Advances in Biological Control and Resistance Genes of Brassicaceae Clubroot Disease-The Study Case of China

Abstract

Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae. It occurs in cruciferous crops exclusively, and causes serious damage to the economic value of cruciferous crops worldwide. Although different measures have been taken to prevent the spread of clubroot disease, the most fundamental and effective way is to explore and use disease-resistance genes to breed resistant varieties. However, the resistance level of plant hosts is influenced both by environment and pathogen race. In this work, we described clubroot disease in terms of discovery and current distribution, life cycle, and race identification systems; in particular, we summarized recent progress on clubroot control methods and breeding practices for resistant cultivars. With the knowledge of these identified resistance loci and R genes, we discussed feasible strategies for disease-resistance breeding in the future.

Full text

Citation: Zhang, C.; Du, C.; Li, Y.;
Wang, H.; Zhang, C.; Chen, P.
Advances in Biological Control and
Resistance Genes of Brassicaceae
Clubroot Disease-The Study Case of
China. Int. J. Mol. Sci. 2023,24, 785.
https://doi.org/10.3390/
ijms24010785
Academic Editor: Andrés J. Cortés
Received: 3 November 2022
Revised: 20 December 2022
Accepted: 21 December 2022
Published: 2 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Review
Advances in Biological Control and Resistance Genes of
Brassicaceae Clubroot Disease-The Study Case of China
Chaoying Zhang, Chunyu Du, Yuwei Li, Huiying Wang, Chunyu Zhang * and Peng Chen *
College of Plant Science, Huazhong Agricultural University, Wuhan 430070, China
*Correspondence: zhchy@mail.hzau.edu.cn (C.Z.); chenpeng@mail.hzau.edu.cn (P.C.)
Abstract:
Clubroot disease is a soil-borne disease caused by Plasmodiophora brassicae. It occurs in
cruciferous crops exclusively, and causes serious damage to the economic value of cruciferous crops
worldwide. Although different measures have been taken to prevent the spread of clubroot disease,
the most fundamental and effective way is to explore and use disease-resistance genes to breed
resistant varieties. However, the resistance level of plant hosts is influenced both by environment
and pathogen race. In this work, we described clubroot disease in terms of discovery and current
distribution, life cycle, and race identification systems; in particular, we summarized recent progress
on clubroot control methods and breeding practices for resistant cultivars. With the knowledge of
these identified resistance loci and R genes, we discussed feasible strategies for disease-resistance
breeding in the future.
Keywords: clubroot disease; Plasmodiophora brassicae; R gene
1. Overview of Clubroot Disease
1.1. The Discovery and Distribution of Clubroot Disease
The pathogen causing clubroot disease in crucifer plants is called Plasmodiophora
brassicae, and belongs to the genus Plasmodiophora in the phylum Protozoa. Clubroot disease
has been found in continental European Brassica plants as early as the 13th century, and
some reports suggest that its presence may be traceable to earlier than the Roman times [
1
].
In 1737, cruciferous clubroot diseases were officially reported in England from the west coast
of the Mediterranean and southern Europe [
2
]. Early Scottish people generally believed
that clubroot disease was caused by poor soil quality or unbalanced fertilizers [
1
]. In 1873,
Russian biologist Michael Woronin first identified the clubroot pathogen and named it
Plasmodiophora brassicae [
3
]. In the late 1960s, Woronin studied the relationship between
the host and the pathogen and described the pathogen’s life cycle and the interaction
mode with the host [
4
]. From the late 19th and early 20th centuries, the clubroot disease
was brought to Canada by European settlers [
5
]. In 2003, clubroot disease was officially
reported on B. rapa and thereafter it became rapidly spread in Canada [
6
,
7
]. The incidence
of clubroot disease in Asia started in Japan and has become a serious economic problem in
Japan and Korea [
8
]. A variety of names have been given to clubroot disease from different
countries and regions, indicating the diversity in the nature of the disease from both the
pathogen side as well as the plant hosts [
9
]. In China, clubroot disease was first found in
Taiwan and Fujian in the early 1920s [
10
], though in recent years it has expanded both to
the north and to the west inland regions during agronomy development [
11
–
13
]. With
the expansion of rapeseed cultivation and especially the modern farming activities, the
incidence of clubroot disease has rapidly increased; provinces with the heaviest incidence
include Sichuan, Hubei, Yunnan, and Anhui (Figure 1) [14].
Int. J. Mol. Sci. 2023,24, 785. https://doi.org/10.3390/ijms24010785 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023,24, 785 2 of 18
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 2 of 19
Figure 1. The distribution of clubroot disease in mainland China (Reprinted with permission from
Ref. [Wang et al 2021]. 2021, Wang, Y.Y.; Yang, Z.Q.; Yang, Q.Y.; Zhang, C.Y.).
1.2. Life History of Plasmodiophora brassicae
Clubroot disease occurs through a specialized parasitism of cruciferous plant roots.
The causative pathogen is Plasmodiophora brassicae, and transmission of the disease occurs
mainly through resting spores on crop residues from an infected field. The resting spores
of P. brassicae can survive the winter and remain viable for a considerable time in a frozen
state, thus posing great difficulties for disease control. The infestation of host roots by P.
brassicae is generally divided into two stages: I. primary infection on root hairs and II.
secondary infection in root cortical cells (Figure 2). The duration of these two phases
varies slightly depending on the physiological subspecies of P. brassicae and the host [15].
A method of fluorescent probe-based confocal microscopy was used to investigate the
root infection process of P. brassicae on Arabidopsis roots (Figure 2) [16]. During the pri-
mary infection (0-7 dpi, dpi = days post infection), resting spores germinated and pro-
duced primary mobile spores; the encapsulated primary mobile spores pierced the host
cell wall to produce mononucleated protozoa in root epidermis (1 dpi). Mononuclear
Plasmodium underwent mitosis and produced multinucleated zoospores (1-3 dpi),
which was then accompanied by cytoplasmic cleavage to produce mononuclear second-
ary spores (3-4 dpi). Secondary free-living spores were released into root hairs or epi-
dermal cells for primary infection (4-7 dpi). In root epidermal cells, the union of two
haploid mononuclear secondary zoosporangium produces diploid mononuclear conidia
(2n zygote), a process featuring an increase in chromatin volume and nucleus size. At 8
dpi, the presence of widespread mononuclear secondary plasmodium in root cortical
cells marked the establishment of secondary infection. During 10-15 dpi, secondary
plasmodium further developed binucleate, tetranucleate, and multinucleate forms, re-
sulting in further occupation of roots by the pathogen and a dramatic increase in the root
volume manifested as swelling symptoms. Upon 24 dpi, mononuclear resting spores
could be found in cortical cells, marking the completion of one life cycle of P. brassicae
(Figure 2C).
By comparison of the infestation process of P. brassicae on different host plants, it is
now clear that P. brassicae host (e.g., rapeseed, cabbage) resistance is mainly determined
by the secondary infection stage, i.e., during the cortical infestation, while non-host (e.g.,
rice, wheat, and barley) resistance acts primarily during the epidermal infestation stage
[17]. Previous studies have provided important references for resolving the mechanisms
Figure 1.
The distribution of clubroot disease in mainland China (Reprinted with permission from
Ref. [14]).
1.2. Life History of Plasmodiophora brassicae
Clubroot disease occurs through a specialized parasitism of cruciferous plant roots.
The causative pathogen is Plasmodiophora brassicae, and transmission of the disease occurs
mainly through resting spores on crop residues from an infected field. The resting spores
of P. brassicae can survive the winter and remain viable for a considerable time in a frozen
state, thus posing great difficulties for disease control. The infestation of host roots by
P. brassicae is generally divided into two stages: I. primary infection on root hairs and
II. secondary infection in root cortical cells (Figure 2). The duration of these two phases
varies slightly depending on the physiological subspecies of P. brassicae and the host [
15
]. A
method of fluorescent probe-based confocal microscopy was used to investigate the root
infection process of P. brassicae on Arabidopsis roots (Figure 2) [
16
]. During the primary
infection (0–7 dpi, dpi = days post infection), resting spores germinated and produced
primary mobile spores; the encapsulated primary mobile spores pierced the host cell wall
to produce mononucleated protozoa in root epidermis (1 dpi). Mononuclear Plasmodium
underwent mitosis and produced multinucleated zoospores (1–3 dpi), which was then
accompanied by cytoplasmic cleavage to produce mononuclear secondary spores (3–4 dpi).
Secondary free-living spores were released into root hairs or epidermal cells for primary in-
fection (4–7 dpi). In root epidermal cells, the union of two haploid mononuclear secondary
zoosporangium produces diploid mononuclear conidia (2n zygote), a process featuring
an increase in chromatin volume and nucleus size. At 8 dpi, the presence of widespread
mononuclear secondary plasmodium in root cortical cells marked the establishment of
secondary infection. During 10–15 dpi, secondary plasmodium further developed binucle-
ate, tetranucleate, and multinucleate forms, resulting in further occupation of roots by the
pathogen and a dramatic increase in the root volume manifested as swelling symptoms.
Upon 24 dpi, mononuclear resting spores could be found in cortical cells, marking the
completion of one life cycle of P. brassicae (Figure 2C).

