Study on bacterial leaf blight (xanthomonas oryzae) of rice

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

FACULTY OF AGRONOMY
-------------***-------------

UNDERGRADUATE THESIS
TITLE: STUDY ON BACTERIAL LEAF BLIGHT
(Xanthomonas oryzae) OF RICE

Student name : BUI HUU QUANG
Class

: K61KHCTT

Student code

: 614114

Department

: PLANT PATHOLOGY

Supervisor

: Assoc.Prof. HA VIET CUONG

HANOI – 2021


DECLAIRE

I hereby declare that this graduation thesis is my own original work and
has not been submitted before to any institution for assessment purposes.
Further, I have acknowledged all sources used and have cited them in the
reference section.
Hanoi, January 2021
Signature

Bui Huu Quang

i


ACKNOWLEDGEMENTS

First of all, I would like to express my deep gratitude to Assoc.Prof.Dr.
Ha Viet Cuong, Lecturer of Plant Disease Department; Dr. Nguyen Van Muoi,
Plant Research Institute, Faculty of Agriculture, Vietnam National Academy of
Agriculture. The two instructors were always by my side, caring, helping,
giving suggestions, commenting and conveying to me valuable experiences
during the graduation thesis.
With the most sincere feelings, I would like to thank all staff of the
Center for Tropical Plant Disease, Center for Conservation and Development
of Plant Genetic Resources, and Plant Research Institute for their enthusiastic
guidance and assistance. has taught enthusiastically about the expertise and
skills of the laboratory along with the support of experimental materials during
the practice of the thesis.
And finally, with all my infinite respect and gratitude, I would like to
extend my parent; is a solid rear for me on the road of life.

Thank you very much!

Bui Huu Quang

ii


TABLE OF CONTENTS
DECLAIRE .............................................................................................................i
ACKNOWLEDGEMENTS ...................................................................................ii
TABLE OF CONTENTS ..................................................................................... iii
LIST OF ABBREVIATIONS ................................................................................ v
LIST OF TABLES ................................................................................................ vi
LIST OF FIG ........................................................................................................vii
SUMMARY ..........................................................................................................ix
PART I: INTRODUCTION ................................................................................ 1
1.1 Introduction ...................................................................................................... 1
1.2.Objectives and requirements ............................................................................ 2
1.2.1 Objectives ...................................................................................................... 2
1.2.2 Requirements................................................................................................. 2
PART II: LITERATURE REVIEW .................................................................. 2
2.1 Status of research on Bacterial leaf blight of rice in the world ........................ 3
2.2 Status of research on Bacterial leaf blight of rice in Vietnam ......................... 3
2.3 Discovery and classFIcation of Xanthomonas oryzae .......................................... 5
2.4 Distribution and economic impact ................................................................... 6
2.5 Pathogen modes of infection, symptoms, and signs ........................................ 8
2.6 Sources of primary inoculum, dissemination and survival .............................. 9
2.7 Control............................................................................................................ 10
2.8 Xanthomonas oryzae pv. oryzae (Xoo).......................................................... 14
2.8.1 Morphology and structural .......................................................................... 14
2.8.2 Genomic ...................................................................................................... 16

2.8.3.Pathogenicity (related genes) genes ............................................................ 18
PART III. MATERIALS AND METHODS .................................................... 21
3.1. Location and time for research ...................................................................... 21
3.1.1. Research location ....................................................................................... 21
3.1.2. Time for research ....................................................................................... 21
3.1.3. Subjects ...................................................................................................... 21
3.2 Materials ......................................................................................................... 21
iii


3.2.1. Media and preparation ............................................................................... 21
3.2.2. Chemicals and buffers ................................................................................ 23
3.2.3. Commercial enzymes and markers ............................................................ 23
3.2.4. Antibiotics and bactericide ......................................................................... 23
3.2.5. Primers ....................................................................................................... 24
3.2.6. Key facilities .............................................................................................. 24
3.3 methods .......................................................................................................... 24
3.3.1 Collection of leaf samples ........................................................................... 24
3.3.2 Bacterial isolation........................................................................................ 25
3.3.3 PCR test ....................................................................................................... 25
3.3.4 Pathogenicity test ........................................................................................ 27
3.3.5. Isolation of Xanthomonas oryzae from rice leaves .................................... 28
3.3.6 Inoculation of Xoo ...................................................................................... 28
3.3.7. Agarose Electrophoresis ............................................................................ 28
PART IV. RESULTS AND DISCUSSION ...................................................... 30
4.1 ISOLATION OF XANTHOMONAS ORYZAE PV. ORYZAE .................. 30
4.1.1 Bacterial leaf blight (BLB) sampling .......................................................... 30
4.1.2 Isolation of Xanthomonas oryzae from collected samples ......................... 31
4.1.3. Identification of Xanthomonas oryzae pv. oryzae by multiplex PCR ....... 33
4.2. MORPHOLOGICAL and biological characteristics of XOO isolates ......... 35

