Performing Department
(N/A)
Non Technical Summary
Developing microbial consortia with potent inhibitoryeffects against the target pathogens is a potentially sustainable alternative to synthetic pesticides.We aim to identify the highly potent antagonistic compounds and their inhibition mechanismagainst the P. ultimum, the causal agent of root rot in corn. Knowledge generated through thisstudy will lead to the development of biocontrol agents and biopesticides that can be tested in the following proposal, where we aim to test the efficacy of the antagonistic agents in fieldexperiments and against other crop pathogens.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
(N/A)
Developmental
50%
Goals / Objectives
Plant-associated microbes remain an untapped resource for discovering biological controls that aregreat alternatives to synthetic pesticides. Discoveries of such agricultural products from a speciesor a mix of species have a great potential to offer solutions for yield losses from plant diseases.Microbial diversity is crucial, especially since some antagonistic compounds are only produced inthe presence of diverse bacterial communities. Our goal is to identify potential inhibitorymechanisms of simplified subcommunities, SC1 and SC2, that are antagonistic toPythium ultimum, the root rot pathogen of corn, and identify the potential inhibitory agents produced by one or all of their members.
Project Methods
Experimental methods1. We aim to look at the hyphal tissues for physical disruptions, such as poorlydeveloped hyphae or stunted growth. P. ultimum will be cultivated on PDB medium at 25 °C fortwo weeks. Results from specific activity 1.1 will reveal combinations of strains with maximuminhibitory potential. Extracts from these select combinations will be used to evaluate theireffectiveness against P. ultimum oospores germination and hyphal growth. We will follow thepreviously developed protocol for microscopy30. Briefly, these extracts will be dissolved in 2%DMSO at varying 2X concentrations (20mg-0.5mg/mL) and mixed with equal volumes of PDAmedium containing 4% agar to give final 1X concentrations (10 mg-0.25mg/mL) in 1% DMSOand 2% agar. This extract will be spread over a glass slide to form a thin layer, and then 5 μl ofoospores suspension (105 oospores/ml) will be placed on the surface. Five replications will be donefor each assay. The slides will then be placed in moist Petri dishes and incubated at 25 °C. Oosporegermination will be checked under a light microscope until total germination on control slides hasoccurred. The percentage of germination inhibition will be calculated from the experimental meanvalues: Inhibition % = 100 x (A-B)/A, where A and B are the percentages of germinated spores inthe control and test samples, respectively.2.Forthe initial analysis, up to ten combinations of the most inhibitory strains and the pure isolatesidentified in Specific Activity 1.1 and 1.2 will be cultured in liquid media (1L) and extracted andanalyzed using routine natural product techniques. An equal volume of ethyl acetate will beadded to each culture, agitated, and separated using a separatory funnel, which will be repeatedthree times. The ethyl acetate extracts will be combined, dried, and subjected to a solventfractionation scheme. The extracts will be dissolved in 10% methanol/water and partitionedsuccessively with hexanes, ethyl acetate, and butanol. Each partition will be dried under reducedpressure, reconstituted in the solvent at 10 mg/ml, and tested against P. ultimum using a diskdiffusion assay (100 and 500 μg per disk, in triplicate). All fractions that show activity (> 20%reduction of pathogen diameter) will be further fractionated. Depending on which partitionfractions are active, several chromatographic approaches can be used, including flash, vacuum,medium pressure, and size exclusion chromatography. In all cases, resulting fractions will beanalyzed by thin-layer chromatography (TLC) and dried under reduced pressure. Theseconsolidated fractions will be reconstituted and tested using either the disk diffusion assay as aboveor a modified broth dilution assay to minimize the compound needed for testing. Active fractionswill be purified using semi-preparative high-performance liquid chromatography (HPLC).Purification and testing will continue iteratively until pure, as determined by 1D 1H NMRspectroscopy. The molecular formula of the compounds will be determined through high-resolution mass spectrometry (ESI or APCI-HRMS). The MS data will be used to develop potentialmolecular formulas. They will be used with the 1D NMR data to search natural product databases(i.e., Dictionary of Natural Products, SciFinder Scholar, ReAxys) for potential matches to knowncompounds. If compounds appear to be new, a full NMR experimental dataset (DEPT, COSY,NOESY or ROESY, HSQC, and HMBC) and UV and IR spectra will be obtained. Theconfiguration of chiral centers will be determined through proton coupling analysis,NOESY/ROESY data, optical rotation, and CD spectral analysis. HPLC and mass spectrometrydata will be compared with the single culture extracts to determine which strains are responsiblefor the compounds isolated from co-cultures. If several antagonistic compounds are identified, wewill select the top three to five compounds to purify and further characterize their inhibitorypotential and combinatorial effects.