JOURNAL OF PLANT PROTECTION RESEARCH
Vol. 56, No. 4 (2016)
DOI: 10.1515/jppr-2016-0062
Occurrence and distribution of nematodes in rice fields in Guilan
province, Iran and the first record of Mylonchulus polonicus
(Stefanski, 1915) Cobb, 1917 (Nematoda: Mononchina)
Soheila Shahabi1, Ahmad Kheiri2*, Farshad Rakhshandehroo3, Salar Jamali4
1 Department
of Plant Pathology, College of Agriculture and Natural Resources, Science and Research Branch,
Islamic Azad University, P.O. Box 14515-775, Tehran, Iran
2 Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, P.O. Box 4111, Karaj,
31587-11167, Iran
3 Department of Plant Pathology, College of Agriculture and Natural Resources, Science and Research Branch,
Islamic Azad University, P.O. Box 14515-775, Tehran, Iran
4 Department of Plant Protection, College of Agriculture, Guilan University, P.O. Box 14115-336, Rasht, Iran
Received: June 27, 2016
Accepted: November 21, 2016
Abstract: The distribution of nematodes was studied in rice fields in Guilan province, Iran, from 2014 to 2016. Nematode biodiversity of 250 soil and root samples was examined. Thirty nematode species were identified morphologically, including plant parasites,
microbivores and mycetophagous and predator species. Molecular techniques were also used for further identification of three plant
parasitic species. Indicators of population were also estimated. Spiral nematodes (Helicotylenchus crenacauda Sher, 1966 and H. digitiformis Ivanova, 1967) and stunt nematode (Tylenchorhynchus agri) were the predominant parasitic species identified. Among other
species, three mononchid species were identified namely Mononchus aquaticus, Mylonchulus sigmaturus and M. polonicus. The species
M. polonicus was found and reported in Iran for the first time. The two plant parasitic species T. agri and Xiphinema index were reported
in association with rice in Iran for the first time. To evaluate the distribution and incidence of Aphelenchoides besseyi (rice white-tip
nematode) in different regions of Guilan province, a total of 255 fresh seed samples were collected/inspected, of which, about 40% of
them were infested with A. besseyi. Of the 16 studied counties, the highest percentage of infected seeds came from fields around the
city of Astara (69.2%) and the second highest infection was observed near the city of Anzali (60%).
Key words: Aphelenchoides besseyi, free-living nematodes, plant-parasitic nematodes, Tylenchorhynchus agri, Xiphinema index
Introduction
Rice (Oryza sativa L.) is one of the primary staple crops
internationally and is the second most important crop in
Iran (Mousanejad et al. 2009). It is susceptible to several
disease agents during its growth (Sayari et al. 2014). This
crop is cultivated in 15 provinces in Iran but at least 69%
of the rice production is found in two northern provinces,
Mazandaran and Guilan. The average rice production
of Guilan province, as the second largest rice producing
province, is about 750,000,000 kg. This amount accounts
for 40% of the country’s production, and 31% of the total
area of the province is under rice cultivation (Mohammadi et al. 2015). Rice is grown traditionally in these areas, using the most common method of rice cultivation
which involves transplanting manually. Currently there
are 230,000 ha of rice paddy fields in Guilan province
(Ashouri 2012). Many genera and species of plant parasitic nematodes are associated with rice, but only some of
these are known or suspected to cause yield loss (Bridge et
al. 2005). Aphelenchoides besseyi Christie, 1942, Hirschman*Corresponding address:
kheiri.ahmad@gmail.com
niella oryzae (Van Breda de Haan 1902) Luc & Goodey,
1963 and Heterodera elachista Ohshima, 1974 are the most
important species which have been found in rice-growing regions of Iran (Kheiri 1971; Minassian and Barooti
1997; Tanha Maafi et al. 2003). A few studies have been
done on the identification of plant parasitic nematodes in
Guilan province (Pedramfar et al. 2001; Soleymanzadeh et
al. 2016), but identification and distribution of nematodes
have not yet been studied in rice fields of Guilan province, even in some recently conducted studies (Aliramaji
et al. 2015; Pedram and Pourjam 2014; Pedram et al. 2015;
Roshan-Bakhsh et al. 2016). The aim of our study was to
perform a comprehensive survey on the identification
and distribution of nematodes across the rice fields of the
province. Since most soil-inhabiting nematode taxa can
be beneficial and may have roles in the decomposition
of organic matter they could be regarded as important
soil quality indicators (Yeates and Coleman 1982; Ingham et al. 1985; Spedding et al. 2004; Ravichandra 2013).
