Objective 1: Purification and processing of 3-DAs from flavonoid over-producing corn line to use as a biopesticide.
Expected outcome: We will grow approximately 2,000 plants of corn flavonoid overproducer line UE for medium-scale isolation and will partially purify 3-DA compounds to use as a biopesticide.
Objective 2: Confirming biological activity and efficacy of isolated 3-DAs.
Expected outcome: A positive result will confirm the bioactivity of 3-DAs as insect feeding deterrents. Further, this objective will help us to understand the mode of action of these compounds.
Objective 3: Testing the efficacy of 3-DAs as a biopesticide in field trials
Expected outcome: The 3-DAs can potentially control FAW and other insect infestations effectively providing us an alternative biopesticide to integrate into IPM as a sustainable pest control approach.
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-DAs) compounds as biopesticides. 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 as UE will be used to isolate large quantities of 3-DAs . 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 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-DAs produced in corn as biopesticides might be beneficial to pollinators.
Materials and Methods for already performed parts of the project:
Collection of corn samples for 3-DAs isolation:
Based on our previous studies, we have identified a maize line that naturally produces an increased amount of 3DA compounds in different parts of the plant, especially in leaf sheath, tassels, corn ear husk and outer layer of seed (pericarp). This maize line is named UE. We grew Approximately 2,000 plants of 3DAs producing maize line UE 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 3-DAs 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 after at room temperature. The 3-DAs in the dried samples are highly stable at normal room temperature allowing easy storage till further use.
Small scale isolation of 3-DAs from field-grown UE corn plant samples to confirm the efficiency of isolation:
We performed small-scale isolation of 3-DAs by boiling the dried and ground UE plant tissues in 2N HCl to extract the flavonoids twice into an equal volume of isoamyl alcohol. The isoamyl alcohol is then evaporated using a rotary evaporator and the residue is re-suspended in methanol with 0.1% HCl. This small-scale isolation procedure yielded approximately 10 mg of 3-DAs from 100 g of dried leaves, that corroborates with the expected outcome.
Medium-scale isolation of 3-DAs from field-grown UE plant samples:
We will use a modified version of the small-scale protocol for medium scale 3-DAs isolation. Penn State’s Behring Fermentation facility will be used to process crop biomass. For batch purification of 3-DAs, 50 kg plant dried corn biomass will be fine ground using a Thomas Wiley grinder to a 1 mm particle size. 3-DAs will be isolated by boiling ground leaf tissue in 2N HCl in a 30L capacity glass vessel followed by extracting the flavonoids into an equal volume of isoamyl alcohol. The isoamyl alcohol will be evaporated to dryness and the residue will be suspended in methanol with 0.1% HCl. The presence of 3-DAs in this methanolic extracts will then be confirmed by 1) Spectrophotometric absorption spectrum of 480 nm. 2) By size-exclusion HPLC from supernatants and its concentration will be measured using an optical density calibration curve (flavonoids absorb at 480-525 nm, depending on chemical modifications). 3) We will work with Penn State Metabolomic facility to characterize 3-DAs using tandem mass spectrophotometry.
Confirming bioactivity of 3-DAs:
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 be 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.
Materials and Methods for upcoming experiments:
Confirming species-specific toxicity:
We will test the bioactivity of 3-DAs on FAW to avoid species-specific toxicity changes because of factors such as stereo-specificity due to large-scale purification. hypothesis for the mechanism of flavonoid toxicity in insects is that flavonoid compounds damage the semipermeable peritrophic membrane (PM) in the midgut, thereby permitting microbes in the food bolus to enter the hemolymph, causing septicemia. To test this hypothesis, 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. Caterpillar weights and mortality will be recorded after 9 days. In our previous results, we have found that larvae fed on diet + 3-DAs (from sorghum) had a 91% mortality rate compared to only 34% for larvae on a control diet (unpublished). The surviving larvae from the 3-DA added diet was also significantly smaller (0.0024+0.0001 grams) than those fed a control diet (0.0342+0.0004 grams) (unpublished). A similar result will confirm the efficacy of these medium-scale isolated 3-DAs from UE line. Preliminary experiments will be carried out to determine the appropriate diameter of FITC-dextran that is unable to penetrate the intact FAW PM. After 24 h, the hemolymph will be extracted and fluorescence will be quantified using a Nikon 80i Epifluorescence microscope. If fluorescence is detected, it will indicate a compromised PM. If exposure to 3-DAs results in a compromised PM, it will be further investigated using scanning electron microscopy in Microscopy Facilities, Huck Institutes of Life Sciences, PSU as a part of a broader project in our lab.
Development of a spray formulation for greenhouse trials:
The 3-DAs can be used either alone or in combination with other active or inactive substances. We have already established an effective mixing of 3-DAs with Tween 80 for our preliminary small experiment to improve surface adhesion of 3-DAs on corn plants. We will further test different anionic detergents other than Tween 80 to select the most effective adhesive substance that can result in the highest efficiency of 3-DAs. We will use sodium bicarbonate, sodium sulfate, and sodium phosphate to increase the antifungal properties of the formulation. The resulting emulsion will be diluted to an appropriate concentration for use. We will evaluate emulsions containing 3-DAs at five concentrations ranging from 0.001% to 10% by weight.
Evaluating the efficacy of 3-DAs as a biopesticide in the controlled condition in the greenhouse: (Currently ongoing)
Greenhouse trial will be done prior to field trials (objective 3) to standardize 3-DAs application as a spray biopesticide and for preliminary evaluation of 3-DAs efficacy. The experiment will be conducted in the College of Agricultural Sciences greenhouses and plant growth facilities, PSU, University Park.
