The use of resistant crop varieties is an inexpensive and environmentally friendly

approach to crop protection. Because some resistance genes are effective only
against particular pest or pathogen subpopulations, it is important to understand the
structure of pest or pathogen populations to determine the best strategy for
deployment of resistance. Information on pathogen population structure would
include knowledge of pathogen diversity, phylogeny, and the partitioning of variation
in time and space. Knowledge of the spatial distribution of pathogen subpopulations,
for example, can aid in the selection of disease resistance sources for a regional crop
breeding program. Unfortunately, detailed information on pathogen populations is
rarely available.
(APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1995, p. 966–971)

Bacterial blight is a widespread and destructive disease of rice in irrigated and rain
fed environments in Asia (17, 25). The disease can cause 30 to 50% yield loss (2, 4,
27). Previous studies have reported assessment of the pathotypic structure of X.
oryzae pv. oryzae on the basis of differential interactions with rice cultivars
containing different resistance genes(1, 3, 6, 7, 9, 17, 21). Molecular techniques have
provided abundant genetic markers that can be used to assess the genetic structure
of field populations of X. oryzae pv. oryzae (13, 15). Molecular probes suitable for
DNA fingerprinting and phylogenetic analysis of X. oryzae pv. oryzae have been
identified and characterized (8, 14, 24). Leach et al. (12) used a probe (pJEL101)
carrying the insertion sequence IS1112 (36), isolated from X. oryzae pv. oryzae (14),
for restriction fragment length polymorphism (RFLP) analysis of a collection of strains
of X. oryzae pv. oryzae from the Philippines.

(APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1994, p. 3275­3283)

They evaluated the diversity of sets of strains collected over defined time
periods and from different regions and determined the relationship between
pathogenic races and phylogeny of the pathogen. Nelson et al. (24) analyzed a
similar set of strains with four mobile, repetitive elements and an avirulence gene,
avrXa10 (8), isolated from X. oryzae pv. oryzae. Of the five probes tested, analysis
with probe IS1112 resulted in the most robust phylogeny. Probe avrXa10, which is of
interest because of its role in host pathogen interactions, gave the least robust
phylogeny (24). Although details of the population structure derived from RFLP data
were probe dependent, the basic phylogenetic structure that emerged from their
study was consistent with that inferred from the study of Leach et al. (12). The broad-
scale spatial partitioning of pathogen variation has implications for crop breeding
programs that serve wide areas and for the utilization of resistant germ plasm in local
breeding and deployment efforts. Furthermore, X. oryzae pv. oryzae is thought to be
seed borne (18), so international tracking of pathogen populations would be useful
for quarantine programs. Although X. oryzae pv. oryzae populations have been or are
being studied in individual countries (12, 23a, 24), comparative studies of molecular
variation at an international level have not been carried out previously. It has also
been showed that the pathogen based on the genetic distance can be divided into
five clusters where the samples collected from south asia showed greatest variation
within a single clusters due to the fact that the variation is because of considerable
variation in environmental condition. Basically the various strains showed the
variation based on geographic location.

. Molecular typing offers a powerful tool for understanding population structure and
evolution and has been used successfully for several plant pathogens (5, 11, 19, 21,
24, 25, 30). Genetic analyses have demonstrated the existence of at least 19 genes
for resistance in rice cultivars (18).
While host resistance is considered to be a sound approach to control the disease,
new races of the pathogen have emerged to overcome deployed resistance (6, 26,
28). Six Philippine races of X oryzae pv. oryzae have been defined (27,29). A race or
pathotype is a group of strains sharing a common phenotype of virulence to a set of
host cultivars, and hence strains of the same race are generally assumed to be
genetically related.

The resistance of rice to its bacterial blight pathogen Xanthomonas oryzae pv. oryzae
(Xoo) has both qualitative and quantitative components that were investigated using
three near-isogenic line sets for four resistance (R) genes (Xa4, xa5, xa13, and Xa21)
and 12 Xoo races (Genetics 159: 757–765 (October 2001))

Although some intriguing structural features have been suggested based on the
amino acid sequence (Yang and Gabriel 1995), no similarities to other proteins with
known biochemical
functions have been identified. The arrangements of the variable regions of all
characterized genes vary and appear to be critical features for the race specificity of
the proteins (Fig. 2).

