Bacterial blight, caused by Xanthomonas oryzae pv. oryzae
(Ishiyama) Swings et al. (Xoo), is the most destructive
disease of rice worldwide which causes significant yield losses annually
(Mew, 1987). Yield loss from this disease can be as high as 50% (Adhikari
et al. 1995). In Punjab and Haryana states of India, major epidemics
occurred in 1979 and 1980; severe kresek was observed and total crop failure
was reported (POS, 1979 &1980; Mew, 1987). Compared with conventional
inbred varieties, rice hybrids are more responsive to nitrogenous fertilizer
and exhibit vigorous growth. These attributes have made most of the rice
hybrids highly sus-ceptible to bacterial blight disease (Mew et al.
1988; Reddy et al. 1998). Cai and Zhong (1980) reported that bacterial
blight is a common disease on hybrid rice in Zhejiang Province in China.
In view of the limited success in chemical control of the disease, the
importance of hostplant resistance has been well recognized. So far, 24
resistance genes have been identified in rice and its wild relatives (Kinoshita,
1995; Lin et al. 1998). The dominant gene Xa21 introgressed
from Oryza longistaminata into O. sativa confers broad-spectrum
resistance to most of the isolates of Xoo, making it a valuable
gene for resistance to bacterial blight disease (Khush et al. 1990).
However, the improvement of hybrid parental lines through conventional
breeding is a time-consuming process and linkage with undesirable characters
is a problem. Song et al. (1995) cloned the Xa21 gene through
map-based strategy. This is the first wellcharacterized disease-resistance
gene that has been characterized in rice. Tu et al. (1998) introduced
the Xa21 gene into elite indica cultivar IR72 via the biolistic
transformation method. The improved transgenic IR72 carrying Xa21
exhibited broad-spectrum resistance in field tests (Tu et al. 2000).
The maintainer line IR58025B (designated here as ML1) was transformed
with bacterial blight resistance gene Xa21 using the particle bombardment
method. We report here the reaction of homozygous T3 transgenic
ML1 lines to two Philippine races of Xoo, race 4 (PXO 61)
and race 6 (PXO 99), and the stable integration of Xa21 in subsequent
generations. A total of 140 T3 homozygous transgenic ML1 plants
from seven different lines were grown in the IRRI CL4 containment green
house. Non-transformed ML1 plants served as the control. Standard agronomic
practices were followed for growing the plants, except that no chemical
pesticides were applied to allow for an optimum evaluation of the resistance
reaction. The plants were inoculated at the maximum tillering stage following
the leaf-clipping technique (Kauffman et al. 1973). For this purpose,
the bacteria were mass-multiplied on peptone sucrose agar or modified
Wakimoto's medium. The bacteria were harvested from 72 h growth and
concentration was adjusted to 108-109 c.f.u./ml
of suspension. In each line, sets of 10 plants were inoculated with race
4 and another 10 plants were inoculated with race 6 of Xoo. Observations
were recorded 14 days after inoculation on lesion length on transgenic
and non-transgenic plants.
All the lines tested were highly resistant to race 4 and possessed a good
level of resistance to race 6 (Figure 1). Philippine race 6 is a highly
virulent race. The mean lesion length against race 4 varied from 4.04
to 5.74 cm in transgenic plants, whereas the average lesion length in
non-transformed ML1 plants was 34.11 cm (Table 1). Against race 6, the
average lesion length varied from 8.79 to 11.16 cm in transgenic plants
compared with 24.97 cm in nontransformed plants. The variation in the
bacterial blight reaction among the lines may be due to differences in
the level of expression of the transgene. Among the different lines tested,
ML1-7-9-1-8 and ML1-7-9-24-2 were the most promising. The bioassay results
supplemented by Southern blot analysis of the Xa21 gene. The presence
of the 3.8-kb band indicated the presence of the Xa21 gene (Figure
2). The study revealed that transgenic ML1 plants carrying the Xa21
gene showed a high level of resistance to both races of the bacterial
blight pathogen. The study also revealed that the increased level of resistance
to the bacterial blight pathogen persists in transgenic plants over generations,
indicating its stable inheritance. This homozygous transgenic maintainer
line can be useful in a hybrid rice development program.
Thanks are due to Dr. Pamela Ronald of UC-Davis for providing the Xa21
gene, the Rockefeller Foundation for partial financial support, and Bill
Hardy for editorial assistance.
Adhikari, T.B., C.M. Vera Cruz, Q. Zhang, R.J. Nelson, D.Z. Skinner, T.W.
Mew and J.E. Leach, 1995. Genetic diversity of Xanthomonas oryzae
pv. oryzae in Asia. Appl. Environ. Microbiol. 61: 966-971.
Cai, S. F. and M.Y. Zhong, 1980. Control experiment of diseases and pests
of hybrid late rice [In Chinese]. Zhejiang Nongye Kexue 4: 166-169.
Kauffman, H.E., A.P.K. Reddy, S.P.Y. Hsieh and S.D. Merca, 1973. An improved
technique for evaluating resistance of rice varieties to Xanthomonas
oryzae. Plant Dis. Rep. 57: 537- 541.
Khush, G.S., E. Bacalangco and T. Ogawa, 1990. A new gene for resistance
to bacterial blight from O. longistaminata. Rice Genet. Newsl.
Kinoshita, T., 1995. Report of committee on gene symbolization, nomenclature
and linkage groups. Rice Genet. Newsl. 12: 9-115.
Lin, X., C., Wang, G. Wen, D. Zhang, Y. Xie and Q. Zhang, 1998. Progress
in map-based cloning of Xa22(t), a new gene for bacterial blight
resistance in rice. Paper presented at the Plant and Animal Genome Conference,
18-22 Jan 1998, San Diego, Calif., USA.
Mew, T.W., 1987. Current status and future prospects of research on bacterial
blight of rice. Annu. Rev. Phytopathol. 25: 359-382.
Mew, T. W., F.M. Wang, J.T. Wu, K.R. Lin and G.S. Khush, 1988. Disease
and insect resistance in hybrid rice. In: Hybrid rice. Manila (Philippines):
International Rice Research Institute. p. 189-200.
POS (1979 & 1980). Production-oriented survey, Directorate of Rice
Research, Hyderabad, India.
Reddy, A.P.K., K. Krishnaiah, Z.T. Zhang and Y. Shen, 1998. Managing vulnerability
of hybrid rice to biotic stresses in China and India. In: Proceedings
of the 3rd International Symposium on Hybrid Rice Technology: Advances
in hybrid rice technology (Virmani, S.S., E.A. Siddiq, and K. Muralidharan,
eds.). Hyderabad, India & International Rice Research Institute, Philippines.
Song, W.Y., G.L. Wang, L. Chen, H-S. Kim. Li-Ya Pi. J. Garner, B. Wang,
T. Holsten, W.X. Zhai, L.H. Zhu, C. Fauquet and P. Ronald, 1995. A receptor
kinase-like protein encoded by the rice disease resistance gene Xa21.
Science 270: 1804-1806.
Tu, J., I. Ona, Q. Zhang, T.W. Mew, G.S. Khush and S.K. Datta, 1998. Transgenic
rice variety IR72 with Xa21 is resistant to bacterial blight. Theor.
Appl. Genet. 97: 31-36.
Tu, J., K. Datta, G.S. Khush, Q. Zhang and S.K. Datta, 2000. Field performance
of Xa21 transgenic indica rice (Oryza sativa L.), IR72.
Theor. Appl. Genet. 101: 15-20.