Morphological and Morphometrical Analysis of Heterodera Spp

Saudi Journal of Biological Sciences (2015) xxx, xxx–xxx

King Saud University Saudi Journal of Biological Sciences

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE Morphological and morphometrical analysis of Heterodera spp. populations in Jordan

Hamzeh A. Laﬁ a,b,*, Luma Al-Banna b, Monther T. Sadder c, Hussein M. Migdadi d a Department of Plant Protection, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia b Department of Plant Protection, Faculty of Agriculture, The University of Jordan, Amman, Jordan c Department of Horticulture and Crop Science, Faculty of Agriculture, The University of Jordan, Amman, Jordan d Department of Plant Production, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia

Received 26 October 2014; revised 3 February 2015; accepted 7 February 2015

KEYWORDS Abstract Phenotypic diversity of five Jordanian populations of cyst nematodes, Heterodera spp. Discriminant analysis; collected from five regions from Jordan (Ar-Ramtha, Madaba, Dana, Al-Karak, and Jerash) was Interspecific variation; investigated. Soil samples were collected from one representative field in each region. Morpho- Mediterranean cereal cyst logical and morphometrical characteristics revealed that Heterodera latipons is dominated in cereal nematode; fields at Ar-Ramtha, Madaba, Dana and Al-Karak regions and Heterodera schachtii in Jerash. Morphometry; Cysts populations from all cereal fields had bifenestrate vulval cone and a strong underbridge. Sugar beet cyst nematode Wherever, cysts of the cabbage population had ambifenestrate vulval cone with long vulval slit. The bullae were absent in Ar-Ramtha, Madaba and Dana populations, but present in Al-Karak and Jerash. Based on 12 morphometrical characters, the first three functions in canonical discriminant analysis accounted 99.3% of the total variation. Distance from dorsal gland duct opening to stylet base, underbridge length, a = L/W (body length/midbody width) and length of hyaline tail tip had strong and significant contributions in the first function. While the second function was strongly influenced by length of hyaline tail, fenestral length, fenestral width and tail length. How- ever, the third canonical discriminate function was found to be influenced by stylet length, fenestral length, a = L/W (body length/midbody width) and underbridge width. The graphical representation of the distribution of the samples showed that the first canonical discriminant function clearly separated H. schachtii from Jerash from other populations. Whereas, H. latipons collected from

* Corresponding author at: Department of Plant Protection, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia. E-mail address: hlaﬁ@ksu.edu.sa (H.A. Laﬁ). Peer review under responsibility of King Saud University.

