority 1 "development of individual tools and tactics needed for pest management systems" for Applied Research projects.Problem area: Corn (a.k.a. maize; Zea mays L.) is an important, multi-use cereal crop in United States' agriculture. Field corn uses include animal feed, biofuel production, and a multitude of food and industrial products such as starch, sweeteners, and oil. In 2018, field corn acreage throughout the United States approached 90 million acres, on par with soybean and far exceeding wheat (USDA, NAAS, 2018).Sweet corn is produced on a smaller acreage than field corn (<500,000 acres in 2018); however, sweet corn is the fourth most consumed vegetable in the United States. Approximately 40% of the United States' sweet corn acreage is dedicated to the fresh market while the remaining production goes towards frozen corn, canned corn, or other processed products (USDA, NASS, 2018). Sweet corn was planted on 42,000 acres in 2018 in Florida, where production value generally exceeds $150 million annually, making Florida the first or second largest producer of fresh market sweet corn in the United States depending on the year. Pennsylvania is another major sweet corn producer with 13,000 acres planted in 2018 (USDA, NASS, 2018).Sorghum, Sorghum bicolor (L.) Moench, was planted on 5.7 million acres in 2017 in the United States, the world's leading sorghum producer. Uses of sorghum include livestock feed and ethanol production (United Sorghum Checkoff Program, http://www.sorghumcheckoff.com/all-about-sorghum).Corn, including field and sweet corn, and sorghum are attacked by numerous insects and all parts of the plant are susceptible to attack. In addition, none of the growth stages ranging from seedling to grain, even during storage, are immune from insect attacks. Thus, producers encounter crop losses due to insect pests, but also diseases, and environmental stresses. Input and labor associated with the use of pesticides and tillage raise production costs. Furthermore, pesticide and fertilizer usage can lead to the potential contamination of the food pipeline and waterways. Consumers respond to these concerns through market choices, which results in additional constraints on producers.Goals: Goals of this project are to use plant natural compounds to develop innate immunity in corn and sorghum against important insect pests. Current methods of controlling insect damage in corn are losing efficacy due to development of resistance and growing public concerns regarding the use of transgenic crops (GMOs) and synthetic chemicals. For example, concerns of insecticide resistance in Corn Silk Fly (CSF) were first raised in 2004. Recent laboratory and field results suggest that the efficacy of pyrethroids may be declining, especially for E. stigmatias. In March 2019, management failures in at least three large fields, resulting in the entire fields not being harvested, were reported in Florida (J. Beuzelin, pers. obs.). Thus, as intense insecticide selection continues to be applied to CSF populations, current pest practices need to be improved to secure the sustainability of CSF management.We propose environmentally friendly natural compounds from corn and sorghum to confer resistance to insect pests and to develop crop germplasm that will facilitate large-scale production of compounds to be used as biopesticides. Although the proposed project is limited to the control of a few insect pests, our preliminary data suggests that the use of plant flavonoids can be extended to control a variety of other pests and pathogens by tailoring their composition and mode of delivery. The proposed project will integrate host plant resistance to provide an additional pest management tactic that will complement current fall armyworm (FAW), and CSF, management.Objectives: We propose a two-pronged approach to combat insect pests in corn and sorghum by developing (1) breeding lines that are high yielding and enriched in flavonoids and (2) large-scale production and deployment of flavonoids compounds as biopesticides.
