Materials and methods
In a two-year survey study (2009–2010), a total of 178 rhizosphere soil samples were collected from the six major sugar beet producing governorates in Syria, west Asia (Fig. 1). These governorates and the related total number of samples collected from each governorate in the two seasons were: Aleppo (26), Ar Raqqah (17), Dair az Zawr (33), Idlib (34), Hamah (41) and Homs (27 samples). Each governorate was visited twice (in the summer season of 2009 and autumn season of 2010). Soil samples were collected directly before and after plant harvest @ 10–15 samples/ha, using sampling augers at a depth of 10–30cm (Soomro et al., 1997). Sampling sites were selected in the fields following a zig-zag pattern. Samples of each ha were thoroughly mixed, and a 2kg composite sample was taken to represent this area. Root samples were also collected to be examined for the presence of white cysts and/or females on the roots. Soil samples were kept in plastic bags while, root samples were kept in paper bags. All samples were labeled, and sent in cool insulated chests to our laboratory in Damascus for nematode extraction, identification and community analysis.
In the laboratory, soil samples were air dried at room temperature, and sieved to exclude stones. Samples were then thoroughly mixed, and three 200g sub-samples were selected from each sample to extract cysts using a modified Fenwick can having an extra water supply near the SCH 527123 of the apparatus (Kort, 1960). Cysts on the second sieve (250μm) were transferred to a filter paper, and were examined and counted using a magnification lens. Root samples were also washed with a gentle stream of tap water on a 250μm sieve, and cysts on the sieves were collected and counted. For each sample, the total number of cysts per soil and roots were accumulated, and expressed as the number of cysts/100g soil. Cysts of several random samples were crushed in water, and the mean number of eggs+J2/100g soil was also calculated.
Community analysis of H. schachtii in the surveyed governorates included: absolute frequency (FO%), mean population density (PD) (Norton, 1978) and prominence value (PV) (Beals, 1960).
The specimens used for the morphological and morphometric studies were obtained from fresh materials. Brown cysts were extracted from the roots and soil at the end of each growing season. Eggs were obtained by squashing the cysts in water and incubated to obtain the newly hatched second-stage juveniles (J2). Other eggs without incubation were used for morphometric studies. The posterior ends (vulvar cones) of cysts which were collected from the roots and soil were cut-off in sterile water, and were cleaned in 30% H2O2 for 3–4min. Cones were then transferred to clean drops of sterile water for 2–3min, then to drops of ethyl alcohol to get-rid of air and water, and mounted in glycerin on glass slides (Hooper, 1970). Cones were finally examined under a compound microscope, and certain morphological and morphometric features were determined. Eggs and J2s were also mounted in TAF (7ml formalin 40%, 2ml triethanolamine, 91ml distilled water) on glass slides, and certain morphometric criteria (Tables 2 and 3) were measured using a compound microscope with a digital camera and a computer system. The obtained morphological and morphometric data of the six populations were compared to each other and referenced to related published data (Subbotin et al., 2010).
Based on Euclidian distance coefficients, a phonological dendrogram was constructed to evaluate the level of phenotypic variation among the six tested H. schachtii populations. Data were then run through multivariate principal component analysis using SPSS 22 (SPSS Inc., 2012) to analyze the relationships between the studied populations and to identify the variables that show the highest multiple correlations with these populations.
Materials and methods