Production and hosting by Elsevier http://dx.doi.org/10.1016/j.sjbs.2015.02.007 1319-562X ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Madaba and Dana were clearly separated in the second function. The results indicated that differences at morphological and morphometrical levels revealed diverse populations of Heterodera spp. in Jordan. ª 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction spp. in Jordan are limited, this study was carried out to assess the interspecific variability among Heterodera spp. using mor- Cyst-forming nematodes (Heterodera spp.) are highly special- phological and morphometrical analysis. ized and economically important soil-borne parasites attacking numerous agricultural crops (Greco et al., 2002). The 2. Materials and methods Mediterranean cereal cyst nematode (MCCN), Heterodera latipons, occurs mainly in Mediterranean region but also in 2.1. Nematode populations Asia and Europe (Abidou et al., 2005; Smiley and Nicol, 2009). In Jordan, the MCCN (H. latipons) was first detected Five Jordanian populations of cyst nematodes, Heterodera spp. in two irrigated wheat fields at low population densities at were collected from major rainfed barley and wheat producing the Jordan Valley (Yousef and Jacob, 1994) and it was first regions (Ar-Ramtha, Madaba, Al-Karak and Dana) and from noted in a rainfed field of barley at Ar-Ramtha region in the irrigated cabbage field in Jerash (Fig. 1). Soil samples were col- Northern part of Jordan in 1996 (Abu Gharbieh and Al-Ban- lected from one representative field in each region. Soil samples na, unpublished data). Later on, barley fields’ surveys revealed were collected in July and August 2006–2008 after harvesting. that Mediterranean cereal cyst nematode was recovered from Each sample weighs about 250 g soil collected from the root Northern and Southern Mediterranean zones and Eastern zone system. Cysts were extracted using sieving and floatation Desert (Al Abed et al., 2004). Incidence and severity studies methods (Shephered, 1986). Eggs were obtained by crushing showed that these nematodes were dominant and severe in the extracted cysts. Whereas, the second-stage juveniles (J2s) Ar-Ramtha and Mafraq areas whereas they were moderate were obtained by subjecting eggs at 10 C to hatch in tap water in Al-Karak area and not found in newly introduced areas such as the south region at Al Mudawarra (Al Abed et al., 2004). Although, sugar beet is not cultivated in Jordan the sugar beet cyst nematode Heterodera schachtii Schmidt had been recorded on cabbage and cauliflower in Jordan (Saleh, 1987). Investigations on the nematode distribution had shown that it is restricted to a small area near the old Roman city of Jerash (Saleh and Qadri, 1989). Nowadays, the genus Heterodera contains more than 60 species. Extensive studies have revealed the presence of several distinct species of Heterodera infecting cereals and grasses within studied populations primarily identified as Heterodera avenae. Presently, the H. avenae complex is considered to con- tain: H. avenae, Heterodera arenaria, Heterodera aucklandica, Heterodera australis, Heterodera filipjevi, Heterodera mani, Heterodera pratensis, and Heterodera ustinovi (Wouts and Sturhan, 1995; Gabler et al., 2000; Sturhan and Krall, 2002; Subbotin et al., 2002). The species H. latipons is considered to form a separate species complex within the H. avenae group (Subbotin et al., 2003). On the other hand, H. schachtii belongs to the H. schachtii sensu stricto group, which also contains Heterodera betae, Heterodera ciceri, Heterodera daverti, Heterodera galeopsidis, Heterodera glycines, Heterodera lespedezae, Heterodera medicaginis, Heterodera rosii and Heterodera trifolii (Subbotin et al., 2000). Knowledge and characterization of genetic variability among and within the cyst nematode populations can provide predictive estimates of genetic variation among these populations and for efficient management and selection of appropriate control strategies (Ganguly and Rao, 2003). Several multivariate analysis methods are used to analyze the genetic variability and to investigate the differences between populations of a nematode species. Since published Figure 1 A map of Jordan showing the five regions where studies related to the morphological variability of Heterodera samples were collected from.

(Al Abed et al., 2009). Vulval cones were prepared as described Second stage juveniles are cylindrical in shape, with slightly by Hesling (1965). J2s were fixed in hot buffered formalin offset anterior part (Fig. 2A–D), and a tapering round tail (Humason, 1972). Permanent mounts of second juvenile stage tip (Fig. 2D). Stylet is strong; with basal knobs shallowly con- were prepared following Seinhorst (1959). cave anteriorly (Fig. 2B). However, the morphometrical data of J2 showed some variability (Table 1). Brown cysts of all 2.2. Morphometrical and morphological characterization populations were variable in size, and mostly lemon-shaped, with a protruding neck and vulvar cone structure (Fig. 2F). For each population, both qualitative and quantitative mor- Cyst wall is dark brown in color, and bearing a zig-zag pattern. phological characteristics of J2s and cysts were tabulated and New cysts mostly enveloped with a chalk-like bloom. Vulval used to identify the species following the original descriptions cones were either bifenestrate with short vulva slit (8.4– and the diagnostic keys of H. Latipons (Franklin, 1969) and H. 10.2 lm) (Fig. 2F) or ambifenestrate with long vulval slit schachtii (Schmidt, 1871). The obtained morphological and (42.7–45 lm) (Fig. 2E). Underbridge was found in the vulval morphometrical data of the five populations were compared cone structures of all the examined populations, while crowded to each other, and referenced to related published data. protruding bullae were present only in some populations Morphometrical data were run through discriminant multi- (Fig. 2G). variate analysis to investigate the separate ability of the five Species of Heterodera were differentiated using morpho- populations based on their morphometrical characters. Data logical and morphometrical features including; seven charac- were analyzed with reference H. latipons of Franklin (1969), ters of J2 (body length, midbody width, a, tail length, reference H. latipons Jordanian population (Madaba region) hyaline tail length, stylet length, and distance between dorsal of Al Abed (2004) and reference H. avenae of Handoo gland duct opening to stylet base), and five characters of the (2002). Canonical discriminant analysis (CDA) was performed brown cysts (underbridge length, underbridge width, fenestral using SPSS 15 (SPSS Inc., 2006). length, fenestral width, vulva slit length, and presence of bullae) (Handoo, 2002). Results based on these morphological and morphometrical 3. Results and discussion characters for target cyst species are given in Table 1. The results showed that the bullae were absent in H. latipons Morphological features of second-stage juveniles (J2) of the populations from Ar-Ramtha, Madaba and Dana regions, five Jordanian populations were almost similar (Fig. 2A). however, it was present regularly in H. latipons population

