Location: Horticultural Crops Disease and Pest Management Research Unit
Project Number: 2072-22000-046-009-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2019
End Date: Aug 15, 2022
Objective:
1. Collect Potato Cyst Nematode (PCN) populations from a diversity of locations worldwide.
2. Characterize the mycobiome of collected PCN populations.
3. Identify fungal biocontrol agents for PCN.
Approach:
Potato cyst nematodes (PCN) (G. rostochiensis, G. pallida, and G. ellingtonae) will be requested from domestic and international collaborators. The following populations will be collected: South America – 6 populations from Peru, Chile, and/ or Bolivia; Central America – 1 population from Costa Rica; Africa – 2 populations from Uganda and Kenya; Europe – 2 populations from Scotland and Belgium; North America – 2 populations from New York and Idaho; and, Other – a few additional populations will be obtained from Asia when collaborators are identified. A more extensive collection from South America is proposed because this is the origin of PCN and it is suspected that there will be a greater chance of finding fungal antagonists of PCN from these locations. From each location at least 100 cysts will be requested and cysts will be sent directly to Purdue under an APHIS permit.
Reference cultures: Cysts will be pooled by population into batches of 25, and suspended in phosphate buffer and gently ground with a micropestle. To isolate culturable fungi, 10 millilitre (mL) of the suspension will be serially diluted with phosphate buffer. Three replicates from each dilution will be plated on several fungal selective media and incubated until showing visible signs of germination. Individual germlings will be excised and subcultured. Stock cultures will be maintained in long term storage. Sanger sequencing of reference cultures: Reference cultures will be amplified and sequenced at the fungal barcoding locus (the internal transcribed spacer region, or ITS). Sequences will be edited and blasted against the NCBI GenBank database to ensure results are consistent with culture morphology. An internal FASTA database will be created for each culture sequence obtained for comparison with the Next-generation sequencing (NGS) data.
Next-generation sequencing: NGS will be used to identify the non-culturable fungi and for comparisons of community composition across PCN populations. Fungi that are prevalent in the South American, but not in the other global populations, will be targeted for use in G. ellingtonae assays.
Fungal filtrates will be prepared by inoculating liquid Potato Dextrose broth with inoculum from the stock cultures and incubated. Cultures will be filtered through gauze to remove the fungal mass; the liquid phase will be sterilized by filtration through a cellulose acetate membrane and stored until use. The fungal filtrates will then be screened against G. ellingtonae in a 96-well assay. Globodera ellingtonae will be used as model system to initially screen the fungal cultures for activity because it is not a regulated nematode, therefore, extensive permitting is not required; G. ellingtonae has been demonstrated to be
most biologically similar to G. rostochiensis. To obtain G. ellingtonae eggs and juveniles, first eggs will be freed from cysts. Some of these eggs will go directly into the assay while juveniles will be hatched from the remaining eggs. Those identified with activity against G. ellingtonae will be further screened against G. pallida and G. rostochiensis in a future research collaboration with scientists in Idaho and New York.
