Methods for Rapid Diagnosis and Field Monitoring of Tombusviruses in Lettuce
Crop Improvement and Protection Research
2012 Annual Report
1a.Objectives (from AD-416):
The goal of this project is to confirm reliability of a newly developed detection method; attempt development of a rapid serological test for tombusviruses causing dieback disease in lettuce (long-term and will depend in part on success of molecular test); determine if it will be possible to monitor levels of tombusviruses in soil using commercial soil nucleic acid testing kits; and develop more cost-effective and reliable methods for greenhouse-based testing for tombusvirus resistance in lettuce.
1b.Approach (from AD-416):
Lettuce will be sampled from a diverse array of dieback infested fields from throughout the Salinas Valley and other regions. We will determine if our new molecular detection method can confirm infection more reliably and timely than previous methods. We will test commercial kits for extracting nucleic acid from soils to determine if it is possible to A) detect TBSV and LNSV from soils, and B) quantify levels from different soils and determine correlation with disease incidence in the fields (multi-year objective). Finally, we plan to test a new method for rapid greenhouse pre-screening of lettuce cultivars/lines for tombusvirus resistance.
Improved molecular detection methods (RT-PCR) for identification of both Lettuce necrotic stunt virus (LNSV)and Tomato bushy stunt virus (TBSV), the two viruses responsible for causing lettuce dieback disease, have been developed. Symptomatic lettuce plants have been sampled from throughout the Salinas Valley and other lettuce producing areas of California, and detection methods have been shown to reliably detect both viruses as confirmed by sequence analysis of RT-PCR amplified products.
Studies were initiated in 2011 through this project to develop a method for screening lettuce germplasm for resistance to tombusvirus infection in either greenhouse or growth chambers. This would allow the lettuce seed industry to reliably screen for resistance year-round and without the variability that occurs under field conditions. Although lettuce can be easily inoculated with both LNSV and TBSV by rubbing leaves with infected sap (mechanical inoculation), this form of inoculation does not result in lettuce dieback symptoms, as infection requires specific, and as yet only partially identified stress factors that allow the virus to move systemically in the plant and induce disease. We attempted numerous treatments to facilitate disease development, and found exposure to 24 hour day length and 29°C induced disease development following mechanical inoculation of leaves with virus sap. This method appears to produce systemic infection and disease development in susceptible lettuce, but not resistant lettuce, although studies are continuing. However, some additional and as yet unidentified conditions are required to obtain consistently high rates of disease development. Research is continuing to clarify optimal conditions for disease screening.