Figure 2 (A) The second stage Juvenile of Heterodera latipons. (B). Stylet-&-DGO of Heterodera latipons. (C) Excretory pore of Heterodera latipons. (D) Anus & Hyaline tail tip of Heterodera latipons. E. Female fenestra of Heterodera schachtii. F. Female underbridge & Vulval slit of Heterodera latipons. G. Bullae of Heterodera schachtii female. (Scale bars: A = 50 lm; B–G = 10 lm).

Table1 Diagnostic data of cysts and second-stage juveniles (J2s) of ﬁve Heterodera spp. Populations collected from Ar-Ramtha, Madaba, Dana, Karak, and Jerash regions, Jordan. Cysts data are average of ﬁve measurements each. J2s data are average of ten measurements each. Heterodera spp. populations Ar-Ramtha Madaba Dana Karak Jerash Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD (Min-Max) (Min-Max) (Min-Max) (Min-Max) (Min-Max) Cysts Underbridge length 98.3 ± 17.6 83.0 ± 37.0 100.0 ± 19.6 88.9 ± 7.7 104.0 ± 13.9 (72–107.2) (84–108) (84–132) (78.4–96) (96–120) Underbridge width 11.9 ± 1.7 15.0 ± 1.2 10.4 ± 3.3 11 ± 1.0 12 (10.7–14.3) (14.4–16.8) (7.2–16.8) (9.6–11.8) À12 Fenestral length 69.4 ± 17.3 54.0 ± 11.9 72.0 ± 17.0 66.2 ± 3.5 57.6 ± 3.4 (57.1–81.6) (45.6–62.4) (60–84) (63.7–68.6) (55.2–60) Fenestral width 24.7 ± 4.7 16.8 19.2 ± 2.0 20.8 ± 2.4 18.4 ± 1.4 (21.4–28.1) À16.8 (16.8–21.6) (19.2–23.5) (16.8–19.2) Semi fenestral length 17.9 18.0 ± 1.7 18.4 ± 1.4 17.4 ± 2.0 18.0 ± 1.7 À17.9 (16.8–19.2) (16.8–19.2) (15.7–19.6) (16.8–19.2) Vulval slit 9.0 ± 0.1 8.4 ± 1.7 10.0 ± 0.7 9.8 43.9 ± 1.1 (8.9–9) (7.2–9.6) (9.6–10.8) À9.8 (42.7–45) Bullae Absent Absent Absent Present Present (Scattered) J2s Body length (L) 482 ± 14.9 533.0 ± 49.6 484.2 ± 21.5 520.6 ± 22.5 488.3 ± 55.6 (470-518) (451-576) (456-504) (490-588) (412-600) Midbody width (W) 20.8 ± 1.6 22.0 ± 1.5 22.4 ± 0.8 21.1 ± 2.9 20.1 ± 1.0 (20–24) (20–23) (22–24) (17–26) (18–22) a = L/W 24.9 ± 1.6 25.0 ± 3.6 21.5 ± 0.7 25.4 ± 2.6 25.0 ± 3.0 (22.5–27.3) (19.6–28.8) (20.7–22.9) (22.5–30) (19.6–30) Distance