4.2.1 Identification of Gram group of Xoo isolates by KOH test ........................ 35
4.2.2 Colony characteristics of Xoo isolates on different media ......................... 37
4.2.3 Starch hydrolysis ......................................................................................... 40
4.2.4 Cellulose hydrolysis .................................................................................... 42
4.2.5 Catalase test ................................................................................................. 44
4.2.6 Hypersensitive reaction (HR) on tobacco leaf ............................................ 46
4.2.7 Reaction of Xoo isolates with antibiotics and bactericide .......................... 47
4.2.8 Pathogenicity test ........................................................................................ 50
PART V. CONCLUSION AND SUGGESTION ............................................. 54
5.1. Conclusion..................................................................................................... 54
5.2. Proposal for further work .............................................................................. 54
REFERENCES:.................................................................................................... 55
iv


LIST OF ABBREVIATIONS
ABBREVIATION

TERM

Ab

Antibody

Ag

Antigen

BLB

Bacterial leaf blight

BLS

Bacterial leaf streak

Bp

Base pair

DNA

Deoxyribonucleic acid

dNTP

Deoxynucleoside triphosphate

EDTA

Ethylene diamine tetra acetic acid

ELISA

Enzyme linked immunosorbent assay

EPS

Capsular extracellular polysaccharide

Ig

Immunoglobulins

OD

Optical Density

ODD

Ouchterlony Double Diffusion

PCR

Plolymerase Chain Reaction

PEG

Polyethylen

PSA

Peptone Sucrose Agar

TAE

Tris – acetate – EDTA

UV

Utra Violet

Xo

Xanthomonas oryzae

Xoc

Xanthomonas oryzae pv. oryzicola

Xoo

Xanthomonas oryzae pv. oryzae

v


LIST OF TABLES

Table 3.1 PCR master mix (25 µL) .......................................................................... 26
Table 3.2 PCR profile .............................................................................................. 26
Table 4.1. Rice bacterial leaf blight samples collected in 2020.............................. 30
Table 4.2. Isolation of Xanthomonas oryzae from diseased rice leaf samples on
mXOS medium ........................................................................................ 32
Table 4.3. Detection of Xanthomonas oryzae pv. oryzae by multiplex PCR .......... 34
Table 4.4. Gram differentiation of Xoo isolates by KOH test ................................. 36
Table 4.5. Colony characteristic of Xoo isolates on different media after 4 days at
28 oC ........................................................................................................ 37
Table 4.6. Starch hydrolysis test of Xoo isolates ..................................................... 41
Table 4.7. Cellulose hydrolysis test of Xoo isolates ................................................ 43

Table 4.8. Catalase test of Xoo isolates ................................................................... 45
Table 4.9. Hypersensitive reaction (HR) of 8 selected Xoo isolates on tobacco leaf
(Nicotiana tabacum cv. K326) ................................................................ 46
Table 4.10. Growth inhibition of antibiotics and bactericide against 2 Xoo isolates
................................................................................................................. 48
Table 4.11: Inoculation result of 5 Xoo isolates on rice seedling ............................ 51

vi


LIST OF FIG

Fig. 2.1. Morphology of Xanthomonas oryzae. (a) Strands and condensed droplets
of ooze consisting of X. oryzae cells coated in extracellular
polysaccharide exuded on to the surface of an infected rice leaf. (b)
Colonies of X. oryzae. pv. oryzae on glucose yeast extract agar. (c)
Scanning electron micrograph of a single X. oryzae pv. oryzae cell (bar,
1.0 ............................................................................................................... 16
Figure 2.2. Genomic map of Xoo strain MAFF311018. .......................................... 17
Figure. 4.1. Symptoms of collected rice bacterial leaf blight samples..................... 31
Figure 4.2. Isolation of Xoo on mXOS medium by 3-ways streaking (A) and dilution
(B) methods. The plates are from sample 1. The plate B is at 102 dilution.
The arrow indicates the Xoo-like colonies .............................................. 33
Figure 4.3. Multiplex PCR of 8 Xoo isolates selected from positive colonies. M1 is
100 bp DNA ladder with denoted reference bands. M2 is 1 kb DNA
ladder (GeneRuler 1 kb, Thermo Scientific). Two bands, 331 and 162 bp,
specific to Xoo pathovar are indicated by arrow. .................................... 35
Figure 4.4. KOH reaction of Xoo isolates depictured from the Xoo-01 isolate ...... 36
Figure 4.5. Culture of 8 Xoo isolates on different media after incubation at 28 oC for
4 days.......................................................................................................... 40

