Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Aug 1, 2020
Project End Date
Jul 31, 2024
Grant Year
Project Director
Chopra, S.
Recipient Organization
408 Old Main
Performing Department
Plant Science
Non Technical Summary
Maize (corn) is a very dependable source of food, feed, and biofuels. Several pests and pathogens attack corn and thus challenge its production. Majority of pests are tackled by the use of synthetic chemicals. These chemical pesticides are unsafe to human and environmental health. Thus, a reduced usage of such harmful chemicals and development of safe and sustainable IPM strategies are needed. Our results show increased mortality and reduced growth of fall army worm (FAW) larvae feeding on certain maize lines. We have discovered that flavonoid compounds expressing in these maize lines are the cause of mortality of FAW larvae. Thus, in this research project we are characterizing the interaction of specific flavonoid compound with FAW larvae in order to understand the mechanism their mortality. Thus, we hypothesize that flavonoid toxicity could be due to: a) their direct effect on salivary and/or gut metabolic processes; b) interference with the larval detox pathway resulting from absence/loss of expression of specific enzymes; c) compromising the integrity of the gut barrier. These hypotheses will be tested through three objectives: 1) Characterize the role of flavonoids in host plant defense. 2) Confirm the role of flavonoids in FAW growth and mortality. 3) Determine microbial diversity associated with maize host plants and their pests.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
Maize is an important, multi-use cereal crop in U.S. agriculture. The major uses of this crop include the production of animal feed, biofuel, and a multitude of food and industrial products such as starch, sweeteners, oil, etc. In 2017, total acreage under cultivation was 90 million acres on par with soybean and far exceeding wheat (U.S. Department of Agriculture, National Agricultural Statistics Service). Accompanying this extensive acreage are a plethora of problems related to pest and pathogen pressure which is exacerbated by environmentally favorable practices such as no-till agriculture and increasing pressure as pest populations increase in number and distribution. All parts of the maize plant are attacked by a myriad of insect pests and no growth stage, ranging from seedling to grain even during storage, is immune from attack. Prominent among the cohort of insects causing damage is the fall armyworm (FAW; Spodoptera frugiperda (J. E. Smith). Being highly cold-sensitive, the adult moths move northward progressively from their overwintering sites in the gulf coast region. Although polyphagous, it has a definite preference for graminaceous hosts, especially corn. It is a leaf feeder in the early larval stages, residing in the whorl. During this stage, voracious feeding may result in stripped leaves and destruction of the meristem, causing plant death. If they survive these early stages, later instars can attack the stem and developing ears resulting in extensive devastation. Yield losses are due to lodging of damaged stems and ear damage. Furthermore, secondary infections by mycotoxigenic fungi such as Aspergillus and Fusarium spp. of the damaged ear leads to mycotoxin contamination, making the seed unfit even for animal consumption.Control measures include diligent scouting to address the issue before it becomes a problem. Insecticide applications are not highly effective because major outbreaks occur later in the year when plants are too tall to be effectively treated with chemicals. FAW can only be effectively controlled while the larvae are small. Furthermore, the larvae reside in the whorl under a plug of frass and are thus protected from the insecticide application. The use of resistant germplasm is a viable option. Since the advent of Bt corn, much success has been achieved in controlling FAW damage over the past two decades. Unfortunately, several instances of survival of FAW larvae on Cry1F expressing transgenic maize have been reported in recent years. Further investigation of this phenomenon confirmed that unexpected pest survival was due to the development of resistance to the Cry1F protein. Development of diverse antagonistic mechanisms that can be deployed against FAW will not only provide us with an arsenal against this pest, it will also delay the development of resistance against any single antagonistic mechanism. The current issue of FAW spread in many geographical locations including the Caribbean, China, India, and Africa should be a wake-up call. This pest is moving quickly and it will not be too long before it spreads throughout the world. We need to start investing in alternate IPM strategies that are simple, effective, durable, least