Int. J. Mol. Sci. 2023,24, 785 3 of 18
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of 19
of plant resistance and developing green and efficient prevention and control strategies
against clubroot disease [18,19].
(A)
(B) (C)
Figure 2. Diagram of the refined life cycle of Plasmodiophora brassicae ( Reprinted with permission
from Ref. [Liu et al 2020]. 2020, Liu, L.J.; Qin, L.; Zhou, Z.; Hendriks, W.; Liu, S.; Wei, Y.) (A) Com-
plete life cycle of Plasmodiophra brassicae; (B) primary zoospore in root epidermis (white arrows); (C)
secondary zoospore (white arrowhead) and a fusion of two zoospores to form a zygote (black ar-
rowhead).
1.3. Identification of Physiological Races of P. brassicae
During an investigation of the pathogenesis of different hosts, researchers found
that P. brassicae has a complex physiological race. That is, pathogens isolated from dif-
ferent regions might have different genetic backgrounds, which result in different disease
phenotypes on a given host plant. Several taxonomy systems have been established for
different P. brassicae species in the world, including (1) the Williams identification system,
(2) the European clubroot differential (ECD) system, and (3) the Sinitic Clubroot differ-
entiation (SCD) system.
1.3.1. Williams Identification System
Figure 2.
Diagram of the refined life cycle of Plasmodiophora brassicae (Reprinted with permission from
Ref. [
16
]). (
A
) Complete life cycle of Plasmodiophra brassicae; (
B
) primary zoospore in root epidermis
(white arrows); (
C
) secondary zoospore (white arrowhead) and a fusion of two zoospores to form a
zygote (black arrowhead).
By comparison of the infestation process of P. brassicae on different host plants, it is
now clear that P. brassicae host (e.g., rapeseed, cabbage) resistance is mainly determined by
the secondary infection stage, i.e., during the cortical infestation, while non-host (e.g., rice,
wheat, and barley) resistance acts primarily during the epidermal infestation stage [
17
].
Previous studies have provided important references for resolving the mechanisms of plant
resistance and developing green and efficient prevention and control strategies against
clubroot disease [18,19].
1.3. Identification of Physiological Races of P. brassicae
During an investigation of the pathogenesis of different hosts, researchers found that
P. brassicae
has a complex physiological race. That is, pathogens isolated from different
regions might have different genetic backgrounds, which result in different disease pheno-
types on a given host plant. Several taxonomy systems have been established for different
P. brassicae species in the world, including (1) the Williams identification system, (2) the

