title: Sensitive molecular diagnostic assays to mitigate the risks of asymptomatic bacterial diseases of plants.
abstract: Our highly concentrated monoculture makes crops vulnerable to pests and diseases. An increase in emerging non-indigenous bacterial diseases poses a real threat to US agriculture. The United States has 100,000 miles of shoreline and 6,000 miles of border, making possible easy introduction of crop pests and diseases. Most threatening to crops are the cross-domain enteric bacteria. In contrast to animals, crops have hundreds of major diseases and development of molecular-based detection protocols for each pathogen is impossible with current technology. Rathayibacter toxicus, a neurotoxin-producing bacterium transmitted by a seed gall nematode, is an example of a high-risk Select Agent. The bacterium infects seeds of grasses without showing any symptoms, often resulting in the death of grazing cattle. A prerequisite for the control of any disease is sensitive detection and proper identification of the causal organism. Detecting bacteria in samples of plants showing symptoms is relatively simple, whereas detection in asymptomatic tissues is difficult due to the extremely low numbers of the target pathogen present. Rapid serological assays work well with symptomatic tissues but not from asymptomatic tissue when bacteria levels are below sensitivity limits. Classical agar-plating assays are 1,000 fold more sensitive then serology or PCR. However, agar plating assays take from 3 to 5 days and require pathogenicity tests to confirm the identity. PCR-based assays allow for rapid, accurate identification but are insensitive due to use of 1 microL sample in comparison to 100 microL used for agar plating. To overcome this disadvantage, an enrichment technique termed BIO-PCR can be used in combination with agar plating for detection with asymptomatic tissues. The key to developing a successful BIO-PCR protocol is to determine the time required for development of pin point-size colonies to appear. For most plant pathogens 15 to 24 hours is sufficient time, whereas for the cross-domain bacteria only 1 to 2 hours is needed. For greater sensitivity, BIO-PCR can be combined with 96-well microliter plates with membranes to detect a single viable cell per 10 mL of an aqueous sample.
authors.affiliation: US Department of Agriculture, Agriculture Research Service, Foreign Disease-Weed Science, Research Unit, Ft. Detrick, Maryland, USA. norman.schaad@ars.usda.gov
authors.forename: N
authors.initials: N
authors.lastname: Schaad
authors.forename: E
authors.initials: E
authors.lastname: Schuenzel
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012307:Risk Factors; D012680:Sensitivity and Specificity