Pectate lyase genes from Radopholus similis and their application in pathotype identification

Abstract

Radopholus similis is a destructive, migratory, and endophytoparasitic nematode. It has two morphologically indistinguishable pathotypes (or physiological races): banana and citrus pathotypes. At present, the only reliable method to differentiate the two pathotypes is testing the infestation and parasitism of nematodes on Citrus spp. via inoculation. However, differences in inoculation methods and conditions adopted by different researchers complicate obtaining consistent results. In this study, the parasitism and pathogenicity of 10 R. similis populations on rough lemon (Citrus limon) seedlings and the tropism and invasion of rough lemon roots were tested. It revealed that populations SWK, GJ, FZ, GZ, DBSR, and YJ were citrus pathotypes, which showed parasitism and pathogenicity on rough lemon and could invade rough lemon roots, whereas populations XIN, ML, HN6, and HL were banana pathotypes, having no parasitism and pathogenicity on rough lemon and they did not invade the rough lemon roots. Four pectate lyase genes (Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-pel-5) belonging to the Class III family from these populations were amplified and analysed. The gene Rs-pel-3 could be amplified from six citrus pathotype populations and was stably expressed in the four developmental stages of the nematode, whereas it could not be amplified from the four banana pathotypes. Rs-pel-3 expression may be related to the parasitism and pathogenicity of R. similis on rough lemon. Hence, it can be used as a molecular marker to distinguish between banana and citrus pathotypes and as a target gene for the molecular identification of these two pathotypes.

Key points
• Four pectate lyase genes (Rs-pels) from Radopholus similis were cloned and analysed.
• The expression of Rs-pels is different in two pathotypes of Radopholus similis.
• A molecular identification method for two pathotypes of Radopholus similis using pectate lyase gene Rs-pel-3 as the target gene was established.

Full text

Vol.:(0123456789)
Applied Microbiology and Biotechnology (2024) 108:298
https://doi.org/10.1007/s00253-024-13124-3
APPLIED GENETICS ANDMOLECULAR BIOTECHNOLOGY
Pectate lyase genes fromRadopholus similis andtheir application
inpathotype identification
SihuaYang1· ShuaiYang1· QianyingLi1· YangLu1· XinHuang1· ChunChen1· ChunlingXu1· HuiXie1
Received: 21 November 2023 / Revised: 6 March 2024 / Accepted: 22 March 2024
© The Author(s) 2024
Abstract
Radopholus similis is a destructive, migratory, and endophytoparasitic nematode. It has two morphologically indistinguish-
able pathotypes (or physiological races): banana and citrus pathotypes. At present, the only reliable method to differentiate
the two pathotypes is testing the infestation and parasitism of nematodes on Citrus spp. via inoculation. However, differ-
ences in inoculation methods and conditions adopted by different researchers complicate obtaining consistent results. In
this study, the parasitism and pathogenicity of 10 R. similis populations on rough lemon (Citrus limon) seedlings and the
tropism and invasion of rough lemon roots were tested. It revealed that populations SWK, GJ, FZ, GZ, DBSR, and YJ were
citrus pathotypes, which showed parasitism and pathogenicity on rough lemon and could invade rough lemon roots, whereas
populations XIN, ML, HN6, and HL were banana pathotypes, having no parasitism and pathogenicity on rough lemon and
they did not invade the rough lemon roots. Four pectate lyase genes (Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-pel-5) belonging
to the Class III family from these populations were amplified and analysed. The gene Rs-pel-3 could be amplified from six
citrus pathotype populations and was stably expressed in the four developmental stages of the nematode, whereas it could
not be amplified from the four banana pathotypes. Rs-pel-3 expression may be related to the parasitism and pathogenicity of
R. similis on rough lemon. Hence, it can be used as a molecular marker to distinguish between banana and citrus pathotypes
and as a target gene for the molecular identification of these two pathotypes.
Key points
• Four pectate lyase genes (Rs-pels) from Radopholus similis were cloned and analysed.
• The expression of Rs-pels is different in two pathotypes of Radopholus similis.
• A molecular identification method for two pathotypes of Radopholus similis using pectate lyase gene Rs-pel-3 as the target
gene was established.
