Final report for GNE19-195
For the environmental health and well-being of mankind, it is essential to adapt safe crop protection strategies to achieve sustainable production of economically important crops such as corn. The flavonoids are predominant secondary metabolites produced in plants that confer resistance to fungal diseases and insect infestations. Our recent work has established that flavonoids such as 3-Deoxyanthocyanidins (3-DAs) can effectively reduce the performance of pathogens and insect pests of corn and sorghum. In this project, we proposed to extract and apply flavonoids as a biopesticide, keeping in line with the overarching goal to develop low-risk suppression tactics, including the use of biopesticides, biological control, and products of both traditional breeding and molecular genetic technology. We identified and characterized a corn genotype designated as U-E that overexpresses several flavonoid compounds, including 3-DAs. Here, we established the insecticidal role of 3-DAs specifically against fall armyworm (Spodoptera frugiperda, FAW), which is one of the major insect pests of corn in the western hemisphere and has recently become an invasive pest in Sub Saharan Africa and Southeast Asia. Our results indicate that these flavonoids can affect the peritrophic membrane, the thin protective lining of the caterpillar midgut. We developed an inexpensive method of isolating and purifying flavonoids from U-E plants. An artificial diet supplemented with flavonoid extract showed increased FAW mortality confirming the efficacy of extracted compounds. Spraying the plants with a formulation developed using this extract significantly reduced FAW feeding and leaf damage. These results cumulatively established the potential use of the U-E breeding line for efficient flavonoid production to use as a FAW deterrent. This project is a step forward to achieve a broader impact of reducing synthetic chemicals in IPM strategies leading to a sustainable and environment-friendly pest management system.
Objective 1: Confirming biological activity and efficacy of 3-DAs against FAW caterpillar
Objective 2: Isolation and purification of 3-DAs from flavonoid over-producing corn breeding line
Objective 3: Testing the efficacy of 3-DAs as FAW feeding deterrent
Numerous insects attack corn. None of the growth stages ranging from seedling to grain, even during storage, is immune from insect attacks. 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.
FAW, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) is predominant among the cohort of insects causing damage in the United States and in different parts of the world (Africa, India, and Egypt in recent incidences). Although FAW is polyphagous, it has a definite preference for grain crop hosts, especially corn. FAW is a voracious leaf feeder in the early larval stages that feed inside the leaf whorl. During this stage, extensive feeding may result in stripped leaves and destruction of the meristem, causing plant death. Later instars can attack the stem and developing ears (corn) or seed heads (sorghum), resulting in extensive yield loss in the form of lodging of the damaged stem, damaged ear, or seed head. Furthermore, secondary infections by mycotoxigenic fungi such as Aspergillus and Fusarium sp. of the damaged corn ear lead to mycotoxin contamination, making the seed unfit for animal and human consumption. FAW in field corn is primarily managed by cultural practices such as early planting. In addition, the adoption of transgenic hybrids producing insecticidal Bt. proteins have been a key FAW management tactic in field corn. However, the observation of FAW populations resistant to transgenic corn producing Cry1F in Puerto Rico in the mid-2000s has increased the concern of Bt. resistance. Limited adoption of transgenics among growers because of consumer concerns results in increased dependence on synthetic chemicals.
Carbamate methomyl is the most commonly used insecticide against FAW along with recently registered products such as Chlorantraniliprole/rynaxypyr. Studies have shown that FAW has developed resistance to several synthetic insecticides. For example, in the USA, FAW resistance has been reported against Carbamates, Organophosphates, and Pyrethroids-Pyrethrins. This suggests the need for an alternative durable strategy for the sustainable control of FAW. The usage of these synthetic pesticides, however, is an integral part of today’s crop protection strategies. Innovations in synthetic combinatorial chemistry have led to tremendous advancements in the development of novel chemicals. However, the industrial-scale manufacturing and deployment of chemical strategies have led to a substantial loss of healthy environments due to atmospheric, ground, and surface water pollution. To reduce the use of synthetic pesticides, there is a need for new tools and tactics for more sustainable IPM strategies by (i) developing pest-resistant cultivars and (ii) developing eco-friendly pesticides that are as efficacious as synthetic chemicals. The proposed project is a motivation towards these two aspects.