Project Methods
1 Evaluation of breeding lines for insect pest resistance. This objective will evaluate novel corn lines for resistance to fall armyworm (FAW) and corn silk fly (CSF) and sorghum lines for resistance to FAW and Sugarcane aphid (SCA). Resistance to CEW in the two crops will also be evaluated. Laboratory and greenhouse evaluations will be conducted at the Penn State University Park campus. Field evaluations will be conducted at three locations: 1. Agronomy Farm near University Park, PA, 2. Montgomery county, PA, and 3. UF/IFAS Everglades Research and Education Center, Belle Glade, FL. These field locations will provide a broad range of environments and insect pest population levels.1.1 Corn and sorghum field evaluations. Field corn, sweet corn and sorghum lines (8 lines/crop) along with control lines (1 susceptible and 1 resistant) will be evaluated under natural insect pest infestations. Field experiments for each crop will evaluate these lines in four-row plots (17 ft long, 20 plants) in a randomized complete block design with four blocks (1 replicate/block). For field corn experiments, MP708 and Tx601 will be included as conventional FAW resistant and susceptible checks, respectively. In addition, lines producing the Bt proteins Cry1A.105 and Cry2Ab2 for resistance to FAW and CEW will be included as commercial standards. Sweet corn experiments will include lines producing Cry1A.105 and Cry2Ab2 as commercial standards with susceptibility and resistance, respectively, to FAW and CEW. All commercial sweet corn hybrids are considered highly susceptible to CSF. For sorghum experiments, Sorghum lines will be included as SCA susceptible and resistant commercial hybrids whereas MNC610 will be used as a FAW resistant standard. Field plots will be managed following regional crop management recommendations except for insects that will be left unmanaged. However, for experiments focusing on FAW and CEW, CSF (sweet corn) and the SCA (sorghum), will be managed with carefully timed pyrethroid and flupyradifurone, respectively, applications. For experiments focusing on CSF (sweet corn) and the SCA (sorghum), FAW and CEW will be managed with chlorantraniliprole applications. Insect injury ratings and infestation levels on a per plant basis will be compared among lines using linear mixed models with the appropriate covariance structure (PROC GLIMMIX, SAS Institute Inc. 2016).1.2 Characterization of developmentally regulated, flavonoid mediated resistance to FAW in corn. Our current results from V4 and V10 stages indicate flavonoids in selected corn lines are toxic to FAW neonates. Initially, insect mortality will be tested in vitro by feeding 45 FAW neonates each on leaf segments placed in diet cups. Leaf segments will be changed regularly to ensure a fresh supply. Larval weight and mortality will be determined at the end of 12 days. Subsequently, experiments will be conducted in situ for the most and least resistant plant growth stages in the greenhouse and the field. For greenhouse experiments, plants will be grown in one-gallon pots containing field topsoil. Each treatment will be replicated three times with four plants per replicate in a randomized complete block design (1 replicate/block). For field experiments, we will rely on natural FAW infestations in Montgomery, PA and Belle Glade, FL where insect pest pressure has historically been high.1.3 Use of sorghum NILs to confirm flavonoid compound(s) responsible for FAW mortality. We have selected lines of sorghum from the diversity panels with high bioactivity against FAW larvae. We have also chemically profiled a panel of sorghum NILs for their differential ability to produce different flavonoids. This will enable us to determine the contribution of each group of flavonoids to host plant resistance against FAW. FAW feeding bioassays will be conducted. Fully expanded sorghum flag leaves will be used in the bioassays since flavonoid concentrations are highest at this stage and induced in response to biotic stress. Damage (wounding) resulting from cutting the leaves to set up the experiment also induces flavonoid biosynthesis. At the beginning and end of each assay, an aliquot of leaves fed to the FAW will be tested for flavonoid concentrations to confirm exposure of larvae to specific flavonoids.2 Processing of over-expression lines for purification of flavonoids2.1 Large scale flavonoid isolation. We will grow approximately 10,000 plants of each of the sorghum and corn overproducers for scaling up the isolation and partial purification of flavonoids. Briefly, the sorghum and corn crop will be harvested separately at a stage when expression of flavonoids is at its peak in different tissues. Whole plants will be harvested using a silage harvester/chopper and these chopped materials will be dried in a forced air blower at 65°C. We will scale up our 100 g dried material lab protocol for 100 kg plant dry biomass and this will be done in collaboration with the Penn State's Behring Fermentation facility2.2 Validation of biological activity and efficacy of purified compounds. We will test bioactivity of flavonoids on pests because factors such as large-scale purification may affect stereo-specificity, causing species-specific toxicity changes. First, we will confirm flavonoids by size-exclusion HPLC from supernatants, measure its concentration using an optical density calibration curve. A final confirmation using LC-MS will be done.2.3 Development of a spray formulation for field trials. The flavonoid compounds can be used either alone or in combination with other active or inactive substances. The resulting emulsion will then be diluted to an appropriate concentration for use. Experiments evaluating the field efficacy of five concentrations of emulsions for control of the FAW and CSF in sweet corn will be conducted at the UF/IFAS Everglades Research and Education Center. Compound concentrations and a non-treated check will be randomized to field plots in a randomized complete block design with four blocks (1 replicate/block). Plots will be 30 ft long and four rows and eight rows wide for FAW and CSF experiments, respectively. For FAW, applications will be initiated at first signs of infestations and will be repeated every 3-4 days for four weeks. For CSF, applications will be initiated at first silk and will be repeated every 2-3 days for three weeks. Experiments comparable in size and application protocol to those conducted for FAW control will be conducted for the SCA in sorghum. Injury and infestation levels at termination of treatment will be determined.

