Molecular Identification of Root-Knot Nematodes: Techniques and Limitations

1. Nematology 100 Lecture 15 Slides

3. Meloidogyne – life cycle

4. Meloidogyne – life cycle

5. Giant Cells Root galling

6. Identification Meloidogyne…. incognita javanica hapla arenaria

7. M. incognita - cantaloupe M. incognita - sugarbeet Symptoms • M. incognita • soybean (resistant – left • susceptible – right) M. incognita - cotton M. hapla - lettuce

8. M. incognita - sweetpotato M. chitwoodi- potato Damage Meloidogyne sp. - carrot

9. Nematicides

10. Crop Rotation M. javanica - tobacco, Zimbabwe

11. Meloidogyne– genetic engineering

12. Systems Management – Columbia Root-knot Nematode M. chitwoodi

13. Morphological identification ofroot-knot nematodes is difficult due to limited differences and requires specialized expertise Dr. Valerie Williamson Perineal pattern for species ID: Requires adult female nematodes and skilled experts; interpretation is subjective.

14. Molecular identification: Isozymes PCR amplification of DNA Isozyme electrophoresis: Females are dissected from roots, squashed and run on a gel. After electrophoresis, gels are stained to determine migration of activity bands of Malate dehydrogenase and esterase then compared to a key with known species. Example of gel: Identification Key: MDH esterase Limitations: Requires live, healthy adult females (not J2s) Within species variability (see M.arenaria) Information available for only limited species and of limited phylogenetic value.

15. PCR amplification of DNA Species specific primers Mitochondrial DNA polymorphisms Isolation of nematode DNA Step 1: Put nematodes into a PCR tube (females, juveniles, eggs) Step 2. Add proteinase K solution to dissolve or disrupt the nematodes Step 3: Add specific primers to DNA and amplify DNA in PCR machine. Step 4: Fractionate the amplified DNA in a gel, take a picture and examine pattern to determine species. http://nematode.unl.edu/diagnostics.htm

16. Species-specific primers amplify DNA of a single species Several groups have developed species-specific primers to identify some important species of root-knot nematodes. M. incognita M. javanica 100bp Ladder ‘- ve’ Control VW4 VW5 Limitations: *Need new species-specific primers for each species to be identified. *Failure to get amplification does not necessarily rule out the target species.

17. Species specific primers, recent paper: “Multiplex PCR for the simultaneous identification and detection of Meloidogyne incognita, M. enterolobii, and M. javanica using DNA extracted directly from individual galls.” Hu et al. (2011) Phytopathology 101:1270-1277 PCR on 4 primer pairs on extracts from galls: rDNA Advantages: Single reaction using extract from gall can distinguish several species. Problems: To detect additional species need to add more primers.

18. PCR amplification of mitochondrial DNA polymorphism to distinguish species Themitochondrial genome is circular and present in high copy number in most cells. Uniparental inheritance. Contains rapidly evolving and conserved regions. The COII/16S region has been particularly useful for species ID. Mitochondrial genome of root-knot nematodes

19. Root-knot nematode species differ in the length of the COII/18S (lrRNA) intergenic region of their mitochondrial genome “PCR with mtDNA Primers can identify the species of single juveniles of RKN” Powers and Harris, J. Nematology 25:1-6 (1993)

20. Agaraose gel of PCR of PCR products with mtDNA Primers M. jav. M inc HinfI digestion Limitations: Getting the 1.7 kb fragments to amplify is tricky. This has limited the utilization of the assay. HinfI digestion of amplification products is required to distinguish Mi and Mj. DraI digestion and/or other amplifications are required to resolve other species.

21. Example of scheme to ID RKN using mtDNA PCR “Incorporating Molecular Identification of Meloidogyne spp. into a large-scale regional nematode survey” Powers et al., (2005) J. Nematology 37:226-235.

22. “Ultimately, rapid and inexpensive DNA barcoding will replace PCR/RFLP as a preferred diagnostic method.” Powers (2005) Molecular BarcodingIdentification based on sequence of a specific DNA fragment from an organism. rDNA regions: 18S, D-loop, ITSMitochondrial DNA regions Specific protein-coding genes (Blaxter Web site)

23. “Molecular detection and identification of root-knot nematodes in Africa” Williamson lab extracts DNA and determines species using species-specific primers and barcoding Preserve in ethanol and ship to California Preserve and ship to Jordan Danny Coyne et al collect egg masses from African samples Characterize, determine host range Chris Pagan Morphological ID L. Al Banna

24. Summary and points to think about The best molecular technique to use depends on goals. Standards for molecular diagnostics need to be better established for RKN. The parthenogenic RKN remain difficult to distinguish. They are very closely related. Are they really species? Molecular tools cannot distinguish host races of RKN species. DNA barcoding may be the best identification tool, even for routine diagnostics.