Current methods of controlling insect damage in corn are losing efficacy due to the development of resistance in insects and growing public concerns regarding the use of transgenic crops (GMOs) and synthetic chemicals. Although the proposed project is limited to the control of FAW only, preliminary data suggests that the use of 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 insect management practices. Beneficiaries of the project include field corn and sweet corn producers in Pennsylvania and the rest of the USA.
This proposed project is part of a larger project that has additional goals to combat insect pests in corn and sorghum by developing (1) breeding lines that are high yielding and enriched in flavonoids and (2) plant metabolic pathway engineering for the large-scale production and deployment of 3-deoxyanthocyanidins (3-DA) compounds as botanical or biopesticide. This approach is novel in that this group of plant compounds has not been previously deployed against insects. In addition, these compounds cannot be economically produced in useful quantities without developing flavonoid over-producing corn inbred line, which we have already achieved. This corn inbred named U-E will be used to isolate large quantities of 3-DA. This approach is expected to play a role in the management of insects in other crop systems as well.
Further, the use of this biopesticide can potentially reduce the accumulation of chemical pesticide residues in the food chain. While these compounds are naturally present in plants and show increased resistance when over-accumulated in the plant body, these act in favor of preserving the biodiversity of beneficial organisms. Flavonoids are plant secondary metabolites that are present both in vegetative parts of the plants and in the flower in pollen and nectar. These chemicals enhance plant defense against pests, while their effects on beneficial insects range from avoidance to no discernible effect on their progeny. Recent research further suggests that secondary compounds in flowers can reduce bacterial pathogens present in pollen and nectar and thus are beneficial to pollinators. Thus, the application of 3-DA produced in corn as biopesticides might be beneficial to pollinators.
Previously we have characterized a natural maize mutant Unstable factor for orange1-1 (Ufo1-1) that over-accumulates orange flavonoids in different parts of maize plants. We have isolated a stable progeny of these plants and developed a maize breeding line that naturally produces an increased level of flavonoids, especially in leaf sheath, tassels, corn ear husk, and outer layer of seed (pericarp). This maize line named Ufo1-1 expresser (U-E) was used in this study.
Rearing FAW caterpillars:
We purchased the FAW eggs from Benzon Research Inc (Carlisle, Pennsylvania, USA). These eggs were derived from a FAW colony originated in USDA-ARS Corn Host Plant Resistance Research Laboratory at Mississippi State University. The eggs were supplied as a layer on a paper towel. We cut the paper towel with egg masses into small pieces and kept these at ~29°C for ~48-72 h inside perforated plastic containers for hatching. The perforated lid of the container was covered with a layer of stretched Parafilm (Parafilm “M”; American National Can, Greenwich, CT, USA) to prevent neonates (newborn caterpillars) from escaping while maintaining breathability. After hatching, we transferred the neonates into one-ounce plastic portion cups. A thin layer (1-2 mm) of 3% agar was added to the portion cup base to provide a moist base for the caterpillars.
Detached leaf assay:
Leaves of Tx601, a FAW susceptible maize genotype, were collected and cut into ~1 inch2 pieces and added to the portion cups to feed the caterpillars until the third instar. The remnants of leaves and caterpillar excreta (frass) were cleaned every day. Newly molted third instar caterpillars were weighed and transferred to a fresh portion cup for detached leaf assay. Leaves of U-E and control maize plants the U-S (Ufo1-1 silent) that do not produce flavonoids in leaves were collected, cut into ~1 inch2 pieces, and were fed to the caterpillar for nine days. The caterpillar body weight and mortality were recorded after the treatment.
Confirming bioactivity of flavonoids:
To understand how the flavonoids affect FAW caterpillar growth and increase mortality, we investigated caterpillars' gut health after feeding flavonoid-rich leaf diets. Our objective was to isolate the peritrophic matrix (PM), a fragile lining of the FAW caterpillars' midgut to observe any damage.
