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e2019-76 | Vol. 51Article | DOI: 10.21307/jofnem-2019-076
JOURNAL OF NEMATOLOGY
The morphological characteristics and phylogenetic analysis 
of Pratylenchus vulnus Taiwan strawberry isolate
Yu-po Lin,1 Wan-chun Lee,2  
Pei-che Chung3 and Jiue-in Yang1, 2*
1Plant Medicine Program, National 
Taiwan University, Taipei, 10617, 
Taiwan. No. 1 Roosevelt Rd.,  
Sec. 4, Da-an Dist., Taipei City, 
10617, Taiwan. 
2Department of Plant Pathology 
and Microbiology, National Taiwan 
University, Taipei, 10617, Taiwan. 
No. 1 Roosevelt Rd., Sec. 4, Da-an 
Dist., Taipei City, 10617, Taiwan. 
3Miaoli District Agricultural  
Research and Extension Station, 
Council of Agriculture, Executive 
Yuan, Miaoli, 36346, Taiwan.  
No. 26, Guannan Rd., Gongguan 
Township, Miaoli County 36346, 
Taiwan. 
*E-mail: jiueinyang@ntu.edu.tw
This paper was edited by  
Zafar Ahmad Handoo.
Received for publication June 24, 
2019.
Abstract
Pratylenchus vulnus was discovered in nematode-distributing fields 
from symptomatic seedling roots and corresponding rhizosphere 
soil on strawberry farms in Taiwan. Microscopic measurements and 
scanning electron microscope observations of both sexes of the 
nematode coincided with the general morphological descriptions 
of the species. Four different types of female tail termini were 
observed, including pointed, digitate, smooth and tapering. 
Molecular analysis of the ribosomal RNA sequence (SSU, ITS and 
LSU regions) and the mitochondria COI gene sequences confirmed 
the species identification. Phylogenetic analysis suggested no 
specific geographic linkage of the Taiwan population to other 
previously reported populations.
Keywords
Pratylenchus vulnus, Morphology, SEM, Phylogenetic analysis, 
Strawberry, Root lesion nematode.
Root lesion nematodes (Pratylenchus sp.) are among 
the most economically damaging phytoparasitic 
nematodes on fruits, tree rootstocks and vegetables 
(Yu et al., 2012). Pratylenchus vulnus was first reported 
in 1951 as a pathogen of multiple trees and vines in 
California, USA (Allen and Jensen, 1951) and was later 
found to infect over 80 plant species (Castillo and 
Vovlas, 2007). In Uruguay, Sri Lanka and Australia, 
serious damage in strawberry (Fragaria × ananassa) 
fields caused by this nematode have been reported 
(Colbran, 1974; Minagawa and Maeso-Tozzi, 1990; 
Mohotti et al., 1997). We here report the discovery 
of one Pratylenchus sp. population in strawberry 
fields in Dahu township of Miaoli, Taiwan in 2017. The 
strawberry crops growing in the nematode-distributing 
fields GS and KDL were obviously stunted when 
discovered. Both fields had been cropping strawberry 
for over 10 years. The soil composition of the area was 
characterized as sandy loam by hydrometer method 
(Bouyoucos, 1951). Nematodes were extracted 
from 100 g ± 5% soil with modified Baermann 
funnel technique (Hooper, 1986). Morphological 
observations and measurements of adults (30 females 
and 30 males) were conducted with a compound 
microscope at magnification of up to 1000× (Table 2). 
The dorsal gland orifice of the female of our isolates 
fit the description of P. vulnus perfectly, with a range 
between 3.0 and 4.42 (means = 3.55), rather than 
1.9 to 3.0 characteristic of P. penetrans (Roman and 
Hirschmann, 1969). All nematodes observed had a 
lateral field composed of four incisures with wider inner 
band but two types of lateral field were observed. The 
first type had an evenly uplifted lateral field (Fig. 1E), 
while the second type carried uplifted and thinner 
outer bands (Fig. 1F). Four different types of female 
tail termini were observed, including pointed, digitate, 
2Pratylenchus vulnus Taiwan isolate characteristics: Lin et al.
Figure 1: The morphological variations within the P. vulnus Taiwan strawberry population. Two 
lateral field types and four tail termini were observed. Photographs were taken at 1000X 
magnification with compound microscope and SEM.
smooth and tapering (Fig. 1A-D). P. vulnus is known 
to express morphological differences when cultured 
under different temperature conditions (Doucet 
et al., 2001). A previous report also characterized 
five P. vulnus groups in Japan by tail morphology 
(Mizukubo 1990). The fact that four different tail 
terminus types were observed in the P. vulnus Taiwan 
population implies this population may have been 
established in the region rather than being recently 
imported.
