PAU, Ludhiana, Punjab, India

Background and objectives
Molecular markers such as RFLP, RAPD and AFLP offer new opportunities in DNA fingerprinting of plant pathogens, crop germplasm and in marker-assisted selection (MAS). The advent of PCR has further increased the efficiency of application of molecular markers to supplement various conventional crop improvement programmes. Knowledge on the genetic variability on host as well as pathogen populations is essential to develop cultivars resistant to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo). The present study was undertaken to (i) characterize variability in Xoo populations prevalent in Punjab State of northern India using PCR based primers pJEL1 and pJEL2, and (ii) to determine the virulence of the different Xoo lineages on isogenic and pyramid lines received from IRRI.

Materials and methods
Xoo infected samples of rice were collected from 58 sites of Punjab. In all, 693 isolates of Xoo were purified and maintained in glycerol at -70C. Two outwardly directed primers (pJEL1 and pJEL2) complementary to sequence ISI112, a repetitive element isolated from Xoo, were used to fingerprint the pathogen populations. A pairwise comparison for isolates was made which generated a matrix of similarity coefficients. These coefficients were grouped by cluster analysis using NTSYSL in PC (Version 1.80). Bootstrap analysis was done using the program Winboot. Isogenic and pyramid lines carrying different R gene(s) were inoculated with Xoo isolates. Disease reaction was recorded by measuring lesion length 16 days after inoculation. A cross was made between PR106 and pyramid line NH56, having resistance genes xa5, xa13 and Xa21. Successive backcrosses were made with PR106 to obtain BC3F1 plants. Plant DNA of parents (PR106 and pyramid line) along with backcross progenies (BC3F2) was isolated. Three sequence tagged sites (STS) markers RG556, RG136 and PB7/PB8 linked to BB resistance genes xa5, xa13 and Xa21, respectively, were used to identify plants with different combinations of resistance genes.

Results and discussion
A total of 693 Xoo isolates were fingerprinted using PCR based primers pJEL1 and pJEL2. On the basis of UPGMA analysis, the isolates were clustered into 51 lineages at 70% similarity index indicating high genetic diversity in the Xoo population prevalent in Punjab (India). The four major lineages (5, 7, 27, 29) collectively represented 38% of Xoo population. The lineages with less than 20 isolates were restricted to specific sites. Isogenic lines IRBB8 and IRBB21 carrying resistance genes xa8 and Xa21, respectively, along with other lines carrying Xa1 Xa4, xa5, Xa7, Xa10, xa13, Xa21, in different combinations, were found to be effective against most widely prevalent Xoo isolates in Punjab. Virulence analysis of representative isolates of each lineage indicated 17 different disease reaction patterns on a set of isogenic lines. These data further supported the high genetic diversity among Xoo population prevalent in northern India as revealed by phylogenetic analysis. Plants having xa5+xa13+Xa21, xa5+xa13, xa5+Xa21, xa13+Xa21 and each gene individually in cultivar PR106 background have been identified using molecular markers. The identified plants were inoculated with BB pathogen. A wide spectrum and higher level of resistance to BB pathogen were observed in cultivar PR106 in the BC3F2 generation. The length of the lesion in pyramid lines after inoculation with specific isolates of Xoo was much shorter than those with individual genes, indicating that gene combinations can provide increased level of resistance to the BB pathogen in rice.