Int. J. Mol. Sci. 2023,24, 785 4 of 18
European clubroot differential (ECD) system, and (3) the Sinitic Clubroot differentiation
(SCD) system.
1.3.1. Williams Identification System
The Williams identification system was established in 1965, and it is still widely used
(Table 1) [
20
]. This system uses four Brassica species, Jersey Queen,Badger Shipper,Lauren-
tian, and Wilhelmsburger, and the classified P. brassicae are divided into 16 physiological
races [20]. For example, when Jersey Queen,Laurentian, and Wilhelmsburger are susceptible
(+) and Badger Shipper is resistant (
−
), the pathogen would be classified as race 1 in the
Williams system.
Table 1. The Williams classification system [20].
Host Plant
Race
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
+ + + + −+ + − − +−+− − − −
Jersey Queen
−+−+− − +− − + + −+ + + −
Badger Shipper
+ + + + − − − + + −+−+− − −
Laurentian
+− − +− − − − + + + + −+−+
Wilhelmsburger
1.3.2. European Clubroot Differential System
The European Clubroot Differential (ECD) system was established in 1975 [
21
]. The
system included 15 plant hosts which can be divided into three major groups: (1) ECD01–
ECD05 was the B. rapa group (AA, 2n = 20). Within this group, ECD01–ECD04 were
rapifera and ECD05 was Pekinesis, and they could be infected by all races and serve as
susceptible controls. (2) ECD06–ECD10 were the Brassica napus L. (AACC, 2n = 28) group;
and (3) ECD11–ECD15, the Brassica oleracea (CC, 2n = 18) group. These materials were
collected from different regions of Europe by individual research groups, which served
as representative host standards to classify different pathogen races. A binary recording
calculation was used to dictate the race of pathogen based on the disease level from the
three groups of hosts (Table 2). For example, if the first group’s (B. rapa group) hosts are all
susceptible, the second group’s (B. napus group) are all resistant, and if only ECD15 of the
third group is susceptible, then according to the binary calculation, 0 + 0 + 0 + 0 + 0 = 0/1 +
2 + 4 + 8 + 16 = 31/0 + 0 + 0 + 0 + 0 + 16 = 16, this pathogen race would be recorded as ECD
0/31/16.
1.3.3. The Sinitic Clubroot Differential (SCD) System
Although the ECD system includes three major types of Brassica species, there are
still considerable regional differences in both the host and pathogen in Asian countries,
such as China, Japan, and Korea. Therefore, a SCD (Sinitic Clubroot Differential) system
was developed for pathogen race identification in mainland China (Table 3). Previous
studies using the Williams system showed that the dominant P. brassicae race in of China
was pathotype 4 [
22
]. However, due to the annual variation in pathogen populations and
difficulty in standardization, a single-spore isolation method was developed recently by
Zhang et al., who characterized P. brassicae strains isolated from nine different locations of
Chinese cabbage cultivation field and obtained a total of 281 single-spore strains belonging
to 15 disease types according to the Williams system, of which disease type 4 accounted for
the largest proportion [
23
]. Bai et al. tested 42 species of P. brassicae on 12 different hosts,
and developed the SCD system [24].

Int. J. Mol. Sci. 2023,24, 785 5 of 18
Table 2. The ECD classification system [22].
Differential
Number Differential Host Binary
Numbers
Decimal
Numbers
2n = 20 (Brassica rapa L. Sensu lato)
ECD 01 ssp. rapifera line aaBBCC 201
ECD 02 ssp. rapifera line AAbbCC 212
ECD 03 ssp. rapifera line AABBcc 224
ECD 04 ssp. rapifera line AABBCC 238
ECD 05 ssp. Pekinensis line Granaat 2416
2n = 38 (Brassica napus L.)
ECD 06 var. napus cv. Nevin line Dc101 201
ECD 07 var. napus cv. Giant line Dc119 212
ECD 08 var. napus line Dc128 224
ECD 09 var. napus cv. Clubrootresistance Dc129 238
ECD 10 var. napus cv. Wilhelmsburger Dc130 2416
2n = 18 (Brassica oleracea L.)
ECD 11 var. capitata cv. Badger Shipper 201
ECD 12 var. capitata cv. Bindsachsener 212
ECD 13 var. capitata cv. Jersey Queen 224
ECD 14 var. capitata cv. Septa 238
ECD 15 var. acephala subvar. Laciniata cv. Verheul 2416
Table 3. The Sinitic clubroot differential (SCD) system [24].
SCD Pb1 Pb2 Pb3 Pb4 Pb5 Pb6 Pb7 Pb8 Pb9 Pb10 Pb11 Pb12 Pb13 Pb14
WIlliams
classification
system
2/4/7/11
4442/4444444444
H08 − −−−−−−−−−−−− +
H03 − − − − − − − − +++−+ +
H01 − − − − − − − +− − +−+ +
H04 − − − − − − +− − +++++
H02 − − − − +++−+ + −+ + −
H05 − − − +−+− − + + −+ + −
H06 − − +−−−−−−−−−−−
H07 −+−−−−−−−−−−−−
H12 + +++++++++++++
Note:
Pb1–Pb14 are different physiological races of clubroot disease; H01–H08 are different resistant hosts of
clubroot disease.
A total of 14 physiological races (Pb1–Pb14) were included in SCD system, with
Pb1 as the dominant race [
24
]. Rui et al. improved the single-cell isolation protocol
and combined the SCD system with the Williams system on strains collected from 11
provinces in China [
25
]. In terms of resistance breeding, disease-level phenotyping is
critical. However, pathogens present in the field always constitute a population and this
population is likely to change from year to year. Therefore, resistance developed towards
a major pathotype may disappear upon long-term use. Since the pathogen could not be
multiplied
in vitro
, it is critical to compare the disease phenotype based on single-spore
method, i.e., a uniform genotype of the pathogen strain. Therefore, a scientific and efficient
taxonomy system is very important, and this system must be in accordance with the
regions to monitor changes possibly involved in field population. Different pathotypes of
P. brassicae identified in the field can also facilitate resistance breeding in laboratory and
greenhouse contexts.

