Morphometric and Molecular Characterization of Isolates of the Root Lesion Nematode, Pratylenchus loosi Infecting Tea in Sri Lanka

The Root-lesion nematode, Pratylenchus loosi Loof inhabits all tea growing regions of Sri Lanka depicting a variety of symptoms and damage severities to affected tea. Though much has been researched on biology and control measures, isolate characterization has not been attempted. Hence, male and female morphometric variations and molecular characterization of P. loosi populations isolated from different agro-ecological regions in Sri Lanka viz. PL1 (Cecilton, Balangoda), PL2 (Delmar, Halgranoya), PL3 (Hapugastenna, Ratnapura) PL4 (Mahadowa, Passara), PL5 (Nawalapitiya) and PL6 (Richiland, Deniyaya) were studied. Female morphometrics of P. loosi showed intraspecific variation and clustered in four groups in Principal Component Analysis where PL1 and PL5 were closely-related while PL3 and PL6 clustered separately with the exception of PL2 and PL4. Sequence analysis of the D2/D3 expansion segments of the 28S rDNA gene of the P. loosi populations revealed that PL3 and PL6 were closelyrelated while PL1, PL4 and PL5 were relatively distant. Sequences of the ITS region of rDNA placed PL3 and PL6 in a single clade. The isolates PL1, PL2, PL4 and PL5 were relatively distantly-related and PL2 and PL4 were relatively distant from the other populations. Molecular characterization further validated the relatedness of PL1 and PL5, PL3 and PL6 and, PL2 and PL4 obtained from the morphometric data. The divergence in P. loosi populations shown in this study supports evidence of intra specific isolates resulting different symptomological expressions and thus implies need of specific management strategies in managing nematode infestations in tea plantations in Sri Lanka.

Hence, male and female morphometric variations and molecular characterization of P. loosi populations isolated from different agro-ecological regions in Sri Lanka viz. PL1 (Cecilton, Balangoda), PL2 (Delmar, Halgranoya), PL3 (Hapugastenna, Ratnapura) PL4 (Mahadowa, Passara), PL5 (Nawalapitiya) and PL6 (Richiland, Deniyaya) were studied. Female morphometrics of P. loosi showed intraspecific variation and clustered in four groups in Principal Component Analysis where PL1 and PL5 were closely-related while PL3 and PL6 clustered separately with the exception of PL2 and PL4. Sequence analysis of the D2/D3 expansion segments of the 28S rDNA gene of the P. loosi populations revealed that PL3 and PL6 were closelyrelated while PL1, PL4 and PL5 were relatively distant. Sequences of the ITS region of rDNA placed PL3 and PL6 in a single clade. The isolates PL1, PL2, PL4 and PL5 were relatively distantly-related and PL2 and PL4 were relatively distant from the other populations. Molecular characterization further validated the relatedness of PL1 and PL5, PL3 and PL6 and, PL2 and PL4 obtained from the morphometric data. The divergence in P. loosi populations shown in this study supports evidence of intra specific isolates resulting different symptomological expressions and thus implies need of specific management strategies in managing nematode infestations in tea plantations in Sri Lanka. , is the major plantation crop grown in Sri Lanka. Plant parasitic nematodes are considered one of the key pests limiting establishment, growth and productivity of tea. Pratylenchus loosi is the most predominant nematode species causing economic damage to tea cultivation in different agro climatic regions in Sri Lanka (Gnanapragasam and Mohotti, 2005). Mohotti (1998 and indicated that there may be several P. loosi isolates after studying populations of the nematodes from Sri Lanka, Japan, Iran and Florida. Mizukubo (1998) introduced the P. loosi species complex which appears to include phylogenetically distinct isolates based on four Japanese isolates and a Sri Lankan isolate. However, possible intraspecific variations of different populations present in different localities in Sri Lanka have not been studied. In the past decades, several molecular tools have been deployed to identify and compare nematodes, at the species level and between populations of the same species. For species of the genus Pratylenchus, a remarkable difference exists in the sizes of the ITS region of rRNA of the nematodes; so far, the difference between the smallest and the largest amplified ITS regions is about 350 bases (Castillo and Vovlas, 2007). In addition, intraspecific variation in the ITS region has been observed in this genus, particularly within populations of Pratylenchus coffeae and Pratylenchus vulnus (Orui, 1996;Uehara et al., 1998;Waeyenberge et al., 2000;Mizukubo et al., 2003, Begum et al., 2019. Sequences of the D2/D3 expansion segment of the 28S rDNA gene has also been used to distinguish between Pratylenchus spp and, has been shown to be an important and strong molecular tool for nematode diagnostics (Handoo et al., 2001;Subbotin et al., 2006;de la Pena et al., 2007).
This study was, therefore, undertaken with the objective of using morphometrics and molecular data to characterize P. loosi populations collected from six geographic locations in Sri Lanka causing varying symptomolgical expressions and damage to tea.