Progress 09/01/19 to 08/31/24

Outputs
Target Audience:Students and postdoctoral researchers connected with other presenters and researchers while attending scientific conferences and to present their research results. Students have also been interacting with industry researchers and getting awareness of the issues in IPM as well as crop management. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student completed his MS thesis and graduated in May 2024. Two undergraduate students participated in learning lab and field based biochemical and breeding methods respectivelly. One post-doctoral fellow learned and performed identification of microbes associated with plants. How have the results been disseminated to communities of interest?Results have been presented at scientific meetings and publications in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?This is a final report of this projects.

Impacts
What was accomplished under these goals? 1. Development and use of near isogenic lines of maize to test the role of flavonoids against insect pests. 2. Developed methods of extraction of flavonoids from maize and sorghum vegetative tissues of high flavonoid lines. 3. Developed and tested flavonoid extracts and their formulations for insect feeding assays. 4. Identified new maize and sorghum germplasm from diversity population for intriduction into the breeding program for high flavonoid lines.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Chatterjee, D., Lesko, T., Peiffer, M., Elango, D., Beuzelin, J., Felton, G. W., & Chopra, S. (2023). Sorghum and maize flavonoids are detrimental to growth and survival of fall armyworm Spodoptera frugiperda. Journal of Pest Science, 96(4), 1551-1567.


Progress 09/01/23 to 08/31/24

Outputs
Target Audience:Students and postdoctoral researchers connected with other presenters and researchers while attending scientific conferences and to present their research results. Students have also been interacting withindustry researchers and getting awareness of the issues in IPM as well as crop management. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student completed his MS thesis and graduated in May 2024. Two undergraduate students participated in learning lab and field based biochemical and breeding methods respectivelly. One post-doctoral fellow learned and performed identification of microbes associated with plants. How have the results been disseminated to communities of interest?Results have been presented at scientific meetings and publications in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?This is a final report ofthis projects.