Int. J. Mol. Sci. 2023,24, 785 6 of 18
1.4. Damage of Clubroot Disease
As mentioned above, P. brassicae infects the roots of cruciferous crops, and secondary
infection results in swollen roots and the loss of root function, which in turn affects the
development of the above-ground parts. Brassica L. is one of the most important genera
in the cruciferous family, which include hundreds of agronomic crops such as rapeseed,
Chinese cabbage, cabbage, shepherd’s purse, radish, turnip, and many others. Many
of them can be infected, but there are species that carry resistance genes and become
immune to the disease. The area for cruciferous crop cultivation is expanding with modern
agronomy techniques; additionally, the resting spores of P. brassicae can survive in soil for
8–12 years or even longer, letting mutations accumulate and contribute the appearance
of new physiological races [
26
]. Warm temperature might be in favor of disease outburst,
although so far no direct evidence has been reported. Modern farming measures also
constitute a major reason for the rapid spreading of clubroot disease in recent years.
Since the clubroot disease infection can occur even in seedlings, the earlier the onset
time, the more severe the disease outcome. At the beginning of the disease, there is no
obvious phenotype on the aboveground parts, but the new leaf growth will be significantly
inhibited. During the middle and late stages, the aboveground parts of the host plant will
have stunted growth, yellowing and wilting starts to occur at the base of the leaves, and
root galls of different sizes, shapes, and locations will be formed [
27
]. With the disease
progression, the root xylem will be destroyed, the leaves and stems will eventually wilt,
and the reduction of the absorption of water and nutrients by the roots will cause a great
loss of yield and in extreme cases no harvest at all [28].
The size and location of the root galls/tumors is the basis for disease level grading.
A four-level grading system of clubroot disease is proposed as follows: level 0-normal
root, no tumor; level 1-no tumor on the main root, small tumor on lateral roots and fibrous
roots; level 2-medium tumor on the main root, large tumor on some lateral roots; level
3-large tumor on the main root and lateral roots, enlargement of the basal part of stem,
stunted plant growth [
29
,
30
]. In addition, Hu reported a disease-grading method especially
developed for rapeseed using a scale of 1–4, which can better reflect and assess the severity
of disease symptoms [31].
Due to the nature of soil-borne disease, temperature, soil pH, and humidity are sig-
nificant factors affecting the germination of resting spores and therefore disease incidence.
Studies have shown that soil temperatures of 18–25
◦
C, humidity of about 60%, and pH
values of 5.4–6.5 are the optimum conditions for spore germination [
32
]. In accordance
with the climate zone, Hubei, Hunan, Yunnan, Anhui, Sichuan, Jilin, Liaoning, northeast
China, southwest China, and Shandong are the main sites of clubroot disease in China [
33
].
The disease affects an area of 3.2–4 million hm
2
per year in China, accounting for more than
one-third of the total cultivation area of cruciferous crops. In an extreme year, the affected
area can reach 9 million hm
2
, with an average yield loss of 20%–30%, and in extreme cases a
loss of more than 60% in the field. Therefore, clubroot disease has become a critical problem
for the rapeseed industry and also a great threat for many vegetables; clubroot disease
has demanded great attention during recent years as a bottleneck agricultural problem in
China [34].
1.5. Control Measures of Clubroot Disease
The control of clubroot disease mainly follows the policy of “prevention-oriented,
integrated control”. It emphasizes the important role of agro-ecological control in disease
management, while coordinating biological and chemical control techniques to ensure max-
imum socio-economic and ecological benefits. The current control measures for clubroot
disease are listed below:
(1)
Field management: P. brassicae is spatially aggregated in soil, with high incidence
at entrances and field margins [
35
]. The viability and longevity of P. brassicae are
closely related to soil properties, and it has been shown that alkaline addition in soil
can reduce the germination rate of dormant spores, decrease root-hair infection, and

Int. J. Mol. Sci. 2023,24, 785 7 of 18
inhibit the maturation of sporangia and Zoosporangium [
36
]. Therefore, increasing
soil pH with lime has been often used for the management of small acreage inci-
dence [
37
–
39
]. In addition, it has been shown that high concentrations of calcium,
boron, and magnesium have important effects on soil inoculum density [
40
]. High
concentrations of calcium are involved in the induction of relevant defense com-
pounds and the induction of host cell death by reducing dormant spore germination
and sporangial development at the same time [
41
,
42
]. High concentrations of boron
could slow down the development of P. brassicae by inhibiting its growth during
primary infection stage [
43
]. Interestingly, clubroot incidence and severity were found
to be affected by the level of total and individual glucosinolates between oilseed rape
cultivars [
44
]. Since different crops have difference impact on agronomic residues
on soil after growth seasons, crop rotation measures have also been used to avoid
pathogen accumulation in open fields.
(2) Chemical control: chemical agents are used to inhibit the germination of resting spores.
Pre-disease control is critical for disease prevention; current measures consist of phar-
maceutical seed dressing, seedbed disinfection, soil fumigation, joint root irrigation,
etc. A few conventional fungicides, such as SDD (sodium dimethyl dithiocarbamate),
thiram, carbendazim, fluazinam, cyazofamid, and pentachloronitrobenzene, have
shown to be effective. In addition, options are provided for combining different chem-
icals together to achieve better results, such as 58% metalaxyl mancozeb (1500 times
dilution) and 75% chlorothalonil (1000 times dilution) for milder years [45].
(3)
Biological control: the soil contains a large number of microorganisms; previous
studies showed that Trichoderma and Streptomyces spp. can suppress P. brassicae in
cauliflower both in greenhouse and in the field [
46
]. Heteroconium chaetospira is
effective against the development of clubroot disease in cabbage at low to moderate
soil moisture [
47
]. Application of formulated biocontrol agents including Bacillus
subtilis and Gliocladium catenulatum could significantly reduce the incidence of clubroot
in Brassica napus L. [
48
]. The endophytic fungus Acremonium alternatum was shown to
suppress clubroot disease in cabbage and Arabidopsis thaliana [49].
(4)
Breeding for disease-resistance cultivars: Field management can reduce disease in-
cidence to some extent, but it requires a lot of labor and at the same time does not
fundamentally solve the problem. Indeed, researchers have tried to quantify the
abundance of clubroot pathogens using the qRT-PCR method, although in most cases
the pathogen is present in a mixed population [
50
]. Comparative bioassays performed
in growth chambers showed that resistance was under selection pressure, and the
use of clubroot-resistant cultivars is recommended when P. brassicae DNA exceeds
1300 genes copies per gram soil [
50
]. Chemical control can be efficient but is costly and
causes environmental pollution. Therefore, breeding for resistant cultivars could pro-
tect the plant and environment together from the disease; this is the most fundamental
and effective way to prevent disease spreading. Resistant genes (R genes) could
be identified from plant materials that are naturally immune. Breeding for disease
resistance using R genes is not only very effective, but also in line with sustainable
development strategies.
2. Plant Immune Pathways and R Genes
2.1. Plant Immune Response Pathways
Plants do not have specific immune cells or a somatic adaptive immune system, such
as that of mammals. During evolution, plants have developed their own immune sys-
tems by recognizing invading pathogens (viruses, bacteria, and fungi) through various
receptors on the cell surface as well as inside the cells [
51
–
53
]. Currently, the plant im-
mune system is constituted by two pathways. (1) The primary immune pathway, also
known as the PTI (pattern-triggered immunity) pathway, is activated by cell-surface pat-
tern recognition receptors (PRR), and recognizes invading pathogenic microorganisms by
microbe-associated molecular pattern (MAMP) or damage-associated molecular pattern

