Introduction
Rice (Oryza sativa L.)
accounts for 1/5 of all calories consumed by people, and is the third most
economically important crop in the world (Izawa and Shimamoto 1996). Rice production is essential in many economies, especially developing economies, such as Ghana. The BLB disease
(BLB), caused by Xanthomonas oryzae pv. Oryzae (Xoo), is
a destructive disease that negatively impacts rice yield throughout the world. Bacterial leaf blight disease has been extensively studied in Asia,
especially compared to Africa where Xoo shows high pathogenic variability
(Séré et
al. 2013). The first
BLB disease incidence reported in many African countries occurred in the 1980s, particularly in West African rice
growing regions (Wonni et al. 2014). This disease is considered an emerging disease
in West Africa and other parts of the
African Continent, and can cause significant crop loss.
This disease is characterized
by a continuous reduction in the yield and quality of the crop. According to Mew et al.
(1993), yield reduction due to a mild BLB infection is about 10–20%,
whereas a severely infected rice field may exhibit a 50% crop loss. During
epidemics, yield losses as high as 80% have been recorded. The severity of crop
infection and resulting crop loss is dependent on the Xoo strain and the rice
variety, growth stage, geographical location and seasonal conditions (Wonni et al.
2016). Infection occurs when bacteria (Xoo) penetrate infection courts (leaves) through wounds or
hydathodes, and then spread through the leaf and
colonize xylem vessels. There are several identified pathovars, and they defeat the plant defense system and induce a
set of diverse pathways that ultimately result in a successful infection and bacterial leaf blight disease.
Primarily, seedlings and adult
plants are infected by the Xoo
pathogen, resulting in wilting of young plants and leaf blight symptoms, a
syndrome referred to as kresek (Wonni et al.
2014). Infected, symptomatic plants also display pale yellow leaves.
Symptoms are typically observed in young plants of susceptible cultivars in the
tropics at tillering stage. At this
infection stage, the infected plants may roll and wilt as well as turn
grey-green. Entire plants may even eventually die (Wu et al. 2011).
Bacterial leaf blight disease incidence has increased recently most likely
owing to the use of intensive agronomic practices that generate conditions
favorable to the development of this disease, such as high rates of nitrogen
fertilizers, close spacing and continuous cropping with susceptible cultivars (Gale et al.
1985).
Antibiotic development and use
to control such a pathogen on a commercial, agricultural scale is unlikely and
impractical (Gnanamanickam et al. 1999). Therefore, the most reliable method of control
is to use genetically resistant cultivars. Several cultivars containing known
resistant loci have been identified (Zheng et al.
2009). Continued identification of diverse BLB resistant and susceptible
cultivars using conventional screening techniques is hindered by environmental
effects. However, known, environmentally independent molecular markers
associated with foliar disease resistance have accelerated the identification
processes of resistant genotypes. To date, more than 38 loci have been
identified as conferring strong resistance against different strains of Xoo in rice (Jeung et al. 2006; Niño‐Liu et al. 2006;
Pradhan et al. 2015; Dilla-Ermita et al.
2017; Nguyen et al. 2018),
which are referred to as resistance or ‘R’
genes.
The recent, exponential
progress in rice genomics research and the successful completion of sequencing
the rice genome is allowing researchers to precisely identify many
agronomically important genes. To date, four of the identified Xa-R genes
have been cloned and extensively studied (Blair
and McCouch 1997; Iyer and McCouch 2004,
2007; Salgotra et al. 2011; Singh et al.
2015). R gene Xa1 confers resistance to race 1 isolates of
Xoo in Japan and Xa2 to the Japanese Xoo strain T7147 (Sakaguchi
1967). Xa4 confers durable
resistance in Asian rice (Mew et al. 1992). xa5 (Iyer
and McCouch 2004) and xa13 (Chu et al.
2006; Antony et al. 2010) are
recessive R genes that only confer
resistance when they are present in their homozygous state, whereas Xa21 is a dominant R gene that confers broad spectrum
resistance against Xoo strains
belonging to different races of the pathogen (Song
et al. 1995).