Impacts
What was accomplished under these goals? 1. Development and use of near isogenic lines of maize to test the role of flavonoids against insect pests. 2. Developed methods of extraction of flavonoids from maize and sorghum vegetative tissues of high flavonoid lines. 3. Developed and tested flavonoid extracts and their formulations for insect feeding assays. 4. Identified new maize and sorghum germplasm from diversity population for intriduction into the breeding program for high flavonoid lines.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Panek, B., Lesko, T., Peiffer, M., Felton, G., and Chopra, S. 2024. Flavonoid Expressing Maize Anti-Herbivory For Fall Armyworm And Corn Earworm Management. Presented at the Gamma Sigma Delta Honors Societys Research Exhibition. March 27, 2024, Penn State University. University Park, Pa
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Panek, B., Lesko, T., Beuzelin, J., Peiffer, M., Felton, G., and Chopra, S. 2023. Evaluation of Hybrid Maize Expressing Flavonoids for Fall Armyworm Control. Presented at the Penn State University Graduate Research Exhibition. March 24, 2023, Penn State University. University Park, PA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lesko T., Chatterjee D., Peiffer M., Felton G., Chopra S 2023. Insecticidal 3-deoxyanthocyanidin Flavonoids from Maize and Sorghum to Manage Fall Armyworm. Presented at PSU Graduate Exhibition March 24 2023 State College, PA
  • Type: Theses/Dissertations Status: Published Year Published: 2024 Citation: Panek B. 2024. Anti-Herbivory For Fall Armyworm And Corn Earworm Management. MS thesis. The Pennsylvania State University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Colvin, C., Chatterjee, D., and Chopra, S. 2024. From Metabolites to Microbes: Paving the Way for Sustainable Maize Crop Improvement. Presented at the Penn State Undergraduate Exhibition, April 17th, 2024, Penn State University. University Park, PA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Loudy, Q., Lesko, T., and Chopra, S. 2024. Exploring the genetic basis of flavonoid accumulation in young maize leaves: Implications for fall armyworm resistance. February 15, 2024. Presented at the 39th Annual Graduate Exposition, March 22nd, 2024, Gamma Sigma Delta Research Exposition


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Consultation with corn breeders and geneticistat Univ of Florida, Gainesville campus to increase the area of testing of our breeding materials developed against fall armyworm. Changes/Problems:We are facing some challenges of flavonoid spray experiments in the field because of the rain that washes away the sprayed material before it is dried on the plant. We are trying to screen several inert additives which help in fast drying of the flavonoid residue on the leaves. We have previously performed these experiments in a greenhouse setting and did not find such issues. Another change in our field-based experimentation will be to use artificial infestation of FAW larvae in a separate field trial. This will allow us to get better count of surviving vs. dead larvae and these results will be informative when comparing those obtained from natural infestations. What opportunities for training and professional development has the project provided?Two graduate students and two undergraduate students received training in field and lab-based assays to study plant-insect interactions. One of the graduate students wrote his thesis and defended to graduate in May 2023. Students also performed purification of plant flavonoids using different solvent systems.One post-doctoral fellow is involved in identification of additional flavonoids from sorghum and maize plant tissues. Researchers from University of Florida and Penn State met monthly via Zoom to exchange experimental details and communicated results and findings. How have the results been disseminated to communities of interest?PI Chopra has given talks at scientific meetings and conferences. Students and researchers have presented posters to deliver the results of their research project. What do you plan to do during the next reporting period to accomplish the goals?We are testing different solvents to extract flavonoids so as to make them water soluble and easy to handle. We will be growing additional field trials and testing spray based effectiveness of flavonoids against fall armyworm. This work will be done in Belle Glade in collaboration with University of Florida. Another sweet corn trial will be undertaken to test the effectiveness of silk flavonoids against corn silk fly.

