Screening Rice Cultivars for Resistance to Bacterial Leaf Blight

Abstract

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious threats to rice production. In this study, screening of rice for resistance to BLB was carried out at two different times and locations; that is, in a greenhouse during winter and in an open field during summer. The pathogenicity of Xoo race K1 was tested on 32 Korean rice cultivars. Inoculation was conducted at the maximum tillering stage, and the lesion length was measured after 14 days of inoculation. Five cultivars, Hanareum, Namcheon, Samgdeok, Samgang, and Yangjo, were found to be resistant in both the greenhouse and open-field screenings. Expression of the plant defense-related genes JAmyb, OsNPR1, OsPR1a, OsWRKY45, and OsPR10b was observed in resistant and susceptible cultivars by qRT-PCR. Among the five genes tested, only OsPR10b showed coherent expression with the phenotypes. Screening of resistance to Xoo in rice was more accurate when conducted in open fields in the summer cultivation period than in greenhouses in winter. The expression of plant defense-related genes after bacterial inoculation could give another perspective in elucidating defense mechanisms by using both resistant and susceptible individuals.

Introduction

Owing to an increasing world population, expected to reach eight billion people by 2020 [14], the world’s most challenging problem is to feed the growing population; as the population increases, food production decreases owing to a lack of suitable land for crop cultivation. However, rice breeders face different environmental challenges, including bacterial pathogens that cause diseases. The Xanthomonas genus causes serious bacterial leaf blight in many crops such as cassava and rice via a gram-negative bacterium [3,27]. In rice, it causes annual yield losses conservatively estimated at 50% [24]. This bacterium causes reduction in total dry matter weight of rice, poor maturation, and broken grain during milling. Bacterial leaf blight (BLB) is a vascular disease that causes a white-yellow or tannish-grey discoloration in the rice crop along the veins, leaf margins, and leaf blades, and these lesions may extend to the sheath [10]. There is some research directed at understanding the principles underlying the interaction between the pathogen and its host, leading to either a compatible or an incompatible disease reaction. When rice is infected by Xanthomonas oryzae pv. oryzae (Xoo), although the symptoms of the disease may be observed at the tillering stage, the disease may continue to increase as the plant grows. It was observed that rice plants at less than 21 days old are more susceptible to the disease and that the bacteria may favor temperatures at 28–34℃ for growth. The improvement of host resistance and the application of chemical and biological measures have been used for the control of BLB [2]. However, owing to the breakdown of resistance in many resistant cultivars, their useful lifespan is only a few years. Therefore, it is important to screen rice for bacterial leaf blight resistance in order to assess the diversity within the germplasm and to provide information about resistance/susceptibility for further use in breeding practice [5]. Moreover, screening for varietal resistance based on artificial inoculation may always be conclusive because of to the presence of adequate inoculation initiating the disease [20]. In the present study, 32 Korean rice cultivars were observed for their reaction to Xoo race K1. Resistance to BLB in a greenhouse during winter and in a rice field during the summer rice cultivation period was screened and compared. Moreover, some genes related to disease response in plants are known; JAmyb is one of the genes associated with reactions to biotic and abiotic stress. The expression of JAmyb suggests its involvement in JA-mediated disease responses [15]. The OsNH1 gene (also known as OsNPR1) is thought to be involved in down-regulation of gene expression, those also involved in photosynthetic activity. The overexpression of OsNPR1/NH1, a rice ortholog of NPR1, reinforces resistance to bacterial blight disease [6,26]. The OsPR10b gene is known as an inducer of biotic and abiotic stress responses, including to pathogen infection, and transcripts of PRP10b also were enhanced by Magnaporthe grisea [19]. OsPR1a is highly responsive to plant wounds by cutting and exogenous application of phytohormones [1]. OsWRKY45 has a role in regulating a defense-related response gene and it gives an enhanced blast resistance in rice [23]. Hence, the expression of these five defense-related genes in rice during Xoo infection was investigated.