expensive and at the same time non-toxic to human and animal health.Usage of agrochemicals has become an integral part of today's crop protection strategies. Innovations in crop protection research in synthetic combinatorial chemistry have led to tremendous advancements in the development of novel chemicals. These large-scale chemical deployment strategies have led to negative consequences on non-target biota due to atmospheric, ground and surface water pollution. Alternatively, a reduction of the use of these harmful synthetic chemical pesticides may be possible by developing sustainable strategies of crop protection. One approach is to develop resistant cultivars and a second is to pursue the development of safer pesticides that are as efficacious as synthetic chemicals. The proposed project is motivated by these two approaches and aims to understand the biological interactions of plant flavonoid compounds with plants and their pests. During the past twenty years of flavonoid research in the Chopra Lab, a genetic pathway has been explored to discover the role of 3-deoxyanthocyanidins (3-DAs) in host plant defense.The goal of this project is to understand the role of plant flavonoids in strengthening the plant defense response by mitigating the insect suppressive cues. The project also aims to explore plant and insect associated microbial diversity which may play a role in weakening/strengthening plant defense mechanisms. The following specific objectives are proposed to understand how plant defense pathway(s) may be affected via the secondary phytochemicals and how these compounds affect microbial diversity associated with plants and pests: 1. Characterize the role of flavonoids in host plant defense against FAW. 2. Confirm the role of flavonoids in FAW growth and mortality. 3. Determine microbial diversity associated with maize host plants and their pests.
Project Methods
Obj. 1. Characterize the role of 3-DAs in host plant defense to FAW. These studies will be done using leaves from field-grown plants of two specific maize lines that have been developed at Penn State in the Chopra laboratory. For objective 1, we will solely focus on the use of a single source of FAW eggs/population from Benzon Research Lab. All field experiments will be performed at Penn State Agronomy Farm, Rock Springs, PA. Initially, mortality will be tested in vitro by feeding 45 neonates each on leaf segments from these growth stages placed in diet cups. Fully expanded leaves of each plant growth stage will be used in the FAW feeding bioassay. We will test the survival of neonate larvae on field-grown plants and leaf growth stages from V4 to VT. These stages are where innate resistance is critical due to the unavailability of effective control measures. Leaf feeding assays will allow us to identify the most resistant/susceptible stages to be used for clip cage experiments. At the beginning and end of each feeding, leaf samples used will be assayed for 3-DA concentrations to confirm their exposure to FAW larvae. 3-DAs will be quantified by measuring absorbance at ?490 nm and expressing concentration as luteolinidin equivalents. Confirmation of compounds will be done via HPLC profiles. All experiments will be repeated in situ in field-grown plants over two growing seasons by placing five neonates on plant leaves in clip cages. For the field experiments, plants will be grown in 17' long rows containing 20 plants each in an RCB design. Ten rows of each genotype will be grown. To obtain sufficient plant tissue for various assays, the experiment will consist of 10 replicates with 3 cages per plant per replicate. Leaf samples collected in all assays will be snap-frozen in liquid nitrogen and stored at -80C until used for gene expression, metabolite and proteomics analysis. All plant defenses will be assessed by qRT-PCR expression assays of standard defense-related maize genes. Expression of maize chalcone synthase (chs) c2 gene will be assayed to correlate with the 3-DA synthesis.Results will establish if the presence/absence of flavonoids in maize leaves affects standard JA-mediated defense responses to FAW feeding. Any deviation from a typical herbivore-induced defense response could indicate the involvement of a flavonoid mediated defensive layer. This aim would thus also provide a mechanistic detail of induced expression of 3-DA during FAW feeding.Obj. 2. Confirm the role of 3-DAs in FAW growth and mortality. To confirm and to perform a dose-dependent response, in vitro bioassays with purified compounds will be carried out. 3-DAs will be isolated from leaves of sorghum line that high expression of 3-DA. LC-MS profiling will confirm the presence of apigeninidin, luteolinidin and methoxylated luteolinidin. Forty-five neonates/treatment will be fed with amended media containing purified 3-DAs