Maize inbred lines MP708 and TX601 will be included as conventional FAW resistant and susceptible checks, respectively . In addition, Dekalb Brand DKC 66-94 and its isogenic line DKC 66-97 producing the Bt. proteins Cry1A.105 and Cry2Ab2 for resistance to FAW and CEW will be included as commercial standards.
Growing the plants:
Seeds will be germinated in polystyrene seed starter greenhouse tray inserts filled with potting mix (Sunshine mix #4 from Sun Gro® Horticulture). The seedlings will be transplanted 10 days after germination into 3.79 – liter pots (PF400; Nursery Supplies Inc., Chambersburg, PA, USA) containing Hagerstown loam soil and will be fertilized once with 10 g of the fertilizer Osmocote plus (15-9-12, Scotts, Marysville, OH, USA).
Potted plants of Tx601 susceptible line will be used to test the effectiveness of 3-DAs as a biopesticide. The solvent used to dissolve 3-DA for developing the spray formulation will be used as a negative control whereas commercially available pesticide Carbamate methomyl and recently registered products such as Chlorantraniliprole or Spinetoram will be used as a positive control.
FAW damage comparisons will be made using resistance genotype Mp708 and Bt. transgenic lines as mentioned above. These resistant plants will be separated from the susceptible Tx601 tests either by barriers in the same house or will be grown in a different house with identical conditions to the house where biopesticide spray will be tested.
Artificial FAW infestation:
We will use the genetic line of FAW, purchased from Benzon Reseach (Carlisle, PA) which originated in USDA-ARS Mississippi. The FAW larvae will be grown from eggs hatched on detached leaves of TX601. The plants of the V8-V9 physiological stage will be challenged by FAW caterpillars. Fifty neonates will be placed in the whorl of all plants and damage rated with the Davis’ 1-9 scale seven days after release and larval larval weight and mortality will be recorded at 12 days after infestation.
Evaluating the efficacy of 3-DAs in the field as biopesticide:
Experiments evaluating the field efficacy of five concentrations of 3-DA emulsions for control of the FAW will be conducted.
Site(s) of field experiment:
For field experiments, we will rely on natural FAW infestations either at Agronomy Farm near University Park, PA, or/and Montgomery County, PA where insect pest pressure has historically been high (See attached later from Andrew Frankenfield, Frankenfield Farm Market).
Randomized complete block design with four blocks (1 replicate per block) will be used. Plots for treatments of different 3-DAs concentrations and non-treated plots will be randomized. Each plot will be 30 feet long with 8 rows. During sowing, the seed-to-seed distance will be kept at 8-10 inches with 20 seeds per row. Each plot will be separated by 3 border rows to reduce edge effects.
The experiment will be repeated on the same plant materials that we will use for the greenhouse trial (see obj 2.2).
Real-time alerts for detection of insect infestation:
PestWatch http://www.pestwatch.psu.edu/sweet_corn.htm, a web-based mapping tool will be used for real-time alerts for FAW infestation in the field. This mapping tool is developed through the efforts of Dr. Shelby Fleischer and other researchers at Penn State and so we have readily available consultation in case of expertise help needed to interpret any observations. This tool will help us to strategize the location of field trials more precisely.
Timing and method of spraying:
We will initiate the 3-DAs application at the first sign of infection followed by repeated application every 3 days for four weeks. For this purpose, we will use a CO2-pressurized backpack-sprayer with a boom equipped TeeJet TP8002VS nozzles (2-nozzles/row) calibrated to deliver 20 GPA at 30 PSI.
Scoring for insect infestation:
Ten plants from each of the two center rows of each plot will be individually evaluated for insect infestation or injury. For FAW, foliar injury to leaves will be rated using the 0-9 Davis scale at the V8-V9 and silking.
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). The effects of experimental treatments will be compared using linear mixed models in PROC GLIMMIX (SAS Institute 2016). Random effects estimating the effects of blocks, experimental units, and sampling units will be included in the models as needed. When differences among treatments are detected, means will be separated using the Tukey-Kramer adjustment (p = 0.05). Multiple contrasts may also be used to compare selected groups of treatments with p-values adjusted using the step-down Bonferroni method to control family-wise error rates (PROC MULTTEST, SAS Institute 2011).
Diet based bioassay show 3-DAs kill FAW.
To test the effectiveness of 3-DAs against FAW larvae, we used diet-incorporation bioassays. FAW larvae were reared on a wheat germ and casein-based artificial diet (BIOSERV, Frenchtown, NJ, USA). The purified 3-DA compounds were added to the diet at a concentration of 0.07μg/ml. Neonates (newborn larvae) were placed in individual plastic cups with 1 cm3 of diet in a growth chamber at 27°C with a 16-h photoperiod. Larval weight and mortality were recorded after 9 days. Larvae fed on a 3-DA supplemented diet showed a 91% mortality compared to only 34% for larvae on the control diet. The surviving larvae on the 3-DA diet were also smaller (2.4 ± 0.1 mg) than those on control diet (34.2 ± 0.4 mg).
Evaluating the efficacy of 3-DAs as a biopesticide in the controlled condition in the greenhouse: (Currently ongoing)
Education & Outreach Activities and Participation Summary
We are preparing our preliminary results to present in the 62nd Annual Maize Genetics Meeting.