Therefore the free living forms of the collected species
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Occurrence and distribution of nematodes in rice fields in Guilan province, Iran and the first record of Mylonchulus polonicus…
of nematodes were also identified. The purposes of this
study were quantifying nematode biodiversity, preparing
information on population dynamics and investigating
the incidence and distribution of A. besseyi in rice growing areas in Guilan province. Three plant parasitic taxa
were also identified by using molecular techniques. The
species, M. polonicus (Stefanski, 1915) Cobb, 1917 was reported in Iran for the first time.
Materials and Methods
During 2014–2016, a total of 250 soil samples were taken
from the rhizosphere of rice at a depth of 5–25 cm, in different regions of rice fields in Guilan province, northern
Iran. The samples came from near almost all the villages of
the province under rice cultivation in 16 counties. The distribution map of the sampling areas is shown in Figure 1.
Each of the 250 samples was composed of 25–30 subsamples collected from 1–2 ha. To deal with variability
in the field, a systematic zigzag pattern was used for collecting sub-samples. Each sample was stored at 4°C
before extracting the nematodes. Nematodes were extracted from soil using the centrifugation sugar-flotation
technique (Jenkins 1964). Nematodes were also extracted
from collected roots (5 g root) by maceration in a waring
blender at 3,000 rpm for 2 min and extracted with a Bearmann funnel. After extraction, samples were evaluated
under a dissecting microscope at 40x magnification. All
nematode genera were counted with the aid of counting
slides and recorded before fixing. Nematode population
421
levels were determined and expressed as numbers per
500 cm3 soil and 5 g root.
The data obtained were subjected to community analysis to determine the ecological index according to Norton (1978) as follows: the absolute frequency = number of
samples containing a genus ×100/number of samples collected; relative frequency = frequency of a genus × 100/
sum of frequency of all genera; absolute density = average
population density (nematodes/100 cm3 soil); relative density = average number of individual genus × 100/average
number of all nematode genera; prominence value = absolute density × absolute frequency square for each certain
nematode genus (absolute frequency); relative prominence
= prominence value of a genus × 100/sum of prominence
values of all genera. After counting, nematode specimens
were killed, fixed, processed in dehydrated glycerin and
mounted on glass slides according to De Grisse (1969).
For the fresh seed samples survey, 255 fresh seed samples from 16 locations were collected. The number of seed
samples for each location is shown in Table 1. Nematodes
were extracted from 50 g of fresh seeds (out of 500 g),
randomly collected from each point, then they were extracted using the Coolen and D’Herde technique (1972).
The extracted nematodes were counted and fixed. Species
characterization in this survey was based on morphological and morphometric characters using available literature and their original descriptions (Hopper and Cairns
1959; Loof and Luc 1990; Nickle and Hooper 1991; Jairajpuri and Ahmad 1992; Hunt 1993; Handoo 2000; Siddiqi
2000; Andrássy 2009; Ahmad and Jairajpuri 2010).
Fig. 1. Distribution map of the samples taken from rice fields in different areas of Guilan province (with the capital city, Rasht),
northern Iran. Sample locations are shown with white squares; triangles indicate location of majorcounties for this study
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422
Journal of Plant Protection Research 56 (4), 2016
Table 1. Incidence of Aphelenchoides besseyi in fresh seed samples of rice in Guilan province
Sampling locations
Number of samples
Amlash
10
Number of seed samples Percent of seed samples
Average numbers of
infested with A. besseyi
infested with A. besseyi A. besseyi per 1,000 seeds
1
10.00
0–38*
Anzali
10
6
60.00
0–760**
Astane
15
18
53.33
0–605**
Astara
13
9
69.20
0–770**
Foman
16
9
56.30
0–515**
Lahijan
15
5
33.33
0–167*
Langrood
12
3
25.00
0–60*
Tavalesh
28
13
46.42
0–560**
Masal
11
2
18.18
0–90*
Rasht
11
6
54.54
0–598**
Rezvanshahr
10
1
10.00
0–98*
Roodbar
28
2
7.00
0–54*
Roodsar
26
9
34.61
0–22*
Shaft
14
5
35.71
0–258*
Siahkal
20
8
40.00
0–560**
Somesara
16
6
37.50
0–480*
*low infestations (0–500 nematodes per 1,000 seeds); **moderate infestations (500−1,000 nematodes per 1,000 seeds)
For molecular identification purposes, three selected
individuals of plant parasitic taxa (A. besseyi, H. oryzae
and H. elachista) were further identified by using molecular markers. Two genomic fragments, 28S rDNA
D2/D3 and internal transcribed spacer (ITS) (ITS1-5.8S-ITS2) regions, were sequenced. Each individual was
transferred to a small drop of AE buffer (10 mM Tris-Cl,
0.5 mM EDTA; pH 9.0, QIAGEN Inc., Valencia CA, USA)
on a clean slide and crushed using a cover slip. The suspension was collected by adding 20 μl AE buffer. DNA
samples were stored at –20°C until used for polymerase
chain reaction (PCR) amplification. Primers used for D2D3 amplification were forward primer D2A (5’-ACAAGTACCGTGAGGGAAAGT-3’) and reverse primer D3B
(5’-TGCGAAGGAACCAGCTACTA-3’) (Nunn 1992).