from anterior end to median bulb 77.6 ± 4.8 80.2 ± 3.8 76.8 ± 2.0 76.0 ± 2.9 80.0 ± 6.1 (71.5–86.2) (73.5–82.3) (73.5–84) (72–79.6) (68.6–90.2) bm = L/distance from lip to median bulb 6.2 ± 0.3 6.6 ± 0.6 6.3 ± 0.2 7.0 ± 0.4 6.2 ± 0.4 (5.9–6.8) (5.9–7.2) (6–6.5) (6.4–7.5) (5.5–7) Tail length 52.6 ± 1.7 50.1 ± 2.7 62.8 ± 9.0 54.8 ± 5.2 64.1 ± 7.1 (49–53.9) (47–53.9) (48–72) (45.6–72) (52.9–72.5) c = L/tail length 9.1 ± 0.4 10.3 ± 1.1 8.4 ± 1.9 9.6 ± 0.7 7.5 ± 0.5 (8.9-9.8) (9.2-11.8) (7-10.5) (8.2-10.7) (6.5-8.4) Body width at anus 14.8 ± 0.3 14.7 16.4 ± 2.2 16.0 ± 3.1 14.6 ± 2.0 (14.7–15.7) À14.7 (14.4–19.6) (12–24) (11.8–16.7) Tail length/anal body width 3.6 ± 0.1 3.4 ± 0.2 4.1 ± 0.6 3.6 ± 0.6 4.3 ± 0.4 (3.3–3.7) (3.2–3.7) (3–4.5) (2.6–5.6) (3.5–4.7) Length of hyaline tail tip 31.3 ± 3.4 31.2 ± 2.8 26.0 ± 2.4 30.8 ± 4.8 33.8 ± 5.8 (27.4–37.6) (29.4–36) (24–28.8) (26.4–64.8) (27.4–49) Stylet length 24.6 ± 1.1 25.3 ± 1.2 24 24.5 ± 1.6 24.3 ± 2.0 (23.5–26.5) (24–26.4) À24 (21.6–26.4) (19.6–26.5) Hyaline tail tip/stylet length 1.3 ± 0.2 1.2 ± 0.1 1.1 ± 0.1 1.3 ± 0.3 1.6 ± 0.2 (1.1–1.6) (1.1–1.4) (1–1.2) (1.1–2.6) (1.3–1.8) Distance (dorsal gland duct opening to stylet base) 5.2 ± 1.2 5.5 ± 0.5 4.0 4.9 ± 0.7 5.5 ± 1.2 (4–7) (5–6) À4 (4–7) (4–7) Head width 9.9 ± 0.3 10.0 ± 0.7 9.6 9.7 ± 0.5 10.0 ± 0.4 (9.8–10.8) (9.8–12) À9.6 (8.8–12) (9.8–10.9) Head height 4.6 ± 0.5 4.9 4.4 4.4 ± 0.6 4.8 ± 0.3 (3.9–4.9) À4.9 À4.4 (3.6–4.9) (3.9–4.9) Distance (anterior end to start of overlapping) 114.2 ± 17.8 119.7 ± 3.6 98.4 ± 5.4 114.6 ± 11.3 102.9 ± 16.2 (98–132) (115.2–127.4) (96–108) (96–132) (88.2–125.4) Distance (anterior end to end of overlapping) 160.7 ± 16.6 176.4 ± 8.7 145.6 ± 19.5 160.0 ± 25.3 164.3 ± 15.8 (137.2–178.4) (166.6–186.2) (132–168) (117.6–192) (137.2–186.2) Distance from anterior end to excretory pore 107.1 ± 5.5 105.5 ± 4.2 104.6 ± 7.8 110.2 ± 6.0 107.9 ± 7.6 (100–112.8) (98–108) (98–117.6) (103.2–122.4) (98–121.5)

