To develop a rapid assay for evaluating the potential impact of Meloidogyne incognita on a crop, we investigated the relationship between its density and gall index in a seedling assay. Forty-eight soil samples were collected from 12 sites of four greenhouses at two depths (surface 0–30 and subsurface 30–45 cm) two times (after disinfection and at harvest of tomato). While the densities of M. incognita were measured with real-time PCR, an assay using cucumber seedlings was conducted to evaluate damage by M. incognita. No or very low damage was observed to cucumber seedlings in all the 13 soils in which no M. incognita was detected. No serious damage was observed in the soils with densities of M. incognita less than 48 J2eq (second-stage juveniles equivalent)/20 g soil, suggesting that this density may be a threshold for damage occurrence. Gall index was significantly correlated (r2 = 0.4586, P < 0.001) with the densities of M. incognita. Meloidogyne incognita was detected in all the 12 subsurface soil samples collected after disinfection and damage to seedlings was observed in 10 out of the 12 samples, suggesting that it is necessary to assess the impact of disinfection not only on surface soil but also subsurface soil. The present results reveal that a diagnosis method of M. incognita using real-time PCR might be a rapid and reliable tool for risk evaluation after disinfection.
The potato rot nematode Ditylenchus destructor is a major threat to garlic production in Aomori Prefecture, Japan. The objectives of this study were i) to design real-time PCR primers specific to D. destructor, and ii) to make calibration curves to evaluate the relationships between the number of D. destructor inoculated to soil or outer skin of garlic and the cycle threshold (Ct) values. Ditylenchus destructor strains were collected from 11 major growing regions in Aomori Prefecture and their ITS regions were sequenced. Strains from different regions showed the same sequence and, thus, real-time PCR primers specific to D. destructor were designed. The specific primers (Ddf and Ddr) matched 100% with 10 out of 11 D. destructor sequences from different countries as well as with the Aomori strains, while there are 7 and 16 bp mismatches in the closest species D. africanus and D. askenasyi, respectively. There were highly significant correlations (soil: y =－1.1221x + 35.225, R2 = 0.9973; outer skin of garlic: y =－1.145x + 35.295, R2 = 0.9883) between the log-transformed numbers of nematodes inoculated (x) and the respective Ct values (y). Based on this calibration curve, the densities of D. destructor in soils were estimated to be 43/10 g of soil in an infested field and 0/10 g of soil in a field without a history of garlic cultivation. These results demonstrated that the presently designed primers are useful to quantify the density of D. destructor in both soil and garlic.
We developed a very simple method for simultaneous detection and discrimination of the principal Pratylenchus and Meloidogyne species in nematode communities collected from Japanese fields. The method consists of a single polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) process using group-specific primers (PNem-F and PNem-R1, 2) constructed from the rDNA-ITS region. DNA from the target species (nine Pratylenchus species and three Meloidogyne species) was amplified with sufficient specificity, and most PCR products could be clearly separated by PAGE using a commercial precast polyacrylamide gel. Although these primers also amplified the DNAs of cyst nematodes and Scutellonema brachyurum, as the sizes of these PCR products were larger than those of the target species, detection and discrimination of the target species bands was not hindered. Even when two or more target species coexisted in a community, these could easily be detected and distinguished, simultaneously and accurately. We confirmed that the combination of a group-specific primer with a community analysis technique was effective for nematode diagnosis. Thus, this method could be a useful tool for the diagnosis of Pratylenchus and Meloidogyne species in Japanese fields.
We developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) markers for the potato cyst nematode (PCN) resistance gene Hero A in tomato. Using these PCR-RFLP markers, we evaluated the presence of Hero A in 22 tomato cultivars. All cultivars displayed complete concordance between their PCN phenotype and the genotype as determined by PCR-RFLP. We confirmed that PCN-resistant tomato cultivars in Japan contained the Hero A gene, whereas susceptible cultivars did not. PCN is a highly important quarantine pest, and the PCR-RFLP markers for Hero A provide a means of testing for resistance without the need to use live nematodes.