Int. J. Mol. Sci. 2023,24, 785 8 of 18
(DAMP). (2) The secondary immune response, also known as the ETI (effector-triggered
immunity) pathway, is triggered by effectors released by pathogens; plants in turn can
evolve resistance genes (R genes) that recognize the effectors and trigger host-immune
responses [
54
] (Figure 3). The most commonly accepted model for PTI-ETI interaction is
the “zigzag” model [
55
]. According to this model, PTI and ETI are temporally and spatially
distinct and mediated by different factors, but they also interact on the molecular level
and there are considerably overlap partners downstream of the ETI and PTI pathways.
PTI is the front line of plant defense against pathogens and stimulates the basal defense,
while ETI is an accelerated and amplified response of PTI and is generally more effective in
preventing further transmission [56].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 19
(DAMP). 2) The secondary immune response, also known as the ETI (effector-triggered
immunity) pathway, is triggered by effectors released by pathogens; plants in turn can
evolve resistance genes (R genes) that recognize the effectors and trigger host-immune
responses [54] (Figure 3). The most commonly accepted model for PTI-ETI interaction is
the “zigzag” model [55]. According to this model, PTI and ETI are temporally and spa-
tially distinct and mediated by different factors, but they also interact on the molecular
level and there are considerably overlap partners downstream of the ETI and PTI path-
ways. PTI is the front line of plant defense against pathogens and stimulates the basal
defense, while ETI is an accelerated and amplified response of PTI and is generally more
effective in preventing further transmission [56].
Figure 3. Schematic diagram of the plant immune system ( Reprinted with permission from Ref.
[Song et al 2021]. 2021, Song, W.; Forderer, A.; Yu, D.; Chai, J.)
2.2. Disease-Resistance (R) Genes and the NBS–LRR Protein Family
Most of the plant disease-resistance genes (R genes) identified so far encode proteins
of the NBS–LRR (Nucleotide Binding Site–Leucine Rich Repeat) family, which are also
known as NLR proteins as the major type for the plant R gene family. The NLR protein
consists of three main components: the variable N-terminal structural domain, the NB
(Nucleotide-Binding) structural domain, and the C-terminal conserved LRR (leu-
cine-rich-repeat) structural domain [57,58]. Based on the characteristics of the N-terminal
structural domain, NLRs are mainly divided into TNL with TIR (Toll-interleukin-1 re-
ceptor) at the N-terminal, and CNL with a CC (coiled-coil) structural domain at the
N-terminal. The CNL class R genes were found in both dicotyledonous and monocoty-
ledonous plants and significantly more than the TNL class. However, TNL class R genes
were detected only in dicotyledons [59–61].
During plant immunity, NLR proteins act as intracellular immune recognition re-
ceptors, recognizing effectors released by pathogens and triggering immune responses
[62–64]. The ways in which plant NLRs are involved in resistance are divided into direct
Figure 3.
Schematic diagram of the plant immune system (Reprinted with permission from Ref. [
3
]).
2.2. Disease-Resistance (R) Genes and the NBS–LRR Protein Family
Most of the plant disease-resistance genes (R genes) identified so far encode proteins
of the NBS–LRR (Nucleotide Binding Site–Leucine Rich Repeat) family, which are also
known as NLR proteins as the major type for the plant R gene family. The NLR protein
consists of three main components: the variable N-terminal structural domain, the NB
(Nucleotide-Binding) structural domain, and the C-terminal conserved LRR (leucine-rich-
repeat) structural domain [
57
,
58
]. Based on the characteristics of the N-terminal structural
domain, NLRs are mainly divided into TNL with TIR (Toll-interleukin-1 receptor) at the
N-terminal, and CNL with a CC (coiled-coil) structural domain at the N-terminal. The
CNL class R genes were found in both dicotyledonous and monocotyledonous plants and