Figure 4.6. Starch hydrolysis test of 8 Xoo isolates show all of them are positive . 42
Figure 4.7. Cellulose hydrolysis test of 8 Xoo isolates showed all of them were
positive. ...................................................................................................... 44
Figure 4.8 Catalase test of Xoo isolates. The Xoo isolates are indicated. ............... 45
Figure 4.9. Hypersensitive reaction (HR) of 8 selected Xoo isolates on tobacco leaf
(Nicotiana tabacum cv. K326) ................................................................. 47
Figure 4.10a. Evaluation of growth inhibition of antibiotics and bactericide at
recommended 10X concentration against 2 Xoo isolates. ...................... 49

vii


Figure 4.10b. Illustration of position and growth inhibition of antibiotics and
bactericide at recommended 10X concentration against Xoo isolates
(Xoo-1) ....................................................................................................... 49
Figure 4.11. XOO-1 isolate infection in rice varieties .............................................. 52
Figure 4.12. XOO-2 isolate infection in rice varieties .............................................. 52
Figure 4.13. XOO-3 isolate infection in rice varieties .............................................. 52
Figure 4.14. XOO-4 isolate infection in rice varieties .............................................. 53
Figure 4.15. XOO-6 isolate infection in rice varieties .............................................. 53

viii


SUMMARY
1. Title: “Study on Bacterial leaf blight of rice
2. Purpose
-This study aimed to detect and evalution of biological, physiological and
pathogenic characteristics of Xanthomonas oryzae pv. Oryzae.
3. Research method

-To collect the infected leaves samples from different locations
-To isolate and identify Xanthomonas oryzae pv. oryzae (Xoo) from
collected samples
-To evaluate the biological characteristics of isolated Xoo isolates
-To evaluate the physiological characteristics of isolated Xoo isolates
-To evaluate the pathogenicity of isolated Xoo isolates
4. Newness, creativity
Leaf blight is caused by bacteria Xanthomonas oryzae pv. oryzae (Xoo)
causes severe damage to rice growing regions across the country, reducing rice
yield by up to 50%. Although some chemicals have been used in recent years,
they cannot control the disease under severe conditions. They also cause
environmental problems, especially water pollution. Therefore, my research
contributes to the evaluation of the pathogenicity of current blight bacteria, while
also preventing the risk of outbreaks.
5. Research results
- Using semi-selective medium, mXOS, several anthomonas oryzae – like
isolates collected from 5 provinced in the Red River Delta were isolated. Based
on Multiplex PCR, 8 Xoo isolates were identified and selected for futher
studies.
- The 8 Xoo isolates have several identical characteristics including: Gram
negative (KOH test), grow slowly on nutrient media, colony being convex,
mucoid, shiny, pale to straw yellow colour in PSA, mWakimoto and YDC
ix


medium, rose pink colour in mXOS, starch hydrolysis positive, cellulose
hydrolysis positive and catalase positive.
- Invitro test on two Xoo isolates, Xoo-1 and X00-9, indicated that
Streptomycin could inhibit growth of Xoo. In contrast, 4 other antibiotics
including chloramphenicol, rifampicin, tetracycline and ampicillin and on

bactericide, kasugamycin from Kasumin 2 SL (Arysta), could not inhibit Xoo,
even at concentrations that are 10 times higher than recommended ones.
6. Contribution to the socio-economic, educational and training aspects
and the applicability of the topic:
- To collect more Xoo isolates in other province.
- To characterize further physiological and particularly pathogenic
characteristics of the collected isolates.
- To apply the antibiotics that are not inhibit Xoo for selection of this
pathovar during isolation.

x


PART I: INTRODUCTION
1.1 INTRODUCTION
Rice has long been the main food crop of Vietnam. With a favorable
tropical monsoon climate and a long-standing wet rice civilization, our country
has gradually become one of the world's leading rice exporting countries. The
climate is good for crops, but also facilitates some rice pests and diseases.
Especially among them, bacterial leaf blight (BLB) disease caused by a
bacteriam, Xanthomonas oryzae pv. oryzae (Xoo) has been one of major
drawbacks to rice production throughout the country. In many cases, BLB
disease cause 100% losses.
In 1995, 1996 forty isolates of Xanthomonas oryzae pv. oryzae have been
collected from different regions in Vietnam by Van E et al. (1999). The isolates
were inoculated on some Vietnamese rice cultivars, nearly isogenic lines and
check varieties to identify pathogenicity of BLB and resistant response to Xoo
isolates. The results suggested the pathogenic diversity of the isolates in
Vietnam seems to be complex despite their simple pathogenic characteristics
to the differential varieties. Noda et al. (1999) collected 52 isolates of Xoo

throughout Vietnam and examined the variability in virulence of these isolates
based on the reactions of the 18 differential varieties. The results showed the
susceptibilities of the local varieties tested to the Vietnamese isolates. These
studies would increase knowledge of pathogen diversity and their virulence.
However, the latest significant survey dates from more than 10 years ago.
Contemporary situation needs to be re-assessed country-wide. Moreover, it’s
unable to access strain collections if they still exist. For many years it was
unclear whether there was any pathogen variation of virulence. Resistance may
change an old pathogen into a new form. There is a need for an accessible
national collection of X. oryzae for further studies of virulence of pathogens.
1