The nucleotide sequence was determined for the genome of Xanthomonas oryzae
pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice
(Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular
chromosome that is G 1 C rich (63.7%). The genome includes 4637 open reading
frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs
for 80% of the predicted Xoo genes were found in the
previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris
(Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes
likely to be associated with pathogenesis include eight with similarity to
Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and
pathogenicity (hrp) genes, genes for exopolysaccharide
production, and genes encoding extracellular plant cell wall-degrading enzymes. The
presence of
these genes provides insights into the interactions of this pathogen with its
gramineous host

The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas

oryzae pv. oryzae strains producing the AvrXa21 elicitor. Xa21 codes for a receptor-
like kinase consisting of
an extracellular leucine-rich repeat domain, a transmembrane domain, and a
cytoplasmic kinase domain. AvrXa21 activity requires the presence of rax (required
for AvrXa21) A, raxB,
and raxC genes that encode components of a type one secretion system. In contrast,
an hrpC _ strain deficient in type three secretion maintains AvrXa21 activity.
Xanthomonas campestris pv.
campestris can express AvrXa21 activity if raxST, encoding a putative
sulfotransferase, and raxA are provided in trans. Expression of rax genes depends on
population density and other
functioning rax genes.
All known species of the genus Xanthomonas, a member of the gamma subdivision of
the Proteobacteria, are plant-associated and most are plant pathogens. Among them,
Xanthomonas oryzae pv. oryzae (Xoo), is a pathogen of the staple crop plant rice
(Oryza sativa). Xoo causes bacterial blight of rice, alongside rice blast caused by the
fungus Magnaporthe grisea. In addition to the importance as a pathogen, the
bacterium is known to be an ideal model for studying plant–pathogen interactions,
race differentiation and evolution of plant pathogens. Therefore, the rice–Xoo
interaction has been studied at the molecular level with special reference to the hrp
genes, encoding the type III secretion system, and the avr genes, encoding Avr
proteins6,20. The diversity of races in Xoo is remarkable: more than 30 races of
different virulence have been reported worldwide25. Because of the variability of
virulence, the breeding of resistant cultivars always confronts difficulties with the
durability of resistance. Race differentiation is associated with diversity of host
resistance genes, and the specificity is controlled in a ‘gene-for-gene’ manner14.
More than 25 rice resistance genes for bacterial blight have been identified, mostly
in Japan and at the International Rice Research Institute13. Recently, genome
structure has been studied in certain plant-pathogenic bacteria as well as in many
industrial and human-pathogenic prokaryotes, and comparative genomics of these
organisms has revealed differences— key to understanding unique
characteristics4,31,33,36 (JARQ 39 (4), 275 – 287 (2005) http://www.jircas.affrc.go.jp)

Host resistance

In the absence of effective chemical or other control agents against the BB pathogen, host resistance has
gained enormous importance in controlling this disease. In rice, the genetics of resistance to several
pathogens has been well characterized.

Resistance of rice plants towards Xoo at different growth stages varies according to host genotypes as
seedling resistance (at seedling stage) and adult plant resistance (at adult stage but susceptible at seedling
stage).

In response to a pathogen, the host plant expresses various degrees of resistance which is usually classified
into two categories namely qualitative resistance and quantitative resistance.

Qualitative resistance is generally controlled by major genes while quantitative resistance is controlled by
polygenic factors.

The pathogen

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the oldest known
diseases and was first noticed by the farmers of Japan in 1884.
All known species of the genus Xanthomonas, a member of the gamma subdivision of the Proteobacteria,
are plant-associated and most are plant pathogens. Among them, Xanthomonas oryzae pv. oryzae (Xoo). In
addition to the importance as a pathogen, the bacterium is known to be an ideal model for studying plant–
pathogen interactions, race differentiation and evolution of plant pathogens.

The disease is known to occur in epidemic proportions in many parts of the world, incurring severe crop
loss of up to 50%.

Crop loss assessment studies have revealed that this disease reduces grain yield to varying levels,depending
on the stage of the crop, degree of cultivar susceptibility and to a great extent, the conduciveness of the
environment in which it occurs.

Though the use of Bordeaux mixture, antibiotics and other copper and mercurial compounds were resorted
to in the early fifties, environmentally safe and stable chemical control agents rendering control at very low
concentrations are yet to be developed.
The pathogen is a yellow, slime-producing, motile, gram negative rod with a polar flagellum and enters the
host normally through wounds or natural openings.
It reaches the vascular tissue, particularly the xylem, from where it multiplies and spreads throughout the
plant.