Many practical markers for
tagging and marker assisted selection are microsatellites
since they are co-dominant, PCR-based, and can detect high levels of
polymorphism. Several DNA marker types, including microsatellites, have been
used to investigate rice cultivars carrying BLB resistance genes, and rice
breeding programs benefit from the identification of resistant germplasm and resistance genes. Therefore, the aim of this
study was to screen different rice ecotype cultivars originating from Ghana for
varietal resistance to BLB disease. The
specific objectives were to: (1) assess the phenotypic response of 10 rice
cultivars following BLB inoculation, (2) conduct a genotyping-based assessment
to determine the presence or absence of resistance alleles Xa2, Xa4, xa5, xa13 and Xa21 using
polymerase chain reaction (PCR) -based molecular markers (STS and SSR), and (3)
investigate the expression of the plant defense-related genes, OsWKY45, OsJAZ8, OsPR1a and OsPR10b among selected rice cultivars.
Materials and Methods
Materials
Ten rice genotypes, six of which are local rice
accessions from Ghana, including Popa Tos13150, IRAT 10, Kabre, Tinsibe,
AGRIC-1 and Krampa White, and four resistant and susceptible controls, were
used to conduct this study (Table 1). Cultivars Tetep (Blair and McCouch 1997) and Jinbeak (Kim et al. 2009) served
as resistant controls, whereas Nampyeong (Fred et al. 2016) and IR661 served as
susceptible controls to Xoo K1
strain. All of the Ghanaian cultivars as well as Tetep and IR661were O.
sativa subsp. indica, whereas as Jinbeak and Nampyeong were O.
sativa subsp. japonica. Samples of all 10 rice cultivars were
provided by the National Agro-biodiversity Center (NAC) in Jeonju, Republic of
Korea.
Table 1:
Characteristics and source of collection of rice genotypes used for the studies
|
Rice
Varieties |
Subspecies |
Country
of Origin |
Source |
|
Tetep |
Indica |
China |
NAC-
South Korea |
|
IR661 |
Indica |
Philippines
|
NAC-
South Korea |
|
Jinbeak |
Japonica |
Korea |
NAC-
South Korea |
|
Nampyeong |
Japonica |
Korea |
NAC-
South Korea |
|
Popa Tos 13150 |
Indica |
Ghana |
NAC-
South Korea |
|
IRAT 10 |
Indica |
Ghana |
NAC-
South Korea |
|
Kabre |
Indica |
Ghana |
NAC-
South Korea |
|
Tinsibe |
Indica |
Ghana |
NAC-
South Korea |
|
AGRIC -1 |
Indica |
Ghana |
NAC-
South Korea |
|
Krampa White |
Indica |
Ghana |
NAC-
South Korea |
Table 2: BLB disease severity and evaluation
scale
|
Disease rating |
Lesion
size (% of leaf length) |
Interpretation
|
|
0 |
0 |
Immune
(I) |
|
1 |
>1-10
% |
Resistant
(R) |
|
3 |
>11-30
% |
Moderate
Resistant (MR) |
|
5 |
>31-50
% |
Moderately
Susceptible (MS) |
|
7 |
>51-75
% |
Susceptible
(S) |
|
9 |
>76-100
% |
Highly
Susceptible (HS) |
Source: (Chaudhary 1996)
Experimental design and growth conditions
This study was conducted under greenhouse
conditions at Kyungpook National University, Daegu,
Republic of Korea. The experiment was a complete randomized design with
three replicates. Seeds of each of the 10 cultivars were sown in Petri dishes, incubated and germinated at ± 25°C for two weeks. Approximately 14 day-old seedlings
were then transplanted into 50 cm diameter plastic pots and kept under greenhouse conditions. Plants
were grown under a
16 h/8 h light and dark cycle at a temperature ranging between 25 and 30°C in the greenhouse.
Pathogen growth and inoculation to plants
The Xoo, K1 strain (K1 race) is a
Korean strain of X. o. pv. Oryzae, and was obtained from the National Agrobiodiversity
Center in Jeonju, Republic of Korea. Bacterial cultures were grown and
incubated on potato sucrose agar (PSA) Petri plates prepared using 5 g Bacto-peptone (Becton, USA), 0.5 g sodium L-glutamate
monohydrate, 5 g sucrose and 8 g Bacto-agar at 30°C overnight. Single colonies
were picked and grown on PSA medium at 30°C overnight. Bacterial counts were
then adjusted to 0.002 CFU/mL by measuring the optical density of the culture
at 600 nm using a spectrophotometer as described previously (Yin et al.
