Nematology 100 Lecture 15 Slides
Jan 19, 2016
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.