The ITS1 region was amplified using forward primer TW81 (5’-GTTTCCGTAGGTGAACCTGC-3’) and
reverse primer AB28 (5’-ATATGCTTAAGTTCAGC
GGGT-3’) as described in Vovlas et al. (2008). The information of PCR is according to Panahandeh et al. (2015).
The PCR products were sequenced in both directions using PCR primers with an ABI 3730XL sequencer (Bioneer
Corporation, South Korea). Newly obtained sequences
for these species were deposited in GenBank database
(accession numbers KX622689, KX622690 and KX622691
for 28S rDNA D2/D3 fragments of A. besseyi, Tylenchorhynchus agri and H. elachista respectively, and KX622692
for ITS of H. elachista).
Results
In this study, thirty species of soil inhabiting nematodes
were identified. Free-living nematodes were more diverse in the number of species (about 70%) compared
with plant parasitic nematodes (about 30%). Tripylla sp.
and Mononchus sp. (with 4.8% and 4.4% frequency) had
the highest populations among free-living nematodes. In
comparison to the populations of other groups, the order
Mononchida Jairajpuri, 1969 (Jairajpuri 1969) and other
beneficial predatory nematodes showed the lowest populations in the studied areas (Table 2).
The results of this survey indicated that the plantparasitic nematodes, Helicotylenchus spp. (33%) and
T. agri (31.4%), were found to be the predominant genera compared with others in the studied fields. Heterodera
elachista (1.79%) was the next most populous and Criconemella paragoodeyi Choi & Geraert, 1975 was the least
populous (0.1%). The rice white-tip nematode, A. besseyi,
was observed at low frequency in soil but had a moderate population in fresh seed samples compared with soil
in the studied areas. Among the 255 fresh seed samples,
113 seed samples (40.4%) were infested with A. besseyi
in the studied area. Of these samples, 33.3% had low infestations (0–500 nematodes per 1,000 seeds) and 67.98%
had moderate infestations (500–1,000 nematodes per
1,000 seeds). Among the studied locations, Astara and
Anzali had the largest percent of infected fresh seed
samples and also the highest level of infection. Roodsar
had the lowest number of infestation (0–22 nematodes
per 1,000 seeds). Fukano (1962) determined the economic
damage threshold density for A. besseyi to be 3,000 live
nematodes per 1,000 seeds, which provides a useful basis
for damage prediction (Bridge et al. 2005). Based on this
threshold, about 67% of the infected fresh seed samples
had more than 500 nematodes per 1,000 seeds, a threshold creating a need for management programs in order to
prevent greater crop losses.
In the present study M. polonicus was found for the
first time in Iran’s nematode fauna. Morphometric data
of the species are given in Table 3. It is characterized
by having a medium sized body, smooth cuticle (under
light microscopy), about 2.5–3.0 μm wide at midbody,
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423
Table 2. List of nematodes identified in rice fields in Guilan province of Iran and some of their ecological features calculated
according to Norton (1978). Root (5 g)/Soil (500 cm3)
Nematode species/character
Absolute
frequency
[%]
Relative
frequency
[%]
Absolute
density
Prominence
value
[%]
Aphelenchoides spp. (Aphelenchoides besseyi, A. bicaudatus)
0.29
0.29
1.60
5.30
Aporcelaimellus obtusicaudatus
0.95
0.95
5.30
5.35
Aquatides aquaticus
1.01
1.02
2.80
10.71
Chronogaster sp.
