Abstract: Bacterial blight caused by Xanthomonas oryzae pv. oryzae has been and is one of the major constraints for rice production by virtue of its greater adaptation and pathotype variations since, its identification in the early seventies. The pathogen population being very dynamic is changing at an accelerated pace and posing a serious challenge to the agricultural sector. The present study was undertaken to understand and gain an insight on the prevalent pathogen population which is a prerequisite for deployment of the right combination of resistant genes to combat the pathogen population. Infected leaf samples were systematically collected from a hotspot (Maruteru) of the disease and were characterized by following both field inoculation pathotyping and at molecular level by DNA fingerprinting. Greater variation was observed in the molecular phenotypes than in virulence patterns. Among genes and their combinations studied, the four gene combination (Xa4+xa5+xa13+Xa21) was found more resistant against the isolates. Such observations open up new strategies and paradigms for aiming at focused breeding programs for more stable and durable resistant cultivars by pyramiding favorable genes together.
INTRODUCTION
Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases prevalent in the major rice growing countries of Asia (Wang et al., 2006). In India, BB is a serious problem under irrigated and high fertilizer input conditions, which are conducive for disease development. Yield losses as high as upto to 80% have been reported under severe epidemics. Breeding for disease resistance is the most effective and economical method for control of BB that has a neutral impact on the environment. The BB is characterized by a high degree of race-cultivar specificity.
The pathogen is known to exhibit pathogenic variation, with diverse pathotypes or races in different rice growing areas. Many resistant genes have been identified and genetically defined in cultivars and germplasm. A clear understanding of the molecular mechanisms in host resistance to pathogens is a prerequisite for better design of control strategies for rice bacterial blight (Dai et al., 2007). There are over 30 reported races of isolates from several countries (Mew, 1987; Noda et al., 1996, 2001; Adhikari et al., 1999b; Shanti et al., 2001). Thirty two different BB resistant genes have been identified from wild and cultivated species of rice and designated in series from Xa1 to xa 32t (Sun et al., 2003, 2004; Iyer and Couch, 2004; Gu et al., 2005; Chu et al., 2006; Jiang et al., 2006a, b; Xiang et al., 2006; Wang et al., 2006; Jin et al., 2007). Several donor genotypes carrying diverse genes for BB resistance have been used to develop BB resistant varieties (Khush et al., 1989). Pyramid lines have displayed higher levels and/or wider spectra of resistance to BB than the parents or NILs with single resistance genes, suggesting synergism and complementary gene action among resistance genes (Adhikari et al., 1999a; Huang et al., 1997; Narayanan et al., 2002; Shanti and Shenoy, 2005; Sundaram et al., 2008). However, the manifestations of resistance genes vary across locations due to geographic adaptations and structuring of the pathogen.
Various molecular tools like Restriction Length Fragment Polymorphism (RFLP) have been developed to determine the pathogen population structure of Xanthomonas oryzae pv. oryzae (Leach et al., 1992; Nelson et al., 1994; Ardales et al., 1996). To expidate the analysis of a large number of isolates a simple and efficient method of DNA fingerprinting based on PCR using primers corresponding to the repetitive element IS1112 have been developed (George et al., 1995). Outwardly directed oligonucleotides complementary to each end of the IS 1112 element were used to fingerprint pathogen strains, resulting in discrimination of different strains both in the Phillipines and India (George et al., 1995, 1997; Shanti et al., 2001).
Though a lot of research has been done on the R genes and pyramiding into different cultivars of interest, there is not much study on the pathogen population especially in India, where there is a diverse geographical variation within the country. Knowledge of the pathogen structure and changes in the races of the contemporary pathogen are very vital areas of research. The present study is part of a regional effort to apply knowledge of pathogen population structure to the deployment of resistance genes. It has been undertaken to identify genes and gene combinations conferring resistance to the contemporary BB pathogen of Maruteru (hotspot) and for deploying of such genes in future breeding programs so as to incorporate durable levels of resistance in promising high yielding varieties.
MATERIALS AND METHODS
Materials Used
All the genotypes used in this study were obtained from Central Rice Research
Institute, Cuttack under the Asian Rice Biotechnology Network (ARBN) (Table
1). The experiments were initiated in kharif 2007 and repeated for three
consecutive kharif seasons. The seeds were sown in nursery beds during first
fortnight of June at Barwale Foundation research farm, Hyderabad, India. Twenty
one day old seedlings were transplanted in the fields with a spacing of 20x20
cm. Standard package of practices were followed for raising the crop.
