Sensitive quantitative detection of Ralstonia solanacearum in soil by the most probable number-polymerase chain reaction (MPN-PCR) method

Abstract

We developed a sensitive quantitative assay for detecting Ralstonia solanacearum in soil by most probable number (MPN) analysis based on bio-PCR results. For development of the detection method, we optimized an elution buffer containing 5g/L skim milk for extracting bacteria from soil and reducing contamination of polymerase inhibitors in soil extracts. Because R. solanacearum can grow in water without any added nutrients, we used a cultivation buffer in the culture step of the bio-PCR that contained only the buffer and antibiotics to suppress the growth of other soil microorganisms. To quantify the bacterial population in soil, the elution buffer was added to 10g soil on a dry weight basis so that the combined weight of buffer, soil, and soil-water was 50g; 5mL of soil extract was assumed to originate from 1g of soil. The soil extract was divided into triplicate aliquots each of 5mL and 500, 50, and 5μL. Each aliquot was diluted with the cultivation buffer and incubated at 35 °C for about 24h. After incubation, 5μL of culture was directly used for nested PCR. The number of aliquots showing positive results was collectively checked against the MPN table. The method could quantify bacterial populations in soil down to 3cfu/10g dried soil and was successfully applied to several types of soil. We applied the method for the quantitative detection of R. solanacearum in horticultural soils, which could quantitatively detect small populations (9.3cfu/g), but the semiselective media were not able to detect the bacteria.