 

Materials and Methods

Plant Materials and Field Trials

Thirty-two rice cultivars were used in this study. Field trials were conducted during the winter of 2014 at an experimental greenhouse at Kyungpook National University, Daegu, and during the summer of 2015 in a field at the Kyungpook National University Experimental Station, Gunwi, Korea. The seeds were force sprouted in an incubator at 28℃ for 3 days and sown in a 50-hole tray. The seed trays were maintained in dark conditions in the greenhouse used for germination for 4 days and seeds were germinated. Fifteen seedlings per cultivar were transplanted in a planting row of paddy field with a planting density of 30 × 15 cm. The amount of fertilizer applied was N-P2O5-K2O = 9.0-4.5-5.7 kg/10 a.

Bacterial Inoculation and Phenotype Analysis

The bacterial pathogen Xoo race K1 was cultured on peptone sucrose agar medium containing 2% sucrose (w/v), 2.5% peptone (w/v), 0.05% K2PO4 (w/v), and 0.025% MgSO4·7H2O (w/v) at pH 7.0. The bacterial culture was suspended in sterile water and adjusted to a concentration of OD600 = 1.0. Five replications of each cultivar were inoculated at the maximum tillering stage by cutting rice leaves using scissors dipped in the suspended solution [12]. The severity of BLB infection was determined by measuring the length of lesions (LL) on infected leaves at 14 days post-inoculation. Plant reactions to BLB were classified as resistant, moderately susceptible, or susceptible depending on LL, and the principle for classification of resistance in this study was determined by the range in disease severity.

Gene Expression Analyses

RNA was isolated from leaf tissue at 4 days post-inoculation using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. A hundred milligram plant sample was flash-frozen in liquid nitrogen, ground by mortar and pestle, and put into a 2 ml microcentrifuge tube. Next, 450 μl of Buffer RLT was added, and the mixture was vortexed and incubated for 1–3 min at 56℃. The lysate was transferred to a QIAshredder homogenizer in a 2 ml collection tube, and centrifuged for 2 min at 13,000 rpm. Following centrifugation, the supernatant was transferred to a new 1.5 ml microcentrifuge tube along with 0.5 ml of 96% ethanol. The mixture was transferred to an RNeasy spin column placed in a 2 ml collection tube, centrifuged for 1 min at 13,000 rpm, and the flow-through was discarded. Similarly, 700 μl of Buffer RW1 was added to an RNeasy spin column, centrifuged for 1 min at 13,000 rpm, and the flow-through was discarded. The RNeasy spin column was washed twice with 500 μl of Buffer RPE followed by centrifugation for 1 min at 13,000 rpm and the flow-through was discarded. Finally, the RNeasy spin column was placed in a 1.5 ml collection tube and the total RNA was suspended in 30 μl of RNase-free water by centrifugation for 1 min at 13,000 rpm. Complementary DNA (cDNA) synthesis was conducted using a cDNA synthesis kit (Philekorea Technology Inc., Korea) by following the manufacturer’s protocol. In a total reaction volume of 20 μl, 2–2.5 μg of total RNA was mixed with 1×cDNA synthesis mix (3 mM MgCl2, 1 mM dNTPs, enhancers, and stabilizers) and 1× MMLV Reverse Transcriptase (RTase). The reaction was carried out at 42℃ for 30 min. To stop the reaction, the reaction mixture was incubated at 70℃ for 10 min to denature the RTase. Finally, cDNA was purified by ethanol precipitation before being suspended in nuclease-free water. Expression profiles of several plant defense-related genes (i.e., JAmyb, OsNH1, OsPR1a, OsPR10b, and OsWRKY45) were generated using the Eco Real-Time PCR System (Illumina, USA). Relative expression levels were determined by comparing the expression level of each gene tested from the treated and control plants and normalized to Actin1 [18]. Primers (Table 1) were designed using Primer3 (http://bioinfo.ut.ee/) set at the following parameters: sequence length 18–25 bp, product size 100–200 bp, GC content 45–55%, annealing temperature 59.5–60.5℃, and bridge exon-exon junctions if the gene contained more than one exon. In total, the reaction mixture was 20 μl and contained 90 ng of cDNA template, 0.4 μM of each forward and reverse primer, and 1× qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystems Ltd., London, UK). All analyses were conducted in triplicates.

