Agrochemical Discovery: Finding New Fungicides From Natural Products

The continuing development of fungicide resistance in plant and human pathogens necessitates the discovery and development of new fungicides. Discovery and evaluation of natural product fungicides is largely dependent upon the availability of miniaturized antifungal bioassays. Essentials for natural product bioassays include, sensitivity to microgram quantities, selectivity to determine optimum target pathogens, and adaptability to complex mixtures. Experimental accuracy and precision must be stable between assays over time. These assays should be relevant to potential pathogen target sites in the natural infection process of the host and applicable to the agrochemical industry. Bioassays should take advantage of current high-throughput technology available to evaluate dose-response relationships, commercial fungicides standards, modes of action, and structure activity studies. As part of a program to discover natural product-based fungicides, two sensitive detection systems were developed for evaluation of antifungal agents as agrochemical plant protectants. Bioautography assays using Colletotrichum acutatum, C. fragariae, and C. gloeosporioides were used to identify antifungal components from plant extracts, eliminate commonly occurring nuisance compounds, and characterize antifungal activity of pure compounds. A microbioassay using conidia from Botrytis cinerea, Colletotrichum spp., Fusarium oxysporum, Phomposis obscurans, and P. viticola in a 96-well microtiter format was also developed to evaluate growth effects of active fractions or pure compounds identified by bioautography. These microbioassays allow for the evaluation of microgram quantities, determine dose-response relationships; compare antifungal activity with fungicide standards of known mode of action. This coupled approach to natural product discovery combines the simple and visual nature of direct bioautography with the rapid, sensitive, and high throughput capabilities of a microtiter system. Ultimately, development of safer plant protectants with selective antifungal activity and low environmental and mammalian toxicity is sought. INTRODUCTION Since the early 1970s, agriculture has struggled with the evolution of pathogen resistance to antimicrobial disease control agents. Increased necessity for repeated chemical applications, development of pesticide cross-resistance, and disease resistance management strategies have overshadowed the use of agricultural chemicals. Scientists are currently attempting to control agricultural pests with fewer effective chemical controls. In addition, the desire for safer pesticides with less environmental toxicity is a major public concern. Particularly desirable is the discovery of totally novel, prototype pesticidal agents representing new chemical classes with different toxicities that operate by different modes of action and, consequently, lack cross-resistance with currently used chemicals. In this respect, evaluating natural products and extracts to identify potential new pesticides offers an approach to discover new chemical entities that have never been synthesized by chemists. Antibiotics, antineoplastics, herbicides, and insecticides often originate from plant Proc. WOCMAP III, Vol. 3: Perspectives in Natural Product Chemistry Eds. K.H.C. Başer, G. Franz, S. Cañigueral, F. Demirci, L.E. Craker and Z.E. Gardner Acta Hort. 677, ISHS 2005 110 and microbial chemical defense mechanisms (Wedge and Camper, 2000). Secondary metabolites, once considered unimportant products, are now thought to mediate chemical defense mechanisms by providing chemical barriers against animal and microbial predators (Agrios, 1997; Wedge and Camper, 2000). This chemical warfare between plants and their pathogens shows promise to provide new natural product leads for biomedical research and agricultural pest control. Increasing evidence suggests that plant cellular defenses may be analogous to “natural” immune response of vertebrates and insects. The biosynthesis of phytoalexins after microbial attack appears to be similar to the phenomenon of immune reaction in vertebrates (Oku, 1997). In addition to cell structural similarities, plant and mammalian defense responses share functional similarities. In mammals, natural immunity is characterized by the rapid induction of gene expression after microbial invasion. A characteristic feature of plant disease resistance is the rapid induction of a hypersensitive response in which a small area of cells containing the pathogen is killed. Other aspects of plant defense include an oxidative burst leading to the production of reactive oxygen intermediates (ROIs), expression of defense related genes, alteration of membrane potentials, increase in lipoxygenase activity, cell wall modifications and production of antimicrobial compounds such as phytoalexins (Dixon et al., 1994). In the mammalian immune response, ROIs induce acute phase response genes by activating the transcription factors NFκB and AP-1 (Kopp and Ghosh, 1994; Schreck and Baeuerle, 1991). In plants, ROIs and salicylic acid regulate pathogen resistance through transcription of resistance gene-mediated defenses. Functional and structural similarities among evolutionarily divergent organisms suggest that the mammalian immune response and the plant pathogen defense pathways may be built from a common template (Baker et al., 1997). Wedge and Camper (2000) hypothesize that similar biosynthetic processes involved in signaling pathogen invasion and stress in plants and animals may account for the physiological cross activity of various pharmacologically active phytochemicals. MATERIAL AND METHODS Biological Evaluation of Antifungal Activity Many experimental approaches have been used to screen compounds and extracts from plants and microorganisms in order to discover new antifungal compounds. However, the focus of this paper is the bioassay methods that we are currently using in our laboratories to examine the antifungal potential of compounds produced by plants, marine and freshwater cyanobacteria, and algae. A uniquely synergistic collaboration involving the discovery of new fungicides from marine natural products is in place between the United States Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit and scientists from the University of Mississippi Department of Pharmacognosy at the Thad Cochran National Center for Natural Products Research. The collaborative team has evaluated numerous extracts and many organisms appear to have evolved direct-acting antifungal chemical defenses, most likely to survive in pathogen-rich ecosystems (Nagle and Wedge, 2002). As part of a program to discover natural product-based fungicides, two sensitive detection systems have been developed for the evaluation of antifungal agents. Standardization of Inoculum Conidia of fungal species are harvested from 7-10 day-old cultures by flooding plates with 5 mL of sterile distilled water and dislodging conidia by softly brushing the colonies with an L-shaped glass rod. Conidial suspensions were filtered through sterile Miracloth (Calbiochem-Novabiochem Corp., La Jolla CA) to remove Mycelia. Conidia concentrations are determined photometrically (Espinel-Ingroff and Kerkering, 1991; Wedge and Kuhajek, 1998) from a standard curve based on the percent of transmittance (%T) at 625 nm and manual hemocytometer counts. Conidial stock suspensions are then adjusted with sterile distilled water to a concentration of 1.0 x 10 conidia/mL. Verwijderd: ¶