Yu Yu Min

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.

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.

Suppression of Meloidogyne incognita in different agricultural soils and possible contribution of soil fauna

A total of 12 soils collected from different agricultural fields, having different backgrounds of organic input, were evaluated for their suppressive potential against Meloidogyne incognita. Second-stage juveniles (J2) of M. incognita were inoculated into the soils and their survival was evaluated. The number of M. incognita J2 5 days after inoculation differed depending on soil and was significantly lower in two soils, suggesting higher suppressiveness against M. incognita in these soils. This was confirmed by an experiment using tomato as a test plant, in which the gall formation was significantly lower in the two soils than in other soils. To estimate the contribution of below-ground biota to the suppressiveness, numbers of nematodes (predator, omnivore, bacterivore and fungivore) and other soil fauna such as tardigrades and rotifers, were counted. Some soil chemical and biological properties were also measured. Results from multiple linear regression analysis suggested that the number of rotifers, microbial activity, soil pH and total C may be involved in the suppression. The relationship between the suppressiveness and soil chemical and biological parameters is discussed.

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

Plant-parasitic nematodes in some economically important crops in Myanmar – species, possible damage and control measures

Rice, pulses and oilseed crops are major exporting crops in Myanmar. Many plant-parasitic nematodes, such as Meloidogyne incognita , M. javanica , M. graminicola , Ditylenchus angustus , Hirschmanniella oryzae , Heterodera cajani and Pratylenchus spp., have been detected in these crops in different cropping patterns and are considered one of the reasons for their low yields. Previous surveys have shown potential impact to yield losses in the crops. This Forum article provides collective information on species of the major plant-parasitic nematodes, possible damage and available control measures to such economically important crops in Myanmar.