Int. J. Mol. Sci. 2023,24, 785 9 of 18
significantly more than the TNL class. However, TNL class R genes were detected only in
dicotyledons [59–61].
During plant immunity, NLR proteins act as intracellular immune recognition recep-
tors, recognizing effectors released by pathogens and triggering immune responses [
62
–
64
].
The ways in which plant NLRs are involved in resistance are divided into direct and indi-
rect effects. Typical of the “gene-for-gene” model is the interaction between the flax rust
resistance fungal gene AvrL567 and the L protein [
65
]. In rice, the Avr-Pita176 protein binds
directly to the Pi-ta LRD region to initiate an immune response against rice blast fungus [
66
].
The TNL family member RPP1 (Recognition of Peronospora parasitica 1) in Arabidopsis
directly and specifically recognizes the ATR1 (Arabidopsis thaliana Recognized 1) effector
variant produced by the foliar oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) to
trigger an immune response [67].
However, most of the NLR proteins are bound to other host proteins before recog-
nizing the effector (Table 4). The TNL protein RPS4 (resistance to Pseudomonas syringae
4) can interact specifically with the transcriptional activator bHLH84 and they mediate
transcriptional regulation downstream of immunity [
68
]. RPS4 can also act in concert with
RRS1 (resistance to Ralstonia solanacearum 1) to confer recognition of Pseudomonas AvrRps4
and Ralstonia PopP2 [
69
,
70
]. In AvrRps4-triggered resistance, RPS4 crosstalks with SNC1.
While SPRF1 acts as a transcriptional repressor, its mutation activates SNC1 and lead to
enhanced resistance [
71
]. The fact that RPS4 can interact with multiple proteins reflects the
structural diversity of the protein, but the underlying mechanism regarding whether there
is competition between multiple factors is not yet fully understood.
CNL protein RPM1 confers resistance to Pseudomonas syringae by recognizing the Pseu-
domonas effector Avrpm1 (ADP-ribosyltransferase) and AvrB through the phosphorylation
of RIN4 during infection [
72
,
73
]. RPS2 (RESISTANT TO P. SYRINGAE2) is activated in
Arabidopsis (At) RIN4 by the Pseudomonas syringae effector AvrRpt2, forming the AvrRpt2–
RIN4–RPS2 defense-activation module [74]. CRT1 encodes a protein with ATPase activity
and is an important mediator of defense signaling triggered by R proteins such as RPS2 [
75
].
Activation of the RPS5 protein requires PBS1 cleavage to trigger ADP–ATP exchange [
76
].
In other dicotyledons, the tobacco mosaic virus-resistant CNL protein NRG1 plays an
important role in the recognition of the TNL proteins Roq1 and RPP1 [
77
]. The CNL protein
Rx1 in potato interacts with NbGlK1 to regulate the binding affinity for DNA [
78
]. In
monocotyledonous species, this indirect action-induced immune response is also prevalent.
In barley, a series of MLA proteins (including MLA1, MLA6, and MLA10), which belong
to CC-type NLRs, interact with RING-type E3 ligases and mediate the resistance to pow-
dery mildew fungi (Blumeria graminis) [
79
,
80
]. In rice, Pigm genes encode a set of NLRs,
including PigmR, which mediates broad-spectrum resistance. Additionally, PIBP1, a CNL
protein containing an RNA-recognition structural domain (RRM), can interact with PigmR
to accumulate in the nucleus in an NLR-dependent manner and directly bind target genes
OsWAK14 and OsPAL1 A/T cis-acting elements of DNA to activate defense against rice
plague [81].
In addition to this, the process of plant immunization is usually accompanied by a
hypersensitivity response (HR) or local programmed cell death (PCD). Therefore, maintain-
ing the homeostasis of plant NLR proteins is critical for balancing between immunity and
growth [
82
–
85
]. The abovementioned studies fully demonstrate that NLR proteins have key
roles in the disease-resistance pathways of different pathogens, both in monocotyledonous
and dicotyledonous plants. By influencing the binding and possible recognition of effectors
by NLR proteins and downstream helper NLRs, different circuits of immune response
pathways involving phytohormones and transcriptional reprogramming are initiated and
motivated, leading to resistance and morphological changes accompanied by with disease
progression. In this sense, NLR-like R genes are the most important gene resources for
disease-resistance breeding.

Int. J. Mol. Sci. 2023,24, 785 10 of 18
Table 4. NLR protein resistance in different crops and their intercrossing proteins.
Host Plant NLR Class NLR Protein NLR-Interacting
Protein (Type) Pathogen Effector Reference
Dicot
Arabidopsis
TNL RPS4
bHLH84 (TF) Pseudomonas syringae AvrRps4 [68]
RRS1 (Paired NLRs) Pseudomonas syringae AvrRps4,
PopP2
[69]
Ralstonia olanacearum [70]
SNC1 SRFR1 (TPR domain) Pseudomonas syringae AvrRps4 [71]
CNL
RPS5 PBS1 (RLCK VII
family kinase) Peronospora parasitica AvrPphB [76]
RPM1
HSP90.2 (Chaperone)
Pseudomonas syringae
AvrRpm1,AvrB
[72]
RIN4 (Unknown) Pseudomonas syringae
AvrRpm1,AvrB
[73]
RPS2
CPR1 (E3 ligase
(F-box)) Pseudomonas syringae — [84]
RIN4 (Unknown) Pseudomonas syringae AvrRpt2 [74]
CRT1 (ATPase
activity) Pseudomonas syringae AvrRpt2 [75]
MUSE13 (TRAF) Pseudomonas syringae AvrRpt2 [83]
Tobacco CNL NRG1 EDS1 (Lipase-like) Tobacco mosaic virus P50 [77]
Potato CNL Rx GLK1 (TF) Potato virus X Coat protein [78]
Monocot Barley CNL
MLA10
WRKY1,WRKY2 (TF)
Blumeria graminis — [80]
MYB6 (TF) Blumeria graminis — [85]
MLR1 (E3 ligase
(RING)) Blumeria graminis —
[79]
MLA1 MLR1 (E3 ligase
(RING)) Blumeria graminis AvrA1
MLA6 MLR1 (E3 ligase
(RING)) Blumeria graminis —
Rice CNL PIBP1 PigmR (TF) Magnaporthe oryzae — [81]
3. Clubroot Resistance (CR) Genes and Resistance Breeding in Brassica Species
Brassica spp. have been domesticated and artificially selected over a long period of
time from three diploid parents, Brassica rapa (AA, 2n = 20), Brassica nigra (BB, 2n = 16),
and Brassica oleracea (CC, 2n = 18). The combination of the three allopolyploid species
gives rise to diverse and rich members of the Brassica genus, including three tetraploid
groups, Brassica napus L. (AACC), Brassica juncea (AABB), and Brassica carinata (BBCC) [
86
].
These hundreds of Brassica species share a high degree of genome structure similarity and
duplication from ancestors, but also have accumulated mutations, gene loss, and functional
divergence, leading to phenotypic differences in disease resistance.
In order to create a new germplasm with disease resistance, a parent with CR genes
needs to be identified and crossed with an elite parent with other desirable agronomy
traits such as a tight shape, high yield, and abiotic stress tolerance. Wide hybridization has
been widely used to innovate CR cultivars using the crossing of species within the Brassica
genus. Marker assisted selection (MAS) facilitates to narrow down the relevant genetic and
chromosomal regions and pinpoint candidate genes. Traditional and molecular breeding
are combined to accelerate the CR breeding process.
3.1. Progress on CR Loci Mapping and CR Gene Identification
B. oleracea,Raphanus sativus L. (RR, 2n = 18), and B. rapa ssp. Rapifera (AA, 2n = 20) are
the main resource materials for clubroot resistance (CR) genes since they are immune to the
clubroot pathogen. The most widely used materials for clubroot disease resistance are the
ECD series of European turnip, especially ECD01, ECD02, ECD03, and ECD04 [
87
]. The
CR loci of European turnips were mainly distributed on chromosomes from the A genome
as quality traits. Some candidate CR genes have been identified by fine mapping and
functional validation. Diederichsen and Sacristan artificially synthesized allotetraploids