I) Peritrophic Matrix Sample Collection:
After feeding treatment, we weighted and immobilized them on ice. On a cold surface, the caterpillars were cut open using a pair of fine scissors. Under the microscope, the inner gut surface was pulled back with fine forceps to expose the PM. The PM samples were collected and immediately fixed by immersing in 1 ml fixative solution (2.5% Glutaraldehyde, 1.5% formaldehyde in 0.1 sodium cacodylate buffer) in 1.5 ml Eppendorf tubes. We stored the samples at 40C until further processing.
II) Scanning Electron Microscopy (SEM):
The fixed samples were rinsed thrice with 0.1 sodium cacodylate buffer and incubated in 1% OsO4 (Osmium tetroxide) in the dark at room temperature for 1-2 h. After incubation, samples were again rinsed three times with 0.1 M sodium cacodylate buffer. Next, the samples were dehydrated in 25%, 50%, 70%, 85%, 95%, 100% ethanol series for 10 mins per ethanol gradient. After dehydration, an EM CPD300 Critical Point Dryer (Leica, Wetzlar, Germany) was used to critically dry the samples. Samples were mounted on SEM stubs using carbon tape. Bal-tec SCD-050 Sputter Coater (Bal-tec AG, Balzers, Liechtenstein) was used to coat the samples with gold particles. The samples were investigated for the signs of internal damages a SIGMA VP-FESEM (SEM) (Zeiss, Oberkochen, Germany). Four biological replicates were used per feeding treatment. Samples were observed using different magnification, and representative images were captured.
Collection of corn samples for flavonoid isolation:
We grew Approximately 2,000 plants of flavonoid-producing maize line U-E at the Pennsylvania State University Agronomy Farm, Rock Springs, PA during the summer of 2019. We have collected the plants at R2 (blister) to R3 (Milk) stage when maximum flavonoid accumulation takes place in the vegetative tissue as well as kernels. The whole corn plants were harvested at these different stages using a silage harvester/chopper and the chopped materials were dried in a forced-air blower dryer at 65°C. Dried materials were stored at room temperature. The flavonoids in the dried samples are highly stable at normal room temperature allowing easy storage till further use.
Isolation of flavonoid-rich extracts from field-grown U-E plants:
We collected biomass from high flavonoid containing plant parts such as corn ear husk and leaf sheath from flavonoid overproducing U-E plants. However, these plant parts are not enriched for 3-DAs that are only produced under biotic stress, such as fungal infection. These biomasses mainly contain a group of flavonoids, flavan-4-ols. As commercial standards for flavan-4-ols is unavailable, we performed a conversion to 3-DAs by adding an equal volume of 2N HCL to the crude extract and boiling for one hour. The extract was centrifuged at 20,000g for 15 minutes after cooling. The supernatant was collected. Further organic extraction was performed by adding equal parts of isoamyl alcohol (IAA) to the supernatant. BUCHI Rotavapor®- R (Brinkmann Instruments, Westbury, NY, USA) was used to evaporate IAA to dryness. We resuspended the resulting pellet in 0.1% HCl/MeOH (ACS grade, BDH Chemicals, USA). The extract was filtered through 0.45-μm Acrodisc LC filters (Gelman Laboratory, Ann Arbor, MI, USA). We measured the extract's concentration at 480 nm using a UVmini-1240 UV-VIS spectrophotometer (Shimadzu, Kyoto, Japan). The extract was secured at four °C for short-term and -20°C for long-term storage.
Artificial Diet Feeding Assay:
To assess the effects of the 3-DA compound on FAW, caterpillars were reared on a wheat germ and casein-based artificial diet with ingredients purchased from BIOSERV (Frenchtown, NJ, USA). Different concentrations of 3-DAs ranging from 0. 01 µg/ml to 1 mg/mL were added to the diet to determine LC50. Neonates were placed in individual plastic cups with a 1 cm3 of diet and maintained in a growth chamber at 27°C with a 16-hr. photoperiod. The mortality rate of FAW larvae and the fresh weight of surviving larvae were measured.
