Location: Crop Genetics Research
Project Number: 6066-21000-053-006-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 22, 2022
End Date: Aug 21, 2027
Objective:
1. Evaluate potentially resistant/tolerant cotton leaf crumple virus (CLCrV) cotton lines in a field endemic for CLCrV to identify resistant/tolerant lines.
2. Monitor the trial area for CLCrV and other viruses potentially damaging to Cotton. Whitefly-infested plant species within and around the field trial as well as the whitefly vector will also be collected and assayed for CLCrV virus.
Approach:
A CLCrV resistance screening field trial will be conducted in a field with a history of high whitefly infestation and high incidence of begomovirus-positive whiteflies based on previous studies. Three putative resistant lines, one susceptible line and a commonly grown cotton cultivar will be planted in three replications in two row plots. The trial will be planted early March and maintained with the normal production practices of the grower. The field trial will be repeated for a second year. Data will be collected as follows:
From first flower bud formation, field plots will be scouted every 2 weeks and whitefly counts taken from 10 randomly selected plants per plot. Whitefly cohorts will be collected with an aspirator at each time point and stored in labeled vials containing 70% ethanol (v/v) until assayed for viruses. Plants in each plot will be rated for disease incidence and symptom severity. Incidence will be assessed as the proportion of plants showing virus-like symptoms out of the total number of plants within the plot. Disease severity will be assessed on the top canopy of every 12th plant per plot on a 1 to 5 scoring scale, where 1 = no symptoms to 5 = severe mosaic with distortion of the entire leaf (modified from Terry, 1975). Leaf tissue samples will be taken, 10 per plot (with or without symptoms), every 2 weeks beginning from first flower bud stage. Total nucleic acid (TNA) will be extracted from plant and insect samples according to Dellaporta (1983). The nucleic acid extracts will be quantified on a NanoDrop spectrophotometer and an aliquot subjected to rolling cycle amplification (RCA) to enrich for circular DNA targets, then screened by PCR using a pair of generic primers targeting a 576 bp fragment of the core coat protein of begomoviruses. The obtained DNA amplicons from two randomly chosen plant and insect samples per plot per time-period will be cloned and Sanger-sequenced. This approach allows us to detect incidence of CLCrV and any other begomovirus species present within an infected sample. The DNA sequences from plant and insect samples will be identified by species and genetic relatedness to corresponding gene fragments of published virus isolates.