2.98
3.07
4.50
19.64
Criconemella paragoodeyi
0.11
0.11
2.00
1.78
Dorylaimoides elegans
Helicotylenchus crenacauda
0.47
0.47
8.00
1.78
24.22
31.00
11.94
60.71
Helicotylenchus digitiformis
8.90
9.70
11.50
23.21
Heterodera elachista
1.79
1.82
6.00
8.90
10.71
Ischiodorylaimus cognatus
1.19
1.20
3.30
Labronemella labiata
0.29
0.29
2.50
3.57
Laimydorus pseudostegnalis
1.49
1.51
3.10
14.20
Leptonchus sp.
0.23
0.23
4.00
1.78
Lindseyus costatus
1.20
1.21
2.60
14.20
Mesodorylaimus litoralis
1.55
1.57
2.60
17.85
Mononchus aquaticus
4.47
4.67
7.50
17.85
Mylonchulus spp. (M. polonicus & M. sigmaturus)
0.169
0.16
3.00
3.00
Oxydirus oxycephalus
0.40
0.47
4.00
3.57
Paractinolaimus decraemerae
0.35
0.35
3.00
5.35
Plectus spp. (P. aquatilis & P. parientinus)
3.20
3.31
11.00
8.90
Rhyssocolpus vinciguerrae
1.40
1.40
12.00
3.57
Tylenchidae (Basiria graminophila, Neopsilen chusmagnidens,
Irantylenchus clavidorus, Filenchus facultativus & F. polyhypnus)
1.61
1.64
2.45
19.64
31.40
45.00
11.19
83.90
1.19
1.20
1.25
28.57
Tylenchorhynchus agri
Thornenema baldum
Tobrilidae (Tobrilus, Eutobrilus)
3.80
3.95
9.20
12.50
Tripyla sp.
4.80
5.04
8.10
17.85
Xiphinema index
0.29
0.29
5.00
1.78
Table 3. Morphometric characters for female Mylonchulus polonicus (Stefanski, 1915) Cobb, 1917 (measurements are in μm except “L” in mm)
Population characters
Present study
Khan et al. 2002
Andrássy, 2009
n
4
7
–
L
1.91±0.4(1.87–1.95)
1.8±0.1(1.6–2.0)
1.6–2.5
1.82
28–38
33.10
a
28.1±1.4(27–29.2)
b
3.6±0.34(3.4–3.8)
c
19±1.4(18–20)
c’
2.1±0.2(2.0–2.3)
36.7±2.1(33–41)
3.8±0.2(3.6–4.3)
17.01±0.5(16–18)
2.8±0.1(2.6–3.0)
Mulvey, 1961
–
3.2–3.7
3.50
16–25
20.10
2.8–4.0
–
62.50
V
62.5±2.4(61–64)
63.5±1.3(61–66)
56–66
Buccal cavity diameter
20.5±1.8(19–22)
19–23
18–22
–
36.7±1.5(35.5–38)
32–36
33–36
–
Buccal cavity length
Pharynx length
505±22.1(490–520)
453–536
480–510
–
Nerve ring from ant. end
194±7.5(188–200)
133–145
–
–
G1
7.4±0.1(7.4–7.6)
–
–
–
G2
9.6±0.1(9.5–9.7)
–
–
–
Anterior genital gonad length
255±4.8(250–260)
–
–
–
Posterior genital gonad length
197.5±2.7(195–200)
–
–
–
Tail length
94±2.1(93–96)
107.5±6.3(95–117)
80–120
–
n – number of nematodes counted; L – nematode total length; a – body length/greatest body diameter; b – body length/distance
from anterior to esophagointestinal valve; c – body length/tail length; c’ – tail length/tail diameter at anus or cloaca; V – %distance
of vulva from anterior; G1 – overall length of the anterior ovary from vulva × 100/body length; G2 – overall length of the posterior
ovary from vulva × 100/body length
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424
Journal of Plant Protection Research 56 (4), 2016
Fig. 2. Female Mylonchulus polonicus: A – head; B – pharyngo-intestine junction; C – vulva region; D – tail; E – whole body
C-shaped open body upon fixation, amphids cup-like
with oval aperture, papillae slightly protruding, wide
buccal cavity with thick walls, a small dorsal claw like
tooth, directed forward and situated in the anterior
half of the buccal cavity, its apex at 71–74% of length of
buccal cavity length from its base, two smaller sub median teeth, the large dorsal tooth apex facing forward,
sub ventral small teeth and irregular rasp-like denticles
in 6–7 rows, vertical walls of prominent buccal cavity
and barrel-shaped buccal capsule with a tapering base
(Fig. 2). A nerve ring encircles the pharynx at the anterior end of the body, transverse vulva, pars refringens
vaginae appearing as two triangular-like sclerotized
pieces, advulval papillae absent, the female reproductive system didelphic-amphidelphic, small ovaries and
reflexed with a single row of oocytes, except at the tip of
the ovary, rectum about equal to the diameter of the anal
body, sphincter present, female tail rather long, ventrally
arcuate, bluntly-conoid and three caudal glands distinct
in tandem (Fig. 2D). Currently, eight species of the genus
Mylonchulus have been reported in Iran [M. brachyuris
(Bütschli, 1873) Cobb, 1917; M. cf. hawaiiensis (Cassidy,
1931) Goodey, 1951; M. kermaniensis Shokoohi, Mehrabi-Nasab and Mirzaei, 2013; M. lacustris (Cobb in Cobb
1915) Andrássy, 1958; M. minor (Cobb, 1893) Cobb, 1916;