Preparing Spray Formulation:
We prepared the spray solution by diluting 41.7 ml 3-DAs extract (0.6 mg ml-1) in 2458.3 ml of 9% ethanol (EtOH) to obtain 16.0 µl ml-1 final concentration. We further prepared the solution with 0.25% Tween20 to improve surfactant property for spraying on plants. Sterile water, 9% EtOH, and 0.25% Tween20 added 9% EtOH were used as controls for experiments. The emulsions were swirled using a magnetic stir bar at room temperature. The spray solutions were stored at room temperature for a shorter duration. For long time storage, the solutions were saved at 40C.
Flavonoid Extract Sprayed Detached Leaf Assay:
We collected the leaves from the TX601 corn plants. After detaching from the plants, leaves were uniformly sprayed with the flavonoid spray solution and air-dried. Next, the leaves were cut into ~1-inch square pieces. The leaf pieces were added to the once-ounce plastic portion cups holding FAW larvae on thin agar beds on the bottom of the cups. Tow feed per 10 larvae for two to three days, fifty grams of leaf pieces were added. Each portion cups contain one third-instar caterpillar. For each treatment, five replicates were added, and a completely randomized design (CRD) was used to arrange the cups. Old leaf pieces and excess excreta (frass) of the insects were cleaned, and new pieces of treated leaves were added every second day. Caterpillar bodyweight and mortality were recorded after seven days of treatment.
Plant-based assay of FAW feeding deterrence by flavonoid (3-DAs) spray:
We grew the maize plants in the Crop and Soil Sciences greenhouse (The Pennsylvania State University, PA, USA). The greenhouse conditions were set for ~26°C temperature and a 16 h light period. 70% field topsoil (Hagerstown silt loam) and 30% potting media (Sun Gro Horticulture, Agawam, MA, USA) were mixed and used to grow the plants in pots. The plants were transferred to a growth chamber (Conviron, Winnipeg, Canada) at the V6 growth stage for the controlled condition during the treatment. The temperature of the growth chamber was set to 27°C for the day and ~14°C for the night. Approximately 65% humidity, 16-hour light (140 mol m−2 s−1), and eight hours dark period growing condition were used.
We recorded the bodyweight of the fifth instar of FAW larvae reared on detached leaf pieces before starting the spray treatment. One caterpillar was released on the leaf whorl per plant. The caterpillars were allowed to adjust to the plant microenvironment for 12 hrs before starting spray application. The three different treatments included 3-DAs solution (3-DAs in 9% EtOH and 0.25% Tween20), solvent (9% EtOH and 0.25% Tween20), and water. A 1.5 liters hand-held pressure pump mister and sprayer was used to uniformly spray the plants every alternate day. Caterpillar body weight and mortality were recorded after seven days of treatment.
Leaves of U-E causes increased mortality of FAW caterpillars:
To test the efficacy of endogenous flavonoids of U-E, we performed a detached leaf assay. The U-E leaves and leaf sheaths visibly show orange flavonoids pigment accumulation. After nine days of feeding, caterpillars fed on U-E showed significantly increased mortality (~40%) than caterpillars fed on U-S leaves (~13%).
Flavonoid-rich diet affects the peritrophic membrane of FAW caterpillars:
We dissected and observed the peritrophic membrane (PM), a thin protective lining of the caterpillar midgut, to understand the mode-of-action of flavonoids against FAW caterpillars. Using scanning electron microscopy, at low magnification, we detected folds and wrinkles on PM of caterpillars fed on U-E. Moreover, at higher magnification, widespread ruptures were found on the PM of U-E-fed caterpillars. The peritrophic membrane protects the hemocoel from the harmful materials present in ingested food. Hemocoel is the primary body cavity of insects that contains a circulatory fluid equivalent to the blood of mammals. Flavonoids, being phenolic compounds, can create oxidative stress-induced damage to the PM. A damaged PM can no longer protect the gut and hemocoel from damage caused by physical, chemical means, and pathogenic microbes, potentially leading to septicemia. This might contribute to the increased mortality of FAW caterpillars.