Although some chemicals have been used in recent years, they cannot
control the disease under severe conditions. They also cause environmental
problems, especially water pollution.
Stemming from the above practical situation, with the permission of the
Department of Plant Disease, Faculty of Agriculture, Vietnam University of
Agriculture, with the guidance of Assoc. Prof. Ha Viet Cuong, I choose the
project: "Study on bacterial leaf blight of rice".
1.2.OBJECTIVES AND REQUIREMENTS
1.2.1 Objectives
This study aimed to detect and evalution of biological, physiological and
pathogenic characteristics of Xanthomonas oryzae pv. oryzae.
1.2.2 Requirements
- To collect the infected leaves samples from different locations
- To isolate and identify Xanthomonas oryzae pv. oryzae (Xoo) from
collected samples
- To evaluate the biological characteristics of isolated Xoo isolates
- To evaluate the physiological characteristics of isolated Xoo isolates

- To evaluate the pathogenicity of isolated Xoo isolates

PART II: LITERATURE REVIEW

2


2.1 STATUS OF RESEARCH ON BACTERIAL LEAF BLIGHT OF
RICE IN THE WORLD
Bacterial leaf blight is not a new disease of rice. Its importance to rice
production in tropical Asia, however, was recognized only after the
introduction of modern cultivars, which are highly responsive to nitrogen
fertilizer. It is now a major rice disease throughout Asia. Bacterial blight is
especially prevalent in irrigated and rainfed lowland rice areas in Asia. In the
last twenty years, epidemics have been reported prior to the introduction of
modern rice. Since the introduction and widespread cultivation of high yielding
but susceptible rice cultivars, bacterial blight has become one of the most
serious diseases of rice in Asia. In Punjab, Haryana, and western Uttar Pradesh
States of India, major epidemics occurred in 1979 and 1980; severe kresek was
observed; and total crop failure was reported. Estimates of yield losses caused
by bacterial blight are not available. The extent of yield loss depends on
locality, season, weather, and cultivar. An increase in the amount of nitrogen
fertilizer application favors disease development and thus causes greater yield
loss. The amount of nitrogen applied did not increase the incidence and severity
of the disease in a resistant cultivar. In recent years the disease has been
observed not only in all major rice producing countries of Asia, but also in the
Sahelian countries of Africa and in South America. As improvement of rice
crops in these regions has gradually taken place without incorporation of proper
resistance through a breeding program, the disease could emerge to threaten
irrigated rice production, as has happened in Asia.

2.2 STATUS OF RESEARCH ON BACTERIAL LEAF BLIGHT OF
RICE IN VIETNAM
Bacterial leaf blight is one of the most destructive bacterial diseases of
rice in Vietnam. The disease was often found in the North, but occasionally
found appearance in the Southern part, especially in Mekong Delta. After 1985,
3


the changes of rice ecosystem and cropping patterns significantly influenced to
disease occurrence in the delta (Van E et al. , 1999). It spread rapidly and
prevailed throughout the rice-planting area because of the widely grown hybrid
rice from China combinations with high yield which susceptible to BLB. The
disease becomes more serious because of the introduction of new cultural
practices, the adoption of high-density direct seeding and high inputs of
nitrogen fertilizers, especially in rainy season (Noda et al. , 1999). However,
no approach is considered as an effective and economic practice to control this
disease. The varietal resistance is considered as a key tool under tropical
conditions. In addition, knowledge of pathogen population structure, coupled
with an understanding of the mechanisms that drive genetic changes in
pathogen populations, is essential to formulate long-term strategies to manage
the disease.
Noda et al. (1999) collected BLB samples in various regions of the
northernVietnam as well as in the Mekong River Delta (MRD) to survey the race
distribution of Xoo. The varietal resistance of the major rice varieties including
local ones to the Vietnamese races was tested. As their results, fifty-two isolates
collected and classified into six pathogenic groups (races) based on their
pathogenicity to the 18 differential varieties. More than 80% of the isolates were
classified into predominant race, which was distributed throughout Vietnam. The
other nine isolates, belonging to five other races, were minor races, the pathogenic
characteristics of these six races were relatively similar. Therefore, the diversity