M. nainitalensis Jairajpuri, 1970; M. paitensis Yeates, 1992;
M. sigmaturus Cobb, 1917] (Farahmand et al. 2009; Ghaderi et al. 2012; Koohkan et al. 2014).
Mylonchulus polonicus closely resembles M. lacustris
but there are some significant differences e.g. it has a larger body (vs. 1.1–1.5 mm) and longer tail (vs. c' = 1.5–2.0).
Andrássy (1992) synonymized M. polonicus with M. montanus described by Mulvey (1961). Our material is in
agreement with the description given by Mulvey (1961)
in both the measurements and morphological characters.
Also a slight difference in value (27–29.2 vs. 33.1) was observed. In comparison with the specimens studied by Andrássy (2009), the Iranian population has no remarkable
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Occurrence and distribution of nematodes in rice fields in Guilan province, Iran and the first record of Mylonchulus polonicus…
425
Astara
Tavalesh
Rezvanshahr
Masal
Anzali
Somesara
Foman
Rasht
Shaft
Astane
Lahijan
Siahkal
Langrood
Roodbar
Amlash
Roodsar
Table 4. Distribution of nematodes identified in rice fields in Guilan province, Iran (with location of sampling). Root (5 g)/Soil
(500 cm3)
Aphelenchoides (A. besseyi & A. bicaudatus )
+
+
+
0
+
+
+
+
+
0
0
+
+
0
+
0
Aporcelaimellus obtusicaudatus
+
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Aquatides aquaticus
+
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
Chronogaster sp.
+
+
0
0
0
0
+
0
0
++
0
0
0
+
+
+
Criconemella paragoodeyi
+
0
0
0
+
0
+
0
0
+
0
+
0
0
0
0
0
+
0
0
0
Nematode species
Dorylaimoides elegans
Helicotylenchus spp.
+++ +++
0
0
0
++
+
++
+++ +++ +++
0
0
0
0
0
0
0
0
++
+
++
++
+
+
+
+
Heterodera elachista
0
0
0
0
0
+
+
0
0
0
0
0
0
0
0
0
Ischiodorylaimus cognatus
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Labronemella labiata
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
0
Laimydorus pseudostegnalis
+
++
+
+
+
+
+
++
+
+
+
+
+
+
+
+
Leptonchus sp.
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Lindseyus costatus
+
+
0
0
0
0
+
0
0
+
0
0
0
0
0
+
Mesodorylaimus litoralis
+
+
+
0
0
+
+
+
+
++
+
+
0
0
0
0
Mononchus aquaticus
++
++
+
+
+
++
++
++
+
+
+
+
+
+
+
+
Mylonchulus spp. (M. polonicus & M. sigmaturus)
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Oxydirus oxycephalus
0
0
0
0
0
0
0
+
0
0
0
0
0
0
0
0
Paractinolaimus decramerae
0
+
0
0
0
0
0
0
0
+
0
0
0
0
0
0
Plectus spp. (P. aquatilis & P. parientinus)
+
+
0
0
+
0
+
+
0
+
0
0
+
+
+
0
Rhyssocolpus vinciguerrae
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Thornenema baldum
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Tobrilidae (Tobrilus, Eutobrilus)
++
+
+
+
+
++
+
0
0
+
+
0
+
0
+
0
+
+
+
+
+
Tripyla sp.