3-DA supplemented-diet based bioassay showed increased mortality and reduced bodyweight of caterpillars:
Although flavonoid-producing maize leaves increased the FAW caterpillar mortality, we aimed to test the effectiveness of 3-DA when extracted, purified, and exogenously applied against FAW caterpillars. For this objective, we used diet-based bioassays where a wheat germ and casein-based artificial caterpillar diet (BIOSERV, Frenchtown, NJ, USA) was supplemented with purified 3-DA compounds a concentration of 0.07μg/ml. The experiment was performed on the newborn caterpillars (neonates). After nine days of feeding, caterpillars on 3-DA supplemented diet had a lower average body weight (~2.5 mg) and ~90% mortality compared to the caterpillars reared on the control wheat gram casein-based diet (~34 mg body weight and ~35% mortality). This result confirmed that the extracted flavonoids purified as 3-DA were as effective and endogenous flavonoids of the U-E leaves.
Increased FAW Mortality in Flavonoid Sprayed Detached Leaf Assay:
Based on the previous results, to further confirm the extract's effectiveness against FAW growth and survival, we performed a bioassay using detached leaf pieces sprayed with flavonoid formulation. Third, instar caterpillars were used for this experiment. Flavonoid extract dissolved in 9% ethanol was used to spread the leaves as treatment. As two separate controls, 9% ethanol and sterile water were used. The results indicated a significant reduction in body weight increase after nine days of feeding. The sprayed leaf diet further caused 40% mortality. In contrast, caterpillars fed on both 9% ethanol and water sprayed leaf pieces did not display any significant difference in body weight gain. A 5% mortality was recorded only among larvae fed on water sprayed leaf, likely due to random chances.
Plant-based bioassay using 3-DA spray showed increased FAW mortality:
To assay the efficacy of the 3-DA extracts of FAW caterpillar in plant microclimate, we sprayed the maize plants artificially infested with fifth instar FAW caterpillars. We precisely added one caterpillar inside the leaf whorl of each plant for controlled monitoring of the experiment. For better adherence to the leaf surface, 0.25% Tween20 was added to the 9% ethanolic solution of extracted 3-DA. After seven days of treatment, by spraying the plants every alternate day, plants sprayed with Tween20 containing the ethanolic solution of flavonoids showed ~50% FAW mortality. In contrast, plants sprayed with just 9% ethanol with Tween20% and plants sprayed with water (controls) showed 0% mortality of caterpillars. At the end of seven days of treatment, the caterpillars on 3-DA treated plants had an average 40 mg bodyweight compared to ~160 mg average bodyweight of caterpillars from plants sprayed with controls. This result further confirms that the above-mentioned 3-DA spray formulation can effectively control FAW caterpillar feeding damage. Damage to the leaves was assayed on a 0-9 scale where 9 indicates the highest feeding damage comprising more than 30 cm elongated lesions. Flavonoid treated plants scored ~3 but compared to ~8 score of control plants.
We have developed and characterized a corn inbred line U-E that overproduces flavonoids. We observed increased mortality of FAW caterpillars in detached leaf assay when fed on U-E compared to the control line. To understand the mode of action of endogenous flavonoids on FAW, we performed scanning electron microscopy of caterpillar midgut. Results revealed that the peritrophic membrane (PM) that protects the midgut from physical and chemical stress was severely damaged when caterpillars were fed on U-E leaves. We developed an inexpensive method to isolate flavonoids-rich extract from U-E for external application as a potential biopesticide. Increased mortality of FAW caterpillars while fed on flavonoid supplemented artificial diet confirmed the efficacy of the extract. We further developed a spray formulation. We observed a significant reduction in FAW feeding and leaf damage when the plants are sprayed with this formulation. These results established the potential use of U-E for efficient small-scale production of flavonoid extract to use as a FAW deterrent.
Education & Outreach Activities and Participation Summary
We presented our results at the 62nd Annual Maize Genetics Meeting:
We presented a poster at the 62nd Annual Maize Genetics Meeting. The Meeting was initially supposed to be held at Kailua-Kona, Hawaii, the USA, on March 12th-15th, 2020, and was later rescheduled as a virtual conference on June 25th-26th due to COVID-19. We presented the poster virtually to 20 participants.