Keywords Radopholus similis· Pathotypes· Molecular identification· Pectate lyase· Tropism
Introduction
Radopholus similis, with a common name, the burrow-
ing nematode, a migratory endophytoparasitic nematode,
is one of the top 10 most damaging plant-parasitic nema-
todes worldwide (Jones etal. 2013). R. similis has over 350
host plants, including bananas, Citrus spp., and ornamental
plants, and is an important factor in reducing the production
of bananas and other horticultural crops worldwide; it is
listed as a quarantine plant pest in most countries (Xie 2006;
Duncan and Mons 2013). Two pathotypes (or physiological
races) of R. similis are generally recognised: the banana and
citrus pathotypes. The banana pathotype parasitises bananas
and many other hosts, but not Citrus spp., whereas the citrus
pathotype parasitises both bananas and Citrus spp. (Valette
etal. 1998). Therefore, the citrus pathotype has a wider host
range and is more harmful than the banana pathotype. The
European and Mediterranean plant Protection Organiza-
tions and European Union classify banana and citrus patho-
types as A1 and A2 quarantine pests, respectively (EPPO
2008). At present, the mechanisms of host specificity and
* Hui Xie
xiehui@scau.edu.cn
1 Laboratory ofPlant Nematology andResearch Center
ofNematodes ofPlant Quarantine, Department
ofPlant Pathology, College ofPlant Protection, South
China Agricultural University, Guangzhou510642,
People’sRepublicofChina
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Applied Microbiology and Biotechnology (2024) 108:298 298 Page 2 of 11
differences in pathogenicity between these two pathotypes
are unclear. Testing the parasitism of nematodes on Citrus
spp. is recognised as the only reliable method for distin-
guishing between these pathotypes.
Pectate lyase (PEL) degrades pectin to produce oligosac-
charides via β-trans elimination and exists widely in plants
and microorganisms (Chen etal. 2019). Pathogenic microor-
ganisms destroy plant cell walls to facilitate their invasion via
secreting PEL to degrade pectin and stimulate plant immune
responses (Shevchik etal. 1998; Ferrari etal. 2013). There-
fore, PEL plays an important role in the interaction between
plant pathogens and their hosts and is an important factor
affecting the host range of pathogens (Filho etal. 2016). Cur-
rently, plant-parasitic nematodes are the only animals known
to produce PEL. The first PEL gene (pel) found in plant-par-
asitic nematodes was Gr-pel-1 from Globodera rostochiensis
(Popeijus etal. 2000). Subsequently, several pel genes were
identified and cloned from sedentary parasitic nematodes
belonging to the genera Heterodera, Globodera, and Meloido-
gyne, all of which belong to the Class III PEL family (Deboer
etal. 2002; Doyle etal. 2002; Huang etal. 2005; Kudla etal.
2007; Vanholme etal. 2007; Zhuo etal. 2011; Peng etal.
2012, 2016; Li etal. 2017). The PEL of sedentary parasitic
plant-parasitic nematodes may play an important role in host
specialization and pathogenic differentiation (Huang etal.
2005; Stare etal. 2011; Sabeh etal. 2019; Tian etal. 2019).
To date, only three pel genes have been identified and cloned
from Bursaphelenchus xylophilus, a migratory parasitic nema-
tode (Kikuchi etal. 2006; Lee etal. 2013); however, the func-
tion and mechanism of these genes and their relationship with
pathogenic differentiation have not been reported.
In this study, five pels from R. similis (Rs-pels) were
screened and cloned using transcriptome data (Huang etal.
2019), and their basic characteristics were studied using bio-
informatics. The pathogenicity and tropism of 10 R. similis
populations from different hosts were tested to determine
their pathotypes against rough lemon (Citrus limon). Then,
the expression of Rs-pels in these populations was analysed
to screen the differential pel genes that could potentially
distinguish between the banana and citrus pathotypes to
establish a molecular identification method for these two
pathotypes.
Materials andmethods
Ten populations of R. similis and their host sources are
shown in Table1. All populations were identified by the
Plant Nematodes Laboratory of South China Agricultural
University, propagated and preserved on carrot callus
(25°C) according to the method described by Fallas and
Sarah (1994). R. similis was isolated from carrot callus by
the method of Stoffelen etal. (1999), soaked in 0.2% strep-
tomycin sulfate for 8 hours, and then washed with sterile
distilled water for five times, and then the nematode sus-
pensions were obtained for subsequent experiments. The
different developmental stages (eggs, juveniles, females,
and males) of R. similis were distinguished by morphologi-
cal characteristics under a Nikon SMZ18 stereomicroscope
(Nikon Corporation, Tokyo, Japan). Females and males have
vulva and spicule respectively.
The C. limon seeds were donated by Prof. Xiaoling Deng
from South China Agricultural University. River sand pur-
chased from Guangzhou Xiankelian Garden Flower Co., Ltd.
(Guangzhou, China) was used as the planting medium. A
glass tube (3 cm × 15 cm) was filled with dry river sand,
accounting for about 1/3 the tube height, and was sterilised
at 125 °C 20 p.s.i (about 137.8 kPa) for 1 h; after cooling
overnight, it was subjected to secondary sterilisation under
the same conditions. After peeling the seed coat, rough
lemon seeds were placed in 0.26% sodium hypochlorite
solution for 30 min, washed with sterile water four times,
laid flat in a Petri dish (d=90 mm) with moist sterilised filter
paper at the bottom, and placed in an incubator at 25 ± 1.5
°C for 3–4 days to accelerate germination in the dark.