Impacts
What was accomplished under these goals? I. We established lab-based methods of extraction of desirable flavonoids from sorghum biomass. We grew several thousand plants and used approximately 1,000 plants of for batch extractions. Whole plants were harvested using a silage harvester and dried in a forced air blower at 65°C. Dried plant tissues were fine ground and 3-DAs were isolated by boiling ground tissue in 2N HCl followed by extracting the flavonoids into an equal volume of isoamyl alcohol. The isoamyl alcohol was evaporated to dryness and the residue suspended in acidified methanol. II. We initiated field testing (at Belle Glade) of the flavonoid extract using sprays on susceptible maize lines when we had 12% FAW infestation. We performed 4 sprays (2/week). Every day for 2 weeks after spraying flavonoid extracts, we scouted for dead or alive FAW larvae as well as FAW leaf damage data/week. Our results are promising and indicate the effectiveness of flavonoid sprays. III. Over the past three years, we have been backcrossing our flavonoid lines (field corn) to accumulate flavonoid biosynthesis and regulatory loci. During 2020 to early 2022, because of COVID-19 restrictions of travel etc., we were not able to perform field screenings. Our field-based testing of maize genetic material against fall armyworm (FAW) has begun now at our collaborators Belle Glade Research Center. A winter nursery was planted in later October to early November 2022. Selected 50 lines were screened to assess the tolerance of these lines against fall armyworm. These lines accumulate varying degree of flavonoids in leaves during different plant growth stages. We selected V3/V4/V5 stages and did three weekly FAW whorl damage ratings on the 40 lines during November 20-30. Foliar damage was recorded from this replicated trial which consisted of 40 plants per replication with three replications. We also collected several dying and surviving FAW larvae and recorded their mortality and body weight. We observed that there FAW mortality was high in flavonoid producers. Several lines in addition showed that FAW larvae also had high incidence of parasitism. These results provided preliminary results that both direct and indirect defense pathways may be active in controlling FAW populations.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Chatterjee, D., Lesko, T., Peiffer, M., Dixon, C., Elango, D., Gafoor, I., Felton, G., Chopra, S. 2020. Increased expression of antifungal and insecticidal flavonoid phytoalexins in specialty maize lines. 62nd Maize Genetics Conference, Virtual. June 25  26.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Panek, B., Lesko, T., Chopra, S. 2022. Breeding Flavonoid Expressing Maize Hybrids for Resistance to Fall Armyworm.Presented at the IPM strategies for FAW management 2022 Venue: Virtual, Zambia. 21-23 September.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Chatterjee, D., Lesko, T., Peiffer, M., Elango, D., Beuzelin, J., Felton, G., Chopra, S. 2022. Sorghum and maize flavonoids are detrimental to the survival of fall armyworm larvae. IPM Approaches Plenary Session: Breeding for FAW resistance and tolerance. Presented at the IPM strategies for FAW management 2022
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lesko, T., Chatterjee, D., Peiffer, M., Felton, G., Chopra, S. 2023. Insecticidal 3-deoxyanthocyanidin flavonoids from maize and sorghum to manage fall armyworm. Presented at The 65th Annual Maize Genetics Conference. March 16-19., 2023. St. Louis, Missouri.
  • Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Effectiveness of Sorghum and Maize Flavonoids Against Pests. Master of Science thesis in Agricultural and Environmental Plant Science. February 2023. Penn State University, University Park, PA. https://etda.libraries.psu.edu/catalog/29553tkl5215
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Chatterjee, D., Chopra, S. 2023. Understanding spatiotemporal regulation of basal endosperm differentiation in maize. In ASPB Mid-Atlantic Section symposium University of Maryland, May 24. Oral talk.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Showalter, L., Lesko., T., Panek, B., Chopra, S. 2023. Breeding Hybrid Maize for Endogenous Flavonoid Expression to Improve Fall armyworm Resistance. Presented at the Gamma Sigma Delta Expo, March 30, 2023, Penn State University.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Elango D, Wang X, Bhatnagar RS, Tan Q, Gaffoor I, Hu Z, Xue W, Roth GW, Morris GP, Reddivari L, Chopra S. Association genetics of early season cold and late season frost tolerance in Sorghum bicolor. Crop Science. 2022 Sep;62(5):1844-65.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Colvin, C., Chatterjee, D., and Chopra, S. 2023. Sugar to pigments: Deciphering sugar, reactive oxygen species, and flavonoid crosstalk using maize unstable factor for orange1. April 10, 2023. Presented at Undergraduate Exhibition, Penn State University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Panek, B., Lesko, T., Beuzelin, J., Peiffer, M., Felton, G., Chopra, S. 2023. Evaluation of hybrid maize expressing flavonoids for fall armyworm control. Presented at The 65th Annual Maize Genetics Conference. March 16-19., 2023. St. Louis, Missouri.