Tylenchorhynchus agri
+++ +++
+
+
+
++
+
++
+++ +++ +++
+
++
+
+
+
+
+
0
++
+
++
++
++
+
++
+
Tylenchidae
(Basiria, Neopsilenchus, Irantylenchus, Filenchus)
0
+
0
0
0
+
+
+
0
0
0
0
0
0
0
0
Xiphinema index
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0 = not recorded; + = present in survey; ++ = common; +++ = widespread. Relative density > 30% = widespread, relative density >
10–30% = common and relative density < 10% = present in survey
differences in morphology and morphometric data. Compared with the data given by Khan and Araki (2002), our
population has slightly smaller ranges for the index “a”
(27–29.2 vs. 33–41) and tail length (93–96 vs. 95–117 μm).
The species, only found in one soil sample (Table 4),
was collected from a rice field next to the Caspian Sea in
Tavalesh county (southwest of the Caspian Sea, 140 km
northwest of Rasht. GPS coordinates: 37°21'54" North,
50°5'34" East).
The current study presents new data on the diversity and distribution of nematode fauna in rice paddies
in Guilan province, Iran. Most of the common species of
plant parasitic nematodes observed in this study were
similar to those reported in a previous survey in rice
fields in Guilan by Pedramfar et al. (2001). However, the
plant parasitic nematodes mentioned below which have
previously been identified in rice include: A. besseyi,
A. bicaudatus (Imamura, 1931) Filipjev & Schuurmans
Stekhoven, 1941; Basiria graminophila Siddiq, 1959; Criconemella paragoodeyi, Filenchus facultative (Szczygiel,
1970) Brzeski, 1982; F. polyhypnus (Steiner & Albin, 1946)
Meyl, 1961; Helichotylenchus crenacauda, H. digitiformis,
Heterodera oryzae Luc & Berdon, 1961 and Tylenchorhynchus annulatus (Cassidy, 1930) Golden, 1971. The two species Heterodera elachista and T. agri were reported for the
first time as associates with rice in Guilan province. The
28S rDNA D2/D3 sequence of the Iranian population of
T. agri, found and sequenced in the present study (morphometric data and comparison with other populations
are given in Table 5, with accession number KX622690)
was identical to the sequence of the same genomic fragment of the species with accession number KJ461559 sequenced by Handoo et al. (2014), and only one nucleotide
difference was observed in the overlapping part of two
sequences, which is a common intra-species variation.
T. agri has already been reported in Iran from the rhizospheres of pomegranate, date and sour lemon (Mojtahedi
et al. 1983; Nowruzi and Barooti 2001).
The species H. elachista has previously been reported
from rice fields in Mazandaran province by Tanha Maafi
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426
Journal of Plant Protection Research 56 (4), 2016
Table 5. Morphometric characters of Tylenchorhynchus agri Ferris, 1963 population (measurements are in μm except ”L” in mm)
Present study
Population
characters
female
Ferris, 1963
male
female
male
female
male
n
12
10
–
10
10
L
737±100(637–810)
695±5(682–700)
700(660–770)
660(540–720)
700(660–770)
660(540–720)
a
29.54±2.5(26–32.3)
31.5±1.5(29.4–33)
30(28–33)
33(29–36)
30(28–33)
33(29–36)
5.4±1.5(4.8–6.8)
5.1±1.5(4.0–6.5)
5.1(4.7–5.5)
5.1(4.3–5.6)
5.1(4.7–5.5)
5.1(4.3–5.6)
18(15–21)
16(15–18)
18(15–21)
16(15–18)
b
c
4
Nickle, 1991
14.3±2.2(13.6–16.1) 14.0±1.0(13.2–14.5)
c’
2.8±0.5(2.1–3.4)
3.0±0.6(2.7–3.7)
2.6
–
–
–
V/T
55.2±3.5(52.8–58)
53±2.5(49–55.3)
56(55–58)
52(48–56)
56(55–56)
52(48–56)
Stylet length
20.8±2.5(19–23)
19.0±1.2(18.6–20.0)
21(20–23)
20(19.5–21)
21(20–23)
20(19.5–21.0)
DGO
24±3(21–26)
–
23–24
–
–
–
–
–
–
–
MB
48.3±4.5(44–52.3)
46.9±3.2(46–51.6)
Excretory pore
96±4.5(92–101)
98.5±6.5(93.2–106)
–
–
92(88–98)
88–105
Oesophagus length
122±10(114–132)
119±10(110–129)
–
–
116(108–128)
111–128
48.5±5.5(42–54)
45.3±4.2(41.8–49.8)
–
–
35(32–39)
–
–
4
–
–
–
–
22(18–26)
–
–
–
–
24(22–25)
–
24(22–25)
–
13.4(12.5–14.4)
–
13.4(12.5–14.4)
Tail
Head annuli
Tail annuli
4
23±2.5(21–25)
Spicules
–
Gubernaculum
–
23±3.5(21–26)
11.5±1.5(10.9–12.4)