Training undergraduate students:
Undergraduate student: Tyler Lesko (Current Ph.D. Candidate, Agricultural and Environmental Plant Science, The Pennsylvania State University)
Tyler joined our Maize genetics lab as an undergraduate through the support of this grant to assist us with sample collection, 3-DA extraction, and quantification. It provided him the opportunity to experience how an academic research lab operates. He gained experience working with numerous techniques and equipment, including insect bioassays, flavonoid extraction, and rotary evaporation. Besides, he was able to gain knowledge on how data is collected and analyzed and was involved in the writing of a scientific paper. This opportunity inspired him to further pursue higher education in graduate school. He enjoyed the experience of working in the lab and decided to join a Ph.D. program in the Pennsylvania State University to continue flavonoid research, explicitly investigating the mechanistic action of toxicity of these flavonoids and whether they can be used as an effective biopesticide for large scale applications.
Undergraduate student: Nicolai Sartori
Nicolai participated in this project and gained experience with planning maize planting in the field research nursery and growing maize plants in the field and greenhouse. He received training in sample collection and rearing of FAW caterpillars.
Undergraduate student: Shuhan Yin
Shuhan participated in this project and gained experience in rearing FAW caterpillars. She was also trained to collect samples from FAW caterpillars for further experiments.
We are currently preparing a journal article on the results gained from this project.
This project helped to gain knowledge on the mode-of-action of maize flavonoids against FAW caterpillars.
We established that flavonoid overproducing non-transgenic breeding line U-E can be utilized for efficient flavonoid extraction.
We developed an inexpensive method of flavonoid (3-DA) extraction from maize biomass.
We demonstrated that spraying maize flavonoid extract can deter FAW feeding as efficiently as endogenous maize flavonoids.
This project's outcomes established the foundation of using maize flavonoid extract as a component of integrated pest management against FAW caterpillar.
Flavonoids are present in a vast array of natural human foods. Our flavonoid-based approach is devoid of GMO and synthetic chemicals, further ensuring the well-being of humans and the environment.
Current measures of controlling insect damage in maize are losing efficacy due to the development of resistance among insects, high costs of pesticides, and growing public concerns regarding the use of transgenic crops/GMOs and synthetic chemicals. Our project is a step toward developing environmentally friendly solutions by using natural maize compounds to deter FAW feeding damage.
The maize 3-DA flavonoids affect FAW caterpillar gut lining:
Our results showed that maize flavonoids affect the peritrophic membrane (PM), a thin protective lining of the caterpillar midgut. PM is a ubiquitous gut structure primarily present among a diverse group of insects. Although the current research is limited to FAW, the results suggest that the use of these compounds can be extended to control a variety of other pests. This translational research project will also lead to a more comprehensive knowledge of the insect herbivore gut and its response to plant defense compounds.
The maize breeding line U-E can be used for inexpensive extraction of 3-DA flavonoids:
The breeding line U-E was derived from a natural maize mutant Unstable factor for orange1-1 (Ufo1-1) that over accumulates flavonoids in different plant parts. The abundance of flavonoids in U-E lead to the development of an inexpensive method to extract 3-DA flavonoids. Being a non-transgenic breeding line, U-E shows the potential to be grown as border rows for extraction of 3-DA to control FAW in small maize fields.
Extracted 3-DA is equally efficient in FAW deterrence as plants overproducing 3-DA:
Results showed that extracted 3-DA is as effective to increase FAW caterpillar mortality as endogenous flavonoids. This broadens the prospect of using 3-DA extract against FAW on other host plants. This project could lead to utilizing this novel flavonoid as potent insect deterrents without being harmful to human health and the environment.
Damage to the peritrophic membrane caused by 3-DA may lead to leakage of harmful microbes in the caterpillar's hemocoel, resulting in increased mortality. Further studies on the effect of flavonoids on caterpillar gut microbial diversity may help us gain additional insight into this plant defense mechanism. We have new working collaborators Dr. Mary Ann Bruns, a microbiologist and Dr. Gary W. Felton, an entomologist in the Pennsylvania State University; and Dr. Julien Beuzelin, an entomologist in the University of Florida, helping us to develop experiments on the effect of flavonoids in FAW gut microbiome.
Additionally, further field experiments for evaluating the persistence of exogenous 3-DA can help move forward to integrate these flavonoid extracts in pest management strategies. Calibration of large-scale production of flavonoids extract may enhance its potential utilization.