in the pathogenicity of the Vietnamese BLB strains may be low based on their
virulence to differential varieties.
In addition, forty isolates of Xanthomonas oryzae pv. oryzae have been
collected from different regions in Vietnam by Van E et al. (1999). The isolates
were inoculated on some Vietnamese rice cultivars and nearly isogenic lines
(NILs) + check varieties to identify pathogenicity of bacterial leaf blight and
4


resistant response to Xoo isolates. Isolates belong to dominant pathogenic
group were widely distributed at different places from the North to the South
Vietnam. Almost the Vietnamese cultivars were susceptible to all testing
isolates. However, some varieties in The Mekong Delta were resistant to a few
isolates which were collected in North Vietnam.
More information on current population structure of Xanthomonas
oryzae pv. oryzae in the northern Vietnam was provided by Furuya et al.
(2012). Strains of Xoo were collected from nine regions in this part of Vietnam
(Fig 2.1) in 2001 and 2002 using PCR-based DNA fingerprinting and virulence
analysis. Their results showed that predominant races between northern and
southern Vietnam represented distinct population of Xoo, indicating substantial
geographical differentiation between the pathogen populations in difference
agroecosystems. The pathotype diversity was influenced by the collection site
and cultivars sampled. Moreover, the diversity in Vietnam may reflect the
relatively broad range of environmental conditions under which rice is
cultivated and the wide use of diverse traditional cultivars.
2.3 DISCOVERY AND CLASSFICATION OF XANTHOMONAS ORYZAE
The species Xanthomonas oryzae includes two pathovars, namely,
oryzae and oryzicola (Swings et al., 1990) that cause two different diseases,
Bacterial Leaf Blight (BLB) and Bacterial Leaf Streak (BLS), respectively.
These bacterial pathogens are closely related organisms and were earlier

named as pathovars of Xanthomonas campestris. Rice is the main host for both
pathogens, which are seed-borne and seed-transmitted. Bacterial leaf blight of
rice (BLB) was first reported in Fukuoka Prefecture, Japan, during 1884 in rice
affected by X. oryzae pv. oryzae. This disease is considered one of the most
serious rice diseases worldwide although it has declined in incidence in Japan
since the mid 1970s. Nevertheless it is still prevalent worldwide. The disease
was reported in South East Asia in the early 1960s, where it is currently
5


widespread, and it still affects the rice crop in its severe form (Goto, 1992). It
has also been reported in several African countries, in Australia, North America
(Lousiana and Texas, US), Central and South America (OEPP/EPPO, 2006a)
but it is only of economic importance in Asia. Bacterial leaf blight of rice was
originally believed to be caused by acidic soil (Sh, 1972). In 1909, masses of
bacteria were isolated from the (acidic) turbid dewdrops of infected rice leaves,
and the disease was reproduced by inoculating healthy leaves with these
dewdrops. Shortly thereafter its etiology as a bacterial disease was established,
and the causal agent was isolated and classified as Bacillus oryzae (Mizukami
& Wakimoto, 1969). The bacterium was renamed Pseudomonas oryzae and
later Xanthomonas oryzae (Ishiyama, 1922). In 1978, it was reclassified as X.
campestris pv. oryzae (Dye, 1978)
Bacterial leaf streak blight of rice BLS was first discovered in the
Philippines in 1918 and named bacterial stripe (Sh, 1972) although it was
erroneously referred to as bacterial blight for several years. In a study in southern
China in 1957, disease was again characterized as distinct from bacterial leaf
blight and called bacterial leaf streak. The causal agent was distinguished from
that of bacterial leaf blight and given the name Xanthomonas oryzicola (Sh,
1972).The pathogen was later renamed X. translucens f. sp. Oryzae (Sh, 1972).
It has also been referred to as X. translucens f. sp. oryzicola and X. campestris

pv. oryzicola (Ou, 1985). In 1990 both pathogens were elevated to their current
status as a new species and named Xanthomonas oryzae pv. oryzae and
Xanthomonas oryzae pv. oryzicola (Swings et al., 1990; Goto, 1992). The
species

resides

within

the

family

Xanthomonadaceae

in

the

Gammaproteobacteria (Niño‐Liu et al., 2006).
2.4 DISTRIBUTION AND ECONOMIC IMPACT
BLB and BLS are endemic to much of Asia and parts of West Africa.
BLB is prevalent in both tropical and temperate areas, and has also been
6


reported in Australia, Latin America and the Caribbean (Mew et al., 1993). By
contrast, BLS is restricted largely to tropical and subtropical Asia, including
southern China, Thailand, Malaysia, India, Viet Nam, the Philippines and
Indonesia, but it also affects rice-growing regions of northern Australia

