The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Genetic Variability among Different Populations of Root Knot Nematodes Based on Their Encumbrance Response to Pasteuria Isolates Using PCR-RFLP

  • Kamran, Muhammad (Plant Pathology Research Institute, Ayub Agricultural Research Institute Faisalabad) ;
  • Javed, Nazir (Department of Plant Pathology, University of Agriculture Faisalabad) ;
  • Ullah, Ihsan (School of Agriculture, Policy and Development, University of Reading) ;
  • Nazir, Shahid (Agricultural Biotechnology Research Institute) ;
  • Jamil, Shakra (Agricultural Biotechnology Research Institute) ;
  • Iqbal, Muhammad Zafar (Agricultural Biotechnology Research Institute) ;
  • Abbas, Huma (Department of Plant Pathology, University of Agriculture Faisalabad) ;
  • Khan, Sajid Aleem (Department of Plant Pathology, University of Agriculture Faisalabad) ;
  • Haq, Muhammad Ehetisham ul (Plant Pathology Research Institute, Ayub Agricultural Research Institute Faisalabad)
  • Received : 2017.12.18
  • Accepted : 2018.09.04
  • Published : 2019.02.01
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Abstract

A great variable response was observed when PP-3 and PP-J encumbered with 116 populations of root knot nematode (RKN) at two different temperatures ($25{\pm}2^{\circ}C$ and $30{\pm}2^{\circ}C$) and concentrations ($10^4$ and $10^5$ spores/ml). The PCR reaction amplified intergenic region between cytochrome oxidase subunit II gene (COII) and large subunit of rRNA gene (lrRNA) of the mitochondrial genome of different RKN species. The primer C2F3 and 1108 identified M. incognita with the highest frequency (52.6%) followed by M. javanica (36.8%) and M. arenaria (10.5%). The sizes of PCR products were 1.7 kb for M. incognita and M. javanica populations while populations of M. arenaria produced 1.1 kb fragment. The digestion with Hinf I yielded three different fragment length patterns on 1.5 % agarose gel. From current research it is concluded that intra-Meloidogyne genetic variability exist in RKN populations which have better encumbrance with P. penetrans.

Keywords

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Fig. 1. Map of Punjab-Pakistan showing the five sampling Dis-tricts.

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Fig. 2. Diagrammatic representation of primer binding sites on the Meloidogyne mitochondrial genome. Primer C2F3 anneals to the coding strand of the cytochrome oxidase subunit II (COII) gene and primer 1108 anneals approximately 450 bp downstream from the start of the lrRNA gene. The intergenic region varies in size among the different Meloidogyne.

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Fig. 3. Gel image of C2F3/1108 amplified PCR product of COII/ LrRNA of mitochondrial genome. The 1.7 kb sizes of PCR products are characteristics of M. incognita and M. javanica while M. arenaria produce 1.1 kb fragment size. M lanes were loaded with 1 kb ladder.

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Fig. 4. Gel image of PCR products from 17 nematode populations restricted with Hinf I enzyme. Lanes showing two fragments of 1.0 and 0.7 kb were related M. javanica whereas lanes with three fragments of 1.0, 0.4 and 0.3 kb were related to M. incognita. The products were resolved on 1.5% agarose gel and stained with ethidium bromide. M lanes were loaded with 1 kb ladder.

Table 1. List of 19 root-knot nematode populations selected on the basis of their encumbrance to two Pasteuria isolates (PP-3 and PP-J) and used in species differentiation using PCR-RFLP marker

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Table 2. Composition of PCR reaction mixtures assembled for characterization of 19 root-knot nematode populations

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Table 3. Temperature cycles used in PCR performed for charac-terization of 19 root-knot nematode populations

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Table 4. Encumbrance of two Pasteuria isolates (PP-3 and PP-J) with different populations of Meloidogyne spp. isolated from tomato

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Table 4. Continued

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Table 5. Encumbrance of two Pasteuria isolates (PP-3 and PP-J) with different populations of Meloidogyne spp. isolated from cucum-ber.

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Table 5. Continued

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