Sterile distilled water (3 mL) was added to each sand-
filled tube to moisten the sand and a 2 cm deep depression
was placed in the sand surface centre; a single seed was
placed in it, and covered with sterile sand. The tubes were
then placed in an incubator at 25 ± 1.5 °C with the light
intensity of 3200–4000 l × (9.5 h light and 14.5 h darkness),
normal watering management, and humidity maintained at
approximately 3% w/w. After 30 days of culture, the rough
lemon seedlings were inoculated.
Determining parasitism andpathogenicity oftested
nematodes torough lemon
As per the method described by Kaplan (1994), rough lemon
seedlings with essentially the same growth after planting for
Table 1 Population information of Radopholus similis
No. Population coding Hosts
1 XIN Zingiber officinale Roscoe
2 ML Crataegus pinnatifida
3SWK Chrysalidocarpus lutescens
4 HN6 Musa AAA Giant Cavendish cv. Baxi
5 HL Maranta arundinacea
6 GJ Citrus reticulata
7 FZ Anthurium andraeanum ‘Pink Champion’
8 GZ Anthurium andraeanum Linden
9 YJ Curcuma longa
10 DBSR Anubias nana
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Applied Microbiology and Biotechnology (2024) 108:298 Page 3 of 11 298
30 days were selected and inoculated nematode suspensions
by a dropper with an inoculation amount of 200 female nem-
atodes (identified under a Nikon SMZ18 stereomicroscope;
Nikon Corporation, Tokyo, Japan) per plant. Inoculation was
performed twice at an interval of 5 days, and 100 nematodes
were inoculated each time. Each population of R. similis
was inoculated for one treatment, and each treatment was
repeated five times; non-inoculated seedlings were used as a
control treatment. Inoculation tests were repeated twice. The
day before inoculation, sterile distilled water was added to
each test tube to wet the sand, and the nematode suspension
was inoculated at a depth of 1 cm underground. To ensure
normal infection of nematodes, plants were not watered for
the first three days after inoculation, and routine manage-
ment was performed. After inoculation with nematodes for
30 days, the inoculated plant root systems were observed
and photographed, plant symptoms were recorded, plant
height and root weight were measured, and the nematodes
were isolated according to Kaplan’s method and counted
using a Nikon SMZ18 stereomicroscope (Nikon Corpora-
tion, Tokyo, Japan).
Tropism andinvasion ofnematodes totherough
lemon root system
The root systems of rough lemon seedlings were cut into
1 cm long segments. According to Čepulytė etal. (2018),
Pluronic F-127 gel system was prepared to conduct the tro-
pism test of R. similis to the rough lemon root. This gel was
liquid at low temperatures (< 15 °C) but solidified into a
gel at higher temperatures (> 15 °C). At the temperature
of 10 °C, 1 mL of Pluronic F-127 gel was absorbed into a
sterile 24-well culture plate. A total of 200 female nema-
todes were picked up from nematode suspensions by a nee-
dle under a Nikon SMZ18 stereomicroscope and added to
each well, gently shaken and mixed, and then a fresh rough
lemon root segment was placed in each well. The culture
plate was sealed and transferred to a dark incubator at 28
°C for culturing. Each nematode population had five wells
(five replicates), and the experiment was repeated twice.
Nematode tropism was observed under a Nikon SMZ18
stereomicroscope after 2, 4, 6, and 8 h of culturing. The
nematode aggregation and invasions around the root incision
were photographed and recorded. In addition, a modified
sodium chlorate-acid fuchsin staining method (Feng 2001)
was used to dye the root segments cultured for 8 h. Stained
root tissues were sliced and photographed under a Nikon
SMZ18 stereomicroscope.
Cloning ofpel genes fromR. similis
A total of 20,000 mixed-stage nematodes of the GJ
population were isolated and collected from a carrot
callus in which R. similis was cultured. The total RNA
of the nematodes was extracted by a Trizol Regent
(Invitrogen, Carlsbad, California, USA). The 5' RACE
and the 3' RACE cDNA templates for RACE amplifi-
cation were synthesized by the SMART RACE cDNA
amplification kit (Clontech, Takara Biotechnology
(Dalian) Co., Ltd., Dalian, China), respectively. The
cDNA template for the open reading frames (ORF)
sequence amplification was synthesized by One-step
gDNA Removal and cDNA synthesis SuperMix Kit
(Transgen, Beijing, China). The total genomic DNA of
R. similis was extracted by HiPure Mollusc DNA Kit
(Magen Co., Ltd., Guangzhou, China).