from Al-Karak and maral in H. schachtii population from Jer- ambifenestrate one in H. schachtii. In the other hand, varia- ash (Table 1). All Heterodera cysts populations had a strong tions were existing in some morphometrics of these three underbridge, with either bifenestrate vulval cone in H. latipons populations (Table 1). Such variations exist in vulval cone populations (Ar-Ramtha, Madaba, Dana and Al-Karak) or measurements of the cysts. For example, the fenestral length

Please cite this article in press as: Lafi, H.A. et al., Morphological and morphometrical analysis of Heterodera spp. populations in Jordan. Saudi Journal of Biological Sciences (2015), http://dx.doi.org/10.1016/j.sjbs.2015.02.007 Morphological and morphometrical analysis of Heterodera spp. populations 5 was the longest in Dana, and shortest in Madaba. There were The second function (accounted 1.1%) was found to be strong- also slight differences between the isolates in semi fenestral ly influenced by length of hyaline tail, fenestral length, fenes- length. The J2s of H. latipons recovered from cysts collected tral width and tail length. The third canonical discriminate from Madaba were the longest, furthermore, distance from function (accounted 0.9%) was found to be influenced by stylet anterior end to start of overlapping and distance from anterior length, fenestral length, a = L/W (body length/midbody end to end of overlapping were also the longest. Whereas, width) and underbridge width (Table 3). These results reveal Dana isolate J2s had the shortest length of hyaline tail tip the value of some characters that can be utilized for the separa- and distance from anterior end to start of overlapping tion of different species within Heterodera genus. Dawabah (Table 1). Both qualitative and quantitative characters of J2s et al. (2012) reported some other characters that can be utilized and vulval cones of the cyst nematode that recovered from for the separation of different populations within H. avenae the three barley geographic regions were agreed with the origi- and in determining the intraspecific variations between these nal H. latipons description of Franklin (1969) and Al Abed populations. These characters include cyst body dimensions, et al., 2004. Measurements of Al-Karak population also agreed J2 midbody width, J2 body width at the anus, J2 head height with H. latipons original description. Sometimes bullae are pre- and J2 ratios like total body length/tail length and tail length/- sent at the level of the underbridge (Franklin, 1969). The mor- body width at the anus. phological characteristics of the Jerash isolate agreed with the The graphical representation of the distribution of the five original H. schachtii description of Schmidt (1871). and referenced populations in the space of the two discriminate The canonical discriminant analysis, performed with the functions (Fig. 3) showed a clear separation of H. schachtii standardized canonical discriminant function coefficients for population (Jerash) and H. avenae reference of Handoo from the 12 morphometrical traits, showed that the first three func- the four H. latipons populations collected in this study together tions accounted 99.3% of the total variation (Table 2). with H. latipons references of Franklin and Al Abed. This The first canonical discriminate function which account separation was due to morphometrics differences in distance 97.3% was strongly influenced by distance from dorsal gland from dorsal gland duct opening to stylet base, underbridge duct opening to stylet base, underbridge length, a = L/W length, a = L/W (body length/midbody width), length of hya- (body length/midbody width) and length of hyaline tail tip. line tail tip, fenestral length, fenestral width and tail length. The cyst population distribution also agreed with the characteristics of bullae, underbridge and fenestra, the bullae were absent or slightly present in the H. latipons populations from Table 2 Eigen values and percent of variability explained by Ar-Ramtha, Madaba, Dana and Al-Karak regions and H. lati- each canonical discriminant function for the five Jordanian pons references of Franklin and Al Abed. Otherwise, it was populations of Heterodera spp. and the references populations present in H. schachtii population from Jerash (Table 1). With of Franklin, Al Abed and Handoo, based on 12 morphmetrical exception of the H. avenae reference of Handoo, all other Het- characters. erodera cysts populations had a strong underbridge, with Function Eigen value % of variance Cumulative% either a bifenestrate vulval cone in H. latipons populations (Ar-Ramtha, Madaba, Dana and Al-Karak) or an ambifenes- 1 837.781 97.3 97.3 trate one in H. schachtii. Abidou et al., 2005 showed specific 2 9.610 1.1 98.4 differentiation between H. latipons and both H. avenae and 3 7.850 0.9 99.3 4 4.937 0.6 99.9 H. filipjevi, which was based on strong and deep underbridge 5 0.361 0.0 99.9 without bullae. Furthermore, Yan and Smiley (2009) reported a good discrimination of H. filipjevi and H. avenae from H.

Table 3 Standardized canonical discriminant function coefﬁcients for the ﬁve Jordanian populations of Heterodera spp. and the references populations of Franklin, Al Abed and Handoo, based on 12 morphometrical characters. Trait Function 12345 Cyst Underbridge length 0.420 0.005 À0.557 À0.790 À0.795 Underbridge width 0.167 0.228 0.757 0.159 0.601 Fenestral length 0.121 0.438 1.131 0.459 0.438 Fenestral width À0.066 0.400 À0.245 0.741 0.700 Vulval slit À0.026 À0.557 À0.349 À0.268 À0.208 Second stage juvenile Body length À0.331 À0.265 À0.232 À0.010 À0.326 Midbody width À0.138 0.125 À0.050 0.011 0.594 a 0.383 0.313 0.883 0.509 À0.679 Tail length 0.000 0.345 0.157 À0.691 0.765 Length of hyaline tail tip 0.329 1.284 À0.944 0.318 À0.168 Stylet length À0.085 À1.262 1.180 0.490 0.156 Distance (dorsal gland duct opening to stylet base) 1.171 À0.026 À0.001 À0.023 À0.027