n – number of nematodes counted; L – nematode total length; a – body length/greatest body diameter; b – body length/
distance from anterior to esophagointestinal valve; c – body length/tail length; c’ – tail length/tail diameter at anus or cloaca;
V/T – distance of vulva from anterior/length of male gonad relative to body length; DGO – dorsal espohageal gland orific;
MB – distance from anterior to median bulb relative to length of oesophagus
Table 6. Morphometric data for female Xiphinema index Thorne and Allen, 1950 (measurements are in μm except ”L” in mm)
Population
characters
Present study
Thorne and
Allen, 1950
Heyns, 1971
Andrassy, 2009
Hunt, 1993
n
9
–
10
–
–
L
2.9±0.3(2.5–3.2)
3.4
3.25(2.9–3.6)
2.9–3.4
3.1(2.91–3.28)
a
56±3.5(52.3–60.4)
58
57(54–61)
54–66
62(58–66)
b
6.6±0.8(5.7–7.5)
7.6
6.6(6.2–8)
6.0–7.6
6.8(6.0–7.7)
c
73.4±10.8(65–84)
76
88(72–98)
75–95
84(75–93)
c’
1.1±0.1(1–1.2)
–
0.9(0.7–1.1)
0.8–1.3
1.12(1.0–1.3)
V
38.4±2.5(37–41)
38
41(40–42)
38–42
39.4(38–40)
Odontostyle
128±7.5(121–134)
–
129(123–134)
120–130
126(119–129)
Odontophore
73.5±5.5(69–78.2)
–
78(74–81)
70–80
70(63–78)
195±11.5(185–209)
–
206(197–215)
–
196(190–206)
Stylet total length
Guiding ring from anterior end
107.8±7.1(102–115)
–
–
–
–
Diam. at mid-body
50.4±4.0(46–54)
–
–
–
–
Anterior genital tract length
318.5±63(252.5–380)
–
–
–
–
Posterior genital tract length
400±45(355–460)
–
–
–
–
Tail
37.3±2.5(36–39)
–
–
–
–
Hyaline portion of tail
18.5±2.5(17–21)
–
–
–
–
n – number of nematodes counted; L – nematode total length; a – body length/greatest body diameter; b – body length/
distance from anterior to esophagointestinal valve; c – body length/tail length; c’ – tail length/tail diameter at anus or cloaca;
V – %distance of vulva from anterior
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Occurrence and distribution of nematodes in rice fields in Guilan province, Iran and the first record of Mylonchulus polonicus…
et al. (2003). In this study the collected population of this
species was sequenced for its 28S rDAN D2/D3 and ITS
fragments (accession numbers KX622691 and KX622692,
respectively). The ITS sequence of our population was
identical to the corresponding sequences of the species deposited into the GenBank (accession numbers
HM560778 and AF498391) (identity = 100%). A 98–99%
identity was observed while comparing with several
other populations of the species accessible in the database. The 28S rDNAD2/D3 sequence of the presently sequenced population of A. besseyi with accession number
KX622689 was also identical to the same genomic fragment of several populations of the species available in the
GenBank (99–100% identity was observed).
Based on morphological and morphometric characters, Xiphinema index Thorne and Allen, 1950 was identified for the first time in association with rice in Iran. Morphometric data and a comparison with other populations
of the species are presented in Table 6.
Discussion
Xiphinema spp. are large nematodes and economically
important pests of several crops. These nematodes not
only directly damage their hosts by direct feeding on
root cells, but also can transmit plant pathogenic viruses.
Several species of Xiphinema Cobb, 1913 have so far been
recorded from the rhizosphere of rice namely X. cavenessi
Luc, 1973 in Côte d’Ivoire; X. insigne Loos, 1949 in India;
X. nigeriense Luc, 1961; and X. orbum Siddiqi, 1963 in India,
and X. oryzae Bos and Loof, 1985 in Nigeria, X. seredouense
Luc, 1975 in Guinea but none of these species are known
to be harmful (Bridge et al. 2005). Furthermore, Xiphinema
ifacolum Luc, 1961 can cause significant yield loss in rice
when it interacts synergistically with other root pathogens. Even alone at low population densities, it can reduce rice yield (Lamberti et al. 1987). Because of the presence of X. index in a number of the surveyed rice fields
in the present study, its tentative effects on rice crop loss
should be further investigated.