Screening and blasting of the transcriptome data of R.
similis (Huang etal. 2019) were performed to obtain the
suspected pels sequences. Primers for RACE amplifica-
tion of pels were designed according to those sequences
(Supplementary Information). The 5' and the 3' terminal
fragments of pels were amplified using these primers,
with the 5' RACE cDNA and the 3' RACE cDNA serving
as templates for amplification, respectively. The ampli-
fied fragments were sequenced and spliced together by
Sangon Biotech Co., ltd (Shanghai, China). The ORFs
of Rs-pels (Rs-pel-1, Rs-pel-2, Rs-pel-3, Rs-pel-4, Rs-
pel-5) were predicted on an ORF finder website (http://
www. ncbi. nlm. nih. gov/ gorf/ orfig. cgi), and a pair of full-
length primers were designed for each Rs-pel (Supple-
mental Material, Table S1) by Primer Premier 6 software
(PREMIER Biosoft International, Palo Alto, CA, USA).
The ORF and genomic DNA sequences of Rs-pels were
amplified using full-length primers, with the cDNA and
genomic DNA serving as templates for amplification,
respectively.
The similarity of Rs-PELs was compared to the NCBI
database using the BlastX tool (http:// blast. ncbi. nlm. nih.
gov/ Blast. cgi). Signalp-4.0 (http:// www. cbs. dtu. dk/ servi
ces/ signa lp-4. 0/) and TMHMMServer v.2.0 (https:// servi
ces. healt htech. dtu. dk/ servi ces/ TMHMM-2. 0/) were used to
predict the signal peptide (SP) and transmembrane domains
of Rs-PELs, respectively. Multiple sequence alignments of
Rs-PEL and other PELs from other nematodes were per-
formed using Mega 6.0 (http:// www. megas oftwa re. net/), and
a phylogenetic tree was constructed using the maximum-
likelihood method.
Expression determination ofRs‑pels inR. similis
populations
Using Rs-pels as the target, the DNA of a single nema-
tode from each population was extracted for polymer-
ase chain reaction (PCR) detection, and the expres-
sion of Rs-pels in different populations was analysed
and determined. The target genes were selected for
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Applied Microbiology and Biotechnology (2024) 108:298 298 Page 4 of 11
identification. To analyse the stable expression of the
target gene, DNA from four developmental stages (eggs,
juveniles, females, and males) was used as a template
for PCR amplification to establish a method for rapid
identification of the pathotypes of R. similis. The PCR
amplification of single nematode DNA was performed
as described by Xu etal. (2016).
Data processing andanalysis
The SPSS 14.0 Software (SPSS Inc., Chicago, IL, USA)
and Excel 2013 Software (Microsoft Inc., Washington,
D.C.) were used for statistical analyses. One-way analy-
sis of variance (ANOVA) was employed for the analysis
of the mean and standard error, and Duncan’s Multiple
Ranger Test (DMRT) was used for multiple comparisons.
The significance of the difference was analysed at the level
of p=0.05. Preliminary analyses showed that there was
no significant difference (p>0.05) between the data from
two runs of each experiment using dependent-sample t
tests and that allowed data from two runs to be combined
for analyses.
Results
Parasitism andpathogenicity of10 R. similis
populations torough lemon
Thirty days after inoculation, the root systems of rough lem-
ons inoculated with the SWK, GJ, FZ, GZ, YJ, and DBSR
populations were weak and had evident brown spots (Fig.1).
Plant height, root length, and root weight of the six treat-
ments were significantly lower than those of the control
(p<0.05) and the number of nematodes in the root and rhizo-
sphere soils was significantly higher than those in the control
(p<0.05). However, the average nematode number in the
roots of the XIN, ML, HN6, and HL inoculation treatments
was only 1–2, and the nematode number in the rhizosphere
soil was less than 10, which was not significantly different
from that of the control (p>0.05). The root length differ-
ence between these four treatments was also not significant,
and the root length and weight of XIN and HN6 inoculation
treatments were not significantly different from those of the
control (p>0.05) (Table2). Therefore, SWK, GJ, FZ, GZ,
YJ, and DBSR populations could parasitise rough lemons
Fig. 1 Symptoms of Citrus limon infected with 200 female nema-
todes of Radopholus similis per plant for 30 days. CK: non-inoculated
rough lemon seedling and root system; XIN, ML, SWK, HN6, HL,
GJ, FZ, GZ, YJ and DBSR: the rough lemon seedlings and root sys-
tems were inoculated with the different populations of R. similis,
which originated from Zingiber officinale Roscoe (XIN), Crataegus
pinnatifida (ML), Chrysalidocarpus lutescens (SWK), Musa AAA
Giant Cavendish cv.Baxi (HN6), Maranta arundinacea (HL), Cit-
rus reticulata (GJ), Anthurium andraeanum ‘Pink Champion’ (FZ),
Anthurium andraeanum Linden (GZ), Curcuma longa (YJ), and Anu-
bias nana (DBSR), respectively
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Applied Microbiology and Biotechnology (2024) 108:298 Page 5 of 11 298
and had clear pathogenicity, whereas XIN, ML, HN6, and
HL populations did not parasitise rough lemons.
