Development of a Culture-independent Real-time Pcr Assay for Detection of Agrobacterium Tumefaciens in Soil

Crown gall disease caused by the bacterium Agrobacterium tumefaciens can cause significant economic loss in both commercial walnut orchards and in nursery operations in California. This results from the fact that, Paradox, one of the most popular walnut rootstocks in California, is extremely susceptible to A. tumefaciens infection and Crown Gall formation. By combining direct soil-DNA extraction with PCR amplification of target DNA followed by resolution of the PCR products on an agarose gel; as few as 200 colony forming units (CFU) of A. tumefaciens cells g soil were detected using virD2 primers. Real-time PCR analysis of the same soil lowered the detection limit to 20 CFU g soil. The real time PCR cycling parameters reported here facilitate A. tumefaciens detection and quantification within 3 hr after soil DNA extraction. Dilution plating of spiked soil samples on the Agrobacterium selective tellurite-1A based medium detected as few as 10 CFU g of soil, 20% of the time. The probability of detection rose to 90% when Agrobacterium populations rose to 10 CFU g -1 soil. The development of these detection tools will facilitate a more detailed examination of A. tumefaciens ecology and pathology, under field conditions.