Study of Plant Parasitic Nematodes and Description of New Record (Rotylenchus alius) Associated with Barley (Hordium vulgare L.) in Khorasan Razavi Province, Northeast Iran

Majid Shahi Bajestani1, Esmat Mahdikhani Moghadam2*, Reza Aghnoum3 and Hamid Rohani2

1Plant Protection, (Nematology), Ferdowsi University of Mashhad, Iran; 2Plant Pathology, Department of Plant Protection (Nematology), Faculty of Agriculture, Ferdowsi University of Mashhad, Iran; 3Seed and Plant Improvement Research Department, Khorasan Razavi Agricultural and Natural Resources Research and Education Center, AREEO, Mashhad, Iran.

Received | October 17, 2020; Accepted | November 17, 2020; Published | December 23, 2020

*Correspondence | Esmat Mahdikhani Moghadam, Plant Pathology, Department of Plant Protection (Nematology), Faculty of Agriculture, Ferdowsi University of Mashhad, Iran; Email: [email protected]

Citation | Bajestani, M.S., Moghadam, E.M., Aghnoum, R. and Rohani, H., 2020. Study of plant parasitic nematodes and description of new record (Rotylenchus alius) associated with barley (Hordium vulgare L.) in Khorasan Razavi Province, Northeast Iran. Pakistan Journal of Nematology, 38(2): 171-178.

DOI | http://dx.doi.org/10.17582/journal.pjn/2020/38.2.171.178

Keywords | Meloidogyne arenaria, Plant parasitic nematode, Rotylenchus alius, Molecular identification

Introduction

Barley (Hordium vulgare L.) is one of the most important cereal crops in the world (Czembor and Czembor, 2007). Over 160 million tons of barley produced annually in the world from about 70 million hectares and it ranks fourth among main cereals in the world after wheat, corn, and rice (Akar et al., 1999). It is one of the most important agricultural crops in Iran. Barley harvest level in the country has been estimated about 3.2 million tons in the agricultural year, 2011-2012 from Khorasan Razavi Province, Iran (https://www.amar.org.ir/english/Iran-Statistical-Yearbook). One of the growth limiting factors in cereals are plant parasitic nematodes. Globally, many parasitic nematodes have so far been reported from barley viz., cereal cyst nematodes (Heterodera spp.), root-knot nematodes (Meloidogyne naasi), seed gall nematode (Anguina tritici), cereal dwarf nematode (Geocenamus brevidens), root lesion nematodes (Pratylenchus spp.) (Luc et al., 2005). This species was also found as a dominant species from barley and wheat fields of Iran (Haji-Hassani et al., 2008). In addition, Criconemella antipolitana was reported as a dominant species in Esfahan province of Iran from barley fields (Jamali et al., 2003). Heterodera avenae (Barooti and Alavi, 2003; Tanha et al., 2007; Ahmadi and Maafi, 2009), Anguina scopoli (Pakniyat and Sahandpoor, 2005), Heterodera latipons (Davis and Venette, 2004), Anguina agrostis were reported from Fars Province in barley fields (Tanha, 2005). Several investigations showed that the most prevalent species was Geocenamus brevidens via, Harat, Shahrebabak, Zobeideh, Modi-abad cities associated with barley fields in Iran (Barooti and Alavi, 2003; Pakniyat and Sahandpoor, 2005; Haji- Hassani et al., 2008; Ahmadi et al., 2014).

Although barley is one of the most important cereals in our country, there is no comprehensive study on the variation of plant parasitic nematodes in barley fields especially in Khorasan Razavi Province. The aim of present investigation was to identify plant parasitic nematodes in barley fields of Khorasan Razavi Province of Iran as the major producer in barley (https://www.amar.org.ir/english/Iran-Statistical-Yearbook).

 

Materials and Methods

Soil and root samples (120) were collected from the rhizospheres of barley along important barley fields in Khorasan Razavi Province, northeast Iran during 2015-2017and after transfer to the laboratory, kept in the refrigerator at 4 °C, until processed. Nematodes were extracted from soil samples by using the Jenkins (1964) method, killed and fixed according to De Grisse (1969). Genera and species were identified based on morphological and morphometerical characters. Perineal pattern of Meloidogyne arenaria (Neal, 1889) Chitwood, 1949, the adult females were mounted according to the method of Hartman and Sasser (1985), and the second instar larvae were identified using Jepson (1987) method. Measurements were taken with an ocular micrometer of “Olympus BH2” Model microscope. Drawings were made by drawing tube attached to the microscope and photographed from some of the best-preserved specimens by camera Olympus BX51. To validate the identification of Meloidogyne species, molecular analysis was performed based on the Ghaderi et al. (2014) method. A single live nematode from pure culture was picked out, then transferred to the small drop of AE buffer (10 mMTris-Cl, 0.5 mM EDTA; pH 9.0) on a clean slide and crushed using a clean slide cover. The suspension was collected by adding 20 µl AE buffer. DNA samples were stored at −20°C until using as PCR templates. The optimal thermo-cycling conditions were adjusted to be: An initial denaturation at 95 °C for 4 min; 35 cycles of amplification (94 °C for 30s; 52 °C for 40s; 72 °C for 80s); followed by a final extension at 72 °C for 10 min Table 1. PCR products were purified and then sequenced by Bioneer Company (South Korea). The phylogenetic tree was drawn by Mega 6 software.