2017).
Three
replicates of fully expanded leaves of well-acclimatized plants were inoculated
with Xoo culture 40 days after germination. Three leaves
per plant were inoculated through the leaf clipping method (Kauffman 1973). A 2 cm piece from each leaf
tip was clipped using a sterile scissor and dipped into the bacterial solution
(0.002 CFU/mL). Negative controls were mock inoculated using only sterile
distilled water. Plants were kept at 35 ± 2°C under greenhouse conditions, and
symptoms development was closely monitored.
Measurement of disease severity and pathogenicity assessment
After inoculation, leaf samples were collected at
three set time points, 4, 10, and 14 days’ post inoculation (dpi), in order to
observe the response of inoculated cultivars to Xoo inoculation. To confirm and evaluate Xoo infection, leaf
extract was spread on PSA medium with cephalexin, and colony counts were
recorded (Wang et al. 1996). The identification of Xoo specific symptoms was performed based on morphological
characteristics previously described (Swings et al. 1990) and further
confirmed through 16srRNA sequencing (Zhang et
al. 2000). The sequencing results are reported in Fig. S1.
Disease severity was scored
using a previously described disease rating scale (Gnanamanickam et al. 1999;
Waheed et al. 2009). Scoring
was performed 14 dpi. Disease symptoms were recorded from the leaf tip to the
base of the blade (Gourieroux et al. 2017). The lesion size
percentage was recorded using the equation below as described by Kauffman (1973).
%201233-1241_files/image002.png){kind=small}
The BLB disease severity scoring was classified using a disease index scale listed in Table
2 (Chaudhary 1996).
STS/SSR markers analysis
Approximately 20-day old leaves were collected
from each cultivar for STS/SSR marker analysis. DNA was extracted using the
CTAB method as described by Goto et al. (1999). The concentration
and quality were checked using NanoQ (Optizen, South Korea). Four previously
reported SSR markers and one STS marker (RM-317,
RM-224, RM-13, xa-13prom and pTA248, respectively) were used to screen 10 rice cultivars for the
absence or presence of five common BLB resistance loci linked to Xa2, Xa4, xa5, xa13 and Xa21 R genes, respectively (Singh et al.
2015). There are 40+ known Xa-R genes, and these were chosen due
to the fact that they tend to confer broader resistance as opposed to
race-specific resistance.
A 20 µL reaction mixture, including 2X F-Star Taq PCR Master mix
(BioFact™, South Korea) and 10 µM of
each marker specific forward and reverse primers, was used to amplify the
selected DNA markers (Applied Biosystems, California, U.S.A.). Additional
information on the markers used is provided in Table S1. The PCR conditions
were as follows: initial polymerase activation at 94.0°C for 2 min followed by
35 cycles of 94.0ºC for 15 s, 58.5ºC–61.4ºC for 30
s (optimized individually for each marker primer) and 72.0ºC for 1 min
30 s with a final extension of 72.0ºC for 5 min. Amplified PCR products were analyzed using gel electrophoresis with a 3%
agarose gel and visualized with a gel documentation system (Uvitec
Cambridge, UK).
Quantitative real-time PCR
(qRT-PCR) analysis
RNA extraction and qRT-PCR
were performed as described in Imran et al. (2018). Briefly, total RNA
was extracted using the TRIzol® reagent method. The quality and
quantity of RNA were checked with agarose gel electrophoresis and NanoQ
(OPTIZEN, South Korea), respectively. Complementary DNA (cDNA) was synthesized
as described by Imran et al. (2018). A two-step real-time PCR reaction was
performed using an Eco TM real-time PCR system (Illumina,
California, U.S.A.) using 2x Real-Time PCR Master mix including SYBR Green I
(BIOFACT, South Korea) with 100 ng of template DNA and 10 nM of each primer in a final volume of 20 µL. The PCR conditions were polymerase activation at 95°C for 15
minutes and concurrent denaturation at 95°C, annealing and extension at 60°C
for 40 s for a total 40 cycles. The primer list is given in Table S2.