Tropism andinvasion of10 R. similis populations
totheroot system ofrough lemon
The tropism test of 10 R. similis populations to the rough
lemon root system showed that the nematodes of all popula-
tions were randomly and uniformly dispersed in the gel when
the root segment was placed in the well, and the nematodes
started to migrate and aggregate to the root segment after 2
h and 4 h. The nematodes of the SWK, GJ, FZ, GZ, DBSR,
and YJ populations were observed to invade the root from
the wound position after 6 h, whereas the nematodes of the
XIN, ML, HN6, and HL populations were still around the
root and did not invade it (Fig.2A). The root tissues treated
for 8 h were dyed with modified sodium chlorate-acid fuch-
sin. Under the stereomicroscope, many nematodes of SWK,
GJ, FZ, GZ, DBSR, and YJ populations were observed in
the root segments, but only one or no nematodes of the XIN,
ML, HN6, and HL populations were found in the root seg-
ments (Fig.2B). The results showed that all the tested popu-
lations tended towards the rough lemon root; however, the
SWK, GJ, FZ, GZ, DBSR, and YJ populations could invade
the rough lemon root, whereas the XIN, ML, HN6, and HL
populations did not invade the rough lemon root.
Cloning andanalysis ofgenes ofthepel family inR.
similis
Five transcripts of suspected pels were obtained via screen-
ing and blasting the transcriptome data of the GJ population
of R. similis. The ORF and DNA genomic sequences of
these genes were obtained using PCR amplification (Fig.3).
After sequencing these fragments, a BLAST comparison
confirmed that the deduced proteins of all five genes had
conserved domains of the PEL family, and these five pels
were named Rs-pel-1, Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-
pel-5. Their ORFs were 843, 819, 873, 771, and 810 bp long,
and the number of encoded amino acids (aa) were 280, 272,
290, 256, and 269 aa, respectively (Table3). ClustalX was
used to compare the amino acid sequences of these five Rs-
PELs with known Class III PEL sequences, and Rs-PEL-2,
Rs-PEL-3, Rs-PEL-4, and Rs-PEL-5 were confirmed to have
four conserved regions unique to the Class III PEL family
(Fig.4) (Shevchik etal. 1997), whereas Rs-PEL-1 did not
have a conserved region unique to Class III PEL.
Sequence analysis of the five Rs-PELs predicted that none
of them had a transmembrane domain. Among them, Rs-
PEL-2, Rs-PEL-4, and Rs-PEL-5 contained signal peptides,
whereas Rs-PEL-1 and Rs-PEL-3 did not have signal peptide
sequences (Table3).
Amino acid sequences of the five Rs-PELs were blasted
against those of other plant-parasitic nematodes, bacteria,
and fungi, and a phylogenetic tree was constructed using the
maximum likelihood method in MEGA software (Fig.5).
The results showed that PELs from fungi and bacteria
clustered into a large category, whereas those from plant-
parasitic nematodes clustered independently into another
large category. The Rs-PELs could be divided into three
categories: first, Rs-PEL-3 and the PELs from four species
of Meloidogyne were clustered into a branch; second, Rs-
PEL-2 and Rs-PEL-5 were clustered with the PELs from
two species of Meloidogyne and species of Heterodera and
Table 2 Effects of Radopholus similis on Citrus limon growth 30 days after inoculation with 200 nematodes and the population density of the
nematode recovered from root
CK, healthy control group; XIN, ML, SWK, HN6, HL, GJ, FZ, GZ, YJ and DBSR, the treatments inoculated with the populations of R. similis
originated from Zingiber officinale Roscoe (XIN), Crataegus pinnatifida (ML), Chrysalidocarpus lutescens (SWK), Musa AAA Giant Caven-
dish cv.Baxi (HN6), Maranta arundinacea (HL), Citrus reticulata (GJ), Anthurium andraeanum ‘Pink Champion’ (FZ), Anthurium andraeanum
Linden (GZ), Curcuma longa (YJ), and Anubias nana (DBSR), respectively
Population Height Root length Root weight Number of nematodes in
rhizosphere soil
Number of nema-
todes in root
Total number
of nematodes
CK 8.86±0.36 f 7.52±0.32 f 0.11±0.01 d 0.00±0.00 a 0.00±0.00 a 0.00±0.00 a
XIN 7.72±0.39 e 6.75±0.38 ef 0.10±0.01 d 6.20±0.49 abc 1.80±0.47 a 8.00±0.58 ab
ML 6.11±0.42 cd 6.26±0.36 de 0.07±0.01 c 3.50±0.45 ab 1.40±0.50 a 4.90±0.67 ab
