Distribution Survey, Species Identification And Quantitative Detection Of Root-Knot Nematodes On Vegetables In Jiangsu Province

Root-knot nematodes (RKN) are obligate endoparasites of plant roots and widely distribute and severely threat the agricultural production in the world. In recent years, the occurrence of root-knot nematodes in our country was increased year by year and seriously affected the crop yield and quality as the rapidly extending the greenhouse vegetable growing and increasing the multiple cropping. The population density of root-knot nematodes in soil is significantly correlated with the damage degree on the host. Therefore, accurately, reliably and rapidly identifying and counting RKN in soil is critically important for the effective control of disease. The distribution and species as well as population densities of root-knot nematodes on vegetables were investigated in9distircts of Jiangsu Province. The methods for real-time PCR assay were developed to detect and quantify Meloidogyne spp. and Meloidogyne incognita, respectively. The methods have been applied in quantitative detection of root-knot nematodes in field samples. The main results are as follows:During2011, the distribution survey and species identification of root-knot nematodes was carried out on vegetables growing in25towns of9districts in Jiangsu Province. The results showed that the average detection rate of root-knot nematodes in collected samples was73.6%. The highest population density of second-stage juveniles (J2s) in sample was reached to2920nematodes per200mL soil. The detection rates of RKN in Suqian and Huai-an were higher with100%when compared to Yancheng with57.1%. Molecular identification by SCAR markers revealed that the RKN species occurred on vegetables in Jiangsu Province included Meloidogyne incognita, M. arenaria, M. javanica and M. hapla. Among them, M. incognita was the dominant species. Most of samples in collected fields were detected with a single population, and only18%samples were mixed with populations of M. incognita and M. arenaria, or M. incognita and M. javanica.In order to rapidly detect and quantify population density of Meloidogyne spp. in soil, a SYBR Green I real-time PCR assay was developed from DNA extracts of soil. A pair of specific primers was designed based on18S rDNA conserved sequences of Meloidogyne spp. The results indicated that the method developed was able to specifically detect Meloidogyne spp. from DNA templates extracted from soil sample, and the standard curve established in this study showed the fine linear relationship between cycle threshold and the logarithmic values of the number of Meloidogyne spp.. The correlation coefficient and amplification efficiency of the equation from standard curve was0.9836and80%, respectively. The sensitivity of the real-time PCR assay was1J2which was10times higher than that of the conventional PCR. The detection of10field samples revealed the significantly positive correlation between the microscope counting and the real-time PCR estimation for the numbers of Meloidogyne spp. in soil.To accurately identify and quantify M. incognita in soil, the real-time PCR assays for eggs and J2s were developed in the study. The specific primers and TaqMan probe were designed according to the SCAR marker sequences of M. incognita. Correlation was analyzed between the nematode numbers obtained by real-time PCR estimation and traditional microscope counting. The results indicated that the primers and probe were highly specific to M. incognita. The established standard curve showed the strong linear relationship between cycle threshold and the logarithmic values from the number of eggs and J2s. The correlation coefficient in the equation from eggs and J2s was0.9771and0.9853, respectively. The amplification efficiencies of the real-time PCR assay for both were90%, and the sensitivity was0.1egg and0.001J2, respectively. The detection of10field samples revealed the significantly positive correlation between the real-time PCR estimation and microscope counting for the numbers of J2s and eggs of M. incognita in soil, and the numbers of J2s determined by two methods was not significantly different.The methods developed in the study can not only rapidly, sensitively and specifically detect population density of Meloidogyne spp. and M. incognita in soil samples, but aslo provide the technical supports for forecasting root-knot nematode disease and making integrated control strategies.