Comparative d2/d3 LSU–rDNA sequence study of some Iranian Pratylenchus loosi populations

The D2/D3 LSU rDNA expansion segment of 13 isolates attaching tea shrubs roots in tea gardens that verified by morphological and morphometrical studies as Pratylenchus loosi Loof, 1960 from Guilan province, North of Iran, were amplified and sequenced. Amplification of the D2/D3 LSU rDNA expansion segments yielded one fragment at over all sequenced isolates as 787 bp in size. The DNA sequences were aligned using Clustral X1.81 together and with three sequences of similar region of P. loosi isolates available in Genbank database (Isolate T from Serilanka and Isolates N1 and N2 from Florida, USA). Also the genetic distance between sequences data were calculated through four methods as following; Uncorrected distance (UC), Jukes-Cantor (JC) Kimura distance (K) and Jin-Neigamma distance (JNG). For generating phyllogenetic trees both Neighbor-joining (NJ) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) were used. The results indicated that very short genetic distance exist among the Iranian isolates and between the Iranian isolates and isolate T from Serilanka whereas the Iranian isolates and isolate T were genetically distinct from isolates N1 and N2. The phyllogenetic analyses revealed relationship not only among Iranian isolates but also between Iranian isolates and isolate T.


INTRODUCTION
The tea root lesion nematode, Pratylenchus loosi Loof, 1960 is considered one of the most important and destructive pathogen attacking tea shrubs roots in tea gardens of North of Iran (Hajieghrari et al., 2005) as well as Serilanka and Japan (Sivapalan et al., 1986).It causes a sever decline of tea shrubs where it infects all commercial tea orchards.In Iran, this species is one of the quarantine pests and were found in rooted tea slips imported from Japan (Maafi, 1993).Nowadays, it has been distributed in some tea growth areas of north Iran (Hajieghrari et al., 2005).*Corresponding author.E-mail: bhajieghrari@uma.ac.ir.Tel : +989143186861.Fax : +984527463417 Abbreviations: LSU, Large subunit; SSU, Small subunit; ITS, Internal transcribed spacer; UC, Uncorrected distance; JC, Jukes-Cantor; K Kimura distance; JNG, Jin-Neigamma distance; NJ, Neighbor-joining; UPGMA, Unweighted Pair Group Method with Arithmetic Mean.
Identification of Pratylenchus species is essential for facility diagnosis of potential pest problems as well as improving prediction about pathogenicity and host range.In the other hand, species identification in the genus Pratylenchus is particularly difficult because of a little morphological diversity exhibition between species.Intraspecific variability of certain morphological characters among genus Pratylenchus used for classical distinguishing species is well known and has been adequately documented (Roman and Hirschmann, 1969;Tarjan and Frederick, 1978).
Biochemical methods such as soluble protein analysis and isozyme markers useful for inter-and intraspecific differentiation of plant parasitic nematodes (Hussay, 1979;Fox and Atkinson, 1986) as well as useful for diagnosis of Pratylenchus species (Payan and Dickson, 1990;Jaumot et al., 1997;Ibrahim et al., 1995;Andres et al., 2000) but these methods are time consuming for culturing of nematode and gathering a sufficiently abundant sample because a large number of individuals are needed for biochemical analyses.Direct examination of the genetic material especially DNA sequence comparison are being used to examine relationship among taxa, even among diverse taxa that cannot readily be compared with morphological analysis (Chaswell-Chen et al., 1993) and a powerful tool to analyze genetic variation (Waeyenberge et al., 2000;Williamson and Westerdahl, 1993).In recent years sequence analysis of coding and non-coding region of nuclear ribosomal DNA (rDNA) have became a popular tools for species and subspecies identification of plant parasitic nematode from many genera (Ferris et al., 1993;Caswell-Chen et al., 1993;Cherry et al., 1997;De Ley et al., 2002) and has been evaluated as a means to clarify phyllogenetic relationships among population of species of nematode (Kaplan et al., 2000) because of highly stability and exhibition a mosaic of conserved and diverse regions (Powers et al., 1997).Each repeat consist of transcribed units (small subunit or SSU or 18S; large subunit or LSU or 28S; 5.8S; internal and external transcribed spacers) and an external non-transcribed or intergenic spacer (Power et al., 1997;De Ley et al., 1999).The D 2 /D 3 expansion domains of the nuclear 28S rDNA subunit are sequence region that has been successfully used for diagnosing Pratylenchus species as well as other phytoparasitic nematodes (Mizuku et al., 1997;Handoo et al., 2001;Inserra et al., 2001).The D 2 /D 3 expansion segments of the 28S rDNA subunit (D 2 /D 3 LSU-rDNA) are the longest expansion fragments in the LSU and are the most rapidly evolving coding region of the rDNA genes (De Ley et al., 2002;Kaplan et al., 2000;Al Banna et al., 2004;Subbotin et al., 2005).It is demonstrated that it is most useful for characterizing species of Pratylenchus and their phyllogenetic relationships (Al-Bana et al., 1997;Mizuku et al., 1997;Duncan et al., 1999;Carta et al., 2001;De Hajieghrari et al. 2459Luca et al., 2004).The purpose of this study was to determine the nucleic acid sequence of D 2 /D 3 fragment of some Iranian isolates and to compare D 2 /D 3 LSU-rDNA homologues amplified for multiple P. loosi isolates available in the Genbank database.