or diet only. Doses/quantities tested will range from 0.01 mg - 1 mg/ml based on the specific growth rate of gut bacteria (section 2.10 and data not shown). A very small quantity of the diet amended with the appropriate amount of 3-DAs will be fed to the larvae each morning for complete ingestion. Larvae will be fed on a regular diet for the rest of the day. Larval weight and mortality will be determined after 12 days. We will test if the mechanism of toxicity is that 3-DAs damage the peritrophic membrane, thereby permitting microbes in the food bolus to enter the hemolymph, causing septicemia. Forty-five neonates/treatment will be transferred to diet with 3-DAs as described above. Controls will consist of larvae fed on a diet or on field-grown TX601 leaves. After eight days of feeding, larvae will be fed with a very small portion of diet laced with polydisperse fluorescein isothiocyanate-labeled dextran (FITC-dextran) to ensure complete consumption.Obj. 3. Determine microbial diversity associated with maize host plants and their pests. We will develop a more extensive list of FAW midgut microbes, as well as the diversity of microbes associated with the two maize genotypes, we propose performing experiments using HTS methods.Maize genotypes and their effect on the diversity of culturable leaf microflora. We will attempt a preliminary test to find out if bacterial diversity differs between maize NILs with/without 3-DAs in their leaves. Leaves (V6 stage) will be sampled from the field at the PSU Agronomy Farm, Rock Springs, PA. Three leaf pieces (each 3 cm2) per replication from each test genotype and B73 (a standard maize line with sequenced genome) will be combined in a tube, homogenized in 600 µl of sterile PBS and 100 µl was then applied to a 2xYT media plate and incubated overnight at 28°C. For each genotype this will be replicated 3 times. After 24 h, individual colonies will be sub-cultured onto 2xYT plates, grown at 28°C and identified using the Bruker Biotyper MALDI-TOF MS. We will also perform HTS of larval regurgitant before and after feeding on maize NILs to capture greater diversity associated with these maize lines. We will assay microbial diversity associated with salivary regurgitant and midgut of FAW.Sample collection and DNA extraction. Caterpillar regurgitant (20 μl/larva) will be collected and stored at −80 °C until needed. For midgut collections, caterpillars will be starved for 2-3 h, surface-sterilized in 10% Coverage Plus NPD (Steris, Mentor, OH, USA), and rinsed twice in sterile water. All DNA extractions will be performed using the Quick-DNA™ Fecal/Soil Microbe Microprep Kit (Zymo Research, Irvine, CA).PCR amplification of 16S rRNA and ITS amplicons. Illumina iTag Polymerase Chain Reactions will be performed using the Earth Microbiome 515F/806R primer pair for 16S rRNA (bacterial diversity) or the ITS1/ITS2 primer pair for ITS (fungal diversity. Amplicons will be generated in 25 μL volumes using Phusion Hi-Fidelity Polymerase (New England BioLabs, Ipswich, MA, USA) containing 0.5 μM of forward and reverse primers and 10 ng of template DNA.Library purification, verification, & sequencing. PCR products will be pooled in an approximate equimolar manner. The pooled PCR products will be run on a 2% agarose gel and bands of expected product length will be cut and DNA purified using the QIAquick Gel Purification Kit (Qiagen, Frederick, MD) and quantified. Each library on the sequencing run will be multiplexed into one sequencing library by normalizing each library's input based on the number of samples to ensure even sequencing and coverage.Processing of sequencing data. Demultiplexed paired-end sequences will be first imported within the QIIME 2 software ( Raw sequences will be subjected to DADA2 for read merging, de-noising, filtration, and chimera removal. To increase the robustness of data analysis and to compare methods, we will process bacterial amplicon sequences using a modified workflow with mothur v. 1.37. For ITS sequences, paired-end reads will be quality filtered and merged at an expected error of less than 1% using USEARCH v10. Open reference ASVs will be picked using the UPARSE algorithm, and taxonomy assignment will be performed using the UNITE database within QIIME-1.9.0.Statistical analyses. Analyses of the bacterial communities will be conducted using non-metric multidimensional scaling (nMDS) ordination and permutation-based multivariate analysis of variance (PerMANOVA). Heat maps will be generated using the 30 most abundant ASVs by performing a log2 [x] transformation. We will use Bray-Curtis similarities generated from standardized data that incorporates a relative abundance of ASVs to assess community structure; Jaccard similarities will be generated using subsampled data incorporating presence/absence to assess composition.