Selection of sampling sites
Sampling sites to collect P. loosi populations to study the morphometrics and molecular data were selected by reviewing history of records maintained by the Nematology Laboratory of the Tea Research Institute, Sri Lanka. Accordingly, six locations representing different elevations with higher P. loosi populations were recognized and sampled in this study (Table 1).

Morphometric characterization of P. Loosi populations
The P. loosi specimens were collected from the sampling sites described in Table 1 and the slides were prepared as per the standard protocols in Seinhorst (1959). For the study, 20 female and 12 male specimens of P. loosi were used for each site. Specimens were observed under OPTIKA B 500 microscope, and all morphometric measurements were done with Optika Vision Pro plus (Version 2.7) software. The measurements taken were as follows;

Data analysis
The data were statistically analysed using SAS 9.1. Principle Component Analysis was used to make an objective assessment of the relative similarity among the populations using fourteen (14) morphometric characters for the females.

Molecular characterization of P. loosi populations
The P. loosi populations used for molecular study were collected from the locations described in Table 1. The molecular work (e.g. PCRs, cloning, sequencing, sequence analyses and DNA homology work) of this study was carried out at the State Agricultural Biotechnology Centre (SABC), Murdoch University, Perth, Australia. DNA extraction from single nematodes was done using phenol chloroform method and methods described by Marek et al. (2014) and Wayenberg et al. (2000).

PCR of the D2/D3 rDNA
Genomic DNA of single adult P. loosi nematodes were used for PCRs. Primers D2-1 (5'-GACCCGTCTTGAAACACGGA-3') and D3-2 (5'-CGGAAGGAACCAGCTACTA-3') were used for amplification of the D2/D3 expansion region of the 28S RNA gene. All PCRs were made up to 20 µl with nuclease-free water and contained 10.0 µl Go Taq Green master mix (Promega Corp, Australia), 1.0 µl each of the primers D2-1 and D3-2 and 2.0 µl of DNA template. Thermal cycling was done using an initial denaturation at 95 °C for 5 min, followed by 50 amplification cycles (denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min) and a final step at 72 °C for 10 min. Amplified products were observed on 1% TAE-buffered agarose gels, stained with SyBr Safe DNA gel stain (Invitrogen, USA) and visualized with a Trans illuminator. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, USA) according to the manufacturer's protocol.
The PCR conditions and the thermal cycling profiles were the same as those used for amplification of the D2/D3 rDNA PCR above. Ligation of the purified PCR products to the pGEM-T cloning vector and transformation of E. coli JM109 with the ligated products were as per the manufacturer's protocol (Promega Corp). This was followed by confirmation of successful ligation by PCR of the colonies, isolation of plasmid DNA, restriction digestion of recombinant plasmids, and sequencing of plasmid DNA using standard molecular biology protocols (Sambrook et al., 1989).

Sequence alignments and analyses
The software, Finch TV 1.4.0 (Geospiza) was used to visualize and edit erroneous sequences. Clustal omega and MEGA 7.0.26 (Kumar et al., 2016) were used to align the edited sequences and to develop phylogenetic trees respectively.

Phylogenetic analyses
Phylogenetic trees were constructed using sequences of the D2/D3 28S rDNA expansion segment and ITS region separately using the Neighbour-Joining and Maximum Parsimony (MP) algorithms. Bootstrap analyses with 1000 replicates were performed to assess the degree of support for each clade on the trees. Sequences of similar regions of the respective rDNA for P. coffeae (GenBank Accession numbers HQ688681.1 and KF974721.1) were used as out-group taxon for the phylogenetic analyses.

RESULTS AND DISCUSSION Morphometric characterization of P. loosi populations
The specimens of males and females conformed closely to the earlier descriptions of P. loosi, but there were noteworthy variations (Tables 2 and 3). Table 2 presents the data compared with morphometrics reported earlier for P. loosi. The data showed that the measurements of the populations studied were in the ranges of previously reported P. loosi and confirmed their taxonomic identity. Although some may be significant, the deviations in the morphometric characters among the populations were acceptable for Pratylenchus species. The least variable characters were considered in the comparison of different P. loosi populations. The results of this study showed evidence of existence of a significant intraspecific variation in the morphometric characters of P. loosi found in Sri Lanka.