MATERIAL AND METHODS
Original DNA sequence data were collected from 13 Iranian tea root lesion nematode isolates that verified by morphological and morphometrical studies as a P. loosi using three Pratylenchus genus diagnostic key (Café-filho and Huang, 1989;Frederick and Tarjan, 1989;Handoo and Goldon, 1989) and original description of P. loosi (Loof, 1960;Seinhorst, 1997).These P. loosi populations were isolated from different geographical location from tea shrubs infested roots of Guilan province, Iran (Table 1).
For DNA extraction, ten individuals from each isolates were handpicked and placed in 10 µl double distilled water on slide glass and cut them into two or more pieces.Nematode pieces in 10 µl double distilled water were transfer into a sterile eppendorf tube containing 8 µl lysis buffer which consist of 500 mM KCl, 100 mM Tris-Cl pH 8.3, 15 mM MgCl2 10 mM DTT, 4.5 % Tween 20 and 0.1% gelatin (Waeyenberge et al., 2000), then 2 µl of proteinase K (600 µl/ml) were added into each samples and were stored at -80° for 10 min for several days.After freezing, the tube were thawed and incubated for 1 h at 65° in water bath followed by 10 min at 95° for denaturing proteinase K before centrifugation for 5 min at 13000 rpm.The supernatant were transferred to PCR reagent mixture.Forward primer D2A 5'-ACA AGT ACC GTG AGG GAA AGT TG -3' and reverse primer D3B 5'-TCG GAA GGA ACC AGC TAC TA -3' (Kaplan et al., 2000;Courtright et al., 2000;Tenente et al., 2004) were used for amplification of the D2/D3 expansion region of the 28S RNA gene.All PCRs consisted of 50 µl reagent mixture containing; 37 µl dd H2O, 5 µl 10X reaction buffer, 1 µl 15 mM MgCl2, 1 µl dNTPs (10 mM), 0.3 µl D2A primers 0.3 µl D3B primers and 0.5 µl (2.5 unit) Taq-polymerase enzyme.The PCR reaction tubes were placed in a palm thermal cycler model GP001, Correbett research, Australia.Thermal cycling was done as follows: an initial denaturetion at 95° for 10 min, 40 amplification cycles (denaturizing at 95° for 30 s, annealing at 60° for 45 s and extension at 72° for 45 s) and a final step at 72° for 10 min.Amplified products were separated on 1% TAE-buffered agarose gels, stained with ethidium bromide and visualized with UV illumination, and then excised from agarose gels using the Qiaquick Gel Extraction Kit (Qiagen Benelux B.V., the Netherlands), cloned into the pGEM-T vector and transformed into JM 109 High Efficiency Competent Cells (Promega, Leiden, the Netherlands).Ten colonies of each population were isolated using blue/white selection and submitted to PCR with vector primers (pGEM-T forward primer 5'GTTTTCCCAGTCACGAC-3' and pGEM-T reverse primer 5'-CAGGAAACAGCTATGAC-3').Amplified products were purified using a Qiaquick PCR Purification Kit (Qiagen Benelux B.V., the Netherlands).DNA fragments were sequenced using the Big Dye Terminator V3.1 Cycle Sequencing Ready Reaction Kit and purified according to manufacturer's instructions (PE Applied Biosystems, Foster City, CA, USA).The resulting products were analyzed using an ABI Prism 310 Genetic analyzer.
The DNA sequences of all P. loosi populations were aligned using Clustal X1.81 (default options) together and with three sequences of P. loosi from Genbank (AF170439 isolate T from Serilanka, AF170438 isolate N2 and AF170437 isolate N1 from Florida, USA reported by Duncan et al., 1999).Also four types of genetic distance analyses were applied to analyze the alignment; uncorrected distance (UC), Jukes cantor (JC), Kimura distance (K) and Jin-Nei gamma distance (JNG).For generating phyllogenetic trees both Neighbor-joining (NJ) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) were used.