Genotypes used in the study were the near Isogenic Lines (NILs) for BB resistance genes in IR 24 genetic background viz., ARBN 120 (Xa4), ARBN 121 (xa5), ARBN 126 (xa13), ARBN 128 (Xa21) and checks including susceptible TN1, Karuna and resistant Malagkit Sung Song. Each NIL carried a defined resistance gene backcrossed into susceptible cultivar IR 24 (Ogawa et al., 1991). In addition, the response of a set of two, three and four gene pyramids ARBN 129 (Xa4+xa5), ARBN 130 (Xa4+xa13), ARBN 131 (Xa4+ Xa 21) ARBN 132 (xa5+xa13), ARBN 133 (xa5+ Xa 21), ARBN 134 (xa13+Xa21), ARBN 135 (xa5+ xa13+ Xa21) and ARBN 137 (Xa4+xa5+xa13+Xa21) in IR 24 background were also evaluated.
Collection of Infected Leaf Samples
Infected leaf samples were collected from farmers’ fields adjoining
Agricultural Research Station (ANGRAU), Maruteru which is a hotspot for bacterial
blight disease in coastal Andhra Pradesh. For this purpose, the infected field
was divided into five hills in the form of a W. Two infected leaf samples were
collected from each hill. This was done in order to cover the entire infected
field. A total of twenty five infected leaf samples were collected from infected
fields.
Isolation and Maintenance of the Pathogen
All strains of Xoo were isolated in Barwale Foundation’s molecular
biology laboratory and maintained on modified Waikimoto’s medium (Karaganilla
et al., 1973) containing 20 g sucrose, 5 g peptone, 0.5 g calcium
nitrate, 1.82 g disodium hydrogen phosphate, 0.05 g ferrous sulphate, 18 g agar-agar
per liter with a pH 6.8 to 7.0. One colony was picked up per leaf sample and
streaked onto a petri plate. For long term storage cultures were maintained
in sterile distilled water at 4°C. When necessary these stored cultures
were revived on modified Waikimoto’s medium.
Evaluation of Resistance
Top five or six leaves of plants at maximum tillering stage were clip-inoculated
with a cell suspension of 108 colony forming units mL-1
prepared from 48 h old cultures (Kauffmann et al.,
1973). For each culture-strain combination, five leaves of a plant were
inoculated per replication. A total of three replications were undertaken. Observations
were recorded 15 days after inoculation and lesion lengths were measured to
the nearest centimeter for classifying the disease response. Individual plants
were classified as resistant (0-4 cm) and susceptible (>4 cm).
Genomic DNA Isolation
For DNA extraction, cultures were grown in 15 mL nutrient broth (per liter
10 g peptone and 3 g beef extract, pH 6.5) at 28°C on a rotary shaker (200
rpm). Genomic DNA of Xoo was extracted from 5 mL nutrient broth cultures
grown overnight. The bacterial cells were pelleted and lysed in 650 μL
extraction buffer [100 mM Tris pH 8, 100 mM EDTA, 250 mM NaCl, 15% SDS (w/v),
1% PVP-40 (w/v)] at 65°C for 30 min. DNA was isolated using a modified method
with 100 μL of potassium acetate (3 M potassium and 5 M acetate) and precipitated
with isoprgopanol (George et al., 1997).
PCR Profiling
Amplification was performed in a 25 μL volume containing 50 pM of the
two IS1112 based primers JEL1 and JEL 2, 20 ng of template DNA, 185 μM
of dNTPs 2 units of Taq polymerase in a standard incubation buffer supplemented
with 10% DMSO (v/v) and 0.75 μL of 1 M Tris HCl (pH 9.5) (George
et al., 1995). The thermo profile used includes 1 min denaturation
at 94°C followed by 30 cycles of denaturation at 94°C/10 sec, annealing
for 1 min at 62°C followed by extension at 65°C for 8 min and a final
extension for 8 min at 65°C using a thermo cycler (MJ Research Inc., USA).
The amplified products were run on 6% polyacrylamide gel, visualized by silver
staining procedure and documented. Banding pattern of each isolate was recorded
in binary form and a dendrogram was constructed employing sequential, agglomerative,
hierarchical and nested unweighted pair group method using arithmetic averages
(SAHN/UPGMA) clustering routine of NTSYS-pc using the unweighted pair-group
method using arithmetic averages.
RESULTS
The single genes and their gene combinations were chosen for this study based on our earlier studies as well as by studies conducted at All India Coordinated Rice Improvement Program (Shanti et al., 2001; Shanti and Shenoy, 2005). The results based on the mean disease reaction observed among the 16 genotypes to isolates exemplified that no single gene confers complete resistance to all the isolates (Table 1). Incidentally, individual genes showed susceptibility as compared to the gene combinations. Among the four single genes used in the study to evaluate their disease spectrum, the dominant gene Xa21 showed a higher degree of resistance being resistant to ten isolates whereas Xa4 showed the lowest degree of resistance being resistant to only five of the twenty five isolates. xa5 and xa13 were found moderately resistant with xa5 being resistant to seven and xa13 resistant to eight isolates. Figure 1 shows the varying resistance/susceptibility reactions of the 25 isolates to the 16 lines.
