Detection of Bacterial Leaf Blight Resistance Genes in Indigenous Glutinous Rice Landraces

Bacterial leaf blight disease (BLB) caused by Xanthomonas oryzae pv is one of the most widespread devastating diseases of rice worldwide. In this study, a total of 86 indigenous glutinous rice landraces were examined for BLB resistant genes included Xa4, xa5, and Xa7 by using molecular markers. The results indicated that 37 samples carrying resistant genes, in which Xa4 was found in 11 samples, xa5 determined in 6 samples, Xa7 was in 19 samples, respectively. However, both of Xa7 and Xa4 were observed in only one sample. The resistant level against 10 bacterial strains carrying the resistant genes was also evaluated. We found that the number 6, 9, and 8 out of the 10 bacterial strains were resistant to the landraces which carried the Xa4, xa5, and Xa7. The bacterial strain number 5 was revealed highly toxic, causing infection of all samples. The agronomical traits, yield and yield components of 37 rice landraces included the resistant genes (Xa4, xa5 and Xa7) were evaluated. Our findings may provide useful genetic sources in indigenous glutinous rice landraces to further develop BLB resistant rice lines via molecular breeding program.


Introduction
Bacterial leaf blights (BLB) disease in rice caused by Xanthomonas oryzae pv.oryzae bacterial, and is one of the most widespread devastating diseases in all rice-cultivating areas of the world. Under the pressure of bacteria infestation, the rice leaves were blighted, severely infected leaves tend to dry promptly, and then the chlorophylls will be non-synthesis, leading to reduce productivity up to 80%. If the disease becomes serious, rice yield can be totally lost. In rice producing countries in Asia, millions of hectares of rice paddies have seriously influenced yearly by infestation of BLB.
Rice (Oryza sativa L.) is the main food and providing daily meals for over 90 million people in Vietnam. According to the report of MARD (2015) [1] BLB was caused severe infestation across the country up to 11.921 ha in both Mekong and Red River Deltas. More seriously, total BLB affected areas in south provinces were 7.069 ha, increased 2.129 ha comparing in 2016 and 110 ha were severely lost [2] (MARD, 2017). Currently, there is no specific treatment for this disease, other synthetic pesticides are being used only to prevent the disease infestation. Therefore, using the resistant varieties are considered as the most feasible solution, which could help for environmentalfriendly and possibly produce safety products instead of using agrochemical pesticides. In order to successfully improve BLB resistant rice varieties, the genetic sources of rice landraces play a key role in controlling this disease.
To date, total 42 resistant genes to BLB have been reported. Among them, there are 14 recessive genes [3,4]. In Vietnam, 4 resistant genes including Xa4, xa5, Xa7, and Xa21 have been reported as the significant resistant genes to pathogenic bacterial strains in the northern Vietnam. These genes have had important role in rice breeding for resistance to BLB. Some markers tightly linked with BLB resistant genes have been identified [5]. Therefore, application of molecular markers to detect genetic resources becomes the way of simple and accurate. Therefore, the objective of this study was to examine a total of 86 Vietnamese indigenous glutinous rice varieties for resistant genes Xa4, xa5, and Xa7 by molecular markers. Subsequently, resistant levels of rice varieties carrying the resistant genes were evaluated by applying infection of the 10 bacterial strains. Besides, some major agronomical traits were evaluated to select the best varieties for glutinous rice for molecular breeding.

Plant Materials
The 86 rice landraces were kindly provided by the Center for Conservation and Development of Crop Genetics Resources (CCD-CGR), Vietnam National University of Agriculture were used in this study. The 10 pathogenic bacterial strains of BLB were collected in the northern Vietnam were used in this study as shown in Table 1. The molecular markers were used to detect the resistant genes Xa4, xa5, and Xa7 including the information of sequence, genes, markers and chromosomes were shown in Table 2.

Methods
Application of molecular marker to detect resistant genes Xa4, xa5 and Xa7

DNA extraction
Total DNA extraction was carried out according to the process of Zheng et al. [9]. The young leaves were chopped at 2 cm and crushed in 800 µl of mixed solution (200mM Tris-HCl pH 8.0 buffer; 25 mM EDTA; 250 mM NaCl, 0.5 SDS) in order to break the cells. Fine hundred μl of 2 JHPR Volume 2 crushing solution were transferred into the test tube (1.5 ml) and added 700μl of mixture (with the ratio of phenol:chloroform:isoamyl alcohol are 25:24:1, respectively). The mixture was slightly shaken and centrifuged for 5 min at 13.000 rpm and 4 o C. The supernatant was then transferred into the new test tube, added 800 µl of ethanol 100%, and centrifuged for 5 min for 13.000 rpm at 4 o C. The precipitated DNA extracts were collected, cleaned by ethanol 70% and naturally dried. The precipitated DNA extracts were dissolved in 50 µl TE and stored at -20 o C for next use.