SWK 6.66±0.37 d 4.85±0.33 ab 0.05±0.01 abc 10.80±0.88 bc 9.50±0.75 b 20.30±0.80cd
HN6 6.86±0.26 de 6.16±0.40 de 0.10±0.01 d 6.10±1.39 abc 2.10±0.55 a 8.20±1.65 ab
HL 5.29±0.26 bc 6.44±0.60 de 0.07±0.00 c 8.30±0.47 abc 2.10±0.60 a 10.40±0.86bc
GJ 4.91±0.28 ab 5.03±0.32 abc 0.04±0.00 ab 10.80±0.63 bc 9.70±0.98 b 20.50±1.46cd
FZ 4.82±0.28 ab 6.02±0.30 cde 0.06±0.01 bc 33.10±5.37 e 20.40±1.99 c 53.50±5.84 e
GZ 4.91±0.26 ab 5.84±0.18 bcde 0.05±0.01 abc 20.00±1.97 d 9.90±1.11 b 29.90±2.69 d
YJ 4.51±0.23 ab 5.58±0.18 bcd 0.05±0.01 abc 13.60±1.55 cd 7.90±0.96 b 21.50±1.54 d
DBSR 4.26±0.30 a 4.27±0.14 a 0.04±0.00 a 12.50±0.91 cd 8.30±1.09 b 20.80±1.21 d
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Applied Microbiology and Biotechnology (2024) 108:298 298 Page 6 of 11
Fig. 2 Tropism of 10 populations of Radopholus similis to the Cit-
rus limon root. A: Tropism of R. similis to the root of C. limon in
Pluronic F-127 gel evenly mixed with 200 female nematodes 6 h
after assay initiation; B: Fuchsin staining of C. limon roots 8 h after
assay initiation by infection with 200 female nematodes; XIN, ML,
SWK, HN6, HL, GJ, FZ, GZ, YJ, and DBSR: the C. limon roots
were infected with populations of R. similis, which originated from
Zingiber officinale Roscoe (XIN), Crataegus pinnatifida (ML), Musa
AAA Giant Cavendish cv.Baxi (HN6), Maranta arundinacea (HL),
Chrysalidocarpus lutescens (SWK), Citrus reticulata (GJ), Anthu-
rium andraeanum ‘Pink Champion’ (FZ), Anthurium andraeanum
Linden (GZ), Curcuma longa (YJ), and Anubias nana (DBSR),
respectively; scale bar = 500 μm
Fig. 3 PCR amplification of
five pectate lyase genes from
Radopholus similis (Rs-pels).
A: Amplification of the ORF
sequences of Rs-pels; B:
Amplification of the genomic
DNA sequences of Rs-pels.
M:DS2000 marker (GDSBio
Co., Ltd., Guangzhou China);
1–5: products of Rs-pel-1,
Rs-pel-2, Rs-pel-3, Rs-pel-4, Rs-
pel-5 amplification respectively
B
1000 bp
2000 bp
A
2000
bp
1000
bp
750bp
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Applied Microbiology and Biotechnology (2024) 108:298 Page 7 of 11 298
Globodera into a branch; third, Rs-PEL-1 and Rs-PEL-4
were clustered with the PELs from the nematodes of Heter-
odera, Globodera, and Aphelenchida into one branch.
Expression ofPEL genes in10 populations ofR.
similis
Single nematode DNA from 10 populations was used
as the template, and sequence amplification of four Rs-
pels of the class III PEL family, i.e., Rs-pel-2, Rs-pel-3,
Rs-pel-4, and Rs-pel-5, was performed. The results
showed that Rs-pel-2, Rs-pel-4, and Rs-pel-5 could be
amplified from the SWK, GJ, FZ, GZ, DBSR, and YJ
populations that parasitised rough lemon, and the XIN,
ML, HN6, and HL populations that did not parasitise
rough lemon. However, the gene Rs-pel-3 could only be
amplified from the six populations parasitizing rough
lemon, showed stable expression in four developmental
stages of these populations, and could not be amplified
from the four non-parasitized rough lemon populations
(Fig.6A). Therefore, Rs-pel-3 could be used as a tar-
get gene to identify the banana and citrus pathotypes of
R. similis and could be identified at all developmental
stages in the citrus pathotype (Fig.6B).
Table 3 Sequence analyses of
pectate lyases of Radopholus
similis
Gene name ORF
length
(bp)
Amino acid
length (aa)
Protein molecu-
lar weight(kDa)
Signal peptide Transmem-
brane domain
Genbank
Rs-pel-1 843 280 30.2 No No MN176119
Rs-pel-2 819 272 28.8 Yes No MN176120
Rs-pel-3 873 290 30.6 No No MN176121
Rs-pel-4 771 256 26.9 Yes No MN176122
Rs-pel-5 810 269 27.8 Yes No MN176123
Fig. 4 Predicting the conserved regions of pectate lyases of the
Class III PEL family from Radophulus similis. Regions I–IV indi-
cate conserved regions characteristic of the Class III pectate lyase
family (Shevchik etal.1997). Highly conserved charged residues are
indicated with asterisks (*), RS_PEL2 to 5 indicate the amino acid
sequences of Rs-PEL-2 to Rs-PEL-5 from R. similis, the species of
the bacterium or the fungus, the gene, the amino acid size, and the
accession number of the aligned sequences are: F_sol_PelA = Fusar-
ium solani f. sp. pisi, PelA, 242 aa (M94692.1); F_sol_PelB = F.