RESULTS
The 13 isolates showed that the qualitative characters of the populations such as number of lip annuli, spermatheca shape and tail shape agreed with original description of P. loosi Loof, 1960.The amplification of the D2/D3 LSU-rDNA expansion segments yielded one fragment at over all isolates as 787 bp in size (Figure 1).Control reaction without nematode DNA template never gives any PCR product.
Shown in Figure 2 are aligned sequences of the D 2 /D 3 expansion segment of LSU-rDNA for Iranian isolates of P. loosi compared with three isolates from the Genbank database (AF170439 isolate T, AF170438 isolate N 2 and AF170437 isolate N 1 ) by using Clustral X 1.81.There is some sequence variability among studied P. loosi isolates within this cluster.The comparisons of the aligned sequences demonstrate that very high sequence similarity was detected with Iranian isolates and isolate T from Serilanka.On the other hand sequence variability was observed within Iranian and American isolates (N 1 and N 2 ) where differences was found not only between Iranian and American isolates but also with in American isolates and isolate T. It is interesting to note that all populations from Iran were replaced by G at position 320 instead of T which is present in P. loosi, isolate T. On the other hand, nucleotide T is missing at position 311 in all Iranian isolates.Within this cluster, sequence divergence within the Iranian isolates ranged from complete identity between P1, P3, P5, P8, P9, P12 and P13 therefore indicating that in these isolates the D 2 /D 3 LSU rDNA expansion segment is completely homogeneous, until from 1 to 3 nucleotide differences between P2, P4, P6, P7, P10 and P11 were detected between some of the Iranian isolates of P. loosi (Table 2).
The genetic distance between sequences data were calculated through four methods as following; uncorrected distance (UC), Jukes-Cantor (JC) Kimura distance (K) and Jin-Neigamma distance (JNG).The results showed very short genetic distance among Iranian isolates and within Iranian isolates and isolate T from Serilanka (less than 0.53% distance).Also the longest distance is between N 1 and N 2 isolates with Iranian isolate and isolate T. Phyllogenetic analyses with Neighbor-Joinig (NJ) and Un-

Substituted nucleotide
Substituting nucleotide weighted Pair Group Method with Arithmetic Mean (UPGMA) yielded very similar topologies for the phyllogenetic relationship of P. loosi isolates by using calculated genetic distances (available on request).Therefore only one phyllogenetic tree are presented (Figure 3).Two mainly clades are particularly strongly supported, one of them includes the N 1 and N 2 isolates (supported with 1.82, 1.79, 1.92 and 1.90% distance calculated with JNG, K, JC and UC, respectively) and any one include Iranian isolates and isolate T (supported with 0% to 0.53% distance analyzed with each four methods) In this clade the most genetic distance based on D 2 /D 3 LSU rDNA were obtained between P2 and P11 isolates (0.53%).The genetic distance between these clades were calculated as 8.87, 8.35, 8.33 and 7.88% distances with JNG, K, JC and UC methods, respectively.
In order to clarify the taxonomic status of tea infesting nematode from Guilan province, we characterize the D 2 /D 3 expansion segment of large submit of nuclear DNA.Sequence dataset demonstrated a very low level of sequence diversity in Iranian isolates of P. loosi and isolate T from Serilanka strongly suggesting extensive genetic homogenization.These result provide evidence to support the proposal that Iranian isolate belong to P. loosi and phyllogenetically relationship exist between Iranian and isolate T from Serilanka; and despite the morphologically similarity of P. loosi populations described from Iran and American isolate, there are substantial D 2 /D 3 sequence difference between them, confirming Duncan et al. (1999) proposal that the American isolates as a undescribed species of Pratylenchus.
The presence of Iranian isolates and T isolate D 2 /D 3 LSU-rDNA nucleotide sequences can be considered as the molecular signature of P. loosi and can be used as an additional tool for close identification of this species from other geographical regions and among other P. loosi-like species.
Al- Banna et al. (1997Banna et al. ( , 2004) ) considered that the D3 expansion segment does not show intra specific variation in Pratylenchus sp.Our also study showed that the D 2 /D 3 LSU rDNA expansion segment is not a suitable region to use for intraspecific variation of P. loosi as well as some other plant parasitic nematodes (Subbotin et al., 2005) because the D 2 /D 3 28S rDNA expansion segment is the most rapidly evolving coding region of the rDNA and is flanked by highly conserved sequences and can distinguish taxa at species level.

Figure 2 .
Figure 2. Sequence alignment of D2/D3 LSU r DNA with Clustral X 1.81 for 13 isolate of P. loosi (P1-P13) in compared with same position of three isolates AF170439 isolate T, AF170438 isolate N2 and AF170437 isolate N1 from Genbank database reported by Duncan et al.(1999).

Figure 3 .
Figure 3. Phyllogenetic tree describing the relationships of Pratylenchus loosi isolates of this study in compared with three isolates AF170439 isolate T, AF170438 isolate N2 and AF170437 isolate N1 from Genbank database reported by Duncan et al. (1999) based on D2/D3 LSU rDNA sequences Jin-Neigamma distance (JNG) and generated by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analyses.

Table 1 .
Origin of the different Pratylenchus loosi isolates used in this study.