Figure 3 Canonical discriminant functions between the five Jordanian populations of Heterodera (1: H. latipons Ar-Ramtha, 2: H. latipons Madaba, 3: H. latipons Dana, 4: H. schachtii Jerash, 5: H. latipons Al-Karak) and the references populations (6: H. latipons Franklin, 7: H. latipons Al Abed-Madaba, 8: H. avenae Handoo), and the two main functions, performed on the basis of 12 morphological characters. latipons and H. schachtii based on PCR-RFLP molecular assay Dababat, A.A. (Eds.), Cereal Cyst Nematodes: Status, Research utilizing six endonucleases. and Outlook. CIMMYT, Ankara, Turkey, pp. 183–188. Our study showed that morphological characters and mor- Dawabah, A.A.M., Al-Hazmi, A.S., Al-Rehiayani, S., Abdel-Maw- phometrical analysis can distinctly separate species and good, A.L., Motawei, M.I., Al-Otayk, S., Sadder, M.T., Elgorban, A.M., Migdadi, H.M., Moustafa, K.A., Al-Doss, A.A., 2012. populations within the Heterodera genus. Furthermore, Morphological and molecular characterization of cereal cyst DNA observations, biological, ecological and biogeographical nematode (Heterodera avenae) populations from arid environ- studies can be done to identify at which taxonomic level ments. Aust. J. Crop Sci. 6, 970–979. populations of cyst-forming nematodes can be separated. Franklin, M.T., 1969. Heterodera latipons n. sp., a cereal cyst The creation of a catalog of morphological characters and nematode from the Mediterranean region. Nematologica 15, 535– morphometrical analysis of cyst forming nematode species 542. would facilitate the identication of species and population. Gabler, C., Sturhan, D., Subbotin, S.A., Rumpenhorst, H.J., 2000. Heterodera pratensis sp. n., a new cyst nematode of the H. avenae complex (nematoda: Heteroderidae). Russ. J. Nematol. 8, 115–126. Acknowledgments Ganguly, A.K., Rao, Uma., 2003. Application of molecular biology in nematology. In: Trivedi, P.C. (Ed.), Advances in Nematology. Authors wish to thank the Deanship of Scientific affairs, Scientific Publication, New Delhi, India, pp. 1–14. University of Jordan. Our extended thank to Agricultural Greco, N., Volvas, N., Troccoli, A., Inserra, R.N., 2002. The Materials Company (Miqdadi) represented in Mister Khalid Mediterranean cereal cyst nematode, Heterodera latipons: a menace Miqdadi. As well authors wish to thank College of Food to cool season cereals of the United States, nematology circular and Agricultural Sciences, Research Center and Deanship of 221. Florida department of agriculture and conservation services Scientific Research, King Saud University, Saudi Arabia for division of plant industry. supporting this work. Handoo, Z.A., 2002. A key and compendium to species of Heterodera avenae group (nematoda: Heteroderidae). J. Nematol. 34, 250–262. Hesling, J.J., 1965. Heterodera: morphology and identification. In: References Southey, J.F. (Ed.), . In: Plant Nematology. Technical Bulletin No. 7. Ministry of Agriculture, Fisheries and Food, London, UK, pp. Abidou, H., Valette, S., Gauthier, J.P., Rivoal, R., El-Ahmed, A., 103–130. Yahyaoui, A., 2005. Molecular polymorphism and morphometrics Humason, G.L., 1972. Animal Tissue Techniques, third ed. W.H. of species of the Heterodera avenae group in Syria and Turkey. J. Freeman and Company, San Francisco, CA, USA. Nematol. 37, 146–154. Saleh, H., 1987. Occurrence of the sugar beet cyst nematode, Al Abed, A., Al-Momany, A., Al Banna, L., 2004. Heterodera latipons Heterodera schachtii in Jordan. Arab Near East Plant Protect. on barley in Jordan. Phytopathol. Mediterr. 43, 311–317. Newslett. 4, 16. Al Abed, A., Al-Momany, A., Al Banna, L., 2009. Epidemiological Saleh, H., Qadri, A.N., 1989. Fungi associated with Heterodera studies on the Mediterranean cereal cyst nematode, Heterodera schachtii in Jordan II. Identity and incidence. Nematol. Mediterr. latipons, attacking barley in Jordan. In: Riley, I.T., Nicol, J.M., 17, 109–112.

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