Body length (L)
The results showed that 'L' of the study population PL 4 is significantly higher (552µm) than that of the other populations. However, the mean 'L' of each of the populations and for all the populations were in the range of body lengths reported by Loof (1960), Duncan et al. (1999), Inserra et al. (2001) and Wu et al. (2002). Also, the average body length of the populations was similar to that reported for P. loosi identified from pasture grasses in central Florida (Inserra et al. 1996).

Distance from vulva to anterior end/ body Length * 100% -(V%)
The distance from the vulva to the anterior end/Body Length * 100% (V%) is the one of the most important morphometric parameters used for characterizing Pratylenchus species. Several studies have demonstrated that V ratio is less modified by biotic or abiotic factors and the ratio is a reliable diagnostic character for the genus.
This was confirmed in our study which indicated a very small coefficient of variation (2.96%) as presented in Table 2. The PL 4 population had a lower V ratio (77.05%) while the PL 3 population had a significantly higher V ratio (84.01%) than the other populations.

Maximum body width
The populations from the PL 2, PL 3, PL 5 and PL 6 locations had a higher maximum body widths compared to P. loosi previously identified by Mohotti (1998) from several locations in Sri Lanka. In contrast, PL 4 population were on average 3 µm thinner. Roman and Hirschmann (1969) and Loof (1991) explained that the appearance of the stylet knobs may change during storage or after mounting in glycerine implying the diagnostic value of this character may not always be reliable. For this reason, detailed work on stylet morphology was not carried out. However, in our study, the average stylet length of all the populations showed low coefficient of variation (6.53%), ranging from 13.47 to 16.48 µm. The data showed that stylet length of all the study populations were in the range of that reported by Loof (1960) and Seinhorst (1977) of P. loosi identified. The PL 3 and PL 4 populations had comparatively longer stylet lengths than those of PL 2 and PL 5. Our study showed that some morphometric features, such as the stylet length and V ratio, were less modified by biotic or abiotic factors and are probably more reliable as diagnostic characters for the Pratylenchus genus.

Male populations of P. loosi
Males were abundant in all the populations studied. Coefficients of variation of all the male characters showed similar trends as in the female characters. Variation in volume and weight were however less in the males of the same population. The data from this study and comparison with existing references are presented in Table 3.

Tail length
The tail length of the males of the populations studied was shorter than those to described by Mohotti (1998) except for the populations from PL 4 and PL 5. Similarly, the tail lengths of males from all the locations studied were shorter than in isolates from Taiwan (Wu et al. 2002).

Body length (L)
The 'L' of the PL 4 populations showed similar trends as in the females with highest 'L' of 436.50 µm. Also, the male 'L' was statistically not significantly different among the populations (P < 0.05). However, the mean values fall within the range in Sri Lankan populations of P. loosi previously described by Mohotti (1998) and generally did not conform to the findings of Handoo and Golden (1989) and Wu et al. (2002).

Maximum body width
Our data revealed that the maximum body width was highest in the PL 4 nematodes (12.83 µm) and lowest in PL 6 nematodes (12.16 µm). The average maximum body width of the nematodes of the six populations were in the range of those described by Mohotti (1998) but not for those described by Wu et al. (2002). For descriptive determination of the P. loosi isolates, morphometry of the female specimens was used ( Figure 1).

Principal component analysis
Fourteen (14) morphometric characters of the female nematodes were used for Principal Component Analysis. The six populations were clearly separated into four groups. The PL 4, Mahadowa population and the PL 2, Delmar populations were in separate groups compared to the other populations. The PL 1 and PL 5 were clustered into one group while PL 3 and PL 6 clustered into another.

Morphometric characterization of P. Loosi populations
The P. loosi specimens were collected from the sampling sites described in Table 1 and the slides were prepared as per the standard protocols in Seinhorst (1959). For the study, 20 female and 12 male specimens of P. loosi were used for each site. Specimens were observed under OPTIKA B 500 microscope, and all morphometric measurements were done with Optika Vision Pro plus (Version 2.7) software. The measurements taken were as follows;

Male
Body length (L), Maximum body width (w), Tail length, Stylet length, Spicule, Body length / maximum body width, Body length / maximum body width, Body length / tail length, Volume (µm 3 ) and weight (µg). De Man's formula for male and female morphometrics were calculated based on Siddiqi (1986).