(Awoderu et al., 1991; Moffett and Croft, 1983; Ou, 1985; Sigee, 1993; Singh
et al., 1980) and recently has become a significant problem in parts of West
Africa (J. Notteghem, personal communication). In the United States, although
an apparent mild outbreak of BB was reported in the late 1980s (Jones et al.,
1989), it was later determined that the bacterium associated with the disease
was not Xoo (Ryba- White et al., 1995). Quarantines for Xoo and Xoc are in
place in the United States and other rice-growing countries where the diseases
are not endemic, but also in places where they are present, to prevent the
introduction of new virulent strains. Xoc is listed in the 2002 Agricultural
Bioterrorism Protection Act of the USA as a potential bioterrorism agent,
necessitating strict biosecurity measures (to limit access to pathogen stock
cultures in research laboratories) in addition to standard biosafety measures (to
prevent release of the pathogen into the environment). Implementation of
biosecurity measures is an unfortunate necessity that somewhat restricts US
research on this important pathogen.
Damage due to BLB increased significantly following the widespread
cultivation of high-yielding and nitrogen-responsive dwarf hybrid varieties of
rice in the 1960s. Prior to the more recent incorporation of resistant varieties and
implementation of strict quarantine measures in Japan, BLB damage there was
reported to range from 20 to 30% and as high as 50% (Sh, 1972). In tropical
countries BLB is even more destructive. Reports from the Philippines, Indonesia
and India estimate that losses due to the kresek syndrome of BLB, which affects
recently transplanted seedlings, have reached 60–75%, depending on weather,
location and rice variety (Reddy et al., 1979; Ou, 1985). In addition to reducing
7


yield, BLB may also affect grain quality by interfering with maturation (Ou,
1985; Goto, 1992). Although documentation does not exist for many areas in
which BLS is present, available reports suggest that yield losses due to this

disease typically range from 0 to 20% depending on the rice variety and climatic
conditions (Ou, 1985). Without strong wind and rain, secondary spread of BLS
is limited and the effect of the disease dwindles rapidly as the growth of new
leaves compensates for damage to infected leaves (Ou, 1985). Under conditions
favourable for spread, however, BLS may affect entire fields and cause damage
comparable with BLB, e.g. reductions in grain weight of up to 32% (Ou, 1985).
Overall, however, BLS is less economically important than BLB. Nevertheless,
BLS is increasing in significance in parts of Asia where hybrid rice varieties are
grow, as these varieties can be particularly susceptible to the pathogen.
2.5 PATHOGEN MODES OF INFECTION, SYMPTOMS, AND SIGNS
X. oryzae pv. oryzae enters either through wounds or hydathodes,
multiplies in the epitheme and moves to the xylem vessels where active
multiplication results in blight on the leaves. The symptoms of the disease
include leaf blight, wilting (kresek), and pale yellow leaves. BLB is
characterized by wavy elongated lesions, which develop along the leaf margins.
They start as small water-soaked stripes from the tips where water pores are
found and rapidly enlarge in length and width, forming a yellow lesion with a
wavy margin along the lead edges. Later on, diseased areas turn white to grey.
These lesions can develop on one or both sides of the leaf and occasionally
along the midribs, and BLB symptoms generally occur from maximum tillering
stage and onwards. In young lesions, drops of bacterial ooze can be observed
early in the morning. On panicles the disease causes grey to light brown lesions
on glumes that result in infertility and low quality of the grains. Kresek is the
result of systemic infection that is common in the tropics in young plants and
during the tillering stage of susceptible cultivars. Leaves of infected plants wilt,
8


roll up, turn grey-green and whither, and entire plants finally die. Surviving
plants look stunted and yellowish. Yellow or pale yellow leaves are due to

systemic infections that appear at tillering stage; the youngest leaves become
uniformly pale yellow or show a broad yellow stripe, and bacteria are found in
the internodes and crowns of affected stems, but not in the leaf itself (Ou, 1985;
Goto, 1992).
2.6 SOURCES OF PRIMARY INOCULUM, DISSEMINATION AND
SURVIVAL
Outbreaks of both BLB and BLS are more likely to occur during the
monsoon season of the south-east Asian and Indian oceans (from June to
September) than at other times of the year (Mew et al., 1993). Wind and rain
disseminate bacteria from infected rice plants and other hosts, as well as
contaminated rice stubble from previous crop seasons—the most important
sources of primary inoculum. Severe epidemics often occur following
typhoons, the fierce winds, wind-blown rain and hail of which both wound rice
plants and disperse bacteria. Bacteria may also be disseminated in irrigation
water, as well as by humans, insects and birds (Ou, 1985; Nyvall et al., 1999).
Other hosts of Xoo include several species of wild rice (O. sativa, O. rufipogon,
O. australiensis ) and a number of gramineous weeds ( Leersia oryzoides and
Zizania latifolia in temperate regions and Leptochloa spp. and Cyperus spp.
in the tropics). Virtually all species of wild rice can serve as hosts for Xoc
(Moffett and Croft, 1983), but other alternative hosts have not been identified.
In a study conducted at the International Rice Research Institute (IRRI) in the
late 1960s, none of several gramineous weeds and crop species inoculated with
Xoc developed disease (Ou, 1985). In temperate regions, Xoo can survive the
winter in the rhizosphere of weeds of the genera Leersia and Zizania as well as
in the base of the stem and the roots of rice stubble (Mizukami & Wakimoto,
1969). In addition, in temperate regions, Xoo can survive in the soil for 1–3
9