PCR performance
PCR performance was carried out with the volume of 20 μl including: 10 μl PCR master mix (2X) provided by Promega company, 1 μl Primer Forward (1.0 pmol/ul), 1 μl Primer Reverse (1.0 pmol/ul), 1 μl total DNA and 7 μl nuclease-Free water. PCR Performances of genes Xa4, xa5, and Xa7 were performed as follows: 94 o C for 4 m, 30 cycles: 94 o C for 1 min, 56 o C for 1 min, 72 o C for 2 min, and 72 o C for 7 min. Electrophoresis of PCR products were performed on agarose gel 1.5%, 100V for 45 min. Gel was dyed with Ethidium Bromide 0.5 µg/ml and observed by UV scanner.

Investigation of rice landraces
The rice samples were grown in the paddy field according to the conventional methods. Each landrace was cultivated in an area of 5 m 2 with the density of 11 cm x 20 cm x 30 cm. The agronomic traits were evaluated following the method of IRRI [10] and QCVN 01-55: 2011/BNNPTNT such as: number of panicles per plant (NPP), filled grains per panicle (FGP), grain weight (GW-P1000), plant height (PH), days to heading (DTH) and some characteristics involving in quality were also recorded.

Experiment of artificial infection
Artificial infection was carried out at the time that rice plant started booting stage by cutting at 3-5 cm of leaf. The bacteria were cultured on Wakimoto medium for 48 h. The concentration of bacterial solution was about 10 8 -10 9 cells/ml. Whole green leaves of each plant were also cut. Resistant ability of each variety was evaluated by measuring the lesion length after 20 days of infection following the IRRI standards [10] as follows: lesion length <8cm: resistance (R); from 8-12 cm: medium (M); and >12 cm: susceptibility (S).

Detection of resistant gene Xa4
The indigenous rice landraces have been cultivated and undergone a long time for domestification. Therefore, they have had many valuable agronomic characteristics such as: high yield potential (grains per panicle), high quality, strong tolerance to abiotic and biotic stresses, especially resistance to pests and diseases. In order to exploit them for rice breeding, it is initially examine for BLB resistant genes. In this research, we investigated the ocurrence of Xa4, xa5 and Xa7 in the 86 rice landraces. According to report of Ton et al. [5], Xa4 gene was determined the resistance to 6/10 leaf blight bacterial strains in the nothern Vietnam. This gene is located on the chromosome 11 which is tightly linked with the marker Npb181 and marker MP [6], respectively. Npb181 marker was used to detect Xa4 gene. PCR product was electrophoresised on agarose gel 1.5%. Rice landraces containing Xa4 gene showed a DNA band with the size of 150 base pair (bp). Otherwise, the landraces with non-carrying Xa4 gene which displayed a DNA band with the size of 120bp fragment as corresponding to the negative line IR24. The result of electrophoresis of PCR products disclosed that 11 glutinous rice varieties which had Xa4 gene in the total of 86 samples as shown in Fig. 1. Recently, Ullah et al [11] reported that Xa4 was detected in Basmati-385 and Basmati 2000 cultivars by using STS markers, which was agreed with our current study. In Vietnam, there are sporadical work examining BLB resistant genes of rice landstrains. In much Journal of Horticulture and Plant Research Vol. 2 effort has been paid on developing the BLB resistant cultivars since 1970 by using Xa4 gene. Some cultivars carrying Xa4 were reported such as CR203, MT [12].

Detection of resistant gene xa5
The recessive gene xa5 is considered as the best resistant gene against BLB. This gene involves in the resistance to 9/10 leaf blight bacterial strains existing in the North of Vietnam [5]. Two markers were previously identified that tightly linking with this gene including RG556 [13] and RM122 [7]. In order to identify the resistant gene xa5 using RG556 marker, PCR products must be cut by restriction enzymes DraI. Otherwise xa5 can be detected directly from PCR products using RM122 marker. Basically, two markers have the same accuracy. Therefore, to save time and cost in this study, RM122 marker was used to detect xa5 gene. The results have shown that the samples carrying the xa5 gene which appears the band with the size of 240 bp, the samples with non-containing the xa5 gene displayed a band with the size of 220 bp, respectively (Fig. 2). Overall, 6 out of 86 rice landraces carrying the xa5 gene were identified.

