Root-knot nematode species
identification using mitochondrial
DNA
What are root-knot nematodes and
why is it important to identify their
species?
Nematodes (roundworms) are tiny
animals
“Four out of five animals on earth are nematode worms” – E. O. Wilson
free living species, animal parasites, plant parasites
Plant parasitic nematodes
Estimated average annual yield loss of the world’s major crops due to
damage by plant parasitic nematodes is 12.3%
Monetary losses due to nematodes exceed $100 billion annually.
Almost all are obligate parasites that feed exclusively from the
cytoplasm of living plant cells.
Diverse group – plant parasitism evolved at least 3 times
Root-knot nematodes are generally regarded as the most
damaging group
Root-knot nematodes
Can infect most crops including tomato, melon,
prunus, cotton, alfalfa, grape, walnut, peanut,
eggplant, wheat, corn, strawberries…….
>70 species of Meloidogyne, but the most
widespread and damaging are: Meloidogyne
incognita, M. javanica, M. arenaria, M. hapla
M. javanica infected-tomato roots
M. incognita infected
carrot
DAMAGE (here on tomato)
Root knot nematode-infected plants appear less vigorous than healthy plants,
may be yellowed, are prone to wilt, and respond poorly to fertilizer.
NCSU R.G. Gardner
See UC ANR pub #3470
Management of Root-knot Nematodes
1. CHEMICAL FUMIGATION
1,3-DICHLOROPROPENE (Telone)
Liquid fumigant for the preplant treatment of soil against
plant-parasitic nematodes using drip irrigation systems.
Use of a tarp seal is mandatory for all applications of this
product.
METAM SODIUM (Vapam, Metam Sodium)
These fumigants are toxic and a prime source of volatile
organic compounds, which are a major air quality issue.
Fumigate only as a last resort when other
management strategies have not been successful
or are not available.
See UC ANR pub #3470
Management of Root-knot Nematodes
2. CULTURAL CONTROL
Some crop varieties are resistant to nematodes. Rotation
with resistant varieties and non-host crops can be almost
as effective as fumigation.
Cultural control requires accurate identification of the
root-knot nematode species present.
See UC ANR pub #3470
Nematode resistance in tomato is mediated by a
single dominant gene, Mi-1
Resistant (Mi)
Susceptible (mi)
The Mi-1 gene
*Was introduced into cultivated tomato from a wild tomato species
in the 1940s at UCD.
*Most processing tomato varieties in California have the Mi gene.
*Confers effective resistance against several species of
root-knot nematode (M. incognita, M. javanica, M. arenaria, but
not M. hapla or M. enterolobii).
Meloidogyne enterolobii
Meloidogyne enterolobii was originally
described from a population collected from
the pacara earpod tree in China in 1983.
In 2001 it was reported for the first time in
the continental USA in Florida.
It has also been reported in South America,
Carribean, Costa Rica, Mexico, Europe,
Africa.
M. enterolobii is now considered as one of
the most important root-knot nematode
species because of its ability to reproduce
on root-knot nematode-resistant tomato,
bell pepper and other economically
important crops.
-Wikipedia
Courtesy: Dr Sebastian Kiewnick, Agroscope
Changins-Wädenswil Research Station (CH)
The most commonly used methods for diagnostic
identification of root-knot nematodes were error-prone and
had limitations
Morphology/microscopy
isozymes
M. incognita
M. javanica
Species-specific primers
The most promising diagnostic was PCR
with mitochondrial DNA Primers
Powers and Harris, J. Nematology 25:1-6 (1993)
Root-knot nematode species differ
in the length and sequence of an
intergenic region of their
mitochondrial genome
This method can identify the
species of single juveniles of RKN
This approach was not broadly
adapted.
Why?
http://nematode.unl.edu/diagnostics.htm
Scheme to identify root-knot nematodes by
mitochondrial DNA PCR
Limitations:
*Too many steps
*Prone to failure
*Not all species can
be distinguished
Powers et al., (2005) J.
Nematology 37:226-235.
This UC-ANR project:
Root-knot nematode species
identification using mitochondrial DNA
First step was a meeting of collaborators and end users to establish project
plan details and desirable end products.
Principal investigators; extension nematologist; CDFA scientists;
collaborator from UC Riverside.
Reliable, robust, validated.