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:1. Graduate and undergraduate students gained an understanding of flavonoids and their role in insect defense pathways. 2. Growers and corn producers were consulted to understand the fall armyworm insect pressure. 3. Collaborated with soil microbiologist and entomologist to establish insect bioassays 4. collaborated with international scientists from FAO, Italy and CIMMYT, Mexico Changes/Problems:As previously noted in last report, the research project was slowed down because of COVID-19 pandemic from March 22 onwards. We may have to request another year's No Cost Extensionto fullfill the goals of our collabprative research project with Univ of Florida. Becauseof COVID-19 travel and work restrictions we were not able to test our new corn breeding lines at the Belle Glade research station. We are now performing these multi-year corn trials starting from October 2022 till March 2025. What opportunities for training and professional development has the project provided?Students and post-doc researchers learned new techniques of IPM. How have the results been disseminated to communities of interest?Research results were published in: 1. Peer reviewed journal 2. Penn State research news What do you plan to do during the next reporting period to accomplish the goals?We will be testing our new maize breeding lines in Belle Glade, FL to assay the FAW tolerance in the field/farm setting

Impacts
What was accomplished under these goals? 1. Insect artifical diet assays were performed to test the effectiveness of flavonoids extracted from sorghum leaves. Neonates were fed artificial diet supplemented with 3-deoxyanthocyanidins (3-DAs) extract. After nine days of feeding, the average body weight for the control group was 4.22 (± 0.435) mg with a 9% mortality. Average body weight declined to 2.31(± 0.288) mg at 1 μg/ml, 1.72 (± 0.216) mg at 2 μg/ml and 0.48 mg at 10 μg/ml concentration. Mortality slightly increased to 11% at 1 μg/ml and 15% at 2 μg/ml, while the 10 μg/ml concentration showed an 88% mortality, and 20 μg/ml concentration killed all neonates feeding on the diet. 2. To further confirm the effectiveness of the exogenous application of 3-DAs in the natural diet of FAW, we used detached leaves of the Tx601 line. Leaves were either sprayed with 3-DAs extract or with the solvent used for dissolving 3-DAs (9% ethanol) or sterile water. Larvae that fed on 3-DAs sprayed leaves had significantly lower mean body weight (108.35 ± 14.268 mg) compared to both the controls of 9% ethanol (163.75 ± 9.226 mg) and sterile water (164.4 ± 10.696 mg) (F = 8.47; df (treatment) = 2; df (error) = 57; P < 0.001). Larvae had 40% mortality when fed 3-DA sprayed leaves, whereas larvae fed control leaves showed ≤ 5% mortality. These results demonstrated that sorghum 3-DA sprayed leaves of a susceptible maize line deterred herbivory by challenging the survival of the larvae. 3. We further tested the effectiveness of the exogenous application of sorghum flavonoid extracts on whole plants in a growth chamber. B73, a maize inbred line that is also susceptible to FAW, was used in this assay. Flavonoid spray increased the mortality of larvae to 50%. The larvae with reduced body weight also visually showed a reduction in body size compared to the healthy larvae. The leaf damage score was ~3 in flavonoid sprayed plants, while plants sprayed with sterile water or solvent had a score of ~8, showing significantly more leaf damage.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Elango, Dinakaran, Xiaoyu Wang, Rohil Sahai Bhatnagar, Qixian Tan, Iffa Gaffoor, Zhenbin Hu, Weiya Xue et al. "Association genetics of early season cold and late season frost tolerance in Sorghum bicolor." Crop Science. 2022
  • Type: Other Status: Published Year Published: 2022 Citation: https://www.psu.edu/news/research/story/flavonoids-sorghum-plants-kill-fall-armyworm-pest-corn-may-protect-crop/
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Chatterjee, Debamalya, Tyler Lesko, Michelle Peiffer, Dinakaran Elango, Julien Beuzelin, Gary W. Felton, and Surinder Chopra. "Sorghum and maize flavonoids are detrimental to growth and survival of fall armyworm Spodoptera frugiperda." Journal of Pest Science (2022): 1-17. https://doi.org/10.1007/s10340-022-01535-y


