Available at: http://digitalcommons.calpoly.edu/theses/1696
Date of Award
MS in Biological Sciences
Dr. Scott J. Steinmaus
Allelopathy occurs when one organism relesases a compound into the environment that affects the functioning of another organism. Scientists have long suspected that alleopathic plant compounds could offer novel, softer chemistries to the ongoing battle of controlling pests in agricultural fields. Strawberry growers rely on toxic fumigants to kill soilborne fungal pests, weeds, nematodes, and insects. Increased regulations have reduced the use of fumigants (including methyl bromide), and strawberry growers need new sustainable pest control solutions. We selected four putative allelochemicals with known fungicidal and herbicidal activity (ferulic acid, gallic acid, juglone, and p-Coumaric acid). We assessed the pesticidal activity of these plant compounds both in agar and in soil on two emerging soilborne fungal pathogens (Macrophomina phaseolina and Fusarium oxysporum f.sp. fragariae), and four annual weeds commonly found in strawberry production fields (Malva parviflora, Melilotus officinalis, Poa annua, and Senecio vulgaris). We also assayed lettuce (Lactuca sativa ‘Inferno’), which served as a positive control plant species due to its sensitivity to phytotoxic compounds. Fitted sigmoidal dose-response curves predicted EC50 and EC75 values for each combination of plant compound and pest.
All plant compounds inhibited the in vitro radial mycelial growth of the two soilborne fungal pathogens in a dose-dependent manner. Fusarium oxysporum f.sp. fragariae was more sensitive to the plant compounds than Macrophomina phaseolina. Average EC50 values for the radial mycelial growth of two F. oxysporum f.sp. fragariae isolates were 75.1 parts per million by weight (ppmw) juglone, 469 ppmw p-Coumaric acid, and 687 ppmw ferulic acid. Average EC50 values for the radial mycelial growth of two M. phaseolina isolates were 196 ppmw juglone, 2869 ppmw p-Coumaric acid, and 5716 ppmw ferulic acid. The three compounds we assayed in vitro also reduced M. phaseolina colony forming unit counts in soil and the EC50 values were 476 ppmw ferulic acid, 612 ppmw juglone, and 827 ppmw p-Coumaric acid. Metconazole, the conventional fungicide control, did not inhibit M. phaseolina colony forming unit counts in soil at its label high rate. The plant compounds required similar or lower rates to inhibit colony forming units that grew from M. phaseolina overwintering structures (microsclerotia) in soil as to inhibit radial mycelial growth in vitro. Based on the EC50 value in soil assays, ferulic acid was the least expensive plant compound to apply on a per acre basis to inhibit M. phaseolina ($74,226). In F.oxysporum f.sp. fragariae soil assays, the compounds induced hormesis at lower rates and may be germination stimulant candidates. Metconazole and the high rates of every compound effectively or completely inhibited F. oxysporum f.sp. fragariae colony forming units in soil.
The plant compounds were more herbicidal than fungicidal in vitro. When combining the in vitro seedling length results for L. sativa, M. parviflora, P. annua, and S. vulgaris the EC50 values differed significantly (p < .0001) and were: 47 ppmw juglone, 120 ppmw p-Coumaric acid, 189 ppmw ferulic acid, and 297 ppmw gallic acid. At least one rate of ferulic acid, juglone, and p-Coumaric acid inhibited the germination of all plant species, while gallic acid only inhibited the germination of P. annua at 1000 ppmw (p < .05). In soil, visible microbial contamination in individual wells of 24-well plates and seed dormancy made it difficult to fit curves to weed seedling length data. The soil assay L. sativa seedling length EC50 values 11 days after initial treatment were slightly higher than in vitro, although plant compounds were in the same order of phytotoxicity: 129 ppmw juglone, 616 ppmw p-Coumaric acid, 644 ppmw ferulic acid, and 1584 ppmw gallic acid. Based on the EC50 value in soil assays, the least expensive compound to inhibit L. sativa seedling length on a per acre basis was gallic acid ($21,676). Germination 26 days after initial soil treatment generally declined in a dose-dependent manner for each compound. There was a direct relationship between plant compound rate and seedling damage in soil with the higher rates of all compounds, except p-Coumaric acid, inducing damage comparable to a conventional herbicide (pendimethalin or oxyfluorfen). Contaminated treatments appeared to be due to an interaction between plant compounds and microorganisms because herbicide and water controls had almost no microbial growth 11 days after initial treatment. Further, there was a significant positive linear relationship between level of contamination in phenolic acid-treated wells (ferulic acid, gallic acid, and p-Coumaric acid, p < .0001) and the in soil rate. This relationship was slightly negative in juglone soil treatments (p = .0167), which may be due to its greater antimicrobial activity than the phenolic acids. We propose that herbicidal effects in soil were due to the joint effect of the plant compounds themselves, and the microbial growth in wells. Microbial growth was either antagonistic or additive to the inhibitory action of the plant compounds.
The plant compounds we assayed were inhibitory of emerging fungal pathogens in strawberry production and common annual strawberry field weeds. Evidence presented in this thesis correlates well with past research that not only found plant compounds to be herbicidal and fungicidal, but also described their modes-of-action (such as the production of reactive oxygen species that causes necrotic lesions on roots, and inhibition of glycolytic enzyme activity that prevents germination), and implicate plant compounds as carbon sources for a variety of microorganisms. Compound prices are currently exorbitant, but may decline as demand increases. Whether or not they provide effective pest control may depend on soil texture, organic matter, microbial diversity, and other edaphic factors.