مطالعه نماتد سیستی غلات Heterodera filipjevi (Madzhidov, 1981) Stelter, 1984 در استان‌های آذربایجان شرقی و اردبیل

Cereal cyst nematodes are among the most important deleterious pests of cereals in Iran and all around the world. Out of these nematodes, four species namely Heterodera avenae, Heterodera filipjevi, Heterodera latipons and Heterodera hordecalis have been reported from Iran. Among these species H. latipons and H. filipjevi are more widely distributed in cereal farms of Iran and the latter is the prevailing species. In order to identify the cereal cyst nematodes in East-Azarbaijan and Ardabil provinces, 200 soil samples were collected from rainfed and irrigated fields of the provinces in 2017. The cysts were extracted using Fenwick method. To identify the species of nematodes, microscopic slides from vulval cones of several cysts were prepared. Also, morphological and morphometrical characteristics of cysts and second-stage juveniles were evaluated. Overall, twenty H. filipjevi geographical populations were selected and PCR reactions conducted using two pairs of universal primers (AB28, TW81 & 18S, 26S) and a specific primer FILI-CoI. The DNA amplification profile was programmed as following: (95˚C for 4 min, 95˚C for 1 min, 55˚C for 1 min, 72˚C for 1 min and 72˚C for 7 min) by 35 cycles. PCR products were loaded on 1% agarose gel and separated by electrophoresis (104V, 45 min), stained with ethidium bromide, visualized on UV transilluminator and photographed. The results revealed that in all populations amplified by two universal primers a band with the length of 1000 bp was produced and in the case of FILI-CoI primer one fragment with approximately 245 bp long amplified. These results were accordant with the previous studies. Moreover, in order to confirm the species identity a fragment of DNA that was amplified by D2-D3 primer was sent for sequencing and the relevant phylogenetic tree drawn using Mr-Bayes Software. The sequencing results confirmed the species identity as H. filipjevi. Also, in order to determine the presence of genetic diversity among studied populations, PCR products of each population were digested with five restriction enzymes (AluI, PstI, BsuRI, TaqI and EcoRI). The digested DNA fragments were run on 1.5 % agarose gel, stained with ethidium bromide, visualized on gel documentation and photographed. RFLP profiles related to the ITS region of rDNA showed no differentiation among studied populations of this nematode