 

Results and Discussion

During the present research, 18 species belong to nine genera were identified on the basis of morphological and molecular identification (Table 2). Among these species, Rotylenchus alius was reported for the first time from Iran. The nematode fauna identified during the study is listed in Table 2.

Rotylenchus alius Van den Berg, 1986 (Figure 1; Table 3)

During the present study, these species were encountered from barley fields of Bajestan county (Southern of Khorasan Razavi Province), as a new record from Iran.

 

 

Female: Body characteristically curved ventrally upon fixation. Lateral field with four incisures incompletely aerolated except at oesophageal region. Lip region conoid with fiveannules, labial framework well developed.

 

Table 1: Sequence of 18s rDNA primer for Meloidogyne arenaria.

Primer name	5ʹ-3ʹ	Gene region	Reference
Forward	GCT TGT CTC AAA GAT TAA GCC	18s	Blaxter in 1998
Reverse	CAT TCT TGG CAA ATG CTT TCG	18s	Blaxter in 1998

 

Table 2: Nematode species and collected areas in barley fields of Khorasan Razavi Provinces of Iran.

S.No.	Nematode species	Collection area
1	Aphelenchus avenae (Bastian, 1865)	Mashhad, Neyshabour, Golbahar, Torbat heydariye, Gonabad
2	Basiria gracilis (Thorne, 1949; Siddiqi, 1963)	Mashhad, Feizabad, Chenaran, Quchan
3	Basiria graminophila (Siddiqi, 1959)	Torbat heydariye, Fariman
4	Boleodorus thylactus (Thorne, 1941)	Mashhad, Torbat heydariye
5	Ditylenchus apus (Brzeski, 1991)	Neyshabour
6	Ditylenchus medians (Thorne and Malek, 1968) Fortuner and Maggenti, 1987	Quchan, Neyshabour, Kalat nadery
7	Geocenamus brevidens (Allen, 1955) Brzeski, 1991	Most areas
8	Geocenamus dobroticus (Budurova et al., 1996)	Bajestan, Kashmar, Golbahar, Quchan, Daregaz
9	Geocenamous graminicola (Kirjanova, 1951)	Mashhad, Torbat jam, Tayebad
10	Geocenamus rugosus (Siddiqi, 1963)	Mashhad, Khaf, Torbat jam and Heydariye
11	Geocenamus tartuensis (Krall, 1959; Brzeski, 1991)	Khaf
12	Geocenamus tenuidens (Thorne and Malek, 1968)	Torbat jam, Fariman
13	Meloidogyne arenaria (Chitwood, 1949)*	Neyshabour
14	Pratylenchus thornei (Sher and Allen, 1953)	Most areas
15	Pratylenchus neglectus (Filipjev and Stekhoven, 1941)	Most areas
16	Pratylenchus penetrans (Cobb, 1917) Filipjev and Stekhoven, 1941	Most areas
17	Rotylenchus alius (Van Den Berg, 1986)**	Bajestan county
18	Zygotylenchus guevarai (Tobar-Jiménez, 1963)	Mashhad, Tayebad, Torbat jam, Bajestan, Fiezabad

*First isolation in barley fields of Iran; **First report from Iran nematode fauna.

 

Stylet slender, stylet knobs flattened. Conus of stylet shorter than telenchium (47.05%). Median bulb ovate to long ovate, excretory pore opposite posterior part of oesophagus. Terminal bulb dorsally overlaps. Hemizonid 1.5 annules long and two annules anterior to excretory pore. Spermatheca round and filled with sperms. Phasmids situated three annules posterior to annus. Tail 117.5-25µm long having 11-13 annules, ventrally curved with a ventral point.

Male: Not found.

General description and morphometric measurements of this species (Table 3) closely fit to the original description of Rotylenchus alius by Van den Berg (1986). However, the (a) parameter was bigger (27.33-29 vs. 25.4-27.4).

Meloidogyne arenaria (Neal, 1889) Chitwood, 1949

Morphometric analyses: The magnitude of morphometric characters is influenced by several factors and can show significant differences not only between species but also among populations within a species (Doucet and Cagnolo, 1998; Carneiro et al., 2008; Skantar et al., 2008). Morphometrics of female, male and juveniles of under studied population were compared with original description of Chitwood (1949) and two other description from Iran (Table 4).