Table 1.Primer sequences of the genes used for quantitative real-time PCR analysis on resistant and susceptible rice cultivars after 14 days of inoculation with Xoo race K1.

 

Results

Inoculations of rice cultivars with Xoo were conducted during two different seasons (i.e., winter and summer 2014) and at two different locations (i.e., greenhouse and open field) with the purpose of determining whether Xoo inoculation in different environments produced the same results. The severity of BLB, indicated by LL on leaves, was significantly lower in the greenhouse than in the open field (Table 2). The range of LL in the greenhouse screening was 0.24–3.33 cm whereas that in the open field screening was 0.32–8.42 (Fig. 1). Therefore, by visual observation of the LL, the reaction to BLB in the greenhouse only classified rice cultivars into two categories, resistant or susceptible. Five cultivars (Hanareum, Namcheon, Samdeok, Samgang, and Yangjo) with LL of less than 1 cm were classified as resistant and the remaining 27 cultivars with LL greater than 1 cm were classified as susceptible.

Table 2.aR: resistant; MS: moderately susceptible; S: susceptible.

Fig. 1.Distribution of lesion length in 32 Korean rice cultivars after 14 d of inoculation with Xoo K1 race in open field cultivation.

Meanwhile, in the open field screening, disease severity was more obvious. Therefore, the reaction to BLB was classified into resistant (0–3 cm), moderately susceptible (3–6 cm), and susceptible (6–9 cm). The principle for classification of resistance in this study was determined by the range in disease severity. The same five cultivars were found to be resistant in both the greenhouse and open field, whereas 23 cultivars were moderately susceptible, and four cultivars were susceptible. It is also noteworthy that cultivar Joan, the most susceptible cultivar in the greenhouse, was only moderately susceptible in the open field, suggesting that greenhouse screening was not as accurate as screening in the open field.

Fig. 2 depicts the phenotypes of the five most resistant (i.e., Hanareum, Namcheon, Samdeok, Samgang, and Yangjo) and susceptible cultivars (i.e., Dongjin1, Hegjinju, Jeogtomi, Jinbu, and Namil) from the field inoculation. Infected leaves had yellow water-soaked lesions at the margins of the leaf blade. The lesions were parallel along the leaf and when joined together could cover the entire leaf surface. Bacterial discharge appeared from young lesions early in the morning and appeared as a milky dewdrop. As the disease progressed, the leaves dried up, showing white lesions, and the margins of the leaf blade became wavy.

Fig. 2.Phenotypes of the five most resistant cultivars (i.e., A. Hanareum; B. Namcheon; C. Samdeok; D. Samgang; E. Yangjo) and five most susceptible cultivars (i.e., F. Dongjin1; G. Hegjinju; H. Jeogtomi; I. Jinbu; J. Namil), after 14 days of inoculation with Xoo K1 race in open field cultivation. Leaves on the left and right in each picture are inoculated and control plants, respectively.

Expression levels of several common genes related to plant defense (i.e., OsNPR1, OsWRKY45, JAmyb, OsPR1a, and OsPR10b) during BLB inoculation were assessed. Owing to the low severity of BLB in greenhouse-grown plants, rice cultivars from the open field were used for gene expression analysis. Five resistant cultivars (i.e., Hanareum, Namcheon, Samdeok, Samgang, and Yangjo) were subjected to qRT-PCR analysis to determine which plant defense-related gene(s) are regulated during BLB infection by comparing the expression levels of the same genes in control and treated plants.