months depending on the soil moisture and acidity, though this is not

considered an important source of inoculum (Ou, 1985). Xoo can overwinter in
piled straw as well; this source of inoculum may acquire importance in areas
where little or no weedy hosts occur (Ou, 1985). In the tropics, high
temperature, humidity and an abundance of host plants typically allow Xoc, and
Xoo, to persist throughout the year (Ou, 1985). Both pathovars can be isolated
easily from seed of infected plants (Sakthivel et al., 2001). Nevertheless,
controversy exists over how long bacteria can survive in stored grain and
whether seed-borne transmission is important.
2.7 CONTROL
Control measures for BB include cultural practices, chemical and
biological control, disease forecasting, and, most importantly, host genetic
resistance, typically major gene resistance. Few studies have been conducted
on control methods for BLS, although many of the same measures used for BB
can be expected to be effective against BLS. As is the case for BB, in practice,
host genetic resistance is the most important control measure for BLS, although
it is so far limited to quantitative resistance (Gnanamanickam et al., 1999;
Sheng et al., 2005; Tang et al., 2000).
Cultural practices useful for BLB control vary depending on the location
and disease incidence records. At the nursery stage, methods include seed
disinfection (see below), proper nursery drainage, and removal of diseased
plants, weeds and debris. Prior to transplanting, fields may be disinfected by
burning rice straw left from the previous season. Weeds are removed from
canals and ridges in order to reduce natural habitats for the pathogen and its
dispersal through irrigation water. At the paddy field stage, judicious
fertilization and proper plant spacing are the most recommended cultural
methods of control (Goto, 1992; Mizukami and Wakimoto, 1969). Fertilization
must avoid an excess of nitrogen as it stimulates rapid vegetative growth of the
10

plant, which favours disease development. Application of fertilizers rich in
potassium and phosphorus, as well as application of agrochemicals at the
maximum tillering to booting stages or after a typhoon or a severe flood are
common practices (Goto, 1992; Ho and Lim, 1979; Mizukami and Wakimoto,
1969).
Chemical control of BLB in rice fields began in the 1950s with the
preventative application of Bordeaux mixture (hydrated lime and copper
sulfate) and the testing of several antibiotics, mercuric and copper compounds.
Laboratory tests determined that streptomycin derivatives and mercuric
compounds were most effective, but they were found to damage rice grains
when sprayed at the heading stage in the field (Mizukami and Wakimoto,
1969). In the 1960s, different kinds of agrochemicals were developed from
repeated field trials and made available on a large commercial scale, mostly in
Japan.

They

were

based

on

Lchloramphenicol,

nickel-

dimethyldithiocarbamate, dithianon and fentiazon. Most were unreliable,
however, owing to variability in sensitivity among the pathogen population
(Gnanamanickam et al., 1999; Mizukami and Wakimoto, 1969; Ou, 1973).

Although seed transmission of the disease is an uncertain source of primary
inoculum, disinfection of rice seeds with mercuric compounds, antibiotic
solutions or hot water is practised in several countries in tropical Asia. In
temperate regions, chemical control of BLB in nurseries and paddy fields
includes the application of probenazole to the paddy water before and after
transplanting the seedlings, in order to inhibit bacterial multiplication and
prevent or retard the disease. Other chemicals such as tecloftalam, phenazine
oxide and nickel dimethyldithiocarbamate are sprayed directly on plants (Goto,
1992; Mizukami and Wakimoto, 1969). However, chemical control of BLB in
the tropical monsoon climate of Asia is impractical, and no truly effective