Female: Species of Meloidogyne can be identified based on female adult morphology, including head structures, perineal patterns, and stylet (Eisenback et al., 1980). The perineal pattern is a valuable morphological feature for identification of the species in genus Meloidogyne (Chitwood, 1949; Eisenback et al., 1980; Hirschmann, 1985). Three difference type of perineal patterns was seen in population under study. Adults with semi protuberance at the end of the body, pyriform, style 575-710 µm, lateral field existed but not clear, conus dorsally curved with range of 12-19 µm.

 

Table 3: Morphometric characters of the Iranian population of Rotylenchus alius and their comparison with type population (Van den Berg, 1986). All measurements are in μm and in the form: mean ± S.D. (range).

Characters	Khorasan Razavi region (Bajestan county)	Van Den Berg, 1986
	Female	Female
Number	8	3
L	881±84.95 (770-990)	600-900
A	28.22±0.68 (27.33-29)	25.4-27.4
B	5.65±0.32 (5.22-5.92)	5-6.8
b’	5.42±0.56 (4.66-5.91)	5.4-7.2
C	40.55±4.5 (32.8-44.61)	33.4-42.1
c’	1.15±0.12 (1-1.25)	1.1-1.4
V	59.80±1.66 (57.47-62.19)	59-60
v’	61.63±2.31 (58.82-65.4)	-
Stylet	28.83± 0.75 (28-30)	26.5-31.3
Pharynx length	142.58±5.96 (133-150)	-
S.E-pore	128.67±1.97 (125-130)	103-121
DGO	16±1.9 (13-18)	11.1-21.6
Mb	52.59±0.78 (51.47-53.25)	-
Head-vulva	526.67±45.02 (470-590)	-
Vulva-anus	333.33±41.64 (282.5-375)	-
Width at vulva	31.25±3.06 (27.5-35)	-
Tail length	22±3.39 (17.5-25)	16.9-25.7
Width at anus	19.08±1.24 (17.5-20)	-
G1	299.42±18.56 (285-325)	-
G2	235±27.88 (187.5-265)

 

Second stage larvae: Body length range 410-512 µm with13-16.5 µm body width, the average stylet length 13.33 µm (12-15), more or less similar length given by and near to studied population with Garcia and Sanchez-Puerta (2012) but it is greater than Chitwood (10 µm). Lateral field with four ridges. The rectum undilated as reported earlier (Esser et al., 1976; Garcia and Sanchez-Puerta, 2012). Tail tip pointed to rounded 50-64.5 µm in length. The alpha (body length/the maximum of body width) 32.42 µm and gamma (Body length/tail length) 8.2 µm.

Male: Body length 1340-1352 µm, body ventrally curved, the mean spicules length 31.75±1.06. Pharynx length 167.5- 170 µm. Overall, all measurements of this population were consistent with original description by Chitwood, 1949.

Populations of root-knot nematodes Meloidogyne arenaria species have been well documented, by Mahdikhani et al. (2003) and Sajjadi and Assemi (2016) from Iran, however, there is no information about the presence of root-knot nematodes in the barley fields of Iran. Morphometric results showed that all measurements were consistent with the original description given by Chitwood (1949) with few variations and differences, can be seen in the present population and earlier studies of Mahdikhani et al. (2003) and Sajjadi and Assemi (2016). These differences can be attributed to the intra species variations due to geography, host type, root structure and vegetation season of host.

 

 

Table 4: Morphometrics characters of Meloidogyne arenaria from Iranian population and their comparison with the type population (Chitwood, 1949) and other population from Iran. All measurements are in μm and in the form: mean ± S.D. (range).

 

Molecular analyses result

Molecular examination of 18S rDNA ribosomal domain in Meloidogyne arenaria verified the morphometric studies. Sequence of 18S ribosomal gene (Sequence of Iran) showed 99 percent resemblance to AB905316 from Japan. For phylogenetic tree, the evolutionary history inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 500 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the taxa analyzed (Saitou and Nei, 1987). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates collapsed. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. The analysis involved 30 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were 579 positions in the final dataset (Figure 2). Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013).

 

Novelty Statement

In this study, Rotylenchus alius was found as a new record from Iran and Basiria gracilis, Boleodorus thylactus, Ditylenchus apus and Meloidogyne arenaria were reported as new host record from barley fields of Iran.

 

Author’s Contribution

M. Shahi-Bajestani and E. Mahdikhani-Moghadam: Study concept and design.

M. Shahi-Bajestani: Drawing, analysis and interpretation of data.

M. Shahi-Bajestani: Drafting of the manuscript.

E. Mahdikhani-Moghadam, R. Aghnoum and H. Rohani: Critical revision of the manuscript for important intellectual content.

Conflict of interest

The authors have declared no conflict of interest.

 

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