Molecular identification and development of real-time PCR quantification methods for plant-parasitic nematodes in blueberry fields of Japan

Abstract

Occurrence of plant parasitic nematodes (PPN) on blueberry has been reported in North America. However, there is no available report on nematodes in blueberry cultivated in Japan, where its cultivation area is increasing, especially in suburban areas. This study aimed to develop quantitative methods using real-time PCR to enable direct quantification and identification of PPN from blueberry field soils in Japan. Nematodes were isolated from soil samples collected from six commercial blueberry fields located in Kanagawa Prefecture and Tokyo Metropolis, Japan. Pratylenchus penetrans, Helicotylenchus dihystera, Meloidogyne incognita, Paratrichodorus renifer, Tylenchorhynchus claytoni, and Criconema mutabile were the PPN identified by molecular methods. Among them, new primer sets for P. renifer, T. claytoni and C. mutabile and a modified primer set for H. dihystera were designed in the internal transcribed spacer 1 region of ribosomal RNA, and calibration curves for direct quantification of these PPN from soil using realtime PCR were obtained. The quantification limits for these nematodes obtained by real-time PCR were 84.4/20 g soil for P. penetrans, 9.1/20 g soil for M. incognita, 10.4/20 g soil for P. renifer, 19.4/20 g soil for C. mutabile, 1.6/20 g soil for T. claytoni, and 23.2/20 g soil for H. dihystera. Moreover, these primers did not amplify DNA extracts of non-target PPN individuals found in the blueberry soils or amplified with very low efficiency (Ct value ≧ 34.3). This study identified plant-parasitic nematodes in blueberry fields in Japan for the first time and developed their molecular quantification methods.