A number of modern approaches to bacterial taxonomy, classification and nomenclature appear promising
especially with the blight pathogen. In 1908, Takaishi found bacterial masses in dew drops of rice leaves
but he did not name the organism.

Bokura in 1911 isolated a bacterium, and after a study of its morphology and physiology, the bacterium was
named Bacillus oryzae Hori and Bokura.

Ishiyama7 studied the disease further and renamed the bacterium Pseudomonas oryzae Iyeda and Ishiyama
according to Migula’s system.

It was later transferred to Bacterium oryzae and subsequently to Xanthomonas oryzae (the name used for
the last 40 years).

According to the revision of the International Code of Nomenclature of Bacteria the committee on
taxonomy of phytopathogenic bacteria of the International Society of Plant Pathology adopted the name
Xanthomonas campestris pv. oryzae Dye.

In 1990, the pathogen was elevated to a species status and was named Xanthomonas oryzae pv. Oryzae.

Transmission of BB pathogen
Irrigation water is considered to contribute to the spread of this disease over large areas of cultivated land,
as it carries the bacterial ooze that drop into rice field water.

However, the role of water as a primary mode of transmission has been disputed as the pathogen survives
only for 15 days in field water36,37.

The BB pathogen is seed-borne, although the extent to which it is transmitted through the seed has been
questioned

Serological methods also serve as sensitive tools for detection of the pathogen. Gnanamanickam et al.41
demonstrated the detection of Xoo in rice seeds inoculated with the pathogen using enzyme-linked immuno
sorbent assay (ELISA), whereby the bacterial colonies that reacted positively to monoclonal antibodies
specific to the bacterium were examined by direct immunofluorescence (IF).

Though ELISA and IF provide conclusive evidence for the presence of the pathogen, neither techniques are
sensitive enough to detect low numbers of the pathogen, which necessitates enrichment42.

Molecular probes, on the other hand, facilitate detection of even low numbers of the pathogen through PCR
analyses43.

However, a serious limitation to their use is that they fail to distinguish live cells from dead ones.

Attempts to control BB through chemicals like Bordeaux mixture with or without sugar, copper–soap
mixture, copper–mercury fungicides were made. Spraying copper oxychloride44 and streptomycin solution
at short intervals was recommended to control this disease45
Chlorinating irrigation water with stable bleaching powder was also reported to be effective in minimizing
the disease46
Synthetic organic bactericides such as nickel dimethyl dithiocarbamate, dithianone, phenazine and
phenazine Noxide were also recommended.
Spraying techlofthalam was more useful than soil application and it translocated readily and inhibited
bacterial multiplication in rice plants48

However, effective and economical chemical control has yet to be developed for this disease. This may be
because the pathogen population is highly variable in its sensitivity to the antibiotics used for control. The
existence and development of drug-resistant strains also pose serious problems in formulating fool-proof
control agents.

An ideal agent for chemical control will be one that functions at low concentration by either killing or
inhibiting the multiplication of the pathogen by blocking an important metabolic pathway.

It should also readily translocate and be stable in the plant system and cause minimal damage to the
environment. Attempts to control BB through chemicals like Bordeaux mixture with or without sugar,
copper–soap mixture, copper–mercury fungicides were made.

Spraying copper oxychloride44 and streptomycin solution at short intervals was recommended to control this
disease45. Chlorinating irrigation water with stable bleaching powder was also reported to be effective in
minimizing the disease46.
Synthetic organic bactericides such as nickel dimethyl dithiocarbamate, dithianone, phenazine and
phenazine Noxide were also recommended.

Spraying techlofthalam was more useful than soil application and it translocated readily and inhibited
bacterial multiplication in rice plants.

Seed treatment with hot water at 57°C for 10 min or disinfecting with mercury compounds was suggested
earlier to eradicate seed-borne inoculum.

Also, reliable forecasting systems are essential to determine the proper time of application of these
chemicals for effective control.

Biological control
Biological control for BB disease has not received much attention, though there has been a report of
bacterial antagonists against the pathogen55.
Breeding for resistance to BB with single major genes has often proved unsuccessful, as their long-term use
results in sub-populations of the pathogen that overcome these resistance genes.

Also, pathogen variation has hindered the development of suitable chemicals as agents of control. In this
light, biological control appears to be a suitable eco-friendly strategy, for disease control and management.

Recent studies have identified rice-associated rhizosphere bacteria antagonistic to Xoo from in vitro assays.
These organisms were able to cause significant disease suppression in field assays suggesting that these
strains can possibly be used as agents of biocontrol.