11


bactericide is commercially available for disease control (Lee et al., 2003; Ou,
1973).
Biological control is an environmentally friendly and costeffective
alternative to chemical control. Bacterial antagonists of Xoo have received
particular attention as biocontrol candidates, largely because of their rapid
growth, easy handling and effective colonization of the rhizosphere (Vasudevan
et al., 2002). In India, about 40 bacterial isolates antagonistic to Xoo were
identified through plate and field assays. Among those antagonists native strains
of the rice-associated rhizobacteria Pseudomonas fluorescens and P. putida
strain V14i (also used in biocontrol of the rice sheath blight pathogen
Rhizoctonia solani) significantly suppressed BLB severity when sprayed on
leaves (Sivamani et al., 1987). For both agents, there was a significant correlation
between endophytic survival in rice tissues and the extent of disease suppression
(Gnanamanickam et al., 1999; Johri et al., 2003). Similarly, different species of
Bacillus have been employed as seed treatment before sowing, root dips prior to
transplanting and foliar sprays in the fields. In at least one study, BLB was

suppressed by almost 60%, and plant height and grain yield increased by twofold (Vasudevan et al., 2002). Although the mechanisms of BLB suppression are
not known, a recent investigation of biocontrol of the rice sheath blight disease
has suggested that a rice systemic resistance response to the agents may be
involved, as has been observed in other systems (Vasudevan et al., 2002).
Despite promising results such as these, biological agents have not seen
widespread use in the control of BLB.
Forecasting of BLB and BLS is difficult because epidemics are
dependent on the rice cultivars and cultural practices in use, in addition to
environmental and geographical conditions. Methods used for forecasting may
include scouting for early disease development and tracking climatic conditions
(Mizukami and Wakimoto, 1969). In temperate locations, monitoring of
12


bacteriophage strains specific for Xoo has been used in forecasting since the
1960s. Under particular agroenvironmental conditions, an increment of
bacteriophage population in irrigation water and paddy fields early in the
planting season correlates well with an increase in bacterial populations and is
used to predict BLB outbreaks.
However, the bacteriophage forecasting system is not practised
extensively in tropical Asia, because rice cultivation is mostly rainfed, limiting
the use of phage detection in paddy fields (Murty and Devadath, 1982;
Wakimoto and Mew, 1979). In general also, disease forecasting has been of
limited utility because chemical control is unavailable or impractical. Xa26).
Curiously, Xa3 is typically effective only in adult plants, but against at least one
race it is effective at all stages of growth. Some genes condition resistance to a
wide spectrum of Xoo races (e.g. Xa21, Xa23), whereas others are effective
against only one or a few races that may be limited to a particular geographical
location (e.g. Xa1). Most R genes to BLB are dominant, but some are recessive
(e.g. xa5, xa13), and some display semidominance (e.g. Xa27). Among the

handful of genes that have been cloned there is remarkable structural diversity
(see below).
Most R genes to BLB have been introgressed into the background of the
susceptible indica cultivar IR24 to develop a set of near isogenic lines (NILs),
and some have been pyramided, either through classical breeding and markerassisted selection or through genetic engineering, to develop new plant types
and NILs (Narayanan et al., 2002; Sanchez et al., 2000; Singh et al., 2001).
Pyramid lines have displayed higher levels and/or wider spectra of resistance
to BLB than the parental NILs with single R genes, suggesting synergism and
complementation among R genes (Adhikari et al., 1999a; Huang et al., 1997;
13


Narayanan et al., 2002). With pyramid lines, it is possible to conduct
quantitative analysis on the effect of each gene and their interactions, but most
importantly, to maximize the performance and durability of genetic resistance.
Resistance of rice to specific Xoo races is governed by both major R genes
with a qualitative effect that condition complete resistance (CR) and polygenes
with a quantitative effect (quantitative trait loci, QTL) that condition partial
resistance (PR) (Koch and Parlevliet, 1991b; Li et al., 2006). A recent study of
the epistatic effects between R genes and QTL for resistance in rice revealed a
complex genetic network in which the interactions between alleles at the rice R
loci and alleles at the corresponding.
Breeding and deployment of resistant cultivars carrying major resistance
(R) genes has been the most effective approach to controlling BLB. To date, 29
R genes to BLB have been identified (see Table 1 for details and references),
mostly from O. sativa ssp. indica cultivars, but some also from japonica varieties,
and from related wild species including O. longistaminata, O. rufipogon, O.
minuta and O. officinalis (Brar and Khush, 1997; Lee et al., 2003). In addition,
several resistance genes or alleles have been produced by mutating cultivated
rice lines, e.g. by treatment with N-methyl-N-nitrosourea or thermal neutron

irradiation, or by somaclonal mutagenesis (Gao et al., 2001; Lee et al., 2003;
Nakai et al., 1988). Some R genes are effective only in adult plants (e.g. Xa21)
whereas most do not seem to be developmentally regulated (e.g. Xa23,
2.8 XANTHOMONAS ORYZAE PV. ORYZAE (XOO)
2.8.1 Morphology and structural
Xanthomonas oryzae (Xo) is a rod-shaped, round-ended, Gram-negative
species. Individual cells vary in length from approximately 0.7 µ m to 2.0 µ m and
14