Data analysis
The data were statistically analysed using SAS 9.1. Principle Component Analysis was used to make an objective assessment of the relative similarity among the populations using fourteen (14) morphometric characters for the females.

Molecular characterization of P. loosi populations
The P. loosi populations used for molecular study were collected from the locations described in Table 1. The molecular work (e.g. PCRs, cloning, sequencing, sequence analyses and DNA homology work) of this study was carried out at the State Agricultural Biotechnology Centre (SABC), Murdoch University, Perth, Australia. DNA extraction from single nematodes was done using phenol chloroform method and methods described by Marek et al. (2014) and Wayenberg et al. (2000).

PCR of the D2/D3 rDNA
Genomic DNA of single adult P. loosi nematodes were used for PCRs. Primers D2-1 (5'-GACCCGTCTTGAAACACGGA-3') and D3-2 (5'-CGGAAGGAACCAGCTACTA-3') were used for amplification of the D2/D3 expansion region of the 28S RNA gene. All PCRs were made up to 20 µl with nuclease-free water and contained 10.0 µl Go Taq Green master mix (Promega Corp, Australia), 1.0 µl each of the primers D2-1 and D3-2 and 2.0 µl of DNA template. Thermal cycling was done using an initial denaturation at 95 °C for 5 min, followed by 50 amplification cycles (denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min) and a final step at 72 °C for 10 min. Amplified products were observed on 1% TAE-buffered agarose gels, stained with SyBr Safe DNA gel stain (Invitrogen, USA) and visualized with a Trans illuminator. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, USA) according to the manufacturer's protocol.

PCR of the ITS region
The partial 18S-ITS1-5.8S-ITS2-partial 28S of the rDNA was amplified from genomic DNA of single adult P. loosi nematodes using the

Morphometric characterization of P. Loosi populations
The P. loosi specimens were collected from the sampling sites described in Table 1 and the slides were prepared as per the standard protocols in Seinhorst (1959). For the study, 20 female and 12 male specimens of P. loosi were used for each site. Specimens were observed under OPTIKA B 500 microscope, and all morphometric measurements were done with Optika Vision Pro plus (Version 2.7) software. The measurements taken were as follows;

Male
Body length (L), Maximum body width (w), Tail length, Stylet length, Spicule, Body length / maximum body width, Body length / maximum body width, Body length / tail length, Volume (µm 3 ) and weight (µg). De Man's formula for male and female morphometrics were calculated based on Siddiqi (1986).

Data analysis
The data were statistically analysed using SAS 9.1. Principle Component Analysis was used to make an objective assessment of the relative similarity among the populations using fourteen (14) morphometric characters for the females.

Molecular characterization of P. loosi populations
The P. loosi populations used for molecular study were collected from the locations described in Table 1. The molecular work (e.g. PCRs, cloning, sequencing, sequence analyses and DNA homology work) of this study was carried out at the State Agricultural Biotechnology Centre (SABC), Murdoch University, Perth, Australia. DNA extraction from single nematodes was done using phenol chloroform method and methods described by Marek et al. (2014) and Wayenberg et al. (2000).

PCR of the D2/D3 rDNA
Genomic DNA of single adult P. loosi nematodes were used for PCRs. Primers D2-1 (5'-GACCCGTCTTGAAACACGGA-3') and D3-2 (5'-CGGAAGGAACCAGCTACTA-3') were used for amplification of the D2/D3 expansion region of the 28S RNA gene. All PCRs were made up to 20 µl with nuclease-free water and contained 10.0 µl Go Taq Green master mix (Promega Corp, Australia), 1.0 µl each of the primers D2-1 and D3-2 and 2.0 µl of DNA template. Thermal cycling was done using an initial denaturation at 95 °C for 5 min, followed by 50 amplification cycles (denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min) and a final step at 72 °C for 10 min. Amplified products were observed on 1% TAE-buffered agarose gels, stained with SyBr Safe DNA gel stain (Invitrogen, USA) and visualized with a Trans illuminator. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, USA) according to the manufacturer's protocol.