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:1. Discussion with corn breeders and geneticistat Univ of Florida, Gainesville campus to increase the area of testing of our breeding materials. 2. Discussion with animal and poultry scientists at Penn State, University Park Campusto test our breeding materials that have high flavonoid content in the seeds. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. This project is providing training to one graduate student and two undergraduate students in plant-insect interactions. More specifically students are receiving training about identification and purification plant natural compounds with a focus on plant flavonoids. 2. One post-doctoral fellow (50% appointment) is involved in identification of plant proteins that are affected by the presenceof flavonoids in plant tissues. 3. Undergraduate students (2) are learning various techniques of rearing and handling insects like fall armyworm, silk fly and corn ear worms. 4. All researchers interacted via Zoom as well as in person. Researchers from University of Florida and Penn State meet monthly to exchange and communicate results.? How have the results been disseminated to communities of interest?1. Presented during meetings; A. Maize Genetics Conference; 2. NESARE meeting 2. Results were discussed with vegetable growers to test the flavonoid extracts from corn and sorghum lines on select vegetables against aphids and borers. 3. A new research aspect of testing of our flavonoidson mushroom fungal infections is being spearheaded by the one of the graduate students. What do you plan to do during the next reporting period to accomplish the goals?1. We are planning to expand the testing sites in areas where fall army worm infestations occur naturally 2. Promising breeding lines will be seed multiplied for yield trials 3. Selected breeding lines and hybrids will be used for small- and large-scale extraction of flavonoid compounds to be tested against different insects.?

Impacts
What was accomplished under these goals? Objective 1.Development of breeding lines that are high yielding and enriched in flavonoids. Obj 1A. Development of genetic/breeding lines: Set 1. These near isogeniclines are being developed fromcrosses that involve selected parental lines carrying transcription factors that regulate expression of flavonoids in various tissues. Currently, four near isogenic lines are in the advanced pipeline and these lines differ in their expression of flavonoids in leaves and seeds. Line A, has no flavonoid expression in any tissue, Line B has expression of flavonoids in seed pericarp, Line C shows flavonoids accumulations in seed endosperm, and Line D has expression in leaves as well as seeds. Set II. This second set of lines are single and double cross F1s developed from high yielding parents combined with breeding lines with higher flavonoid content. Obj 1B. Fall armyworm infestation assays to test the effectiveness of flavonoids Both the Sets I and II are being tested for their response to fall armyworm infeeding assays in the lab at Penn State University. In addition these lines and hybrids are being grown inreplicated trails for testing for fall armyworm infestations in the fiield at University of FL research station at Belle Glade (in collaboration with co-PI Julien Beuzelin). Detached Leaf Assay:V6 stage leaf samples were collected from each genotype, and equal-sized pieces of leaves were placed into each cup with a single neonate. Feeding larvae were kept at 21°C with a 12 h light-dark cycle. The leaves were changed every 3rd day. Larvae weight and mortality were measured after 12 days of feeding. The larvae were considered dead if unable to move and shift to their ventral surface by themselves after being placed on the dorsal surface.On day 13, larval weight and mortality were determined. The larval mass was reduced, and larvae suffered high levels of mortality inflavonoid(+) line compared to those fed the flavonoid (-) isogenic line. Significant differences have been observed for larval weight between afterfeeding. These results are being further confirmed in additional independent experiments. Objective 2.Development of large-scale production and deployment of flavonoids compounds as biopesticides. Obj 2 A. Small scale extraction of compounds and their effectiveness. These assays are being performed using artificial diet assays to test the LD50 dosage of the flavonoid and identification of potent flavonoid compound(s) that are responsible for larval mortality.Neonates were reared on a wheat germ and casein-based artificial diet (Jacob and Chippendale 1971) with ingredients purchased from BIOSERV (Frenchtown, NJ, USA). Purified 3-DAs compounds were added to the diet for a final concentration of 0.07 μg ml-1 (Luteolinidin equivalent). Neonates were placed in individual plastic cups with a 1 cm3of freshly prepared diet and maintained in a growth chamber at 28°C with a 16 hours light period. Larvae weights and mortality were recorded after nine days.Neonates fed on flavonoids extract supplemented diet had a more than 50%mortality rate compared to only 34% for larvae on the control diet. The surviving larvae from the extract supplemented diet were also significantly smallerthan those on the control diet. These results are further conformed using additional biological replications.?

