Nematology 100 Lecture 15 Slides Meloidogyne

Nematology 100 Lecture 15 Slides

Meloidogyne – life cycle

Meloidogyne – life cycle

Giant Cells Root galling

Identification Meloidogyne…. incognita javanica hapla arenaria

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

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

Nematicides

Crop Rotation M. javanica - tobacco, Zimbabwe

Meloidogyne – genetic engineering

Systems Management – Columbia Root-knot Nematode M. chitwoodi

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

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 J 2 s) Within species variability (see M. arenaria) Information available for only limited species and of limited phylogenetic v

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

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 100 bp Ladder VW 4 VW 5 ‘- ve’ Control Limitations: *Need new species-specific primers for each species to be identified. *Failure to get amplification does not necessarily rule out the target species.

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: r. DNA Advantages: Single reaction using extract from gall can distinguish several species. Problems: To detect additional species need to add more primers.

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

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

Agaraose gel of PCR products with mt. DNA Primers M inc M. jav. Hinf. I digestion Limitations: Getting the 1. 7 kb fragments to amplify is tricky. This has limited the utilization of the assay. Hinf. I digestion of amplification products is required to distinguish Mi and Mj. Dra. I digestion and/or other amplifications are required to resolve other species.

Example of scheme to ID RKN using mt. DNA PCR “Incorporating Molecular Identification of Meloidogyne spp. into a large-scale regional nematode survey” Powers et al. , (2005) J. Nematology 37: 226235.

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

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

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.