solani f. sp. pisi, PelB, 242 aa (U13051); F_sol_PelC = F. solani f.
sp. pisi, PelC, 215 aa (U13049); F_sol_PelD = F. solani f. sp. pisi,
PelD, 233 aa (U13050); E_car_PelB = Erwinia carotovora, PelB,
347 aa (X79232)
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Applied Microbiology and Biotechnology (2024) 108:298 298 Page 8 of 11
Discussion
Two pathotypes, the banana and citrus pathotypes of R. simi-
lis are generally believed to exist with different host ranges
and be morphologically indistinguishable. In this study, the
parasitism and pathogenicity of 10 R. similis populations to
rough lemon were determined using the method described
by Kaplan etal. (1994) and the Pluronic F-127 gel system
(Čepulytė etal. 2018; Wang etal. 2009). The results showed
that six populations could parasitise rough lemon, whereas
four populations could not. Four Rs-pels of the Class III
PEL family (Rs-pel-2, Rs-pel-3, Rs-pel-4, Rs-pel-5) could be
amplified from six populations of parasitised rough lemon,
whereas only three Rs-pel of the Class III PEL family (Rs-
pel-2, Rs-pel-4, Rs-pel-5) could be amplified from four
populations of non-parasitised rough lemon, and Rs-pel-3
was stably expressed in four developmental stages of popu-
lations of parasitised rough lemon. Rs-pel-3 was proven to
be used as a target gene to distinguish the banana and citrus
pathotypes of R. similis.
Kaplan and Opperman (1997) conducted parasitism and
pathogenicity experiments on R. similis in citrus using the
method described by Kaplan etal. (1994). They reported
that if fewer than 10 nematodes were isolated from the
Fig. 5 Maximum-likelihood phylogenetic trees of pectate lyases of
Radopholus similis (Rs-PELs) and other organisms. Phylogenetic
tree for proteins with conserved domains of pectate lyases from cyst
nematodes, root-knot nematodes, Aphelenchus, Bursaphelenchus,
bacteria, and fungi generated by MEGA6.0. The Rs-PELs amino acid
sequences were marked in bold, and each sequence was followed by
its accession number in GenBank
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Applied Microbiology and Biotechnology (2024) 108:298 Page 9 of 11 298
rhizosphere (including in root and rhizosphere soil), the
population would not parasitise citrus, whereas if more
than 100 nematodes were isolated from the rhizosphere, the
population would parasitise citrus (Kaplan and Opperman
1997). However, the parasitism of a population of 10–100
nematodes isolated from the rhizosphere has not been clearly
defined by Kaplan and Opperman (1997). Goo and Sipes
(1997) proposed that if low numbers of nematodes were
recovered from the root, it suggested a low level of nematode
reproduction might have occurred on the plant, indicating it
was a poor host; if a few nematodes were recovered from the
media but not from the root, it suggested that these nema-
todes might have been survivors from the inoculation, so
the plant was considered a non-host or a very poor host. In
this study, 30 days after rough lemons were inoculated with
SWK, GJ, FZ, GZ, DBSR, and YJ populations of R. similis
nematodes were isolated from each inoculated root system.
The average number of nematodes in the roots was 8–20, and
the total number of nematodes isolated from the rhizosphere
of each inoculation treatment was more than 20. The roots of
rough lemons inoculated with these six populations showed
clear symptoms of damage, and the average nematode num-
ber in the root and rhizosphere soil was significantly higher
than that of the control treatment and the other four popula-
tions. However, 30 days after rough lemon inoculation with
XIN, ML, HN6, and HL populations, only 1–5 nematodes
were isolated from seven inoculated root systems, and no
nematodes were isolated from the other three inoculated root
systems. The average nematode number in the roots was only
one or two, and the total number of nematodes isolated from
the rhizosphere was less than 10 in each inoculation treat-
ment, both of which were not significantly different from
Fig. 6 DNA amplification of pectate lyase genes of Radopholus simi-
lis (Rs-pels) in 10 populations. A: DNA amplification of Rs-pels in
10 populations of R. similis; B: DNA amplification of Rs-pel-3 in dif-
ferent populations and developmental stages of R. similis; XIN, ML,
SWK, HN6, HL, GJ, FZ, GZ, YJ, and DBSR: single nematode DNA
from populations of R. similis, which originated from Zingiber offici-
nale Roscoe (XIN), Crataegus pinnatifida (ML), Musa AAA Giant
Cavendish cv.Baxi (HN6), Maranta arundinacea (HL), Chrysalido-
carpus lutescens (SWK), Citrus reticulata (GJ), Anthurium andrae-
anum ‘Pink Champion’ (FZ), Anthurium andraeanum Linden (GZ),
Curcuma longa (YJ), and Anubias nana (DBSR), respectively; M:
DS2000 marker (GDSBio Co., Ltd., Guangzhou China); Fe: female;
Ma: male; J2: second stage juvenile; Eg: egg
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Applied Microbiology and Biotechnology (2024) 108:298 298 Page 10 of 11
those of the control. The roots of rough lemons inoculated
with these four populations did not show symptoms of dam-
age (Fig.1).
