PREVALENCE AND SPECIES IDENTIFICATION OF ROOT-LESION NEMATODE IN POTATO Amro Mahran

PREVALENCE AND SPECIES IDENTIFICATION OF ROOT-LESION NEMATODE IN POTATO
SOILS OF MANITOBA, CANADA
Amro Mahran1,2, Mario Tenuta1, Fouad Daayf2, and Tracy Shinners-Carnelley3
1Department
of Soil Science, 2 Department of Plant Science, University of Manitoba, Winnipeg MB Canada R3T 2N2, 3Manitoba Agriculture, Food, and Rural Initiatives, Carman.
Contact Information: [email protected] or [email protected]
Introduction:
Results:
•
The root-lesion nematode, Pratylenchus spp. (Cobb) Chitwood and
Oteifa is a serious pest of potato (Solanum tuberosum) throughout most
potato producing areas of North America.
•
•
Yield reduction to potato varies with species of the root-lesion
nematode.
•
The species of Pratylenchus dominant in potato fields in the eastern
provinces of Canada (Prince Edward Island, New Brunswick, Quebec,
Ontario) is P. penetrans.
•
The root lesion-nematode was present in 6 of the 21 potato fields
sampled, with population ranging from 45-631 nematode/kg soil
(Table 1)
The morphometrics of the females showed that the species present is
P. neglectus (Table 2).
Amplification of the D3 expansion region yielded a single PCR
product with the length of approximately 340 bp. (Fig. 1).
•
• Sequences from the D3 expansion region were ambiguous, while for
the nested PCR products , 50-60 bp at the beginning and at the end of
each sequence were ambiguous as well.
In Manitoba, Canada’s second largest potato producing province,
surveys conducted in 1991, and 2002 to 2004 showed that Pratylenchus
spp. was present in 33% of a total of 135 fields sampled with
populations ranging from 4 to 5,300 nematodes/kg soil.
•
Sequences from the nested PCR product (Fig. 2) matched sequences
of P. neglectus partial 26S rRNA gene in the Genebank (Table 3).
•
None of the surveys identified the species of Pratylenchus present in
Manitoba.
•
Table 2 : Morphometrics of Pratylenchus spp. females found in the 6
potato fields in Manitoba in summer 2005.
Table 1 : Mean Population , frequency of occurrence per field, maximum
population and STDEV of means of the root-lesion nematode in 6 of the
21 potato fields in Manitoba collected in summer 2005.
Objectives:
Field
code
The objective of this study was to determine the prevalence and
species identification of Pratylenchus spp. in Manitoba potato fields
using morphometric characters and molecular techniques (PCR), which
will help to predict risks of crop damage based on pre-plant nematode
population.
Mean
Population /
Kg. soil
STDEV
Of
Field
Means
1
57
4
286
9
Character
Field
1
Field
4
Field
9
Field
13
Field
14
Field 20
P.
neglectus
a (ratio)
21.31
22.14
21.21
21.77
21.21
21.46
16.5 - 32.2
range1
Frequency of
Occurrence in
field sections
Maximum
Population / Kg.
soil in field
sections
b (ratio)
5.29
5.16
4.71
5.2
5.3
5.12
4.9 - 7.8
c (ratio)
21.06
21.4
22.62
21.77
22.62
21.11
13.8 - 26.8
L mm
0.44
0.42
0.42
0.43
0.42
0.43
0.32 - 0.59
113
1/4
226
S µm
13.5
12.5
13.75
13.33
11.87
12.33
15 - 19
201
4/4
509
V%
80.93
81.09
83.79
81.49
83.24
81.6
75.5 - 86.6
11
23
1/4
45
13
158
316
1/4
631
14
47
54
2/5
94
20
230
152
3/3
402
•
a (ratio) = Body Length/Greatest body width.
b (ratio) = Body Length/Distance from anterior end to junction of Esophagus.
c (ratio) = Body length/ Tail length (from the anus to the posterior end).
L = Mean length in millimeters.
S = Stylet length in micro meters.
V % = Vulva in relation to the head % of body length.
1
Materials and Methods:
1 Kb
1 - Survey and Morphometrics:
Field
13
Field
4
Field
20
Field
code
Primer
PCR
produc
t size
13
PNEG
301
4
PNEG
267
20
PNEG
258
300 bp
200 bp
100 bp
2 Fields
Fig. 1. Amplification of the D3 region
of using D3A forward primer and D3B
reverse primer.
2 Fields
1 Field
Fig. 2. Chromatograph of the sequence of the
nested PCR product for the field no. 13.
Matches
Score
Evalue
Identities
P. neglectus partial 26S rRNA
339
bits
2e-90
171/171
(100%)
P. neglectus partial 26S rRNA
345
bits
3e-92
174/174
(100%)
P. neglectus partial 26S rRNA
345
bits
3e-92
174/174
(100%)
gene, clone 24 (AJ545028.1)
gene, clone 24 (AJ545028.1)
gene, clone 24 (AJ545028.1)
Discussion:
16 Fields
1- In summer 2005, potato
fields (21) suspected to have
Early Dying symptoms of plants
were chosen for nematode
analysis.
Measurements from Roman and Hirschmann 1969.
Table. 3. Nested PCR product size, matches from the Genebank, score, E-values and identities
2 - Fields were divided into
three to five sections
according to the total area of
the field and soil samples were
collected from each section.
3 - Nematodes were
extracted using Cobb
decanting and sieving
method then the population
per kg. soil was determined.
4- Fifteen females per
field section were chosen
and characters used for
species differentiation
were measured.
2 - Molecular Diagnostics:
•
Approximately, one hundred nematodes were hand picked from the 3
field sections having the highest Pratylenchus population and their
DNA was extracted.
•
The D3 expansion region of the 26S rDNA was amplified following AlBanna et al. (1997) and its products were sequenced.
•
Nested PCR using species-specific primers following Al-Banna et al.
(2004) was performed, the PCR products were sequenced.
•
A Nucleotide-nucleotide BLAST search in the Genebank for the PCR
product sequences was performed to compare these sequences with
those present in the Genebank.
The results obtained from the morphometrics agreed with those from the
molecular diagnosis. Amplification by PCR using species-specific primers
resulted in bands only with the P. neglectus and not with other Pratylenchus
species specific primers used. The presence of P. neglectus rather than P.
penetrans is encouraging to producers in Manitoba. P. neglectus has a higher
threshold level of individuals in soil without causing disease. Additionally it also
does not interact synergistically with Verticillium spp. to the same degree as P.
penetrans to cause Early Dying Disease of Potato.
•
Sequencing the D3 region of a 100 nematodes showed ambiguous results,
which could indicate that there is a mixture of Pratylenchus species in our
fields. Of the 6 species specific primers used only that for P. neglectus gave
positive result indicating other species of Pratylenchus to be present that we
don’t have primers to select. Sequencing the D3 region from single nematodes
is needed to verify the presence of other species and the ratio of each species
present.
•
References:
•
Al-Banna, L., V. Williamson, and S. L. Gardner. 1997. Molecular Phylogenetics and Evolution 7:94-102.
•
Al-Banna, L., A. T. Ploeg, V. M. Williamson, and I. Kaloshian. 2004. Journal of Nematology 36(2):142-146.
•
Roman, J., and H. Hirschmann, 1969. Journal of Nematology, 1: 363-386.