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Evaluation of bacterial blight resistance in rice lines carrying multiple resistance genes and Xa21 transgenic lines

Prashant Swamy, Ajay N. Panchbhai, Priti Dodiya, Vaishali Naik, S.D. Panchbhai, Usha B. Zehr, Kasi Azhakanandam and Bharat R. Char

Mahyco Research Center, Dawalwadi, Maharashtra Hybrid Seeds Company Limited, Aurangabad-Jalna Road, PO Box 76, Jalna
431 203, India. Email:


A major disease of rice, bacterial blight (BB), is caused by Xanthomonas oryzae pv. oryzae (Xoo). Six distinct Indian isolates of Xanthomonas oryzae were tested on rice genetic backgrounds carrying single or multiple BB resistance genes. Three BB resistance genes were introgressed into a susceptible proprietary line, MH2R, by marker-assisted selection, which resulted in a gain of resistance. In addition, Pusa Basmati 1 (PB1) transgenic lines carrying the BB resistance gene, Xa21, and IR72-Xa21 transgenic lines were evaluated after inoculation with the 6 Xoo isolates. An evaluation of the efficacy of the Xa21 transgene against Indian isolates is described. The results indicate that introducing multiple BB resistance genes by marker-assisted selection is an effective strategy in breeding for BB resistance against Indian isolates. Genetic diversity among the BB isolates was also studied in order to provide a basis for a more detailed analysis of pathogen virulence.

Key words

bacterial blight, marker-assisted selection, resistance genes, rice, transformation


Bacterial blight (BB) is a serious disease of rice caused by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), resulting in significant crop yield losses in the range of 20 to 40% (Sonti 1998). Studies of the genetics of BB resistance have resulted in the identification of more than 20 resistance (R) genes, and the development of donor lines carrying major R genes. A number of these donor lines have been used in rice breeding programs around the world. The Xa21 gene was transferred from the wild species Oryza longistaminata through wide hybridization with IR24, resulting in the near-isogenic line, IRBB21 (Khush et al. 1990). In tests for disease resistance, IRBB21 has been reported to be resistant to many Xoo strains from the Philippines and India (Khush et al. 1990).

Breeding for durable resistance against BB is one of the most effective ways to control the disease. Pyramiding multiple R genes in a single line confers wide-spectrum and durable resistance. This has been achieved by a number of groups using marker-assisted selection. Singh et al. (2001) used molecular markers to introduce xa5, xa13 and Xa21 into PR106, a BB susceptible line widely grown in the Punjab. A three-gene combination appeared to be the most effective, with Xa21 contributing the largest component of resistance. In this study, we compared the degree of resistance of lines bearing single or multiple BB resistance against six genetically diverse Indian Xoo isolates. Using marker-assisted selection, up to three resistance genes were introgressed into a hybrid rice parental line and the resulting progeny lines evaluated against the pathogen. In addition, a large number of transgenic lines carrying Xa21 were generated to test the efficacy of the transgene against the six isolates, and compared in terms of resistance to transgenic IR72 bearing the Xa21 gene. We describe here the results on lines found to be most effective against the Xoo isolates tested, and discuss the possible optimum combination of resistance genes to provide durable resistance against Indian isolates.

Results and Discussion

Survey of Xa genes with molecular markers

Fourteen MH parental lines were screened for the presence of xa5, xa13 and Xa21 using DNA markers RM122, RG136 and pTA248 respectively. Two lines, MH2R and MH3R, showed the presence of the xa5 gene whereas all other lines evaluated were negative for the presence of the R genes tested. MH2R was chosen for further breeding due to its preferred agronomic characters. We also surveyed a number of other rice lines for the presence of the xa5, xa13 and Xa21 BB resistance genes (Table 1). These were IRBB5, IRBB21, IRBB56, IRBB57, IRBB58, IRBB59 and IRBB60, which have variable numbers of Xa- genes as described in Table 2, and TN1, a susceptible variety. Transgenic lines carrying Xa21 were also evaluated along with their corresponding non-transgenic lines. The lines analysed were IR72 and transgenic IR72-Xa21 (Tu et al. 2000), Pusa Basmati 1 and transgenic Pusa Basmati-Xa21. The presence of the appropriate Xa genes was confirmed in the IRBB lines. IR72 carries an endogenous xa4 gene, while Pusa Basmati and TN1 were negative for xa5, xa13 and Xa21.

