/subfamilies; molecular analyses based on the D2-D3 region, for example, provide support for the placement of Richtersia within the Selachinematidae (De Ley et al ., 2005). Morphology-based hypotheses about the phylogenetic relationships within the family Selachinematidae, however, remain largely untested using
about the definition and delimitation of pratylenchid genera. Recent molecular and phylogenetic studies using sequences of the D2-D3 expansion regions of 28S rRNA (Subbotin et al. , 2006 ; Trinh et al. , 2009 ) and 18S rRNA (Holterman et al. , 2006 ; Bert et al. , 2008 ) have indicated that
Nematology , 2005, Vol. 7(2), 285-293 Multiple displacement amplification (MDA) of total genomic DNA from Meloidogyne spp. and comparison to crude DNA extracts in PCR of ITS1, 28S D2-D3 rDNA and Hsp90 Andrea M. S KANTAR ∗ and Lynn K. C ARTA USDA-ARS Nematology Laboratory, 10300 Baltimore Avenue
; Nguyen et al ., 2004; Decraemer & Hunt, 2013; Ghaderi et al ., 2014, 2016).
In recent years, intra- and interspecific variation in sequences of D2-D3 expansion segments of 28S rRNA and ITS genes, as well as phylogeny, have been studied to examine the relationships among paratylenchids, especially in
the D2D3 expansion fragments of 28S rRNA gene, and tested alternative hypotheses of nematode evolution and classifications previously proposed based on morphology.
Materials and methods
Nematode isolates were collected from different plants and localities in Iran
Nematology , 2005, Vol. 7(6), 927-944 Phylogeny of Criconematina Siddiqi, 1980 (Nematoda:
Tylenchida) based on morphology and D2-D3 expansion segments of the 28S-rRNA gene
sequences with application of a secondary structure model Sergei A. S U B B OT I N 1 , 2
, ∗ , Nicola V OVLAS 3
Paratylenchus (Kashi et al ., 2009; Van den Berg et al ., 2014; Wang et al ., 2016a). The ribosomal internal transcribed spacer (ITS) 1 and 2, D2-D3 expansion segment of large subunit 28S rRNA and small subunit 18S rRNA gene served as good molecular markers to understand the phylogenetic relationships of
fragment of the 28S rDNA gene containing the D2-D3 region was amplified using primers D2F: 5′-CCTTAGTAACGGCGAGTGAAA-3′ (forward) and 536: 5′-CAGCTATCCTGAGGAAAC-3′ (reverse) (Nguyen, 2007). The PCR mixture of each sample consisted of 2.5 μ l of 10 × PCR buffer, 2.5 μ l dNTPs (2 mM), 1.25 μ l Taq
. similis based on the D2-D3 of rDNA from diseased plant. Our results indicate that the R. similis LAMP assay is sensitive and specific and has the potential to be developed into a user friendly field diagnostic technology.
Materials and methods
). All reported Pratylenchus species in Iran, except for P. loosi and P. pseudocoffeae Mizukubo, 1992, were identified based only on morphology and morphometrics. Populations of P. loosi isolated from tea plantations were studied and compared with other pratylenchids using the D2-D3 expansion