The two gene combinations performed better than single genes. Interestingly, Xa4+Xa21 combination performed well (resistant to 20 isolates). This is in complementation with the earlier studies showing that Xa4 though referred to as a defeated gene, it was found that this gene when in combination with Xa21 showed higher level of resistance (Jeung et al., 2006). The next best combination was xa5+ Xa21 and xa5+xa13 showing resistance to eighteen of the twenty five isolates inoculated upon. The gene combination Xa4+xa13 showed resistance to seventeen isolates. The gene combination having the least resistance was xa13+Xa21 showing resistance to fifteen isolates only.
| Table 1: | Reaction of rice genotypes to isolates of Xanthomonas oryzae pv. oryzae |
| Fig. 1: | Reaction of isolates against near isogenic lines and check cultivars |
| Fig. 2: | Virulence spectrum of the 25 isolates |
The three gene combinations showed some degree of susceptibility being susceptible to four isolates, but the four gene combination showed complete resistance to all 25 isolates (<1.0 cm lesion length) inoculated upon.
Figure 2 shows the virulence spectrum of the isolates used in the study. No isolate showed complete virulence. All the isolates showed varying degrees of resistance and susceptibility. The most virulent isolate was Xoo 22 which was very virulent against twelve out of the thirteen different combinations (single, two, three and four gene). Only the four gene combination could not be knocked down. The next most virulent isolates were Xoo 19 and Xoo 21 showing virulence to nine lines. The least virulent isolates were Xoo 1, Xoo 5 and Xoo 6 which could cause disease only in the susceptible checks. They could not breakdown the resistance of even the single gene differentials.
Pathotyping Studies
Virulence analysis exhibited a high level of diversity among the different
isolates. Using a differential set consisting of near isogenic lines and checks
the twenty five strains were grouped into pathotypes. Field inoculations on
the near isogenic lines and the check cultivars showed a total of seven pathotypes
based on the resistant and susceptibility spectrum to each of the near isogenic
line containing the individual genes (Table 2). Of these pathotypes
XA 7 was the most prevalent with fourteen isolates falling into this category.
This pathotype was the most virulent also by knocking down all the individual
genes. The next most prevalent pathotype was XA 2 having four isolates in this
class. The XA 1 had three isolates in its group and all the other pathotypes
were represented by one isolate each. The isolates in XA 1 were the least virulent
unable to knock down any of the single genes used in this study.
DNA Fingerprinting Studies
DNA fingerprinting of 25 isolates using the primer IS1112 showed distinct
banding patterns among groups (Fig. 3). DNA profiles consisted
of 40 scorable bands. Subsequent SAHN/UPGMA clustering of pathogen strains grouped
them into 17 lineages at 95% level of similarity (Fig. 4).
The maximum number of isolates in one lineage was four. At 82%, level of similarity
there was no difference and all the isolates formed one group.
DNA fingerprinting showed 17 lineages and 18 haplotypes at 95% level of similarity. Field evaluation and virulence analysis detected seven pathotypes. Table 3 shows the comparison between molecular studies and field studies used in classifying the isolates. The relationship between haplotypes, lineages and pathotypes appears to be a complex one, as observed in earlier studies (Shanti et al., 2001; Shanti and Shenoy, 2005; Nayak et al., 2008).
| Table 2: | Reaction of isolates to the host differentials |
| Table 3: | Comparison of lineages (DNA fingerprinting) versus pathotpyes (field studies) |
| Fig. 3: | DNA profile of Xoo isolates studied on PAGE using IS1112 primer |
Thus, although DNA fingerprinting is useful in revealing genetically related strains, there is no perfect prediction for virulence characterization. An example of this is Xoo 5, 13 and 16 belong to one lineage at the molecular level, but at the field Xoo 5 belonged to XA 4 and Xoo 13 and 16 belonged to XA 8. Thus, one should judiciously integrate field studies and molecular studies and make the right choice of isolates to be used in breeding programs.
| Fig. 4: | SHAN/UPGMA Clustering of Xoo isolates based on DNA profiles using the primer IS1112 |
DISCUSSION
In the present study, the potential genes and their combinations conferring resistance to the contemporary BB pathogen have been identified. In addition the most prevalent and virulent pathogen population has been identified which in turn paves the way for the right choice of genes to be deployed.
Evaluation of single genes has enabled us to analyze the risk potential involved in deploying single genes for incorporation of resistance e.g., Xa21, had a broad spectrum of resistance (Wang et al., 1996) and presumably effective in many regions. Earlier reports also show that higher frequencies of virulent strains against the gene were noticed (Shanti et al., 2001). Such observations pave the path for selection of suitable gene combinations for pyramiding for longer lasting and durable resistance.