Table 1. Rice lines tested for bacterial blight reaction against Xoo isolates


R genes present








Xa4, xa5, xa13


Xa4, xa5, Xa21


Xa4, xa13, Xa21


xa5, xa13, Xa21


Xa4, xa5, xa13, Xa21








Xa4, Xa21


xa5, xa13, Xa21

Marker-assisted selection for evaluation of BB resistance

An F2 population derived from a cross between MH2R and IRBB60 was subjected to PCR analysis for the presence of marker(s) linked to xa5, xa13 and Xa21. Four different DNA markers were used for this. Genomic DNA was extracted from 384 F2 plants and used for PCR amplification to test for the presence of the Xa21 gene using the marker pTA248. Out of the 384 individuals tested, 71 were found to be homozygous for the Xa21 locus, which was not different than expected as determined by chi-square analysis. These 71 plants were checked for the presence of xa13 using the marker RG136, which was found to be in the homozygous condition in 17 plants. The presence of the xa5 gene was confirmed with marker RM122 in all 17 plants as expected, as the gene is present in both MH2R and IRBB60 in the homozygous condition.

Generation of transgenic Pusa Basmati-Xa21 (PB-Xa21)

Transgenic Pusa Basmati1 rice plants were generated via particle bombardment of rice calli with the plasmid pAHXa21 (Song et al., 1995). Twenty-four independent transgenic events were obtained and analysed by PCR to determine the presence of the transgene. Of these, 10 lines were subjected to Southern blot analysis. Due to the presence of multiple Xa21-like genes in the rice genome, the Nos terminator sequence was used as a probe. Digestion with EcoRV, a unique restriction site within the gene cassette, revealed distinct patterns of hybridisation in the 10 lines analysed with sites of integration varying from 1 to 4. Digestion of the genomic DNA of these 10 lines with EcoRI releases the Nos terminator, which was also confirmed by Southern blot. RT-PCR analysis of individual plants of six PB-Xa21 lines at the T1 generation revealed that all lines tested were expressing the xa21 gene at the transcriptional level.

Genetic characterisation of BB isolates

The rice lines included in the study were inoculated with six Xoo isolates (Table 2) at the stage of maximum tillering. These six isolates (designated Xoo1-6) were subjected to amplified fragment length polymorphism (AFLP) analysis (Vos et al. 1995) using two different EcoRI and MseI primer combination pairs, and were found to be distinguishable from each other on the basis of polymorphic DNA bands, suggesting that the isolates are genetically distinct. Cluster analysis of the AFLP data revealed that Xoo1 and Xoo6, though collected from different geographical locations, are the most closely related amongst the isolates with a correlation coefficient of 0.7. Xoo2 was found to be the most genetically distinct isolate of the isolates included in this study.

