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 J2s)
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
100bp
Ladder
VW4 VW5
‘- 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:
rDNA
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/16S 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/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.
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.
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 37:226235.
“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.
rDNA regions: 18S, 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.