Detection of resistant Xa7 gene
The Xa7 gene is dominant gene and considered as responsible for greater resistance level to 8/10 bacterial strains in the northern Vietnam [5]. Some markers have been found that tightly linking with this resistant gene such as P3 marker [8], RM5509 marker [13], M5 marker [14]. In Vietnam, P3 marker is widely used to screen and detect the Xa7 resistant gene. By using this marker, PCR products of the landraces containing Xa7 gene which were shown a band with the size of 262 bp after electrophoresis, the varieties with non-containing this gene appears a band with the 4 JHPR Volume 2 size of 297 bp (Fig. 3). The result of electrophoresis has shown that 19/86 glutinous rice varieties having Xa7 gene. Only one rice landrace, Pe con Lam (lua nep) carrying both Xa4 and X7 genes (Table 3). Previously, some single dominant genes codes for resistance to Xo, were reported in some varieties such as Di Huong HD, Dai Duc 65, Canh Nong HB, Mai Khua TB, IR20 and IR22 [12].

Evaluation of BLB resistant samples
Currently, there were over ten existing bacterial strains in the north of Vietnam [3]. These strains are being stored at Faculty of Biotechnology, Vietnam National University of Agriculture. In this research, 10 bacterial strains were used to examine resistant level to BLB. The results demonstrated that the landraces containing Xa4 gene had similar resistant level to IRBB4 control variety (containing Xa4 gene). These landraces had resistant activity against 6/10 bacterial strains including the strains number 2, 5, 6, 7, 9, and 10, respectively. The other landraces containing xa5 resistant gene which had similar resistant level to control variety (carrying xa5 gene). These landraces were resistant against 9/10 bacterial strains except the bacterial strain number 5. The containing Xa7 resistant gene also had similar resistant ability to control variety (carrying Xa7 gene). These varieties were resistant to 8/10 bacterial strains, they were infected by only the two strains including number 4 and 5, respectively.
In general, almost the rice landraces with non-containing resistant genes to BLB were seriously infected by all the 10 bacterial strains or some strains out of the 10 studied strains (

Agronomic traits of some landraces carrying resistant genes
In order to effectively exploit some major agronomical characteristics of each genetic source were evaluated including some major traits: days to heading (DTH), plant height (PH), a number of panicle per plant (NPP), number of grains per panicle (NGP), grain weight (GW-P1000), individual yield (IY) were evaluated. The results showed that the 37 rice landraces containing resistant genes to BLB, then were evaluated their agronomic traits in spring season crop, 2016 to provide further comprehensive information for effective exploitation these genetic resources as shown in Table 4. Regarding the growth duration, the growth duration of 37 samples was ranged from 131 to 146 days. In which, 17 landraces were grouped into short growth duration (115 to 135 days). The other landraces were grouped into medium growth duration (136 to 160 days). For plant height, there were 7 rice landraces in medium height (90 to 125 cm). The other landraces were rather high with the height of more than 125 cm. The number of panicles per plant was ranged from 4.0 to 7.6 panicles. The number of grains per panicle was from 60.2 to 210 grains/panicle. Some rice landraces had extremely big panicles such as: Lua nep Pelenh, Pe con lam, Lo cam and nep roi. For the total grains (P1000), the glutinous rice landraces mostly had very high weight, some samples had P1000 up to over 36 g, especially was 40.3 g found in Nep rau. In the case of individual yield, it was ranged from 7.5 g to 17.3 g, which was shown significantly high. This is an extremely valuable material sources, which should be exploited and managed for future rice breeding program.

Conclusions
Among the total of 86 rice landraces, 11 samples carrying Xa4 gene, 6 landraces were detected xa5 gene, 19 landraces possessed Xa7 gene and 1 rice landrace carried both of the Xa4 and Xa7 genes were identified by using molecular markers.The evaluation of resistant level of rice varieties to BLB using 10 bacterial strains indicated that Xa4 gene involved in resistance to 6/10 strains, Xa5 gene involved in resistance to 9/10 strains and Xa7 gene involved in resistance to 8/10 strains of bacteria. The bacterial strain number 5 had the strongest toxicity.The evaluation of agronomic traits of the 37 samples included the resistant genes rice landraces disclosed that they are precious source of materials for glutinous rice breeding program of resistance to BLB disease.