Statistical analysis
Table 3:
Marker results of 10 accessions screened for BLB resistance. Cultivars are
listed based on lesion % for both the control and Ghanaian cultivars from
lowest to highest. Positive (+) and negative (-) signs indicate the presence
and absence of Xa R genes in a particular cultivar,
respectively
|
Category |
Rice Subspecies |
Accession Number |
Local Name |
BLB resistance genes |
||||
|
Xa2 |
Xa4 |
xa5 |
xa13 |
Xa21 |
||||
|
Controls |
|
|
|
|
|
|
|
|
|
Resistant |
japonica |
|
Jinbeak |
- |
- |
+ |
- |
- |
|
Resistant |
indica |
IT 102103 |
Tetep |
+ |
- |
+ |
- |
- |
|
Susceptible |
indica |
IT 001944 |
IR661 |
- |
- |
+ |
- |
- |
|
Susceptible |
japonica |
|
Nampyeong |
- |
+ |
- |
- |
- |
|
Ghanaian cultivars |
|
|
|
|
|
|
|
|
|
Resistant |
indica |
IT 226965 |
Popa |
- |
- |
+ |
- |
- |
|
Susceptible |
indica |
IT 283479 |
Krampa White |
+ |
+ |
- |
- |
- |
|
Susceptible |
indica |
IT 226946 |
Kabre |
- |
+ |
- |
- |
- |
|
Susceptible |
indica |
IT 267919 |
AGRIC -1 |
+ |
- |
- |
- |
- |
|
Susceptible |
indica |
IT 214850 |
IRAT 10 |
- |
- |
+ |
- |
- |
|
Highly susceptible |
indica |
IT 226964 |
Tinsibe |
+ |
- |
+ |
- |
- |
|
Approx. size (bp) |
|
|
|
154 |
160 |
139 |
498 |
982 |
Resistance and susceptibility to Xoo strain K1
GraphPad Prism
7.03 (GraphPad, California, U.S.A.) was used to
perform an analysis of variance (ANOVA) on the experimental data. Means were
separated using least significant difference (LSD) at a 5% probability level.
DNA banding profiles were recorded as present or absent. Relative expression
levels were determined by comparing treated and control plants both normalized
to OsUBI.
Results
Genotypic screening for BLB resistance locus Xa
The presence or absence of resistant locus Xa was evaluated in all cultivars
through marker genotyping and is shown in Table 3. Tested cultivars received a
negative score if no Xa R gene was
present for each corresponding SSR or STS marker, a non-specific band was
amplified, or a band size corresponding to susceptible genotypes was present.
When the presence of an expected size SSR or STS band was present, cultivars
were scored positive. The genotyping results revealed that Ghanaian cultivars,
Tinsibe, AGRIC-1 and Krampa White, carry Xa2; Kabre and Krampa White carry Xa4; and Popa and IRAT10 carry xa5. However, none of the 10 cultivars showed the presence of xa13 and Xa21(Fig. S2;
Table 3). Tetep, the O. sativa subsp. indica resistant control harbors Xa2 and xa5, and the susceptible IR661 only carried xa5. Interestingly, Jinbaek, the O.
sativa subsp. japonica resistant
control, only harbored xa5 out of the
five BLB R genes screened for as
well, while the susceptible Nampyeong carries Xa4.
Symptoms development in selected rice cultivars to Xoo bacterial inoculation
All of the 10 cultivars mentioned in Table 1 were
screened in a pilot experiment, particularly for their response to Xoo inoculation. Based
on the initial screening results (Fig. S3), five cultivars were
selected, which included Tetep, Jinbaek, Popa, Tinsibe and AGRIC-1, for further
testing. The results suggest that the Ghanaian cultivar Tinsibe was the most
susceptible cultivar tested, and it showed severe symptom development (Fig. 1A)
followed by AGRIC-1. The known South Korean cultivar Jinbaek showed the most
resistance to Xoo infection followed
by Tetep (Fig. 1A). The Ghanaian cultivar Popa also showed resistance to Xoo K1 strain, and a similar response
was observed in Tetep (Fig. 1A).
The initial symptoms that
appeared as colonies were circular, convex and light yellow that looked smooth.