No single gene was able to provide complete resistance to all the isolates. This is in line with our earlier studies conducted to study the pathogen population in Eastern India (Shanti et al., 2001) and studies conducted elsewhere. The two gene combination and three gene combinations conferred a broader spectrum of resistance as compared to the single genes. Earlier studies conducted (Adhikari et al., 1999a; Shanti et al., 2001; Shanti and Shenoy, 2005; Joseph et al., 2004; Sundaram et al., 2008; Suh et al., 2009) have shown similar trends. It has been found that the presence of Xa21 in combination with genes Xa4, xa5 and xa13 performed better than when treated alone. Though Xa 4 individually did not perform well in complementation with Xa21 it showed enhanced resistance. The three gene combination without Xa4 showed susceptibility to four isolates whereas the four gene combination (Xa4+xa5+xa13+Xa21) has consistently shown positive results thereby proving that it is the right combination for gene pyramiding to combat the contemporary bacterial blight pathogen in South India.
DNA fingerprinting analysis has opened up new avenues for plant pathologists across the world since, this comprehensive analysis enables us to select representative pathogen strains to identify the appropriate resistance genes for use in the region. There is no need to inoculate thousands of isolates which is very tedious and cumbersome. Once a set of representative isolates have been identified these can serve to inoculate the advanced breeding lines in any marker-assisted selection program. This saves on time, labor, money and seasons for forwarding of advanced material.
All the isolates have been collected from farmers’ fields from one area only, but at the molecular as well as the field level they are showing a large amount of variability. This is clearly indicative of the fact that many races or strains or pathotypes of Xoo can occur in a given location, of which a particular strain may be more virulent. All the isolates which are weakly susceptible and unable to breakdown the resistance of the differentials can be avoided and only the very and moderately virulent ones can be incorporated in the breeding programs in order to get the best results.
Xoo 22 showed very high virulence by breaking down the three gene combination also. Two other isolates Xoo 16 and Xoo 21 though not as virulent as Xoo 22 were also able to defeat the three gene combination. At the molecular level Xoo 21 and Xoo 22 belong to the same lineage. At the field level all three belong to the same pathotype. In an asexually propagated pathogen, like bacteria in absence of recombination it acquires favorable mutations one at a time. By accumulating favorable mutations in series, they tend to develop co-adaptive gene complexes. Once a favorable combination of genes has evolved, asexual reproduction and selection ensures the maintenance of the rapid dissemination of that particular combination. There is every possibility that all the isolates belonging to XA 7 will very soon evolve and breakdown the three gene combination also. Hence, the four gene combination is more apt for providing durable and longer lasting resistance.
The information gained in this study has significant implications for regional gene deployment. Though, lot of research has been conducted on pyramiding there is not much information available on the pathogen spectrum. The pathogen’s ability to rapidly overcome major genes (Mew, 1987; Mew et al., 1992) is one of the major challenging areas of research for deployment of R gene containing lines in order to maximize the durability spectrum in the target location (Adhikari et al., 1999a). The recent advances in cloning and characterization of major genes (Dai et al., 2007) have enabled incorporation of major genes for resistance to specific races of the pathogen.
In this study, a set of NILs having single, two, three and four gene combinations have been evaluated. Field inoculated data indicated the effectiveness of some of the individual genes with broad spectrum of resistance shown by Xa21 followed by xa13. Among the two gene combinations the broadest spectrum of resistance was shown by Xa4+ Xa21 followed by the combinations xa5+xa13 and xa5+ Xa 21. The three gene combination showed a broad spectrum of resistance being susceptible only to four isolates. The resistance spectrum of the two, three and four gene combinations may presumably be due to the complementary action of the resistance genes (Yoshimura et al., 1995). Similar observations were made in studies conducted in China and Nepal (Zheng et al., 1998; Adhikari et al., 1999a; Ming et al., 2006; Sundaram et al., 2008).
In conclusion, the present study indicates that the effectiveness of genes vary in different regions. For longer lasting and durable resistance regional information of individual genes (Shanti and Shenoy, 2005; Nayak et al., 2008; Suh et al., 2009) is a prerequisite which in turn can pave the way for deployment of the right gene combinations. In addition the intensified pathogen studies have shown the prevalence of one particular pathotype. Representative isolates from each group can be used to inoculate the advanced breeding lines and test their response to the disease. These results shed light on the right kind of gene combinations to be used in the target areas so as to insulate the losses caused due to BB and combat the pathogen population using broad spectrum and durable resistance.
ACKNOWLEDGMENT
The authors are thankful to the Management and Executive Director, Barwale Foundation for all their encouragement and support.