Nematology 100

Nematology 100

Lecture 15 Slides

Meloidogyne – life cycle

Meloidogyne – life cycle

Giant Cells

Root galling

Identification

Meloidogyne….incognita javanicahapla arenaria

M. incognita- sugarbeet

M. incognita- cantaloupe

M. hapla- lettuce

M. incognita- soybean

(resistant – leftsusceptible – right)

M. incognita- cotton

Symptoms

Damage

M. chitwoodi - potato M. incognita - sweetpotato

Meloidogyne sp.- carrot

Nematicides

Crop Rotation

M. javanica- tobacco, Zimbabwe

Meloidogyne – genetic engineering

Systems Management – Columbia Root-knot Nematode

M. chitwoodi

Perineal pattern for species ID: Requires adult female nematodes and skilled experts; interpretation is subjective.

Morphological identification of root-knot nematodes is difficult due to limited differences and requires specialized expertise Dr. Valerie Williamson

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.

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.

Identification Key:Example of gel:

Molecular identification:IsozymesPCR amplification of DNA

MDH

esterase

PCR amplification of DNA

Species specific primers

Mitochondrial DNA polymorphisms

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

Isolation of nematode DNA

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.

Limitations:*Need new species-specific primers for each species to be identified.*Failure to get amplification does not necessarily rule out the target species.

100bp

Ladder VW4 VW5 ‘- ve’ Control

M. javanica

M. incognita

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

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

rDNA

PCR on 4 primer pairs on extracts from galls:

Mitochondrial genome of root-knot nematodes

The mitochondrial 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.

PCR amplification of mitochondrial DNA polymorphism to distinguish species

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)

Agaraose gel of PCR of PCR products with mtDNA Primers

M inc M. jav.

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.

HinfI digestion

“Incorporating Molecular Identification of Meloidogyne spp. into a large-scale regional nematode survey”

Powers et al., (2005) J. Nematology 37:226-235.

Example of scheme to

ID RKN using

mtDNA PCR

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)

“Ultimately, rapid and inexpensive DNA barcoding will replace PCR/RFLP as a preferred diagnostic method.”

Powers (2005)

“Molecular detection and identification of root-knot nematodes in Africa”

Danny Coyne et al collect egg masses from African samplesCharacterize, determine host range

Preserve in ethanol and ship to California

Williamson lab extracts DNA and determines species using species-specific primers and barcoding

Morphological IDL. Al Banna

Preserve and ship to Jordan

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.