It took 3–5 dpi for the yellow pigment on the leaf
to appear in the form of a curl. Symptom development was
further quantified by measuring lesion length. The disease score was calculated
as the percentage of the lesion length of each cultivar relative to the total
leaf length. Jinbaek was observed to be the most resistant cultivar and
exhibited a shorter lesion length at all time-points (Fig. 1A), whereas Tinsibe
was the most susceptible, showing significantly longer lesion length compared
to other cultivars (Fig. 1A). At 4 dpi, there was no significant difference in
lesion length among cultivars. However, at 10 dpi, the invasion of the pathogen
accelerated and was traceable through the development of symptoms in
susceptible genotypes. Jinbaek had a mean lesion length of 0.29 cm, while
Tinsibe had one of 4.33 cm (Fig. 1B). Similarly, at 14 dpi Tinsibe showed the
longest mean lesion length (8.52 cm), and Jinbaek showed the shortest mean
lesion length (0.41 cm). Among all Ghanaian cultivars, Popa was the most
resistant with a mean 0.9 cm lesion length at 14 dpi compared to Tinsibe and
AGRIC-1 having 7.8 and 4.2 cm mean lesion lengths, respectively (Fig. 1B).
Pathogenicity assessment of Xoo
To assess the pathogenicity of Xoo K1 strain in different cultivars, the disease severity percentage was
calculated to give a better understanding of the individual varietial responses
to Xoo K1 infection. The
results showed that Tetep (4.9%), Jinbeak (1.4%) and Popa (3.1%) had the lowest
disease severities (percentage of leaf length affected), and were subsequently
classified as resistant based on the standard scoring proposed by Chaudhary (1996). Moderate susceptibility was
observed in AGRIC-1 (31.5%), whereas Tinsibe (77.9%) was highly susceptible
(Fig. 2).
%201233-1241_files/image004.jpg)
Fig. 1:
Response of different rice
cultivars towards attempted Xoo infection. (A) Symptom development after 14 dpi. The leaves on the right are inoculated with Xoo, whereas those on the
left were mock inoculations only
containing sterile water. (B)
Quantification of lesion length in cm of select cultivars. Each data
point is the mean of three replicates. Error bars indicates means ± S.E. (n =
3)
%201233-1241_files/image006.jpg)
Fig. 2:
Disease severity scoring by percentage of five representative cultivars. The scale rating used based on Chaudhary
(1996). All the data points represent the mean of three replicates. Error bars
represent ± SE
Gene expression (transcript levels) of pathogenesis-related genes
To determine whether disease severity or resistance
is related to pathogenesis-related (PR) genes, the expression of known PR genes
was evaluated in the selected cultivars 4 dpi with Xoo. This time point was selected since it was when the cultivars
typically started to show symptoms of Xoo
infection (Fig. 1). The
results showed that a jasmonic acid (JA)-mediated BLB resistant gene, OsJAZ8, was up-regulated in Jinbaek and
AGRIC-1 and down-regulated in Popa and Tinsibe compared to control plants (Fig.
3A). Tetep did not show any significant difference in OsJAZ8 gene expression (Fig. 3A). Similarly, the expression of OsWRKY45, another disease-related gene,
was up-regulated only in AGRIC-1 and down-regulated in all other tested
cultivars (Fig. 3B). Furthermore, the expression of salicylic acid (SA)-pathway
related genes, OsPR1a and OsPR10b, were evaluated, and the results showed that Jinbaek and
Tinsibe have increased transcript accumulation of OsPR1a and the other cultivars had a decrease in expression level
compared to control plants (Fig. 3C). Transcript accumulation of OsPR10b was highest in Tetep followed by
Tinsibe and Jinbaek and down-regulated in Popa (Fig. 3D).
%201233-1241_files/image008.jpg)
Fig. 3: Early transcript accumulation at 0, 6, 12,
24, and 48 h after infection with Xoo of (A) OsPR10b and (B) OsWRKY45. Plants of three selected cultivars were inoculated with Xoo and the expression of the indicated genes was measured overtime relative to OsUBI expression
through qRT-PCR. Values are means ± SE of three
replicates. Five leaf blades were pooled to make one replicate with three
replicates in total
Early transcript accumulation of
OsWRKY45 and OsPR10b in selected cultivars
To
understand the early response of two important biotic stress related genes, OsWRKY45 and OsPR10b, to Xoo
infection, three cultivars, Tetep (resistant control indica), Tinsibe
(susceptible) and Jinbaek (resistant control japonica), were selected
and inoculated with Xoo K1 strain.