In addition, the nematodes of SWK, GJ, FZ, GZ, DBSR,
and YJ populations were observed to be attracted to and
invaded the rough lemon roots, whereas the nematodes of
XIN, ML, HN6, and HL populations were only attracted
to but did not invade the rough lemon roots in the Pluronic
F-127 gel system (Fig.2). Plant root exudates can lead to
tropism of plant-parasitic nematodes in both host and non-
host plants (Sasakicrawley 2013; Hu etal. 2017); however,
they can only induce nematodes to infect host plants and not
non-host plants. Therefore, we considered that rough lemon
was a host plant for the SWK, GJ, FZ, GZ, DBSR, and YJ
populations, but not for the XIN, ML, HN6, and HL popu-
lations. This indicated that the SWK, GJ, FZ, GZ, DBSR,
and YJ populations belonged to the citrus pathotype (or cit-
rus race), whereas the XIN, ML, HN6, and HL populations
belonged to the banana pathotype (or banana race).
Many studies have shown that pel is an important factor
affecting host range. Different types of PELs recognise dif-
ferent sequences of methylated and unmethylated galactu-
ronate sites with different chemical and enzyme properties
(Herron etal. 2000; Filho etal. 2016), which may result in
different pathogen host ranges. In this study, five pels from
R. similis (Rs-pel-1, Rs-pel-2, Rs-pel--3, Rs-pel-4, Rs-pel-5)
were screened and cloned based on R. similis transcriptome
data, four of which belonged to the Class III family (Rs-
pel-2, Rs-pel-3, Rs-pel-4, Rs-pel-5). The amplification from
genomic DNA and analysing gene expression of pels in the
10 R. similis populations indicated that the gene Rs-pel-3
could be amplified from six populations that parasitised
rough lemon (SWK, GJ, FZ, GZ, DBSR, and YJ), but could
not be amplified from four populations that did not para-
sitise rough lemon (XIN, ML, HN6, and HL). Therefore,
the gene Rs-pel-3 was absent in the genomes of non-citrus
parasitic populations, and the expression of Rs-pel-3 may
be related to R. similis parasitism in Citrus spp.. Reportedly,
the difference in pel expression in sedentary plant-parasitic
nematodes is associated with their host range (Stare etal.
2011; Sabeh etal. 2019; Tian etal. 2019). Stare etal. (2011)
reported that the difference in sequence polymorphism and
gene copy of the pel-2 gene from Globodera was associated
with the variation in the host range of G. rostochiensis, G.
pallida, and G. tabacum, while Sabeh etal. (2019) consid-
ered that the difference expression of pel-1 was related to
the host range of G. rostochiensis, G. pallida, G. tabacum,
and G. mexicana. Tian etal. (2019) reported differences in
pel expression between tobacco and soybean pathotypes of
Heterodera glycines. This study revealed the relationship
between the gene Rs-pel-3 and host specialisation of R. simi-
lis and confirmed the difference between the two R. similis
pathotypes at the molecular level. It is the first time that the
difference in pel expression from migratory plant-parasitic
nematodes may also be related to their host range.
Moreover, this study confirmed that the gene Rs-pel-3
was stably expressed in eggs, juveniles, females, and males
of six populations of R. similis parasitising rough lemon.
Therefore, Rs-pel-3 can not only be used as a molecular
marker to distinguish citrus from banana pathotypes of R.
similis, but also as a target gene to develop molecular iden-
tification methods for the quick and accurate identification
of R. similis pathotypes.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00253- 024- 13124-3.
Acknowledgments HX and CX conceived and designed the research.
SY, SY, QL, YL, XH, and CC conducted experiments. SY analyzed
data. SY and HX wrote the manuscript. All authors read and approved
the manuscript.
Funding This study was funded by the Special Provincial Project
of Rural Revitalization Strategy in Guangdong Province (2022-92)
and the Guangdong Basic and Applied Basic Research Foundation
(2021A1515011273).
Declarations
Ethical approval No specific permissions were required for the nema-
todes used in this study, and these nematodes were plant pests and not
protected by the government.
Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
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otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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