The expression of JAmyb in resistant cultivars was not correlated with the phenotype (Fig. 3). Its expression was higher in the cultivars Samdeok, Samgang, and Yangjo compared with control plants, but was lower in the cultivars Hanareum and Namcheon. The expression levels of OsNPR1 were unexpectedly low in all resistant cultivars (Fig. 4), suggesting that this gene is not essential for BLB resistance. However, this does not mean that OsNPR1 down-regulates a reaction to the pathogen because the relative expression in OsNPR1 appeared randomly. The expression levels of OsPR1a in the resistant cultivar group were similar patterns to those noted in JAmyb. This gene was highly expressed in the cultivar Yangjo, whereas its expression levels in other cultivars were either lower or slightly higher than in control plants (Fig. 5). The expression levels of OsWRKY45 in resistant cultivars were not correlated with the phenotype. Except for one cultivar (i.e., Namcheon), OsWRKY45 expression levels were lower than those in the control plants (Fig. 6). The expression levels of OsPR10b in all resistant cultivars were higher than those in control plants (Fig. 7). The gene was expressed twice as much, except in Yangjo, where its expression level was more than 9 times higher than in the control plants. This expression pattern indicates a positive correlation with the phenotype. This finding indicates that OsPR10b is essential for resistance to BLB. To confirm this, expression levels of OsPR10b were also observed in the five most susceptible cultivars (i.e., Dongjin1, Hegjinju, Jeogtomi, Namil, and Seolgaeng). As a result, the expression levels of OsPR10b did not change significantly and were either similar (i.e., Seolgaeng), lower (i.e., Hegjinju, Jeogtomi, and Namil), or slightly higher (i.e., Dongjin1) compared with those of the control plants (Fig. 8).

Fig. 3.Relative expression of JAmyb in five resistant Korean rice cultivars after 4 days of inoculation with Xoo race K1. Striped bars represent control plants and bars in white represent treated plants. Han-C: Hanareum control; Han-T: Hanareum treated; Nam-C: Namcheon control; Nam-T: Namcheon treated; Sam-C: Samdeok control; Sam-T: Samdeok treated, Sag-C: Samgang control; Sag-T: Samgang treated; Yan-C: Yangjo control; Yan-T: Yangjo treated.

Fig. 4.Relative expression of OsNPR1 in five resistant Korean rice cultivars after 4 days of inoculation with Xoo race K1. Striped bars represent control plants and bars in white represent treated plants. Han-C: Hanareum control; Han-T: Hanareum treated; Nam-C: Namcheon control; Nam-T: Namcheon treated; Sam-C: Samdeok control; Sam-T: Samdeok treated, Sag-C: Samgang control; Sag-T: Samgang treated; Yan-C: Yangjo control; Yan-T: Yangjo treated.

Fig. 5.Relative expression of OsPR1a in five resistant Korean rice cultivars after 4 days of inoculation with Xoo race K1. Striped bars represent control plants and bars in white represent treated plants. Han-C: Hanareum control; Han-T: Hanareum treated; Nam-C: Namcheon control; Nam-T: Namcheon treated; Sam-C: Samdeok control; Sam-T: Samdeok treated, Sag-C: Samgang control; Sag-T: Samgang treated; Yan-C: Yangjo control; Yan-T: Yangjo treated.

Fig. 6.Relative expression of OsWRKY45 in five resistant Korean rice cultivars after 4 days of inoculation with Xoo race K1. Striped bars represent control plants and bars in white represent treated plants. Han-C: Hanareum control; Han-T: Hanareum treated; Nam-C: Namcheon control; Nam-T: Namcheon treated; Sam-C: Samdeok control; Sam-T: Samdeok treated, Sag-C: Samgang control; Sag-T: Samgang treated; Yan-C: Yangjo control; Yan-T: Yangjo treated.

Fig. 7.Relative expression of OsPR10b in five resistant Korean rice cultivars after 4 days of inoculation with Xoo race K1. Striped bars represent control plants and bars in white represent treated plants. Han-C: Hanareum control; Han-T: Hanareum treated; Nam-C: Namcheon control; Nam-T: Namcheon treated; Sam-C: Samdeok control; Sam-T: Samdeok treated, Sag-C: Samgang control; Sag-T: Samgang treated; Yan-C: Yangjo control; Yan-T: Yangjo treated.