Progress 08/01/21 to 07/31/22

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 entomologists and a soil microbiologist for the identification insect and plant associated microbes. Changes/Problems:Since March 2020, COVID-19 related restrictions have slowed down certain aspects of laboratory research. These restrictions have caused delays including obtaining supplies from vendors. We may request for no cost extension in the future. What opportunities for training and professional development has the project provided?Students, post-doctoral, and technical researchers pparticiated and learned laboratory and field based techniques. How have the results been disseminated to communities of interest?Researchers exchanged experiemntal results, wrote manuscript, and published research paper in peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?Research is being performed to accomplish aspects of goals 2 and 3.

What was accomplished under these goals? Accomplishments are presented under each project goals: 1. Characterize the role of flavonoids in host plant defense against FAW. We have now demonstrated that sorghum and maize flavonoids affect survival of FAW larvae. Larvae reared on an artificial diet supplemented with sorghum flavonoids showed significant mortality and decreased body weight. When sprayed on leaves of susceptible maize lines, flavonoid extract effectively reduced growth and increased mortality of FAW larvae. Extracts were obtained from the high flavonoid containing biomass comprising dried leaf tissue from a sorghum line. 3-DAs were extractedby the addition of an equal volume of 2 N HCl and boiling for one hour. After cooling, the solution was centrifuged at 20,000 xg for 15 min, and the supernatant was collected. An organic extraction with an equal volume of isoamyl alcohol (IAA) was performed twice on the supernatant. Aliquots of extracted 3-DAs were routinely checked using HPLC as explained and presented previously (Kariyat et al. 2019). Purified commercial standards of luteolinidin and apigeninidin were used to measure the concentration of the 3-DA extracts using a UVmini-1240 UV-VIS spectrophotometerat 480 nm. We performed diet assays to test the effectiveness of flavonoids extracted from sorghum leaves. Neonates were fed artificial diet supplemented with 3-DA extract. After nine days of feeding, the average body weight for the control group was 4.22 (± 0.435) mg with a 9% mortality. The lowest two concentrations tested, 0.02 μg/ml and 0.14 μg/ml, showed similar average weights to the control at 3.87 (± 0.544) mg and 4.60 (± 0.762) mg with similar mortality rates of 8% and 9% respectively (F = 6.06; df (treatment) = 5; df (error) = 453; P < 0.001). 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. Confirm the role of flavonoids in FAW growth and mortality. Weinvestigated the larval mortality when reared on maize lines overproducing flavonoids compared to their near-isogenic wild type lines. The detached leaf assays showed significantly high mortality of larvae that were fed on flavonoid producer lines compared to wild type.The peritrophic membrane that protects the midgut was severely damaged in larvae fed on leaves of flavonoid producer lines compared to wild type. The effectiveness of the flavonoids as feeding-deterrents by endogenous expression and exogenous application demonstrates the eco-friendly potential for the management of FAW larvae. 3. Determine microbial diversity associated with maize host plants and their pests. To determine leaf-associated microbes before feeding and how larval feeding changes the leaf-associated microbes, Sanger sequencing of the 16s rRNA gene will beused. DNA was extracted fromleaves using the Quick-DNA™ Fecal/Soil Microbe Microprep Kit. Briefly. Further cloning and sequencing will be performed.