Simple, reproducible transferable.
Adaptable to different sample types
Open ended (can identify new species)
We focused on the variable region of the
mitochondrial genome
16.5 kb
We collected samples of DNA isolated from
characterized strains and species of Root-knot
Nematodes from different parts of the world
USA: M. incognita, M. javanica, M. arenaria, M. hapla (3), M. chitwoodi
France: M. arenaria
Costa Rica: M. enterolobii
Brazil (R. Carneiro): M. arenaria, M. enterolobii, M. incognita (3), M. javanica,
M. hapla, M. arabicida, M. ethiopica, M. hispanica, M. inornata, M.
izalcoensis, M. morocciensis, M. paranaensis (2), M. petunia, M. exigua
We determined the DNA sequence of
the variable region of 24 isolates
representing 15 different species
tRNAHis
AT region
Intergenic spacer
COII
l-rRNA
C2F2
1108
MORF
1
C2F2 /1108
(1640 bp)
MTHIS
MORF/MTHIS
(743 bp)
743
TRNAH
737
(H-790)
MRH106
M -1086
1294
(H-1182)
M-1163
TRNAH /MRH106
(557 bp)
Based on the sequence data we developed a
strategy to distinguish major RKN species
undigested
MnlI digests
HinfI digests
500 bp
M
Mi
Mj
VW6 VW4
Ma
F
Me
BR17
Mh
VW9
M
Mi Mj Ma
VW6 VW4 F
Me
Mh
BR17 VW9
M
Mi
Mj
VW6 VW4
Ma
F
Me Mh
BR17 VW9
Key to species identification based on Mitochondrial DNA
patterns of characterized strains
Pattern
F1
uncut
F1
Hinf1
F1
Mnl1
F2
uncut
A
556
445,112
340,140
214
arenaria, morocciensis
B
556
396,112
340, 217
743
incognita
B’
556
396,112
340, 217
636
incognita V1
C/H
556
445,112
417,140
743
arenaria V1
D
556
556
340, 140
743
javanica
G
556
445,112
340, 140
743
arenaria V2, ethiopica,
hispanica, inornata,
petunia
K
723
723
583, 140
NP
enterolobii
Species
The following species produced patterns that did not match any of the above:
M. arabicida, M. chitwoodi, M. exigua, M. hapla, M. izalcoensis, M. paranaensis
Our protocol can reliably identify the species of
major nematode species from a range of sample
types
males
juveniles
females
egg masses collected from African
field isolates, preserved in
ethanol, and shipped to California
Root pieces
picture from: Castagnone-Sereno, Heredity (2006)
Species identification of samples from Africa
country
# of
samples
A
B
(Mi)
C/H
D
(Mj)
G
K
(Me)
novel
Tanzania
15
1
3
0
11
0
0
0
Kenya
15
1
7
0
5
0
0
2
Nigeria
50
0
36
0
5
0
9
0
Benin
23
0
7
0
4
3
8
0
Hap B (M. incognita) and G (M. javanica) were most common.
Hap K (M. enterolobii) was found in W. Africa, but not E. Africa.
Two samples from Kenya showed novel pattern intermediate between M.
incognita and M. arenaria
Summary points
Our assay is robust, easy and can distinguish all the common RKN species found
in California.
Our assay easily detects Meloidogyne enterolobii.
Our assay can be applied to ethanol-preserved egg masses (or females).
DNA sequence of the amplified region can provide information on new,
uncommon or invasive species.
Nematode barcode
(courtesy Mark Blaxter)
These findings have been published in
Pagan et al. (2015) Phytopathology
Credits
Research team:
Valerie Williamson, UCD, PI
Steve Nadler, UCD, Co-PI
Chris Pagan, UCD, Junior Specialist
Collaborators:
Becky Westerdahl, UCD, Extension Nematologist
Ke Dong, Pest Diagnostics Branch, CDFA
Sergei Subbotin, Pest Diagnostics Branch, CDFA
Brad Hyman, UCR, Biology
Provided DNA samples: Regina Carneiro, Embrapa, BRAZIL; J. Starr, Texas; A.
Elling, Washington State; D. Humphries, Costa Rica; L. Zuretto, France
Samples from Africa: Danny Coyne, IITA, George Kariuki, Kenyatta Univ.