Int. J. Mol. Sci. 2023,24, 785 11 of 18
using ECD04 and Brassica oleracea var. capitata Linnaeus, and identified three CR dominant
loci, Pb-Bn1,Pb-Bn2, and Pb-Bn3 [88].
CR loci identified so far from the ECD series include Crr1 (A08), Crr2 (A01), Crr3
(A03), Crr4 (A06), CRa (A03), CRb (A03), CRc (A02), CRk (A02), Rcr1 (A03), PbBa3.1 (A03),
PbBa3.2 (A03), PbBa3.3 (A03), and PbBa8.1 (A08) (Figure 4). As shown in Figure 4, most of
the CR loci were present on chromosome A03 of the A genome [
89
]. Crr3,CRk,PbBa3.3,
CRd, and BraCRc loci are located on upper part of A03 and referred as the “A03-1 cluster”;
CRa,CRb,CRbki,BraA3P5X/G.CRa/bKato1.1,BraA3P5X/G.CRa/bKato1.2,Rcr1,Rcr2,Rcr4 and
BraCRa are located on the lower arm of chromosome A03 and therefore named as “A03-2
cluster” [
90
]. Among all the CR loci and CR gene candidates, only CRa and Crr1a have
been successfully cloned and functionally validated [91,92].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 12 of 19
was found between the Rsa10025569 locus and disease resistance in a BC1F1 population
[97].
Arabidopsis, a model crop in the Cruciferae family, is a good model for analyzing the
resistance mechanisms of P. brassicae. The earliest analyses of Arabidopsis resistance to
clubroot disease were mainly performed with multiple metabolic pathways [98–100], and
the observation of the natural response of Arabidopsis to clubroot disease in various loca-
tions [101–103]. Currently, in addition to the identification of the gene RPB1, located on
chromosome 1, involved in clubroot disease resistance [104], Jubault et al. identified four
additive QTL loci, Pb-At5.23, Pb-At5.1, Pb-At1, and Pb-At4, and all of these alleles for re-
sistance were derived from the parent Bur-0 [105]. In addition to this, the homology of
well-defined resistance genes on the Arabidopsis genome was used to further design de-
signer markers for fine targeting [106].
In the typical Brassica radish, Kamei et al. identified Crs1, and found that the ge-
nomic region around Crs1 and the genomes around Crr3 on turnip (B. rapa.) share a
common ancestor [94]. Gan et al. identified five QTL loci, RsCr1, RsCr2, RsCr3, RsCr4, and
RsCr5, associated with clubroot disease resistance, with RsCr1 being homologous to the
well-defined locus Crr1 [107]. Recently, Gan et al. identified a new locus RsCr6 on chro-
mosome 8 and screened for possible resistance candidate genes R120263140 and
R120263070 [108].
The investigation of the resistance mechanism of different crops to clubroot disease
can help us further understand the resistance loci as well as provide a solid theoretical
basis for breeding against clubroot disease.
Figure 4. Physical mapping of CR loci ( Reprinted with permission from Ref. [Yang et al 2022].
2022, Yang, Z.; Jiang, Y.; Gong, J.; Li, Q.; Dun, B.; Liu, D.; Yin, F.; Yuan, L.; Zhou, X.; Wang, H.; et al.)
3.2. Genomic and Molecular Markers Associated with Clubroot Resistance
Figure 4. Physical mapping of CR loci (Reprinted with permission from Ref. [89]).
However, a single resistant variety cannot maintain stable resistance over a long time.
Convergent breeding by aggregating disease-resistance genes from different sources in
a single material is expected to improve the broad spectrum and persistence of disease
resistance in varieties and become a more practical and effective breeding model [
93
]. In
order to do this, more loci and CR genes need to be identified from different germplasms
that are immune to the disease. The nature and relationship between these loci need to be
evaluated before their utilization as gene resources for resistance breeding.
In 2010, Kamei et al. crossed Japanese radish (CR donor) with Chinese susceptible
radish to construct a mapping population with 18 linkage groups; they used AFLP and
SSR markers to identify a region of 554 Mb [
94
]. Matsumoto et al. obtained pure lines with
high resistance by mounting three CR genes (CRa,CRk, and CRc), and demonstrated that
disease resistance can be elevated by mounting CR genes [
95
]. By SNP mapping and RNA
sequencing, Huang et al. identified two possible CR genes (Bra019410 and Bra019413)
from Rcr2 loci in cabbage [
96
]. Recently, Rsa10003637 and RSA1005569/Rsa10025571

