Characterization of cereal cyst nematodes in wheat using morphometrics, SCAR markers, RFLP, and rDNA-ITS sequence analyses

In this study, morphological and molecular characterizations of twenty-four Heterodera populations (cereal cyst nematodes, CCNs) collected from wheat production fields in Turkey were carried out. Light microscopy, species-specific markers, RFLP, and ITS sequencing were used to identify the nematode populations. The obtained CCN populations were identified as Heterodera avenae, H. filipjevi, and H. latipons according to the morphometric analysis, which was confirmed by the molecular techniques. The ITS region sequencing analysis confirmed the species identification, and phylogenetic analysis of this region grouped the populations with representative Heterodera populations from different origin countries deposited in GenBank. The simulation of four restriction enzymes, Alul, PstI, BsuRI (HaeIII), and Rsal, employed the ITS sequences of isolates to discriminate the Turkish Heterodera populations. ITS-RFLP patterns produced by endonuclease enzymes provided variations among Heterodera species. There was no intraspecific variation in populations of each Heterodera species in the ITS-RFLP analyses. The species-specific primers, AvenF-COI/AvenR-COI, HfF/HfR, and H-LatF/H-LatR, yielded 109 bp, 646 bp, and 204 bp products for H. avenae, H. filipjevi, and H. latipons populations, respectively. This is the first research to provide conclusive diagnostic tests for cyst nematode populations isolated from Turkey. These assays provide a sensitive, practical, and quick method for detecting Heterodera species and, therefore, have the potential to be utilized in the early identification of populations and monitoring of infestations without morphometric studies.