Erika Sato

Development of a real-time PCR method for the potato-cyst nematode Globodera rostochiensis and the root-knot nematode Meloidogyne incognita

Abstract The primers PCN280f and NEPCN398r were designed for the quantitative detection of the potato-cyst nematode Globodera rostochiensis using real-time polymerase chain reaction (PCR). One, five, 50, 125 and 250 individuals of the second-stage juveniles (J2) of G. rostochiensis were mixed with various stages of vermiform Caenorhabditis elegans to make a total of 500 individuals and DNA was extracted from the nematode mixture. There was a significant correlation (r 2 = 0.9355, P < 0.001) between the threshold cycle values and the number of G. rostochiensis added. When nematodes were extracted from soils artificially infested with G. rostochiensis to various degrees and real-time PCR was conducted using DNA templates from the nematodes extracted, there was a highly significant correlation in the numbers of G. rostochiensis J2 from the real-time PCR method and morphological identification. Real-time PCR sensitively detected a single G. rostochiensis J2 out of 1,000 individuals of free-living nematodes. Similarly, real-time PCR primers RKNf and RKNr were designed for the detection of the root-knot nematode Meloidogyne incognita. This study demonstrated that the real-time PCR assay for the potato-cyst nematode and the root-knot nematode provides a sensitive and reliable means for the rapid quantification of these vermiform pests.

A novel nematode diagnostic method using the direct quantification of major plant-parasitic nematodes in soil by real-time PCR

We have developed a direct quantification method using real-time PCR for various plant-parasitic nematodes. Firstly, specific primers were designed for the root-knot nematode Meloidogyne incognita, the root-lesion nematode Pratylenchus penetrans, the potato cyst nematode Globodera rostochiensis and the soybean cyst nematode Heterodera glycines. A DNA extraction method was then developed beginning with 20 g of soil, a relatively large amount of soil but a necessary amount in the consideration of heterogeneous distribution of nematodes in soil. To estimate the density of the target nematode in soil, calibration curves for each plant-parasitic nematode were obtained by inoculating different numbers of the target nematode and then extracting DNA from the soils. The detection limit was 4-5 nematodes (20 g soil)−1. This method was applied to nematode diagnostics. Soil sampling was done when transplanting of radish and sweet potato in fields was taking place, and the density of plant-parasitic nematodes was measured using both the Baermann funnel extraction method and real-time PCR methods. In some soils, P. penetrans and M. incognita were not found with the Baermann method but were detected with the real-time PCR method. At harvest, damage to crops was evaluated and its relationship with initial densities was investigated. The real-time PCR method more precisely predicted damage to radish and sweet potato by nematodes and was considered to be a powerful tool in the diagnosis of nematode diseases.

Relationships between the damage to radish caused by the root-lesion nematode Pratylenchus penetrans, its density prior to cultivation and the soil nematode community structure evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis

Abstract A preliminary survey using 20 conventionally farmed fields in which fumigants have been applied every year showed that the root-lesion nematode Pratylenchus penetrans was distributed both in the upper (0–30 cm) and lower (30–60 cm) soil layers. In six of the 20 fields, P. penetrans was detected in the lower layers exclusively, suggesting that the most appropriate depth to sample soil is 0–60 cm to estimate the relationship between the density of P. penetrans and its damage to radish. There was a highly significant correlation (r = 0.923) between the density of P. penetrans in the 0–60 cm depth and the number of spots on a radish. No damage to radish was observed in soils with <2.5 individuals of P. penetrans per 20 g soil before cultivation. However, in cases in which the density of P. penetrans was 3.4–6.2 individuals per 20 g soil, the number of spots on a radish showed more variation (0–131.5 per radish) and there was no significant correlation between them. The nematode community structure of soils with 3.4–8 individuals of P. penetrans per 20 g soil, evaluated by polymerase chain reaction-denaturing gradient gel electrophoresis, was significantly different (anova, PC2, P < 0.05) between soils with low (0–42) and high (more than 80) damage levels, suggesting that radish damage might be predicted on the basis of the prevailing soil nematode community structure.

Development of a direct quantitative detection method for Meloidogyne incognita in sandy soils and its application to sweet potato cultivated fields in Tokushima prefecture, Japan

A real-time PCR (quantitative PCR: qPCR)-based detection method of the root-knot nematode Meloidogyne incognita was developed for sandy soils, the major soil type in sweet potato cultivated fields in Tokushima prefecture, Japan. Different numbers (5, 20, 80, 200 and 500) of second-stage juveniles (J2) were artificially added into 20 g of an air-dried sandy soil not containing M. incognita . To make homogenous samples, soil was homogenised by two different ways (ground with either a mortar and pestle or ball mill) and then 0.5 g of the soil was used for DNA extraction. There was a strong negative correlation in each homogenisation method between the cycle threshold number (Ct) and inoculated numbers of M. incognita J2. The Ct values were consistently lower and their variations among replicates were smaller in the samples ground with ball mill, suggesting that grinding with ball mill may be suitable for the preparation of soil for DNA extraction. Sandy soils were collected from sweet potato fields in Tokushima prefecture at the transplanting and harvesting times. Damage to sweet potato caused by M. incognita was also evaluated in some of the fields. At the transplanting time, no M. incognita was extracted in all the soils by the Baermann funnel method, while detection in the qPCR method ranged from zero to 4 210 000 J2 equivalent (20 g soil) –1 . Heavy damage was observed in fields with more than 500 equivalent M. incognita J2 (20 g soil) –1 . By contrast, very few galls were observed in fields with fewer than four individuals (20 g soil) –1 . At harvest, zero to >1000 individuals of M. incognita was counted by the Baermann method and there was a significant correlation in estimated numbers of M. incognita between the two methods. However, the estimated numbers were 15 times higher in the qPCR method than in the Baermann method. These results indicate that direct quantification of M. incognita based on the qPCR method might enable a sensitive diagnosis to predict damage by the nematode.