Int. J. Mol. Sci. 2023,24, 785 12 of 18
were identified as CR loci from radish; a significant correlation was found between the
Rsa10025569 locus and disease resistance in a BC1F1 population [97].
Arabidopsis, a model crop in the Cruciferae family, is a good model for analyzing
the resistance mechanisms of P. brassicae. The earliest analyses of Arabidopsis resistance
to clubroot disease were mainly performed with multiple metabolic pathways [
98
–
100
],
and the observation of the natural response of Arabidopsis to clubroot disease in various
locations [
101
–
103
]. Currently, in addition to the identification of the gene RPB1, located
on chromosome 1, involved in clubroot disease resistance [
104
], Jubault et al. identified
four additive QTL loci, Pb-At5.23,Pb-At5.1,Pb-At1, and Pb-At4, and all of these alleles
for resistance were derived from the parent Bur-0 [
105
]. In addition to this, the homology
of well-defined resistance genes on the Arabidopsis genome was used to further design
designer markers for fine targeting [106].
In the typical Brassica radish, Kamei et al. identified Crs1, and found that the genomic
region around Crs1 and the genomes around Crr3 on turnip (B. rapa.) share a common
ancestor [
94
]. Gan et al. identified five QTL loci, RsCr1,RsCr2,RsCr3,RsCr4, and RsCr5,
associated with clubroot disease resistance, with RsCr1 being homologous to the well-
defined locus Crr1 [
107
]. Recently, Gan et al. identified a new locus RsCr6 on chromosome
8 and screened for possible resistance candidate genes R120263140 and R120263070 [108].
The investigation of the resistance mechanism of different crops to clubroot disease
can help us further understand the resistance loci as well as provide a solid theoretical basis
for breeding against clubroot disease.
3.2. Genomic and Molecular Markers Associated with Clubroot Resistance
With the development of sequencing technologies, different omics have been used
on combinations of different hosts and pathogens to understand plant–pathogen inter-
actions [
109
,
110
]. Yu et al. performed QTL analysis on resistant cultivars to P. brassicae
and mapped three QTLs on chromosomes A02, A03, and A08; one QTL, Rcr4 on chromo-
some A03, was responsible for resistance to pathotypes 2, 3, 5, 6, and 8 in the Williams
system [
111
]. The QTLs on chromosomes A02 and A08 were named Rcr8 and Rcr9 respec-
tively, and two TNL genes were identified from genomic regions between Bra020936 and
Bra020861 around Rcr9 loci in B. rapa [
111
]. On the other hand, proteome and metabolome
studies showed differential expression of proteins in lipid metabolism, plant defense,
cell-wall repair, hormone production, and signal transduction in response to P. brassicae
infestation [112].
Marker-assisted breeding has been extensively used for clubroot-resistance breeding.
With more genomic information released, genomic sequence has been used more frequently
for the development of new markers. Zheng et al. identified five molecular markers
for pathotype identification, which can distinguish race P11 from P4, P7, and P9, and
similarly P9 from P4, P7, and P11 [
113
]. Lei et al. validated the genetic stability of two
co-dominant markers CRaEX04-1 and CRaEX04-3 associated with the CRa gene in cabbage
using 57 resistant varieties and two genetic populations [
114
]. Jiang et al. studied the
CR locus found in resistant “Kc84R” and identified BnERF034 as one of the CR genes on
chromosome A03 [
115
]. Indeed, many CR loci have been reported, including the ones
recently identified by Wang et al., for two QTLs on A03 and A08, conferring resistance to
pathotypes 3H, 3A, and 3D in turnip [
116
]. The development of markers and identification
of genomic regions responsible for clubroot resistance laid an important foundation for
marker-assisted breeding for the generation of resistant cultivars with durable resistance to
clubroot disease.
3.3. Progress on CR Breeding for Clubroot Disease
In 2015, Gao et al. performed disease phenotyping on twenty germplasm of Brassica
napus L; they found that Huayouza 9 and Huashuang 3 had strong resistance [
117
]. Using
molecular marker-assisted selection, PbBa8.1 locus of turnip ECD04 was transferred into the
elite B. napus variety Huashuang 5 to create clubroot-resistant line H5R, which was immune

Int. J. Mol. Sci. 2023,24, 785 13 of 18
to most of the race 4 pathogens in China [
118
]. A dominant CR gene CRd was successfully
transferred from the self-incompatible line “85–74” to the conventional varieties “W3”
and “Zhong Shuang 11”, resulting in two new germplasms, “W3R” and “Zhong Shuang
11R”, respectively [
119
]. In 2021, Li Qian et al. successfully developed the first hybrid
oilseed rape variety “Huayouza 62R”, by hybrid combination of a sterile line Huayouza
62R with a resistant cabbage donor parent Bing409R [
80
]. Huayouza 62R contains two
disease-resistance loci, PbBa8.1 and CRb, and showed excellent performance on field trial in
disease areas [
120
]. Hou et al. used “Hua Resistant No. 5” as the source material and Ogu
CMS (cytoplasmic male sterility) recovery line RF04 as the recipient, and created a kale-type
spring oilseed rape immune to race 4 pathogens [
121
]. In an attempt to test the contribution
of different CR genes and feasibility to promote the resistance by multiple CR genes in one
germplasm, Nadil performed hybridization between kale type 409R containing CRa gene
with kale type rape 305R containing PaBa8.1, and selected progeny with two CR loci. The
plants with two CR loci displayed good additive effect for disease resistance, supporting a
valid basis for the gene-mounting strategy for CR breeding [122].
In addition to breeding for clubroot disease resistance in oilseed rape, Sun Chaohui
et al. developed an early maturing variety of cabbage “Anxiu” with resistance to clubroot
disease in several trials in Shandong [
123
]. Yang et al. obtained a hybrid F1 of B. oleracea
×
B. napus rape carrying both clubroot disease-resistance genes and Ogura CMS-recovery
genes through distant crosses and embryo rescue [
124
]. He et al. developed 14 new
disease-resistant cabbage varieties using heterozygous crossbreeding and molecular marker-
assisted selection techniques to meet the production needs of Yunnan cabbage [
125
]. A
generation hybrid, “Jingchun CR3”, with resistance to clubroot disease and tolerance to the
shoots of cabbage was created by crossing two self-incompatible lines, CR1572 and CR1582,
by Yu Yangjun et al. [126].
4. Conclusions
Clubroot disease is considered as a “cancer” for Brassica species; the fast spreading of
the disease as well as the risk of losing resistance over time calls for a deeper understanding
of the Plasmodiophora pathogen and the host pathways leading to disease resistance. In
this review, we covered a basic background of the disease distribution and the pathogen’s
nature, with a greater focus on the plant ETI pathways and the roles of NLR proteins as R
genes for clubroot disease. We strongly believe that with the identification and isolation
of more CR (clubroot resistance) genes, more resistance materials could be developed to
provide a better safeguard for the agronomy industry of Brassica species.
Author Contributions:
C.Z. (Chaoying Zhang) and C.D. prepared figures and drafted the manuscript,
and Y.L. and H.W. helped to revise the manuscript. C.Z. (Chunyu Zhang) and P.C. conceived the
study, participated in its coordination. All authors have read and agreed to the published version of
the manuscript.
Funding:
This work was supported by the National Natural Science Foundation of China (U20A2034
and 31871659) and China Agriculture Research System (CARS-12) to CZ.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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