Fig. 8.Relative expression of OsPR10b in five susceptible Korean rice cultivars after 4 days of inoculation with Xoo race K1. Striped bars represent control plants and bars in white represent treated plants. Doj-C: Dongjin1 control; Doj-T: Dongjin1 treated; Heg-C: Hegjinju control; Heg-T: Hegjinju treated; Jeg-C: Jeogtomi control; Jeg-T: Jeogtomi treated; Nam-C: Namil control; Nam-T: Namil treated; Seg-C: Seolgaeng control; Seg-T: Seolgaeng treated.

 

Discussion

Bacterial leaf blight of rice has been reported in several parts of the world with high incidence and severity [25]. Therefore, strategies adapted to particular environments must be developed to avoid possible epidemics. Among these many different control strategies, host-plant resistance is an important control measure. Knowledge of varietal resistance is important for selecting cultivars with durable resistance to the disease [3,5,21]. In this study, 32 rice cultivars were screened for resistance to Xoo race K1, the causal agent of BLB. Experiments were conducted in a greenhouse during winter 2014 and in the open field during summer 2015. Based on phenotypic data and visual observations of disease symptoms among the 32 rice cultivars screened, only five cultivars showed resistance to Xoo race K1 (i.e., Hanareum, Namcheon, Samdeok, Samgang, and Yangjo) in both the greenhouse and the open field. The relatively low severity observed in the greenhouse screening compared with the open field could be due to environmental factors such as day/night temperature difference, humidity, and wind, which significantly affect the activity of Xoo. During their growth and development stages, plants are subjected to a wide variety of biotic and abiotic stressors. As a result, they develop defense mechanisms because of their sessile nature. During an attack from a pathogen, plants are protected by defensive signaling pathways that alert the plant [13]. Numerous genes that are essential during pathogen infection or wounding are expressed in a variety of plant species in response to pathological attacks. Five genes (i.e., JAmyb, OsPR10b, OsPR1a, OsNPR1, and OsWRKY45) were used to identify gene expression in cultivars during screening by qRT-PCR. Among these genes, only OsPR10b exhibited expression levels with a positive correlation to the phenotype (Figs. 7 and 8). In resistant cultivars, OsPR10b expression levels were higher than in the control plants, whereas in susceptible cultivars the expression levels in treated plants were relatively similar to those in the control plants. The relatively inconsistent expression of JAmyb [15] in the resistant cultivars compared with control plants indicated that resistance to Xoo race K1 in this study is not directly through the jasmonate (JA)-signaling pathway. It is known that the activation of the JA-dependent signaling pathway is generally induced during an attack by a necrotrophic pathogen [9]. In the case of biotrophic pathogens like Xoo, it is normal to assume that the salicylic acid (SA)-dependent signaling pathway is triggered in order to activate a hypersensitive response [4]. However, the expression of OsWRKY45 [22], OsPR1a [1], and OsNPR1 [8,31], which are involved in the SA-signaling pathway, also occurred in inconsistent patterns. This was especially noted with OsNPR1, which is a key regulator of SA-mediated resistance, where its transcript levels in all resistant cultivars were lower than those in their control counterparts. SA and JA are endogenous signal molecules required in the induction of plant immunity, in which they interact antagonistically [28-30]. The induction of OsPR10b in the resistant cultivar in this study might be attributed to its characteristic. In rice inoculated with the fungus M. grisea, the induction of OsPR10b took place later than that of OsPR10a, which started at 2 days post-inoculation [19]. Plant defense mechanisms are a result of a broad, complex, and interconnected network of genes, particularly in the interaction of rice and Xoo where race-specific resistance [11] and allelic interaction [17] between genes exist. Our results here provide insight and information in assessing gene expression by utilizing resistant and susceptible individuals as the test subjects. Our results reveal that some genes, including OsNPR1, were randomly expressed and did not show any significant correlation with the phenotype in resistant and susceptible groups; however, among the tested genes, only OsPR10b showed coherent expression in both resistant and susceptible individuals. Given this result, OsPR10b can be inferred as the gene having the most significance as a specific resistance trait to Xoo race K1 compared with other genes.