Quantification of Pratylenchus penetrans in radish fields using a combination method of soil compaction and real-time PCR to determine the economic threshold

The objective of this study was to compare the economic threshold (ET) of Pratylenchus penetrans in radish fields of andosols using the Baermann method and the combination method of soil compaction and real-time polymerase chain reaction (PCR). Soil samples were collected from 26 plots at depths of 0 to 30 and 30 to 60 cm before seeding of radishes. The number of P. penetrans in each sample was estimated by the Baermann method and the combination method. No P. penetrans was detected in 13 plots by the Baermann method, while the number of plots in which no P. penetrans was detected was only two by the combination method. The number of spots caused by P. penetrans on radishes harvested from the plots was also counted. It was difficult to determine the ET of P. penetrans estimated by the Baermann method, based on whether severe damage (more than 10 spots on average per radish) was seen. However, the ET of P. penetrans estimated by the combination method was determined at 5.3 J2 equivalents per 20 g dry soil. In plots with P. penetrans densities lower than the ET as evaluated by the combination method, the ratio of plots with no damage was 87%. The results suggest that the combination method has an advantage in the estimation of damage to radish by nematode.

Effects of the density of root-lesion nematode (Pratylenchus penetrans), soil chemical and microbial properties on the damage to Japanese radish

The density of Pratylenchus penetrans in soil is known to be a major determinant in the severity of damage to Japanese radish, but soil chemical and microbial properties may also affect the severity. This study investigated the relationships between damage and the density of P. penetrans, soil chemical properties, such as pH (H2O), EC, available phosphate, NO3–N, NH4–N, total carbon and nitrogen, and microbial properties, such as microbial biomass and fungal and bacterial community structures, in 48 different plots in a field for 2 years. The density of P. penetrans estimated with real-time PCR was most significantly correlated with the damage to radish in both 2010 and 2011. The damage to radish caused by P. penetrans and the nematode density were negatively correlated with total carbon in soil in both years, suggesting that soil management leading to increased soil C could decrease the density of P. penetrans and hence the damage. Multiple regression models including the density of P. penetrans and the soil properties suggested that damage to radish was predictable with a higher probability for some of the soil chemical properties, such as total C and N, and the density of P. penetrans. The community structures of soil fungi and Pseudomonas spp. on the radish surface were different depending on the levels of damage to radish and the densities of P. penetrans. The results suggested that the damage to radish and the density of P. penetrans might be affected by the soil microbial community structure.

A multiplex real-time PCR assay for the simultaneous quantification of the major plant-parasitic nematodes in Japan

Multiplex real-time PCR assays were developed to quantify multiple species of Meloidogyne incognita, Pratylenchus penetrans, Globodera rostochiensis and Heterodera glycines in soil. The probes specific for P. penetrans and H. glycines are labelled with a fluorescence molecule, FAM, and those for M. incognita and G. rostochiensis with ROX. The primers and probes are species-specific to P. penetrans, but group-specific to the other species. DNA was extracted from suspensions containing each nematode and multiplex Cycleave® PCR assays were done for pairs of P. penetrans and M. incognita, P. penetrans and G. rostochiensis, or G. rostochiensis and H. glycines. The results revealed that the target nematode, except for H. glycines, was quantified in the presence of less than 100 times that of the other nematode (competitor), but underestimated in the presence of 1000 times the competitor. Such underestimation was solved by the use of SYBR Green I real time PCR assays targeting a single species. Multiplex PCR assay for P. penetrans and M. incognita was done using environmental DNA (eDNA) extracted from a soil naturally infested with the nematodes. Results quantified both species. Multiplex assay using eDNA may enable a sensitive and simultaneous detection of P. penetrans and M. incognita or P. penetrans and G. rostochiensis in soil although caution is needed in case the existing ratio is biased to one of the species.

Effects of Anaerobically Digested Slurry on Meloidogyne incognita and Pratylenchus penetrans in Tomato and Radish Production

Since effective disposable way of anaerobically digested biogas slurry is expected, ADS was applied to soil to evaluate its effects on nematode damage. Damage index of tomato by root-knot nematode was significantly (𝑃<.05) lower and the growth better in pots applied with ADS (100 and 200 mg NH

Rhizoctonia blight of turnip green caused by Rhizoctonia solani AG-4 HG-III

Severe wilt was found on seedlings of turnip green (Brassica rapa L., perviridis group) in Ibaraki Prefecture, Japan, from May to September 2013. A fungus repeatedly isolated from diseased plants was identified as Rhizoctonia solani AG-4 HG-III based on culture morphology, anastomosis and sequencing of the internal transcribed spacer region on ribosomal DNA (rDNA-ITS region). Pathogenicity of this isolate to turnip green was confirmed based on inoculation test. Because Rhizoctonia blight of turnip green by AG-4 HG-III has not been reported so far, we propose including it as one of the pathogens causing this disease.