A series of experiments to evaluate the performance of Pseudomonas putida strain V14i (a proven
biocontrol agent against the sheath blight pathogen Rhizoctonia solani) in suppressing BB disease was also
conducted.
The results of these experiments have shown that among the different ways of application of the bacterial
bio control agent, its application as a foliar spray offered maximum suppression of BB disease severity56. A
direct correlation was also observed between the endophytic survival of P. putida in rice tissues and the
extent of disease suppression indicating that biological control may be a strategy worth pursuing for the
management of BB disease.

Host – Pathogen Interactions

Host plant resistance is an economically and ecologically sound approach for
protecting crops from many pests. Unfortunately, resistance has often proven short-
lived in farmer’s fields. Resistance may be overcome by newly evolved pest types or
by preexisting pest types that were not utilized in screening the resistant lines. Thus,
the breakdown of resistance is essentially a problem of microbial evolution.

The structure of the avr gene family from Xanthomonas is striking in that the middle
third of the encoded protein of each member is a repeated sequence of 34 amino
acids (Fig. 1). The number of repeats in an individual gene varies from 13.5 copies for
avrb6 from X. campestris pv. malvacearum to 25.5 copies in avrXa7 (Bonas et al
1989, De Feyter et al 1993, Hopkins et al 1992), and are highly conserved with the
exception of amino acids at positions 12 and 13. The sequence of positions 12 and 13
of each repeat is referred to as the variable region (Fig. 1). All members of the gene
family encode relatively large polypeptides in the range of 120 kd. . Some
deletions in repeats of avrBs3 resulted in the loss of avirulence activity on pepper
plants containing the Bs3 gene for resistance and, concomitantly, gained new
avirulence activity on
tomato (Herbers et al 1992). Upon replacement of the repeat region of avrXa10 with
the region of avrXa7 and avrBs3, the resulting phenotype was found in our laboratory
to confer specificity that corresponds to the source of the repeat domain (Fig. 2). The
avrBs3 repeat domain in avrXa10 conferred a resistant phenotype to X.c. pv.
Vesicatoria on pepper containing the Bs3 gene, and a hypersensitive reaction (HR)
response to Xoo, which normally does not give an HR on the nonhost plant pepper,
when inoculated on pepper (Fig. 2). The avrXa7 repeat in avrXa10 conferred
specificity for the resistance gene Xa7 and lost specificity for Xa10.
The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas
oryzae pv. oryzae strains producing the AvrXa21 elicitor. Xa21 codes for a receptor-
like kinase consisting of
an extracellular leucine-rich repeat domain, a transmembrane domain, and a
cytoplasmic kinase domain. AvrXa21 activity requires the presence of rax (required
for AvrXa21) A, raxB,
and raxC genes that encode components of a type one secretion system. In contrast,
an hrpC _ strain deficient in type three secretion maintains AvrXa21 activity.
Xanthomonas campestris pv.
campestris can express AvrXa21 activity if raxST, encoding a putative
sulfotransferase, and raxA are provided in trans. Expression of rax genes depends on
population density and other
functioning rax genes.

Therefore, the rice–Xoo interaction has been studied at the molecular level with
special reference to the hrp genes, encoding the type III secretion system, and the
avr genes, encoding Avr proteins6,20. The diversity of races in Xoo is remarkable:
more than 30 races of different virulence have been reported worldwide25. Because
of the variability of virulence, the breeding of resistant cultivars always confronts
difficulties with the durability of resistance. Race differentiation is associated with
diversity of host resistance genes, and the specificity is controlled in a ‘gene-for-
gene’ manner14. More than 25 rice resistance genes for bacterial blight have been
identified, mostly in Japan and at the International Rice Research Institute13

Previous studies have reported assessment of the pathotypic structure of X. oryzae

pv. oryzae on the basis of differential interactions with rice cultivars containing
different resistance genes(1, 3, 6, 7, 9, 17, 21). Molecular techniques have provided
abundant genetic markers that can be used to assess the genetic structure of field
populations of X. oryzae pv. oryzae (13, 15). Molecular probes suitable for DNA
fingerprinting and phylogenetic analysis of X. oryzae pv. oryzae have been identified
and characterized (8, 14, 24). Leach et al. (12) used a probe (pJEL101) carrying the
insertion sequence IS1112 (36), isolated from X. oryzae pv. oryzae (14), for restriction
fragment length polymorphism (RFLP) analysis of a collection of strains of X. oryzae
pv. oryzae from the Philippines.