Gene expression was measured at 0, 6, 12, 24 and 48 h post Xoo infection. The expression of OsPR10b was up-regulated only in the resistant cultivars Tetep and
Jinbeak, particularly at 12 h post inoculation, whereas it was down-regulated
in the susceptible cultivar Tinsibe. These findings indicate that OsPR10b is positively correlated with
plant resistance against Xoo and that
the resistance of Tetep and Jinbaek may be due to an early increase in the
expression of OsPR10b (Fig.
4A). Furthermore, the expression of OsWRKY45
was down-regulated in response to Xoo
inoculation in all cultivars at all-time points (Fig. 4B).
Discussion
Verdier et al. (2012) hypothesized that African wild rice germplasm likely contains novel resistance
loci and can serve as valuable genetic resources for identifying and deploying
new R genes. To better understand the
available resistance in Ghanaian rice germplasm, six West African rice
cultivars were evaluated first phenotypically for their response to the Korean Xoo
K1 strain and then genotyped with five of the more common Xa-R gene markers.
In the current study, three of the Ghanaian rice cultivars, Krampa White,
Tinsibe and AGRIC-1, harbored the Xa2 R gene like the resistant control
Tetep. However, while Krampa White and Tetep had a resistant phenotype, Tinsibe
and AGRIC-1 were susceptible to Xoo K1 strain. While Xa2 may
function as and R gene in Asian cultivars, this inconsistency in
phenotypic response to Xoo infection in the West African cultivars
evaluated suggests that it does not function in the same way in that genetic
background.
The Xa4 gene is reportedly one of the most widely studied resistance
genes in many Asian rice breeding programs, and it confers durable resistance
in many commercial rice cultivars (Mew et al. 1993). It has been mapped
on to chromosome 11 with a linkage distance of 1.0 cM to the widely used,
reliable RM-224 marker (Sun et al. 2003). Our results revealed the presence of Xa4 in two resistant Ghanaian cultivars,
Kabre and Krampa White, and in the Korean susceptible, control japonica cultivar
Nampyeong. While Nampyeong showed mild susceptibility to Xoo, the
African cultivars Kabre and Krampa White may owe their resistance status to the
presence of Xa4, and it could be of use in future BLB resistance
breeding. However, Kabre and Krampa White showed some susceptibility during
initial screening. This inconsistency may be due to a masking effect of other
genes in these cultivars or any number of epistatic interactions, reducing the
expression of their resistance. It could also be due to the virulent nature of
the K1 Xoo strain used in our study,
and Xa4 may not impart complete
resistance to the Xoo K1 race in
these cultivars. Still, Xa4 may
confer resistance to other Xoo races.
The SSR marker RM-13 was used
to screen for resistant gene xa5,
which maps at a linkage distance of 17.9 cM (Blair
and McCouch 1997). This potential resistance gene is frequently present
in indica cultivars. Blair and McCouch (1997) studied
microsatellites and sequence-tagged sites diagnostic for the rice BLB resistance
gene xa5 in 122 rice accessions, and they found that for all of the genotypes evaluated, xa5
donor and recurrent parents had indica backgrounds. xa5
was absent from all of the japonica
genotypes from South Korea. Furthermore, Busto et al. (1990) showed that the xa5 gene is more pronounced among
isozyme group II, which is a distinct
group derived from the indica subspecies. This study suggested
that the center of origin of xa5 is
likely the Indian subcontinent (Nepal,
Pakistan, India and Bangladesh), especially
given that xa5 has not been
found in any varieties from South Korea, Japan, Taiwan or the Philippians (Busto et al.
1990). In the current study, this marker was identified in the West
African cultivars Popa, IRAT 10 and Tinsibe, as well as both the resistant and
susceptible indica controls Tetep and
IR661. Since the marker occurred in the most resistant and susceptible
cultivars tested, xa5 likely does not play a role in the resistance
phenotype to Xoo K1 strain.
xa13 gene is fully
recessive, conferring resistance only in the homozygous state (Khush and Angeles 1999; Chu et al. 2006). Gene expression studies using pathogen-induced
subtractive cDNA library analysis have
revealed that some defense responsive genes activated in xa13-mediated resistance are not controlled by dominant R genes (Wen
et al. 2003). For the
resistance to detectable, the cultivars tested would have needed to be in a
homozygous state. Unfortunately, the evaluation of the presence of the
resistance gene xa13, which maps at a distance of 3.7 cM,
resulted in a single band approximately 280 bp in all the rice cultivars
studied (Fig. S2). No polymorphism was detected in any of the cultivars, which indicates that xa13 was absent or it could not be detected by the xa-13prom (SSR) due to some recombination in
the 3.7 cM region between the xa13
locus and marker.
