A Nylon Membrane Bag Assay for Determination of the Effect of Chemicals on Soilborne Plant Pathogens in Soil

Abstract

A new nylon membrane bag (NMB) assay was developed for studies to determine the effect of chemicals added to soil on survival of soilborne plant pathogens. The rapid and effective assay can be used to study organisms for which there are no selective media or for which a selective medium is expensive or difficult to prepare. This assay consists of placing pathogens inside a bag made of small-pore (0.22-μm) nylon filtration membrane, which is placed in soil and later retrieved to determine survival of the pathogens on nonselective media. Chemicals but not other microorganisms can enter the bag from the soil. Using this assay, Streptomyces scabies, Fusarium oxysporum f. sp. lycopersici race 3, and Ralstonia solanacearum were successfully recovered from soil after 72 h as demonstrated by growth on a semiselective Streptomyces medium (S. scabies) or nonselective potato dextrose agar medium (F. oxysporum f. sp. lycopersici race 3 and R. solanacearum) with minimal microbial contamination. Addition of acetic acid (200 mM) to soil killed 100% of S. scabies. SPK (a mixture of organic chemicals) at a concentration of 1,500 mg kg–1 of soil killed 83.3% of F. oxysporum f. sp. lycopersici race 3 culture plugs, 100% of F. oxysporum f. sp. lycopersici race 3 spores, and 97.2% of R. solanacearum cells. SPK at 1,000 mg kg–1 of soil killed 50% of F. oxysporum f. sp. lycopersici race 3 culture plugs, 68.2% of F. oxysporum f. sp. lycopersici race 3 spores, and 12% of R. solanacearum. Benlate (500 to 1,500 mg kg–1 of soil) did not kill the culture plugs of F. oxysporum f. sp. lycopersici race 3 but reduced the growth rate of F. oxysporum f. sp. lycopersici race 3. Benlate (500, 1,000, and 1,500 mg kg–1 of soil) reduced F. oxysporum f. sp. lycopersici race 3 spore germination by 39.4, 49.3, and 50.4%, respectively. Streptomycin sulfate (1,500, 800, 400, and 200 mg kg–1 of soil) caused 75.3, 21, 11.9, and 0.9% mortality, respectively, of R. solanacearum.