  • 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.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2022 Citation: Elango, D., Wang, X., Bhatnagar, R.S., Tan, Q., Gaffoor, I., Hu, Z., Xue, W., Roth, G.W., Morris, G.P., Reddivari, L. and Chopra, S., Association genetics of early season cold and late season frost tolerance in Sorghum bicolor. Crop Science.

Progress 08/01/20 to 07/31/21

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.? Changes/Problems:There are no major changes in the project. However, the pace of the project has been slowed down because of restrictions imposed during COVID-19 and some of these restrictions (especially indoors) are continuing. We will revisit the progress in the coming years to evaluate and may request a couple of years' no-cost extensions. What opportunities for training and professional development has the project provided?During 2020 and till today COVID-19 has hampered professional activities to a greater extentent. However, students were encouraged to attend the following online activities as well as Zoom meetings with professionals and mentors. 1. One graduate and one undergraduate student attended Zoom meetings organized by faculty to disseminate knowledge around plant breeding and entomological techniques. 2. Both the students attended online workshops to learn microscopytechniques. 3. Students attend regular weekly laboratory meetings and mentorship sessions organized by project faculty participants. 4. Students were provided laboratory, greenhouse, and field-based training to handle plants and insects for performing feeding assays, flavonoid extractions, and quantifications. How have the results been disseminated to communities of interest?Results obtained so far were presented at the maize genetic 2021 conference (online). See publications for details of this presentation. What do you plan to do during the next reporting period to accomplish the goals?We will be working towards accomplishing the goals of the project by performing experiments for objective 2:Confirm the role of flavonoids in FAW growth and mortality. We plan to perform the following sets of experiments:? i. Flavan-4-ols, and Total Phenolics Quantification. ii.Peritrophic Matrix (PM) Sample Collection and Scanning Electron Microscopy. iii. Testing of Flavonoids-Rich Extract for Exogenous Application.

What was accomplished under these goals? During this reporting, period the following objectivewas investigated: 1. Characterize the role of flavonoids in host plant defense against FAW. Plant Material:Two groups of flavonoid-producing maize Near-isogenic lines (NILs) were selected for this study. The first group consisted of transgenic and transgene negative segregant, near-isogenic lines. The second group consisted of NILs of maize mutants.TheseNILs induce overaccumulation of flavonoid compounds. These two sets of maize NILs were grown at The Pennsylvania State University Agronomy farm at Russell E. Larson Agricultural Research Center, Rock Springs, PA, USA. Seeds were sown during the second week of May when the soil temperatures have reached 60oC or higher. Young plants at the V6-V7 stage were used for detached leaf-feeding assays as well as extraction of plant flavonoids. Plants showing any sign of substantial insect feeding damage were excluded from sampling. Plants of the inbred line B73 were grown in the Plant Science Department greenhouse at The Pennsylvania State University, PA, USA. Insects. FAW eggs were obtained from Benzon Research Inc (Carlisle, PA, USA). A paper towel with egg masses was cut into small pieces and kept in perforated plastic containers covered with a layer of stretched Parafilm (Parafilm "M"; American National Can, Greenwich, CT, USA) at ~29°C for ~48-72 h for hatching. Upon hatching, neonates were individually transferred to 30-ml plastic portion cups containing a food source. Results obtained from these bioassays were very encouraging. We are further repeating them to confirm the role of flavonoids in causing mortality of caterpillars of fall armyworm.


  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Chatterjee, Debamalya; Lesko, Tyler; Peiffer, Michelle; Dixon, Cullen; Elango, Dinakaran; Gafoor, Iffa; Felton, Gary W; Chopra, Surinder. Increased expression of antifungal and insecticidal flavonoid phytoalexins in specialty maize lines. Maize Genetics Conference 2020. Held via Zoom.