Molecular analysis of the ribosomal RNA and 
mitochondrial gene sequences of the extracted 
nematodes confirmed their species as P. vulnus 
(Table 1). The rDNA LSU region (D2A/D3B: ACAAGTA 
CCGTGAGGGAAAGTTG/TCGGAAGGAACCAGCT 
ACTA) (Nunn, 1992), rDNA ITS region (TW81/AB28: 
GTTTCCGTAGGTGAACCTGC/ATATGCTTAAGTTCA 
GCGGGT) (Amiri et al., 2002; Subbotin et al., 2001), 
rDNA SSU region (SSU18A/SSU26R: AAAGATTAAG 
CCATGCATG/CAT TCT TGGCA A ATGCT T TCG) 
(Eyualem and Blaxter, 2003) and mtDNA COI region 
(JB3/JB4.5: TTTTTTGGGCATCCTGAGGTTTAT/TAAA 
GAAAGAACATAATGAAAATG) (Derycke et al., 2010) 
of the Pv-GS and Pv-KDL are 99.04, 95.96, 99.66 
and 99.24% identical, respectively. Both Pv-GS and 
Pv-KDL isolates had maximum 100% similarity of 
their rDNA LSU sequences to P. vulnus (GenBank 
accession: U47547.1). The Pv-KDL mtDNA COI 
region sequences of isolates were 99.74 to 100% 
similar to other sequences of P. vulnus available 
in the database (GenBank accessions KY424094, 
KY424095, KY828312, KY828317, KY424096-7 
and KX349427), and only 81.08% identical to the 
second most closely related species, P. scribneri 
(GenBank accession: KY424089.1). The rDNA 
SSU region sequence analysis provided a similar 
result as Pv-KDL and are 99.42% (GenBank 
accession: KY424163) to 99.88% (GenBank 
accession: KY424164) identical to all available 
P. vulnus sequences in GenBank, and only 94.48% 
3JOURNAL OF NEMATOLOGY
Table 1. GenBank sequence deposit information of multiple gene regions of P. vulnus 
Taiwan isolates from strawberry fields GS and KDL.
P. vulnus sequences GenBank deposit information
rDNA mtDNA
Isolate location LSU (730 bp) ITS (669 bp) SSU (883 bp) COI (441 bp)
GS MG372808 MG372806.1 MG372807 MN431203
KDL MK713641 MK713613 MK713614 MK764689
Figure 2: The phylogenetic tree of the combined partial 28S D2-D3 region and complete ITS 
region of the P. vulnus rDNA sequences. Morphologically similar species, Pratylenchus penetrans 
F1 isolate and of P. kumamotoensis Chilgok isolate, were used as out groups. Each node is 
marked with the isolate code, species and origin. The number at the fork represents the 
percentage of the bootstrap tested with 10,000 times for the indicating result.
similar to P. kumamotoensis (GenBank accession: 
AB905295.1). The rDNA ITS sequences also 
separated the Pv-KDL from P. kumamotoensis 
(GenBank accession: KT175521.1) clearly with a 
very low 86.22% similarity. Phylogenetic analysis of 
the rDNA ITS and LSU regions combined suggested 
the nematodes from the two fields belong to one 
population (Fig. 2) and are isolated from the rest 
of the previous populations from other geographic 
regions (Table 2).
4Pratylenchus vulnus Taiwan isolate characteristics: Lin et al.
Table 2. Measurements of the Taiwan P. vulnus population morphological characteristics.
Female (n = 30) Male (n = 30)
Characteristics Mean SD m. Range Mean SD m. Range
L (μ m) 704.01 74.85 573.46–839.91 587.02 46.83 492.76–678.1
K (μ m) 25.97 4.06 17.76–36.18 18.47 1.47 15.49–21.50
Stylet (μ m) 14.5 1.1 12.6–16.5 13.76 0.73 12.18–15.40
Tail (μ m) 32.87 3.93 24.35–41.10 28.74 4.07 22.28–39.80
a (ratio) 27.47 3.15 22.74–34.30 31.85 2.53 26.83–36.62
b (ratio) 6.1 0.6 4.87–7.16 5.7 0.47 4.88–6.95
b’ (ratio) 4.86 0.42 4.07–5.59 4.51 0.37 3.92–5.26
c (ratio) 21.57 2.38 17.83–27.46 20.71 2.68 13.51–23.99
c’ (ratio) 2.29 0.31 1.59–2.86 2.29 0.38 1.77–3.83
V (%) 78.34 1.67 73.84–81.09 – – –
Spicules (μ m) – – – 16.88 1.89 12.06–20.15
DGO (μ m) 3.55 0.32 3.00–4.42 3.41 0.33 2.77–4.31
Our discovery of the existence and population 
dynamics of P. vulnus in Taiwan implies a new threat 
to the 1,639 million NTD (ca. $52 million) strawberry 
industry. Currently, no nematicide is registered for 
root-lesion nematode suppression in strawberry. 
Related cultural and physical control options are 
currently undergoing evaluation by agricultural 
extension agencies to prevent serious damage.
Acknowledgments
The authors thank Dr. Zeng-Yei Hseu (Soil Survey and 
Contamination Remediation Laboratory, National Taiwan 
University) for the soil characterization assistance. The 
authors also thank Dr. Shiuh-Feng Shiao (Laboratory 
of Insect Systematics, National Taiwan University) and 
Ms. Shiang-Jiuun Chen (Department of Life Science, 
National Taiwan University) for SEM assistance. This 
research was funded by the Council of Agriculture, 
Executive Yuan, Taiwan, Grant No. 107AS-1.2.7-ST-aA. 
The authors declare no conflict of interest.
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