Project Number: 8042-21000-283-12-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2018
End Date: Sep 30, 2019
1. Identify non-pathogenic Streptomyces strains that are antagonistic to M. chitwoodi. 2. Screen most promising strains for plant-protective potential against M. chitwoodi infection in a soil-based, greenhouse pot assay. 3. Screen most promising strains for the ability to protect against M. hapla to identify strains that can inhibit, in soil, this second most important potato root-knot nematode species. 4. Determine whether chitinase activity of the Streptomyces strains is responsible for the antagonism to M. chitwoodi and M. hapla.
1. 60 non-pathogenic Streptomycetes (already in culture collection) will be cultured in the Clarke lab and shipped to the Gleason lab. Dr. Gleason will travel to the Beltsville Agricultural Research Center for a project initiation meeting and hands-on training for handling the Streptomycetes. Cell extract solutions of culture filtrates for each strain will be collected. Culture filtrates will be added to tubes containing 1ml of H2O and 200 nematode juveniles. The nematodes will be monitored at 24 and 48 hours and the number of living and dead nematodes will be quantified. Four replicate tubes will be measured for each culture filtrate extract. Tubes without added culture filtrate will be used as the control. In a parallel experiment, culture filtrate will be added to purified nematode eggs and egg hatch rate will be quantified 72 hours after the treatment. 2. Five to twenty Streptomyces strains that exhibit nematode antagonism in Approach 1 will be cultured in the Clarke lab and dry-coated onto Russet Burbank (which is susceptible to root-knot nematode) seed tubers. These coated-potato seed tubers (and Streptomyces-absent control-coated tubers) will be express shipped to the Gleason lab and planted into pots with sand. After 4 weeks, pots will be inoculated with 1000 M. chitwoodi eggs. At 4 weeks, post-inoculation, the potato roots will be stained with acid fuchsin, which dyes nematodes pink, and the number of nematodes and their life-stages will be assessed. Two to three Streptomyces strains that do not exhibit M. chitwoodi antagonism in culture (Approach 1) will be used as negative controls. 3. One to five strains that protect potato from M. chitwoodi infection (Approach 2) will be tested for antagonism against M. hapla using the same protocol described in Approach 1 for the M. chitwoodi in culture antagonism assay. Any of the strains that are antagonistic to M. hapla will then be coated onto tubers and sent to the Gleason lab and screened using the same protocol as Approach 2. 4. The Clarke lab will PCR amplify and sequence the chitinase gene(s) from several of the putative biocontrol Streptomycetes. After the pot assays, we will disrupt the chitinase gene(s) in the most promising biocontrol strain using the standard Streptomyces integration vector pIJ773. This strain will be tested for chitinase activity and sent to the Gleason lab to be tested for antagonism to M. chitwoodi and M. hapla.