In addition, no amplicon specifically visualized to Xa21, which encodes a leucine-rich repeat receptor-like kinase,
were detected for any of the 10 rice cultivars evaluated. Therefore, both xa13 and Xa21 were determined to be absent from all the accessions
evaluated. Similar results were also reported on Indian rice genotypes used in
other breeding programs (Davierwala et al. 2001; Singh et al. 2013). Meanwhile, Song
et al. (1995) showed that rice
cultivars carrying Xa21 are able to
induce an effective defense response to multiple strains of the bacterial Xoo pathogen. Moreover, many genes that
are required for Xa21 gene activation
mediated immunity have been identified in Xoo
(Shen et
al. 2002).
Out of six
Ghanaian local cultivars, only two of them harbored more than one of the
resistance genes screened for. Tinsibe possessed Xa2 and xa5, and Krampa
White harbored Xa2 and Xa4.
Interstingly, Gonzalez et al. (2007)
reported that Xa2 and Xa4 have race-specific resistances to
African Xoo strains. These
observations are similar to those reported by Ullah
et al. (2012) that showed that
out of 52 materials, only 10 basmati rice landraces
had multiple resistance genes. All Ghanaian cultivars harbored one or two genes
(Xa2, Xa4, and xa5), and the
presence of Xa-R genes already
in local Ghanaian backgrounds, either individually or in combination, may be
useful for providing resistance against African strains of Xoo (Verdier et al. 2012).
The most resistant control,
Jinbeak, possessed xa5 (Table 3). One
study regarding the response of Jinbaek to BLB reported that this rice cultivar
exhibited resistance against Xoo K1,
K2, K3, and K3a infection (Kim et al. 2009). The authors also
found that Jinbaek carries Xa3 in addition to xa5. Therefore, the reported resistance phenotypic response of
Jinbaek to K1 infection in the current study supports that xa5 may have some race specificity to K1 strains, which likely
contributes to much of its resistance to BLB.
These findings indicated that local African cultivars as well as other landrace
cultivars conserved by farmers show potential for discovering previously
unknown resistant lines useful in future breeding programs. Many cultivars
showed the presence of one or more genes responsible for Xoo resistance. However, most of the cultivars were found to be
susceptible to Xoo K1 strain
infection indicating that these Xa-R
genes may be non-functional or race specific. Screening these cultivars against
Xoo strains occurring in Western Africa would better determine the level
of field resistance these cultivars have.
Based on initial screening,
five cultivars were selected to be evaluated for their response to Xoo. Similar disease and symptom
development observations consistent with those of this study were made by Kauffman (1973) and Noor et al. (2006) who reported that the Xoo appearance occurs within 4–5 dpi in the form of leaf curling.
None of the cultivars were found without lesions at 14 dpi, which indicates
that none of the genotypes were completely resistant or immune to Xoo. This also validates that the K1
strain of Xoo used in this study was
virulent.
In addition, the resistant
cultivars, Jinbaek, Tetep and Popa, showed symptom development at a later stage
(i.e., 11 dpi), which suggests that
the pathogen is able to infect and start causing damage in susceptible
cultivars earlier, whereas relatively resistant varieties seem to initially
inhibit the bacteria from causing infection or disease. In a similar study, Singh et al.
(2013) reported that the first symptoms of BLB appeared 7 dpi in
moderately susceptible cultivars. AGRIC-1 showed moderate susceptibility, which
is consistent with the findings by Agaba et al. (2015). The
Ghanaian cultivar Popa shows phenotypic resistance to BLB at 14 dpi,
making it a good candidate for inclusion in BLB resistance breeding programs.