Helicotylenchus spp. were the most prevalent plantparasitic nematodes in the studied area. The economic
importance of these nematodes on rice crop loss has not
been well studied. Helicotylenchus spp. are often considered as mild pathogens (Norton 1974) and can increase
susceptibility to other plant pathogenic fungi in order to
gain access to the host root cells.
Tylenchorhynchus spp. are common nematodes in rice
fields throughout the world, and their damage is accentuated by an aggregation phenomenon known as ‘swarming’ (Joshi and Hollis 1976). Tylenchorhynchus annulatus
has the widest distribution and is the main species found
in irrigated rice fields. Other less commonly reported
species of the genus in rice fields are T. annulatus; T. brassicae Siddiqi, 1961; T. clarus Allen, 1955; T. clavicaudatus
Seinhorst, 1963; T. claytoni Cobb, 1913; T. crassicaudatus
Siddiqi, Mukherjee & Dasgupta, 1982; T. elegans Siddiqi, 1961; T. karnalensis Saha, Singh, Lal & Kaushal, 2002
T. mashoodi Siddiqi & Basir, 1959 and T. nudus Allen, 1955
(Khan et al. 1990; Haidar et al. 1996; Khan and Shaukat
2000). However, none of these species have been shown
427
to cause remarkable damages to rice in the field (Bridge et
al. 2005). The high population density of T. agri recovered
in this study emphasizes the need for further studies on
its potential pathogenicity and crop losses on rice.
Heterodera elachista has an important role in yield
loss in rice and despite low levels of infection with this
species, management programs against it should be applied. A. besseyi causes white=tip disease in rice and it has
worldwide distribution in rice fields. It causes up to 60%
crop losses in various infested regions (Bridge et al. 2005).
The nematode was first reported in Iran by Kheiri (1971)
and is widely distributed in the rice growing areas in
northern parts of the country including Guilan province
(Talachian and Akhiani 1976; Elahinia and Mahdavian
1998; Jamali et al. 2006).
Based on our results, about 40.4% of collected
fresh seed samples of rice were infested with A. besseyi
and about 27.1% of fresh seed samples had more than
500 nematodes per 1,000 seeds. Due to the economic importance of rice in Guilan province and the devastating
effects of this nematode species on susceptible cultivars,
management methods should be considered to reduce
nematode population levels in these areas.
Free-living nematodes have very important and beneficial roles in the decomposition of organic material and
the recycling of nutrients in soil. They serve as important
environmental indicators, and the predator forms could
potentially be bio-control agents against parasitic forms.
In the present survey, based on morphological and morphometric characteristics, a number of free living nematodes were identified and their distribution and population density were studied. The identified species were
as follows: Aporcelaimellus obtusicaudatus (Bastian, 1865)
Altherr, 1968; Aquatides aquaticus (Thorne, 1930) Heyns,
1968; Chronogaster sp.; Dorylaimoides elegans Thorne &
Swanger, 1936; Eutobrilus sp.; Ischiodorylaimus cognatus Andrássy, 1983; Labronemella labiate Andrássy, 1985;
Laimydorus pseudostagnalis (Micoletzky, 1927) Siddiqi,
1969; Leptonchus sp.; Lindseyus costatus Ferris & Ferris,
1973; Mesodorylaimus litoralis Loof, 1969; Mononchus aquaticus Coetzee, 1968; Mylonchulus polonicus; Mylonchulus
sigmaturus; Oxydirus oxycephalus (De Man, 1885) Thorne,
1939; Paractinolaimus decraemerae Pedram, Niknam, Vinciguerra, Ye & Robbins, 2010; Plectus aquatilis Andrassy,
1985; Plectus parientinus Bastian, 1865; Rhyssocolpus vinciguerrae Pedram, Porurjam, Robbins, Ye & Pena-Santigo,
2011; Thornenema baldum (Thorne, 1939) Andrássy, 1959;
Tobrilus sp. and Tripyla sp.
All of the abovementioned free living nematodes, except L. costacus, were first found in the rhizosphere of rice
in Iran by Coomans and Kheiri in 1986. Earlier, they had
been reported in the rhizosphere of other plants from the
country (Ghaderi et al. 2012).
Acknowledgements
The authors are grateful to Prof. Nathaniel A. Mitkowski
(Professor in the Science Department at the University of
Rhode Island, Kingston, RI) for his helpful comments and
valuable suggestions in revising the manuscript.
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428
Journal of Plant Protection Research 56 (4), 2016
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