title: A high-throughput screening assay to identify bacterial antagonists against Fusarium verticillioides.
abstract: A high-throughput antagonistic assay was developed to screen for bacterial isolates capable of controlling the maize fungal phytopathogen Fusarium verticillioides. This assay combines a straightforward methodology, in which the fungus is challenged with bacterial isolates in liquid medium, with a novel approach that uses the plant lectin wheat germ agglutinin (WGA) coupled to a fluorophore (Alexa-Fluor® 488) under the commercial name of WGA, Alexa Fluor® 488 conjugate. The assay is performed in a 96-well plate format, which reduces the required laboratory space and streamlines quantitation and automation of the process, making it fast and accurate. The basis of our assay is that fungal biomass can be assessed by WGA, Alexa Fluor® 488 conjugate staining, which recognizes the chitin in the fungal cell wall and thus permits the identification of potential antagonistic bacteria that inhibit fungal growth. This principle was validated by chitin-competition binding assays against WGA, Alexa Fluor® 488 conjugate; confocal laser microscopy confirmed that the fluorescent WGA, Alexa Fluor® 488 conjugate binds to the chitin of the fungal cell wall. The majority of bacterial isolates did not bind to the WGA, Alexa Fluor® 488 conjugate. Furthermore, including washing steps significantly reduced any bacterial staining to background levels, even in the rare cases where bacterial isolates were capable of binding to WGA. Confirmatory conventional agar plate antagonistic assays were also conducted to validate our technique. We are now successfully employing this large-scale antagonistic assay as a pre-screening step for potential fungal antagonists in extensive bacteria collections (on the order of thousands of isolates).
authors.affiliation: Departamento de Biotecnología Agrícola, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional Unidad Sinaloa, Instituto Politécnico Nacional (CIIDIR Unidad Sinaloa, IPN), Col. San Joachin, Guasave Sinaloa, México.
authors.forename: Alejandro Miguel
authors.initials: AM
authors.lastname: Figueroa-López
authors.forename: Jesús Damián
authors.initials: JD
authors.lastname: Cordero-Ramírez
authors.forename: Francisco Roberto
authors.initials: FR
authors.lastname: Quiroz-Figueroa
authors.forename: Ignacio Eduardo
authors.initials: IE
authors.lastname: Maldonado-Mendoza
mesh_terms: D000898:Antibiosis; D001419:Bacteria / Q000302:isolation & purification*; D018407:Bacterial Physiological Phenomena; D005670:Fusarium / Q000254:growth & development*; D057166:High-Throughput Screening Assays / Q000379:methods*; D003313:Zea mays / Q000382:microbiology
keywords: Bacterial antagonism; Confocal Laser Scanning Microscopy; Fluorescence bioassay; High-throughput screen; WGA, Alexa Fluor® 488 conjugate