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Wu B, Chang H, Marini R, Chopra S, Reddivari L. Characterization of Maize Near-Isogenic Lines With Enhanced Flavonoid Expression to Be Used as Tools in Diet-Health Complexity. Frontiers in plant science. 2021 Jan 18;11:2249. 10.3389/fpls.2020.619598
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Chatterjee, D., Lesko, T., Peiffer, M., Dixon, C., Elango, D., Gafoor, I., Felton, G.W., Chopra, S. Increased expression of antifungal and insecticidal flavonoid phytoalexins in specialty maize lines. Maize Genetics Conference 62, 97.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Cloutier M, Chatterjee D, Elango D, Cui J, Bruns MA, Chopra S. Sorghum root flavonoid chemistry, cultivar, and frost stress effects on rhizosphere bacteria and fungi. Phytobiomes Journal. 2020 Aug 12:PBIOMES-01.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Lesko,T., Chatterjee, D., Kimberly, P., Beyer, D., Chopra, S. (2021). Effectiveness of Sorghum Compounds as Biofungicides in Mushroom Cultivation. Presented at ISMS e-Congress, September 14th-17th online via zoom.


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:Collaborators in University of Gainesville, Florida Changes/Problems:In 2020, the research was disrupted because of lockdown during COVID-19 in March 2020 and in the following months. We anticipate requesting a no-cost extension beyond the current end date of the project. What opportunities for training and professional development has the project provided?Graduate students participated in writing and publication of the research articles How have the results been disseminated to communities of interest?Journal publications What do you plan to do during the next reporting period to accomplish the goals?1.We will plant trials of maize trials at 3 sites during the summer of 2021: a. Belle Glade, FL. B. University Park, PA. c. Montgomery county. These accessions will be evaluated for fall armyworm and other corn borers. 2.To continue flavonoid purification and improve the large-scale extraction process. 3.Perform insect survival assays using diets prepared from artificial diet with and without flavonoids.

Impacts
What was accomplished under these goals? (1) A sweet corn and field corn trial wasgrownat the field site of Belle Glade, FloridaonFeb 10,2020. The purpose of these trials was to evaluatebreeding lines for their response to fall armyworm and silk fly attacks. In March, COVID-19 led to lockdown which caused disruption in the research and the field trial was left unattended. No data could be recorded. A second field trial of the same material that was planted on Feb 10, 2020, was attempted again onNovember 5, 2020.Unfortunately, the November trial was also unsuccessful because of devastating rainstorms. No data could be collected from the Nov 2020 trial also (2) Large-scale production and deployment of flavonoids compounds as biopesticides. Protocols have been standardized for the extraction of the compounds and these small-scale methods will be further tried at a larger scale. (3) Sorghum near-isogeniclines were evaluated for the flavonoid profile as well as the presence/absence of wax. These materials were evaluated using a GWAS approach and will be used in the project further to test the role of these compounds against sugarcane aphid.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Elango D, Xue W, Chopra S. Genome-wide association mapping of epi-cuticular wax genes in Sorghum bicolor. Physiology and Molecular Biology of Plants. 2020 Aug;26(8):1727-37.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Cloutier M, Chatterjee D, Elango D, Cui J, Bruns MA, Chopra S. Sorghum root flavonoid chemistry, cultivar, and frost stress effects on rhizosphere bacteria and fungi. Phytobiomes Journal. 2020 Aug 12:PBIOMES-01.