PCR of the ITS region
The partial 18S-ITS1-5.8S-ITS2-partial 28S of the rDNA was amplified from genomic DNA of single adult P. loosi nematodes using the

Morphometric characterization of P. Loosi populations
The P. loosi specimens were collected from the sampling sites described in Table 1 and the slides were prepared as per the standard protocols in Seinhorst (1959). For the study, 20 female and 12 male specimens of P. loosi were used for each site. Specimens were observed under OPTIKA B 500 microscope, and all morphometric measurements were done with Optika Vision Pro plus (Version 2.7) software. The measurements taken were as follows;

Male
Body length (L), Maximum body width (w), Tail length, Stylet length, Spicule, Body length / maximum body width, Body length / maximum body width, Body length / tail length, Volume (µm 3 ) and weight (µg). De Man's formula for male and female morphometrics were calculated based on Siddiqi (1986).

Data analysis
The data were statistically analysed using SAS 9.1. Principle Component Analysis was used to make an objective assessment of the relative similarity among the populations using fourteen (14) morphometric characters for the females.

Molecular characterization of P. loosi populations
The P. loosi populations used for molecular study were collected from the locations described in Table 1. The molecular work (e.g. PCRs, cloning, sequencing, sequence analyses and DNA homology work) of this study was carried out at the State Agricultural Biotechnology Centre (SABC), Murdoch University, Perth, Australia. DNA extraction from single nematodes was done using phenol chloroform method and methods described by Marek et al. (2014) and Wayenberg et al. (2000).

PCR of the D2/D3 rDNA
Genomic DNA of single adult P. loosi nematodes were used for PCRs. Primers D2-1 (5'-GACCCGTCTTGAAACACGGA-3') and D3-2 (5'-CGGAAGGAACCAGCTACTA-3') were used for amplification of the D2/D3 expansion region of the 28S RNA gene. All PCRs were made up to 20 µl with nuclease-free water and contained 10.0 µl Go Taq Green master mix (Promega Corp, Australia), 1.0 µl each of the primers D2-1 and D3-2 and 2.0 µl of DNA template. Thermal cycling was done using an initial denaturation at 95 °C for 5 min, followed by 50 amplification cycles (denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min) and a final step at 72 °C for 10 min. Amplified products were observed on 1% TAE-buffered agarose gels, stained with SyBr Safe DNA gel stain (Invitrogen, USA) and visualized with a Trans illuminator. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, USA) according to the manufacturer's protocol.

PCR of the ITS region
The partial 18S-ITS1-5.8S-ITS2-partial 28S of the rDNA was amplified from genomic DNA of single adult P. loosi nematodes using the In summary, the female morphometrics of the six populations of P. loosi measured in this study indicates there is intraspecific variation in the nematodes (Table 2 and Figure 1). However, the PCA results did not clearly separate the populations into clusters based on their geographic locations. Generally, the specimens could be confirmed as P. loosi species as their morphometric features were well within the ranges of typical measurements reported for this species. The slight variations in the morphometric parameters allowed the six populations to be clustered into four distinct groups. The results revealed that PL 1 and PL 5 populations are closely-related, as were the PL 3 and PL 6 populations. However, the separation of PL 2 and PL 4 populations into individual separate groups indicate they may be morphometrically different from the other four populations.  Table 5. Representative sequences from all the locations best matched with P. loosi with sequence identities ranging from 96 % -100%. The individual sequences from each location showed some intra-species variation depicting potential isolates of P. loosi.

Phylogenetic analysis of D2/D3 sequences
The Neighbor-Joining trees constructed using the sequences of D2/D3 28S rDNA expansion segment are shown in Figure 2. The five populations were clustered into different phylogenetic clades with nematodes from populations PL 6 and PL 3 closelyrelated to each other than the others. Sequences of the nematodes of the PL 3 and PL 6 populations were grouped with EF446995.1, a sequence of P. loosi isolated from Iran but were more distantly-related to AF170439.1 P. loosi isolate T which was described as a Sri Lankan isolate. The rest of the populations existed as well separated Genbank are presented in Table 5. The sequences from the same location were in the same clade confirming there were no sequence differences among P. loosi isolates from the same location; sequences with the same number PL designation (e.g. PL5.1 and PL5.2) were from the same location.

Morphometric characterization of P. Loosi populations CONCLUSIONS
Overall results showed evidence of existence of morphotypes in the six P. loosi populations infesting tea in Sri Lanka. It also showed some divergence in the molecular data of the nematodes. Whether these divergence amount to differences in infestation patterns and severity and persistence of the nematodes in different regions of Sri Lanka with varied soil and environmental factors needs further investigation. Results of such investigations are important as they will indicate whether specific management strategies are required to control damage caused by the nematodes in each of the teagrowing areas in Sri Lanka.