Plants have evolved disease resistance in response to
pathogen attack by activating systems controlled through various signaling
pathways. Diverse regulatory pathways have been identified and are possible
targets for plant genetic manipulation for disease resistance. Both JA- and SA-signaling pathways have been identified for
the use of mediating responses against these infectious diseases in plants. The
JA-induced OsJAZ8 was up-regulated in
AGRIC-1 and Jinbaek, both cultivars at least somewhat resistant to BLB, whereas
it was down-regulated in the susceptible cultivar Tinsibe (Fig. 3A). The
induced expression of OsJAZ8 may be
due to the inoculation method that consists of cutting the leaf that induced a
wound response, a JA-related process. The inclusion of healthy, uninoculated
controls in future studies will assist in determining if inoculation method is
a factor affecting expression. Conversely, in the well-studied resistant
cultivar Jinbaek, a reduction in the expression of OsWRKY45 was observed, which may be due to the negative regulation
of disease by OsWRKY45 consistent
with that described by Huangfu et al. (2016). Other reports have
confirmed that SA-mediated plant defense
signaling pathways are present in rice (Silverman et
al. 1995; Yang et al. 2004).
Pathogenesis-related
proteins, whose production and accumulation have
been reported to be a vital component of the active plant defense repertoire, are increasingly studied as
important factors in disease resistance (Agrawal
et al. 2001). Results of the
current study showed increased transcript accumulation of OsPR1a in Jinbaek and Tinsibe and of OsPR10b in Tetep followed by Tinsibe (Fig. 3C, D). OsPR10b was likely mainly responsible
for the SA-induced PR gene expression owing to the fact that the transcript
accumulation of OsPR10b was almost 10
times higher than that of OsPR1a
expression. This hypothesis is also consistent with other studies (Jwa et al. 2001).
The increased expression of PR-related genes in Tinsibe was unexpected and
inconsistent with the observed susceptible phenotypic response of this
cultivar. However, this increased OsPR10b
accumulation may be due to delayed induced expression.
Fred et al. (2016)
conducted a comprehensive screening of various rice genotypes for BLB
resistance to Korean K1 strain, and the findings reported showed that the
phenotype recorded during the experiment and the expression patterns of OsNPR1, OsPR1a, OsWRKY45, etc. were not well correlated. The
transcript accumulation of these genes was also found to be unexpectedly
down-regulated in resistant cultivars leading Fred
et al. (2016) to conclude that
OsPR10b was the only defense-related
gene with a coherent transcriptional pattern correlated with the observed
phenotype in resistant and susceptible genotypes. Given findings from previous
studies, the early response of susceptible and resistant cultivars to Xoo was determined by studying OsPR10b and OsWRKY45 expression (Fig. 4A, B). It was confirmed that Tinsibe
showed a reduction in transcript accumulation of OsPR10b overtime, while Tetep and Jinbaek showed an increase in transcript accumulation at
early time points (Fig. 4A). This indicates that PR-related gene expression
shows early response to infection, and it subsequently returns to the basal
level to reduce cellular metabolism and store energy for other processes.
Conclusion
This study identified resistant and susceptible
Ghanaian rice cultivars to Xoo K1 strain
infection. Popa exhibited the highest resistance level to Xoo among all Ghanaian genotypes. These findings suggest that Popa
is a promising candidate cultivar that may be widely utilized in the Ghanaian
agricultural system and may contribute to improving BLB disease management.
Furthermore, Popa could also be included in plant breeding programs in Ghana
using available modern breeding technologies.
In addition, Xa4 may
account for some resistance in Ghanaian rice cultivars to Xoo, but
marker and phenotypic data were largely inconsistent. While the markers
screened for in this study may not provide much insight into resistance status
of West African rice cultivars, it highlights the diversity of R genes
responsible for resistance, and suggests that resistance in West African rice
may rely on Xa-R genes or alleles as yet unreported. This unique West
African germplasm will likely be useful in future works determining different
modes of action/ mechanisms of resistance, novel resistance alleles or loci,
and/or different epistatic interactions related to vast differences in
genotypic background.
Acknowledgements
We are thankful to the Korea International
Cooperation Agency (KOICA) for support.
Author
Contributions
EF, NCH and HHK: conducted the experiments; EF: wrote the
manuscript; NKR and WNJ: helped in the experiments; NKR, QMI, BGM, and AH:
analyzed the data and reviewed the manuscript for its technical content’ BWY:
designed and supervised the study, and mobilized funding.
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