Table 2. Xanthomonas oryzae pv. oryzae isolates tested




Madhya Pradesh, India


Haryana, India


Haryana, India


Andhra Pradesh, India


Andhra Pradesh, India


Andhra Pradesh, India

Inoculation of rice lines with BB isolates

The 17 F2 plants derived from a cross between IRBB60 and MH2R, and homozygous for 3 Xa genes, were also individually inoculated with the same isolates. Lesion length recordings were taken at 21 days after inoculation (d.a.i.). Plants carrying multiple genes were highly resistant, and displayed a hypersensitive response (HR). Further, F2 plants that were homozygous for xa5, xa13 and Xa21 were as resistant as the parental line used in the cross, IRBB60, demonstrating the effectiveness of resistance gene pyramiding. Near-isogenic lines containing the single R genes Xa4 or Xa21 were less effective than those containing multiple R genes. IRBB4 was susceptible to Xoo2, Xoo4, Xoo5 and Xoo6 (8.77, 19.05, 12.2 and 16.44 cm average lesion lengths respectively). IRBB21 performed better, and was moderately resistant to Xoo4 and Xoo6 and resistant to the other isolates. IRBB56, IRBB58, IRBB59 and IRBB60 were resistant to all six isolates, with the latter two lines displaying the highest overall degree of resistance. Interestingly, IRBB57, which lacks xa13, was susceptible to Xoo2, Xoo4, Xoo5 and Xoo6 (11.35, 8.18, 6.83 and 13.0 cm average lesion lengths respectively) suggesting an important role for this R gene against the Xoo isolates tested. Pusa Basmati-Xa21 transgenic lines failed to show resistance against all the isolates tested. These lines often had lesions covering the entire inoculated leaf leading to plant death at 21 d.a.i... Transgenic IR72-Xa21 displayed resistance against Xoo1 and Xoo5, moderate resistance to Xoo2, Xoo3 and Xoo6 and was susceptible to Xoo4. This enhanced resistance against BB over the PB-Xa21 lines may be due to the combined effect of Xa4 and Xa21 genes. The parental line MH2R displayed resistance to Xoo1 but was susceptible to the other isolates.

The main goal in breeding for BB resistance is to achieve durable resistance in the field. In this study, two approaches were taken to develop and evaluate BB resistance – stacking of multiple BB resistance genes by marker-assisted selection and a transgenic approach in which Xa21, one of two cloned BB resistance genes, was introduced into Pusa Basmati1, a genetic background lacking known major BB resistance genes. Plants were challenged with a number of genetically distinct BB isolates from the Indian subcontinent. A consistent finding was that lines which carried two or more BB resistance genes showed a higher degree of resistance over lines, both non-transgenic and transgenic, containing single BB resistance genes. The single Xa gene containing lines IRBB4 and IRBB21 were susceptible and moderately resistant to the more virulent Xoo isolates used in this study (Xoo4 and Xoo6) respectively. These results agree with those of studies done with BB isolates from Punjab, India in which it was observed that combinations of R genes provide a broader spectrum of resistance to the disease (Singh et al. 2001).

In order to bring about durable BB resistance in elite rice lines, marker-assisted selection has proved to be invaluable. We were interested in bringing in multiple BB resistance genes into proprietary hybrid parent lines. We found that the line MH2R, containing xa5, was susceptible to the isolates Xoo2, Xoo4, Xoo5 and Xoo6. However, upon marker-aided introgression of xa13 and Xa21 into MH2R using IRBB60 as the source of resistance genes, xa5-, xa13- and Xa21-homozygous F2 individuals showed clear resistance against the same Xoo isolates, providing an example of the value of this approach. Another goal of this study was to evaluate the efficacy of the Xa21 transgene against the Xoo1-6 isolates. From the results obtained here it appears the Xa21 gene alone is ineffective against some virulent isolates (Xoo4, Xoo5 and Xoo6) and thus must be deployed in genetic backgrounds that contain other BB resistance genes. This is supported by the data obtained with transgenic IR72-Xa21, which contains an endogenous Xa4 gene. IR72-Xa21 proved to be resistant or moderately resistant to the pathogen isolates tested, except for isolate Xoo4, which showed virulence. IR72 was susceptible against most of the isolates tested. Pusa Basmati1 and its transgenic derivative PB-Xa21 appeared to be much more susceptible to the virulent Xoo isolates than IRBB21, suggesting a difference in either levels of expression of the Xa21 gene or contributions from the IRBB genetic background. RT-PCR confirmed the transcriptional activity of the Xa21 transgene in a number of PB-Xa21 lines.

A major challenge is developing broad-spectrum resistance to BB in the Indian subcontinent due to the diverse agro-climactic zones where rice is cultivated, as well as the number of genetically distinct virulent Xoo strains from different geographical areas of the region. Based on the diverse, though limited, number of isolates used in this study we suggest that at a minimum, the use of a three-gene combination such as xa5 with xa13 and Xa21 would be desirable to achieve durable and broad-spectrum resistance. Careful field evaluation of pyramided lines obtained by MAS of BB resistance genes is needed to confirm these results. Further marker-assisted backcrossing of IRBB60 x MH2R F2 plants would be needed to select for the desired agronomic traits.


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