Simultaneous detection and quantification of Phytophthora nicotianae and P. cactorum, and distribution analyses in strawberry greenhouses by duplex real-time PCR

Abstract

Phytophthora nicotianae and P. cactorum cause Phytophthora rot of strawberry. A duplex real-time PCR technique for simultaneous detection and quantification of the two pathogens was developed. Species-specific primers for P. nicotianae and P. cactorum were designed based on the internal transcribed spacer regions (ITS) of rDNA and the ras-related protein gene Ypt1, respectively. TaqMan probes were labeled with FAM for P. nicotianae and HEX for P. cactorum. Specificities were demonstrated using 52 isolates, including various soil-borne pathogens. Sensitivities for P. nicotianae and P. cactorum DNAs were 10 fg and 1 pg, respectively. The technique was applied to naturally infested soil and root samples; the two pathogens were detected and the target DNA concentrations were quantified. Significant correlations of DNA quantities in roots and the surrounding soils were found. The minimum soil DNA concentration predicting the development of disease symptoms was estimated as 20 pg (g soil)(-1). In three strawberry greenhouses examined, the target DNA concentrations ranged from 1 to 1,655 pg (g soil)(-1) for P. nicotianae and from 13 to 233 pg (g soil)(-1) for P. cactorum. The method proved fast and reliable, and provides a useful tool to monitor P. nicotianae and P. cactorum in plants or soils.

Fig. 1

Detection limits, standard curves, correlation coefficients and amplification efficiencies assessed for Phytophthora nicotianae and P. cactorum. Total DNA from the two species was mixed together and serially diluted to yield final concentrations ranging from 1 ng μL⁻¹ to 1 fg μL⁻¹ before duplex real-time PCR amplification.

Fig. 2

Correlations between DNA quantities in soils and roots for P. nicotianae and P. cactorum. Roots and soils surrounding the roots were collected from the diseased strawberry plants and the surrounding soils in a strawberry planting greenhouse. The DNA extracts were applied in the duplex real-time PCR for the quantifications of P. nicotianae and P. cactorum. Significance level: * = 5%, ** = 1%.

Fig. 3

Distributions of Phytophthora nicotianae and P. cactorum in Saga and Gifu strawberry greenhouses. The soil DNA extracts were applied in duplex real-time PCR for the detection of P. nicotianae and P. cactorum. N = north side of strawberry greenhouse; S=south side; nic=P. nicotianae; cac=P. cactorum.